Enhancer_id Pubmed Enhancer_symbol Reference_genome Chromosome Start_position End_position Species Biosample_name Experiment_class Enhancer_experiment Enhancer_tar_ex_De Enhancer_type Target_gene target_gene_strong_experiment target_gene_weak_experiment target_gene_experiment_description target_gene_other_name Disease DO mesh Enhancer_function Enhancer_function_experiment En_function_ex_de TF_name TF_other_name Experiment TF_experiment_de SNP_id SNP_position SNP_experiment Distance_from_TSS Distance_from_TSS_AB enhancer_MID TSS n_Distance_from_TSS E_01_001 28652246 -- hg19 chr6 86160197 86176777 Human Melanoma Low+High throughput "ChIP-seq,ChIP-qPCR" A ChIP-qPCR approach revealed prominent binding of c-Jun at the AP-1 sites #1-2 and #5-6 (Fig. 3E) coinciding with enhancer regions (H3K27Ac) identified by the ENCODE ChIP-seq project (Fig. 3D). Enhancer NT5E CRISPR/Cas9 "qPCR,ChIP-qPCR" "Next, we devised a CRISPR/Cas9 approach to clarify the functional relevance of the individual c-Jun/AP-1 binding sites.c-Jun/AP-1 induces CD73 expression via binding to an intronic enhancer.Strong enrichment of AP-1 site mutagenesis frequencies in the CD73 low subfractions (52% versus 4% in CD73 lowest 10% versus highest 10%) was observed only in the cell population transfected with the sgRNA against the c-Jun/AP-1 site #5 (Fig. 3G-H) being consistent with reduced CD73 surface expression (Fig. S4A)." "CALJA,CD73,E5NT,NT,NT5,NTE,eN,eNT" Melanoma DOID:1909 D008545 The intronic Enhancer of the CD73 gene controls its c-Jun/AP-1-dependent transcriptional activation downstream of mitogenic MAPK and inflammatory cytokine signaling. "ChIP-qPCR,CRISPR/Cas9" "Taken together, we identified a critical c-Jun/AP-1 binding site in an intronic enhancer of the CD73 gene that controls its c-Jun/AP-1-dependent transcriptional activation downstream of mitogenic MAPK and inflammatory cytokine signaling." JUN "AP-1,AP1,c-Jun,cJUN,p39" ChIP-qPCR We looked for potential c-Jun/AP-1¨Cbinding sites in the CD73 gene using ENCODE ChIP-Seq data.A ChIP-qPCR approach revealed prominent binding of c-Jun at the AP-1 sites #1¨C2 and #5¨C6 coinciding with Enhancer regions (H3K27Ac)identified by the ENCODE ChIP-seq project. -- -- -- Intron 9186 E_01_002 26751173 ER¦Áenh588 hg19 chr11 69329684 69330954 Human "MCF-7,T-47D" Low+High throughput "ChIP-seq,CRISPR/Cas9,Luciferase Reporter Assay,Western blot,Competitive Proliferation Assay" "To demonstrate the generalizability of our screening approach, we designed a dropout screen to identify novel ER¦Á-bound enhancers. We started by cloning ER¦Áenh588 WT region in a reporter vector and verified that it has strong ER¦Á¨Cdependent transcription-enhancing activity, since mutations in the ER¦Á binding site completely abolish the enhancer activity and response to 17¦Â-estradiol (Supplementary Fig. 4b). Next, we examined endogenous ER¦Á binding at ER¦Áenh588 region by ChIP-seq and observed a substantial decrease in both MCF-7 and T47D cells expressing sgRNA-ER¦Áenh588 (Fig. 3e). Because CCND1 is a puttive target of ER¦Áenh588, we verified the endogenous expression of ER¦Áenh588 eRNAs and CCND1 mRNA and protein in MCF-7 and T47D cells transduced with sgRNA-ER¦Áenh588.Reassuringly, we found that the expression of eRNA, mRNA and protein is significantly decreased (P < 0.01; about twofold) in both cell lines (Fig. 3f¨Ch). These results indicate that ER¦Áenh588 is a bona fide enhancer element that regulates the expression of CCND1. " Enhancer CCND1 -- "ChIP-seq,Western blot,qPCR" "Next, we examined endogenous ER¦Á binding at ER¦Áenh588 region by ChIP-seq and observed a substantial decrease in both MCF-7 and T47D cells expressing sgRNA-ER¦Áenh588 (Fig. 3e).Because CCND1 is a putative target of ER¦Áenh588, we verified the endogenous expression of ER¦Áenh588 eRNAs and CCND1 mRNA and protein in MCF-7 and T47D cells transduced with sgRNA-ER¦Áenh588. Reassuringly, we found that the expression of eRNA, mRNA and protein is significantly decreased (P < 0.01; about twofold) in both cell lines (Fig. 3f¨Ch). These results indicate that ER¦Áenh588 is a bona fide enhancer element that regulates the expression of CCND1.Next, we assessed the dependency of ER¦Áenh588 and CCND1 endogenous expression on estrogen signaling by qPCR (Fig. 3i). As expected, we confirmed that both ER¦Áenh588 eRNA and CCND1 mRNA are upregulated in MCF-7 cells upon treatment with 17¦Â-estradiol. However, we found that sgRNAenh588 severely compromises the induction of eRNA expression and completely abolishes CCND1 mRNA activation in MCF-7 cells. These results suggest that the activation of CCND1 expression by estrogen in breast cancer cells requires a fully active ER¦Áenh588 enhancer element." "BCL1, D11S287E, PRAD1, U21B31" Breast Cancer DOID:1612 D001943 "The phenotypic outcome of disrupting ER¦Áenh588 activity shows that MCF-7 cells transduced with sgRNAenh58 display a ~2.5-fold reduction in S-phase entry, compared to control transduced cells, due to decreased CCND1 expression." Flow Cytometry "Finally,as CCND1 is a crucial component of the G1-S phase transition, we examined the phenotypic outcome of disrupting ER¦Áenh588 activity Figure 3j shows that MCF-7 cells transduced with sgRNAenh58 display a ~2.5-fold reduction in S-phase entry, compared to control transduced cells, due to decreased CCND1 expression. " ESR1 "ER,ESR,ESRA,ESTRR,Era,NR3A1" "Luciferase Reporter Assay,ChIP-seq" "We started by cloning ER¦Áenh588 WT region in a reporter vector and verified that it has strong ER¦Á¨Cdependent transcription-enhancing activity, since mutations in the ER¦Á binding site completely abolish the enhancer activity and response to 17¦Â-estradiol (Supplementary Fig. 4b). Next, we examined endogenous ER¦Á binding at ER¦Áenh588 region by ChIP-seq and observed a substantial decrease in both MCF-7 and T47D cells expressing sgRNA-ER¦Áenh588 (Fig. 3e). " -- -- -- >2KB 125553 E_01_003 26252164 NEC1 Enhancer hg19 chr2 223164458 223164688 Human Melanoma Low throughput "Phylogenetic Footprinting,Luciferase Reporter Assay" "Through phylogenic footprinting and in silico promoter analys islands of homology within the proximal 1.6 Kb PAX3 promo referred to as either NCE1 and NCE2 ([Milewski et al., 2004], shown schematically and by sequence in Fig. 4A,B) or sites I and II [Natoli et al., 1997]. Two FOX long sites were identified in the PAX3 promoter, one in NCE1 (F1) and another in NCE2 (F2). To determine if these FOXD3 sites are active in melanoma cells, PAX3pm constructs containing intact or mutated F1 and F2 sites were transfected into A375 and SKMEL-28 melanoma cell lines (Fig. 6C,D). While mutation of the F1 site reduced reporter expression to 78.5% ¡À18.6% (A375) and 82.1% ¡À19.4% (SKMEL-28) of the PAX3pm levels, this reduction was not significant. However, if the F2 or both F1 and F2 sites were mutated, the reporter levels dropped to 51.1% ¡À13.8% and 45.6% ¡À11.6% for A375 cells and 32.4% ¡À9.8% and 37.5% ¡À10.2% of the PAX3pm vector levels, respectively. This supports that FOXD3 can drive PAX3 expression from both the F1 and F2 enhancers, but the F2 enhancer is the site utilized in melanoma cells." Enhancer PAX3 -- Luciferase Reporter Assay "PAX3pm constructs with or without mutated F1 and/or F2 sites were also transfected into 293T cells in the presence or absence of a FOXD3 expression vector (Fig. 5D). FOXD3 was able to drive reporter expression at significant levels when either or both sites were present in comparison to promoter vector alone. However, when both sites were mutated, the ability of FOXD3 to drive this construct was attenuated." "CDHS,HUP2,WS1,WS3" Melanoma DOID:1909 D008545 The PAX3 gene contains two FOX binding motifs within highly conserved enhancer regulatory elements that are essential for neural crest development. "ChIP,PCR" The PAX3 gene contains two FOX binding motifs within highly conserved Enhancer regulatory elements that are essential for neural crest development. FOXD3 "AIS1,Genesis,HFH2,VAMAS2" EMSA "EMSA analysis for FOXD3 binding of PAX3 promoter sequences in vitro. Labeled probe containing the PAX3 promoter F1 site (lanes 1-3) or F2 site (lanes 4-6) were mixed with 293T lysate without (lanes 1, 4) or with (lanes 2, 3, 5, 6) exogenous FOXD3 protein. Binding of the labeled probe was inhibited with cold probe competition (lanes 3, 6)." -- -- -- >2KB 99968 E_01_004 26252164 NEC2 Enhancer hg19 chr2 223164846 223165065 Human Melanoma Low throughput "Phylogenetic Footprinting,Luciferase Reporter Assay" "Through phylogenic footprinting and in silico promoter analys islands of homology within the proximal 1.6 Kb PAX3 promo referred to as either NCE1 and NCE2 ([Milewski et al., 2004], shown schematically and by sequence in Fig. 4A,B) or sites I and II [Natoli et al., 1997]. Two FOX long sites were identified in the PAX3 promoter, one in NCE1 (F1) and another in NCE2 (F2). To determine if these FOXD3 sites are active in melanoma cells, PAX3pm constructs containing intact or mutated F1 and F2 sites were transfected into A375 and SKMEL-28 melanoma cell lines (Fig. 6C,D). While mutation of the F1 site reduced reporter expression to 78.5% ¡À18.6% (A375) and 82.1% ¡À19.4% (SKMEL-28) of the PAX3pm levels, this reduction was not significant. However, if the F2 or both F1 and F2 sites were mutated, the reporter levels dropped to 51.1% ¡À13.8% and 45.6% ¡À11.6% for A375 cells and 32.4% ¡À9.8% and 37.5% ¡À10.2% of the PAX3pm vector levels, respectively. This supports that FOXD3 can drive PAX3 expression from both the F1 and F2 enhancers, but the F2 enhancer is the site utilized in melanoma cells." Enhancer PAX3 -- Luciferase Reporter Assay "PAX3pm constructs with or without mutated F1 and/or F2 sites were also transfected into 293T cells in the presence or absence of a FOXD3 expression vector (Fig. 5D). FOXD3 was able to drive reporter expression at significant levels when either or both sites were present in comparison to promoter vector alone. However, when both sites were mutated, the ability of FOXD3 to drive this construct was attenuated." "CDHS,HUP2,WS1,WS3" Melanoma DOID:1909 D008545 The PAX3 gene contains two FOX binding motifs within highly conserved enhancer regulatory elements that are essential for neural crest development. "ChIP,PCR" The PAX3 gene contains two FOX binding motifs within highly conserved Enhancer regulatory elements that are essential for neural crest development. FOXD3 "AIS1,Genesis,HFH2,VAMAS2" EMSA "EMSA analysis for FOXD3 binding of PAX3 promoter sequences in vitro. Labeled probe containing the PAX3 promoter F1 site (lanes 1-3) or F2 site (lanes 4-6) were mixed with 293T lysate without (lanes 1, 4) or with (lanes 2, 3, 5, 6) exogenous FOXD3 protein. Binding of the labeled probe was inhibited with cold probe competition (lanes 3, 6)." -- -- -- >2KB 100351 E_01_005 26751173 p53enh3507 hg19 chr6 36631690 36640853 Human BJ Low+High throughput "ChIP-seq,CRISPR/Cas9,Luciferase Reporter Assay,siRNA,GRO-seq" "We identified several functional enhancer elements and characterized the role of two of them in mediating p53 (TP53) and ERa (ESR1) gene regulation. To build a CRISPR-Cas9 single guide RNA (sgRNA) library, we followed a strategy that enabled us to target ¡Ö90% of p53-bound enhancers ( (Fig. 1b and Supplementary Notably, two independent sgRNAs targeted a putative enhancer located ~10 kb upstream of CDKN1A (formerly known as p21), which is a key effector of p53-dependent OIS (Fig. 1e; p53enh3507). Another top-scoring sgRNA mapped to a known p53-responsive element that is located proximal to the transcription start site of CDKN1A (Fig. 1e; p53enh3508). Next, we assessed the enhancer capacity of the p53enh3507 region by cloning it into a reporter vector and verified that it strongly induces transcription (Fig. 2a). We also observed a substantial reduction in enhancing activity upon siRNA-mediated knockdown of p53 (Fig. 2b). We obtained similar results for the p53enh3508 enhancer element (Supplementary Fig. 2b,c). " Enhancer CDKN1A -- Luciferase Reporter Assay "Next, we assessed the enhancer capacity of the p53enh3507 region by cloning it into a reporter vector and verified that it strongly induces transcription (Fig. 2a). We also observed a substantial reduction in enhancing activity upon siRNA-mediated knockdown of p53 (Fig. 2b). We obtained similar results for the p53enh3508 enhancer element (Supplementary Fig. 2b,c). Interestingly,concurrent disruption of TP53enh3507 and TP53enh3508 resulted in further reduction of CDKN1A expression,suggesting an independent regulation by both Enhancer elements. " "CAP20,CDKN1,CIP1,MDA-6,P21,SDI1,WAF1,p21CIP1" -- -- -- The p53enh3507 is an endogenous Enhancer of CDKN1A and disruption of this region causes bypass of senescence in p53-WT cells. "BrdU Assay,¦Â-galactosidase Assay" "Upon oncogene activation, one major function of p53 is to activate an irreversible cell-cycle arrest program named oncogene-induced senescence (OIS). We performed BrdU labeling and senescence-associated ¦Â-galactosidase (¦Â-gal) assays and found that sgRNA-p53enh3507 and sgRNA-p53enh3508 caused OIS bypass and continuous cell proliferation (Fig. 1g,h and Supplementary Fig. 2a) Collectively, these results demonstrate that p53enh3507 is an endogenous Enhancer of CDKN1A and disruption of this region causes bypass of senescence in p53-WT cells.Moreover, cooperative action of p53enh3507 (distal Enhancer of CDKN1A-deCD-KN1A) and p53enh3508 (proximal Enhancer of CDKN1A-peCDKN1A) is required to activate CDKN1A expression and initiate OIS." TP53 "BCC7,BMFS5,LFS1,P53,TRP53" Luciferase Reporter Assay "The same assay as in a, only that cells were co-transfected with control, or p53-targeting short interfering RNA (si-Cont.; si-p53). A reporter vector containing the enhancer region p53-BER4 was used as a positive control for p53-dependency. The efficiency of p53 knockdown was determined by immunoblot analysis." -- -- -- >2KB 7964 E_01_006 26751173 p53enh3508 hg19 chr6 36640690 36643070 Human BJ Low+High throughput "ChIP-seq,CRISPR/Cas9,Luciferase Reporter Assay,siRNA" "We present two distinct CRISPR-Cas9 genetic screens to identify and characterize functional enhancers in their native context. Our strategy is to target Cas9 to transcription factor binding sites in enhancer regions. We identified several functional enhancer elements and characterized the role of two of them in mediating p53 (TP53) and ERa (ESR1) gene regulation. To build a CRISPR-Cas9 single guide RNA (sgRNA) library, we followed a strategy that enabled us to target ¡Ö90% of p53-bound enhancers ( (Fig. 1b and Supplementary Notably, two independent sgRNAs targeted a putative enhancer located ~10 kb upstream of CDKN1A (formerly known as p21), which is a key effector of p53-dependent OIS (Fig. 1e; p53enh3507). Another top-scoring sgRNA mapped to a known p53-responsive element that is located proximal to the transcription start site of CDKN1A (Fig. 1e; p53enh3508). Next, we assessed the enhancer capacity of the p53enh3507 region by cloning it into a reporter vector and verified that it strongly induces transcription (Fig. 2a). We also observed a substantial reduction in enhancing activity uponsiRNA-mediated knockdown of p53 (Fig. 2b). We obtained similar results for the p53enh3508 enhancer element (Supplementary Fig. 2b,c). " Enhancer CDKN1A -- Luciferase Reporter Assay "Next, we assessed the enhancer capacity of the p53enh3507 region by cloning it into a reporter vector and verified that it strongly induces transcription (Fig. 2a). We also observed a substantial reduction in enhancing activity upon siRNA-mediated knockdown of p53 (Fig. 2b). We obtained similar results for the p53enh3508 enhancer element (Supplementary Fig. 2b,c). Interestingly,concurrent disruption of TP53enh3507 and TP53enh3508 resulted in further reduction of CDKN1A expression,suggesting an independent regulation by both Enhancer elements. " "CAP20,CDKN1,CIP1,MDA-6,P21,SDI1,WAF1,p21CIP1" -- -- -- Study found that sgRNA-p53enh3507 and sgRNA-p53enh3508 caused OIS bypass and continuous cell proliferation CRISPR/Cas9 "Upon oncogene activation, one major function of p53 is to activate an irreversible cell-cycle arrest program named oncogene-induced senescence (OIS). We performed BrdU labeling and senescence-associated ¦Â-galactosidase (¦Â-gal) assays and found that sgRNA-p53enh3507 and sgRNA-p53enh3508 caused OIS bypass and continuous cell proliferation (Fig. 1g,h and Supplementary Fig. 2a) Collectively, these results demonstrate that p53enh3507 is an endogenous Enhancer of CDKN1A and disruption of this region causes bypass of senescence in p53-WT cells.Moreover, cooperative action of p53enh3507 (distal Enhancer of CDKN1A-deCD-KN1A) and p53enh3508 (proximal Enhancer of CDKN1A-peCDKN1A) is required to activate CDKN1A expression and initiate OIS." TP53 "BCC7,BMFS5,LFS1,P53,TRP53" Luciferase Reporter Assay "The same assay as in a, only that cells were co-transfected with control, or p53-targeting short interfering RNA (si-Cont.; si-p53). A reporter vector containing the enhancer region p53-BER4 was used as a positive control for p53-dependency. The efficiency of p53 knockdown was determined by immunoblot analysis." -- -- -- >2KB 2356 E_01_007 26374622 -- hg19 chr21 36180372 36181392 Human "Leukemia Cell Lines,K-562" Low throughput "Luciferase Reporter Assay,DNaseI hypersensitivity" "We searched 510 bp P1 promoter, 218bp P2 promoter, 237bp +23 enhancer, and 392bp intron 5.2 enhancer of RUNX1, 109bp upstream regulatory element (URE) and 296bp URE distal of PU.1, 443bp promoter of CEBPA, and 324bp promoter of CEBPE for single nucleotide polymorphisms (SNPs) in UCSC genome browser.510bp RUNX1 intron 5.2 enhancer contains H3K27Ac mark which is often found near active regulatory elements, and DNaseI hypersensitivity clusters which are another mark for transcription active regions." Enhancer RUNX1 -- Luciferase Reporter Assay "510bp RUNX1 intron 5.2 enhancer contains H3K27Ac mark which is often found near active regulatory elements, and DNaseI hypersensitivity clusters which are another mark for transcription active regions.We next cloned 510bp RUNX1 intron 5.2 enhancer with either rs2249650 minor allele (A) and rs2268276 major allele (G) or rs2249650 major allele (G)and rs2268276 minor allele (A) into pGL3-promoter vector. Then luciferase reporter plasmids were transfected into leukemia cell lines K562 to measure the reporter activities. 510bp reporter with rs2249650 A allele and rs2268276 G allele (L1A2G) showed significantly higher promoter activities than the reporter with rs2249650 G allele and rs2268276 A allele (Figure 2b, left)." "AML1,AML1-EVI-1,AMLCR1,CBF2alpha,CBFA2,EVI-1,PEBP2aB,PEBP2alpha" Acute Myeloid Leukemia DOID:9119 D015470 Rs2249650 and rs2268276 at RUNX1 intron 5.2 enhancer were found to be associated with acute myeloid leukemia susceptibility. "Luciferase Reporter Assay,EMSA" "We next narrowed 510bp enhancer down into 69 bp (20bp+rs2249650(A/G)-rs2268276(G/A)+20bp) DNA and cloned these nucleotides into pGL3-promoter to construct 1A2G, 1G2A, 1A2A, and 1G2G reporters and measured their activities. Both 1A2A and 1G2G reporters had medium luciferase activity. The activity of 1A2A reporter was not significantly different from that of 1A2G reporter whereas the activity of 1G2G was significantly different from that of 1A2G. Thus, rs2249650 contributed to reporter activities more than rs2268276." SPI1 "OF,PU.1,SFPI1,SPI-1,SPI-A" EMSA "We checked the 69bp DNA and found that a classical consensus PU.1 binding sequence (GAGGAA) is located between rs2249650 and rs2268276 (Figure 1b). We adopted supershift to test if the differentially bound proteins in EMSA is PU.1. Indeed, PU.1 antibodies resulted in the lost of interested protein (Figure 3b)." "rs2249650,rs2268276" "34808689, 34808717 " "Luciferase Reporter Assay,EMSA" Intron 20785 E_01_008 26766440 -- hg19 chr8 23448386 23454886 Human Human CD34+ Hematopoietic Stem/Progenitor Cell Low+High throughput "CRISPR/Cas9,ChIP-seq" "The enhancer cluster upstream of the SLC25A37 gene, consisting of three distinct constituent enhancers as measured by H3K4me1 and H3K27ac ChIP-seq, is defined as an erythroid-specific super-enhancer in both human (A5 ProE) and mouse (G1ER) erythroid cells (Figure 3A; Table S4). To define the regulatory components of the SLC25A37 super-enhancer, we asked whether the function of each constituent enhancer depends on the activity of neighboring enhancers in situ. We employed site-directed loss-of-function analysis of the SLC25A37 super-enhancer constituents using CRISPR/Cas9-mediated genomic engineering.We focused on the orthologous mouse super-enhancer in the murine G1E/G1ER erythroid cell model (Welch et al., 2004). We designed sequence-specific guide RNAs (or sgRNAs) flanking each constituent enhancer (E1, E2 and E3) or the promoter (P) (Figure 3B). Upon transfection into undifferentiated G1E cells together with an SpCas9 expressing construct, we screened and obtained multiple independent single cell-derived clones containing bi-allelic deletion of each enhancer (Figure S2B,C). Surprisingly, knockout of individual enhancers confer markedly varying effects on Slc25a37 expression. Specifically, while deletion of E1 or E2 individually only modestly or slightly impairs Slc25a37 activation during differentiation, respectively, E3 deletion abolishes its activation resulting in a 15-fold decrease in expression (48h after ¦Â-estradiol treatment; Figure 3C). " Enhancer SLC25A37 CRISPR/Cas9 ChIP-seq "The enhancer cluster upstream of the SLC25A37 gene, consisting of three distinct constituent enhancers as measured by H3K4me1 and H3K27ac ChIP-seq, is defined as an erythroid-specific super-enhancer in both human (A5 ProE) and mouse (G1ER) erythroid cells (Figure 3A; Table S4). To define the regulatory components of the SLC25A37 super-enhancer, we asked whether the function of each constituent enhancer depends on the activity of neighboring enhancers in situ. We employed site-directed loss-of-function analysis of the SLC25A37 super-enhancer constituents using CRISPR/Cas9-mediated genomic engineering.We focused on the orthologous mouse super-enhancer in the murine G1E/G1ER erythroid cell model (Welch et al., 2004). Surprisingly, knockout of individual enhancers confer markedly varying effects on Slc25a37 expression. Specifically, while deletion of E1 or E2 individually only modestly or slightly impairs Slc25a37 activation during differentiation, respectively, E3 deletion abolishes its activation resulting in a 15-fold decrease in expression (48h after ¦Â-estradiol treatment; Figure 3C). " "HT015,MFRN,MFRN1,MSC,MSCP,PRO1278,PRO1584,PRO2217" -- -- -- GATA2-to-GATA1 switch is prevalent at dynamic enhancers and drives erythroid enhancer commissioning. CRISPR/Cas9 "While the E2 and E3 Enhancers are indispensable for maximal Gata2 activation in stem/progenitor cells, the E1 Enhancer is required to maintain Gata2 repression in committed erythroid cells. " "GATA1,TAL1" "ERYF1,GATA-1,GF-1,GF1,NF-E1,NFE1,XLANP,XLTDA,XLTT,SCL,TCL5,bHLHa17,tal-1" ChIP-seq "Strikingly, while only 3% (22,570 of 707,718) of the GATA1 motif-matched loci are covered by GATA1 ChIP-seq at a genome scale in ProEs, 34% of identified GATA1 motifs are covered by GATA1 within the Enhancer context.Similar pattern is observed for another principal erythroid regulator TAL1." -- -- -- >2KB 65273 E_01_009 26766440 -- hg19 chr8 23433011 23439511 Human Human CD34+ Hematopoietic Stem/Progenitor Cell Low+High throughput "CRISPR/Cas9,ChIP-seq" "The enhancer cluster upstream of the SLC25A37 gene, consisting of three distinct constituent enhancers as measured by H3K4me1 and H3K27ac ChIP-seq, is defined as an erythroid-specific super-enhancer in both human (A5 ProE) and mouse (G1ER) erythroid cells (Figure 3A; Table S4). To define the regulatory components of the SLC25A37 super-enhancer, we asked whether the function of each constituent enhancer depends on the activity of neighboring enhancers in situ. We employed site-directed loss-of-function analysis of the SLC25A37 super-enhancer constituents using CRISPR/Cas9-mediated genomic engineering.We focused on the orthologous mouse super-enhancer in the murine G1E/G1ER erythroid cell model (Welch et al., 2004). We designed sequence-specific guide RNAs (or sgRNAs) flanking each constituent enhancer (E1, E2 and E3) or the promoter (P) (Figure 3B). Upon transfection into undifferentiated G1E cells together with an SpCas9 expressing construct, we screened and obtained multiple independent single cell-derived clones containing bi-allelic deletion of each enhancer (Figure S2B,C). Surprisingly, knockout of individual enhancers confer markedly varying effects on Slc25a37 expression. Specifically, while deletion of E1 or E2 individually only modestly or slightly impairs Slc25a37 activation during differentiation, respectively, E3 deletion abolishes its activation resulting in a 15-fold decrease in expression (48h after ¦Â-estradiol treatment; Figure 3C). " Enhancer SLC25A37 CRISPR/Cas9 ChIP-seq "The enhancer cluster upstream of the SLC25A37 gene, consisting of three distinct constituent enhancers as measured by H3K4me1 and H3K27ac ChIP-seq, is defined as an erythroid-specific super-enhancer in both human (A5 ProE) and mouse (G1ER) erythroid cells (Figure 3A; Table S4). To define the regulatory components of the SLC25A37 super-enhancer, we asked whether the function of each constituent enhancer depends on the activity of neighboring enhancers in situ. We employed site-directed loss-of-function analysis of the SLC25A37 super-enhancer constituents using CRISPR/Cas9-mediated genomic engineering.We focused on the orthologous mouse super-enhancer in the murine G1E/G1ER erythroid cell model (Welch et al., 2004). Surprisingly, knockout of individual enhancers confer markedly varying effects on Slc25a37 expression. Specifically, while deletion of E1 or E2 individually only modestly or slightly impairs Slc25a37 activation during differentiation, respectively, E3 deletion abolishes its activation resulting in a 15-fold decrease in expression (48h after ¦Â-estradiol treatment; Figure 3C). " "HT015,MFRN,MFRN1,MSC,MSCP,PRO1278,PRO1584,PRO2217" -- -- -- GATA2-to-GATA1 switch is prevalent at dynamic enhancers and drives erythroid enhancer commissioning. CRISPR/Cas9 "While the E2 and E3 Enhancers are indispensable for maximal Gata2 activation in stem/progenitor cells, the E1 Enhancer is required to maintain Gata2 repression in committed erythroid cells. " "GATA1,TAL1" "ERYF1,GATA-1,GF-1,GF1,NF-E1,NFE1,XLANP,XLTDA,XLTT,SCL,TCL5,bHLHa17,tal-1" ChIP-seq "Strikingly, while only 3% (22,570 of 707,718) of the GATA1 motif-matched loci are covered by GATA1 ChIP-seq at a genome scale in ProEs, 34% of identified GATA1 motifs are covered by GATA1 within the Enhancer context.Similar pattern is observed for another principal erythroid regulator TAL1." -- -- -- >2KB 49898 E_01_010 26766440 -- hg19 chr8 23422136 23432636 Human Human CD34+ Hematopoietic Stem/Progenitor Cell Low+High throughput "CRISPR/Cas9,ChIP-seq" "The enhancer cluster upstream of the SLC25A37 gene, consisting of three distinct constituent enhancers as measured by H3K4me1 and H3K27ac ChIP-seq, is defined as an erythroid-specific super-enhancer in both human (A5 ProE) and mouse (G1ER) erythroid cells (Figure 3A; Table S4). To define the regulatory components of the SLC25A37 super-enhancer, we asked whether the function of each constituent enhancer depends on the activity of neighboring enhancers in situ. We employed site-directed loss-of-function analysis of the SLC25A37 super-enhancer constituents using CRISPR/Cas9-mediated genomic engineering.We focused on the orthologous mouse super-enhancer in the murine G1E/G1ER erythroid cell model (Welch et al., 2004). We designed sequence-specific guide RNAs (or sgRNAs) flanking each constituent enhancer (E1, E2 and E3) or the promoter (P) (Figure 3B). Upon transfection into undifferentiated G1E cells together with an SpCas9 expressing construct, we screened and obtained multiple independent single cell-derived clones containing bi-allelic deletion of each enhancer (Figure S2B,C). Surprisingly, knockout of individual enhancers confer markedly varying effects on Slc25a37 expression. Specifically, while deletion of E1 or E2 individually only modestly or slightly impairs Slc25a37 activation during differentiation, respectively, E3 deletion abolishes its activation resulting in a 15-fold decrease in expression (48h after ¦Â-estradiol treatment; Figure 3C). " Enhancer SLC25A37 CRISPR/Cas9 ChIP-seq "The enhancer cluster upstream of the SLC25A37 gene, consisting of three distinct constituent enhancers as measured by H3K4me1 and H3K27ac ChIP-seq, is defined as an erythroid-specific super-enhancer in both human (A5 ProE) and mouse (G1ER) erythroid cells (Figure 3A; Table S4). To define the regulatory components of the SLC25A37 super-enhancer, we asked whether the function of each constituent enhancer depends on the activity of neighboring enhancers in situ. We employed site-directed loss-of-function analysis of the SLC25A37 super-enhancer constituents using CRISPR/Cas9-mediated genomic engineering.We focused on the orthologous mouse super-enhancer in the murine G1E/G1ER erythroid cell model (Welch et al., 2004). Surprisingly, knockout of individual enhancers confer markedly varying effects on Slc25a37 expression. Specifically, while deletion of E1 or E2 individually only modestly or slightly impairs Slc25a37 activation during differentiation, respectively, E3 deletion abolishes its activation resulting in a 15-fold decrease in expression (48h after ¦Â-estradiol treatment; Figure 3C). " "HT015,MFRN,MFRN1,MSC,MSCP,PRO1278,PRO1584,PRO2217" -- -- -- GATA2-to-GATA1 switch is prevalent at dynamic enhancers and drives erythroid enhancer commissioning. CRISPR/Cas9 "While the E2 and E3 Enhancers are indispensable for maximal Gata2 activation in stem/progenitor cells, the E1 Enhancer is required to maintain Gata2 repression in committed erythroid cells. " "GATA1,TAL1" "ERYF1,GATA-1,GF-1,GF1,NF-E1,NFE1,XLANP,XLTDA,XLTT,SCL,TCL5,bHLHa17,tal-1" ChIP-seq "Strikingly, while only 3% (22,570 of 707,718) of the GATA1 motif-matched loci are covered by GATA1 ChIP-seq at a genome scale in ProEs, 34% of identified GATA1 motifs are covered by GATA1 within the Enhancer context.Similar pattern is observed for another principal erythroid regulator TAL1." -- -- -- >2KB 41023 E_01_011 26656844 MYC-LASE hg19 chr8 129166547 129190290 Human "A549,NCI-H358,NCI-H2009" Low+High throughput "ChIP-seq,Luciferase Reporter Assay,3C-qPCR,CRISPR/Cas9,siRNA" "We performed H3K27ac ChIPseq in two additional lung adenocarcinoma cell lines, NCI-H2009 and NCI-H358, the lung squamous cell carcinoma cell line HCC95 and the small cell lung carcinoma cell line NCI-H2171 and validated that MYC-LASE is part of a lung adenocarcinoma¨Cspecific super-enhancer." Super-Enhancer MYC 3C -- "We performed chromosome conformation capture (3C) assays in A549 and Ishikawa cells and found that MYC-LASE physically interacted with the MYC promoter only in A549 cells and, reciprocally, that MYC-ECSE physically interacted with the MYC promoter only in Ishikawa cells" "MRTL,MYCC,bHLHe39,c-Myc" Lung Adenocarcinoma DOID:3910 D000077192 Two distinct focal amplifications of super-enhancers 3¡ä to MYC in lung adenocarcinoma (MYC-LASE) and endometrial carcinoma (MYC-ECSE) are physically associated with the MYC promoter and correlate with MYC overexpression. "3C,ChIP-seq" "We performed chromosome conformation capture (3C) assays in A549 and Ishikawa cells and found that MYC-LASE physically interacted with the MYC promoter only in A549 cells and, reciprocally,that MYC-ECSE physically interacted with the MYC promoter only in Ishikawa cells (Fig. 2e). In addition, tumors with amplification of MYC alone or MYC-LASE or MYC-ECSE alone had higher MYC expression than tumors lacking either amplification (Fig. 2f). These results suggest that both MYC-LASE and MYC-ECSE drive MYC expression through lineage-specific chromatin loops." -- -- -- -- -- -- -- >2KB 430105 E_01_012 24413736 -- hg19 chr5 51773619 51857504 Human "HEK-293,¦Â-Cell" Low+High throughput "RNA-seq,ChIP-seq,ChIP,EMSA,PCR,4C,Luciferase Reporter Assay" "To define enhancer clusters, we first created 1,000 iterations of randomized C3 sites in the mappable genome of individual chromosomes. We then calculated for each chromosome the 25th percentile of inter-site distances of randomized C3 sites (Supplementary Fig. 6a). Next,we defined clusters of islet C3 sites as any group of ¡Ý3 C3 sites in which all adjacent C3 sites were separated by less than the abovementioned 25th percentile distance for randomized sites in the same chromosome. The distribution of islet C3 clusters differed from that of clusters generated with randomized C3 sites (Supplementary Fig. 6b)" Enhancer ISL1 4C -- interactions between the ISL1 promoter and the C3-3 enhancer tested in zebrafish transgenics and located >1 Mb away (green star) "ISLET1,ist-1" Type 2 Diabetes Mellitus DOID:9352 D003924 Dysregulation of islet enhancers is relevant to the mechanisms underlying type 2 diabetes. Luciferase Reporter Assay We next attempted to define functional enhancer variants within established GWAs loci that might be causal for T2D susceptibility. These results illustrate how islet accessible chromatin maps can pinpoint functional cis-regulatory variants that are strong candidates for a causal role in driving T2D-association signals. "FOXA2,MAFB,NKX2-2" "HNF3B,TCF3B,DURS3,KRML,MCTO,NKX2.2,NKX2B" ChIP-seq "To determine the genomic binding sites of these islet transcription factors, we used chromatin immunoprecipitation and sequencing(ChIP-seq) in duplicate human islet samples and identified 3,911-32,747 high-confidence sites per transcription factor. " -- -- -- >2KB 1136605 E_01_013 26656844 -- hg19 chr13 73880690 73990596 Human BICR-31 Low+High throughput "ChIP-seq,Luciferase Reporter Assay,3C-qPCR,CRISPR/Cas9,siRNA" "The focal amplification on chr13q identified in head and neck squamous cell carcinoma (HNSC) (~110 kb, chr13:73880690-73990596) and esophageal carcinoma (ESCA) (~162 kb, chr13:73880413-74042621)" Super-Enhancer KLF5 -- ChIP-seq "The expression of KLF5, but not KLF12, is significantly higher in HNSC tumors with KLF5-HNSE amplification, suggesting that KLF5 is the target gene." "BTEB2,CKLF,IKLF" Esophageal Carcinoma DOID:1107 -- -- -- -- -- -- -- -- -- -- -- >2KB 302714 E_01_014 24413736 -- hg19 chr6 170855462 170856787 Human "HEK-293,¦Â-Cell" Low+High throughput "RNA-seq,ChIP-seq,ChIP,EMSA,PCR,4C,Luciferase Reporter Assay" "To define enhancer clusters, we first created 1,000 iterations of randomized C3 sites in the mappable genome of individual chromosomes. We then calculated for each chromosome the 25th percentile of inter-site distances of randomized C3 sites (Supplementary Fig. 6a). Next,we defined clusters of islet C3 sites as any group of ¡Ý3 C3 sites in which all adjacent C3 sites were separated by less than the abovementioned 25th percentile distance for randomized sites in the same chromosome. The distribution of islet C3 clusters differed from that of clusters generated with randomized C3 sites (Supplementary Fig. 6b)" Enhancer TBP 4C "RNA-seq,ChIP-seq,ChIP,EMSA,PCR,Luciferase Reporter Assay" islet-specific gene loci showed an increased density of islet transcription factor-bound active enhancer (C3) accessible chromatin sites. "GTF2D,GTF2D1,HDL4,SCA17,TFIID" Type 2 Diabetes Mellitus DOID:9352 D003924 Sequence variants associated with type 2 diabetes and fasting glycemia are enriched in these clustered islet enhancers and identify trait-associated variants that disrupt DNA binding and islet enhancer activity. Luciferase Reporter Assay "Transcription factor-bound C3 sites were also cloned in Gateway-adapted PGL4.23 and cotransfected in triplicate wells with pRL in MIN6 and 3T3 cells, and luciferase activity was measured after 48 hr. Results were expressed as luciferase/renilla ratios in vectors carrying putative Enhancers, relative to the ratio in empty PGL4.23 vector." "FOXA2,MAFB,NKX2-2" "HNF3B,TCF3B,DURS3,KRML,MCTO,NKX2.2,NKX2B" ChIP-seq "To determine the genomic binding sites of these islet transcription factors, we used chromatin immunoprecipitation and sequencing(ChIP-seq) in duplicate human islet samples and identified 3,911-32,747 high-confidence sites per transcription factor. " rs58692659 37775652 "Luciferase Reporter Assay,EMSA" >2KB 7295 E_01_015 26656844 -- hg19 chr13 73880413 74042621 Human Esophageal Carcinoma Low+High throughput "ChIP-seq,Luciferase Reporter Assay,3C-qPCR,CRISPR/Cas9,siRNA" "The focal amplification on chr13q identified in head and neck squamous cell carcinoma (HNSC) (~110 kb, chr13:73880690-73990596) and esophageal carcinoma (ESCA) (~162 kb, chr13:73880413-74042621)" Super-Enhancer KLF5 -- ChIP-seq "Similarly, the ESCA amplicon also harbors a super-enhancer based on the H3K27ac ChIP-seq profile of esophageal cells and ESCA tumors with this amplicon exhibited a trend towards increased KLF5 expression" "BTEB2,CKLF,IKLF" Lung Adenocarcinoma DOID:3910 D000077192 "Copy number gain of the e3 Enhancer region drives MYC overexpression, which contributes to tumorigenic phenotype" "ChIP-seq,CRISPR/Cas9,PCR" "Finally, repression of the e3 Enhancer led to a significant decrease in both anchorage-independent and clonogenic growth, suggesting that activity of the e3 Enhancer is critical for the tumorigenicity of lung adenocarcinoma cells. These results suggest that copy number gain of the e3 Enhancer region drives MYC overexpression, which contributes to tumorigenic phenotype." "NFE2L2,CEBPB" "HEBP1,IMDDHH,NRF2,C/EBP-beta,IL6DBP,NF-IL6,TCF5" "ChIP-seq,siRNA,Luciferase Reporter Assay" "Short interfering RNA (siRNA)-mediated knockdown of NFE2L2 and CEBPB in A549 cells led to a significant reduction in e3-driven luciferase reporter activity as compared to exposure to control siRNAs. Binding of NFE2L2 and CEBP¦Â to the super-Enhancer was subsequently confirmed by ChIP-seq, with the greatest enrichment at the e3 constituent Enhancer." -- -- -- >2KB 54786797 E_01_016 26656844 -- hg19 chr13 27523026 27544353 Human Colon Crypt Cell Low+High throughput "ChIP-seq,Luciferase Reporter Assay,3C-qPCR,CRISPR/Cas9,siRNA" "Additional focal amplification peaks on chromosome 13q in colorectal carcinoma (CRC) (~21 kb, chr. 13: 27,523,026¨C27,544,353) and chromosome 20q in liver hepatocellular carcinoma (~22 kb,chr. 20: 48,997,377¨C49,019,434) were identified. ChIP-seq profiling of H3K27ac in colon crypt cells31 and in the hepatocellular carcinoma cell line HepG2 (ref. 20) demonstrated that these amplicons contain super-enhancers." Super-Enhancer USP12 -- ChIP-seq "Right, log2-transformed expression level (RPKM) of USP12 in CRC tumors with focal amplification of USP12-CCSE (USP12 colorectal carcinoma super-enhancer) alone (n = 6) and tumors without the amplification (n = 127). (Fig. 1d)" "UBH1,USP12L1" Colorectal Carcinoma DOID:0080199 D015179 -- -- -- -- -- -- -- -- -- -- >2KB 106596 E_01_017 26656844 -- hg19 chr20 48997377 49019434 Human Hep G2 Low+High throughput "ChIP-seq,Luciferase Reporter Assay,3C-qPCR,CRISPR/Cas9,siRNA" "Additional focal amplification peaks on chromosome 13q in colorectal carcinoma (CRC) (~21 kb, chr. 13: 27,523,026¨C27,544,353) and chromosome 20q in liver hepatocellular carcinoma (~22 kb,chr. 20: 48,997,377¨C49,019,434) were identified. ChIP-seq profiling of H3K27ac in colon crypt cells31 and in the hepatocellular carcinoma cell line HepG2 (ref. 20) demonstrated that these amplicons contain super-enhancers." Super-Enhancer PARD6B -- ChIP-seq "Right, log2-transformed expression level (RPKM) of PARD6B in liver hepatocellular carcinoma tumors with focal amplification of PARD6B-LCSE (PARD6B liver carcinoma superenhancer) alone (n = 7) and tumors without the amplification (n = 245). (Fig. 1e)" PAR6B Hepatocellular Carcinoma DOID:684 D006528 -- -- -- -- -- -- -- -- -- -- >2KB 339674 E_01_018 26673704 -- hg19 chr7 117038917 117040917 Human Caco-2 Low throughput "RT-qPCR,4C,ChIP,CRISPR/Cas9" "Two pairs of gRNAs ?anking the ?20.9 kb CTCF site and the intron 11 enhancer core were identifed using the CRISPR Design Program" Enhancer CFTR CRISPR/Cas9 -- Here we use a combination of CRISPR/Cas9 editing of cis-regulatory elements and siRNA-mediated depletion of architectural proteins to determine the relative contribution of structural elements and enhancers to the higher order structure and expression of the CFTR locus. "ABC35,ABCC7,CF,CFTR/MRP,MRP7,TNR-CFTR,dJ760C5.1" -- -- -- -- -- -- "FOXA1,FOXA2,HNF1A,CDX2" "HNF3A,TCF3A,HNF3B,TCF3B,HNF-1A,HNF1,HNF4A,IDDM20,LFB1,MODY3,TCF-1,TCF1,CDX-3/AS, CDX3, CDX2" "4C,ChIP,CRISPR/Cas9" "Approximately 1.2 encompass_x0002_ing the binding sites for critical transcription factors driving the intron 11 Enhancer (FOXA1/2,HNF1 and CDX2)." -- -- -- >2KB 80099 E_01_019 26673704 -- hg19 chr7 117099048 117099450 Human Caco-2 Low throughput "RT-qPCR,4C,ChIP,CRISPR/Cas9" "Two pairs of gRNAs ?anking the ?20.9 kb CTCF site and the intron 11 enhancer core were identifed using the CRISPR Design Program" Enhancer CFTR CRISPR/Cas9 -- Here we use a combination of CRISPR/Cas9 editing of cis-regulatory elements and siRNA-mediated depletion of architectural proteins to determine the relative contribution of structural elements and enhancers to the higher order structure and expression of the CFTR locus. "ABC35,ABCC7,CF,CFTR/MRP,MRP7,TNR-CFTR,dJ760C5.1" -- -- -- The -20.9 kb site primarily plays a structural role at the locus and in its absence other CTCF sites can at least partially compensate for its function. "CRISPR/Cas9,ChIP,RT-qPCR" Next we performed RT-qPCR to measure CFTR expression levels in the ?20.9 kb deletion clones in comparison to WT clones and showed that the minor increase (1.25-fold) was not signiicant.This suggests that the ?20.9 kb site primarily plays a structural role at the locus and in its absence other CTCF sites can at least partially compensate for its function. "FOXA1,FOXA2,HNF1A,CDX2" "HNF3A,TCF3A,HNF3B,TCF3B,HNF-1A,HNF1,HNF4A,IDDM20,LFB1,MODY3,TCF-1,TCF1,CDX-3/AS, CDX3, CDX2" "4C,ChIP,CRISPR/Cas9" "Approximately 1.2 encompass_x0002_ing the binding sites for critical transcription factors driv_x0002_ing the intron 11 Enhancer (FOXA1/2,HNF1 and CDX2)." -- -- -- >2KB 20767 E_01_020 26673704 -- hg19 chr7 117228051 117229440 Human Caco-2 Low throughput "RT-qPCR,4C,ChIP,CRISPR/Cas9" "Two pairs of gRNAs ?anking the ?20.9 kb CTCF site and the intron 11 enhancer core were identifed using the CRISPR Design Program" Enhancer CFTR CRISPR/Cas9 -- Here we use a combination of CRISPR/Cas9 editing of cis-regulatory elements and siRNA-mediated depletion of architectural proteins to determine the relative contribution of structural elements and enhancers to the higher order structure and expression of the CFTR locus. "ABC35,ABCC7,CF,CFTR/MRP,MRP7,TNR-CFTR,dJ760C5.1" -- -- -- "Deletion of a strong enhancer in intron 11 has little impact on the 3D architecture, but represses CFTR gene expression." "CRISPR/Cas9,RT-qPCR" "Approximately 1.2 kb encompassing the binding sites for critical transcription factors driving the intron 11 enhancer (FOXA1/2, HNF1 and CDX2).The del11 clones show a significant reduction in RNAP II occupancy at the promoter, intron 1, 10 and 11, suggesting a global loss of the polymerase across the CFTR locus.The intron 11 enhancer participates in recruitment of RNAPII to the CFTR promoter as well as to intronic regions." "FOXA1,FOXA2,HNF1A,CDX2" "HNF3A,TCF3A,HNF3B,TCF3B,HNF-1A,HNF1,HNF4A,IDDM20,LFB1,MODY3,TCF-1,TCF1,CDX-3/AS, CDX3, CDX2" "4C,ChIP,CRISPR/Cas9" "Approximately 1.2 encompass_x0002_ing the binding sites for critical transcription factors driv_x0002_ing the intron 11 Enhancer (FOXA1/2,HNF1 and CDX2)." -- -- -- Intron 108730 E_01_021 26656844 MYC-ECSE (MYC endometrial carcinoma super-Enhancer) hg19 chr8 129543949 129554294 Human Endometrial Carcinoma Cell Lines Ishikawa Low+High throughput "ChIP-seq,Luciferase Reporter Assay,3C-qPCR,CRISPR/Cas9,siRNA" "The endometrial carcinoma peak (chr. 8: 129,543,949¨C129,554,294) encompasses a 10-kb noncoding region that harbors a super-enhancer,as defined by the H3K27ac ChIP-seq profile of the endometrial carcinoma cell line Ishikawa, which we refer to as MYC-ECSE(MYC endometrial carcinoma super-enhancer)." Super-Enhancer MYC 3C -- "We performed chromosome conformation capture (3C) assays in A549 and Ishikawa cells and found that MYC-LASE physically interacted with the MYC promoter only in A549 cells and, reciprocally, that MYC-ECSE physically interacted with the MYC promoter only in Ishikawa cells" "MRTL,MYCC,bHLHe39,c-Myc" Endometrial Carcinoma DOID:2871 D016889 Two distinct focal amplifications of super-enhancers 3¡ä to MYC in lung adenocarcinoma (MYC-LASE) and endometrial carcinoma (MYC-ECSE) are physically associated with the MYC promoter and correlate with MYC overexpression. "3C,ChIP-seq" "We performed chromosome conformation capture (3C) assays in A549 and Ishikawa cells and found that MYC-LASE physically interacted with the MYC promoter only in A549 cells and, reciprocally,that MYC-ECSE physically interacted with the MYC promoter only in Ishikawa cells. In addition, tumors with amplification of MYC alone or MYC-LASE or MYC-ECSE alone had higher MYC expression than tumors lacking either amplification. These results suggest that both MYC-LASE and MYC-ECSE drive MYC expression through lineage-specific chromatin loops." -- -- -- -- -- -- -- >2KB 800808 E_01_022 29045844 -- hg19 chr8 98253530 98257713 Human Mantle Cell Lymphoma (MCL) Low+High throughput "ChIP-seq,ChIP-qPCR,4C-seq" "These candidate enhancers (E1 and E2, Figure 2A) are the most highly acetylated elements (based on H3K27ac chromatin immunoprecipitation sequencing [ChIP-seq]) near MYC in SP-49 cells" Enhancer MYC 4C -- "To identify interactions between E1, E2,and the MYC promoter in MCL cells, we performed 4C-Seq on Rec-1 cells treated for 3 days with GSI or vehicle control(DMSO).4C-seq data for Rec-1 cells showing interactions between E1, E2, and the MYC promoter. " "MRTL,MYCC,bHLHe39,c-Myc" Small B Cell Lymphoma -- D015451 "Notch stimulates MYC expression in B cells through the E1 and E2 sites, referred to as the B cell Notch-dependent MYC enhancer (B-NDME)." "qRT-PCR,ChIP-seq" "To test the effect of co-repressing the E1 and E2 modules,KRAB-dCAS9-P2A-mCherry lines were co-transduced with E1- and E2-targeting sgRNA lentiviruses. This led to decreased MYC expression (Figure 5B) and cell proliferation (Figure 5C) in SP-49 and Granta-519 cells, but not in MYC-rearranged Jeko-1 cells. Thus, Notch stimulates MYC expression in B cells through the E1 and E2 sites, referred to as the B cell Notch-dependent MYC enhancer (B-NDME)." "RBPJ,IRF8,POU2AF1,MYBL2" "AOS3, CBF1, IGKJRB, IGKJRB1, KBF2, RBP-JK, RBPSUH, SUH, csl, RBPJ,H-ICSBP,ICSBP,ICSBP1,IMD32A,IMD32B,IRF-8,BOB1,OBF-1,OBF1,OCAB,B-MYB,BMYB" "ChIP-qPCR,ChIP" Our data therefore suggest that CLL and MCL cell lines with an intact MYC locus require RBPJ-NICD (or RBPJ-EBNA2) binding to drive MYC expression via the E1/E2 enhancers. -- -- -- >2KB 30492692 E_01_023 29045844 -- hg19 chr8 98310330 98314427 Human Mantle Cell Lymphoma (MCL) Low+High throughput "ChIP-seq,ChIP-qPCR,4C-seq" "These candidate enhancers (E1 and E2, Figure 2A) are the most highly acetylated elements (based on H3K27ac chromatin immunoprecipitation sequencing [ChIP-seq]) near MYC in SP-49 cells" Enhancer MYC 4C -- "To identify interactions between E1, E2,and the MYC promoter in MCL cells, we performed 4C-Seq on Rec-1 cells treated for 3 days with GSI or vehicle control (DMSO).4C-seq data for Rec-1 cells showing interactions between E1, E2, and the MYC promoter. " "MRTL,MYCC,bHLHe39,c-Myc" Small B Cell Lymphoma -- D015451 "Notch stimulates MYC expression in B cells through the E1 and E2 sites, referred to as the B cell Notch-dependent MYC enhancer (B-NDME)." "qRT-PCR,ChIP-seq" "To test the effect of co-repressing the E1 and E2 modules,KRAB-dCAS9-P2A-mCherry lines were co-transduced with E1- and E2-targeting sgRNA lentiviruses. This led to decreased MYC expression (Figure 5B) and cell proliferation (Figure 5C) in SP-49 and Granta-519 cells, but not in MYC-rearranged Jeko-1 cells. Thus, Notch stimulates MYC expression in B cells through the E1 and E2 sites, referred to as the B cell Notch-dependent MYC enhancer (B-NDME)." "RBPJ,IRF8,POU2AF1,MYBL2" "AOS3, CBF1, IGKJRB, IGKJRB1, KBF2, RBP-JK, RBPSUH, SUH, csl, RBPJ,H-ICSBP,ICSBP,ICSBP1,IMD32A,IMD32B,IRF-8,BOB1,OBF-1,OBF1,OCAB,B-MYB,BMYB" "ChIP-qPCR,ChIP" Our data therefore suggest that CLL and MCL cell lines with an intact MYC locus require RBPJ-NICD (or RBPJ-EBNA2) binding to drive MYC expression via the E1/E2 enhancers. -- -- -- >2KB 30435935 E_01_024 26751080 -- hg19 chr17 26077792 26078792 Human A549 Low throughput 3C "Lastly, our 3C assay was able to detect a long DNA loop between the hiNOS enhancer and core promoter site, and ChIP loop assay confirmed that p300 binds to AP--1 and RNA pol II proteins. " Enhancer NOS2 3C -- "Lastly, our 3C assay was able to detect a long DNA loop between the hiNOS enhancer and core promoter site." "HEP-NOS,INOS,NOS,NOS2A" -- -- -- The coactivator p300 mediates cytokine-induced hiNOS transactivation by forming a distant DNA loop between its enhancer and core promoter region. Luciferase Reporter Assay These findings indicate that the cytokine-induced Enhancer region at -5 kb to -6 kb is required for p300-mediated increase in CM-induced hiNOS promoter activity. JUND AP-1 ChIP These findings are consistent with p300 mediated hiNOS transactivation at the hiNOS Enhancer mainly through the upstream AP-1 site at -5.3 kb by forming a DNA-Protein complex. -- -- -- >2KB 5499 E_01_025 19386363 CD200 distal Enhancer hg19 chr3 112045517 112046117 Human HUVEC Low throughput "Luciferase Reporter Assay,ChIP,PCR,DNase I Hypersensitivity Assay" "Using a DNase I hypersensitivity assay, we identified a distal enhancer region responsible for inducible expression of CD200.Generation of a luciferase reporter construct containing both CD200 core promoter and potential enhancer region" Enhancer CD200 -- Site-Directed Mutagenesis "The potential regulatory region (?5677/?5077) enhanced CD200 promoter activity after stimulation of IFN- and TNF-? but this function was abolished without C/EBP¡¦ binding site in the core promoter Site-directed mutagenesis was performed to generate four mutant constructs" "MOX1,MOX2,MRC,OX-2" "Graft Rejection,Autoimmune Diseases,Spontaneous Abortion" -- "D006084,D001327,D000022" "All the above findings suggest that IFN-¦Ã and TNF-¦Á induce CD200 expression through a 5 upstream Enhancer and that NF-¦ÊB, STAT1 and IRF-1 play pivotal roles in this process." ChIP "These results suggest that NF-kB, STAT1 and IRF-1 interact with the Enhancer region containing the corresponding binding sites following stimulation with IFN-¦Ã and TNF-¦Á." "NFKB1,STAT1,IRF1" "CVID12,EBP-1,KBF1,NF-kB,NF-kB1,NF-kappa-B1,NF-kappaB,NFKB-p105,NFKB-p50,NFkappaB,p105,p50,CANDF7,IMD31A,IMD31B,IMD31C,ISGF-3,STAT91,IRF-1,MAR" "qPCR,ChIP" "The results of ChIP and PCR suggest that NF-kB, STAT1 and IRF-1 interact with the Enhancer region containing the corresponding binding sites following stimulation with IFN-r_x0002_and TNF-a. " -- -- -- >2KB 5976 E_01_026 24413736 -- hg19 chr6 37772587 37778315 Human "HEK-293,¦Â-cell" Low+High throughput "RNA-seq,ChIP-seq,ChIP,EMSA,PCR,4C,Luciferase Reporter Assay" "To define enhancer clusters, we first created 1,000 iterations of randomized C3 sites in the mappable genome of individual chromosomes. We then calculated for each chromosome the 25th percentile of inter-site distances of randomized C3 sites (Supplementary Fig. 6a). Next,we defined clusters of islet C3 sites as any group of ¡Ý3 C3 sites in which all adjacent C3 sites were separated by less than the abovementioned 25th percentile distance for randomized sites in the same chromosome. The distribution of islet C3 clusters differed from that of clusters generated with randomized C3 sites (Supplementary Fig. 6b)" Enhancer ZFAND3 -- Luciferase Reporter Assay "For example, at the ZFAND3 T2D locus discovered in East Asians44, SNP rs58692659 forms part of an array of variants that was highly-correlated with the reported lead SNP (rs9470794) (1KG CHB (Han Chinese in Beijing, China) and JPT (Japanese in Tokyo, Japan) r2 = 0.96), and maps to a C3 element bound by multiple islet transcription factors within an enhancer cluster (Fig. 6a). This variant altered sequence-specific DNA binding of a key islet-enriched transcription factor, NEUROD1, and abolished enhancer activity in ¦Â-cells (Fig. 6b,c). " TEX27 Type 2 Diabetes Mellitus DOID:9352 D003924 Sequence variants associated with type 2 diabetes and fasting glycemia are enriched in these clustered islet enhancers and identify trait-associated variants that disrupt DNA binding and islet enhancer activity. Luciferase Reporter Assay "Transcription factor-bound C3 sites were also cloned in Gateway-adapted PGL4.23 and cotransfected in triplicate wells with pRL in MIN6 and 3T3 cells, and luciferase activity was measured after 48 hr. Results were expressed as luciferase/renilla ratios in vectors carrying putative Enhancers, relative to the ratio in empty PGL4.23 vector." "FOXA2,MAFB,NKX2-2" "FOXA2,MAFB,NKX2-2" ChIP-seq "To determine the genomic binding sites of these islet transcription factors, we used chromatin immunoprecipitation and sequencing(ChIP-seq) in duplicate human islet samples and identified 3,911-32,747 high-confidence sites per transcription factor. " -- -- -- >2KB 51008158 E_01_027 18202151 hRLD2 Enhancer hg19 chr13 43111372 43112072 Human MG-63 Low throughput "EMSA,ChIP,RT-PCR" "Previous work in our laboratory using chromatin immunoprecipitation (ChIP) techniques led to the identification of five evolutionarily conserved enhancer regions located at 16, 22, 60, 69, and 76 kb upstream of the mouse Rankl transcriptional start site (TSS) (24). An assessment of the transcriptional activity of each of these enhancer regions in transfection assays revealed that only the most distal, located at 76 kb, was capable of mediating response to 1,25-(OH)2D3 (24)." Enhancer TNFSF11 -- EMSA "These experiments define the cis acting element that mediates 1,25-(OH)2D3 response in the hRLD2 region of the RANKL gene. " "CD254,ODF,OPGL,OPTB2,RANKL,TNLG6B,TRANCE,hRANKL2,sOdf" Osteopetrosis DOID:13533 D010022 "Activity at this region in response to 1,25(OH)2D3 was associated with a significant increase in histone acetylation as well as the enhanced recruitment of RNA polymerase II." ChIP "Importantly, activity at this region in response to 1,25(OH)2D3 was associated with a significant increase in histone acetylation as well as the enhanced recruitment of RNA polymerase II. Both likely reflect the primary role of this enhancer in human RANKL gene expression." VDR "NR1I1,PPP1R163" "ChIP-seq,siRNA" "To evaluate this possibility, we treated MG63 cells with either nontargeting or previously validated hVDR small interfering RNA (siRNA) for 48 h and then subjected the cells to ChIP analysis using antibodies to VDR, RXR, or IgG. The ability of the VDR siRNA to suppress VDR mRNA levels is documented in Fig. 4A The results in Fig. 4B reveal that VDR siRNA treatment does produce a decrease in the level of occupancy of both VDR and RXR at the hRLD1 enhancer but not at the TSS. Interestingly, this reduction in unliganded VDR at the hRLD1 enhancer also caused an approximate 50% reduction in the basal level of human RANKL mRNA expression in MG63 cells as well. " -- -- -- >2KB 25149 E_01_028 19467240 -- hg19 chr17 26072592 26074592 Human Hep G2 Low throughput "Luciferase Reporter Assay,PCR,EMSA,ChIP" "Taken together, this work identifies a far-upstream functional Oct-1 enhancer motif at -10.2 kb in the hiNOS promoter that regulates cytokine-induced hiNOS gene transcription, and further underscores the tight control mechanisms regulating expression of the human iNOS gene." Enhancer NOS2 -- "EMSA,ChIP" "Taken together, this work identifies a far-upstream functional Oct-1 enhancer motif at -10.2 kb in the hiNOS promoter that regulates cytokine-induced hiNOS gene transcription, and further underscores the tight control mechanisms regulating expression of the human iNOS gene." "HEP-NOS,INOS,NOS,NOS2A" -- -- -- -- -- -- POU2F1 "OCT1,OTF1,oct-1B" "EMSA,ChIP" "To examine whether the Oct-1 protein can interact with the potential Oct-1 binding site at ?10.2 kb of hiNOS in vitro, we carried out electrophoretic mobility shift assays (EMSAs) with nuclear extracts from HepG2 cells.Chromatin immunoprecipitation assay confirms that Oct-1 specifically binds to the motif at ?10.2 kb in the hiNOS promoter in vivo." -- -- -- >2KB 10199 E_01_029 19708858 HREs Enhancer hg19 chr3 48598405 48598558 Human TE-671 Low throughput "Luciferase Reporter Assay,RT-PCR" "This activation was conserved in constructs with the 3' FLR fragment placed upstream of the luciferase gene, indicating an enhancer function for HRE1.To test their role as enhancers, we cloned several fragments of the 3 'FLR of the hUcn2 gene downstream of the firefly luciferase gene in (a) the pGL3 ?746 bp vector containing the functional hUcn2 promoter, and (b) the pGL3control vector containing an internal SV40 promoter (Figure 2A). " Enhancer UCN2 -- "Luciferase Reporter Assay,Transfection" "To test their role as enhancers, we cloned several fragments of the 3 ' FLR of the hUcn2 gene downstream of the firefly luciferase gene in (a) the pGL3 ?746 bp vector containing the functional hUcn2 promoter, and (b) the pGL3control vector containing an internal SV40 promoter (Figure 2A). TE--671 cells were transfected with the different constructs and exposed to normoxia or hypoxia for 12 h. Hypoxia significantly increased luciferase expression, specifically in the constructs containing the fragment 3 'FLR3 (Figure 2A)." "SRP,UCN-II,UCNI,UR,URP" Myocardial Ischemia -- D017202 "Hypoxia induces hUcn2 expression via a specific HRE in the 3 FLR of the hUcn2 gene, which interacts with the transcription factor HIF1¦Á." Luciferase Reporter Assay "Similar to the results obtained with constructs that contained the 3' FLR3 variants downstream of the luciferase gene, the hypoxia- and CPX-induced luciferase activation was strongest in the 3' FLR3 containing both wild-type HRE1 and HRE2." HIF1A "HIF-1-alpha,HIF-1A,HIF-1alpha,HIF1,HIF1-ALPHA,MOP1,PASD8,bHLHe78" ELISA "To prove that HIF1¦Á binds to HRE1 and HRE2 in the 3_x0002_FLR of the hUcn2 gene, we performed HIF1¦Á binding and competition ELISA assays. After 12 h of hypoxia, binding of HIF1¦Á to the EPO HRE was stronger than under normoxic conditions." -- -- -- <2KB 668 E_01_030 19773398 SF-1 Enhancer hg19 chrx 30317939 30318739 Human HEK-293 Low throughput "Luciferase Reporter Assay,PCR" Luciferase reporter gene activity in transiently transfected HEK293 cells. Relative luciferase activity refers to the activity of the firefly reporter gene normalized to Renilla luciferase internal control for transfection efficiency. Firefly reporter constructs contained the DAX1 conserved noncoding ¡°enhancer¡± elements upstream of a minimal thymidine kinase promoter driving the firefly reporter. Enhancer NR0B1 -- "Luciferase Reporter Assay,PCR" Our results therefore strongly suggest the existence of an SF-1 binding site 4kb upstream of NR0B1 that acts as an enhancer of DAX1 expression. "AHC,AHCH,AHX,DAX-1,DAX1,DSS,GTD,HHG,NROB1,SRXY2" X-Linked Congenital Adrenal Hypoplasia DOID:0080156 D000075262 A reporter construct lacking this Enhancer element upstream of NR0B1 was unresponsive to SF-1 transcriptional activation. Luciferase Reporter Assay "SF-1 acted as an activator on the wild-type construct, whereas the mutant construct,lacking the SF-1 consensus binding site, showed no response to SF-1. " NR5A1 "AD4BP,ELP,FTZ1,FTZF1,POF7,SF-1,SF1,SPGF8,SRXX4,SRXY3,hSF-1" Luciferase Reporter Assay Luciferase reporter gene activity in transiently transfected HEK293 cells. Relative luciferase activity refers to the activity of the firefly reporter gene normalized to Renilla luciferase internal control for transfection efficiency. The mutant CNE is unresponsive to the transcription enhancement seen in the presence of cotransfected SF-1. -- -- -- >2KB 4199 E_01_031 20061391 MnSOD Enhancer hg19 chr6 160101620 160102231 Human PC-3 Low throughput "RT-PCR,Luciferase Reporter Assay,EMSA,ChIP" "For reporter gene activity, a luciferase assay was performed by co-transfecting the cells with the p53 expression vector and vector containing the enhancer (I2E) and the promoter (?3400/+24, ?555/+24, or ?210/+24) of the human MnSOD gene in the pGL3 reporter vector." Enhancer SOD2 -- "Luciferase Reporter Assay,EMSA,PCR,ChIP" These results suggest that the positive effect of p53 on MnSOD expression is dependent on the presence of the intronic enhancer element of the MnSOD gene.FIGURE 3 "IPO-B,IPOB,MNSOD,MVCD6,Mn-SOD" -- -- -- The positive effect of p53 on MnSOD expression is dependent on the presence of the intronic Enhancer element of the MnSOD gene Luciferase Reporter Assay Addition of the Enhancer element to the MnSOD promoter construct led to the bi-directional effect of p53 as observed with the endogenous MnSOD gene.These results suggest that the positive effect of p53 on MnSOD expression is dependent on the presence of the intronic Enhancer element of the MnSOD gene. TP53 "BCC7,BMFS5,LFS1,P53,TRP53" "PCR,EMSA,ChIP" "The promoter fragment amplified after p53 overexpression was decreased when the ChIP was preformed with the Sp1 antibody.These results are consistent with the DNA-binding data obtained by EMSA, suggesting that p65 binding to the MnSOD enhancer may contribute to the induction of MnSOD gene transcription." -- -- -- Intron 1831 E_01_032 20061391 MnSOD Enhancer hg19 chr6 160101890 160102231 Human PC-3 Low throughput "Luciferase Reporter Assay,EMSA,PCR,ChIP" "For reporter gene activity, a luciferase assay was performed by co-transfecting the cells with the p53 expression vector and vector containing the enhancer (I2E) and the promoter (?3400/+24, ?555/+24, or ?210/+24) of the human MnSOD gene in the pGL3 reporter vector." Enhancer SOD2 -- "Luciferase Reporter Assay,EMSA,PCR,ChIP" These results suggest that the positive effect of p53 on MnSOD expression is dependent on the presence of the intronic enhancer element of the MnSOD gene.FIGURE 3 "IPO-B,IPOB,MNSOD,MVCD6,Mn-SOD" -- -- -- -- -- -- SP1 SP1 "PCR,EMSA,ChIP" "The promoter fragment amplified after p53 overexpression was decreased when the ChIP was preformed with the Sp1 antibody.These results are consistent with the DNA-binding data obtained by EMSA, suggesting that p65 binding to the MnSOD enhancer may contribute to the induction of MnSOD gene transcription." -- -- -- Intron 1966 E_01_033 19285986 Di hg19 chr16 67519201 67519833 Human "H295R,N38" Low throughput "Transgenic mice,Luciferase Reporter Assay" "Schematic representation of the genomic region encompassing the AgRP and the ATPase locus. Expression constructs for regions A, B, C, and D were directionally cloned into pGL3-basic luciferase vector.Regions C and D were evaluated in the same fashion. Region D (but not region C) contained enhancer elements in both neuronal and somatic cell types (Fig. 1a). Region D was therefore divided into three overlapping sub-regions: Di(-18/+312)(+2727/+3359), Dii(-18/+312) (+3341/+3941), and Diii(-18/+312)(+3943/+4575). Sub-regions Di and Diii (but not Dii) had enhancer properties for the AgRP basal promoter in both h295R and N38 cell lines (Fig. 1b).Fig. 1. In vitro determination of enhancer elements" Enhancer AGRP -- "Transgenic mice,Luciferase Reporter Assay" "Region D (but not C) increased activity of the human AgRP promoter, equally in the human adrenocortical NCI-h295R and the mouse clonal hypothalamic N38 cell lines. " "AGRT,ART,ASIP2" -- -- -- -- -- -- -- -- -- -- -- -- -- >2KB 3044 E_01_034 19285986 Dic hg19 chr16 67519609 67519833 Human "H295R,N38" Low throughput "Transgenic mice,Luciferase Reporter Assay" "Di and Diii were further engineered into three overlapping sub-regions each and the experiments were performed in the N38 and h295R mouse hypothalamic cells. Sub-region Dic had the most significant enhancer effect on AgRP basal promoter in both cell lines " Enhancer AGRP -- "Transgenic mice,Luciferase Reporter Assay" "Region D (but not C) increased activity of the human AgRP promoter, equally in the human adrenocortical NCI-h295R and the mouse clonal hypothalamic N38 cell lines. " "AGRT,ART,ASIP2" -- -- -- -- -- -- -- -- -- -- -- -- -- >2KB 3248 E_01_035 19285986 Diii hg19 chr16 67520417 67521049 Human "H295R,N38" Low throughput "Transgenic mice,Luciferase Reporter Assay" "Schematic representation of the genomic region encompassing the AgRP and the ATPase locus. Expression constructs for regions A, B, C, and D were directionally cloned into pGL3-basic luciferase vector.Regions C and D were evaluated in the same fashion. Region D (but not region C) contained enhancer elements in both neuronal and somatic cell types (Fig. 1a). Region D was therefore divided into three overlapping sub-regions: Di(-18/+312)(+2727/+3359), Dii(-18/+312) (+3341/+3941), and Diii(-18/+312)(+3943/+4575). Sub-regions Di and Diii (but not Dii) had enhancer properties for the AgRP basal promoter in both h295R and N38 cell lines (Fig. 1b). 1. In vitro determination of enhancer elements" Enhancer AGRP -- "Transgenic mice,Luciferase Reporter Assay" "Region D (but not C) increased activity of the human AgRP promoter, equally in the human adrenocortical NCI-h295R and the mouse clonal hypothalamic N38 cell lines. " "AGRT,ART,ASIP2" -- -- -- -- -- -- -- -- -- -- -- -- -- >2KB 4260 E_01_036 19285986 Diiia hg19 chr16 67520417 67520638 Human "AtT-20,N38" Low throughput "Transgenic mice,Luciferase Reporter Assay,Transfection" "Di and Diii were further engineered into three overlapping sub-regions each and the experiments were performed in the N38 and h295R mouse hypothalamic cells. " Enhancer AgRP-ATP6V0D1 -- "Luciferase Reporter Assay,Transgenic mice" Schematic of transgenes and bioluminescence of offspring representing the two enhancers. (Fig. 3) "AGRT,ART,ASIP2,ATP6D,ATP6DV,P39,VATX,VMA6,VPATPD" -- -- -- -- -- -- -- -- -- -- -- -- -- >2KB 4054 E_01_037 19285986 Diiib hg19 chr16 67520620 67520851 Human H295R Low throughput "Luciferase Reporter Assay,Transfection" Di and Diii were further engineered into three overlapping sub-regions each and the experiments were performed in the N38 and h295R mouse hypothalamic cells. Enhancer AgRP-ATP6V0D2 -- "Luciferase Reporter Assay,Transgenic mice" Schematic of transgenes and bioluminescence of offspring representing the two enhancers. (Fig. 3) "AGRT,ART,ASIP2,ATP6D2,VMA6" -- -- -- -- -- -- -- -- -- -- -- -- -- >2KB 4262 E_01_038 20093418 FKBP51 Enhancer hg19 chr6 35574362 35576362 Human A549 Low throughput "RT-qPCR,ChIP" "According to our quantitative ChIP and enhancer assays, there are at least three major distal gene regions that significantly bind GR and also function as strong GR-regulated enhancers in isolation." Enhancer FKBP4 -- "ChIP,Luciferase Reporter Assay" Our quantitative chromatin immunoprecipitation scans and enhancer activity analyses indicate that activation of the FKBP51 locus by glucocorticoids in vivo is triggered by the loading of GR to enhancers at about 34 kb 5 and about 87 kb 3 of the transcription start site. "FKBP51,FKBP52,FKBP59,HBI,Hsp56,PPIase,p52" -- -- -- -- -- -- CTCF MRD21 ChIP We used the genome-wide CTCF-binding information from the CTCF-binding site database for choosing the regions to be ChIP scanned with anti-CTCF antibody. -- -- -- >2KB 32671255 E_01_039 20093418 FKBP51 Enhancer hg19 chr6 35505138 35511492 Human "A549,VCaP" Low throughput "RT-qPCR,ChIP" "According to our quantitative ChIP and enhancer assays, there are at least three major distal gene regions that significantly bind GR and also function as strong GR-regulated enhancers in isolation." Enhancer FKBP4 -- "ChIP,Luciferase Reporter Assay" Our quantitative chromatin immunoprecipitation scans and enhancer activity analyses indicate that activation of the FKBP51 locus by glucocorticoids in vivo is triggered by the loading of GR to enhancers at about 34 kb 5 and about 87 kb 3 of the transcription start site. "FKBP51,FKBP52,FKBP59,HBI,Hsp56,PPIase,p52" -- -- -- The holo-GR is capable of activating transcription and evoking changes in chromatin structure through distant-acting Enhancers. ChIP Our quantitative chromatin immunoprecipitation scans and Enhancer activity analyses indicate that activation of the FKBP51 locus by glucocorticoids in vivo is triggered by the loading of GR to Enhancers at about 34 kb 5' and about 87 kb 3' of the transcription start site. CTCF MRD21 ChIP "Interestingly,our ChIP assays from the A549 cells indicate a significant enrichment of both the CTCF and the cohesin subunit RAD21 at regions approximately 48 kb 5' and about 105kb 3' of the FKBP51 TSS, which border the region en_x0002_compassing the GR-regulated Enhancers." -- -- -- >2KB 32604208 E_01_040 20093418 FKBP51 Enhancer hg19 chr6 35616283 35622638 Human "A549,VCaP" Low throughput "RT-qPCR,ChIP" "According to our quantitative ChIP and enhancer assays, there are at least three major distal gene regions that significantly bind GR and also function as strong GR-regulated enhancers in isolation." Enhancer FKBP4 -- "ChIP,Luciferase Reporter Assay" Our quantitative chromatin immunoprecipitation scans and enhancer activity analyses indicate that activation of the FKBP51 locus by glucocorticoids in vivo is triggered by the loading of GR to enhancers at about 34 kb 5 and about 87 kb 3 of the transcription start site. "FKBP51,FKBP52,FKBP59,HBI,Hsp56,PPIase,p52" -- -- -- the holo-GR is capable of activating transcription and evoking changes in chromatin structure through distant-acting Enhancers. ChIP Our quantitative chromatin immunoprecipitation scans and Enhancer activity analyses indi_x0002_cate that activation of the FKBP51 locus by glucocorticoids in vivo is triggered by the loading of GR to Enhancers at about 34 kb 5_x0001_ and about 87 kb 3_x0001_ of the transcription start site. CTCF MRD21 ChIP "Interestingly,our ChIP assays from the A549 cells indicate a significant enrichment of both the CTCF and the cohesin subunit RAD21 at regions approximately 48 kb 5' and about 105kb 3' of the FKBP52 TSS, which border the region en_x0002_compassing the GR-regulated Enhancers." -- -- -- >2KB 32715354 E_01_041 20093418 FKBP51 Enhancer hg19 chr6 35636622 35642872 Human "A549,VCaP" Low throughput "RT-qPCR,ChIP" "According to our quantitative ChIP and enhancer assays, there are at least three major distal gene regions that significantly bind GR and also function as strong GR-regulated enhancers in isolation." Enhancer FKBP4 -- "ChIP,Luciferase Reporter Assay" Our quantitative chromatin immunoprecipitation scans and enhancer activity analyses indicate that activation of the FKBP51 locus by glucocorticoids in vivo is triggered by the loading of GR to enhancers at about 34 kb 5 and about 87 kb 3 of the transcription start site. "FKBP51,FKBP52,FKBP59,HBI,Hsp56,PPIase,p52" -- -- -- the holo-GR is capable of activating transcription and evoking changes in chromatin structure through distant-acting Enhancers. ChIP Our quantitative chromatin immunoprecipitation scans and Enhancer activity analyses indi_x0002_cate that activation of the FKBP51 locus by glucocorticoids in vivo is triggered by the loading of GR to Enhancers at about 34 kb 5_x0001_ and about 87 kb 3_x0001_ of the transcription start site. CTCF MRD21 ChIP "Interestingly,our ChIP assays from the A549 cells indicate a significant enrichment of both the CTCF and the cohesin subunit RAD21 at regions approximately 48 kb 5' and about 105kb 3' of the FKBP53 TSS, which border the region en_x0002_compassing the GR-regulated Enhancers." -- -- -- >2KB 32735640 E_01_042 20952403 RET Enhancer hg19 chr10 43569296 43572684 Human SK-N-BE Low throughput "Luciferase Reporter Assay,PCR" We then performed chromatin immunoprecipitation (ChIP) experiments to verify the presence of RAR¦Á Enhancer RET -- "ChIP,qRT-PCR" "In order to study possible chromatin changes occurring during RET gene activation, we determined by a series of ChIP assays coupled with qRT¨CPCR, the histone modification state at the RET regulatory regions after different times of RA treatment." "CDHF12,CDHR16,HSCR1,MEN2A,MEN2B,MTC1,PTC,RET-ELE1" -- -- -- -- -- -- "EZH2,DNMT1" "ENX-1,ENX1b,KMT6,KMT6A,WVS,WVS2,EZH2,ADCADN,AIM,CXXC9,DNMT,HSN1E,MCMT,m.HsaI" "ChIP,RT-PCR" EZH2 and DNMT1 binding to RET regulatory regions.SK-N-BE cells were treated for the indicated times with 10 mM RA.ChIP experiments were performed using anti-EZH2 and anti-DNMT1 antibodies and bound DNA was quantitated by real time PCR. -- -- -- <2KB 1526 E_01_043 20952659 CCL2 Enhancer hg19 chr17 32579684 32579693 Human "HEK-293,HEK-MOR Cells" Low throughput "ChIP,PCR" ChIP analysis shows that DAMGO administration induces binding of p65 to the enhancer region of the CCL2 promoter. Enhancer CCL2 -- "ChIP,PCR" ChIP analysis shows that DAMGO administration induces binding of p65 to the enhancer region of the CCL2 promoter. "GDCF-2,HC11,HSMCR30,MCAF,MCP-1,MCP1,SCYA2,SMC-CF" -- -- -- -- -- -- NFKB1 "CVID12,EBP-1,KBF1,NF-kB,NF-kB1,NF-kappa-B1,NF-kappaB,NFKB-p105,NFKB-p50,NFkappaB,p105,p50" "ChIP,PCR" "Based on these studies and our pre_x0002_liminary experiments, we designed PCR primers to span both of the NF-KB-binding elements in the CCL2 Enhancer region for PCR analysis following ChIP." -- -- -- >2KB 2606 E_01_044 21051589 androgen Enhancer hg19 chr19 50850114 50850982 Human LNCaP Low throughput "Luciferase Reporter Assay,RT-PCR" "The luciferase activity of the pPSABHE reporter vector, which contains the promoter (2589 to 241) and androgen enhancer regions (24801 to 23933) of the PSA gene, increased by 8--fold after IL--6 treatment. " Enhancer KLK3 -- Luciferase Reporter Assay "The luciferase activity of the pPSABHE reporter vector, which contains the promoter (2589 to 241) and androgen enhancer regions (24801 to 23933) of the PSA gene, increased by 8--fold after IL--6 treatment." "APS,KLK2A1,PSA,hK3" Prostate Cancer DOID:10283 D011471 "Results from 5'-deletion reporter assays revealed that the effects of IL-6 appear to be mediated via an androgen Enhancer region (24801 to 23933), which is dependent on the signal transducer and activator of the transcription 3 (STAT3) pathway." Luciferase Reporter Assay "Moreover, we also found that IL-6 treatment did not further enhance the stimulatory effect of transient overexpression of STAT3 on luciferase activity of the pPSABHE reporter vector, suggesting that the STAT3 signaling pathway, which is dependent on the Enhancer region of PSA gene, is the major pathway involved in the IL-6¨Cregulated expression of the PSA gene in LNCaP cells." STAT3 "ADMIO,ADMIO1,APRF,HIES" Luciferase Reporter Assay Transient gene expression assays of cells cotransfected with the pPSABHE reporter vector and the STAT3 expression vector revealed that transient overexpression of STAT3 significantly upregulated the luciferase activity of the pPSABHE reporter vector in an androgen-independent manner. -- -- -- >2KB 507622 E_01_045 21056086 -- hg19 chr1 23881789 23881796 Human "Immortalized Murine Gonadotrope L¦ÂT2 Cell,Ovarian Cancer Cell" Low throughput "qRT-PCR,Luciferase Reporter Assay,ChIP" "To determine if these base pairs also play a role in BMP2induction of the human ID3 promoter, we introduced mutations comparable to Mut4 and Mut5 in the murine Id3 promoter into the minimal hID3 promoter--reporter construct (?653/+402 ID3--luc) (Fig. 6(B)). " Enhancer ID3 -- ChIP We identified the same sequence in the murine gene (?3283/?3276) (Fig. S8) and observed through ChIP analysis that BMP2 stimulated recruitment of SMAD1 to this region of the endogenous Id3 gene in L?T2 cells "HEIR-1,bHLHb25" -- -- -- A more distal Enhancer was shown to mediate BMP4-induction of the human ID3 gene in ovarian cancer cells. ChIP "We identified the same sequence in the murine gene (?3283/?3276) and observed through ChIP analysis that BMP2 stimulated recruitment of SMAD1 to this region of the endogenous Id3 gene in LT2 cells. Mutation of the distal BRE in the longer murine Id3 reporter greatly reduced, but did not abolish, BMP2 induction." BMP2 "BDA2A, SSFSC, BMP2" ChIP "We identified the same sequence in the murine gene (?3283/?3276) (Fig. S8) and observed through ChIP analysis that BMP2 stimulated recruitment of SMAD1 to this region of the endogenous Id3 gene in L¦ÂT2 cells (Fig. S7A). Muta_x0002_tion of the distal BRE in the longer murine Id3 reporter greatly reduced, but did not abolish, BMP2 induction." -- -- -- >2KB 2627 E_01_046 21087445 -- hg19 chr22 24236392 24237411 Human "CEMC7A,THP-1,293T,SW982" Low throughput "Luciferase Reporter Assay,PCR,EMSA,DNaseI-seq" " DNase I hypersensitivity was used to identify potential hypersensitive sites (HS) across the MIF gene locus. Reporter gene assays were performed in different human cell lines with constructs containing the native or mutated HS element. Following phylogenetic and transcription factor binding profiling, electrophoretic mobility shift assay (EMSA) and RNA interference were performed and the effects of incubation with mithramycin, an antibiotic that binds GC boxes, were also studied. An HS centred on the first intron of MIF was identified. The HS acted as an enhancer in human T lymphoblasts (CEMC7A), human embryonic kidney cells (HEK293T) and human monocytic cells (THP-1), but not in a fibroblast-like synoviocyte (FLS) cell line (SW982) or cultured FLS derived from rheumatoid arthritis (RA) patients." Enhancer MIF -- "Luciferase Reporter Assay,PCR,EMSA,DNaseI-seq" " DNase I hypersensitivity was used to identify potential hypersensitive sites (HS) across the MIF gene locus. Reporter gene assays were performed in different human cell lines with constructs containing the native or mutated HS element. Following phylogenetic and transcription factor binding profiling, electrophoretic mobility shift assay (EMSA) and RNA interference were performed and the effects of incubation with mithramycin, an antibiotic that binds GC boxes, were also studied. An HS centred on the first intron of MIF was identified. The HS acted as an enhancer in human T lymphoblasts (CEMC7A), human embryonic kidney cells (HEK293T) and human monocytic cells (THP-1), but not in a fibroblast-like synoviocyte (FLS) cell line (SW982) or cultured FLS derived from rheumatoid arthritis (RA) patients." "GIF,GLIF,MMIF" Rheumatoid Arthritis DOID:7148 D001172 -- -- -- SP1 SP1 "Luciferase Reporter Assay,EMSA" "In order to determine whether Sp1 is recruited to the MIF intron 1 Enhancer, EMSA was performed using CEMC7A nuclear extract and a radiolabelled DNA probe of the 5¡äregion of the intronic sequence.We were able to demonstrate that Sp1 bound this sequence, as competition with excess cold Sp1 consensus sequence and addition of Sp1-specific antibody resulted in the abrogation of a shifted complex." -- -- -- <2KB 338 E_01_047 21310710 -- hg19 chr18 60786011 60788011 Human JURKAT Low throughput "3C,ChIP,RT-PCR" "With chromosome conformation capture (3C) and chromatin immunoprecipitation (ChIP) assays we demonstrated that the mbr could physically interact with BCL2 promoter through SATB1-mediated chromatin looping, which was required for epigenetic modifications of the promoter, CREB accessibility and high expression of the BCL2 gene." Enhancer BCL2 3C "ChIP,RT-PCR" "With chromosome conformation capture (3C) and chromatin immunoprecipitation (ChIP) assays we demonstrated that the mbr could physically interact with BCL2 promoter through SATB1-mediated chromatin looping, which was required for epigenetic modifications of the promoter, CREB accessibility and high expression of the BCL2 gene." "Bcl-2,PPP1R50" -- -- -- -- -- -- "SATB1,CEBPB,EP300" "SATB1,C/EBP-beta,IL6DBP,NF-IL6,TCF5,KAT3B,MKHK2,RSTS2,p300" "Western blot,3C,RT-PCR" "Our data showed that a reduction of the interaction between SBS1 and mbr induced by knockdown of SATB1 was significantly correlated with a decrease in the BCL2 mRNA level , suggesting that SATB1-mediated mbr-promoter interaction was required for transcriptional activity of the gene." -- -- -- >2KB 3567 E_01_048 21327791 LCT Enhancer hg19 chr2 136531405 136531505 Human Caco-2 Low throughput "Transfection,Luciferase Reporter Assay" "The LCT enhancer activity has previously been characterized by transfection experiments using a 450 bp fragment. These fragments were cloned at a distance upstream (approximately 3 Kb) of the 1,085 bp LCT promoter in a luciferase reporter gene plasmid, transfected into the intestinal cell line Caco--2 and compared to the full--length 450 bp enhancer construct." Enhancer LCT -- "Transfection,Luciferase Reporter Assay" "Caco--2 cells were transfected with luciferase reporter gene constructs containing either the LCT promoter alone or the LCT promoter with deleted enhancer fragments using either the African LP --14010*C variant (--14010*C; --13910*C) or the European LP --13910*T variant (--14010*G; --13910*T),or the ancestral variants (--14010*G; --13910*C) (Fig. 2)." "LAC,LPH,LPH1" -- -- -- -- -- -- "HNF1A,POU2F1" "HNF-1A,HNF1,HNF4A,IDDM20,LFB1,MODY3,TCF-1,TCF1,OCT1,OTF1,oct-1B" EMSA "This indicates that the Oct-1 binding to the -14010*C variant is influenced by the downstream sequence that covers the HNF1a site. The small differences in the Oct-1 binding to the -14010 allelic variants in EMSAs we have found, might be expected to be more pronounced in adult intestine where the pattern of transcription.factor expression and activity allows higher expression than in the Caco-2 cell line." -- -- -- >2KB 13954 E_01_049 21348942 -- hg19 chr12 100693355 100695355 Human MCF-7 Low throughput "ChIP,Western blot,siRNA,qPCR" The reaction was performed at a combined annealing/extension temperature of 60?¡ãC for 40 cycles using primers designed to specifically amplify the sequence containing the +571 ERE half©\site and two regions covering ER©\binding sites named enhancer?1 and enhancer?2 located at ?168 and ?206?kb in the PR regulatory regions. Enhancer RB1 -- ChIP ChIP analysis of PR regulatory regions in MCF--7 cells.Distal ER--binding sites and a proximal half--ERE located 571 bp downstream of the PRB tsp are highlighted with black diamonds. "OSRC,PPP1R130,RB,p105-Rb,pRb,pp110" -- -- -- -- -- -- KDM5A "RBBP-2,RBBP2,RBP2" Western blot Overexpression of the wild-type and mutated JARID1A forms after transfection of the corresponding plasmids in MCF-7 cells was confirmed at the protein level by western blot analysis. -- -- -- >2KB 51816473 E_01_050 21348942 -- hg19 chr12 100731355 100733355 Human MCF-7 Low throughput "ChIP,Western blot,siRNA,qPCR" The reaction was performed at a combined annealing/extension temperature of 60?¡ãC for 40 cycles using primers designed to specifically amplify the sequence containing the +571 ERE half©\site and two regions covering ER©\binding sites named enhancer?1 and enhancer?2 located at ?168 and ?206?kb in the PR regulatory regions. Enhancer RB1 -- ChIP ChIP analysis of PR regulatory regions in MCF--7 cells.Distal ER--binding sites and a proximal half--ERE located 571 bp downstream of the PRB tsp are highlighted with black diamonds. "OSRC,PPP1R130,RB,p105-Rb,pRb,pp110" -- -- -- -- -- -- KDM5A "RBBP-2,RBBP2,RBP2" Western blot Overexpression of the wild-type and mutated JARID1A forms after transfection of the corresponding plasmids in MCF-7 cells was confirmed at the protein level by western blot analysis. -- -- -- >2KB 51854473 E_01_051 21355081 -- hg19 chr10 131265598 131265656 Human "Human Bronchial Epithelial Cell,Lung cancer-derived Cell Lines,Glioblastoma Cell Lines" Low throughput "Luciferase Reporter Assay,PCR" This study provides strong evidence that the A allele of a MGMT promoter-enhancer SNP (rs16906252) is a key determinant in the acquisition of MGMT methylation in lung carcinogenesis. Enhancer MGMT -- "Luciferase Reporter Assay,PCR" This study provides strong evidence that the A allele of a MGMT promoter-enhancer SNP (rs16906252) is a key determinant in the acquisition of MGMT methylation in lung carcinogenesis. MGMT Lung Adenocarcinoma DOID:3910 D000077192 -- -- -- -- -- -- -- rs16906252 131265545 "Luciferase Reporter Assay,PCR" <2KB 180 E_01_052 21378152 WT1 Enhancer hg19 chr11 32403489 32405489 Human Human Podocyte Cell Line AB8/13 Low throughput "qRT-PCR,Luciferase Reporter Assay" We tested the effect of TGF-beta1 on a human WT1 enhancer located ?4.3 kb upstream of the transcription start site using a reporter vector WTA. Enhancer WT1 -- "qRT-PCR,Luciferase Reporter Assay" " TGF-beta1 did not alter luciferase activity of the reporter construct for a human WT1 promoter but reduced that for a human WT1 5¡ä enhancer construct, suggesting that TGF-beta1 may regulate WT1 expression by altering the 5¡ä enhancer activity." "AWT1,GUD,NPHS4,WAGR,WIT-2,WT33" -- -- -- -- -- -- SMAD4 "DPC4,JIP,MADH4,MYHRS" Luciferase Reporter Assay "The luciferase activity of p3TP-lux, a reporter construct for Smadbinding element, was determined after incubation with 5 ng/mL TGFbeta1 for 24 h. TGF-beta1 increased the luciferase activity by ~12-fold in the presence of the NTC shRNA, while this increase was largely prevented by Sh-Smad4. The luciferase activity of p3TP-lux was normalized to beta-galactosidase activity of pCMV-beta-gal and shown as relative values to that of pGL2 basic." -- -- -- >2KB 4831 E_01_053 21385855 -- hg19 chr6 135459326 135461326 Human K-562 Low throughput "Luciferase Reporter Assay,PCR,ChIP" " Based on transient expression of a luciferase reporter plasmid, the DNA fragment encompassing the 3-bp deletion polymorphism has enhancer-like activity that is further augmented by the introduction of the 3-bp deletion." Enhancer HBG2 -- "Luciferase Reporter Assay,PCR,ChIP" "In an initial attempt to demonstrate functionality of the DNA fragment surrounding the 3-bp deletion polymorphism, DNA fragment either without or with the 3-bp deletion (Figure 3A) was ligated to an expression vector consisting of 1.4 kb HBG2 proximal promoter and luciferase reporter gene. " "HBG-T1,TNCY" -- -- -- -- -- -- "RUNX1,GATA" "AML1,AML1-EVI-1,AMLCR1,CBF2alpha,CBFA2,EVI-1,PEBP2aB,PEBP2alpha,GATA" Luciferase Reporter Assay "Effect of mutation of transcription factor binding sites in the 58-bp DNA fragment with the 3-bp deletion on its enhancer-like activity.The luciferase activity of the plasmid with the 58-bp DNA fragment was normalized to be 1.0. Mutation of the E-box, RUNX1, or 3_x0004_ GATA binding site down-regulates the enhancer-like activity to approximately 50%. Mutation of the 5_x0004_ GATA binding site does not decrease the enhancer-like activity of the DNA fragment. " rs9399137 135419018 "Luciferase Reporter Assay,PCR,ChIP" >2KB 130185906 E_01_054 21402921 -- hg19 chr12 8217947 8219702 Human "HeLa,THP-1" Low throughput "ChIP-qPCR,ChIP" " To test this hypothesis, we chose the top 10 genes specifically induced by TNF-¦Á in THP-1 cells, but not HeLa cells, from microarray data (Fig. 1B), and verified five of them (CCL3, CCL4, IL12B, C3AR1, and GPR84) had PU.1 peaks as well as THP-1¨Cspecific p65 binding sites within 30 kb of their promoters (Fig. 4A)." Enhancer C3AR1 -- "ChIP-chip,RT-qPCR,ChIP-qPCR" " To test this hypothesis, we chose the top 10 genes specifically induced by TNF-¦Á in THP-1 cells, but not HeLa cells, from microarray data (Fig. 1B), and verified five of them (CCL3, CCL4, IL12B, C3AR1, and GPR84) had PU.1 peaks as well as THP-1¨Cspecific p65 binding sites within 30 kb of their promoters (Fig. 4A)." "AZ3B,C3AR,HNFAG09" -- -- -- "The diversity of transcriptional programs in mammalian cells arises, at least in part,is from preexisting enhancers that are established by cell-specific transcription factors." ChIP These results strongly support the model that combinatorial binding of THP-1¨Cspecific TFs PU.1 and C/EBP¦Á synergistically create a distinct group of enhancers and mediate selection of a subset of monocyte/macrophage-specific NF-¦ÊB target genes for specific induction by allowing the binding of p65 to these locations for productive gene expression. "SPI1,CEBPB,NFKB1" "OF,PU.1,SFPI1,SPI-1,SPI-A,C/EBP-beta,IL6DBP,NF-IL6,TCF5,CVID12,EBP-1,KBF1,NF-kB,NF-kB1,NF-kappa-B1,NF-kappaB,NFKB-p105,NFKB-p50,NFkappaB,p105,p50" "ChIP-qPCR,ChIP" "Strikingly, we found that all these genes gained responsiveness to TNF-¦Á stimulation only when both PU.1 and C/EBP¦Á were coexpressed in HeLa cells (Fig. 4B). To test the chromatin events near these genes, we further identified eight THP-1¨Cspecific p65 locations (P1¨CP8; Fig. 4A) around these five genes and performed ChIP/quantitative PCR (qPCR) to detect p74 occupancy and active chromatin." -- -- -- >2KB 9386 E_01_055 21402921 -- hg19 chr12 54757502 54758950 Human "HeLa,THP-1" Low throughput "ChIP-qPCR,ChIP" " To test this hypothesis, we chose the top 10 genes specifically induced by TNF-¦Á in THP-1 cells, but not HeLa cells, from microarray data (Fig. 1B), and verified five of them (CCL3, CCL4, IL12B, C3AR1, and GPR84) had PU.1 peaks as well as THP-1¨Cspecific p65 binding sites within 30 kb of their promoters (Fig. 4A)." Enhancer GPR84 -- "ChIP-chip,RT-qPCR,ChIP-qPCR" " To test this hypothesis, we chose the top 10 genes specifically induced by TNF-¦Á in THP-1 cells, but not HeLa cells, from microarray data (Fig. 1B), and verified five of them (CCL3, CCL4, IL12B, C3AR1, and GPR84) had PU.1 peaks as well as THP-1¨Cspecific p65 binding sites within 30 kb of their promoters (Fig. 4A)." "EX33,GPCR4" -- -- -- "The diversity of transcriptional programs in mammalian cells arises, at least in part,is from preexisting enhancers that are established by cell-specific transcription factors." ChIP These results strongly support the model that combinatorial binding of THP-1¨Cspecific TFs PU.1 and C/EBP¦Á synergistically create a distinct group of enhancers and mediate selection of a subset of monocyte/macrophage-specific NF-¦ÊB target genes for specific induction by allowing the binding of p65 to these locations for productive gene expression. "SPI1,CEBPB,NFKB1" "OF,PU.1,SFPI1,SPI-1,SPI-A,C/EBP-beta,IL6DBP,NF-IL6,TCF5,CVID12,EBP-1,KBF1,NF-kB,NF-kB1,NF-kappa-B1,NF-kappaB,NFKB-p105,NFKB-p50,NFkappaB,p105,p50" "ChIP-qPCR,ChIP" "Strikingly, we found that all these genes gained responsiveness to TNF-¦Á stimulation only when both PU.1 and C/EBP¦Á were coexpressed in HeLa cells (Fig. 4B). To test the chromatin events near these genes, we further identified eight THP-1¨Cspecific p65 locations (P1¨CP8; Fig. 4A) around these five genes and performed ChIP/quantitative PCR (qPCR) to detect p75 occupancy and active chromatin." -- -- -- <2KB 1998 E_01_056 21402921 -- hg19 chr17 34411749 34415000 Human "HeLa,THP-1" Low throughput "ChIP-qPCR,ChIP" " To test this hypothesis, we chose the top 10 genes specifically induced by TNF-¦Á in THP-1 cells, but not HeLa cells, from microarray data (Fig. 1B), and verified five of them (CCL3, CCL4, IL12B, C3AR1, and GPR84) had PU.1 peaks as well as THP-1¨Cspecific p65 binding sites within 30 kb of their promoters (Fig. 4A)." Enhancer CCL3 -- "ChIP-chip,RT-qPCR,ChIP-qPCR" " To test this hypothesis, we chose the top 10 genes specifically induced by TNF-¦Á in THP-1 cells, but not HeLa cells, from microarray data (Fig. 1B), and verified five of them (CCL3, CCL4, IL12B, C3AR1, and GPR84) had PU.1 peaks as well as THP-1¨Cspecific p65 binding sites within 30 kb of their promoters (Fig. 4A)." "G0S19-1,LD78ALPHA,MIP-1-alpha,MIP1A,SCYA3" -- -- -- "The diversity of transcriptional programs in mammalian cells arises, at least in part,is from preexisting enhancers that are established by cell-specific transcription factors." ChIP These results strongly support the model that combinatorial binding of THP-1¨Cspecific TFs PU.1 and C/EBP¦Á synergistically create a distinct group of enhancers and mediate selection of a subset of monocyte/macrophage-specific NF-¦ÊB target genes for specific induction by allowing the binding of p65 to these locations for productive gene expression. "SPI1,CEBPB,NFKB1" "OF,PU.1,SFPI1,SPI-1,SPI-A,C/EBP-beta,IL6DBP,NF-IL6,TCF5,CVID12,EBP-1,KBF1,NF-kB,NF-kB1,NF-kappa-B1,NF-kappaB,NFKB-p105,NFKB-p50,NFkappaB,p105,p50" "ChIP-qPCR,ChIP" "Strikingly, we found that all these genes gained responsiveness to TNF-¦Á stimulation only when both PU.1 and C/EBP¦Á were coexpressed in HeLa cells (Fig. 4B). To test the chromatin events near these genes, we further identified eight THP-1¨Cspecific p65 locations (P1¨CP8; Fig. 4A) around these five genes and performed ChIP/quantitative PCR (qPCR) to detect p75 occupancy and active chromatin." -- -- -- >2KB 2226 E_01_057 21402921 -- hg19 chr17 34416207 34419359 Human "HeLa,THP-1" Low throughput "ChIP-qPCR,ChIP" " To test this hypothesis, we chose the top 10 genes specifically induced by TNF-¦Á in THP-1 cells, but not HeLa cells, from microarray data (Fig. 1B), and verified five of them (CCL3, CCL4, IL12B, C3AR1, and GPR84) had PU.1 peaks as well as THP-1¨Cspecific p65 binding sites within 30 kb of their promoters (Fig. 4A)." Enhancer CCL3 -- "ChIP-chip,RT-qPCR,ChIP-qPCR" " To test this hypothesis, we chose the top 10 genes specifically induced by TNF-¦Á in THP-1 cells, but not HeLa cells, from microarray data (Fig. 1B), and verified five of them (CCL3, CCL4, IL12B, C3AR1, and GPR84) had PU.1 peaks as well as THP-1¨Cspecific p65 binding sites within 30 kb of their promoters (Fig. 4A)." "G0S19-1,LD78ALPHA,MIP-1-alpha,MIP1A,SCYA3" -- -- -- "The diversity of transcriptional programs in mammalian cells arises, at least in part,is from preexisting enhancers that are established by cell-specific transcription factors." ChIP These results strongly support the model that combinatorial binding of THP-1¨Cspecific TFs PU.1 and C/EBP¦Á synergistically create a distinct group of enhancers and mediate selection of a subset of monocyte/macrophage-specific NF-¦ÊB target genes for specific induction by allowing the binding of p65 to these locations for productive gene expression. "SPI1,CEBPB,NFKB1" "OF,PU.1,SFPI1,SPI-1,SPI-A,C/EBP-beta,IL6DBP,NF-IL6,TCF5,CVID12,EBP-1,KBF1,NF-kB,NF-kB1,NF-kappa-B1,NF-kappaB,NFKB-p105,NFKB-p50,NFkappaB,p105,p50" "ChIP-qPCR,ChIP" "Strikingly, we found that all these genes gained responsiveness to TNF-¦Á stimulation only when both PU.1 and C/EBP¦Á were coexpressed in HeLa cells (Fig. 4B). To test the chromatin events near these genes, we further identified eight THP-1¨Cspecific p65 locations (P1¨CP8; Fig. 4A) around these five genes and performed ChIP/quantitative PCR (qPCR) to detect p75 occupancy and active chromatin." -- -- -- >2KB 2182 E_01_058 21402921 -- hg19 chr17 34429460 34432613 Human "HeLa,THP-1" Low throughput "ChIP-qPCR,ChIP" " To test this hypothesis, we chose the top 10 genes specifically induced by TNF-¦Á in THP-1 cells, but not HeLa cells, from microarray data (Fig. 1B), and verified five of them (CCL3, CCL4, IL12B, C3AR1, and GPR84) had PU.1 peaks as well as THP-1¨Cspecific p65 binding sites within 30 kb of their promoters (Fig. 4A)." Enhancer CCL3 -- "ChIP-chip,RT-qPCR,ChIP-qPCR" " To test this hypothesis, we chose the top 10 genes specifically induced by TNF-¦Á in THP-1 cells, but not HeLa cells, from microarray data (Fig. 1B), and verified five of them (CCL3, CCL4, IL12B, C3AR1, and GPR84) had PU.1 peaks as well as THP-1¨Cspecific p65 binding sites within 30 kb of their promoters (Fig. 4A)." "G0S19-1,LD78ALPHA,MIP-1-alpha,MIP1A,SCYA3" -- -- -- "The diversity of transcriptional programs in mammalian cells arises, at least in part,is from preexisting enhancers that are established by cell-specific transcription factors." ChIP These results strongly support the model that combinatorial binding of THP-1¨Cspecific TFs PU.1 and C/EBP¦Á synergistically create a distinct group of enhancers and mediate selection of a subset of monocyte/macrophage-specific NF-¦ÊB target genes for specific induction by allowing the binding of p65 to these locations for productive gene expression. "SPI1,CEBPB,NFKB1" "OF,PU.1,SFPI1,SPI-1,SPI-A,C/EBP-beta,IL6DBP,NF-IL6,TCF5,CVID12,EBP-1,KBF1,NF-kB,NF-kB1,NF-kappa-B1,NF-kappaB,NFKB-p105,NFKB-p50,NFkappaB,p105,p50" "ChIP-qPCR,ChIP" "Strikingly, we found that all these genes gained responsiveness to TNF-¦Á stimulation only when both PU.1 and C/EBP¦Á were coexpressed in HeLa cells (Fig. 4B). To test the chromatin events near these genes, we further identified eight THP-1¨Cspecific p65 locations (P1¨CP8; Fig. 4A) around these five genes and performed ChIP/quantitative PCR (qPCR) to detect p75 occupancy and active chromatin." -- -- -- >2KB 15436 E_01_059 21402921 -- hg19 chr2 113595983 113598617 Human "HeLa,THP-1" Low throughput "ChIP-qPCR,ChIP" Premarked enhancers and promoters predict cell-specific p65 binding.(Fig.2) Enhancer IL1B -- "ChIP-qPCR,ChIP" Premarked enhancers and promoters predict cell-specific p65 binding.(Fig.2) "IL-1,IL1-BETA,IL1F2,IL1beta" -- -- -- "The diversity of transcriptional programs in mammalian cells arises, at least in part,is from preexisting enhancers that are established by cell-specific transcription factors." ChIP These results strongly support the model that combinatorial binding of THP-1¨Cspecific TFs PU.1 and C/EBP¦Á synergistically create a distinct group of enhancers and mediate selection of a subset of monocyte/macrophage-specific NF-¦ÊB target genes for specific induction by allowing the binding of p65 to these locations for productive gene expression. "SPI1,CEBPB,NFKB1" "OF,PU.1,SFPI1,SPI-1,SPI-A,C/EBP-beta,IL6DBP,NF-IL6,TCF5,CVID12,EBP-1,KBF1,NF-kB,NF-kB1,NF-kappa-B1,NF-kappaB,NFKB-p105,NFKB-p50,NFkappaB,p105,p50" "ChIP-qPCR,ChIP" "Strikingly, we found that all these genes gained responsiveness to TNF-¦Á stimulation only when both PU.1 and C/EBP¦Á were coexpressed in HeLa cells (Fig. 4B). To test the chromatin events near these genes, we further identified eight THP-1¨Cspecific p65 locations (P1¨CP8; Fig. 4A) around these five genes and performed ChIP/quantitative PCR (qPCR) to detect p75 occupancy and active chromatin." -- -- -- >2KB 9964 E_01_060 21402921 -- hg19 chr2 113597789 113600339 Human "HeLa,THP-1" Low throughput "ChIP-qPCR,ChIP" Premarked enhancers and promoters predict cell-specific p65 binding.(Fig.2) Enhancer IL1B -- "ChIP-qPCR,ChIP" Premarked enhancers and promoters predict cell-specific p65 binding.(Fig.2) "IL-1,IL1-BETA,IL1F2,IL1beta" -- -- -- "The diversity of transcriptional programs in mammalian cells arises, at least in part,is from preexisting enhancers that are established by cell-specific transcription factors." ChIP These results strongly support the model that combinatorial binding of THP-1¨Cspecific TFs PU.1 and C/EBP¦Á synergistically create a distinct group of enhancers and mediate selection of a subset of monocyte/macrophage-specific NF-¦ÊB target genes for specific induction by allowing the binding of p65 to these locations for productive gene expression. "SPI1,CEBPB,NFKB1" "OF,PU.1,SFPI1,SPI-1,SPI-A,C/EBP-beta,IL6DBP,NF-IL6,TCF5,CVID12,EBP-1,KBF1,NF-kB,NF-kB1,NF-kappa-B1,NF-kappaB,NFKB-p105,NFKB-p50,NFkappaB,p105,p50" "ChIP-qPCR,ChIP" "Strikingly, we found that all these genes gained responsiveness to TNF-¦Á stimulation only when both PU.1 and C/EBP¦Á were coexpressed in HeLa cells (Fig. 4B). To test the chromatin events near these genes, we further identified eight THP-1¨Cspecific p65 locations (P1¨CP8; Fig. 4A) around these five genes and performed ChIP/quantitative PCR (qPCR) to detect p75 occupancy and active chromatin." -- -- -- >2KB 11728 E_01_061 21402921 -- hg19 chr5 158755252 158761634 Human "HeLa,THP-1" Low throughput "ChIP-qPCR,ChIP" " To test this hypothesis, we chose the top 10 genes specifically induced by TNF-¦Á in THP-1 cells, but not HeLa cells, from microarray data (Fig. 1B), and verified five of them (CCL3, CCL4, IL12B, C3AR1, and GPR84) had PU.1 peaks as well as THP-1¨Cspecific p65 binding sites within 30 kb of their promoters (Fig. 4A)." Enhancer IL12B -- "ChIP-chip,RT-qPCR,ChIP-qPCR" " To test this hypothesis, we chose the top 10 genes specifically induced by TNF-¦Á in THP-1 cells, but not HeLa cells, from microarray data (Fig. 1B), and verified five of them (CCL3, CCL4, IL12B, C3AR1, and GPR84) had PU.1 peaks as well as THP-1¨Cspecific p65 binding sites within 30 kb of their promoters (Fig. 4A)." "CLMF,CLMF2,IL-12B,IMD28,IMD29,NKSF,NKSF2" -- -- -- "The diversity of transcriptional programs in mammalian cells arises, at least in part,is from preexisting enhancers that are established by cell-specific transcription factors." ChIP These results strongly support the model that combinatorial binding of THP-1¨Cspecific TFs PU.1 and C/EBP¦Á synergistically create a distinct group of enhancers and mediate selection of a subset of monocyte/macrophage-specific NF-¦ÊB target genes for specific induction by allowing the binding of p65 to these locations for productive gene expression. "SPI1,CEBPB,NFKB1" "OF,PU.1,SFPI1,SPI-1,SPI-A,C/EBP-beta,IL6DBP,NF-IL6,TCF5,CVID12,EBP-1,KBF1,NF-kB,NF-kB1,NF-kappa-B1,NF-kappaB,NFKB-p105,NFKB-p50,NFkappaB,p105,p50" "ChIP-qPCR,ChIP" "Strikingly, we found that all these genes gained responsiveness to TNF-¦Á stimulation only when both PU.1 and C/EBP¦Á were coexpressed in HeLa cells (Fig. 4B). To test the chromatin events near these genes, we further identified eight THP-1¨Cspecific p65 locations (P1¨CP8; Fig. 4A) around these five genes and performed ChIP/quantitative PCR (qPCR) to detect p75 occupancy and active chromatin." -- -- -- >2KB 16653 E_01_062 21402921 -- hg19 chr5 158783552 158790360 Human "HeLa,THP-1" Low throughput "ChIP-qPCR,ChIP" " To test this hypothesis, we chose the top 10 genes specifically induced by TNF-¦Á in THP-1 cells, but not HeLa cells, from microarray data (Fig. 1B), and verified five of them (CCL3, CCL4, IL12B, C3AR1, and GPR84) had PU.1 peaks as well as THP-1¨Cspecific p65 binding sites within 30 kb of their promoters (Fig. 4A)." Enhancer IL12B -- "ChIP-chip,RT-qPCR,ChIP-qPCR" " To test this hypothesis, we chose the top 10 genes specifically induced by TNF-¦Á in THP-1 cells, but not HeLa cells, from microarray data (Fig. 1B), and verified five of them (CCL3, CCL4, IL12B, C3AR1, and GPR84) had PU.1 peaks as well as THP-1¨Cspecific p65 binding sites within 30 kb of their promoters (Fig. 4A)." "CLMF,CLMF2,IL-12B,IMD28,IMD29,NKSF,NKSF2" -- -- -- "The diversity of transcriptional programs in mammalian cells arises, at least in part,is from preexisting enhancers that are established by cell-specific transcription factors." ChIP These results strongly support the model that combinatorial binding of THP-1¨Cspecific TFs PU.1 and C/EBP¦Á synergistically create a distinct group of enhancers and mediate selection of a subset of monocyte/macrophage-specific NF-¦ÊB target genes for specific induction by allowing the binding of p65 to these locations for productive gene expression. "SPI1,CEBPB,NFKB1" "OF,PU.1,SFPI1,SPI-1,SPI-A,C/EBP-beta,IL6DBP,NF-IL6,TCF5,CVID12,EBP-1,KBF1,NF-kB,NF-kB1,NF-kappa-B1,NF-kappaB,NFKB-p105,NFKB-p50,NFkappaB,p105,p50" "ChIP-qPCR,ChIP" "Strikingly, we found that all these genes gained responsiveness to TNF-¦Á stimulation only when both PU.1 and C/EBP¦Á were coexpressed in HeLa cells (Fig. 4B). To test the chromatin events near these genes, we further identified eight THP-1¨Cspecific p65 locations (P1¨CP8; Fig. 4A) around these five genes and performed ChIP/quantitative PCR (qPCR) to detect p75 occupancy and active chromatin." -- -- -- >2KB 45166 E_01_063 21402921 -- hg19 chr5 158787331 158794352 Human "HeLa,THP-1" Low throughput "ChIP-qPCR,ChIP" " To test this hypothesis, we chose the top 10 genes specifically induced by TNF-¦Á in THP-1 cells, but not HeLa cells, from microarray data (Fig. 1B), and verified five of them (CCL3, CCL4, IL12B, C3AR1, and GPR84) had PU.1 peaks as well as THP-1¨Cspecific p65 binding sites within 30 kb of their promoters (Fig. 4A)." Enhancer IL12B -- "ChIP-chip,RT-qPCR,ChIP-qPCR" " To test this hypothesis, we chose the top 10 genes specifically induced by TNF-¦Á in THP-1 cells, but not HeLa cells, from microarray data (Fig. 1B), and verified five of them (CCL3, CCL4, IL12B, C3AR1, and GPR84) had PU.1 peaks as well as THP-1¨Cspecific p65 binding sites within 30 kb of their promoters (Fig. 4A)." "CLMF,CLMF2,IL-12B,IMD28,IMD29,NKSF,NKSF2" -- -- -- "The diversity of transcriptional programs in mammalian cells arises, at least in part,is from preexisting enhancers that are established by cell-specific transcription factors." ChIP These results strongly support the model that combinatorial binding of THP-1¨Cspecific TFs PU.1 and C/EBP¦Á synergistically create a distinct group of enhancers and mediate selection of a subset of monocyte/macrophage-specific NF-¦ÊB target genes for specific induction by allowing the binding of p65 to these locations for productive gene expression. "SPI1,CEBPB,NFKB1" "OF,PU.1,SFPI1,SPI-1,SPI-A,C/EBP-beta,IL6DBP,NF-IL6,TCF5,CVID12,EBP-1,KBF1,NF-kB,NF-kB1,NF-kappa-B1,NF-kappaB,NFKB-p105,NFKB-p50,NFkappaB,p105,p50" "ChIP-qPCR,ChIP" "Strikingly, we found that all these genes gained responsiveness to TNF-¦Á stimulation only when both PU.1 and C/EBP¦Á were coexpressed in HeLa cells (Fig. 4B). To test the chromatin events near these genes, we further identified eight THP-1¨Cspecific p65 locations (P1¨CP8; Fig. 4A) around these five genes and performed ChIP/quantitative PCR (qPCR) to detect p75 occupancy and active chromatin." -- -- -- >2KB 49052 E_01_064 21402921 -- hg19 chr1 203273730 203275395 Human "HeLa,THP-1" Low throughput "ChIP-qPCR,ChIP" Premarked enhancers and promoters predict cell-specific p65 binding.(Fig.2) Enhancer BTG2 -- "ChIP-qPCR,ChIP" Premarked enhancers and promoters predict cell-specific p65 binding.(Fig.2) "APRO1,PC3,TIS21" -- -- -- "The diversity of transcriptional programs in mammalian cells arises, at least in part,is from preexisting enhancers that are established by cell-specific transcription factors." ChIP These results strongly support the model that combinatorial binding of THP-1¨Cspecific TFs PU.1 and C/EBP¦Á synergistically create a distinct group of enhancers and mediate selection of a subset of monocyte/macrophage-specific NF-¦ÊB target genes for specific induction by allowing the binding of p65 to these locations for productive gene expression. "SPI1,CEBPB,NFKB1" "OF,PU.1,SFPI1,SPI-1,SPI-A,C/EBP-beta,IL6DBP,NF-IL6,TCF5,CVID12,EBP-1,KBF1,NF-kB,NF-kB1,NF-kappa-B1,NF-kappaB,NFKB-p105,NFKB-p50,NFkappaB,p105,p50" "ChIP-qPCR,ChIP" "Strikingly, we found that all these genes gained responsiveness to TNF-¦Á stimulation only when both PU.1 and C/EBP¦Á were coexpressed in HeLa cells (Fig. 4B). To test the chromatin events near these genes, we further identified eight THP-1¨Cspecific p65 locations (P1¨CP8; Fig. 4A) around these five genes and performed ChIP/quantitative PCR (qPCR) to detect p75 occupancy and active chromatin." -- -- -- <2KB 100 E_01_065 21419113 -- hg19 chr1 226065482 226067482 Human Perinodal Cells Low throughput "PCR,Transgenic mice,Immunostaining" We have now identified a transcriptional enhancer (ANE) in the human LEFTY1 gene that exhibits marked L>R asymmetric activity in perinodal cells of the mouse embryos.the enhancer activity of each fragment was examined in transgenic mouse embryos. Enhancer LEFTY1 -- "PCR,Transgenic mice,Immunostaining" We have now identified a transcriptional enhancer (ANE) in the human LEFTY1 gene that exhibits marked L>R asymmetric activity in perinodal cells of the mouse embryos. "LEFTB,LEFTYB" -- -- -- -- -- -- FOXH1 "FAST-1,FAST1" X-Gal Staining Assay "The approximate location of FoxH1 binding sites is indicated by red ovals. Restriction sites: E, EcoRI; Bam, BamHI; BgI, BglI; BX, BstXI; BgII, BglII; Sma, SmaI; Xb, XbaI; Sac, SacI. (B¨CE) Ventral views of X-gal¨Cstained embryos harboring the indicated lacZ transgenes. (F¨CI) Magnified views of the node region of X-gal¨Cstained embryos harboring hNPE7.5-lacZ." -- -- -- >2KB 7499 E_01_066 21424707 -- hg19 chr1 204123719 204123725 Human Calu-6 Low throughput "Luciferase Reporter Assay,PCR" "To test the functional relevance of enhCRE, we fused it to RENMin (construct enhCRE--RENMin). Both cAMP agonists increased the transcriptional activity of enhCRE--RENMin.construct enhCRE--RENMin contains human renin enhancer CRE (enhCRE) fused to RENMin. RLA, relative luciferase activity" Enhancer REN -- Luciferase Reporter Assay "To test the functional relevance of enhCRE, we fused it to RENMin (construct enhCRE--RENMin). Both cAMP agonists increased the transcriptional activity of enhCRE--RENMin.construct enhCRE--RENMin contains human renin enhancer CRE (enhCRE) fused to RENMin. RLA, relative luciferase activity" HNFJ2 -- -- -- -- -- -- CAMP "CAP-18,CAP18,CRAMP,FALL-39,FALL39,HSD26,LL37" "Luciferase Reporter Assay,ChIP,qRT-PCR" "Construct RENMin represents the minimal human renin promoter (-199 to +23) which contains the cAMP target sequence CNRE at ?135 to ?107. The PPAR¦Ã agonist rosiglitazone (Rosi, 500 nM for 20 h) was applied as positive control;RLA, relative luciferase activity." -- -- -- <2KB 221 E_01_067 21445863 -- hg19 chr21 35982223 35983306 Human Acute T-Cell Leukaemia Cell Low throughput "Luciferase Reporter Assay,PCR" "To test the putative enhancer activity of RE1 and RE2, we made a number of genetic constructs with a pGL3--Basic vector, which contains the firefly Luciferase gene." Enhancer RUNX1 -- Luciferase Reporter Assay The promoters were cloned upstream from the Luciferase gene and candidate enhancer elements-downstream of the gene in direct genomic orientation and reverse genomic orientation relative to the corresponding promoter. "AML1,AML1-EVI-1,AMLCR1,CBF2alpha,CBFA2,EVI-1,PEBP2aB,PEBP2alpha" -- -- -- -- -- -- -- -- -- -- -- -- -- >2KB 177332 E_01_068 21445863 -- hg19 chr21 36141738 36142821 Human Acute T-Cell Leukaemia Cell Low throughput "Luciferase Reporter Assay,PCR" "To test the putative enhancer activity of RE1 and RE2, we made a number of genetic constructs with a pGL4--Basic vector, which contains the firefly Luciferase gene." Enhancer RUNX1 -- Luciferase Reporter Assay The promoters were cloned upstream from the Luciferase gene and candidate enhancer elements-downstream of the gene in direct genomic orientation and reverse genomic orientation relative to the corresponding promoter. "AML1,AML1-EVI-1,AMLCR1,CBF2alpha,CBFA2,EVI-1,PEBP2aB,PEBP2alpha" -- -- -- -- -- -- -- -- -- -- -- -- -- >2KB 17817 E_01_069 21445905 -- hg19 chr2 136582836 136583741 Human "Caco-2,Cheek Cell" Low throughput "Luciferase Reporter Assay,PCR,EMSA" "In transient transfection experiments using Caco©\2 cells (a human colorectal adenocarcinoma cell line that mimics intestinal epithelial cells) and lactase promoter/luciferase gene constructs, small DNA fragments containing either the C?13,910 or T?13,910 variants of the enhancer region were shown to significantly increase the expression of the reporter gene when included in the constructs. " Enhancer MCM6 -- "Luciferase Reporter Assay,PCR," Map of the region of human chromosome 2q21 containing the human lactase (LCT) and minichromosome maintenance ©\6 (MCM6) genes (panel a). "MCG40308,Mis5,P105MCM" -- -- -- -- -- -- POU2F1 "OCT1,OTF1,oct-1B" EMSA "In addition, the transcription factor Oct-1 was shown to bind more strongly to DNA fragments containing the T213,910 variant than the C213,910 variant in electrophoretic mobility shift assays." rs4988235 136608646 PCR >2KB 13906 E_01_070 21464207 -- hg19 chr1 101402363 101412724 Human HUVEC Low+High throughput "Luciferase Reporter Assay,PCR,ChIP,ChIP-qPCR,ChIP-seq" "ChIP-seq revealed three distinct STAT6 binding regions in the VCAM-1 locus (kb ?16, ?11, and +15, relative to the TSS) (Fig. 7A)." Enhancer VCAM1 -- "Luciferase Reporter Assay,PCR,ChIP,ChIP-qPCR,ChIP-seq" "Moreover, our strategy led to the identification of a novel functionally important STAT6 binding site within 16 kb upstream of the VCAM-1 gene." "CD106,INCAM-100" Atherosclerosis DOID:1936 D050197 -- -- -- STAT6 "D12S1644,IL-4-STAT,STAT6,STAT6C" "ChIP-seq,PCR,Luciferase Reporter Assay" Genome-wide survey of IL-4-mediated STAT6 binding from sequential chromatin_x0002_immunoprecipitation with deep sequencing (chromatin immunoprecipitation sequencing [ChIP-seq]) in endothelial cells revealed regions of transient and sustained transcription factor binding. -- -- -- >2KB 222349 E_01_071 21494683 -- hg19 chr1 64083756 64083861 Human SK-N-MC Low throughput "ChIP,PCR" "To investigate histone modification and transcription factor binding, we performed chromatin immunoprecipitation (ChIP) assays using a ChIP assay kit (Millipore, Billerica, MA, USA) according to the manufacturer's protocol. " Enhancer ROR1 -- ChIP-seq "The ROR1 SNPs flanked a strong peak of H3K4me1 (Figure 4A) and the PLCB1 SNPs resided near an H3K4me1 peak and two CTCF binding sites (Figure 5A). H3K4me1, often with H3K27ac, is a chromatin marker for enhancers [29]. CTCF is an insulator-binding element that can interfere with nearby enhancers." "NTRKR1,dJ537F10.1" Insomnia -- D007319 -- -- -- PAX6 "AN,AN1,AN2,ASGD5,D11S812E,FVH1,MGDA,WAGR" "ChIP,PCR" "Cross ChIP PCR between PAX6 and CTCF. PAX6 ChIP DNA was detected in the two CTCF sites, and CTCF ChIP DNA was detected in the PAX6 site." -- -- -- >2KB 155880 E_01_072 21494683 -- hg19 chr1 64087305 64087660 Human SK-N-MC Low throughput "ChIP,PCR" "To investigate histone modification and transcription factor binding, we performed chromatin immunoprecipitation (ChIP) assays using a ChIP assay kit (Millipore, Billerica, MA, USA) according to the manufacturer's protocol. " Enhancer ROR1 -- ChIP-seq "The ROR1 SNPs flanked a strong peak of H3K4me1 (Figure 4A) and the PLCB1 SNPs resided near an H3K4me1 peak and two CTCF binding sites (Figure 5A). H3K4me1, often with H3K27ac, is a chromatin marker for enhancers [29]. CTCF is an insulator-binding element that can interfere with nearby enhancers." "NTRKR1,dJ537F10.1" Insomnia -- D007319 -- -- -- PAX6 "AN,AN1,AN2,ASGD5,D11S812E,FVH1,MGDA,WAGR" "ChIP,PCR" "Cross ChIP PCR between PAX6 and CTCF. PAX6 ChIP DNA was detected in the two CTCF sites, and CTCF ChIP DNA was detected in the PAX6 site." -- -- -- >2KB 152206 E_01_073 21494683 -- hg19 chr20 8684437 8686556 Human SK-N-MC Low throughput "ChIP,PCR" "To investigate histone modification and transcription factor binding, we performed chromatin immunoprecipitation (ChIP) assays using a ChIP assay kit (Millipore, Billerica, MA, USA) according to the manufacturer's protocol. " Enhancer PLCB1 -- ChIP-seq "The ROR1 SNPs flanked a strong peak of H3K4me1 (Figure 4A) and the PLCB1 SNPs resided near an H3K4me1 peak and two CTCF binding sites (Figure 5A). H3K4me1, often with H3K27ac, is a chromatin marker for enhancers [29]. CTCF is an insulator-binding elementhat can interfere with nearby enhancers." "EIEE12,PI-PLC,PLC-154,PLC-I,PLC-beta-1,PLC154,PLCB1A,PLCB1B" Insomnia -- D007319 -- -- -- PAX6 "AN,AN1,AN2,ASGD5,D11S812E,FVH1,MGDA,WAGR" "ChIP,PCR" "Cross ChIP PCR between PAX6 and CTCF. PAX6 ChIP DNA was detected in the two CTCF sites, and CTCF ChIP DNA was detected in the PAX6 site." -- -- -- >2KB 572586 E_01_074 21494683 -- hg19 chr20 8687417 8688741 Human SK-N-MC Low throughput "ChIP,PCR" "To investigate histone modification and transcription factor binding, we performed chromatin immunoprecipitation (ChIP) assays using a ChIP assay kit (Millipore, Billerica, MA, USA) according to the manufacturer's protocol. " Enhancer PLCB1 -- ChIP-seq "The ROR1 SNPs flanked a strong peak of H3K4me1 (Figure 4A) and the PLCB1 SNPs resided near an H3K4me1 peak and two CTCF binding sites (Figure 5A). H3K4me1, often with H3K27ac, is a chromatin marker for enhancers [29]. CTCF is an insulator-binding elementt can interfere with nearby enhancers." "EIEE12,PI-PLC,PLC-154,PLC-I,PLC-beta-1,PLC154,PLCB1A,PLCB1B" Insomnia -- D007319 -- -- -- PAX6 "AN,AN1,AN2,ASGD5,D11S812E,FVH1,MGDA,WAGR" "ChIP,PCR" "Cross ChIP PCR between PAX6 and CTCF. PAX6 ChIP DNA was detected in the two CTCF sites, and CTCF ChIP DNA was detected in the PAX6 site." -- -- -- >2KB 575168 E_01_075 21494683 -- hg19 chr20 8718344 8719933 Human SK-N-MC Low throughput "ChIP,PCR" "To investigate histone modification and transcription factor binding, we performed chromatin immunoprecipitation (ChIP) assays using a ChIP assay kit (Millipore, Billerica, MA, USA) according to the manufacturer's protocol. " Enhancer PLCB1 -- ChIP-seq "The ROR1 SNPs flanked a strong peak of H3K4me1 (Figure 4A) and the PLCB1 SNPs resided near an H3K4me1 peak and two CTCF binding sites (Figure 5A). H3K4me1, often with H3K27ac, is a chromatin marker for enhancers [29]. CTCF is an insulator-binding elementcan interfere with nearby enhancers." "EIEE12,PI-PLC,PLC-154,PLC-I,PLC-beta-1,PLC154,PLCB1A,PLCB1B" Insomnia -- D007319 -- -- -- PAX6 "AN,AN1,AN2,ASGD5,D11S812E,FVH1,MGDA,WAGR" "ChIP,PCR" "Cross ChIP PCR between PAX6 and CTCF. PAX6 ChIP DNA was detected in the two CTCF sites, and CTCF ChIP DNA was detected in the PAX6 site." -- -- -- >2KB 606228 E_01_076 21527503 -- hg19 chr1 33353796 33354196 Human HC11 Mammary Epithelium Low throughput "Luciferase Reporter Assay,PCR,ChIP" "As detected by chromatin immunoprecipitation assays, treatment of cells with the progestin agonist R5020 induced a rapid recruitment (5 min) of PR to the proximal promoter (?235 bp) and distal enhancer (?6 kb upstream of transcription start site) of ¦Â-casein" Enhancer "TMEM54,¦Â-casein gene" -- ChIP "In this study, we used HC-11 cells and ChIP assays to examine the influence of progesterone and PR on cooperative interactions and dynamics of the various transcription factors and coregulatory proteins that interact with the promoter and enhancer of the endogenous ¦Â-casein gene." "BCLP,CAC-1,CAC1" -- -- -- -- -- -- "STAT5B,CEBPB,YY1" "STAT5,C/EBP-beta,IL6DBP,NF-IL6,TCF5,DELTA,GADEVS,INO80S,NF-E1,UCRBP,YIN-YANG-1" "PCR,ChIP" "Primers for PCR amplification of the proximal promoter span a 170-bp region (-190 to -20 from the transcription start site) containing binding sites for Stat5a,C/EBP,YY1,and GRE/progestin response element (PRE) half-sites, whereas a 400-bp region (-6400 to -6000) of the distal Enhancer was amplified that contains binding sites for Stat5,C/EBP,and other factors." -- -- -- >2KB 6197 E_01_077 21533051 -- hg19 chr8 128747315 128748315 Human HCT 116 Low+High throughput "ChIP,PCR,ChIP-seq" A diagram of novel ?-catenin binding regions (labeled A through F) and characterized Wnt responsive enhancers (labeled 5¡ä WRE and 3¡ä WRE) that were identified in the screen.Real-time PCR analysis of DNA elements precipitated using ?-catenin specific antibodies in ChIP assays conducted in HCT116 (black bars) or HEK293 (gray bars). Enhancer MYC q3C -- "Using quantitative chromatin conformation capture, I show that the distal WREs are aligned with the MYC promoter through large chromatin loops." "MRTL,MYCC,bHLHe39,c-Myc" Colorectal Cancer DOID:9256 D015179 The architecture of the MYC promoter is comprised of distal elements. 3C These findings suggest that the architecture of the MYC promoter is comprised of distal elements that are juxtaposed through large chromatin loops and that -catenin/TCF4 complexes utilize this conformation to activate MYCexpression in colon cancer cells. TCF4 "E2-2,FECD3,ITF-2,ITF2,PTHS,SEF-2,SEF2,SEF2-1,SEF2-1A,SEF2-1B,SEF2-1D,TCF-4,bHLHb19" ChIP-seq "In the present study, I characterize five novel ¦Â-catenin/TCF4 binding sites that were identified using a ChIP-Seq screen for ¦Â-catenin binding regions in the human HCT116 colon cancer cell line." rs6983267 128413305 "qRT-PCR,3C" <2KB 499 E_01_078 21533051 -- hg19 chr8 128749415 128750415 Human HCT 116 Low+High throughput "ChIP,PCR,ChIP-seq" A diagram of novel ?-catenin binding regions (labeled A through F) and characterized Wnt responsive enhancers (labeled 5¡ä WRE and 3¡ä WRE) that were identified in the screen.Real-time PCR analysis of DNA elements precipitated using ?-catenin specific antibodies in ChIP assays conducted in HCT116 (black bars) or HEK293 (gray bars). Enhancer MYC q3C -- "Using quantitative chromatin conformation capture, I show that the distal WREs are aligned with the MYC promoter through large chromatin loops." "MRTL,MYCC,bHLHe39,c-Myc" Colorectal Cancer DOID:9256 D015179 The architecture of the MYC promoter is comprised of distal elements. 3C These findings suggest that the architecture of the MYC promoter is comprised of distal elements that are juxtaposed through large chromatin loops and that ?-catenin/TCF4 complexes utilize this conformation to activate MYC expression in colon cancer cells. TCF4 "E2-2,FECD3,ITF-2,ITF2,PTHS,SEF-2,SEF2,SEF2-1,SEF2-1A,SEF2-1B,SEF2-1D,TCF-4,bHLHb19" ChIP-seq "In the present study, I characterize five novel ¦Â-catenin/TCF4 binding sites that were identified using a ChIP-Seq screen for ¦Â-catenin binding regions in the human HCT116 colon cancer cell line." rs6983267 128413305 "qRT-PCR,3C" <2KB 1601 E_01_079 21533051 -- hg19 chr8 128346390 128347994 Human HEK-293 Low+High throughput "ChIP,PCR,ChIP-seq" A diagram of novel ?-catenin binding regions (labeled A through F) and characterized Wnt responsive enhancers (labeled 5¡ä WRE and 3¡ä WRE) that were identified in the screen.Real-time PCR analysis of DNA elements precipitated using ?-catenin specific antibies in ChIP assays conducted in HCT116 (black bars) or HEK293 (gray bars). Enhancer MYC q3C -- "Using quantitative chromatin conformation capture, I show that the distal WREs are aligned with the MYC promoter through large chromatin loops." "MRTL,MYCC,bHLHe39,c-Myc" -- -- -- -- -- -- -- -- -- -- -- -- -- >2KB 401122 E_01_080 21533051 -- hg19 chr8 128327941 128330615 Human HEK-293 Low+High throughput "ChIP,PCR,ChIP-seq" A diagram of novel ?-catenin binding regions (labeled A through F) and characterized Wnt responsive enhancers (labeled 5¡ä WRE and 3¡ä WRE) that were identified in the screen.Real-time PCR analysis of DNA elements precipitated using ?-catenin specific antibs in ChIP assays conducted in HCT116 (black bars) or HEK293 (gray bars). Enhancer MYC q3C -- "Using quantitative chromatin conformation capture, I show that the distal WREs are aligned with the MYC promoter through large chromatin loops." "MRTL,MYCC,bHLHe39,c-Myc" -- -- -- -- -- -- -- -- -- -- -- -- -- >2KB 419036 E_01_081 21533051 -- hg19 chr8 128299198 128301336 Human HEK-293 Low+High throughput "ChIP,PCR,ChIP-seq" A diagram of novel ?-catenin binding regions (labeled A through F) and characterized Wnt responsive enhancers (labeled 5¡ä WRE and 3¡ä WRE) that were identified in the screen.Real-time PCR analysis of DNA elements precipitated using ?-catenin specific antibin ChIP assays conducted in HCT116 (black bars) or HEK293 (gray bars). Enhancer MYC q3C -- "Using quantitative chromatin conformation capture, I show that the distal WREs are aligned with the MYC promoter through large chromatin loops." "MRTL,MYCC,bHLHe39,c-Myc" -- -- -- -- -- -- -- -- -- -- -- -- -- >2KB 448047 E_01_082 21533051 -- hg19 chr8 128296257 128297326 Human HEK-293 Low+High throughput "ChIP,PCR,ChIP-seq" A diagram of novel ?-catenin binding regions (labeled A through F) and characterized Wnt responsive enhancers (labeled 5¡ä WRE and 3¡ä WRE) that were identified in the screen.Real-time PCR analysis of DNA elements precipitated using ?-catenin specific antib ChIP assays conducted in HCT116 (black bars) or HEK293 (gray bars). Enhancer MYC q3C -- "Using quantitative chromatin conformation capture, I show that the distal WREs are aligned with the MYC promoter through large chromatin loops." "MRTL,MYCC,bHLHe39,c-Myc" -- -- -- -- -- -- -- -- -- -- -- -- -- >2KB 451522 E_01_083 21536859 -- hg19 chr21 39823183 39825183 Human "T-acute lymphoblastic leukemia (T-ALL),Molt4,REH,416B" Low+High throughput "Luciferase Reporter Assay,PCR,ChIP,ChIP-PCR,ChIP-seq" The ERG stem cell enhancer is active in human T-cell progenitors and in T-ALL.Graphs at right show these peaks of enrichment confirmed by ChIP-PCR. Enhancer "ERG " q3C "qPCR,ChIP-PCR" "Taken together, these results show that genomic sequences in the human ERG locus corresponding to the mouse Erg +85 enhancer are in an active configuration in CD34+ stem/progenitors, pro-T, and T-ALL cells." "erg-3,p55" T-Cell Acute Lymphoblastic Leukemia DOID:5602 D054218 -- -- -- "GATA2,LMO2,SCL,LYL1" "DCML,IMD21,MONOMAC,NFE1B,LMO-2,RBTN2,RBTNL1,RHOM2,TTG2,SCL,bHLHa18" ChIP-PCR "ChIP-PCR analysis of TF binding. The enhancer has active chromatin marks (AcH3) in 416B blood progenitors as well as in T-ALL cells. There is strong LMO2 binding in all cell types, whereas SCL and LYL1 enrichments are more pronounced in T-ALL cells. The Ets factors, FLI1 and ERG, are also enriched in T-ALL cells. Consistent with their expression profiles, Gata2 is enriched at the _x0001_85 enhancer in 416B progenitors, whereas GATA3 is enriched in T-ALL cells. " -- -- -- >2KB 85001 E_01_084 21551367 -- hg19 chr17 32565596 32566796 Human U-87MG Low throughput "Luciferase Reporter Assay,PCR,ChIP,EMSA,3C" Schematic of CCL2 locus showing regions that were tested in the reporter assays. Enhancer CCL2 3C "PCR,qPCR" "The ¨C16.5 ECR physically interacts with CCL2 proximal promoter.To detect such physical interactions we used the 3C assay. In the 3C assay, interaction between widely separated regulatory elements is detected. " "GDCF-2,HC11,HSMCR30,MCAF,MCP-1,MCP1,SCYA2,SMC-CF" -- -- -- -- -- -- "NFKB1,SP1,CEBPB" "CVID12,EBP-1,KBF1,NF-kB,NF-kB1,NF-kappa-B1,NF-kappaB,NFKB-p105,NFKB-p50,NFkappaB,p105,p50,SP1,C/EBP-beta,IL6DBP,NF-IL6,TCF5" "ChIP,EMSA" The EMSA and the pulldown experiments demonstrated in vitro interaction of the ¨C16.5 ECR with NF-kB and c/EBPb. We used ChIP assay to determine whether the ¨C16.5 ECR can bind NF-kB and c/EBPb in vivo. Sheared chromatin that was obtained from U87MG cells (treated or not treated with TNF-a) was immmu_x0002_noprecipitated with Abs directed against p65 and p50 subunits of NF-kB and c/EBPb or with control Abs. -- -- -- >2KB 16099 E_01_085 21556051 UBE2C Enhancer hg19 chr20 44430820 44433428 Human "LNCaP,PC-3" Low throughput "RT-PCR,Luciferase Reporter Assay,ChIP,3C" "we performed quantitative 3C assays for the UBE2C locus in LNCaP and PC-3 cells, in order to identify potential PC-3-specific UBE2C enhancers." Enhancer UBE2C 3C "RT-PCR,Western blot" 3C assays were performed at the UBE2C locus to measure crosslinking frequencies between PC-3-specific UBE2C enhancers and the UBE2C promoter "UBCH10,dJ447F3.2" Prostate Cancer DOID:10283 D011471 -- -- -- "FOXA1,GATA2,POU2F1,ETS1" "HNF3A,TCF3A,DCML,IMD21,MONOMAC,NFE1B,ETS-1,EWSR2,c-ets-1,p54,OCT1,OTF1,oct-1B" ChIP "To examine whether these transcription factors were differentially recruited to E1,E2 and E3 on chromatin, chromatin immunoprecipitation (ChIP) assays were performed using specific antibodies against FoxA1, GATA2, Oct1 and ETS1 in LNCaP and PC-3 cells treated with or without DHT." -- -- -- >2KB 9090 E_01_086 21556051 UBE2C Enhancer hg19 chr20 44441255 44444472 Human "LNCaP,PC-3" Low throughput "RT-PCR,Luciferase Reporter Assay,ChIP,3C" "we performed quantitative 3C assays for the UBE2C locus in LNCaP and PC-3 cells, in order to identify potential PC-3-specific UBE2C enhancers." Enhancer UBE2C 3C "RT-PCR,Western blot" 3C assays were performed at the UBE2C locus to measure crosslinking frequencies between PC-3-specific UBE2C enhancers and the UBE2C promoter "UBCH10,dJ447F3.2" Prostate Cancer DOID:10283 D011471 -- -- -- "FOXA1,GATA2,POU2F1,ETS1" "HNF3A,TCF3A,DCML,IMD21,MONOMAC,NFE1B,ETS-1,EWSR2,c-ets-1,p54,OCT1,OTF1,oct-1B" ChIP "To examine whether these transcription factors were differentially recruited to E1,E2 and E3 on chromatin, chromatin immunoprecipitation (ChIP) assays were performed using specific antibodies against FoxA1, GATA2, Oct1 and ETS1 in LNCaP and PC-3 cells treated with or without DHT." -- -- -- <2KB 1650 E_01_087 21556051 UBE2C Enhancer hg19 chr20 44478646 44484559 Human "LNCaP,PC-3" Low throughput "RT-PCR,Luciferase Reporter Assay,ChIP,3C" "we performed quantitative 3C assays for the UBE2C locus in LNCaP and PC-3 cells, in order to identify potential PC-3-specific UBE2C enhancers." Enhancer UBE2C 3C "RT-PCR,Western blot" 3C assays were performed at the UBE2C locus to measure crosslinking frequencies between PC-3-specific UBE2C enhancers and the UBE2C promoter "UBCH10,dJ447F3.2" Prostate Cancer DOID:10283 D011471 -- -- -- "FOXA1,GATA2,POU2F1,ETS1" "HNF3A,TCF3A,DCML,IMD21,MONOMAC,NFE1B,ETS-1,EWSR2,c-ets-1,p54,OCT1,OTF1,oct-1B" ChIP "To examine whether these transcription factors were differentially recruited to E1,E2 and E3 on chromatin, chromatin immunoprecipitation (ChIP) assays were performed using specific antibodies against FoxA1, GATA2, Oct1 and ETS1 in LNCaP and PC-3 cells treated with or without DHT." -- -- -- >2KB 40389 E_01_088 21556051 UBE2C Enhancer hg19 chr20 44413081 44426646 Human "PC-3,DU-145" Low throughput "RT-PCR,Luciferase Reporter Assay,ChIP,3C" "we performed quantitative 3C assays for the UBE2C locus in LNCaP and PC-3 cells, in order to identify potential PC-3-specific UBE2C enhancers." Enhancer UBE2C 3C "RT-PCR,Western blot" 3C assays were performed at the UBE2C locus to measure crosslinking frequencies between PC-3-specific UBE2C enhancers and the UBE2C promoter "UBCH10,dJ447F3.2" Prostate Cancer DOID:10283 D011471 -- -- -- "FOXA1,GATA2,POU2F1,ETS1" "HNF3A,TCF3A,DCML,IMD21,MONOMAC,NFE1B,ETS-1,EWSR2,c-ets-1,p54,OCT1,OTF1,oct-1B" ChIP "To examine whether these transcription factors were differentially recruited to E1,E2 and E3 on chromatin, chromatin immunoprecipitation (ChIP) assays were performed using specific antibodies against FoxA1, GATA2, Oct1 and ETS1 in LNCaP and PC-3 cells treated with or without DHT." -- -- -- >2KB 21350 E_01_089 21665992 -- hg19 chr20 62593247 62595247 Human "SK-N-AS,Hep G2" Low throughput "Luciferase Reporter Assay,EMSA,PCR" "To gain insight into the biological significance of rs2275294,luciferase reporter plasmids corresponding to a genomic DNA fragment containing rs2275294 were constructed and a luciferase assay using the human neuroblastoma cell line SK--N--Be(2)C was performed. Constructs containing the ALS--susceptibility allele (C allele) of rs2275294 showed lower enhancer activity than those containing the nonsusceptibility allele, indicating that the SNP affects the ZNF512B transcription level (Fig. 2A). We then examined the allelic difference in the binding of genomic DNA containing rs2275294 to nuclear proteins by the electrophoretic mobility shift assay (EMSA)." Enhancer ZNF512B -- "Luciferase Reporter Assay,EMSA" "To gain insight into the biological significance of rs2275294,luciferase reporter plasmids corresponding to a genomic DNA fragment containing rs2275294 were constructed and a luciferase assay using the human neuroblastoma cell line SK--N--Be(2)C was performed. Constructs containing the ALS--susceptibility allele (C allele) of rs2275294 showed lower enhancer activity than those containing the nonsusceptibility allele, indicating that the SNP affects the ZNF512B transcription level (Fig. 2A). We then examined the allelic difference in the binding of genomic DNA containing rs2275294 to nuclear proteins by the electrophoretic mobility shift assay (EMSA)." GM632 Amyotrophic Lateral Sclerosis DOID:332 D000690 -- -- -- -- -- -- -- rs2275294 62594247 "Luciferase Reporter Assay,EMSA,PCR" >2KB 6191 E_01_090 21666600 GATA Enhancer hg19 chr4 101576220 101578659 Human Endothelial Cells (ECs) Low+High throughput "ChIP-seq,ChIP-qPCR" "Subsequently, we searched the GATA2 binding within the locus. From the duplicate ChIP-seqs, GATA2 associations were commonly detected at ?139 kbp and at a weaker level at ?132 kbp (Figure 5A). ChIP¨CqPCR validation resulted in significant bindings of GATA2 to ?132 and ?139 kbp regions (Figure 5B). To verify which region from the ?132 or ?139 kbp is epigenetically designed as an enhancer, we performed ChIP¨CqPCR analysis with antibodies against active PolII, H3Ac, H4Ac, and p300. As shown in Figure 5B, compared with the negative control region at ?84 kbp, only the ?139 kbp region showed strong merged binding by p300, H3Ac, H4Ac, and active PolII. " Enhancer EMCN -- "ChIP-seq,ChIP-qPCR" "To verify which region from the ?132 or ?139 kbp is epigenetically designed as an enhancer, we performed ChIP¨CqPCR analysis with antibodies against active PolII, H3Ac, H4Ac, and p300. As shown in Figure 5B, compared with the negative control region at ?84 kbp, only the ?139 kbp region showed strong merged binding by p300, H3Ac, H4Ac, and active PolII. Moreover, the comparative genome analysis among mammalian species resulted in sharp homologous signals at ?139 kbp (Figure 5A, bottom). Collectively, these findings suggest that the ?139 kbp region is the main GATA2-binding enhancer within the endomucin locus." "EMCN2,MUC14" -- -- -- Endothelial-speci?c GATA2-mediated endomucin gene expression was regulated by the endothelial-speci?c chromatin loop with a GATA2-associated distal enhancer and core promoter. "ChIP-seq,3C" "By using the ChIP-seq with epigenetic histone modications and chromatin conformation capture assays; we elucidated the mechanistic regulation of endothelial-specic GATA2-mediated endomucin gene expression, that was regulated by the endothelial-specic chromatin loop with a GATA2-associated distal enhancer and core promoter. " GATA2 "DCML,IMD21,MONOMAC,NFE1B" "ChIP,ChIP-seq,3C,PCR,siRNA" ChIP analysis with GATA2 antibody. Immunoprecipitated DNA were quantified with primer pairs described in Supplementary Table SIV. -- -- -- >2KB 260943 E_01_091 21681857 NAGS Enhancer hg19 chr17 42078932 42079032 Human Ureagenesis Low throughput Luciferase Reporter Assay "The remaining change, a homozygous C¨CA transversion 3,064--bp upstream of the NAGS translation start codon (c.--3064C > A) (Supp. Fig. S1), was determined to be in the recently identified hepatic nuclear factor 1 (HNF--1)bindingsiteofaconservedNAGSregulatoryregion(Fig.4).Transcription factor binding sites in the NAGS enhancer.Transcription factors HNF--1 and NF--Y bind with in the human NAGS enhancer." Enhancer NAGS -- Luciferase Reporter Assay "The functional effect of the mutation on NAGS transcription was examined using a luciferase reporter assay, driven by the NAGS enhancer with either the wild--type, mutated, or consensus HNF--1 binding sites.Cells were cotransfected with vector pGL4.74, containing Renilla luciferase hRluc, as a transfection efficiency control." "AGAS,ARGA" -- -- -- -- -- -- HNF1A "HNF-1A,HNF1,HNF4A,IDDM20,LFB1,MODY3,TCF-1,TCF1" Luciferase Reporter Assay "The functional effect of the mutation on NAGS transcription was examined using aLuciferase assay,driven by the NAGS Enhancer with either the wild-type,mutated,or consensus HNF-1 binding sites. Reporter plasmids included the NAGS wild-type promoter with the wild-type Enhancer,4.10WT,the Enhancer containing the patient¡¯s mutation -3064C>A,4.10Pat,or the Enhancer containing the consensus HNF-1 binding site,4.10Con (Fig. 5A)." -- -- -- >2KB 3049 E_01_092 21689639 -- hg19 chr12 7940709 7941994 Human HEK-293 Low throughput Luciferase Reporter Assay "Genome sequence analysis of the enhancer region of the human NANOG gene showed the presence of two conserved Tcf/Lef--binding motifs: CAAAGA at positions --1206 to --1190 (designated as Lef #1) and an inverted GTTTCA sequence at ?1020 to ?1004 (designated as Lef #2) (Fig. 1A). To investigate whether Tcf/Lef elements located in the enhancer region play a role in the regulation of NANOG gene transcription, we isolated a 1286--bp fragment of the enhancer region of the human NANOG gene and subcloned it into a firefly luciferase reporter vector, yielding pNanog--Luc(--1286/--1)." Enhancer NANOG -- Luciferase Reporter Assay "To investigate whether Tcf/Lef elements located in the enhancer region play a role in the regulation of NANOG gene transcription, we isolated a 1286--bp fragment of the enhancer region of the human NANOG gene and subcloned it into a firefly luciferase reporter vector, yielding pNanog--Luc(--1286/--1). " NANOG -- -- -- -- -- -- LEF1 "LEF-1,TCF10,TCF1ALPHA,TCF7L3" ChIP ChIP experiments demonstrated that b-catenin and Lef1 bind to the Enhancer region of the NANOG gene. Our present findings would appear to indicate that the Tcf/Lef element in the Enhancer region of the Human NANOG gene functions in transcriptional activation of the Human NANOG gene. -- -- -- <2KB 639 E_01_093 21711161 -- hg19 chr17 62028378 62028924 Human "HEK-293,HeLa" Low throughput Luciferase Reporter Assay "Upstream HS III sequences contained in a 574 bp fragment were previously shown to act as effective enhancers of minimal TKp activity in transfected human HEK293 and HeLa cells.To assess for enhancer--blocking activity, the same HS V fragments were inserted between the HS III enhancer and the downstream TKp and firefly luciferase (Luc) reporter gene. Significant decreases in enhancer activity were observed in both the forward and reverse orientations in both cell lines (p<0.05, n=5¨C6)(Fig. 2)" Enhancer GH1 -- "Luciferase Reporter Assay,Transfection" "Upstream HS III sequences contained in a 574 bp fragment were previously shown to act as effective enhancers of minimal TKp activity in transfected human HEK293 and HeLa cells.To assess for enhancer--blocking activity, the same HS V fragments were inserted between the HS III enhancer and the downstream TKp and firefly luciferase (Luc) reporter gene. Significant decreases in enhancer activity were observed in both the forward and reverse orientations in both cell lines (p<0.05, n=5¨C6)(Fig. 2)" "GH,GH-N,GHB5,GHN,IGHD1A,IGHD1B,IGHD2,hGH-N" -- -- -- HS V sequences contain efficient Enhancer-blocking activity Luciferase Reporter Assay "To assess for enhancerblocking activity, the same HS V fragments were inserted between the HS III enhancer and the downstream TKp and firefly luciferase (Luc) reporter gene" "YY1,CTCF" "DELTA,GADEVS,INO80S,NF-E1,UCRBP,YIN-YANG-1," ChIP "In terms of enhancer-blocking activity, the decrease in either CTCF or Yy1 was associated with a significant *50% increase or rescue of enhancer activity in the case of HSVR, and*80% when siRNA for both CTCF and Yy1 were used." -- -- -- >2KB 34099 E_01_094 21798992 -- hg19 chr8 117978845 117979466 Human "¦ÂTC-3,¦ÁTC-6" Low throughput "Luciferase Reporter Assay,PCR,Transfection" "we have identified a conserved islet beta cell-specific enhancer in SLC30A8 intron 2 (Figs. 4 & 5). " Enhancer SLC30A8 -- "Luciferase Reporter Assay,PCR,Transfection" "To determine whether the +16579 and +16954 region of intron 2 in the human SLC30A8 gene also represents a transcriptional enhancer, this region was isolated using PCR and ligated 5' of the ?150/+3 G6PC2-luciferase fusion gene described above. Luciferase expression directed by this fusion gene was then analyzed by transient transfection of ¦ÂTC-3 and ¦ÁTC-6 cells." "ZNT8,ZnT-8" Type 2 Diabetes Mellitus DOID:9352 D003924 -- -- -- -- -- -- -- rs62510556 118164272 "Luciferase Reporter Assay,PCR" Intron 16645 E_01_095 21868451 -- hg19 chr5 134350424 134352424 Human MCF-7 Low throughput "ChIP,Luciferase Reporter Assay,3C" "Genetic locus from the UCSC genome browser showing the natural 5 to 3 orientation of the PITX1 gene with the location of the identified ER binding regions, the proximal promoter and enhancer, and the location of the primers used in ChIP and chromosome conformation capture experiments." Enhancer PITX1 -- "Luciferase Reporter Assay,Transfection" "ER regulation of PITX1 involves interaction of the ER-binding distal enhancer with the promoter of the PITX1 gene via intrachromosomal looping.We next determined whether the PITX1 enhancer and promoter regions were estrogen responsive by cloning these regions from genomic DNA into luciferase reporters, and we conducted transient transfection assays." "BFT,CCF,LBNBG,POTX,PTX1" -- -- -- -- -- -- ESR1 "ER,ESR,ESRA,ESTRR,Era,NR3A1" ChIP "Recruitment,monitored by ChIP,of ER and PITX1 to ER binding sites that contain a consensus PITX1 binding site in vehicle and 10 nM E2-treated 231ER cell at 45 min (panel D) or to ER binding sites that lack a consensus PITX1 binding site in vehicle and 10 nM E2-treated 231ER cell at 45 min." -- -- -- >2KB 11999 E_01_096 22160855 -- hg19 chr6 31130679 31130761 Human NT2 Low throughput "ChIP,qPCR" "Chromatin immunoprecipitation analysis of the four regions in the proximal promoter (PP), proximal enhancer (PE1 and PE2),and distal enhancer (DE) unraveled consistent histone marks associated with transcription.Oct4 gene expression is dependent on at least three upstream cis-regulatory regions including proximal pro_x0002_moter (PP) located within the first 250 bp of the tran_x0002_scription initiation site, proximal enhancer (PE) located 1.3-1.6 kb upstream, and distal enhancer (DE) located 2.5 kb upstream." Enhancer POU5F1 -- RT-PCR "To verify induction of differentiation, mRNA level of the stemness marker gene, Oct4, was evaluated in both kinds of cell cultures, using RT-PCR (Fig. 1b) as well as quantitative real-time.As expected,expression of Oct4 gene was dominant in undifferentiated embryonal cells,and rapidly declined following RA-induced differentiation." "OCT3, OCT4, OTF-3, OTF3, OTF4, Oct-3, Oct-4" -- -- -- -- -- -- -- -- -- -- -- -- -- <2KB 1394 E_01_097 22160855 -- hg19 chr6 31130445 31130606 Human NT2 Low throughput "ChIP,qPCR" "Chromatin immunoprecipitation analysis of the four regions in the proximal promoter (PP), proximal enhancer (PE1 and PE2),and distal enhancer (DE) unraveled consistent histone marks associated with transcription.Oct4 gene expression is dependent on at least three upstream cis-regulatory regions including proximal pro_x0002_moter (PP) located within the first 250 bp of the tran_x0002_scription initiation site, proximal enhancer (PE) located 1.3-1.6 kb upstream, and distal enhancer (DE) located 2.5 kb upstream." Enhancer POU5F1 -- RT-PCR "To verify induction of differentiation, mRNA level of the stemness marker gene, Oct4, was evaluated in both kinds of cell cultures, using RT-PCR (Fig. 1b) as well as quantitative real-time.As expected,expression of Oct4 gene was dominant in undifferentiated embryonal cells,and rapidly declined following RA-induced differentiation." "OCT3, OCT4, OTF-3, OTF3, OTF4, Oct-3, Oct-4" -- -- -- -- -- -- -- -- -- -- -- -- -- <2KB 1588 E_01_098 22160855 -- hg19 chr6 31129568 31129735 Human NT2 Low throughput "ChIP,qPCR" "Chromatin immunoprecipitation analysis of the four regions in the proximal promoter (PP), proximal enhancer (PE1 and PE2),and distal enhancer (DE) unraveled consistent histone marks associated with transcription.Oct4 gene expression is dependent on at least three upstream cis-regulatory regions including proximal pro_x0002_moter (PP) located within the first 250 bp of the tran_x0002_scription initiation site, proximal enhancer (PE) located 1.3-1.6 kb upstream, and distal enhancer (DE) located 2.5 kb upstream." Enhancer POU5F1 -- RT-PCR "To verify induction of differentiation, mRNA level of the stemness marker gene, Oct4, was evaluated in both kinds of cell cultures, using RT-PCR (Fig. 1b) as well as quantitative real-time.As expected,expression of Oct4 gene was dominant in undifferentiated embryonal cells,and rapidly declined following RA-induced differentiation." "OCT3, OCT4, OTF-3, OTF3, OTF4, Oct-3, Oct-4" -- -- -- -- -- -- -- -- -- -- -- -- -- >2KB 2462 E_01_099 22212979 PSA Enhancer hg19 chr19 51354804 51355203 Human LNCaP Low throughput "qRT-PCR,ChIP" "Chromatin immunoprecipitation (ChIP) on the promoters and enhancers of prostate specific antigen (PSA) and transmembrane protease, serine(TMPRSS2) genes was performed.For PSA, in addition to the AREs located 131 and 150 bp from the tran_x0002_scription start site (TSS), an enhancer region 4 kb upstream of the TSS has been documented.For the TMPRSS2 gene, the AREs in the regulatory region are located 13.5 kb [27,38] and 700 bp up_x0002_stream of the TSS." Enhancer KLK3 -- "ChIP-qPCR,ChIP" "Chromatin immunoprecipitation(ChIP) on the promoters and enhancers of prostate specific antigen (PSA) and transmembrane protease, serine 2(TMPRSS2) genes was performed. qRT-PCR was used to measure the levels of PSA and TMPRSS2 transcripts.We performed ChIP-qPCR analyses on these selected genomic sites of PSA and TMPRSS2." "APS,KLK2A1,PSA,hK3" -- -- -- -- -- -- AR "AIS8,DHTR,HUMARA,HYSP1,KD,NR3C4,SBMA,SMAX1,TFM" ChIP-qPCR "At the 1 hr time point, AR loading onto the TMPRSS2 enhancer (Fig. 3A) was already significantly (P < 0.001, t-test) higher in LNCaP-ARhi cells than in the control cells. However, the TMPRSS2 promoter did not significantly recruit AR, at least in the 4 hr time (Fig. 3B).ChIP-qPCR was performed to assess the AR loading." -- -- -- >2KB 3166 E_01_100 22212979 TMPRSS2 Enhancer hg19 chr21 42822120 42822800 Human LNCaP Low throughput "qRT-PCR,ChIP" "Chromatin immunoprecipitation (ChIP) on the promoters and enhancers of prostate specific antigen (PSA) and transmembrane protease, serine(TMPRSS2) genes was performed.For PSA, in addition to the AREs located 131 and 150 bp from the tran_x0002_scription start site (TSS), an enhancer region 4 kb upstream of the TSS has been documented.For the TMPRSS2 gene, the AREs in the regulatory region are located 13.5 kb [27,38] and 700 bp up_x0002_stream of the TSS." Enhancer TMPRSS2 -- "ChIP-qPCR,ChIP" "Chromatin immunoprecipitation(ChIP) on the promoters and enhancers of prostate specific antigen (PSA) and transmembrane protease, serine 2(TMPRSS2) genes was performed. qRT-PCR was used to measure the levels of PSA and TMPRSS2 transcripts.We performed ChIP-qPCR analyses on these selected genomic sites of PSA and TMPRSS2." "PP9284,PRSS10" -- -- -- -- -- -- AR "AIS8,DHTR,HUMARA,HYSP1,KD,NR3C4,SBMA,SMAX1,TFM" ChIP-qPCR "At the 1 hr time point, AR loading onto the TMPRSS2 enhancer (Fig. 3A) was already significantly (P < 0.001, t-test) higher in LNCaP-ARhi cells than in the control cells. However, the TMPRSS2 promoter did not significantly recruit AR, at least in the 4 hr time (Fig. 3B).ChIP-qPCR was performed to assess the AR loading." -- -- -- >2KB 14017 E_01_101 22253448 -- hg19 chr20 55417234 55417304 Human "NK-92,U-2 OS" Low throughput "Luciferase Reporter Assay,EMSA,RT-PCR,ChIP" "We won_x0002_dered whether the essential and enhancer elements of the NCR1 promoter were tissue-specific. Thus, we ran luciferase assays using the 5_x0004_ truncation constructs in the U2OS cell line.Thus, the essential region acts primarily as a pan-tissue promoter. On the other hand, the -200 to -270 region acts as an enhancer or suppressor, depending on the cellular context." Enhancer NCR1 -- "Luciferase Reporter Assay,EMSA,RT-PCR,ChIP" "We wondered whether either RUNX1 or RUNX3 can indeed bind the predicted motifs in the NCR1 promoter. To test this possibility, electrophoretic mobility shift assays (EMSA) were performed. Here we focus on the proximal upstream region of the human NCR1 gene. We identify two cis-regulatory elements in this region and describe a role of runt related transcription factor (RUNX) proteins, especially RUNX3, in regulating NCR1 expression." "CD335,LY94,NK-p46,NKP46" -- -- -- This latter regulatory element contains a runt related-transcription factor (RUNX) recognition motif that preferentially binds RUNX3. Interfering with RUNX proteins using a dominant negative form results in decreased Ncr1 expression. "qPCR,ChIP" "We found two key cis-regulatory elements in the immediate vicinity upstream of the gene. One element acts as an essential promoter, whereas the other acts as a tissue-dependent enhancer/repressor. This latter regulatory element contains a runt related-transcription factor (RUNX) recognition motif that preferentially binds RUNX3. Interfering with RUNX proteins using a dominant negative form results in decreased Ncr1 expression. " "RUNX1,RUNX3" "AML1,AML1-EVI-1,AMLCR1,CBF2alpha,CBFA2,EVI-1,PEBP2aB,PEBP2alpha,AML2,CBFA3,PEBP2aC" "RT-PCR,EMSA,ChIP" "To test this possibility, electrophoretic mobility shift assays (EMSA) were performed. We tested the two RUNX sites separately. Probes spanning the putative RUNX binding site in the Enhancer were shifted by NK92 nuclear extract.To determine whether RUNX binding to the NCR1 promoter is NK-specific, ChIP was performed in other cell lines." -- -- -- <2KB 238 E_01_102 22260630 -- hg19 chr9 132491985 132492534 Human HFL-1 Low throughput "DNaseI-seq,ChIP,RT-PCR" "We identified two DNase I-hypersensitive sites within the proximal promoter near the Egr-1 element and a novel distal site near ? 8.6 kb. Functional analysis of the distal site revealed two elements that co-operate with basal promoter expression and a stimulus-dependent enhancer.. A specific binding site for C/EBP¦Â (CCAAT/enhancer-binding protein ¦Â) in the enhancer was directly responsible for inducible enhancer activity." Enhancer PTGES -- "DNaseI-seq,ChIP,RT-PCR" "We therefore hypothesized that, apart from the proximal promoter harbouring a potentially important Egr-1 site, there must be other cis-acting regulatory elements required to recapitulate the full cytokine-dependent induction of mPGES-1 expression. Our results demonstrate the identification and characterization of a novel IL-1¦Â (interleukin 1¦Â)-responsive distal enhancer element controlled through C/EBP¦Â (CCAAT/enhancer-binding protein ¦Â) which contributes to the induction of endogenous mPGES-1." "MGST-IV,MGST1-L1,MGST1L1,MPGES,PGES,PIG12,PP102,PP1294,TP53I12,mPGES-1" Lung Cancer DOID:1324 D008175 Interaction between Egr-1 and C/E¦Â highlights the proximal promoter co-operation with a novel distal Enhancer element can regulate inducible mPGES-1 expression. "ChIP,RT-PCR" Knockout/knockdown studies established a functional role for C/E¦Â in mPGES-1 gene regulation and the documented interaction between Egr-1 and C/E¦Â highlights the proximal promoter co-operation with a novel distal Enhancer element in regulating inducible mPGES-1 expression. "EGR1,CEBPB" "AT225,G0S30,KROX-24,NGFI-A,TIS8,ZIF-268,ZNF225,C/EBP-beta,IL6DBP,NF-IL6,TCF5" ChIP ChIP (chromatin immunoprecipitation) analysis demonstrated constitutive Egr-1 binding to the promoter and induced RNA polymerase II and C/EBP¦Â binding to the promoter and Enhancer respectively. -- -- -- >2KB 8354 E_01_103 22265404 -- hg19 chr14 90847327 90849327 Human "MCF-7,K-562,HeLa,HCT 116,Nb4" Low+High throughput "3C-qPCR,Luciferase Reporter Assay,ChIP-seq" "To examine potential enhancer activity of promoters, we per_x0002_formed luciferase reporter gene assays, a commonly used method for promoter and enhancer characterization." Enhancer CALM1 3C-qPCR Luciferase Reporter Assay "In another example (Figure 5F), the promoter of CALM1 interacts with an enhancer element 15 kb upstream and connects to the promoter of C14orf102 further upstream in 65kb. Both RNA-Seq data and the H3K4me3/me1 log ratio indi_x0002_cated that the CALM1 promoter was strong, whereas the C14orf102 promoter was weak and enhancer-like. The luciferase reporter gene assay showed marginal enhancement to the CALM1 promoter reporter gene activity by the native CALM1 enhancer and the C14orf102 promoter individually." "CALML2,CAM2,CAM3,CAMB,CAMC,CAMI,CAMIII,CPVT4,DD132,LQT14,PHKD,caM" Congenital Limb Deformity -- D017880 -- -- -- -- -- -- -- -- -- -- >2KB 15000 E_01_104 22303449 -- hg19 chr5 44385749 44386845 Human Human Embryos Low throughput "ChIP,EMSA" "In conjunction, we identified a novel enhancer containing a highly conserved ISL1 consensus binding site within the FGF10 first intron. ChIP and EMSA demonstrated its direct occupation by ISL1. Transcription mediated by ISL1 from this FGF10 intronic element was enhanced by the presence of GATA4 and TBX20 cardiac transcription factors. Finally, transgenic mice confirmed that endogenous factors bound the human FGF10 intronic enhancer to drive reporter expression in the developing cardiac outflow tract." Enhancer FGF10 -- "ChIP,EMSA" "Using chromatin immunoprecipitation (ChIP) of microdissected embryonic human hearts, we demonstrated that at Carnegie stages 14¨C15 (33¨C36 dpf), ISL1 bound to and enriched a 327 bp FGF10-Int1 fragment (Fig. 2A). In contrast, ISL1 did not occupy FGF10-Pr1 or FGF10-Pr2. Acetylated histone H4 did bind both the ISL1 and FGF10 promoters at CS14-15, confirming that the chromatin around these two promoters is transcriptionally active in the human heart at these stages." FGF10 -- -- -- Endogenous factors bound the human FGF10 intronic enhancer to drive reporter expression in the developing cardiac outflow tract. "ChIP,EMSA,Transgenic mice" "Finally, transgenic mice confirmed that endogenous factors bound the human FGF10 intronic enhancer to drive reporter expression in the developing cardiac outflow tract." "ISL1,GATA4,TBX20" "ISLET1,Isl-1,ASD2,TACHD,TOF,VSD1,ASD4" "ChIP,EMSA" ChIP and EMSA demonstrated its direct occupation by ISL1. Transcription mediated by ISL1 from this FGF10 intronic element was enhanced by the presence of GATA4 and TBX20 cardiac transcription factors. -- -- -- >2KB 81201 E_01_105 22337891 -- hg19 chr11 101981958 101982137 Human "HCT 116,SW620,SW480,RKO" Low throughput "ChIP,RT-PCR,Luciferase Reporter Assay" "To test whether ¦Â-catenin/TCF4 complexes were responsible for enhancer activity, we mutated the consensus TCF motif to CGCTGAT.In transfected cells, levels of luciferase expressed from the YAP reporter were _x0001_60% of the lev_x0002_els observed with the YAP (WT) reporter.To test whether the residual activity of the YAP reporter was due to a second ¦Â-catenin binding site, cells were treated with the GSK3_x0001_inhibitor BIO. BIO treatment increased both pGL3-YAP (WT) and pGL3-YAP reporter activities suggesting that the YAP enhancer element contains a second, unidentified ¦Â-catenin binding site." Enhancer YAP1 -- "ChIP,RT-PCR,Luciferase Reporter Assay" "Because ¦Â-catenin is recruited to chromatin through interactions with TCF4, we next tested whether TCF4 bound to the YAP gene using ChIP assays.Here we demonstrate that ¦Â-catenin/TCF4 complexes bind a DNA enhancer element within the first intron of the YAP gene to drive YAP expression in CRC cells. As such, reducing ¦Â-catenin expression in CRC cells using shRNAs leads to decreased YAP mRNA and protein levels." "COB1,YAP,YAP2,YAP65,YKI" Colorectal Cancer DOID:9256 D015179 ¦Â-Catenin/TCF4 complexes directly regulate YAP gene expression through a novel DNA Enhancer element. "ChIP-seq,ChIP" Here we demonstrate that ¦Â-Catenin/TCF4 complexes bind a DNA enhancer element within the first intron of the YAP gene to drive YAP expression in CRC cells. TCF4 "E2-2,FECD3,ITF-2,ITF2,PTHS,SEF-2,SEF2,SEF2-1,SEF2-1A,SEF2-1B,SEF2-1D,TCF-4,bHLHb19" "ChIP,Luciferase Reporter Assay" "To determine whether ¦Â-catenin/TCF4 bound a transcriptionally active YAP gene locus, we precipitated RNA polymerase II (RNAP) in ChIP assays and found that RNAP bound YAP." -- -- -- Intron 857 E_01_106 22370642 ARE Enhancer hg19 chr22 35766960 35768960 Human HeLa Low throughput "qPCR,ChIP" "Further_x0002_more, chromatin immunoprecipitation showed that RAC3 bound tightly to the ARE enhancer region of the HO-1 promoter via Nrf2 binding. These data suggest that Nrf2 activation is modulated and directly controlled through interactions with the RAC3 protein in HeLa cells." Enhancer HMOX1 -- "qPCR,ChIP" "To determine the effects of RAC3 on the expression of HO-1, an Nrf2 target protein, HeLa cells were transfected with Nrf2 and various concentrations of HA-RAC3 plasmids. First, we measured the protein and mRNA levels of HO-1. The results showed that transfection with HA-RAC3 increased the HO-1 protein (Figure 1a) and mRNA levels (Figure 1b) in a dose_x0002_dependent manner in the presence of a yellow fluorescent protein (YFP)-Nrf2 construct." "HMOX1D,HO-1,HSP32,bK286B10" Breast Cancer DOID:1612 D001943 -- -- -- NFE2L2 "HEBP1,IMDDHH,NRF2" ChIP "To test this hypothesis,we conducted a chromatin immunoprecipitation (ChIP) assay to eluci_x0002_date whether the RAC3 protein was detectable on the ARE sequences bound to Nrf2." -- -- -- >2KB 9099 E_01_107 22383952 -- hg19 chr17 42078865 42079198 Human Hep G2 Low throughput "PCR,ChIP,Luciferase Reporter Assay" "Reporter assays to compare the effect of the enhancer in liver,intestine and lung cells, included data that were normalized to the reporter expression driven by the NAGS promoter. While the NAGS enhancer (4.10PromEnh) increased expression of the reporter gene by 50% in liver derived cells (Figure 2A), expression of the luciferase gene did not increase in the intestine or lung derived cells (Figure 8) suggesting that the enhancer may determine tissue specificity of NAGS expression." Enhancer NAGS -- "ChIP,Luciferase Reporter Assay" "We subsequently confirmed that these regions function as promoter and enhancer and that the enhancer is most effective in liver cells. Avidin-agarose protein_x0002_DNA pull-down assays have been used to confirm binding of Sp1 and CREB within the NAGS promoter and Hepatic Nuclear Factor 1 (HNF-1) and NF-Y within the enhancer regions. Chromatin immunoprecipitation (ChIP) and quantitative real_x0002_time PCR have been used to independently verify that Sp1 and CREB bind to the promoter region, and HNF-1 and NF-Y bind to the enhancer region. We also used 59RACE analysis to identify multiple transcription start sites for NAGS that may be species and tissue specific." "AGAS,ARGA" -- -- -- Two regions of high conservation upstream of the translation start of the NAGS gene. Reporter assays confirmed that these regions represent promoter and enhancer and that the enhancer is tissue specific. Luciferase Reporter Assay "Reporter assays to compare the effect of the enhancer in liver, intestine and lung cells, included data that were normalized to the reporter expression driven by the NAGS promoter. While the NAGS enhancer (4.10PromEnh) increased expression of the reporter gene by 50% in liver derived cells (Figure 2A), expression of the luciferase gene did not increase in the intestine or lung derived cells (Figure 8) suggesting that the enhancer may determine tissue specificity of NAGS expression. When HNF-1" "SP1,HNF1A,NF-Y" "SP1,HNF-1A,HNF1,HNF4A,IDDM20,LFB1,MODY3,TCF-1,TCF1,CBF-A, CBF-B,HAP2,NF-YA" "ChIP,qPCR" "Thus,Pull-down and ChIP assays confirmed that Sp1 and CREB bind along the NAGS promoter and HNF-1 and NF-Y bind along the Enhancer." -- -- -- >2KB 2999 E_01_108 22408256 -- hg19 chr9 136141944 136147321 Human "K-562,KATO III" Low throughput "Luciferase Reporter Assay,PCR" "To verify the enhancer potential of these sites using luciferase reporter assays,PCR-amplified fragments or genomic DNAs from the HG-1 clone encompassing them were subcloned upstream of the ABO proximal promoter sequence in luciferase reporter plasmids. Transient transfections into K562 cells were carried out using these reporter vectors. DNA regions used in the reporter plasmids are denoted as regions -8.8,-5.5,-3.8,+2.0,+5.8, and +11.5." Enhancer ABO -- "Luciferase Reporter Assay,PCR" "On the basis of DNase I¨Chypersensitive sites in and up_x0002_stream of ABO in K562 cells, in the pres_x0002_ent study, we prepared reporter plasmid constructs including these sites. Subsequent luciferase assays indicated a novel positive regulatory element in intron 1.This element was shown to enhance ABO promoter activity in an erythroid cell¨Cspecific manner." "A3GALNT,A3GALT1,GTB,NAGAT" -- -- -- -- -- -- GATA1 "ERYF1,GATA-1,GF-1,GF1,NF-E1,NFE1,XLANP,XLTDA,XLTT" "Luciferase Reporter Assay,EMSA" This element was shown to enhance ABO promoter activity in an erythroid cell¨Cspecific manner. Electrophoretic mobility¨Cshift assays demonstrated that it bound to the tissue-restricted transcription fac_x0002_tor GATA-1. -- -- -- >2KB 14071 E_01_109 22645302 -- hg19 chr12 8036824 8048824 Human "HEK-293,HeLa" Low+High throughput "ChIP-PCR,RT-PCR,3C-ChIP,ChIP-seq" "To examine whether histone enhancers mark changes under hypoxia, we compared the HIF1 binding sites and H3K27ac under normoxia and hypoxia on a genome-wide scale. The number of total reads and uniquely mapped sequences of the ChIP-seq for histone marks are shown in Table S4 in the supplemental material. H3K27ac sites overlapped at 12,498 sites between the state of normoxia (14,679 sites) and hypoxia (16,366 sites) on a genome-wide scale.The results of the HIF1 ChIP-seq analysis provided data of hypoxic enhancer 2 (kbp -24 from the TSS) at the SLC2A3 loci." Enhancer SLC2A3 3C "ChIP-PCR,ChIP" We elucidated that both the chromatin conformational structure and histone modification change under hy_x0002_poxic conditions and enhance the expression of SLC2A3 based on the combined results of chromatin conformation capture (3C) and ChIP assays. KDM3A is recruited to the SLC2A3 locus in an HIF1-dependent manner and demethylates H3K9me2 so as to upregulate its expression.These findings provide novel insights into the interaction between HIF1 and KDM3A and also the epigenetic regulation of HIF1. GLUT3 -- -- -- The enhancer 2 was functionally active under hypoxic condition "ChIP-PCR,Luciferase Reporter Assay" "The experimental data described above showed that distal enhancer 2 was functionally active under hypoxic conditions, suggesting that enhancer 2 has an especially close proximity to the promoter of SLC2A3 under hypoxic conditions. " HIF1A "HIF-1-alpha,HIF-1A,HIF-1alpha,HIF1,HIF1-ALPHA,MOP1,PASD8,bHLHe78" ChIP Genome-wide analysis of HIF1 binding sites (chromatin immunoprecipitation[ChIP] with deep sequencing) of endothelial cells clarified that HIF1 mainly binds to the intergenic regions distal from tran_x0002_scriptional starting sites under both normoxia and hypoxia. -- -- -- >2KB 28999 E_01_110 22645302 -- hg19 chr12 8035824 8037824 Human "HEK-293,HeLa" Low+High throughput "ChIP-PCR,RT-PCR,3C-ChIP,ChIP-seq" "To examine whether histone enhancers mark changes under hypoxia, we compared the HIF1 binding sites and H3K27ac under normoxia and hypoxia on a genome-wide scale. The number of total reads and uniquely mapped sequences of the ChIP-seq for histone marks are shown in Table S4 in the supplemental material. H3K27ac sites overlapped at 12,498 sites between the state of normoxia (14,679 sites) and hypoxia (16,366 sites) on a genome-wide scale.The results of the HIF1 ChIP-seq analysis provided data of hypoxic enhancer 2 (kbp -24 from the TSS) at the SLC2A3 loci." Enhancer SLC2A3 3C "ChIP-PCR,ChIP" We elucidated that both the chromatin conformational structure and histone modification change under hy_x0002_poxic conditions and enhance the expression of SLC2A3 based on the combined results of chromatin conformation capture (3C) and ChIP assays. KDM3A is recruited to the SLC2A3 locus in an HIF1-dependent manner and demethylates H3K9me2 so as to upregulate its expression.These findings provide novel insights into the interaction between HIF1 and KDM3A and also the epigenetic regulation of HIF1. GLUT3 -- -- -- HIF1 is necessary to keep the close proximity of chromatin conformation at the kbp 35 regions of SLC2A3 loci "ChIP-seq,3C-ChIP,ChIP-PCR" "This result indicated that HIF1 is necessary to keep the close proximity of chromatin conformation at the kbp 35 regions of SLC2A3 loci. Taking all of the ndings together, under normoxia, the kbp 35 region functions as an enhancer to induce the expression of SLC2A3 at a relatively low level and retains chromatin conformation also under normoxia." HIF1A "HIF-1-alpha,HIF-1A,HIF-1alpha,HIF1,HIF1-ALPHA,MOP1,PASD8,bHLHe78" ChIP Genome-wide analysis of HIF1 binding sites (chromatin immunoprecipitation[ChIP] with deep sequencing) of endothelial cells clarified that HIF1 mainly binds to the intergenic regions distal from tran_x0002_scriptional starting sites under both normoxia and hypoxia. -- -- -- >2KB 34999 E_01_111 22665440 -- hg19 chr17 69107686 69108045 Human LNCaP Low throughput "Luciferase Reporter Assay,ChIP,3C-qPCR,ChIP-qPCR" "To determine if the rs1859962 PCa risk LD block harbors enhancers, we examined the H3K4me1 ge_x0002_nome-wide distribution defined by ChIP-seq assays in LNCaP and VCaP PCa cells.We show that there are five enhancers within the target genomic segment, hereafter referred to as E1¨CE5." Enhancer SOX9 -- qPCR "This chromatin loop was confirmed through TaqMan probe¨Cbased qPCR assays with a probe targeting the SOX9 gene (Fig. 2C). The chromatin interaction was further confirmed by sequencing the PCR product (Supplemental Fig. S7). Further_x0002_more, this chromatin loop appears to be specific to SOX9, because no other loop could be detected between E1 and any other genes(MAP2K6, KCNJ16, KCNJ2) found within this ;3-Mb window." "CMD1,CMPD1,SRA1,SRXX2,SRXY10" -- -- -- -- -- -- "AR,FOXA1" "AIS8, DHTR, HUMARA, HYSP1, KD, NR3C4, SBMA, SMAX1,TFM,HNF3A,TCF3A" "ChIP,Luciferase Reporter Assay" "In agreement, the luciferase assay in LNCaP cells depletedor not of AR using siRNA reveals that AR is required for the increased transcriptional response associated with the rs8072254 variant A allele.The FKH PWM, defined based on thousands of FOXA1 binding sites identified by ChIP-seq assays, reveals that the tenth position is invariably an A residue." rs8072254 69107816 "Luciferase Reporter Assay,ChIP" >2KB 1009294 E_01_112 22665440 -- hg19 chr17 69108466 69108765 Human LNCaP Low throughput "Luciferase Reporter Assay,ChIP,3C-qPCR,ChIP-qPCR" "To determine if the rs1859962 PCa risk LD block harbors enhancers, we examined the H3K4me1 ge_x0002_nome-wide distribution defined by ChIP-seq assays in LNCaP and VCaP PCa cells.We show that there are five enhancers within the target genomic segment, hereafter referred to as E1¨CE5." Enhancer SOX9 -- qPCR "This chromatin loop was confirmed through TaqMan probe¨Cbased qPCR assays with a probe targeting the SOX9 gene (Fig. 2C). The chromatin interaction was further confirmed by sequencing the PCR product (Supplemental Fig. S7). Further_x0002_more, this chromatin loop appears to be specific to SOX9, because no other loop could be detected between E1 and any other genes(MAP2K6, KCNJ16, KCNJ2) found within this ;3-Mb window." "CMD1,CMPD1,SRA1,SRXX2,SRXY10" -- -- -- -- -- -- "AR,FOXA1" "AIS8, DHTR, HUMARA, HYSP1, KD, NR3C4, SBMA, SMAX1,TFM,HNF3A,TCF4A" "ChIP,Luciferase Reporter Assay" "In agreement, the luciferase assay in LNCaP cells depletedor not of AR using siRNA reveals that AR is required for the increased transcriptional response associated with the rs8072254 variant A allele.The FKH PWM, defined based on thousands of FOXA1 binding sites identified by ChIP-seq assays, reveals that the tenth position is invariably an A residue." rs1859961 69108655 "Luciferase Reporter Assay,ChIP" >2KB 1008544 E_01_113 22771493 -- hg19 chr8 124323890 124325890 Human LNCaP Low throughput "qPCR,Luciferase Reporter Assay,ChIP,EMSA,3C" "Taken together, these data demonstrate that the poten_x0002_tial ARBS identified within the distal enhancer region of ATAD2 gene indeed interacts strongly and specifically with AR in vitro and suggest that it could be a good candidate as a functional ARBS in vivo.To ascertain whether the ARBS identified within the ATAD2 enhancer region is functional in cells in vivo, we performed ChIP using formaldehyde cross-linked LNCaP cells. " Enhancer ATAD2 -- "ChIP,EMSA,RT-qPCR" "In addition, we analyzed chromatin immunoprecipita_x0002_tion (ChIP)-seq data from the literature to identify genes that might share the same regulation mechanism as ATAD2.With this in mind, we aimed first at checking whether ATAD2 might be a direct target gene of AR. To test this hypothesis, we analyzed ATAD2 gene response to androgens in LNCaP cells in the presence of cycloheximide (CHX), a protein synthesis inhibitor." "ANCCA,CT137,PRO2000" Prostate Cancer DOID:10283 D011471 -- -- -- "AR,E2F1" "AIS8, DHTR, HUMARA, HYSP1, KD, NR3C4, SBMA, SMAX1, TFM,E2F-1,RBAP1,RBBP3,RBP3" EMSA "To check the binding of AR to this putative ARBS, we first performed EMSA experiments with bacterially produced AR-DNA-binding domain (DBD) purified to homogeneity,siE2F1 and siAR transfection showed the very efficient and specific knockdown of E2F1 and AR, respectively, at both mRNA and protein levels. " -- -- -- >2KB 7199 E_01_114 22771493 -- hg19 chr8 124321890 124323890 Human LNCaP Low throughput "qPCR,Luciferase Reporter Assay,ChIP,EMSA,3C" "Taken together, these data demonstrate that the poten_x0002_tial ARBS identified within the distal enhancer region of ATAD2 gene indeed interacts strongly and specifically with AR in vitro and suggest that it could be a good candidate as a functional ARBS in vivo.To ascertain whether the ARBS identified within the ATAD2 enhancer region is functional in cells in vivo, we performed ChIP using formaldehyde cross-linked LNCaP cells. " Enhancer ATAD2 -- "ChIP,EMSA,RT-qPCR" "In addition, we analyzed chromatin immunoprecipita_x0002_tion (ChIP)-seq data from the literature to identify genes that might share the same regulation mechanism as ATAD2.With this in mind, we aimed first at checking whether ATAD2 might be a direct target gene of AR. To test this hypothesis, we analyzed ATAD3 gene response to androgens in LNCaP cells in the presence of cycloheximide (CHX), a protein synthesis inhibitor." "ANCCA,CT137,PRO2000" Prostate Cancer DOID:10283 D011471 E2F1 interacts with AR on the promoter of ATAD2 through a chromatin loop to activate transcription when stimulated by androgens. "ChIP-seq,qRT-PCR,Re-ChIP" "Moreover, Re-ChIP experiments demonstrated that AR and E2F1 interact in their promoter region. This mechanism of regulation by the RB/ E2F1/AR axis might, thus, be a general mechanism for several androgen-regulated genes in prostate cells." "AR,E2F1" "AIS8, DHTR, HUMARA, HYSP1, KD, NR3C4, SBMA, SMAX1, TFM,E2F-1,RBAP1,RBBP3,RBP3" EMSA "To check the binding of AR to this putative ARBS, we first performed EMSA experiments with bacterially produced AR-DNA-binding domain (DBD) purified to homogeneity,siE2F1 and siAR transfection showed the very efficient and specific knockdown of E2F0 and AR, respectively, at both mRNA and protein levels. " -- -- -- >2KB 9199 E_01_115 22771493 -- hg19 chr8 124319890 124321890 Human LNCaP Low throughput "qPCR,Luciferase Reporter Assay,ChIP,EMSA,3C" "Taken together, these data demonstrate that the poten_x0002_tial ARBS identified within the distal enhancer region of ATAD2 gene indeed interacts strongly and specifically with AR in vitro and suggest that it could be a good candidate as a functional ARBS in vivo.To ascertain whether the ARBS identified within the ATAD2 enhancer region is functional in cells in vivo, we performed ChIP using formaldehyde cross-linked LNCaP cells. " Enhancer ATAD2 -- "ChIP,EMSA,RT-qPCR" "In addition, we analyzed chromatin immunoprecipita_x0002_tion (ChIP)-seq data from the literature to identify genes that might share the same regulation mechanism as ATAD2.With this in mind, we aimed first at checking whether ATAD2 might be a direct target gene of AR. To test this hypothesis, we analyzed ATAD4 gene response to androgens in LNCaP cells in the presence of cycloheximide (CHX), a protein synthesis inhibitor." "ANCCA,CT137,PRO2000" Prostate Cancer DOID:10283 D011471 -- -- -- "AR,E2F1" "AIS8, DHTR, HUMARA, HYSP1, KD, NR3C4, SBMA, SMAX1, TFM,E2F-1,RBAP1,RBBP3,RBP3" EMSA "To check the binding of AR to this putative ARBS, we first performed EMSA experiments with bacterially produced AR-DNA-binding domain (DBD) purified to homogeneity,siE2F1 and siAR transfection showed the very efficient and specific knockdown of E2F1 and AR, respectively, at both mRNA and protein levels. " -- -- -- >2KB 11199 E_01_116 23159876 bone Enhancer hg19 chr17 48258757 48260757 Human C2C12 Low throughput Luciferase Reporter Assay "The Col1a1 promoter analysis showed the existence of two evolutionarily conserved regions containing pairs of Sp1 sites.These regions are located one at 1700 pb from the transcription start, known as bone enhancer(BE) and where the regulation of Col1a1 expression by Osx has been described,and another region located in the proximal promoter region." Enhancer COL1A1 -- "Luciferase Reporter Assay,PCR" "To further confirm the relevance of p38 in the regu_x0002_lation of Col1a1 by Osx, we studied the effect of a constitutive active form of MKK6, the MAPKK activating p38, combined with Osx on the ac_x0002_tivation of Col1a1 promoter by Osx. The overexpression of Osx or MKK6 activated both the full-length (?2483pCol1a1) as well the proximal promoter reporter constructs. Moreover, expression of Osx and MKK6 together had an additive effect on the activation of both reporter constructs." "EDSARTH1,EDSC,OI1,OI2,OI3,OI4" -- -- -- -- -- -- RUNX2 "AML3,CBF-alpha-1,CBFA1,CCD,CCD1,CLCD,OSF-2,OSF2,PEA2aA,PEBP2aA" Luciferase Reporter Assay "Induction of long Osx transcriptional activity by Runx2. C2C12 cells were co-transfected with mock, short or long Osx constructs and/or Runx2 construct overnight, and serum-starved for 24 h. Luciferase activity was measured and normalized against ¦Â-galactosidase activity." -- -- -- <2KB 1699 E_01_117 27980063 hg19 chr1 24601612 24626812 Human Raji lymphoblastic B cells Low+High throughput "ChIP-seq,Western blot" "ChIP analyses of BRD4 and RNAPII occupancy revealed that decreased BRD4 occupanc on enhancers in response to JQ1 or OTX015 treatment leads to a signiicant concomitant reduction in RNAPII occupancy on these regions (Figure 4H, I and K, L). While BRD4 occupancy was not significantly affected around the Np63 TSS, the potential enhancers displayed a substantial reduction in the occupancy of both BRD4 and RNAPII (Figure 4HandI). Most importantly, BRD4 inhibition resulted in the down regulation of the putative TP63-- and GRHL3--associated eRNAs (Figure 4J and N)." Enhancer GRHL3 -- ChIP "ChIP analyses of BRD4 and RNAPII occupancy revealed that decreased BRD4 occupanc on enhancers in response to JQ1 or OTX015 treatment leads to a significant concomitant reduction in RNAPII occupancy on these regions (Figure 4H, I and K, L).Altogether, these studies demonstrate an enhancer--associated function of BRD4 in modulating the expression of TP63 and GRHL3. " "SOM,TFCP2L4,VWS2" Breast Cancer DOID:1612 D001943 "This Enhancer is located in a region where a long-range chromatin interaction among the promoters and promoter-Enhancer of several genes has been described, possibly affecting their expression simultaneously." Luciferase Reporter Assay "In the present work, by integrating several strategies, we have uncovered the variant most strongly associated with MS in the region. This variant also affects the activity of an Enhancer in an allele-dependent and orientation-dependent manner and correlates with the expression of five genes of the locus." "FOXO,GRHL3" "FOXO,SOM,TFCP2L4,VWS2" Western blot Western blot analyses of MCF10A protein ysates showing total p63 and GRHL3 expression with 100 nM and 1 _x0002_M of FOXO1 inhibitor AS1842856 (FOXO1i) for 24 h after removal of EGF and Insulin (-EGF/-Ins) for 48 h. -- -- -- >2KB 31599 E_01_118 23184392 -- hg19 chr15 90288698 90290698 Human Embryonic Stem Cell Low+High throughput "ChIP-seq,qRT-PCR,Luciferase Reporter Assay" "In addition, a third conserved region is evident from these analyses located -4 to -5.4 kb upstream of the ATG, which had been described as an enhancer for MesP1 expression in nascent mesoderm, primitive streak and cranial mesoderm .We therefore speculated that the cardiovascular-specific region of the human MesP1 promoter resides within the proximal -3.4 kb region possibly providing a tool to purify cardiovascular precursors from differentiating ES cells." Enhancer MESP1 -- Luciferase Reporter Assay "Recently,MesP1 promoter EGFP-driven based FACS sorting of mesodermal cell types was described using a 5.6-kb promoter fragment encompassing the entire regulatory region of MesP1." bHLHc5 -- -- -- -- -- -- -- -- -- -- -- -- -- >2KB 3399 E_01_119 23273978 P53BER4 Enhancer hg19 chr1 181104024 181104549 Human MCF-7 Low+High throughput "qRT-PCR,4C,ChIP-seq,Luciferase Reporter Assay,ChIP" "Interestingly, many of these regions possess conserved p53-binding sites and all known hallmarks of enhancer regions. We demonstrate that these p53-bound enhancer regions (p53BERs) indeed contain enhancer activity and interact intra_x0002_chromosomally with multiple neighboring genes to convey long-distance p53-dependent transcription regulation." Enhancer MDM2 -- ChIP "To identify possible target genes of p53-dependent enhancer domains, we applied chromosome conformation capture (4C) technology in combination with next-generation sequencing.). To confirm that these genes are indeed regulated by p53, we treated cells with the MDM2-inhibitor Nut_x0002_lin-3 and analyzed mRNA levels of one prominent neighboring gene for each p53BER." "ACTFS,HDMX,hdm2" -- -- -- p53BERs produce enhancer RNAs (eRNAs) that are required for efficient transcriptional enhancement of interacting target genes and induction of a p53-dependent cell-cycle arres "qRT-PCR,4C,ChIP" "We show that eRNAs are functional ncRNAs involved in transcription enhancement of genes interacting with the Enhancer they are produced from. Thus, our results imply that p53 can regulate transcription of multiple distantly located genes through binding to Enhancers and that eRNAs produced from these Enhancers are involved in transcrip_x0002_tion enhancement." TP53 "BCC7,BMFS5,LFS1,P53,TRP53" ChIP "Importantly, the enhancer activity of all p53BERs studied here was p53 dependent, as cotransfection of a p53 knockdown vector (p53KD) significantly inhibited enhancer activity." -- -- -- >2KB 111902336 E_01_120 23273978 P53BER2 Enhancer hg19 chr9 118706130 118707280 Human MCF-7 Low+High throughput "qRT-PCR,4C,ChIP-seq,Luciferase Reporter Assay,ChIP" "Interestingly, many of these regions possess conserved p53-binding sites and all known hallmarks of enhancer regions. We demonstrate that these p53-bound enhancer regions (p53BERs) indeed contain enhancer activity and interact intra_x0002_chromosomally with multiple neighboring genes to convey long-distance p53-dependent transcription regulation." Enhancer MDM2 -- ChIP "To identify possible target genes of p53-dependent enhancer domains, we applied chromosome conformation capture (4C) technology in combination with next-generation sequencing.). To confirm that these genes are indeed regulated by p53, we treated cells with the MDM2-inhibitor Nut_x0002_lin-3 and analyzed mRNA levels of one prominent neighboring gene for each p53BER." "ACTFS,HDMX,hdm2" -- -- -- p53BERs produce enhancer RNAs (eRNAs) that are required for efficient transcriptional enhancement of interacting target genes and induction of a p53-dependent cell-cycle arres "qRT-PCR,4C,ChIP" "We show that eRNAs are functional ncRNAs involved in transcription enhancement of genes inter_x0002_acting with the Enhancer they are produced from. Thus, our results imply that p53 can regulate transcription of multiple distantly located genes through binding to Enhancers and that eRNAs produced from these Enhancers are involved in transcrip_x0002_tion enhancement." TP53 "BCC7,BMFS5,LFS1,P53,TRP53" ChIP "Importantly, the enhancer activity of all p53BERs studied here was p53 dependent, as cotransfection of a p53 knockdown vector (p53KD) significantly inhibited enhancer activity." -- -- -- >2KB 49504754 E_01_121 23302769 -- hg19 chr11 8301851 8303851 Human T-acute lymphoblastic leukemia (T-ALL) Low throughput "ChIP,RT-PCR,Luciferase Reporter Assay" "The LMO1 promoters display bivalent histone marks in multiple blood lineages including T-cells, and a 3¡¯ flanking region at LMO1 ? 57 contains a transcriptional enhancer that is active in developing blood cells in transgenic mouse embryos. The LMO1 promoters become activated in T-ALL together with the 3¡¯ enhancer, which is bound in primary T-ALL cells by SCL/TAL1 and GATA3." Enhancer LMO1 -- "ChIP,RT-PCR,Luciferase Reporter Assay" "To investigate whether lack of expres_x0002_sion in the haematopoietic system may be due to active repression of the two LMO1 promoters, we analysed ChIP-Sequencing data released to the public domain by the NIH Roadmap Epigeno_x0002_mics Mapping Project." "RBTN1,RHOM1,TTG1" T-Cell leukemia DOID:715 D015458 -- -- -- "TAL1,GATA3" "SCL,TCL5,bHLHa17,tal-1,HDR,HDRS" "ChIP,qPCR" "ChIP assays on T-ALL cells from the same primagraft (X31) using antibodies against SCL/TAL1and GATA3. Analysis by quantitative PCR demonstrates significant binding of both SCL/TAL1 and GATA3 to the LMO1 +57 enhancer, with no binding to either of the two LMO1 promoters." -- -- -- >2KB 57001 E_01_122 23443045 -- hg19 chr9 21816751 21818751 Human Human 2BS Diploid Fibroblast Cells Low throughput "ChIP-qPCR,3C,ChIP,Luciferase Reporter Assay,EMSA" "Finally, a systematic survey of putative FOXA1 binding sites in the p16INK4a genomic region revealed an B150 kb distal element that could loop back to the promoter and potenti ate p16INK4a expression.To further investigate the possibility that this 150 kb element can function as authentic enhancer, we inserted a stretch of genomic sequence centred on this site into the luciferase vector containing the p16INK4a promoter and co-transfected these constructs with FOXA1 expression plas_x0002_mids into HeLa cells." Enhancer CDKN2A -- "qPCR,ChIP,Luciferase Reporter Assay" "To determine whether FOXA1 was required for p16INK4a activation in replicative exhaustion and OIS, we knocked down FOXA1 using lentiviral shRNA and examined the resultant expression change of p16INK4a. As expected, induc_x0002_tion of p16INK4a protein at late passage was impaired in 2BS cells which integrated with lentiviral shRNA for FOXA1 in middle-aged stage." "ARF,CDK4I,CDKN2,CMM2,INK4,INK4A,MLM,MTS-1,MTS1,P14,P14ARF,P16,P16-INK4A,P16INK4,P16INK4A,P19,P19ARF,TP16" -- -- -- "An B150 kb distal element could loop back to the promoter and potentiate p16INK4a expression. DNA variant at the 150 kb enhancer in?uences FOXA1-dependent p16INK4a activation." "Luciferase Reporter Assay,EMSA" "Importantly, mutation of FOXA1 binding site at promoter region completely abolished the induced luciferase activity, underscoring that the function of -150 kb Enhancer was dependent on its communication with the promoter bridged by FOXA1.DNA variant at the 150 kb enhancer in?uences FOXA1-dependent p16INK4a activation." FOXA1 "HNF3A,TCF3A" "ChIP,Luciferase Reporter Assay,EMSA" "Of note,-2 histone positioning site contains the FOXA1¡¯s binding motif identified through ChIP, luciferase reporter assay and EMSA as stated above." -- -- -- >2KB 149999 E_01_123 23598529 -- hg19 chr2 191834162 191834795 Human Mm96 Low throughput "Luciferase Reporter Assay,ChIP,PCR" "Our previous analysis of the STAT1 gene recognized a region downstream of the promoter and enhancer regions strongly repressing transcription of luciferase gene repor_x0002_ters (20). In the present study, we define an RE-1 element within this repressor region, to which REST bound and repressed transcription. In the absence of REST, transcriptional repression of the STAT1 gene was dramatically reduced." Enhancer STAT1 -- "Luciferase Reporter Assay,ChIP,PCR" "Analysis of REST and STAT1 expression levels in human melanoma cells showed that REST is commonly expressed in melanomas and, together with transcription_x0002_al activation and chromatin immunoprecipitation (ChIP) assay, shows that REST negatively regulates STAT1 expression, contributing to the nonresponsiveness of mel_x0002_anomas to IFN." "CANDF7,IMD31A,IMD31B,IMD31C,ISGF-3,STAT91" -- -- -- -- -- -- -- -- -- -- -- -- -- <2KB 718 E_01_124 23631855 -- hg19 chr12 48369059 48369213 Human SW 1353 Low throughput "PCR,ChIP,Luciferase Reporter Assay" "In addition, CTS increased the association between Smad2/3 and the COL2A1 enhancer on chromatin in SW1353 cells (Fig. 3C). In stretched cells, DNA fragment that contained the COL2A1 enhancer was coimmunoprecipitated with Smad2/3, probably via complex for_x0002_mation among Smad2/3-SOX9-the SOX9-binding site, and was amplified by PCR using specific primers for the COL2A1 enhancer." Enhancer COL2A1 -- "PCR,ChIP,Luciferase Reporter Assay" Chromatin immunoprecipitation revealed that CTS increased Smad2/3 interaction with the CCN2 promoter and the COL2A1 enhancer. Our results suggest that CTS epigenetically stimulates CCN2 transcription via TGF-¦Â1 release associated with Smad2/3 activation and enhances COL2A1 expression through the complex formation between SOX9 and Smad2/3. "ANFH,AOM,COL11A3,SEDC,STL1" -- -- -- -- -- -- SOX9 "CMD1,CMPD1,SRA1,SRXX2,SRXY10" "Western blot,ChIP" "In Western blot(WB)analysis,CTS treatment(2h)did not influence total a mounts of endogenous Smad2/3 and SOX9 in each whole cell lysate(B,20 ¦Ìg/lane). However,Smad2/3 detected in the nuclear fraction was increased by CTS(C,nucleus,20 ¦Ìg/lane). In IP analys is using an anti-SOX9 antibody,CTS increased the association between phosphorylated Smad2/3 and SOX9 in the nuclear fraction derived from stretched SW1353 cells." -- -- -- >2KB 2389 E_01_125 23636943 -- hg19 chr11 72897942 72912333 Human MCF-7 Low+High throughput "3C,ChIP-seq,ChIP-qPCR" "We integrated the data from our GRO-seq assays with data from a variety of other genomic assays (e.g., ChIP-seq, DNase-seq, ChIA-PET) using a novel computational pipeline to provide a comprehensive and global view of ESR1 enhancers and their regulation by E2 in MCF-7 cells. Together, our studies have shed new light on the activity of ESR1 at its enhancer sites and provide new insights about en_x0002_hancer function in general, including the potential roles of en_x0002_hancer transcription." Enhancer P2RY2 -- "ChIP-seq,ChIP-qPCR" "As shown in the GRO-seq browser tracks in Figure 1A, transcription of the P2RY2 gene and a region around ERBS1 is up-regulated rapidly in a short time course of treatment with 17b-estradiol.The transcripts from ERBS1(Fig. 1A), as well as ERBSs 2¨C5 (Supplemental Fig. 2), are produced bidirectionally from both strands of DNA,reminiscent of the enhancer RNAs (eRNAs) described previously (Kim et al. 2010), and the transcribed regions are associated with RNA Pol II and pre_x0002_viously identified transcription start sites (TSSs)." "HP2U,P2RU1,P2U,P2U1,P2UR,P2Y2,P2Y2R" -- -- -- -- -- -- FOXA1 "HNF3A,TCF3A" 3C-PCR "3C-PCR assay showing E2-induced looping between ERBS1 and the P2RY2 gene. The lowercase letters correspond to the primers denoted by orange arrows shown in panel A. The assays were conducted in the presence (experimental) or absence (control) of DNA ligase, as indicated. Digested and ligated bacterial artificial chromosome (BAC) DNA spanning the entire P2YR2 locus was used as a PCR control." -- -- -- >2KB 24204 E_01_126 23644027 -- hg19 chr5 172648612 172649177 Human Leukocytes Low throughput "PCR,Luciferase Reporter Assay" "The wild type and variant upstream enhancers (566 bp) were generated with PCR, and then subcloned into BamHI and SalI sites of a luciferase gene expression vector with human NKX2-5 gene promoter, which has been previously reportedll expression constructs were confirmed by direct sequencing.HEK-293 cells were cultured in DMEM with 10% fetal bovine serum,1% glutamine and 1% penicillin/streptomycin under 5% CO2 at 37 ¡ãC." Enhancer NKX2-5 -- "PCR,Luciferase Reporter Assay" "To determine the effects of the DSVs on transcriptional activity of the enhancer, wild type and variant enhancers were subcloned upstream to the NKX2-5 gene promoter in the luciferase gene expression vector, which has been previously constructed and reported,to generate expression constructs pGL3-WT, pGL3-17483557Ins,pGL3-17483564T and pGL3-17483576G. These constructs were then transiently transfected into HEK-293 cells and luciferase activities were measured. The results were shown in Fig. 2. Expression levels of pGL3-17483564T and pGL3-17483576G were significantly decreased compared to pGL3-WT." "CHNG5,CSX,CSX1,HLHS2,NKX2.5,NKX2E,NKX4-1,VSD3" Congenital Heart Disease DOID:1682 D006330 The DSVs within the upstream Enhancer of the NKX2-5 gene may contribute to a small number of VSD. Luciferase Reporter Assay "To determine the effects of the DSVs on transcriptional activity of the Enhancer,wild type and variant Enhancers were subcloned upstream to the NKX2-5 gene promoter in the Luciferase gene expression vector,which has been previously constructed and reported. Collectively, these results indicated that the activity of the upstream enhancer of the NKX2-5 gene was decreased by the DSVs g.17483564C>T and g.17483576C>G." -- -- -- -- -- -- -- >2KB 10211 E_01_127 23659698 -- hg19 chr12 21439733 21439973 Human HeLa Low throughput "DNaseI-seq,PCR" "To find the enhancers of the IFN¦Ã gene,different 240-bp fragments of the gene were inserted between the GFP gene and the Alu tandem repeats in the C1-Alu14 plasmid.GFP expression in transfected cells was assessed using fluores_x0002_cence microscopy.We found that five different 240-bp fragments displayed strong enhancer activity.One enhancer was located in an upstream sequence of the IFN¦Ã gene (?480~?720 bp,FUIFN3F3R);two enhancers (IFN4F4R and IFN6F6R) were located in intron 1;another two enhancers (IFN21F21R and IFN22F22R) were located in a noncoding region downstream of the IFN¦Ã gene. The percentages of the GFP-positive cells for the five enhancers were 6.9%, 2.6%, 6.3%, 7.7%, and 4.9%,respectively.The ratio of the percentage of the GFP-positive cells induced by the inserting fragments compared with that of C1-Alu14 reflects enhancer activity." Enhancer IFNG -- DNaseI-seq "To determine which fragments of the IFN¦Ã gene activate the GFP reporter gene, the IFN¦Ã gene (4972 bp) and its upstream (720 bp) and downstream (308 bp) sequences were divided into 25 fragments, in which each fragment was 240 bp long." "IFG,IFI" -- -- -- These enhancers may be targets of IFN gene expression regulation. "PCR,Fuorescence Microscopy" "We found that five different 240-bp fragments (FUIFN3F3R, IFN4F4R, IFN6F6R, IFN21F21R, and IFN22F22R) and two 60-bp core sequences (IFN6-2F2R and IFN21-3-4F3-4R) derived from the IFN_x0001_gene contain Enhancers that can activate the GFP reporter gene. These enhancers may be targets of IFN gene expression regulation." -- -- -- -- -- -- -- >2KB 47108696 E_01_128 23659698 -- hg19 chr12 68549287 68549545 Human HeLa Low throughput "DNaseI-seq,PCR" "To find the enhancers of the IFN¦Ã gene,different 240-bp fragments of the gene were inserted between the GFP gene and the Alu tandem repeats in the C1-Alu14 plasmid.GFP expression in transfected cells was assessed using fluores_x0002_cence microscopy.We found that five different 240-bp fragments displayed strong enhancer activity.One enhancer was located in an upstream sequence of the IFN¦Ã gene (?480~?720 bp,FUIFN3F3R);two enhancers (IFN4F4R and IFN6F6R) were located in intron 1;another two enhancers (IFN21F21R and IFN22F22R) were located in a noncoding region downstream of the IFN¦Ã gene. The percentages of the GFP-positive cells for the five enhancers were 6.9%, 2.6%, 6.3%, 7.7%, and 4.9%,respectively.The ratio of the percentage of the GFP-positive cells induced by the inserting fragments compared with that of C1-Alu14 reflects enhancer activity." Enhancer IFNG -- DNaseI-seq "To determine which fragments of the IFN¦Ã gene activate the GFP reporter gene, the IFN¦Ã gene (4972 bp) and its upstream (720 bp) and downstream (308 bp) sequences were divided into 25 fragments, in which each fragment was 240 bp long." "IFG,IFI" -- -- -- These enhancers may be targets of IFN gene expression regulation. "PCR,Fuorescence Microscopy" "We found that five different 240-bp fragments (FUIFN3F3R, IFN4F4R, IFN6F6R, IFN21F21R, and IFN22F22R) and two 60-bp core sequences (IFN6-2F2R and IFN21-3-4F3-4R) derived from the IFN_x0001_gene contain Enhancers that can activate the GFP reporter gene. These enhancers may be targets of IFN gene expression regulation." -- -- -- -- -- -- -- <2KB 867 E_01_129 23659698 -- hg19 chr12 68549796 68550055 Human HeLa Low throughput "DNaseI-seq,PCR" "To find the enhancers of the IFN¦Ã gene,different 240-bp fragments of the gene were inserted between the GFP gene and the Alu tandem repeats in the C1-Alu14 plasmid.GFP expression in transfected cells was assessed using fluores_x0002_cence microscopy.We found that five different 240-bp fragments displayed strong enhancer activity.One enhancer was located in an upstream sequence of the IFN¦Ã gene (?480~?720 bp,FUIFN3F3R);two enhancers (IFN4F4R and IFN6F6R) were located in intron 1;another two enhancers (IFN21F21R and IFN22F22R) were located in a noncoding region downstream of the IFN¦Ã gene. The percentages of the GFP-positive cells for the five enhancers were 6.9%, 2.6%, 6.3%, 7.7%, and 4.9%,respectively.The ratio of the percentage of the GFP-positive cells induced by the inserting fragments compared with that of C1-Alu14 reflects enhancer activity." Enhancer IFNG -- DNaseI-seq "To determine which fragments of the IFN¦Ã gene activate the GFP reporter gene, the IFN¦Ã gene (4972 bp) and its upstream (720 bp) and downstream (308 bp) sequences were divided into 25 fragments, in which each fragment was 240 bp long." "IFG,IFI" -- -- -- These enhancers may be targets of IFN gene expression regulation. "PCR,Fuorescence Microscopy" "We found that five different 240-bp fragments (FUIFN3F3R, IFN4F4R, IFN6F6R, IFN21F21R, and IFN22F22R) and two 60-bp core sequences (IFN6-2F2R and IFN21-3-4F3-4R) derived from the IFN_x0001_gene contain Enhancers that can activate the GFP reporter gene. These enhancers may be targets of IFN gene expression regulation." -- -- -- -- -- -- -- <2KB 1377 E_01_130 23659698 -- hg19 chr12 68553503 68553754 Human HeLa Low throughput "DNaseI-seq,PCR" "To find the enhancers of the IFN¦Ã gene,different 240-bp fragments of the gene were inserted between the GFP gene and the Alu tandem repeats in the C1-Alu14 plasmid.GFP expression in transfected cells was assessed using fluores_x0002_cence microscopy.We found that five different 240-bp fragments displayed strong enhancer activity.One enhancer was located in an upstream sequence of the IFN¦Ã gene (?480~?720 bp,FUIFN3F3R);two enhancers (IFN4F4R and IFN6F6R) were located in intron 1;another two enhancers (IFN21F21R and IFN22F22R) were located in a noncoding region downstream of the IFN¦Ã gene. The percentages of the GFP-positive cells for the five enhancers were 6.9%, 2.6%, 6.3%, 7.7%, and 4.9%,respectively.The ratio of the percentage of the GFP-positive cells induced by the inserting fragments compared with that of C1-Alu14 reflects enhancer activity." Enhancer IFNG -- DNaseI-seq "To determine which fragments of the IFN¦Ã gene activate the GFP reporter gene, the IFN¦Ã gene (4972 bp) and its upstream (720 bp) and downstream (308 bp) sequences were divided into 25 fragments, in which each fragment was 240 bp long." "IFG,IFI" -- -- -- These enhancers may be targets of IFN gene expression regulation. "PCR,Fuorescence Microscopy" "We found that five different 240-bp fragments (FUIFN3F3R, IFN4F4R, IFN6F6R, IFN21F21R, and IFN22F22R) and two 60-bp core sequences (IFN6-2F2R and IFN21-3-4F3-4R) derived from the IFN_x0001_gene contain Enhancers that can activate the GFP reporter gene. These enhancers may be targets of IFN gene expression regulation." -- -- -- -- -- -- -- >2KB 5080 E_01_131 23659698 -- hg19 chr12 68553762 68554012 Human HeLa Low throughput "DNaseI-seq,PCR" "To find the enhancers of the IFN¦Ã gene,different 240-bp fragments of the gene were inserted between the GFP gene and the Alu tandem repeats in the C1-Alu14 plasmid.GFP expression in transfected cells was assessed using fluores_x0002_cence microscopy.We found that five different 240-bp fragments displayed strong enhancer activity.One enhancer was located in an upstream sequence of the IFN¦Ã gene (?480~?720 bp,FUIFN3F3R);two enhancers (IFN4F4R and IFN6F6R) were located in intron 1;another two enhancers (IFN21F21R and IFN22F22R) were located in a noncoding region downstream of the IFN¦Ã gene. The percentages of the GFP-positive cells for the five enhancers were 6.9%, 2.6%, 6.3%, 7.7%, and 4.9%,respectively.The ratio of the percentage of the GFP-positive cells induced by the inserting fragments compared with that of C1-Alu14 reflects enhancer activity." Enhancer IFNG -- DNaseI-seq "To determine which fragments of the IFN¦Ã gene activate the GFP reporter gene, the IFN¦Ã gene (4972 bp) and its upstream (720 bp) and downstream (308 bp) sequences were divided into 25 fragments, in which each fragment was 240 bp long." "IFG,IFI" -- -- -- These enhancers may be targets of IFN gene expression regulation. "PCR,Fuorescence Microscopy" "We found that five different 240-bp fragments (FUIFN3F3R, IFN4F4R, IFN6F6R, IFN21F21R, and IFN22F22R) and two 60-bp core sequences (IFN6-2F2R and IFN21-3-4F3-4R) derived from the IFN_x0001_gene contain Enhancers that can activate the GFP reporter gene. These enhancers may be targets of IFN gene expression regulation." -- -- -- -- -- -- -- >2KB 5338 E_01_132 23675462 -- hg19 chr21 43770915 43772915 Human MCF-7 Low throughput "RT-PCR,ChIP" "To determine whether MSK1 co-occupies the enhancer and UPE with H3S10ph, we performed sequential ChIP assays (re_x0002_ChIP) with mononucleosomes prepared from TPA- treated and formaldehyde-cross linked MCF-7 cell lysates.Together these results show that either MSK1 or MSK2 recruited to the TFF1 UPE and enhancer phopshorylates H3 at S10, leading to the binding of 14-3-3e/f and recruitment of BRG1." Enhancer TFF1 -- "RT-PCR,ChIP" "Here, we used the high-resolution chromatin immunoprecipi_x0002_tation (ChIP) assay to determine the MSK1, MSK2 and H3S10ph distribution along the regulatory and coding regions of the TFF1 gene in response to ERK-MAPK signaling.With this in mind, we performed ChIP assays with antibodies against H3S10phK14ac to determine the distribution of this dual H3 modification along the TFF1 gene._x0002_" "BCEI,D21S21,HP1.A,HPS2,pNR-2,pS2" -- -- -- MSK1 and MSK2 belong to different multiprotein complexes but both mediate chromatin remodeling that is required at the Enhancer and UPE for TPA-induced initiation of TFF1 expression in MCF-7 breast cancer epithelial cells. Re-ChIP "An AP3-like (activator protein 3) enhancer element, located 2.4 kb upstream of PLG-TSS, is involved in PLG transcriptional regulation [20]. In our study we could show that a construct comprising the entire upstream region, containing the AP3 response element (2.4 kb-PLG) was able to further elevate basal luciferase activity 1.7-fold above that of PLG minimal promoters." "RPS6KA5,RPS6KA4 " "MSK1, MSPK1, RLPK,MSK2, RSK-B, S6K-alpha-4" ChIP "we used the high-resolution chromatin immunoprecipitation (ChIP) assay to determine the MSK1, MSK2 and H3S10ph distribution along the regulatory and coding regions of the TFF1 gene in response to ERK-MAPK signaling." -- -- -- >2KB 10475 E_01_133 23872150 AP3 Enhancer hg19 chr6 161119825 161121825 Human "Hep G2,MCF-7" Low throughput "Luciferase Reporter Assay,ChIP,EMSA,PCR" "An AP3-like (activator protein 3) enhancer element, located 2.4 kb upstream of PLG-TSS, is involved in PLG transcriptional regulationIn summary we demonstrated that estrogen regulates plasminogen promoter activity. The 5¡¯-flanking region of the PLG gene con_x0002_tains several cis-acting elements that are responsive to estrogen.An enhancer located at -11.5 kb confers a dramatic estrogen re_x0002_sponse in reporter assays." Enhancer PLG -- "Luciferase Reporter Assay,PCR" "In our study we could show that a construct comprising the entire upstream region, containing the AP3 re_x0002_sponse element (2.4 kb-PLG) was able to further elevate basal luciferase activity 1.7-fold above that of PLG minimal promoters.The observed increase corresponds well to previously published results [20] and the 2.4 kb-PLG construct represents the most active contiguous promoter fragment analysed in this study. Surprisingly, with E2 stimulation this effect decreased by 54% down to the activity level of the PLG core promoter,although no ERE could be identified within this region." PLG Hypoplasminogenemia -- -- "An AP3-like (activator protein 3) enhancer element, located 2.4 kb upstream of PLG-TSS, is involved in PLG transcriptional regulation." Luciferase Reporter Assay "An AP3-like (activator protein 3) enhancer element, located 2.4 kb upstream of PLG-TSS, is involved in PLG transcriptional regulation [20]. In our study we could show that a construct comprising the entire upstream region, containing the AP3 response element (2.4 kb-PLG) was able to further elevate basal luciferase activity 1.7-fold above that of PLG minimal promoters." "DHII,ERE" "anon-EST:Posey114,anon-WO02059370.45,CG13094,DH31,DH-II,dh31,DH31,DH[31],DH[[31],Dh[[31]],DH[[31]],Dmel\CG13094,Drm-DH[[31]],drome-DH31,Drome-DH[[31]],ERE" Luciferase Reporter Assay Transcriptional effect of the DHII and/or ERE (_x0003_11.5 kb) enhancers on the heterologous crystallin promoter (cryst) driven luciferase activity in HepG2. -- -- -- >2KB 2399 E_01_134 23872150 ERE Enhancer hg19 chr6 161110725 161112725 Human "Hep G2,MCF-7" Low throughput "Luciferase Reporter Assay,ChIP,EMSA,PCR" "An AP3-like (activator protein 3) enhancer element, located 2.4 kb upstream of PLG-TSS, is involved in PLG transcriptional regulationIn summary we demonstrated that estrogen regulates plasminogen promoter activity. The 5¡¯-flanking region of the PLG gene con_x0002_tains several cis-acting elements that are responsive to estrogen.An enhancer located at -11.5 kb confers a dramatic estrogen re_x0002_sponse in reporter assays." Enhancer PLG -- "Luciferase Reporter Assay,PCR" "In our study we could show that a construct comprising the entire upstream region, containing the AP3 re_x0002_sponse element (2.4 kb-PLG) was able to further elevate basal luciferase activity 1.7-fold above that of PLG minimal promoters.The observed increase corresponds well to previously published results [20] and the 2.4 kb-PLG construct represents the most active contiguous promoter fragment analysed in this study. Surprisingly, with E2 stimulation this effect decreased by 54% down to the activity level of the PLG core promoter,although no ERE could be identified within this region." PLG Hypoplasminogenemia -- -- The intergenic region between both genes comprises several transcription-regulatory regions with Enhancer sequences that increase the basal activity of the PLG core promoter. Luciferase Reporter Assay An estrogen response element located 11.5 kb upstream of the PLG transcription start site is able to convey a dramatic estrogen-dependent elevation of PLG-minimal promoter driven reporter gene expression. "DHII,ERE" "anon-EST:Posey114,anon-WO02059370.45,CG13094,DH31,DH-II,dh31,DH31,DH[31],DH[[31],Dh[[31]],DH[[31]],Dmel\CG13094,Drm-DH[[31]],drome-DH31,Drome-DH[[31]],ERE" Luciferase Reporter Assay Transcriptional effect of the DHII and/or ERE (_x0003_11.5 kb) enhancers on the heterologous crystallin promoter (cryst) driven luciferase activity in HepG2. -- -- -- >2KB 11499 E_01_135 23873758 -- hg19 chr12 48368959 48369159 Human "HeLa,HEK-293" Low throughput "PCR,ChIP" "Real©\time PCR (qPCR) reactions for ChIP and ChIP reChIP were carried out using primers flanking the COL2A1 promoter start site(¨C167) to (¨C65), see illustration in Figure 6A. Additional analyses were performed on the Sox9©\binding enhancer region of COL2A1(?2259) to (?2430). As a negative control, a distal set of primers flanking the intron 38 (?24148) to (?24368) of the COL2A1 gene was used, based on the protocol by Furlan©\Magarill and colleagues." Enhancer COL2A1 -- "PCR,ChIP" "To understand the regulation of the active COL2A1 promoter, we performed ChIP assays using specific primers flanking the promoter start site and enhancer site within the first intron (illustrated in Fig. 6A). We found enhanced occupancy of the DNA©\binding transcription factor SP1 on the promoter start site of 3D cultured cells, whereas passaged cells (P5) showed a slight but insignificant reduction of SP1 (Fig. 6B). Additionally, passaging significantly decreased Sox9 occupancy on the enhancer site." "ANFH,AOM,COL11A3,SEDC,STL1" -- -- -- "Although Sox9 (SRY©\related high mobility group©\Box gene 9) has been shown to regulate the expression of the COL2A1 gene by binding the Enhancer within the first intron,numerous additional coactivators (eg, LSox5, Sox6, PGC1a, Notch) are involved in activa" "PCR,ChIP" "3D cultures also exhibited augmented mRNA and protein levels of the transcription factors SP1 and Sox9 (Fig. 5A. B), which are known to regulate COL2A1 expression by binding the DNA-motifs of the promoter and Enhancer regions, respectively." "SP1,SOX9" "SP1,CMD1,CMPD1,SRA1,SRXX2,SRXY10" ChIP "Chromatin immunoprecipitation (ChIP) analyses of 3D cultures showed augmented levels of the DNA©\binding transcription factor SP1, and the histone methyltransferase Set7/9, on the COL2A1 promoter site." -- -- -- Intron 2312 E_01_136 23884959 PSA Enhancer hg19 chr8 57069398 57069482 Human LNCaP Low throughput "ChIP,qPCR,qRT-PCR" "The PSA gene contains HREs at its enhancer region in addition to the androgen-responsive elements. ChIP analyses using HIF-1¦Á antibody identified the HIF-1¦Á occu_x0002_pancy on the enhancer region of the PSA gene in response to hypoxia but not on the promoter region. DHT treatment further increased HIF-1¦Á occupancy on the enhancer under hypoxia (Figure?1D). ChIP anal_x0002_yses using AR antibody showed that more AR binds to the enhancer than to the promoter, and that hypoxia augments DHT-induced AR occupancy." Enhancer KLK3 -- "ChIP,qRT-PCR" "Knockdown of JMJD1A and JMJD2C increased the occurrence of H3K9me2 and me3 on the PSA gene, respectively, thus, blocking AR-induced tran_x0002_scriptional activity.These findings indicated that androgen_x0002_induced PSA gene is a target regulated by JMJD1A and JMJD2C. " "APS,KLK2A1,PSA,hK3" -- -- -- -- -- -- -- -- -- -- -- -- -- >2KB 5711270 E_01_137 24040411 ECR18 Enhancer hg19 chr19 57566145 57571006 Human "HeLa,HEK-293" Low throughput "Luciferase Reporter Assay,PCR,3C,ChIP" "The observed interaction is most prominent in brain, but was also detected in testis. Histone modification and DNA methylation on ECR18 show no allele bias, implying that this region is likely functional on both alleles. In vitro assays also reveal ECR18 as a potential enhancer or repressor for the promoter of Peg3. Overall, these results indicate that the promoters of several imprinted genes in the Peg3 domain interact with one evolutionarily conserved region, ECR18, and further suggest that ECR18 may play key roles in the transcription and imprinting control of the Peg3 domain as a distant regulatory element." Enhancer PEG3 3C "Luciferase Reporter Assay,PCR" "In the current study, the chromatin conformation capture (3C) method was utilized to detect potential interactions of these ECRs with the imprinted genes. According to the results, one region, ECR18, located 200-kb upstream of Peg3 interacts with the two promoter regions of Peg3 and Zim2. The observed interaction is most prominent in brain, but was also detected in testis. " "PW1,ZKSCAN22,ZNF904,ZSCAN24" -- -- -- ECR18 may play key roles in the transcription and imprinting control of the Peg3 domain as a distant regulatory element. "3C,ChIP" "In the current study, we performed a series of Chromatin Conformation Capture (3C) and Chromatin Immunoprecipitation (ChIP) analyses to further confirm this prediction. Overall, the current study identifies one region, ECR18, as a key regulatory region for the transcription and imprinting of the Peg3 domain." -- -- -- -- -- -- -- >2KB 247132 E_01_138 24086551 -- hg19 chr1 1083900 1085900 Human HMLE Low+High throughput "qRT-PCR,Luciferase Reporter Assay,ChIP-seq,ChIP-qPCR" "We have analyzed the miR-200b~200a~429 locus for epigenetic modifications in breast epithelial and mesenchymal cell lines using chromatin immunoprecipitation assays and DNA methylation analysis. We discovered a novel enhancer located approximately 5.1kb upstream of the miR-200b~200a~429 transcriptional start site. This region was associated with the active enhancer chromatin signature comprising H3K4me1, H3K27ac, RNA polymerase II and CpG dinucleotide hypomethylation. " Enhancer GIHCG -- "qRT-PCR,Luciferase Reporter Assay,ChIP-qPCR" "Luciferase reporter assays revealed the upstream enhancer stimulated the transcription of the miR-200b~200a~429 minimal promoter region approximately 27-fold in breast epithelial cells. Furthermore, we found that a region of the enhancer was transcribed, producing a short, GC-rich, mainly nuclear, non-polyadenylated RNA transcript designated miR-200b eRNA." GIHCG -- -- -- "The presence of a novel Enhancer contributes to miR-200b~200a~429 transcriptional regulation in epithelial cells. " Luciferase Reporter Assay "Luciferase reporter assays revealed the upstream enhancer stimulated the transcription of the miR-200b~200a~429 minimal promoter region approximately 27-fold in breast epithelial cells. Furthermore, we found that a region of the enhancer was transcribed, producing a short, GC-rich, mainly nuclear, non-polyadenylated RNA transcript designated miR-200b eRNA. Over-expression of miR-200b eRNA had little effect on miR-200b~200a~429 promoter activity and its production did not correlate with miR-200b~200a~429 gene expression." -- -- -- -- -- -- -- -- -- E_01_139 24212882 -- hg19 chr10 23502416 23510031 Human Embryonic Stem Cell Low throughput "ChIP,PCR,3C,EMSA" "In the tenth family a 7.6kb deletion was identified by long range PCR, and sequence analysis showed that the deleted region (chr10:23502416-23510031) included the entire putative enhancer." Enhancer PTF1A 3C ChIP-seq "To assess if this enhancer truly targets PTF1A, we performed chromatin conformation capture (3C) experiments. This demonstrated that the enhancer region establishes direct interactions with the PTF1A promoter in human pancreatic progenitor cells." "PACA,PAGEN2,PTF1-p48,bHLHa29,p48" Pancreatic Agenesis DOID:0050877 -- -- -- -- "FOXA2,PDX1" "HNF3B,TCF3B,GSF,IDX-1,IPF1,IUF1,MODY4,PAGEN1,PDX-1,STF-1" EMSA "Electrophoretic mobility shift assay showing high affinity, sequence-specific interaction of FOXA2 with a double stranded oligonucleotide containing the wild type (WT) sequence, but not the 363 A>G mutation (MUT).Electrophoretic mobility shift assay showing sequence-specific interaction of PDX1 with oligonucleotide containing the wild type (WT) sequence, but not the 446 A>C mutation (MUT). The retardation signal was suppressed by unlabelled consensus high-affinity binding site for PDX1, and supershifted with antibodies recognizing PDX1." -- -- -- >2KB 24765 E_01_140 24256810 -- hg19 chr11 111150916 111155431 Human "HCT 116,SW480,Colorectal Cancer" Low+High throughput "PCR,ChIP-seq,Luciferase Reporter Assay,Transfection,EMSA" "One potentially active promoter region and several candidate enhancer regions were identified using high-resolution genome wide ChIP-seq profiles for histone modifications including H3K4me1, H3K4me3 and H3K9/14ac (Figure 1A and Supple_x0002_mentary Material, Fig. S1), which we generated for the SW480 and HCT-116 CRC cell lines." Enhancer C11orf53 -- ChIP-seq "This is manifest in normal human colon tissues as eQTL for three genes, C11orf53, C11orf92 and C11orf93. Using bicolor EMSA, we further showed that protein binding is markedly dif_x0002_ferent for the sequences containing the two alleles of rs7130173,with binding affinity of the C allele roughly four times greater than the A allele." C11orf53 Colorectal Cancer DOID:9256 D015179 -- -- -- -- -- -- -- rs7130173 111154072 "ChIP-seq,Luciferase Reporter Assay,EMSA" >2KB 26468 E_01_141 24285714 -- hg19 chr8 127047315 127049315 Human Acute Myeloid Leukemia Cell Low+High throughput "ChIP,ChIP-seq,Luciferase Reporter Assay,4C,ChIP-qPCR,3C,qPCR" "To account for this context-specific function, we identify a cluster of lineage-specific enhancers located 1.7 Mb downstream from Myc that are occupied by SWI/SNF as well as the BET protein Brd4. Brg1 is required at these distal elements to maintain transcription factor occupancy and for long-range chromatin looping interactions with the Myc promoter. Notably,these distal Myc enhancers coincide with a region that is focally amplified in ~3% of acute myeloid leukemias." Enhancer MYC -- "ChIP,ChIP-seq,ChIP-qPCR" "To explain this observation, we hypothesized that Brg1 regulates Myc transcription in leukemia cells by occupying cell type-specific regulatory elements. We evaluated this possibility by performing chromatin immunoprecipitation (ChIP) followed by next_x0002_generation sequencing (ChIP-seq) analysis of Brg1, his_x0002_tone H3 Lys 4 trimethylation (H3K4me3), and histone H3 Lys 27 acetylation (H3K27ac) in RN2 leukemia cells." "MRTL,MYCC,bHLHe39,c-Myc" Acute Leukemia DOID:12603 -- -- -- -- "SMARCA4,CEBPA,CEBPB,ERG,LMO2" "BAF190,BAF190A,BRG1,CSS4,MRD16,RTPS2,SNF2,SNF2L4,SNF2LB,SWI2,hSNF2b,C/EBP-alpha,CEBP,C/EBP-beta,IL6DBP,NF-IL6,TCF5,erg-3,p55,LMO-2,RBTN2,RBTNL1,RHOM2,TTG2" "ChIP-qPCR,shRNA" "Since all of these TFs are expressed in RN2 cells (Supplemental Fig. 13), we anticipated that these factors would occupy E1¨CE5 in leukemia as well. Indeed, we detected occupancy of Lmo2, PU.1, and Erg at various subsets of the E1¨CE5 enhancers in RN2 using ChIP-qPCR (Fig. 5I¨CK). Additionally, we found that the hematopoietic TFs Cebp¦Á and Cebp¦Â, both of which are highly expressed in RN2 cells, also occupied the E1¨CE5 elements (Fig. 5L,M; Supplemental Fig. 13).While expression of several regulators was perturbed upon Brg1 knockdown, Myc was among the most down-regulated genes identified (P < 0.01) (Fig. 3A). Hoxa9, which is a direct target of the MLL-AF9 oncoprotein, was also down-regulated (P < 0.01), albeit to a lesser extent than Myc (Fig. 3A). Gene signatures linked to Myc and Hoxa9 function were globally suppressed following Brg1 knockdown, further confirming an effect on these two pathways (Fig. 3B,C; Supplemental Fig. 5)." -- -- -- >2KB 1699999 E_01_142 24288367 -- hg19 chr21 43781591 43782591 Human "MCF-7,MDA-MB-231,293T" Low throughput "qRT-PCR,ChIP" "TFF1 has three EREs, and ERE3 at -9.9 kb upstream of transcriptional start site (TSS) is the major ER binding site and considered to be the major enhancer element. Our chromatin immunoprecipitation (ChIP) assay showed that the enhancer region of the TFF1 gene (ERE3,-9.9 kb) was highly occupied by MLL1 and that the recruitment of MLL1 to this region was further enhanced after E2 treatment in MCF-7 cells (Figure 2A).We validated these findings by a ChIP scanning assay,which surveyed from _x0002_10 kb upstream through 5 kb downstream of the TSS of the TFF1 gene. The distal enhancer region of TFF1 was found to be highly occupied by MLL1." Enhancer TFF1 -- "qRT-PCR,ChIP" "When FLAG-tagged MLL1-CX or MLL1-CXPB was over_x0002_expressed in MCF-7 cells by transient transfection, quan_x0002_titative ChIP assays performed with anti-FLAG antibody demonstrated the occupancy by both MLL1-CX and MLL1-CXPB on ERE3 (but not ERE2 or ERE1) of the TFF1 gene, indicating that the 40 kDa MLL1-CX fragment is sufficient for recruitment to the enhancer region in vivo (Figure 3F) with the same genomic binding specificity as wild-type MLL1." "BCEI,D21S21,HP1.A,HPS2,pNR-2,pS2" Mixed-Lineage Leukemia 1 -- -- Mixed-lineage leukemia 1 (MLL1) protein is a key determinant that maintains permissive chromatin structure of the TFF1 Enhancer region. "ChIP,NOMe-seq" "Mixed-lineage leukemia 1 (MLL1) protein is a key determinant that maintains permissive chromatin structure of the TFF1 enhancer region. MLL1 occupies the TFF1 enhancer region and methylates H3K4 before hormone stimulation. In vitro, MLL1 binds directly to the CpG-rich region of the TFF1 enhancer, and its binding is dependent on hypomethylation of DNA. Furthermore, the depletion of MLL1 in MCF-7 cells results in a dramatic decrease of chromatin accessibility and recruitment of FOXA1 and ERa to the enhancer element." KMT2A "ALL-1,CXXC7,HRX,HTRX1,MLL,MLL1,MLL1A,TRX1,WDSTS" "qPCR,ChIP" "FLAG-tagged MLL1-CX or MLL1-CXPB was transiently expressed in the MCF-7 cells, which were then grown in hormone-free media for 48 h and then treated with 100nM E2 for 1 h before performing the ChIP assay with anti-FLAG antibody. Precipitated DNA was analyzed by qPCR with primers representing the ER binding sites of the TFF1 genes." -- -- -- <2KB 299 E_01_143 24288367 -- hg19 chr21 43772291 43774291 Human "MCF-7,MDA-MB-231,293T" Low throughput "qRT-PCR,ChIP" "TFF1 has three EREs, and ERE3 at -9.9 kb upstream of transcriptional start site (TSS) is the major ER binding site and considered to be the major enhancer element. Our chromatin immunoprecipitation (ChIP) assay showed that the enhancer region of the TFF1 gene (ERE3,-9.9 kb) was highly occupied by MLL1 and that the recruitment of MLL1 to this region was further enhanced after E2 treatment in MCF-7 cells (Figure 2A).We validated these findings by a ChIP scanning assay,which surveyed from _x0002_10 kb upstream through 5 kb downstream of the TSS of the TFF1 gene. The distal enhancer region of TFF1 was found to be highly occupied by MLL1." Enhancer TFF1 -- "qRT-PCR,ChIP" "When FLAG-tagged MLL1-CX or MLL1-CXPB was over_x0002_expressed in MCF-7 cells by transient transfection, quan_x0002_titative ChIP assays performed with anti-FLAG antibody demonstrated the occupancy by both MLL1-CX and MLL1-CXPB on ERE3 (but not ERE2 or ERE1) of the TFF1 gene, indicating that the 40 kDa MLL1-CX fragment is sufficient for recruitment to the enhancer region in vivo (Figure 3F) with the same genomic binding specificity as wild-type MLL2." "BCEI,D21S21,HP1.A,HPS2,pNR-2,pS2" Mixed-Lineage Leukemia 1 -- -- Mixed-lineage leukemia 1 (MLL1) protein is a key determinant that maintains permissive chromatin structure of the TFF1 Enhancer region. "ChIP,NOMe-seq" "Mixed-lineage leukemia 1 (MLL1) protein is a key determinant that maintains permissive chromatin structure of the TFF1 enhancer region. MLL1 occupies the TFF1 enhancer region and methylates H3K4 before hormone stimulation. In vitro, MLL1 binds directly to the CpG-rich region of the TFF1 enhancer, and its binding is dependent on hypomethylation of DNA. Furthermore, the depletion of MLL1 in MCF-7 cells results in a dramatic decrease of chromatin accessibility and recruitment of FOXA1 and ERa to the enhancer element." KMT2A "ALL-1,CXXC7,HRX,HTRX1,MLL,MLL1,MLL1A,TRX1,WDSTS" "qPCR,ChIP" "FLAG-tagged MLL1-CX or MLL1-CXPB was transiently expressed in the MCF-7 cells, which were then grown in hormone-free media for 48 h and then treated with 100nM E2 for 1 h before performing the ChIP assay with anti-FLAG antibody. Precipitated DNA was analyzed by qPCR with primers representing the ER binding sites of the TFF1 genes." -- -- -- >2KB 9099 E_01_144 24288367 -- hg19 chr21 43771491 43773491 Human "MCF-7,MDA-MB-231,293T" Low throughput "qRT-PCR,ChIP" "TFF1 has three EREs, and ERE3 at -9.9 kb upstream of transcriptional start site (TSS) is the major ER binding site and considered to be the major enhancer element. Our chromatin immunoprecipitation (ChIP) assay showed that the enhancer region of the TFF1 gene (ERE3,-9.9 kb) was highly occupied by MLL1 and that the recruitment of MLL1 to this region was further enhanced after E2 treatment in MCF-7 cells (Figure 2A).We validated these findings by a ChIP scanning assay,which surveyed from _x0002_10 kb upstream through 5 kb downstream of the TSS of the TFF1 gene. The distal enhancer region of TFF1 was found to be highly occupied by MLL1." Enhancer TFF1 -- "qRT-PCR,ChIP" "When FLAG-tagged MLL1-CX or MLL1-CXPB was over_x0002_expressed in MCF-7 cells by transient transfection, quan_x0002_titative ChIP assays performed with anti-FLAG antibody demonstrated the occupancy by both MLL1-CX and MLL1-CXPB on ERE3 (but not ERE2 or ERE1) of the TFF1 gene, indicating that the 40 kDa MLL1-CX fragment is sufficient for recruitment to the enhancer region in vivo (Figure 3F) with the same genomic binding specificity as wild-type MLL3." "BCEI,D21S21,HP1.A,HPS2,pNR-2,pS2" Mixed-Lineage Leukemia 1 -- -- Mixed-lineage leukemia 1 (MLL1) protein is a key determinant that maintains permissive chromatin structure of the TFF1 Enhancer region. "ChIP,NOMe-seq" "Mixed-lineage leukemia 1 (MLL1) protein is a key determinant that maintains permissive chromatin structure of the TFF1 enhancer region. MLL1 occupies the TFF1 enhancer region and methylates H3K4 before hormone stimulation. In vitro, MLL1 binds directly to the CpG-rich region of the TFF1 enhancer, and its binding is dependent on hypomethylation of DNA. Furthermore, the depletion of MLL1 in MCF-7 cells results in a dramatic decrease of chromatin accessibility and recruitment of FOXA1 and ERa to the enhancer element." KMT2A "ALL-1,CXXC7,HRX,HTRX1,MLL,MLL1,MLL1A,TRX1,WDSTS" "qPCR,ChIP" "FLAG-tagged MLL1-CX or MLL1-CXPB was transiently expressed in the MCF-7 cells, which were then grown in hormone-free media for 48 h and then treated with 100nM E2 for 1 h before performing the ChIP assay with anti-FLAG antibody. Precipitated DNA was analyzed by qPCR with primers representing the ER binding sites of the TFF1 genes." -- -- -- >2KB 9899 E_01_145 24321386 -- hg19 chr17 72860066 72860183 Human "HeLa,H295R,KGN" Low throughput "ChIP,Luciferase Reporter Assay,PCR,EMSA" "Next, histone modifi- cation in the SF-1-induced cells was examined via ChIP assays, using antibodies against two active enhancer markers, histone H3K27ac and H3K4me2, to determine alterations of chromatin state within the region. The results clearly showed that the signals of these active enhancer markers around the SF-1 binding region increased along with the transduction of SF-1 (Fig. 1D and E), suggesting that the transduction of SF-1 led to the active state of chromatin within the FDXR intronic region." Enhancer FDXR -- "ChIP,Luciferase Reporter Assay,PCR" "Genome-wide analysis of differentiated cells, using a combination of DNA microarray and promoter tiling array analyses, showed that FDXR is a novel SF-1 target gene. In this study,the transcriptional regulatory mechanism of FDXR was examined in steroidogenic cells." "ADR,ADXR,ANOA" -- -- -- -- -- -- NR5A1 "AD4BP,ELP,FTZ1,FTZF1,POF7,SF-1,SF1,SPGF8,SRXX4,SRXY3,hSF-1" "ChIP,qPCR" "Closed and open squares represent Flag-tagged SF-1 (FlagSF-1)- and GFP (Control)-transduced MSCs, respectively; real-time PCR analysis of immunoprecipitated chromatin performed using primers for indicated genomic region; and expressed as a percentage of input." -- -- -- <2KB 1507 E_01_146 24332044 -- hg19 chr11 110716521 110832306 Human B-Cell Lymphoma Cell Lines Low+High throughput "ChIP-seq,ChIP-qPCR" "As predicted, BRD4 load is asymmetrically distributed throughout the genome at enhancer sites. Completely unexpected is the magnitude by which BRD4 load varies among active enhancer regions (Figure 4E). Only a small subset of BRD-loaded enhancers, 285/18330(1.6%), account for 32% of all of the BRD4 enhancer binding in the cell.The BRD4-loaded enhancers in the Ly1 DLBCL cell line are considerably larger than typical enhancer elements, resembling the super-enhancers we recently described with Richard Young." Super-Enhancer POU2AF1 -- ChIP-seq "Notably, the top two gene loci with BRD4-loaded enhancers, POU2AF1 (which encodes the OCA-B transcriptional co-activator protein) and BCL6, and additional genes with disproportionally BRD4-loaded enhancers such as PAX5 and IRF8 (Figure 4E), are essential for B-cell fate determination and germinal center formation." "BOB1,OBF-1,OBF1,OCAB" Diffuse Large B-Cell Lymphoma DOID:0050745 D016403 "These super-Enhancers prove particularly sensitive to bromodomain inhibition,explaining the selective effect of BET inhibitors on oncogenic and lineage-specific transcriptional circuits. " "ChIP-seq,ChIP-qPCR" "These super-enhancers prove particularly sensitive to bromodomain inhibition, explaining the selective effect of BET inhibitors on oncogenic and lineage-specific transcriptional circuits. Functional study of genes marked by super-enhancers identifies DLBCLs dependent on OCA-B and suggests a strategy for discovering unrecognized cancer dependencies." E2F1 "E2F-1,RBAP1,RBBP3,RBP3" "ChIP-seq,ChIP-qPCR" "Then, we assessed the genome-wide localization of E2F1 and the representative BET protein, BRD4, also by ChIP-Seq using the respective antibodies. Rank-ordering of all transcriptionally active promoters based on H3K4me3 enrichment and RNA Pol II occupancy identifies pervasive binding of BRD4 and E2F1 to active promoter elements." -- -- -- >2KB 448565 E_01_147 24332044 -- hg19 chr3 188892648 189232665 Human B-Cell Lymphoma Cell Lines Low+High throughput "ChIP-seq,ChIP-qPCR" "As predicted, BRD4 load is asymmetrically distributed throughout the genome at enhancer sites. Completely unexpected is the magnitude by which BRD4 load varies among active enhancer regions (Figure 4E). Only a small subset of BRD-loaded enhancers, 285/18330(1.6%), account for 32% of all of the BRD4 enhancer binding in the cell.The BRD4-loaded enhancers in the Ly1 DLBCL cell line are considerably larger than typical enhancer elements, resembling the super-enhancers we recently described with Richard Young." Enhancer BCL6 -- ChIP-seq "Notably, the top two gene loci with BRD4-loaded enhancers, POU2AF1 (which encodes the OCA-B transcriptional co-activator protein) and BCL6, and additional genes with disproportionally BRD4-loaded enhancers such as PAX5 and IRF8 (Figure 4E), are essential for B-cell fate determination and germinal center formation." "BCL5A,BCL6,LAZ3,ZBTB27,ZNF51" Diffuse Large B-Cell Lymphoma DOID:0050745 D016403 "These super-Enhancers prove particularly sensitive to bromodomain inhibition,explaining the selective effect of BET inhibitors on oncogenic and lineage-specific transcriptional circuits. " "ChIP-seq,ChIP-qPCR" "These super-enhancers prove particularly sensitive to bromodomain inhibition, explaining the selective effect of BET inhibitors on oncogenic and lineage-specific transcriptional circuits. Functional study of genes marked by super-enhancers identifies DLBCLs dependent on OCA-B and suggests a strategy for discovering unrecognized cancer dependencies." E2F1 "E2F-1,RBAP1,RBBP3,RBP3" "ChIP-seq,ChIP-qPCR" "Then, we assessed the genome-wide localization of E2F1 and the representative BET protein, BRD4, also by ChIP-Seq using the respective antibodies. Rank-ordering of all transcriptionally active promoters based on H3K4me3 enrichment and RNA Pol II occupancy identifies pervasive binding of BRD4 and E2F1 to active promoter elements." -- -- -- >2KB 1623493 E_01_148 24332857 -- hg19 chr1 226085596 226087967 Human Embryonic Stem Cell Low+High throughput "ChIP-seq,siRNA" "Importantly, the SMAD DNA binding partner FOXH1, a major specifier of ME, is found near TSO elements, and upon fate specification£¬we show that TSO is disrupted with subsequent SMAD-FOXH1 induction of ME. These studies define switch-enhancer elements and provide a framework to understand how cellular context dictates interpre_x0002_tation of the same morphogen signal in development." Enhancer LEFTY1 -- "ChIP-seq,PCR" "We first verified binding of TEAD4 and SMAD2 to the CTGF promoter and the LEFTY1 enhancer, respectively (Fig_x0002_ure S3B), and confirmed dramatic reduction in SMAD2 genome occupancy upon SB431542 treatment, as illustrated for the LEFTY1 locus (Figure S3B, lower panel). Inclusion of this control increased our ChIP-seq sensitivity, as reflected by more SMAD binding sites identified here when compared to others." "LEFTB,LEFTYB" -- -- -- -- -- -- "POU5F1,SOX2,NANOG" "OCT3,OCT4,OTF-3,OTF3,OTF4,Oct-3,Oct-4,ANOP3,MCOPS3,NANOG" "ChIP,PCR" "To verify the association of these transcription factors with the endogenous NANOG promoter,we performed ChIP." -- -- -- >2KB 12801 E_01_149 24362753 -- hg19 chr14 77264349 77265491 Human HEK-293 Low+High throughput "ChIP-seq,Luciferase Reporter Assay,PCR" "Using reporter constructs we identified potential promoter/enhancer elements between the transcription start site and 1142 bp upstream. Using 184 post mortem temporal lobe samples we replicated the reported negative effect of age, and after genotyping tagging SNPs we found one (rs981471) showing a significant correlation with the gene¡¯s expression and another(rs8014408) showing an interaction with age, the rare C allele being correlated with higher expression and faster decline." Enhancer NGB -- "ChIP-seq,Luciferase Reporter Assay,PCR" "Real time PCR quantification of the NGB transcript gave consistent amplification from cDNA with little variation within triplicates. It was clear however that the NGB transcript is at low abundance in the adult human temporal lobe, as the detection threshold (Ct) in our qPCR analysis was reached at cycle 26.5 compared to cycle 19 for the ACTB gene at the same cDNA input. This is an estimated 180-fold difference depending on amplification efficiency. " NGB Alzheimer's Disease DOID:10652 D000544 -- -- -- -- -- -- -- rs981471 77629017 Luciferase Reporter Assay >2KB 466913 E_01_150 24374176 -- hg19 chr15 37116298 37122452 Human Embryonic Stem Cell Low+High throughput "ChIP-qPCR,ChIP-seq,ChIP" "Based on these results, we identified sequence d as a MB Meis2 enhancer, and hereafter designate it as MBE. We went on to look for accumulation of histone H3K27 acetylation (H3K27ac) and H3K4 monomethylation (H3K4me1), which demarcate active and potential enhancers, respectively, at MBE by ChIP-seq." Enhancer MEIS2 -- "ChIP-qPCR,ChIP-seq" "In wild-type mice, Meis2 showed distinctive expres_x0002_sion patterns at 11.5 dpc in FB, MB, pharyngeal arches, spinal cord, neural crest, and somites. In Ring1 mutants, however,we found atypical derepression of Meis2 in facial and cephalic mesenchyme, heart primordium, LM, and somatic mesoderm,coupled with its downregulation in FB and MB (Figure 1C). These results indicate that PcG is involved in both repression and activation of Meis2 in a tissue-specific manner." "CPCMR,HsT18361,MRG1" -- -- -- -- -- -- RNF2 "BAP-1,BAP1,DING,HIPI3,RING1B,RING2" "ChIP-qPCR,ChIP-seq" "RING1B ChIP-seq data (GSE48464) over 10 Mb of the mouse genome (mm9) around Meis2 in FB,MB, and LM at 11.5 dpc.Binding of H3K27ac and RING1B in 11.5 dpc MB of WT and Ring1 mutant (Ring1 mut) revealed by ChIP-quantitative PCR (ChIP-qPCR) analysis." -- -- -- >2KB 63846 E_01_151 24510239 -- hg19 chr13 95865319 95866199 Human "Umbilical Vein Endothelial Cell,ESCC" Low throughput Luciferase Reporter Assay "Luciferase reporter gene assays verified that the ABCC4 intron 4 sequence, which was annotated as an enhancer element by ENCODE(chr13: 94,663,320-94,664,200), also exhibited enhancer activity in multiple cancer cell types.However,the resulting intensities were much higher in two ESCC cells, KYSE140 and EC0156£¬which suggests that the enhancer activity within this region is rela_x0002_tively specific to ESCC. " Enhancer ABCC4 -- Luciferase Reporter Assay "GRAIL analysis highlighted potential cancer-associated func_x0002_tions of the genes from the four CNV regions with frequencies >5%.ABCC4 had strong associations with transport regulation, immunol_x0002_ogy and multiple cancer-related signaling pathways.Furthermore, conditional logistic regression analysis showed that variations in ABCC4copy number were associated with ESCC after adjusting for age, sex and lifestyle." "MOAT-B,MOATB,MRP4" Esophageal Squamous Cell Carcinoma -- D000077277 -- -- -- CRE CRE Western blot Western blot analysis showed that ABCC4 was effectively depleted following siRNA transfection. -- -- -- >2KB 193677 E_01_152 24512546 -- hg19 chr8 128709135 128745673 Human "AN3 CA,RL95-2,HEC-1-B" Low+High throughput "ChIP,ChIP-PCR,ChIP-seq,qRT-PCR" "Interestingly, we showed that FOXA1 and AR more evidently bound to the MYC enhancer regions as compared to MYC promoter regions. These results could be attributed to other co-regulators involved in this binding process. Since TCF7L2, a protein mediating DNA looping for long-distance interactions of distal en_x0002_hancers and proximal promoters, physically interacts with FOXA1 and AR and mediates the transcription of MYC in breast cancer [19], future investigation will be needed to clarify which co-regulators are involved in FOXA1/AR binding to the enhancer regions upstream of MYC in EC cells." Enhancer MYC -- "ChIP,ChIP-PCR,qRT-PCR" "Our ChIP assays showed that both FOXA1 and AR could bind to all the five putative FOXA1-AR-binding regions in MFE-296 cells. More_x0002_over, both FOXA1 and AR bound most greatly to the Enh-1 (enhancer 1) region among the five binding regions (Figure 4D). Our ChIP data together with our co_x0002_immunoprecipitation data suggested that FOXA1 forming protein complex with AR might bind to FOXA1-AR over_x0002_lapping binding regions upstream of MYC, leading to MYC activation in EC cells." "MRTL,MYCC,bHLHe39,c-Myc" Endometrial Cancer DOID:1380 D016889 -- -- -- "FOXA1,AR" "HNF3A,TCF3A,AIS,AR8,DHTR,HUMARA,HYSP1,KD,NR3C4,SBMA,SMAX1,TFM" "ChIP,ChIP-PCR,ChIP-seq,qRT-PCR" "Our ChIP assays showed that both FOXA1 and AR could bind to all the five putative FOXA1-AR-binding regions in MFE-296 cells. More_x0002_over, both FOXA1 and AR bound most greatly to the Enh-1 (Enhancer 1) region among the five binding regions." -- -- -- >2KB 20910 E_01_153 24512546 -- hg19 chr8 128392788 128433173 Human "AN3 CA,RL95-2,HEC-1-B" Low+High throughput "ChIP,ChIP-PCR,ChIP-seq,qRT-PCR" "Interestingly, we showed that FOXA1 and AR more evidently bound to the MYC enhancer regions as compared to MYC promoter regions. These results could be attributed to other co-regulators involved in this binding process. Since TCF7L2, a protein mediating DNA looping for long-distance interactions of distal en_x0002_hancers and proximal promoters, physically interacts with FOXA1 and AR and mediates the transcription of MYC in breast cancer [19], future investigation will be needed to clarify which co-regulators are involved in FOXA1/AR binding to the enhancer regions upstream of MYC in EC cells." Enhancer MYC -- "ChIP,ChIP-PCR,qRT-PCR" "Our ChIP assays showed that both FOXA1 and AR could bind to all the five putative FOXA1-AR-binding regions in MFE-296 cells. More_x0002_over, both FOXA1 and AR bound most greatly to the Enh-1 (enhancer 1) region among the five binding regions (Figure 4D). Our ChIP data together with our co_x0002_immunoprecipitation data suggested that FOXA1 forming protein complex with AR might bind to FOXA1-AR over_x0002_lapping binding regions upstream of MYC, leading to MYC activation in EC cells." "MRTL,MYCC,bHLHe39,c-Myc" Endometrial Cancer DOID:1380 D016889 -- -- -- "FOXA1,AR" "HNF3A,TCF3A,AIS,AR8,DHTR,HUMARA,HYSP1,KD,NR3C4,SBMA,SMAX1,TFM" "ChIP,ChIP-PCR,ChIP-seq,qRT-PCR" "Our ChIP assays showed that both FOXA1 and AR could bind to all the five putative FOXA1-AR-binding regions in MFE-296 cells. More_x0002_over, both FOXA1 and AR bound most greatly to the Enh-1 (Enhancer 1) region among the five binding regions." -- -- -- >2KB 335333 E_01_154 24564208 -- hg19 chr4 74586223 74591223 Human AC16 Low+High throughput "RT-qPCR,ChIP-seq" "We have recently developed a computational approach for identifying functional enhancers based on these pat_x0002_terns of transcription in GRO-seq data.Using this approach, we identified 1,146 sites of paired intergenic eRNA production in AC16 cells.Metagene analyses of ChIP-seq data from adult human heart, fetal human heart, and human skeletal muscle myotubes(HSMM) for the 1,146 putative enhancers showed ex_x0002_pected patterns of enrichment for well characterized en_x0002_hancer features, such as p300, H3K4me1,and H3K27ac.Remarkably, the putative enhancers identi_x0002_fied in AC16 cells by GRO-seq match well with enhan_x0002_cer features in the ChIP-seq data from related,but distinct, cell types." Enhancer IL8 -- ChIP-seq "For example, at the proinflammatory gene IL8, we observed two sites of TNF¦Á-induced eRNA pro_x0002_duction about 15¨C20 kb upstream of the TSS (Figure 6A).Both sites showed TNF¦Á-dependent accumulation of GRO-seq and Pol II ChIP-seq signals, while only the more proximal of the two sites was bound by NF-¦ÊB.These enhancers and their associated eRNAs may play an essential role in TNF¦Á-dependent activation of the nearby IL8 gene." "GCP-1, GCP1, IL8, LECT, LUCT, LYNAP, MDNCF, MONAP, NAF, NAP-1, NAP1" Heart Disease DOID:114 D006331 -- -- -- -- -- -- -- -- -- -- >2KB 17500 E_01_155 24594601 -- hg19 chr8 128230172 128243000 Human "HCT 116,RKO" Low throughput "qRT-PCR,3C" "The enhancer region showed long-range in_x005f_x0002_teraction with MYC (?335 kb from the region),suggesting that the enhancer region could regulate CARLo-5 (?180 kb from the region) expression by enhancing its transcription through direct interaction. To test this possibility, we performed chro_x0002_mosome conformation capture (3C) analysis using genomic DNA from CRC-derived HCT116 and RKO cells.The results of 3C analysis show that the enhancer region physically interacts with CARLo-5." Enhancer CCAT1 3C qRT-PCR These results demonstrate that the enhancer region may regulate expression of CARLo-5 by direct interaction with the active regulatory 5¡ä promoter region of CARLo-5.These results suggest that the cancer-associated variant rs6983267 in MYC enhancer could regulate CARLo-5 expression through long-range interaction with the active regulatory region of its promoter. "CARLO5,CARLo-5,onco-lncRNA-40" Colorectal Cancer DOID:9256 D015179 "The MYC Enhancer region physically interacts with the active regulatory region of the CARLo-5 promoter, suggesting long-range interaction of MYC Enhancer with the CARLo-5 promoter regulates CARLo-5 expression." "3C,qRT-PCR,siRNA" "We also found the MYC enhancer region physically interacts with the active regulatory region of the CARLo-5 promoter, suggesting long-range interaction of MYC enhancer with the CARLo-5 promoter regulates CARLo-5 expression. Finally, we demonstrate that CARLo-5 has a function in cell-cycle regulation and tumor development." -- -- -- -- rs6983267 128413305 "qRT-PCR,3C" >2KB 16960 E_01_156 24662484 -- hg19 chr8 119677099 119681977 Human "HT-29,HCT 116" Low throughput "qRT-PCR,3C,ChIP" "The human 8q24 gene desert contains multiple enhancers that form tissue-specific long-range chromatin loops with the MYC oncogene, but how chromatin looping at the MYC locus is regulated remains poorly understood. Here we demonstrate that a long noncoding RNA (lncRNA), CCAT1-L, is transcribed specifically in human colorectal cancers from a locus 515 kb upstream of MYC. This lncRNA plays a role in MYC transcriptional regulation and promotes long-range chromatin looping. " Super-Enhancer MYC 3C "qRT-PCR,ChIP" "Importantly, we found that knock_x0002_down of CCAT1-L greatly reduced the transcription of nascent MYC RNA (Figure 2C), further suggesting that CCAT1-L regulates MYC expression at the transcriptional level. Nevertheless, while the reduction on the steady_x0002_state MYC mRNA that we observed was modest, it is possible that this at least partly reflects the known com_x0002_plexity of MYC regulation." "MRTL,MYCC,bHLHe39,c-Myc" Colorectal Cancer DOID:9256 D015179 The CCAT1-L locus is located within a strong super-enhancer and is spatially close to MYC "3C,RT-PCR" "Here we demonstrate that a long noncoding RNA (lncRNA), CCAT1-L, is transcribed specifically in human colorectal cancers from a locus 515 kb upstream of MYC. This lncRNA plays a role in MYC transcriptional regulation and promotes long-range chromatin looping. Importantly, the CCAT1-L locus is located within a strong super-enhancer and is spatially close to MYC. Knockdown of CCAT1-L reduced long-range interactions between the MYC promoter and its enhancers." "TCF4,CTCF" "E2-2,FECD3,ITF-2,ITF2,PTHS,SEF-2,SEF2,SEF2-1,SEF2-1A,MRD21SEF2-1B,SEF2-1D,TCF-4,bHLHb19" ChIP-seq Analyses of the available ChIP-seq datasets [39] re_x0002_vealed that TCF4 and CTCF are highly enriched in this 8q24 region in HCT116 cells (Figure 6A). Extensive TCF4 binding in the chromatin region between MYC-515 and MYC-335 was consistent with the notion that this chromatin region is a super-Enhancer that can recruit transcriptional factors. rs6983267 128413305 "qRT-PCR,3C" >2KB 9068776 E_01_157 24667089 -- hg19 chr8 141161900 141175436 Human Lymphoblastoid Cell Lines Low throughput "RT-PCR,ChIP,Luciferase Reporter Assay,3C,DNaseI-seq" "Using 3C, we identify mutually exclusive approximately 58 and 500 kb chromatin loops in adult frontal cortex between a novel brain-specific enhancer, marked by H3K4me1 and H3K27ac, with the KCNK9 and PEG13 promoters which we propose regulates brain-specific expression." Enhancer KCNK9 3C "RT-PCR,ChIP" "Utilizing allele-specific RT-PCR, bisulphite sequencing, chromatin immunoprecipitation and chromosome conformation capture (3C) we show the reciprocal expression of the novel, paternally expressed, PEG13 non-coding RNA and maternally expressed KCNK9 genes in brain, and the biallelic expression of flanking transcripts in a range of tissues." "K2p9.1,KT3.2,TASK-3,TASK3" Intellectual Disability DOID:1059 D008607 -- -- -- CTCF MRD21 3C-qPCR 3C-qPCR assays were performed on cerebellar samples and interaction frequencies were determined between a constant HindIII site located within the unmethylated CTCF-cohesin binding site within the KCNK9 promoter and other HindIII sites throughout the locus. We identified strong interactions between the KCNK9 promoter constant fragment with the PEG13-DMR and the CTCF-cohesin site in the enhancer region located within intron 17 of TRAPPC9. -- -- -- >2KB 555588 E_01_158 24667606 -- hg19 chr17 60983452 60984775 Human "HEK293FT,FLP-IN T-REX 293 CELLS" Low throughput "qRT-PCR,ChIP,Luciferase Reporter Assay" "Here we tested whether dTALEs can be used to target genes whose expression is controlled by Wnt/¦Â-catenin signaling.TALE DNA binding domains were engineered to recognize sequences adjacent to Wnt responsive enhancer elements (WREs) that control expression of axis inhibition protein 2 (AXIN2) and c-MYC (MYC).The TALE-SIDs repressed luciferase reporter activity, bound their genomic target sites, and repressed AXIN2 and MYC expression in HEK293 cells. We generated a novel HEK293 cell line to determine whether the TALE-SIDs could function downstream of oncogenic Wnt/¦Â-catenin signaling. " Enhancer AXIN2 -- "qRT-PCR,ChIP,Luciferase Reporter Assay" "The AXIN2 luciferase reporter plasmid was generated by PCR-amplifying a 787-bp fragment of the AXIN2 gene from genomic HCT116 DNA that includes the TALE binding sites. The PCR product was sub-cloned into the pGL3 basic vector as a KpnINheI fragment. To generate the pcDNA5/FRT/TO-¦Â-cateninS45F-estrogen receptor (ER) expression plasmid, ¦Â-cateninS45F cDNA was PCR-amplified from pcDNA3-¦Â-catenin." "AXIL,ODCRCS" Colorectal Cancer DOID:9256 D015179 -- -- -- SID SID "ChIP,qRT-PCR" "We conducted ChIP assays with FLAG antibodies in transiently transfected cells and found that both TALE1-SID and TALE2-SID displayed enriched binding to their target sites within AXIN2 relative to a control site that mapped within the 3¡¯untranslated region (Fig. 2A, B). A qRT-PCR analysis of transcripts found that cells transfected with both TALE-SIDs contained 5-fold lower levels of AXIN2 expression relative to control. " -- -- -- >2KB 2540566 E_01_159 24667606 -- hg19 chr17 60986269 60989232 Human "HEK293FT,FLP-IN T-REX 293 CELLS" Low throughput "qRT-PCR,ChIP,Luciferase Reporter Assay" "Here we tested whether dTALEs can be used to target genes whose expression is controlled by Wnt/¦Â-catenin signaling.TALE DNA binding domains were engineered to recognize sequences adjacent to Wnt responsive enhancer elements (WREs) that control expression of axis inhibition protein 2 (AXIN2) and c-MYC (MYC).The TALE-SIDs repressed luciferase reporter activity, bound their genomic target sites, and repressed AXIN2 and MYC expression in HEK293 cells. We generated a novel HEK293 cell line to determine whether the TALE-SIDs could function downstream of oncogenic Wnt/¦Â-catenin signaling. " Enhancer AXIN2 -- "qRT-PCR,ChIP,Luciferase Reporter Assay" "The AXIN2 luciferase reporter plasmid was generated by PCR-amplifying a 787-bp fragment of the AXIN2 gene from genomic HCT116 DNA that includes the TALE binding sites. The PCR product was sub-cloned into the pGL3 basic vector as a KpnINheI fragment. To generate the pcDNA5/FRT/TO-¦Â-cateninS45F-estrogen receptor (ER) expression plasmid, ¦Â-cateninS45F cDNA was PCR-amplified from pcDNA3-¦Â-catenin." "AXIL,ODCRCS" Colorectal Cancer DOID:9256 D015179 -- -- -- SID SID "ChIP,qRT-PCR" "The MYC luciferase transgene, which contains approximately 8.6-kb of genomic DNA that encompasses MYC, was generated stepwise with two PCR fragments amplified from a bacterial artificial chromosome (BAC) that harbors human MYC." -- -- -- >2KB 2536929 E_01_160 24667606 -- hg19 chr8 128816178 128817335 Human "HEK293FT,FLP-IN T-REX 293 CELLS" Low throughput "qRT-PCR,ChIP,Luciferase Reporter Assay" "Here we tested whether dTALEs can be used to target genes whose expression is controlled by Wnt/¦Â-catenin signaling.TALE DNA binding domains were engineered to recognize sequences adjacent to Wnt responsive enhancer elements (WREs) that control expression of axis inhibition protein 2 (AXIN2) and c-MYC (MYC).The TALE-SIDs repressed luciferase reporter activity, bound their genomic target sites, and repressed AXIN2 and MYC expression in HEK293 cells. We generated a novel HEK293 cell line to determine whether the TALE-SIDs could function downstream of oncogenic Wnt/¦Â-catenin signaling. " Enhancer MYC -- "qRT-PCR,ChIP,Luciferase Reporter Assay" "The MYC luciferase transgene, which contains approximately 8.6-kb of genomic DNA that encompasses MYC, was generated stepwise with two PCR fragments amplified from a bacterial artificial chromosome (BAC) that harbors human MYC. The firefly luciferase gene was inserted in-frame into the second exon of MYC. " "MRTL,MYCC,bHLHe39,c-Myc" Colorectal Cancer DOID:9256 D015179 -- -- -- SID SID "ChIP,qRT-PCR" "using ChIP assays and qRT-PCR analysis, we found that TALE3-SID bound its target sequence and repressed MYC transcription in transiently transfected HEK293 cells." -- -- -- >2KB 68443 E_01_161 24671955 -- hg19 chr5 139970046 139972237 Human Peripheral Blood Monocytes Low+High throughput "RT-PCR,ChIP,Luciferase Reporter Assay,ChIP-seq" "To extend the analysis of regulatory sites to putative enhancers, we also carried out ChIP sequencing for 2 histone marks, namely H3K4me1 and H3K27ac,that were previously associated with enhancers.30-34 H3K27 is a major substrate for the coactivators p300 and CBP and its acetylation marks active enhancers, whereas H3K4me1 is generally associated with distal regulatory elements, including poised enhancers." Enhancer CD14 -- "RT-PCR,Luciferase Reporter Assay" "On this basis, we sought an explanation for the differential ex_x0002_pression of CD14 in classical monocytes. Studies in transgenic mice by Zhang and coworkers established that a region of 80 kb surrounding the human CD14 gene is sufficient to direct its monocyte specific expression,42 whereas smaller constructs recapitulated human liver expression but failed to direct monocyte-specific expression.The genomic interval downstream of the CD14 gene contained a number of H3K27ac-marked sites specific for classical,CD14-expressing monocytes.These sites frequently overlapped with bidirectional enhancers identified using the FANTOM5 expression atlas44 as well as binding sites for PU.1 or C/EBPb in total monocytes,18 which are key factors in establishing distal regulatory sites in these cells." CD14 -- -- -- -- -- -- -- -- -- -- -- -- -- >2KB 40170 E_01_162 24671955 -- hg19 chr5 139974521 139975981 Human Peripheral Blood Monocytes Low+High throughput "RT-PCR,ChIP,Luciferase Reporter Assay,ChIP-seq" "To extend the analysis of regulatory sites to putative enhancers, we also carried out ChIP sequencing for 2 histone marks, namely H3K4me1 and H3K27ac,that were previously associated with enhancers.30-34 H3K27 is a major substrate for the coactivators p300 and CBP and its acetylation marks active enhancers, whereas H3K4me1 is generally associated with distal regulatory elements, including poised enhancers." Enhancer CD14 -- "RT-PCR,Luciferase Reporter Assay" "On this basis, we sought an explanation for the differential ex_x0002_pression of CD14 in classical monocytes. Studies in transgenic mice by Zhang and coworkers established that a region of 80 kb surrounding the human CD14 gene is sufficient to direct its monocyte specific expression,42 whereas smaller constructs recapitulated human liver expression but failed to direct monocyte-specific expression.The genomic interval downstream of the CD14 gene contained a number of H3K27ac-marked sites specific for classical,CD14-expressing monocytes.These sites frequently overlapped with bidirectional enhancers identified using the FANTOM5 expression atlas44 as well as binding sites for PU.1 or C/EBPb in total monocytes,18 which are key factors in establishing distal regulatory sites in these cells." CD14 -- -- -- -- -- -- -- -- -- -- -- -- -- >2KB 36061 E_01_163 24671955 -- hg19 chr5 139985479 139987671 Human Peripheral Blood Monocytes Low+High throughput "RT-PCR,ChIP,Luciferase Reporter Assay,ChIP-seq" "To extend the analysis of regulatory sites to putative enhancers, we also carried out ChIP sequencing for 2 histone marks, namely H3K4me1 and H3K27ac,that were previously associated with enhancers.30-34 H3K27 is a major substrate for the coactivators p300 and CBP and its acetylation marks active enhancers, whereas H3K4me1 is generally associated with distal regulatory elements, including poised enhancers." Enhancer CD14 -- "RT-PCR,Luciferase Reporter Assay" "On this basis, we sought an explanation for the differential ex_x0002_pression of CD14 in classical monocytes. Studies in transgenic mice by Zhang and coworkers established that a region of 80 kb surrounding the human CD14 gene is sufficient to direct its monocyte specific expression,42 whereas smaller constructs recapitulated human liver expression but failed to direct monocyte-specific expression.The genomic interval downstream of the CD14 gene contained a number of H3K27ac-marked sites specific for classical,CD14-expressing monocytes.These sites frequently overlapped with bidirectional enhancers identified using the FANTOM5 expression atlas44 as well as binding sites for PU.1 or C/EBPb in total monocytes,18 which are key factors in establishing distal regulatory sites in these cells." CD14 -- -- -- -- -- -- -- -- -- -- -- -- -- >2KB 24737 E_01_164 24671955 -- hg19 chr5 139987854 139989680 Human Peripheral Blood Monocytes Low+High throughput "RT-PCR,ChIP,Luciferase Reporter Assay,ChIP-seq" "To extend the analysis of regulatory sites to putative enhancers, we also carried out ChIP sequencing for 2 histone marks, namely H3K4me1 and H3K27ac,that were previously associated with enhancers.30-34 H3K27 is a major substrate for the coactivators p300 and CBP and its acetylation marks active enhancers, whereas H3K4me1 is generally associated with distal regulatory elements, including poised enhancers." Enhancer CD14 -- "RT-PCR,Luciferase Reporter Assay" "On this basis, we sought an explanation for the differential ex_x0002_pression of CD14 in classical monocytes. Studies in transgenic mice by Zhang and coworkers established that a region of 80 kb surrounding the human CD14 gene is sufficient to direct its monocyte specific expression,42 whereas smaller constructs recapitulated human liver expression but failed to direct monocyte-specific expression.The genomic interval downstream of the CD14 gene contained a number of H3K27ac-marked sites specific for classical,CD14-expressing monocytes.These sites frequently overlapped with bidirectional enhancers identified using the FANTOM5 expression atlas44 as well as binding sites for PU.1 or C/EBPb in total monocytes,18 which are key factors in establishing distal regulatory sites in these cells." CD14 -- -- -- -- -- -- -- -- -- -- -- -- -- >2KB 22545 E_01_165 24671955 -- hg19 chr5 139990822 139992831 Human Peripheral Blood Monocytes Low+High throughput "RT-PCR,ChIP,Luciferase Reporter Assay,ChIP-seq" "To extend the analysis of regulatory sites to putative enhancers, we also carried out ChIP sequencing for 2 histone marks, namely H3K4me1 and H3K27ac,that were previously associated with enhancers.30-34 H3K27 is a major substrate for the coactivators p300 and CBP and its acetylation marks active enhancers, whereas H3K4me1 is generally associated with distal regulatory elements, including poised enhancers." Enhancer CD14 -- "RT-PCR,Luciferase Reporter Assay" "On this basis, we sought an explanation for the differential ex_x0002_pression of CD14 in classical monocytes. Studies in transgenic mice by Zhang and coworkers established that a region of 80 kb surrounding the human CD14 gene is sufficient to direct its monocyte specific expression,42 whereas smaller constructs recapitulated human liver expression but failed to direct monocyte-specific expression.The genomic interval downstream of the CD14 gene contained a number of H3K27ac-marked sites specific for classical,CD14-expressing monocytes.These sites frequently overlapped with bidirectional enhancers identified using the FANTOM5 expression atlas44 as well as binding sites for PU.1 or C/EBPb in total monocytes,18 which are key factors in establishing distal regulatory sites in these cells." CD14 -- -- -- -- -- -- -- -- -- -- -- -- -- >2KB 19485 E_01_166 24671955 -- hg19 chr5 140009726 140011735 Human Peripheral Blood Monocytes Low+High throughput "RT-PCR,ChIP,Luciferase Reporter Assay,ChIP-seq" "To extend the analysis of regulatory sites to putative enhancers, we also carried out ChIP sequencing for 2 histone marks, namely H3K4me1 and H3K27ac,that were previously associated with enhancers.30-34 H3K27 is a major substrate for the coactivators p300 and CBP and its acetylation marks active enhancers, whereas H3K4me1 is generally associated with distal regulatory elements, including poised enhancers." Enhancer CD14 -- "RT-PCR,Luciferase Reporter Assay" "On this basis, we sought an explanation for the differential ex_x0002_pression of CD14 in classical monocytes. Studies in transgenic mice by Zhang and coworkers established that a region of 80 kb surrounding the human CD14 gene is sufficient to direct its monocyte specific expression,42 whereas smaller constructs recapitulated human liver expression but failed to direct monocyte-specific expression.The genomic interval downstream of the CD14 gene contained a number of H3K27ac-marked sites specific for classical,CD14-expressing monocytes.These sites frequently overlapped with bidirectional enhancers identified using the FANTOM5 expression atlas44 as well as binding sites for PU.1 or C/EBPb in total monocytes,18 which are key factors in establishing distal regulatory sites in these cells." CD14 -- -- -- -- -- -- -- -- -- -- -- -- -- <2KB 581 E_01_167 24703844 -- hg19 chr1 9996966 9997243 Human "GM12878,CD4+ T Cell" Low+High throughput "Hi-C,4C,Luciferase Reporter Assay,ChIP-seq" "By hierarchical clustering of the histone modification profiles,Alu elements, but not the other three transposons, cluster together with RefSeq genes, putative enhancers,and DNase I hypersensitive sites (DHSs).Although we removed TSS -10 and +5 kb regions from DHS,the DHS regions may still contain not only enhancers but also some promoters,as shown by the high level of H3K4me3,a modification exclusively localized to promoters. H3K4me3 is excluded on all four transposons,consistent with their underrepresentation at promoter regions." Enhancer LZIC -- Luciferase Reporter Assay "Two examples of Alus with specific P300, H3K27ac, and H3K4me1 peaks in one tissue but not in other tissues are shown in Figures 2D and 2E. Because P300, H3K27ac, and H3K4me1 peaks do not coexist on other Alus and control re_x0002_peats in the same region, the high active modification levels are unlikely to be conferred by genomic background." LZIC -- -- -- -- -- -- -- -- -- -- -- -- -- >2KB 14935 E_01_168 24703844 -- hg19 chr3 11249688 11250280 Human "GM12878,CD4+ T Cell" Low+High throughput "Hi-C,4C,Luciferase Reporter Assay,ChIP-seq" "By hierarchical clustering of the histone modification profiles,Alu elements, but not the other three transposons, cluster together with RefSeq genes, putative enhancers,and DNase I hypersensitive sites (DHSs).Although we removed TSS -10 and +5 kb regions from DHS,the DHS regions may still contain not only enhancers but also some promoters,as shown by the high level of H3K4me3,a modification exclusively localized to promoters. H3K4me3 is excluded on all four transposons,consistent with their underrepresentation at promoter regions." Enhancer HRH1 -- Luciferase Reporter Assay "Two examples of Alus with specific P300, H3K27ac, and H3K4me1 peaks in one tissue but not in other tissues are shown in Figures 2D and 2E. Because P300, H3K27ac, and H3K4me1 peaks do not coexist on other Alus and control re_x0002_peats in the same region, the high active modification levels are unlikely to be conferred by genomic background." "H1-R,H1R,HH1R,hisH1" -- -- -- -- -- -- -- -- -- -- -- -- -- >2KB 71206 E_01_169 24703906 -- hg19 chr3 184276287 184278287 Human "Crc Cell,HT-29" Low throughput "Luciferase Reporter Assay,ChIP,qRT-PCR" "Importantly, the stimulatory capacity of the ?2.3-kb ECR differed widely among the CRC cell lines and was most pronounced in LS174T cells with highest levels of endogenous EPHB3. Thus, the EPHB3 ?2.3-kb ECR functions as a cell type-specific transcriptional enhancer. Consistent with its evolutionary conservation, also the corresponding region from the mouse EphB3 gene has enhancer properties and exhibits a remarkably similar cell-type specificity in CRC cell lines.We probed these features by chromatin immunoprecipitation (ChIP) and formaldehyde_x0002_assisted isolation of regulatory elements (FAIRE)." Enhancer EPHB3 -- "ChIP,qRT-PCR" "Next, we performed ChIP experiments to investigate the distri_x0002_bution of the histone modifications H3K4me1 and H3K27ac,the acetyltransferase p300, and the Wnt pathway effector TCF7L2 at the EPHB3 locus.Significantly,its cell type-specific activity matches the expression of the endogenous EPHB3 gene in CRC cell lines, suggesting that differences in enhancer function underlie differential EPHB3 expression in CRC." "EK2,ETK2,HEK2,TYRO6" -- -- -- -- -- -- ASCL2 "ASH2,HASH2,MASH2,bHLHa45" Luciferase Reporter Assay we generated luciferase-reporter constructs with point mutations in the TBE and the binding sites for RBPJ and ETS factors. -- -- -- >2KB 2299 E_01_170 24778216 -- hg19 chr19 51358995 51369041 Human "LNCaP,VCaP" Low throughput "Luciferase Reporter Assay,RT-PCR,ChIP,3C" "Because the induction of eRNA is strongly correlated with the enhancer looping to target gene promoters£¬it is possible that the KLK3 enhancer element is able to interact with the KLK2 promoter. To address this, we used the chromosome conformation capture (3C) assay to study the spatial interaction of the KLK3/2 loci. It should be noted that the KLK2 promoter contains an ARE I that shares over 80% sequence homology with the ARE I in the KLK3 promoter." Enhancer KLK3 3C RT-PCR "In fact, the transcription of KLK3 eRNA (KLK3e) was demon_x0002_strated by GRO-seq data (10) and confirmed by our reverse transcription¨Cquantitative PCR (RT-qPCR) analyses in andro_x0002_gen-dependent LNCaP and VCaP, and androgen-independent LNCaP-abl cells (21)." "APS,KLK2A1,PSA,hK3" Prostate Cancer DOID:10283 D011471 KLK3e carries the core enhancer element derived from the androgen response element III (ARE III).KLK3e facilitates the spatial interaction of the KLK3 Enhancer and the KLK2 promoter and enhances long-distance KLK2 transcriptional activation. "RT-PCR,3C,RIP,3C-qPCR" "Kallikrein-related peptidase 3 (KLK3), which codes for prostate-specific antigen (PSA), is a well-known AR-regulated gene and its upstream enhancers produce bi- directional enhancer RNAs (eRNAs), termed KLK3e. Here, we demonstrate that KLK3e facilitates the spatial interaction of the KLK3 enhancer and the KLK2 promoter and enhances long-distance KLK2 transcriptional activation. KLK3e carries the core enhancer element derived from the androgen response element III (ARE III), which is required for the interaction of AR and Mediator 1 (Med1). Furthermore, we show that KLK3e processes RNA-dependent enhancer activity depending on the integrity of core enhancer elements. " AR "AIS8,AR,DHTR,HUMARA,HYSP1,KD,NR3C4,SBMA,SMAX1,TFM" RT-PCR "The effect of siKLK3e on AR-regulated gene expression was determined by RT-qPCR.our data show that KLK3e selectively regulates the expression of AR-regulated genes that do not reside on the same chromosome,implying a transregulatory function of KLK3e on AR dependent gene expression." -- -- -- >2KB 5848 E_01_171 24788237 -- hg19 chr12 54357811 54358445 Human "KYSE-30,KESY-150,ESCC" Low throughput "Luciferase Reporter Assay,PCR" "Because the ESCC susceptibility SNP rs920778 is located within the HOTAIR intron 2 and previous H3K4me1 and H3K4me3 modi_x0002_fication data suggest that there might be a potential enhancer in this intron,we therefore examined the enhancer activity of this region by a set of luciferase reporter gene constructs in human ESCC KYSE30 and KYSE150 cells These results indicated that there may be a negative regulatory element between +1463bp and +1718bpfrom the transcriptional start site and the core region of this intronic enhancer might exist between +1719bp and +2353bp from the tran_x0002_scriptional start?site." Enhancer HOTAIR -- "Luciferase Reporter Assay,PCR" "We hypothesized that the functional single nucleotide polymorphisms (SNP) in HOTAIR may affect HOTAIR expression and/or its function and, thus, ESCC risk. Therefore, we examined the association between three haplotype_x0002_tagging SNPs (htSNP) across the whole HOTAIR locus and ESCC risk as well as the functional relevance of an ESCC susceptibility SNP rs920778." "HOXAS,HOXC-AS4,HOXC11-AS1,NCRNA00072" Esophageal Squamous Cell Carcinoma -- D000077277 The identification of an ESCC susceptibility SNP rs920778 that regulates the expression of lncRNA HOTAIR via a novel intronic Enhancer. "ChIP,Luciferase Reporter Assay,PCR" "Interestingly, the ESCC susceptibility SNP rs920778 in this Enhancer has a genotype-specific effect on lncRNA HOTAIR expression. Our observations also support the hypothesis that functional genetic variants influencing lncRNA regulation may explain a part of ESCC genetic basis." -- -- -- -- rs920778 54360232 "Luciferase Reporter Assay,PCR" >2KB 2036 E_01_172 24797517 -- hg19 chr6 36632237 36634237 Human Human Diploid BJ Fibroblast Cell Lines Low+High throughput "ChIP-qPCR,ChIP,ChIP-seq" "On the other hand,only 4% are positive for H3K4me3, indicating that these sites are likely to represent regulatory enhancer elements.Furthermore, we validated by ChIP-qPCR that p53 and JMJD3 display an IR responsive recruitment to several putative enhancer elements.We confirmed that these sites possess features of active enhancers; they are DNase I positive£¬show binding of p300 and have enrichment of H3K4me1 and H3K27ac but not H3K4me3 and H3K27me3.We also observed that exposure to IR correlated with an increased binding of p300 and subsequently acetylation of H3K27,which is in agreement with data demonstrating that p53 interacts with p300." Enhancer CDKN1A -- ChIP-qPCR "Examples of p53 (before and after IR treatment) and JMJD3 (before and after IR treatment), H3K4me3 and DNase I-seq tracks at two putative enhancer elements located 11 kb upstream of CDKN1A (upper panel) or 19 kb upstream of GML (lower panel).Corresponding ChIP-qPCR validations of the binding of p53, JMJD3 and p300 as well as the levels of histone modifications H3K4me1, H3K4me3, H3K27ac and H3K27me3 at the two distal binding sites listed above." "CAP20,CDKN1,CIP1,MDA-6,P21,SDI1,WAF1,p21CIP1" -- -- -- -- -- -- TP53 "BCC7,BMFS5,LFS1,P53,TRP53" ChIP-seq "To understand if JMJD3 and p53 also display genomic co_x0002_localization, we performed global mapping of JMJD3 and p53 binding sites in telomerase-immortalized human BJ diploid fibroblasts by chromatin immunoprecipitation followed by se_x0002_quencing (ChIP-seq)." -- -- -- >2KB 10999 E_01_173 24797517 -- hg19 chr8 143896217 143898217 Human Human Diploid BJ Fibroblast Cell Lines Low+High throughput "ChIP-qPCR,ChIP,ChIP-seq" "On the other hand,only 4% are positive for H3K4me3, indicating that these sites are likely to represent regulatory enhancer elements.Furthermore, we validated by ChIP-qPCR that p53 and JMJD3 display an IR responsive recruitment to several putative enhancer elements.We confirmed that these sites possess features of active enhancers; they are DNase I positive£¬show binding of p300 and have enrichment of H3K4me1 and H3K27ac but not H3K4me3 and H3K27me3.We also observed that exposure to IR correlated with an increased binding of p300 and subsequently acetylation of H3K27,which is in agreement with data demonstrating that p53 interacts with p300." Enhancer GML -- ChIP-qPCR "Examples of p53 (before and after IR treatment) and JMJD3 (before and after IR treatment), H3K4me3 and DNase I-seq tracks at two putative enhancer elements located 11 kb upstream of CDKN1A (upper panel) or 19 kb upstream of GML (lower panel).Corresponding ChIP-qPCR validations of the binding of p53, JMJD3 and p300 as well as the levels of histone modifications H3K4me1, H3K4me3, H3K27ac and H3K27me3 at the two distal binding sites listed above." LY6D -- -- -- -- -- -- TP53 "BCC7,BMFS5,LFS1,P53,TRP53" ChIP-seq "To understand if JMJD3 and p53 also display genomic co_x0002_localization, we performed global mapping of JMJD3 and p53 binding sites in telomerase-immortalized human BJ diploid fibroblasts by chromatin immunoprecipitation followed by se_x0002_quencing (ChIP-seq)." -- -- -- >2KB 18999 E_01_174 24842713 -- hg19 chr16 86212040 86271919 Human Lung Low+High throughput "ChIP-seq,qRT-PCR,ChIP-qPCR" "Using chromatin immunoprecipitation with high-throughput sequencing (ChIP-seq) of histone H3K27 acetylation (H3K27ac), a mark of active enhancers,we first identified enhancer regions in MYCN-amplified Kelly and nonamplified SH-SY5Y cells." Super-Enhancer FOXF1 -- shRNA "Here we investigate whether inhibition of transcriptional CDKs can be exploited to disrupt aberrant MYC-driven transcription, using the deregulated expression of MYCN as a model. The MYCN protein shares most of the physical properties of MYC and is considered functionally interchangeable, based on the similarity of their transcriptional programs, the cellular phenotypes they induce, and the ability of MYCN to replace MYC during murine development." "ACDMPV,FKHL5,FREAC1" -- -- -- -- -- -- "MITF,YY1" "CMM8,COMMAD,MI,WS2,WS2A,bHLHe32,DELTA,GADEVS,INO80S,NF-E1,UCRBP,YIN-YANG-1" ChIP-qPCR "Binding of RNA Polymerase II, similarly tested with ChIP-qPCR in LP89, is relatively high at the alternative promoter and region B¡ä¡ä, which has several features consistent with an enhancer element, e.g. high H3K27Ac enrichment low H3K4Me3 enrichment and binding of MITF and YY1." -- -- -- >2KB 302152 E_01_175 24916375 -- hg19 chr9 124638991 124641160 Human LP89 Low+High throughput "ChIP-seq,ChIP-qPCR" "We profiled the chromatin structure of the BNC2 region to identify potential regulatory elements using chromatin immune-precipitation (ChIP) of H3K27Ac (an active-enhancer mark) in combination with next generation sequencing (ChIP-seq) and ENCODE data. We provide evidence that a region upstream from the canonical BNC2 promoter acts as an enhancer regulating the expression of BNC2, and this transcriptional regulation is dependent on the allelic status of an LD partner of the pigmentation-associated SNP rs10756819." Enhancer BNC2 -- "ChIP-seq,RT-qPCR" "We confirmed this finding by RT¨CqPCR analysis ofBNC2-expression levels atthree different loca_x0002_tions within the gene, using one primer set targeting mRNA upstream of the alternative promoter, and two primer sets targeting the mRNA downstream of the alternative promoter. " BSN2 -- -- -- -- -- -- -- -- -- -- -- -- -- >2KB 108230576 E_01_176 24997565 -- hg19 chr3 32993199 32996133 Human T Cell Low+High throughput "ChIP-seq,PCR" "We performed 120 high-throughput whole-genome ChIP-seq assays, which generated ~2.8 ¡Á 109 reads covering~1 ¡Á 1011 bases across three cell types (Supplementary Table 1), and computationally analyzed this data set to identify enhancers associated with CD4+ memory T cell differentiation as well as asthma." Enhancer CCR4 -- ChIP-seq "We found clear evidence of cell type¨Cspecific cis_x0002_regulatory DNA elements in noncoding sequences: for instance, compared to naive T cells,the TH2 cell subset displayed extensive enrichment of H3K4me2 at promoters and cis_x0002_regulatory regions in the extended CCR4 and CCR6 loci." "CC-CKR-4,CD194,CKR4,CMKBR4,ChemR13,HGCN:14099,K5-5" Asthma DOID:2841 D001249 -- -- -- -- -- -- -- -- -- -- <2KB 1601 E_01_177 24997565 -- hg19 chr3 33000399 33003599 Human T Cell Low+High throughput "ChIP-seq,PCR" "We performed 120 high-throughput whole-genome ChIP-seq assays, which generated ~2.8 ¡Á 109 reads covering~1 ¡Á 1011 bases across three cell types (Supplementary Table 1), and computationally analyzed this data set to identify enhancers associated with CD4+ memory T cell differentiation as well as asthma." Enhancer CCR4 -- ChIP-seq "We found clear evidence of cell type¨Cspecific cis_x0002_regulatory DNA elements in noncoding sequences: for instance, compared to naive T cells,the TH2 cell subset displayed extensive enrichment of H3K4me2 at promoters and cis_x0002_regulatory regions in the extended CCR4 and CCR6 loci." "CC-CKR-4,CD194,CKR4,CMKBR4,ChemR13,HGCN:14099,K5-5" Asthma DOID:2841 D001249 -- -- -- -- -- -- -- -- -- -- >2KB 8934 E_01_178 24997565 -- hg19 chr3 34181595 34184395 Human T Cell Low+High throughput "ChIP-seq,PCR" "We performed 120 high-throughput whole-genome ChIP-seq assays, which generated ~2.8 ¡Á 109 reads covering~1 ¡Á 1011 bases across three cell types (Supplementary Table 1), and computationally analyzed this data set to identify enhancers associated with CD4+ memory T cell differentiation as well as asthma." Enhancer CCR4 -- ChIP-seq "We found clear evidence of cell type¨Cspecific cis_x0002_regulatory DNA elements in noncoding sequences: for instance, compared to naive T cells,the TH2 cell subset displayed extensive enrichment of H3K4me2 at promoters and cis_x0002_regulatory regions in the extended CCR4 and CCR6 loci." "CC-CKR-4,CD194,CKR4,CMKBR4,ChemR13,HGCN:14099,K5-5" Asthma DOID:2841 D001249 -- -- -- -- -- -- -- -- -- -- >2KB 1189930 E_01_179 24997565 -- hg19 chr3 34201595 34204662 Human T Cell Low+High throughput "ChIP-seq,PCR" "We performed 120 high-throughput whole-genome ChIP-seq assays, which generated ~2.8 ¡Á 109 reads covering~1 ¡Á 1011 bases across three cell types (Supplementary Table 1), and computationally analyzed this data set to identify enhancers associated with CD4+ memory T cell differentiation as well as asthma." Enhancer CCR4 -- ChIP-seq "We found clear evidence of cell type¨Cspecific cis_x0002_regulatory DNA elements in noncoding sequences: for instance, compared to naive T cells,the TH2 cell subset displayed extensive enrichment of H3K4me2 at promoters and cis_x0002_regulatory regions in the extended CCR4 and CCR6 loci." "CC-CKR-4,CD194,CKR4,CMKBR4,ChemR13,HGCN:14099,K5-5" Asthma DOID:2841 D001249 -- -- -- -- -- -- -- -- -- -- >2KB 1210064 E_01_180 25081415 -- hg19 chr12 48368875 48369590 Human "10T1/2,MC615 Cells,MCF-7" Low throughput "RT-PCR,ChIP,Luciferase Reporter Assay,EMSA" "Thus, several binding sites of the intronic enhancer have been shown to interact with transcription factors, such as Sox-9,Sox-6, and L-Sox-5, acting cooperatively and consequently activating expression of COL2A1 in 10T1/2 and MC615 cells.Therefore, in order to validate our in vitro hypoth_x0002_esis and to determine whether these transcription factors were effectively able to bind in vivo to Col2a1-specific enhancer,we performed ChIP assays in primary chondrocytes using primers mapping the +2,383/+2,611-bp region of COL2A1 first intron. This region includes a Sox-9 cis-element (+2,384/+2,404 bp), one HMG binding site (+2,388/+2,394 bp), and one Sp1/Sp3 binding sequence (+2,440/+2,485 bp) but does not harbor ERE cis-sequence." Enhancer COL2A1 -- "ChIP,EMSA" "Altogether, these EMSA assays indicate that 17¦Â-E2 and hER¦Á66 enhance transcription of COL2A1 by increasing the binding activity of Sp1 and to a lesser extent of Sp3, not only to COL2A1 promoter but also to COL2A1-specific enhancer." "ANFH,AOM,COL11A3,SEDC,STL1" Osteoarthritis DOID:8398 D010003 "Several binding sites of the intronic Enhancer have been shown to interact with transcription factors, such as Sox-9,Sox-6,and L-Sox-5, acting cooperatively and consequently activating expression of COL2A1 in 10T1/2 and MC615 cells." "ChIP,EMSA" "By contrast to transient transfection data obtained with reporter construct pGL2-3.316 kb, these last results suggest that Sox-9/Sox-6/L-Sox-5, as well as the COL2A1 enhancer region, might also participate in the anabolic effect of 17¦Â-E2 on COL2A1 in primary and dedifferentiated chondrocytes since the hormone significantly increases their DNA-binding activity to this region." "SP1,SP3,SOX9,SOX6" "SP1,SPR2,CMD1,CMPD1,SRA1,SRXX2,SRXY10,HSSOX6,SOXD" "ChIP,RT-PCR,EMSA" "To identify what protein partners could be involved in the ER¦Á66 recruitment, we performed ReChIP experiments using an anti-ER¦Á antibody. Our data showed that ER¦Á66 was bound indirectly to Col2a1 Enhancer through protein¨Cprotein interactions with Sp1, Sp3, and Sox-9 transcription factors in primary as well as dedifferentiated articular chondrocytes." -- -- -- Intron 2486 E_01_181 25194570 -- hg19 chr8 130179643 130181490 Human "HPB-ALL,JURKAT,T-acute lymphoblastic leukemia (T-ALL)" Low+High throughput "ChIP-seq,ChIP,Luciferase Reporter Assay,3C,PCR" "This highly conserved regulatory element, hereby named N-Me for NOTCH MYC enhancer, is located within a broad super-enhancer region +1.47 Mb from the MYC transcription initiating site,interacts with the MYC proximal promoter and induces orientation-independent MYC expression in reporter assays.Moreover, luciferase reporter assays showed strong, orientationindependent activation of reporter constructs containing this enhancer in association with a ?2.5 kb MYC proximal promoter21 in JURKAT T-ALL cells,which express high levels of constitutively active NOTCH1 protein 22, but not in Daudi and Raji B-cell lineage cells." Enhancer MYC -- "Luciferase Reporter Assay,PCR" "Consistent with this hypothesis, chromatin configuration 3C analysis of the MYC locus demonstrated the association of this enhancer with proximal regulatory sequences in the MYC promoter.Moreover, histological analysis of multiple tissues including those in which NOTCH signaling plays important developmental roles such as breast epithelium, skin, and intestine showed no alterations (Supplementary Fig. 7) and RT-PCR analysis showed no effects of N_x0002_Me deletion in Myc expression." "MRTL,MYCC,bHLHe39,c-Myc" T-Cell Acute Lymphoblastic Leukemia DOID:5602 D054218 -- -- -- NOTCH1 "AOS5,AOVD1,TAN1,hN1" "ChIP,ChIP-seq" "Interestingly, chromatin immunoprecipitation (ChIP) followed by next generation sequencing (ChIP-seq) analysis of NOTCH1 chromatin binding sites in HPB-ALL T-ALL cells revealed a prominent 1 kb NOTCH1 peak in chromosome 8q24 located within the common 40 Kb segment duplicated in all these eight leukemia cases. " -- -- -- >2KB 1432253 E_01_182 25214635 -- hg19 chr10 6064657 6065397 Human T Cell Low throughput "EMSA,Luciferase Reporter Assay" "We first tested for enhancer activity by introducing ~200 bp fragments centered around each SNP (Fig. 6A) into a luciferase reporter vector, driven by SV40 or IL2RA minimal promoters, and transfecting them into Jurkat T cells.The higher enhancer function of the T allele was dependent on activation of the Jurkat cells (Fig. 6C), suggesting that enhancer activity is boosted upon cell activation by interactions affected by the single base change at rs12251836." Enhancer IL2RA -- GWAS "Together, these results point to functional effects of at least two independent associations in the IL2RA locus, which may have balancing effects. We surmise that the associated variant rs12251836 is mainly active in acutely triggered T effector cells, but not in Treg cells, since IL2RA mRNA in unstimulated CD4+ T cells would predominantly come from CD25hi Treg cells, and this association was not apparent in unstimulated cells. " "CD25,IDDM10,IL2R,IMD41,TCGFR,p55" Autoimmune Diseases -- D001327 -- -- -- YY1 "DELTA,GADEVS,INO80S,NF-E1,UCRBP,YIN-YANG-1" EMSA The allele-specific binding of YY1 was also confirmed by EMSA. rs12251836 6091281 "Luciferase Reporter Assay,EMSA" >2KB 12371 E_01_183 25248036 -- hg19 chr2 217918268 217921910 Human Breast Cancer Cell Low+High throughput "Luciferase Reporter Assay,ChIP-seq,3C,PCR" "The strongest candidate for causality, SNP rs4442975, flanks a transcriptional enhancer that physically interacts with the promoter of IGFBP5. Furthermore, we demonstrate that rs4442975 is associated with allele_x0002_specific FOXA1 binding, chromatin looping and IGFBP5 expression. Our data suggest that the g-allele of rs4442975 confers increased breast cancer susceptibility through reduced IGFBP5 expression." Enhancer IGFBP5 3C -- "This SNP flanks a transcriptional enhancer that physically interacts with the promoter of IGFBP5 (encoding insulin-like growth factor-binding protein 5) and displays allele-specific gene expression, FOXA1 binding and chromatin looping. Evidence suggests that the g-allele confers increased breast cancer susceptibility through relative downregulation of IGFBP5, a gene with known roles in breast cell biology." IBP5 Breast Cancer DOID:1612 D001943 -- -- -- FOXA1 "HNF3A,TCF3A" "qPCR,ChIP-seq" "To assess occupancy of FOXA1 in vivo, we conducted ChIP followed by allele-specific quantitative PCR (qPCR) in the heterozygous BT474 breast cancer cell line." rs4442975 217920769 "ChIP-seq,ChIP,qPCR,ChIP-qPCR,3C" >2KB 383262 E_01_184 25259561 DHS-44 Enhancer hg19 chr7 117075514 117075933 Human Human Bronchial Epithelial Cell Low throughput "Luciferase Reporter Assay,EMSA,RT-PCR,ChIP" "To confirm that DHS-44kb contained an enhancer of CFTR expression, the 0.6-kb DHS fragment (hg 19, chr7:117075400¨C117076000) was PCR amplified and inserted into the enhancer site of the pGL3B 245 vector.These data suggest that the enhancer activity of the DHS-44kb element is specific to airway epithelial cells." Enhancer CFTR -- "RT-PCR,ChIP" "To determine whether the SFN_x0002_triggered antioxidant response regulates CFTR expression directly through the DHS-44(279) cis-element, ChIP was used to measure the enrichment levels of the constitutive ARE binding factor Bach1, the SFN-induced ARE binding factor Nrf2, and their common partner MafK at this site." "ABC35,ABCC7,CF,CFTR/MRP,MRP7,TNR-CFTR,dJ760C5.1" -- -- -- -- -- -- "NFKB1,BACH1" "CVID12,EBP-1,KBF1,NF-kB,NF-kB1,NF-kappa-B1,NF-kappaB,NFKB-p105,NFKB-p50,NFkappaB,p105,p50,BACH-1,BTBD24" EMSA "EMSAs were performed with 32P-labeled, double-stranded DNA probes for 44-I, 44-II, and 44-III,Moreover, when Bach1 was overexpressed in 16HBE14o- cells by transient transfection of a mouse Bach1 cDNA clone, a strong supershift was seen with the Bach1 antibody and probe 44-I." -- -- -- >2KB 44292 E_01_185 25263592 -- hg19 chr19 2482923 2484923 Human HEK-293 Low throughput "ChIP,qRT-PCR,3C" "we performed Chromosome Conformation Capture (3C) to quantitatively measure the chromosomal interactions in the regions surrounding the Arc gene.As expected, the eRNA-producing Arc enhancer was the most prominent genomic locus that interacts with the Arc promoter in an activity-dependent manner." Enhancer GADD45B 3C ChIP "To corroborate our findings from 3C analysis,we next examined the effect of eRNA knockdown on the binding levels of the Mediator and cohesin complexes at both the Arc and Gadd45b enhancers as well as promoters.Chromatin immunoprecipitation(ChIP) analysis of a common Mediator complex subunit Med1 and a cohesin subunit RAD21 shows that both the promoters and enhancers of Arc and Gadd45b genes are inducibly occupied by the Mediator/cohesin complex upon membrane depolarization, but eRNA knockdown has no effect on their occupancy. " "GADD45BETA,MYD118" -- -- -- -- -- -- "GADD45B,ARC,C-FOS" "GADD45BETA,MYD118,Arg3.1,hArc,AP-1,C-FOS,p55" shRNA "In NIH3T3 cells, NELF was transiently released from the promoters of Arc, c-fos, and Egr1 at 30 min after serum stimulation. However, knockdown of Arc eRNA by LNA caused the retention of NELF only at the Arc promoter during serum stimulation, which is consistent with the results from the shRNA-mediated knockdown experiment,Interestingly, shRNAmediated knockdown of Arc and Gadd45b eRNAs blocked the NELF release from their corresponding promoters even during membrane depolarization." -- -- -- >2KB 7801 E_01_186 25267720 -- hg19 chr10 43568986 43571244 Human Neural Crest Cells Low throughput "RT-PCR,Luciferase Reporter Assay" "To determine the probable function of SRY on the transactivation of RET by NKX2-1 and PAX3, we had exam_x0002_ined its effects in a reporter system with a luciferase gene directed by a 3.7 kb promoter (23531 to +196) of the human RET gene(27), harboring both distal and proximal enhancers." Enhancer RET -- Luciferase Reporter Assay "However, when either SOX10 or PAX3 was co-transfected with NKX2-1, significant exacerbations of the RET1.5-luciferase activities were observed (Fig. 2D), suggesting potential interactions and collaborations between these transcription factors and NKX2-1 on regulation of RET gene." "CDHF12,CDHR16,HSCR1,MEN2A,MEN2B,MTC1,PTC,RET-ELE1" Hirschsprung Disease DOID:10487 D006627 -- -- -- "PAX3,NKX2-1" "CDHS,HUP2,WS1,WS3,BCH,BHC,NK-2,NKX2.1,NKX2A,NMTC1,T/EBP,TEBP,TITF1,TTF-1,TTF1" "Luciferase Reporter Assay,GST Pull-down Assay" "SOX10 and SRY competitive interactions with NKX2-1and PAX3 demonstrated by GST pull-down assays,Transfection with either the pM-NKX2-1 or pM-PAX3 expression vectors with the UAS-luciferase reporter showed that the GAL4-BD was indeed capable bringing these two transcription factors to the UAS site, thereby transactivating the UAS-luciferase reporter in the transfected Neuro-2A cells. " -- -- -- >2KB 2401 E_01_187 25267720 -- hg19 chr10 43571244 43572713 Human Neural Crest Cells Low throughput "RT-PCR,Luciferase Reporter Assay" "To determine the probable function of SRY on the transactivation of RET by NKX2-1 and PAX3, we had exam_x0002_ined its effects in a reporter system with a luciferase gene directed by a 3.7 kb promoter (23531 to +196) of the human RET gene(27), harboring both distal and proximal enhancers." Enhancer RET -- Luciferase Reporter Assay "However, when either SOX10 or PAX3 was co-transfected with NKX2-1, significant exacerbations of the RET1.5-luciferase activities were observed (Fig. 2D), suggesting potential interactions and collaborations between these transcription factors and NKX2-1 on regulation of RET gene." "CDHF12,CDHR16,HSCR1,MEN2A,MEN2B,MTC1,PTC,RET-ELE1" Hirschsprung Disease DOID:10487 D006627 -- -- -- "PAX3,NKX2-1" "CDHS,HUP2,WS1,WS3,BCH,BHC,NK-2,NKX2.1,NKX2A,NMTC1,T/EBP,TEBP,TITF1,TTF-1,TTF1" "Luciferase Reporter Assay,GST Pull-down Assay" "SOX10 and SRY competitive interactions with NKX2-1and PAX3 demonstrated by GST pull-down assays,Transfection with either the pM-NKX2-1 or pM-PAX3 expression vectors with the UAS-luciferase reporter showed that the GAL4-BD was indeed capable bringing these two transcription factors to the UAS site, thereby transactivating the UAS-luciferase reporter in the transfected Neuro-2A cells. " -- -- -- <2KB 537 E_01_188 25303440 -- hg19 chr7 94002773 94005073 Human Fibroblasts Low throughput Luciferase Reporter Assay "TGF¦Â is a potent inducer of the human procollagen type I ¦Á2 chain gene ( COL1A2). We have recently described in detail the in vivo mechanism underlying the transcriptional control of COL1A2, through a complex interaction between its distal enhancer and proximal promoter, in response to TGF¦Â. Reporter gene constructs containing the wild-type human enhancer region and the heterologous thymidine kinase (TK) promoter, lacking a Smad-dependent T RE (21.1/18.8pLAC-TK), were used to assess the T¦ÂRE in the FUE region. The results indicated that the COL1A2-FUE was activated by TGF¦Â in both normal and scleroderma fibroblasts." Enhancer COL1A2 -- "Luciferase Reporter Assay,EMSA,ChIP,siRNA" "Gene activation was determined by assessing the interaction of transcription factors with the COL1A2 Enhancer using transient transfection of reporter gene constructs,electrophoretic mobility shift assays,chromatin immunoprecipitation analysis,and RNA interference involving knockdown of individual AP-1 family members." "EDSARTH2,EDSCV,OI4" Systemic Sclerosis DOID:418 D012595 "Binding of JunB to the COL1A2 enhancer was observed, with its coalescence directed by activation of gene transcription through the proximal promoter." ChIP "RNA polymeraseII also coprecipitated with the F2 Enhancer sequence,despite not being able to bind the Enhancer directly,thereby demonstrating the interaction between the transcriptional machinery in the promoter with the factors bound to the F2 region." "JUNB,JUND" "AP-1,AP1,C-JUN, CJUN,P39" "EMSA,ChIP" "Supershift assays using specific antibodies (Figure 2B) revealed that normal nuclear extracts bound c-Jun and, to a lesser extent, JunD. In SSc, c-Jun binding was greatly reduced, and JunB, which was previously absent in normal nuclear extracts, accounted for 2 of the shifted bands. JunD binding was more pronounced in SSc extracts (Figure 2B). These gel-shift findings suggest that in SSc dermal fibroblasts or in normal nuclea extracts treated with TGF , JunB binding replaces c-Jun binding on the AP-1 site in the upstream enhancer.These results were confirmed using ChIP assays,which demonstrated the association of the F2 region in the FUE with the proximal promoter and the transcriptional machinery." -- -- -- >2KB 19949 E_01_189 25381333 -- hg19 chr22 42411333 42413643 Human Human Primary Hepatocytes Low throughput "4C,ChIP" "To search for regions that interact with the CYP2D6 promoter, we performed 4C assays with the CYP2D6 promoter as an anchor.With the exception of R1 and R6, peak signals were all associated with an annotated gene locus (gene body, promoter or upstream region, R2, R3, R4, R5 and R7), suggesting cross-regulation between genes. R1 corresponds to the previously identi?ed downstream enhancer region (7), while R6 appears to be novel. To test whether any of these regions could serve as an enhancer, we performed ChIP assays with an antibody against P300, a universal transcription co-factor that binds to enhancer elements. R1 and R2 displayed the highest signals in both HepG2 and hepatocytes (Fig. 1B and C), consistent with R1 serving as an enhancer for CYP2D6, while R2 corresponds to the 3¡ä end of CYP2D6, indicative of active transcription of CYP2D6 in both cells. R6 showed low ChIP signals in both cells, arguing against an enhancer role." Enhancer CYP2D6 4C ChIP "To search for regions that interact with the CYP2D6 promoter, we performed 4C assays with the CYP2D6 promoter as an anchor.With the exception of R1 and R6, peak signals were all associated with an annotated gene locus (gene body, promoter or upstream region, R2, R3, R4, R5 and R7), suggesting cross-regulation between genes. R1 corresponds to the previously identi?ed downstream enhancer region (7), while R6 appears to be novel. To test whether any of these regions could serve as an enhancer, we performed ChIP assays with an antibody against P300, a universal transcription co-factor that binds to enhancer elements. R1 and R2 displayed the highest signals in both HepG2 and hepatocytes (Fig. 1B and C), consistent with R1 serving as an enhancer for CYP2D6, while R2 corresponds to the 3¡ä end of CYP2D6, indicative of active transcription of CYP2D6 in both cells. R6 showed low ChIP signals in both cells, arguing against an enhancer role." "CPD6,CYP2D,CYP2D7AP,CYP2D7BP,CYP2D7P2,CYP2D8P2,CYP2DL1,CYPIID6,P450-DB1,P450C2D,P450DB1" -- -- -- "A region 115 kb downstream of CYP2D6 as Enhancer for CYP2D6, containing two completely linked SNPs, rs133333 and rs5758550, associated with enhanced transcription." "DNaseI Accessibility Assay,qPCR,CRISPR/Cas9" "To determine the relative chromatin accessibility along the R1 region, we performed DNaseI accessibility assays in both HepG2 and primary human hepatocytes, followed by real-time PCR (12). As expected, the region surrounding rs5758550 (A5) showed the highest accessibility in both cell types (Fig. 4), consistent with its role as enhancer.Among three highly linked SNPs in this region, we identify rs5758550 as the causative variant that regulates enhancer activity, with the minor allele rs5758550G displaying higher activity than the major allele rs5758550A. Moreover, using CRISPR-mediated genomic deletion in HepG2 cells, we demonstrate that deletion of the enhancer region surrounding rs5758550 by 70% decreased CYP2D6 mRNA level >2-fold." -- -- -- -- rs133333 42412365 "4C,ChIP,Luciferase Reporter Assay,PCR" >2KB 110012 E_01_190 25381333 -- hg19 chr22 42411544 42415106 Human Human Primary Hepatocytes Low throughput "4C,ChIP" "To search for regions that interact with the CYP2D6 promoter, we performed 4C assays with the CYP2D6 promoter as an anchor.With the exception of R1 and R6, peak signals were all associated with an annotated gene locus (gene body, promoter or upstream region, R2, R3, R4, R5 and R7), suggesting cross-regulation between genes. R1 corresponds to the previously identi?ed downstream enhancer region (7), while R6 appears to be novel. To test whether any of these regions could serve as an enhancer, we performed ChIP assays with an antibody against P300, a universal transcription co-factor that binds to enhancer elements. R1 and R2 displayed the highest signals in both HepG2 and hepatocytes (Fig. 1B and C), consistent with R1 serving as an enhancer for CYP2D6, while R2 corresponds to the 3¡ä end of CYP2D6, indicative of active transcription of CYP2D6 in both cells. R6 showed low ChIP signals in both cells, arguing against an enhancer role." Enhancer CYP2D6 4C ChIP "To search for regions that interact with the CYP2D6 promoter, we performed 4C assays with the CYP2D6 promoter as an anchor.With the exception of R1 and R6, peak signals were all associated with an annotated gene locus (gene body, promoter or upstream region, R2, R3, R4, R5 and R7), suggesting cross-regulation between genes. R1 corresponds to the previously identi?ed downstream enhancer region (7), while R6 appears to be novel. To test whether any of these regions could serve as an enhancer, we performed ChIP assays with an antibody against P300, a universal transcription co-factor that binds to enhancer elements. R1 and R2 displayed the highest signals in both HepG2 and hepatocytes (Fig. 1B and C), consistent with R1 serving as an enhancer for CYP2D6, while R2 corresponds to the 3¡ä end of CYP2D6, indicative of active transcription of CYP2D6 in both cells. R6 showed low ChIP signals in both cells, arguing against an enhancer role." "CPD6,CYP2D,CYP2D7AP,CYP2D7BP,CYP2D7P2,CYP2D8P2,CYP2DL1,CYPIID6,P450-DB1,P450C2D,P450DB1" -- -- -- -- -- -- -- -- -- -- rs4822082 42414198 "4C,ChIP,Luciferase Reporter Assay,PCR" >2KB 109175 E_01_191 25381333 -- hg19 chr22 42411769 42414470 Human Human Primary Hepatocytes Low throughput "4C,ChIP" "To search for regions that interact with the CYP2D6 promoter, we performed 4C assays with the CYP2D6 promoter as an anchor.With the exception of R1 and R6, peak signals were all associated with an annotated gene locus (gene body, promoter or upstream region, R2, R3, R4, R5 and R7), suggesting cross-regulation between genes. R1 corresponds to the previously identi?ed downstream enhancer region (7), while R6 appears to be novel. To test whether any of these regions could serve as an enhancer, we performed ChIP assays with an antibody against P300, a universal transcription co-factor that binds to enhancer elements. R1 and R2 displayed the highest signals in both HepG2 and hepatocytes (Fig. 1B and C), consistent with R1 serving as an enhancer for CYP2D6, while R2 corresponds to the 3¡ä end of CYP2D6, indicative of active transcription of CYP2D6 in both cells. R6 showed low ChIP signals in both cells, arguing against an enhancer role." Enhancer CYP2D6 4C ChIP "To search for regions that interact with the CYP2D6 promoter, we performed 4C assays with the CYP2D6 promoter as an anchor.With the exception of R1 and R6, peak signals were all associated with an annotated gene locus (gene body, promoter or upstream region, R2, R3, R4, R5 and R7), suggesting cross-regulation between genes. R1 corresponds to the previously identi?ed downstream enhancer region (7), while R6 appears to be novel. To test whether any of these regions could serve as an enhancer, we performed ChIP assays with an antibody against P300, a universal transcription co-factor that binds to enhancer elements. R1 and R2 displayed the highest signals in both HepG2 and hepatocytes (Fig. 1B and C), consistent with R1 serving as an enhancer for CYP2D6, while R2 corresponds to the 3¡ä end of CYP2D6, indicative of active transcription of CYP2D6 in both cells. R6 showed low ChIP signals in both cells, arguing against an enhancer role." "CPD6,CYP2D,CYP2D7AP,CYP2D7BP,CYP2D7P2,CYP2D8P2,CYP2DL1,CYPIID6,P450-DB1,P450C2D,P450DB1" -- -- -- -- -- -- -- -- -- -- rs5758550 42412711 "4C,ChIP,Luciferase Reporter Assay,PCR" >2KB 109380 E_01_192 25434007 -- hg19 chr1 98509888 98511451 Human Neuroblastoma Cell Lines Low+High throughput Luciferase Reporter Assay "SNP 1:g.98515539A>T is within an ENCODE-annotated neuronal cell (NH-A and SKNSH) speci?c enhancer and promoter ~3.6 kb upstream of MIR137 (Figure 1A).We hypothesized that the risk allele of 1:g.98515539A>T reduced enhancer activity by interfering with YY1 binding. To test the hypothesis, we cloned the putative enhancer sequence (the transcribed minus strand) ?anking 1:g.98515539A>T into a luciferase reporter gene vector (pGL3-promoter) (Figure 2A; Table S6). As expected from the ENCODE functional annotation, the cloned sequence exhibited robust enhancer activity as indicated by luciferase expression in a human neuroblastoma cell line (SH-SY5Y), but not in HeLa cells. " Enhancer MIR137 3C -- "SNP 1:g.98515539A>T is within an ENCODE--annotated neuronalcell(NH--Aand SKNSH) specificenhancer and promoter ~3.6 kb upstream of MIR137.We therefore performed the 3C assay 58¨C60 in SH--SY5Y cells to examine whether the 1:g.98515539A>T site can physically interact with core promoters of DPYD and LOC729987 (Figure 4A). We identified specific physical interaction of the enhancer sequence flanking 1:g.98515539A>T with other putative regulatory sequences upstream of MIR137/MIR2682, but not with the core promoters of DPYD or LOC729987 (Figure 4B). This suggests the functional 1:g.98515539A>T might influence expression of MIR137/MIR2682, but not of DPYD or LOC729987. " "MIRN137,miR-137" Schizophrenia DOID:5419 D012559 "A rare enhancer SNP, 1:g.98515539A>T, is associated with SZ and BP at MIR137/MIR2682 locus, with risk alleles decreasing MIR137/MIR2682 expression" "Luciferase Reporter Assay,EMSA,3C" "SNP 1:g.98515539A>T is within an ENCODE-annotated neuronal cell (NH-A and SKNSH) speci?c enhancer and promoter ~3.6 kb upstream of MIR137. Together, our reporter gene assay, EMSA, and the 3C experiment on the rare SNP 1:g.98515539A>T suggest a mechanism of reduced expression of MIR137/MIR2682 as contributing to SZ risk at this locus." YY1 "DELTA,GADEVS,INO80S,NF-E1,UCRBP,YIN-YANG-1" "Luciferase Reporter Assay,EMSA,3C" "We hypothesized that the risk allele of 1:g.98515539A>T reduced enhancer activity by interfering with YY1 binding. To test the hypothesis, we cloned the putative enhancer sequence (the transcribed minus strand) ?anking 1:g.98515539A>T into a luciferase reporter gene vector (pGL3-promoter) (Figure 2A; Table S6). As expected from the ENCODE functional annotation, the cloned sequence exhibited robust enhancer activity as indicated by luciferase expression in a human neuroblastoma cell line (SH-SY5Y), but not in HeLa cells. The enhancer activity of the cloned sequence was reduced ~50% by the risk allele A relative to the major allele T (we refer to the nucleotide on the minus strand hereinafter for the functional study section) of 1:g.98515539A>T(Figure 2A). We further carried out an electrophoresis mobility shift say (EMSA) to examine whether the reduced enhancer tivity by allele A of 1:g.98515539A>T correlated with tered DNA binding to TFs in SH-SY5Y cells. We found hat the YY1 motif-?anking DNA probe carrying the risk lele A had a much weaker YY1 binding capacity than he probe with the major allele T (Figure 3A). We identi?ed speci?c physical interaction of the enhancer sequence ?anking 1:g.98515539A>T with other putative regulatory sequences upstream of MIR137/MIR2682, but not with the core promoters of DPYD or LOC729987 (Figure 4B). This suggests the functional 1:g.98515539A>T might in?uence expression of MIR137/MIR2682, but not of DPYD or LOC729987." rs1198588 98515539 "Luciferase Reporter Assay,EMSA,3C" <2KB 955 E_01_193 25434007 -- hg19 chr1 98514701 98515055 Human Neuroblastoma Cell Lines Low+High throughput "Luciferase Reporter Assay,3C,PCR,EMSA" "SNP 1:g.98515539A>T is within an ENCODE--annotated neuronal cell (NH--A and SKNSH) speci?c enhancer and promoter ~3.6 kb upstream of MIR137 (Figure 1A).We hypothesized that the risk allele of 1:g.98515539A>T reduced enhancer activity by interfering with YY1 binding. To test the hypothesis, we cloned the putative enhancer sequence (the transcribed minus strand) ?anking 1:g.98515539A>T into a luciferase reporter gene vector (pGL3--promoter) (Figure 2A; Table S6). As expected from the ENCODE functional annotation, the cloned sequence exhibited robust enhancer activity as indicated by luciferase expression in a human neuroblastoma cell line (SH--SY5Y), but not in HeLa cells. " Enhancer MIR137 3C -- "SNP 1:g.98515539A>T is within an ENCODE--annotated neuronalcell(NH--Aand SKNSH) specificenhancer and promoter ~3.6 kb upstream of MIR137.We therefore performed the 3C assay 58¨C60 in SH--SY5Y cells to examine whether the 1:g.98515539A>T site can physically interact with core promoters of DPYD and LOC729987 (Figure 4A). We identified specific physical interaction of the enhancer sequence flanking 1:g.98515539A>T with other putative regulatory sequences upstream of MIR137/MIR2682, but not with the core promoters of DPYD or LOC729987 (Figure 4B). This suggests the functional 1:g.98515539A>T might influence expression of MIR137/MIR2682, but not of DPYD or LOC729987. " "MIRN137,miR-137" Schizophrenia DOID:5419 D012559 -- -- -- YY1 "DELTA,GADEVS,INO80S,NF-E1,UCRBP,YIN-YANG-1" EMSA EMSA for YY1 binding. A doublestranded oligonucleotide (29 bp; on minus strand) flanking the YY1-binding site and 1:g.98515539A>T (allele T or A; AGAGGTGCTGTGAACACACAGCCATTTTC t/a TAGCAGCTTTTTGACTG TATGTTACCATA) was incubated with the nuclear extracts of neuroblastoma (SH-SY5Y) cells. rs1198588 98515539 "Luciferase Reporter Assay,EMSA,3C" >2KB 3253 E_01_194 25434007 -- hg19 chr1 98516486 98522430 Human Neuroblastoma Cell Lines Low+High throughput "Luciferase Reporter Assay,3C,PCR,EMSA" "SNP 1:g.98515539A>T is within an ENCODE--annotated neuronal cell (NH--A and SKNSH) speci?c enhancer and promoter ~3.6 kb upstream of MIR137 (Figure 1A).We hypothesized that the risk allele of 1:g.98515539A>T reduced enhancer activity by interfering with YY1 binding. To test the hypothesis, we cloned the putative enhancer sequence (the transcribed minus strand) ?anking 1:g.98515539A>T into a luciferase reporter gene vector (pGL3--promoter) (Figure 2A; Table S6). As expected from the ENCODE functional annotation, the cloned sequence exhibited robust enhancer activity as indicated by luciferase expression in a human neuroblastoma cell line (SH--SY5Y), but not in HeLa cells. " Enhancer MIR137 3C -- "SNP 1:g.98515539A>T is within an ENCODE--annotated neuronalcell(NH--Aand SKNSH) specificenhancer and promoter ~3.6 kb upstream of MIR137.We therefore performed the 3C assay 58¨C60 in SH--SY5Y cells to examine whether the 1:g.98515539A>T site can physically interact with core promoters of DPYD and LOC729987 (Figure 4A). We identified specific physical interaction of the enhancer sequence flanking 1:g.98515539A>T with other putative regulatory sequences upstream of MIR137/MIR2682, but not with the core promoters of DPYD or LOC729987 (Figure 4B). This suggests the functional 1:g.98515539A>T might influence expression of MIR137/MIR2682, but not of DPYD or LOC729987. " "MIRN137,miR-137" Schizophrenia DOID:5419 D012559 -- -- -- YY1 "DELTA,GADEVS,INO80S,NF-E1,UCRBP,YIN-YANG-1" EMSA EMSA for YY1 binding. A doublestranded oligonucleotide (29 bp; on minus strand) flanking the YY1-binding site and 1:g.98515539A>T (allele T or A; AGAGGTGCTGTGAACACACAGCCATTTTC t/a TAGCAGCTTTTTGACTG TATGTTACCATA) was incubated with the nuclear extracts of neuroblastoma (SH-SY5Y) cells. rs1198588 98515539 "Luciferase Reporter Assay,EMSA,3C" >2KB 7833 E_01_195 25434007 -- hg19 chr1 98517055 98517909 Human Neuroblastoma Cell Lines Low+High throughput "Luciferase Reporter Assay,3C,PCR,EMSA" "SNP 1:g.98515539A>T is within an ENCODE--annotated neuronal cell (NH--A and SKNSH) speci?c enhancer and promoter ~3.6 kb upstream of MIR137 (Figure 1A).We hypothesized that the risk allele of 1:g.98515539A>T reduced enhancer activity by interfering with YY1 binding. To test the hypothesis, we cloned the putative enhancer sequence (the transcribed minus strand) ?anking 1:g.98515539A>T into a luciferase reporter gene vector (pGL3--promoter) (Figure 2A; Table S6). As expected from the ENCODE functional annotation, the cloned sequence exhibited robust enhancer activity as indicated by luciferase expression in a human neuroblastoma cell line (SH--SY5Y), but not in HeLa cells. " Enhancer MIR137 3C -- "SNP 1:g.98515539A>T is within an ENCODE--annotated neuronalcell(NH--Aand SKNSH) specificenhancer and promoter ~3.6 kb upstream of MIR137.We therefore performed the 3C assay 58¨C60 in SH--SY5Y cells to examine whether the 1:g.98515539A>T site can physically interact with core promoters of DPYD and LOC729987 (Figure 4A). We identified specific physical interaction of the enhancer sequence flanking 1:g.98515539A>T with other putative regulatory sequences upstream of MIR137/MIR2682, but not with the core promoters of DPYD or LOC729987 (Figure 4B). This suggests the functional 1:g.98515539A>T might influence expression of MIR137/MIR2682, but not of DPYD or LOC729987. " "MIRN137,miR-137" Schizophrenia DOID:5419 D012559 -- -- -- YY1 "DELTA,GADEVS,INO80S,NF-E1,UCRBP,YIN-YANG-1" EMSA EMSA for YY1 binding. A doublestranded oligonucleotide (29 bp; on minus strand) flanking the YY1-binding site and 1:g.98515539A>T (allele T or A; AGAGGTGCTGTGAACACACAGCCATTTTC t/a TAGCAGCTTTTTGACTG TATGTTACCATA) was incubated with the nuclear extracts of neuroblastoma (SH-SY5Y) cells. rs1198588 98515539 "Luciferase Reporter Assay,EMSA,3C" >2KB 5857 E_01_196 25453756 -- hg19 chr12 98559923 98563615 Human Induced Pluripotent Stem Cells Low+High throughput "3C,Luciferase Reporter Assay" "We identified 4 SNPs (rs2159100, rs12315711, rs11062170, rs4765905) that are in perfect LD with rs758170 and rs1006737, and lie within two predicted enhancers (spanning 1.4Kb and 1.85Kb), ?185Kb downstream from the gene¡¯s TSS (Figure 5A, Figure 5B). To identify whether the predicted enhancer region might be capable of forming promoter¨Cenhancer loops, we mapped its physical interaction with the CACNA1C TSS in human dorsolateral prefrontal cortex (n = 6) and hiPSC derived-neurons by chromosome conformation capture (3C) assay. Of the 17 restriction site regions tested, only one displayed an increased interaction with the TSS in human postmortem tissue (Primer #4 in Figure 5A¨CD). This enhancer region includes rs2159100 (MAF = 0.35, GWAS P value = 1.1 ¡Á 10?10) and rs12315711 (MAF=0.35, GWAS P value = 1.1 ¡Á 10?10) and demonstrates increased interaction frequency with the CACNA1C promoter (F (16, 83) = 5.52, P = 8 ¡Á 10?8). In addition to being in an enhancer region, rs2159100 also co-localizes with a 600bp DHS region. Thus, we examined its effect on transcriptional activity in in vitro experiments. Compared to the reference rs2159100 C allele, the risk T variant is associated with decreased transcriptional activity in HEK-293 and Neuro-2a cells (42% (P < 0.0001) and 23% (P < 0.05) reduction in luciferase activity, respectively) (Figure 5E)." Enhancer CACNA1C 3C Luciferase Reporter Assay "We identified 4 SNPs (rs2159100, rs12315711, rs11062170, rs4765905) that are in perfect LD with rs758170 and rs1006737, and lie within two predicted enhancers (spanning 1.4Kb and 1.85Kb), ?185Kb downstream from the gene¡¯s TSS (Figure 5A, Figure 5B). To identify whether the predicted enhancer region might be capable of forming promoter¨Cenhancer loops, we mapped its physical interaction with the CACNA1C TSS in human dorsolateral prefrontal cortex (n = 6) and hiPSC derived-neurons by chromosome conformation capture (3C) assay. Of the 17 restriction site regions tested, only one displayed an increased interaction with the TSS in human postmortem tissue (Primer #4 in Figure 5A¨CD). This enhancer region includes rs2159100 (MAF = 0.35, GWAS P value = 1.1 ¡Á 10?10) and rs12315711 (MAF=0.35, GWAS P value = 1.1 ¡Á 10?10) and demonstrates increased interaction frequency with the CACNA1C promoter (F (16, 83) = 5.52, P = 8 ¡Á 10?8). In addition to being in an enhancer region, rs2159100 also co-localizes with a 600bp DHS region. Thus, we examined its effect on transcriptional activity in in vitro experiments. Compared to the reference rs2159100 C allele, the risk T variant is associated with decreased transcriptional activity in HEK-293 and Neuro-2a cells (42% (P < 0.0001) and 23% (P < 0.05) reduction in luciferase activity, respectively) (Figure 5E)." "CACH2,CACN2,CACNL1A1,CCHL1A1,CaV1.2,LQT8,TS,TS.LQT8" Schizophrenia DOID:5419 D012559 -- -- -- -- -- -- -- -- -- -- >2KB 96481817 E_01_197 25453756 -- hg19 chr12 98562692 98566384 Human Induced Pluripotent Stem Cells Low+High throughput 3C "We identified 4 SNPs (rs2159100, rs12315711, rs11062170, rs4765905) that are in perfect LD with rs758170 and rs1006737, and lie within two predicted enhancers (spanning 1.4Kb and 1.85Kb), ?185Kb downstream from the gene¡¯s TSS (Figure 5A, Figure 5B).?To identify whether the predicted enhancer region might be capable of forming promoter¨Cenhancer loops, we mapped its physical interaction with the CACNA1C TSS in human dorsolateral prefrontal cortex (n = 6) and hiPSC derived-neurons by chromosome conformation capture (3C) assay.?Of the 17 restriction site regions tested, only one displayed an increased interaction with the TSS in human postmortem tissue (Primer #4 in Figure 5A¨CD).?This enhancer region includes rs2159100 (MAF = 0.35, GWAS P value = 1.1 ¡Á 10?10) and rs12315711 (MAF=0.35, GWAS P value = 1.1 ¡Á 10?10) and demonstrates increased interaction frequency with the CACNA1C promoter (F (16, 83) = 5.52, P = 8 ¡Á 10?8).?The rs2159100/rs12315711 3C interaction was confirmed in hiPSC derived-neurons (Figure 5D).?" Enhancer CACNA1C 3C -- "We identified 4 SNPs (rs2159100, rs12315711, rs11062170, rs4765905) that are in perfect LD with rs758170 and rs1006737, and lie within two predicted enhancers (spanning 1.4Kb and 1.85Kb), ?185Kb downstream from the gene¡¯s TSS (Figure 5A, Figure 5B).?To identify whether the predicted enhancer region might be capable of forming promoter¨Cenhancer loops, we mapped its physical interaction with the CACNA1C TSS in human dorsolateral prefrontal cortex (n = 6) and hiPSC derived-neurons by chromosome conformation capture (3C) assay.?Of the 17 restriction site regions tested, only one displayed an increased interaction with the TSS in human postmortem tissue (Primer #4 in Figure 5A¨CD).?This enhancer region includes rs2159100 (MAF = 0.35, GWAS P value = 1.1 ¡Á 10?10) and rs12315711 (MAF=0.35, GWAS P value = 1.1 ¡Á 10?10) and demonstrates increased interaction frequency with the CACNA1C promoter (F (16, 83) = 5.52, P = 8 ¡Á 10?8).?The rs2159100/rs12315711 3C interaction was confirmed in hiPSC derived-neurons (Figure 5D).?" "CACH2,CACN2,CACNL1A1,CCHL1A1,CaV1.2,LQT8,TS,TS.LQT8" Schizophrenia DOID:5419 D012559 -- -- -- -- -- -- -- -- -- -- >2KB 96484586 E_01_198 25453903 -- hg19 chr14 97410723 97443618 Human "HeLa,293T" Low+High throughput "shRNA,RNA-seq,qRT-PCR,3C,Immunohistochemistry" "Finally, we sought to address the impact of altered cis-regulatory element activity on the transcriptional landscape of Ewing sarcoma. We performed RNA sequencing (RNA-seq) in the Ewing sarcoma lines before and after EWS-FLI1 knock-down. By mapping EWS-FLI1 distal elements to the nearest expressed genes, we observed a strong relationship between changes in enhancer activity and changes in proximal gene expression (p < 10?10). We confirmed a subset of regulated gene targets by qRT-PCR. We hypothesized that direct regulatory targets of EWS-FLI1 might represent attractive therapeutic targets in Ewing sarcoma and thus ranked target genes by combined changes in chromatin and expression (Fig. 5B). Another top candidate is VRK1, a cell cycle dependent tyrosine kinase involved in G2-M transition (Valbuena et al., 2011). VRK1 is proximal to an EWS-FLI1 dependent enhancer that is active in Ewing sarcoma cell lines and primary tumors and induced de novo by the fusion protein in MSCs (Fig. 5C). Chromatin conformation studies (3C) confirmed the long distance interaction between the EWS-FLI1-bound enhancer and the VRK1 promoter in both SKNMC and A673 cells (Fig. 5D and E, respectively)." Enhancer VRK1 3C "Immunohistochemistry,shRNA" "VRK1 is proximal to an EWS-FLI1 dependent Enhancer that is active in Ewing sarcoma cell lines and primary tumors and induced de novo by the fusion protein in MSCs Chromatin conformation studies (3C) confirmed the long distance interaction between the EWS-FLI1-bound enhancer and the VRK1 promoter in both SKNMC and A673 cells (Fig. 5D and E, respectively). VRK1 protein expression was confirmed in 15/15 primary Ewing sarcoma samples analyzed by immunohistochemistry, which revealed strong VRK1 signals in virtually all cells (Fig.6A and Fig. S5B). EWS-FLI1 knock-down markedly reduced VRK1 expression in the cell lines, while EWS-FLI1 induction was sufficient to up-regulate this kinase in MSCs (Fig. 6B and S5C)." "PCH1,PCH1A" Ewing Sarcoma DOID:3369 D012512 -- -- -- EWSR1 "EWS, EWS-FLI1, bK984G1.4£¬EWSR1" "qRT-PCR,3C,shRNA" "We performed RNA sequencing (RNA-seq) in the Ewing sarcoma lines before and after EWS-FLI1 knock-down.We confirmed a subset of regulated gene targets by qRT-PCR (Fig. S5A). VRK1 is proximal to an EWS-FLI1 dependent enhancer that is active in Ewing sarcoma cell lines and primary tumors and induced de novo by the fusion protein in MSCs (Fig. 5C).Chromatin conformation studies (3C) confirmed the long distance interaction between the EWS-FLI1-bound enhancer and the VRK1 promoter in both SKNMC and A673 cells (Fig. 5D and E, respectively). VRK1 protein expression was confirmed in 15/15 primary Ewing sarcoma samples analyzed by immunohistochemistry, which revealed strong VRK1 signals in virtually all cells (Fig.6A and Fig. S5B). EWS-FLI1 knock-down markedly reduced VRK1 expression in the cell lines, while EWS-FLI1 induction was sufficient to up-regulate this kinase in MSCs (Fig. 6B and S5C)." -- -- -- >2KB 122894438 E_01_199 25505291 -- hg19 chr1 23881244 23881761 Human Human Monocytes/Macrophages Low throughput Luciferase Reporter Assay "Using ECR browser (http://ecrbrowser.dcode.org/), we found two conserved regions,ECR1 located between -3177 and -2660 bp upstream of transcription start and ECR2 located between +4517 and 4662 bp downstream of the gene (sequences provided in Supplemental Table 1). ECR1 overlaps with the enhancer described by Shepherd et al. (32), whereas ECR2 has not been described to date. To test the impact of identi?ed ECRs on the regulation of Id3 expression, we cloned them into a pGL3basic vector together with about a 1-kb fragment of the Id3 promoter (Fig. 1A). Obtained plasmids were transfected using nucleofection into primary monocyte-derived macrophages cultivated in the presence of IL-4 and dexamethasone for 6 d. Cells after transfection were further cultivated for 24 h and then stimulated with TGF- 1forthenext 24 h. Cells were harvested and luciferase activity was measured. Without TGF- 1 stimulation, all reporter constructs showed low promoter activity. Stimulation of macrophages with TGF- 1ledto a 2-fold upregulation of the promoter activity. Addition of ECR1 or ECR2 led to a 5-fold increase of promoter activity. Reporter plasmid containing both of them showed nearly 12-fold upregulation of promoter activity, indicating that ECRs have an additive effect (Fig. 1B)." Enhancer ID3 -- Luciferase Reporter Assay "To test the impact of identi?ed ECRs on the regulation of Id3 expression, we cloned them into a pGL3basic vector together with about a 1-kb fragment of the Id3 promoter (Fig. 1A). Obtained plasmids were transfected using nucleofection into primary monocyte-derived macrophages cultivated in the presence of IL-4 and dexamethasone for 6 d. Cells after transfection were further cultivated for 24 h and then stimulated with TGF- 1forthenext 24 h. Cells were harvested and luciferase activity was measured. Without TGF- 1 stimulation, all reporter constructs showed low promoter activity. Stimulation of macrophages with TGF- 1ledto a 2-fold upregulation of the promoter activity. Addition of ECR1 or ECR2 led to a 5-fold increase of promoter activity. Reporter plasmid containing both of them showed nearly 12-fold upregulation of promoter activity, indicating that ECRs have an additive effect (Fig. 1B)." "HEIR-1,bHLHb25" Cardiometabolic Disorders -- -- -- -- -- -- -- -- -- -- -- -- >2KB 2917 E_01_200 25505291 -- hg19 chr1 23888938 23889083 Human Human Monocytes/Macrophages Low throughput Luciferase Reporter Assay "Using ECR browser (http://ecrbrowser.dcode.org/), we found two conserved regions,ECR1 located between -3177 and -2660 bp upstream of transcription start and ECR2 located between +4517 and 4662 bp downstream of the gene (sequences provided in Supplemental Table 1). ECR1 overlaps with the enhancer described by Shepherd et al. (32), whereas ECR2 has not been described to date. To test the impact of identi?ed ECRs on the regulation of Id3expression, we cloned them into a pGL3basic vector together with about a 1-kb fragment of the Id3 promoter (Fig. 1A). Obtained plasmids were transfected using nucleofection into primary monocyte-derived macrophages cultivated in the presence of IL-4 and dexamethasone for 6 d. Cells after transfection were further cultivated for 24 h and then stimulated with TGF- 1forthenext 24 h. Cells were harvested and luciferase activity was measured. Without TGF- 1 stimulation, all reporter constructs showed low promoter activity. Stimulation of macrophages with TGF- 1ledto a 2-fold upregulation of the promoter activity. Addition of ECR1 or ECR2 led to a 5-fold increase of promoter activity. Reporter plasmid containing both of them showed nearly 12-fold upregulation of promoter activity, indicating that ECRs have an additive effect (Fig. 1B)." Enhancer ID3 -- Luciferase Reporter Assay "To test the impact of identi?ed ECRs on the regulation of Id3 expression, we cloned them into a pGL3basic vector together with about a 1-kb fragment of the Id3 promoter (Fig. 1A). Obtained plasmids were transfected using nucleofection into primary monocyte-derived macrophages cultivated in the presence of IL-4 and dexamethasone for 6 d. Cells after transfection were further cultivated for 24 h and then stimulated with TGF- 1forthenext 24 h. Cells were harvested and luciferase activity was measured. Without TGF- 1 stimulation, all reporter constructs showed low promoter activity. Stimulation of macrophages with TGF- 1ledto a 2-fold upregulation of the promoter activity. Addition of ECR1 or ECR2 led to a 5-fold increase of promoter activity. Reporter plasmid containing both of them showed nearly 12-fold upregulation of promoter activity, indicating that ECRs have an additive effect (Fig. 1B)." "HEIR-1,bHLHb25" Cardiometabolic Disorders -- -- -- -- -- -- -- -- -- -- -- -- >2KB 4591 E_01_201 25522777 MMP9 Enhancer hg19 chr20 44637014 44637468 Human Hepatocellular Carcinoma Low throughput Luciferase Reporter Assay "The MMP9 promoter contains two AP-1 sites (located at -79 bp and -533 bp), a Sp1 site (located at -560 bp) and an NF-¦ÊB site (located at -600 bp). To determine whether regulation of MMP9 is related to these cis-acting regulatory elements for transcription factors, several constructs with deletions or mutations were used and have been described in Materials and methods. Relative Luciferase activities of pGL3-MMP9-WT (containing the potential Enhancer element) and several mutant derivatives of the MMP9 Enhancer region for all motifs were transfected in HCCLM3 cell treated without or with Salin (5-20 ?mol/l). Experimental cells were transfected with reporter vectors that included the tandem repeat of AP-1, NF-¦ÊB or Sp-1 binding sites. Noteworthy, luciferase activity in the cells with the AP-1 construct was significantly reduced by treatment with Salin at 10-20 ?mol/l, whereas luciferase activity containing the NF-¦ÊB or Sp-1 binding site construct showed no statistically significant changes in the cells treated with Salin (Fig. 3A). These results showed that both AP-1 sites in the MMP9 promoter were essential for Salin-regulated MMP9 enhancer region activity." Enhancer MMP9 -- Luciferase Reporter Assay "The MMP9 promoter contains two AP-1 sites (located at -79 bp and -533 bp), a Sp1 site (located at -560 bp) and an NF-¦ÊB site (located at -600 bp). To determine whether regulation of MMP9 is related to these cis-acting regulatory elements for transcription factors, several constructs with deletions or mutations were used and have been described in Materials and methods. Relative Luciferase activities of pGL3-MMP9-WT (containing the potential Enhancer element) and several mutant derivatives of the MMP9 Enhancer region for all motifs were transfected in HCCLM3 cell treated without or with Salin (5-20 ?mol/l). Experimental cells were transfected with reporter vectors that included the tandem repeat of AP-1, NF-¦ÊB or Sp-1 binding sites. Noteworthy, luciferase activity in the cells with the AP-1 construct was significantly reduced by treatment with Salin at 10-20 ?mol/l, whereas luciferase activity containing the NF-¦ÊB or Sp-1 binding site construct showed no statistically significant changes in the cells treated with Salin (Fig. 3A). These results showed that both AP-1 sites in the MMP9 promoter were essential for Salin-regulated MMP9 enhancer region activity." "CLG4B,GELB,MANDP2,MMP-9" Hepatocellular Carcinoma DOID:684 D006528 -- -- -- JUND AP-1 Western blot The results revealed that the AP-1 site is critical for Salin-regulated MMP9 Enhancer activity. Western blot analysis indicated that the expression of the components of AP-1 (JunB and JunD) was regulated by Salin (5-20 ¦Ìmol/l). -- -- -- <2KB 305 E_01_202 25604083 -- hg19 chr17 69487560 69491400 Human HEK-293 Low throughput "Luciferase Reporter Assay,Transgenic mice" "Finally, we performed transfections of reporter gene constructs under control of the minimal Sox9 promoter and additional XYSR fragments into various cell lines.For this, the 32.5 kb region was subdivided into 17 overlapping fragments,F1 to F17 (?gure 3). Whereas none of the conserved elements CNE1¨C7 showed Sertoli cell-speci?c activity (not shown),of all 24 constructs tested, F8 showed the highest luciferase activities in both Sertoli-like cell lines (see online supplementary ?gure S5A, B).In contrast, the same fragment had little in?uence on luciferase activity in both HEK293 and neuro-2a control cell lines (see online supplementary ?gure S5C, D). Even though F7 and F9 overlap with F8 (?gure 3 and online supplementary ?gure S6), their luciferase activities are weak in TM-4 and unremarkable in NT2/D1. Thus F8 appeared to be the best candidate for a testis-speci?c enhancer of the SOX9 gene." Enhancer SOX9 -- "Luciferase Reporter Assay,Transgenic mice" "Finally, we performed transfections of reporter gene constructs under control of the minimal Sox9 promoter and additional XYSR fragments into various cell lines.For this, the 32.5 kb region was subdivided into 17 overlapping fragments,F1 to F17 (?gure 3). Whereas none of the conserved elements CNE1¨C7 showed Sertoli cell-speci?c activity (not shown),of all 24 constructs tested, F8 showed the highest luciferase activities in both Sertoli-like cell lines (see online supplementary ?gure S5A, B).In contrast, the same fragment had little in?uence on luciferase activity in both HEK293 and neuro-2a control cell lines (see online supplementary ?gure S5C, D). Even though F7 and F9 overlap with F8 (?gure 3 and online supplementary ?gure S6), their luciferase activities are weak in TM-4 and unremarkable in NT2/D1. Thus F8 appeared to be the best candidate for a testis-speci?c enhancer of the SOX9 gene." "CMD1,CMPD1,SRA1,SRXX2,SRXY10" Disorders Of Sexual Development -- D012734 -- -- -- "SF1,WT1" "BBP, D11S636, MBBP, ZCCHC25, ZFM1, ZNF162,AWT1, GUD, NPHS4, WAGR, WIT-2, WT33" Luciferase Reporter Assay "Such an enhancer should be under control of SRY, but could possibly also be in?uenced in its activity by other transcription factors present in the early gonad and essential for gonadal differentiation, including SF1 (also known as NR5A1), WT1, and GATA4. To test this assumption, a luciferase reporter containing F8 in front of the ?-globin TATA box was co-transfected in neuro-2a cells with expression plasmids for these factors. Of all factors tested, only SRY elicited a substantial 10-fold increase in luciferase activity. WT1 stimulated reporter activity two fold, whereas SF1 and GATA4 were completely ineffective (?gure 4A)." -- -- -- >2KB 627680 E_01_203 25622893 hCNS1 Enhancer hg19 chr17 61927353 61928827 Human "GC-spd(ts),Hepa 1-6,293T" Low throughput "PCR,Luciferase Reporter Assay" "We first investigated enhancer activity of hCNS1. Because TSS of TCAM1P--I and TCAM1P--III was +401 and three TSSs of TCAM1P--II were close to +401 (Fig. 2E), we cloned about 2--kb sequence upstream of +401 as a major promoter of the TCAM1P gene and connected it to the luciferase gene (TCAM1P--Pro--luc). hCNS1 was amplified by genome PCR and cloned to upstream of the TCAM1P promoter (hCNS1--Pro--luc) or to downstream of the luciferase gene(Pro--luc--hCNS1 and Pro--luc--rev hCNS1).The activity of hCNS1--Pro--luc was significantly increased in all three cell lines compared to the construct without hCNS1 (Fig. 3A). " Enhancer TCAM1P -- Luciferase Reporter Assay "We also examined the luciferase activity in linearized constructs. If hCNS1 was actually a DPE, it might drive the transcription of the entire sequence in a circular plasmid, which might affect the luciferase gene expression.To prevent hCNS1 from driving the luciferase gene transcription in the sense direction, we linearized Pro--luc--hCNS1 and Pro--luc--rev hCNS1 constructs by cutting the immediate downstream sequence of hCNS1 and rev hCNS1. As a result, hCNS1 still significantly increased TCAM1P promoter activity in HEK293T cells, and the fold--increases were around 3 in both constructs compared to the linearized TCAM1P--Pro--luc construct (Fig. 3B). These data showed that hCNS1 could function as an enhancer for the TCAM1P gene." TCAM1 -- -- -- -- -- -- -- -- -- -- -- -- -- >2KB 6285 E_01_204 25628225 -- hg19 chr17 6285838 6586838 Human Human Liver Cell Low throughput "Luciferase Reporter Assay,EMSA,ChIP" "Utilizing cell-based Luciferaseassays,electrophoretic mobility shift assays,and Chromatin immunoprecipitation assays,we identified and functionally characterized two Enhancer modules located upstream of the SLC13A5 gene transcription start site that are associated with regulation of PXR-mediated SLC13A5 induction." Enhancer SLC13A5 -- "Luciferase Reporter Assay,EMSA,ChIP" "Utilizing cell-based Luciferaseassays,electrophoretic mobility shift assays,and Chromatin immunoprecipitation assays,we identified and functionally characterized two Enhancer modules located upstream of the SLC13A5 gene transcription start site that are associated with regulation of PXR-mediated SLC13A5 induction." "EIEE25,INDY,NACT,mIndy" Steatosis -- -- -- -- -- NR1I2 "BXR,ONR1,PAR,PAR1,PAR2,PARq,PRR,PXR,SAR,SXR" EMSA "Utilizing in vitro EMSA,we found that heterodimer of PXR and the retinoid X receptor binds specifically to DR4-1 and DR4-2 at intensities equal to or exceeding that of the everted-repeat (ER6) element from CYP3A4 promoter." -- -- -- >2KB 151693 E_01_205 25628225 -- hg19 chr17 6565038 6567038 Human Human Liver Cell Low throughput "Luciferase Reporter Assay,EMSA,ChIP" "Utilizing cell-based Luciferaseassays,electro_x0002_phoretic mobility shift assays,and Chromatin immunoprecipitation assays,we identified and functionally characterized two Enhancer modules located upstream of the SLC13A5 gene transcription start site that are associated with regulation of PXR-mediated SLC13A5 induction." Enhancer SLC13A5 -- "Luciferase Reporter Assay,EMSA,ChIP" "Utilizing cell-based Luciferaseassays,electro_x0002_phoretic mobility shift assays,and Chromatin immunoprecipitation assays,we identified and functionally characterized two Enhancer modules located upstream of the SLC13A5 gene transcription start site that are associated with regulation of PXR-mediated SLC13A5 induction." "EIEE25,INDY,NACT,mIndy" Fatty Liver Disease DOID:9452 D005234 -- -- -- NR1I2 "BXR,ONR1,PAR,PAR1,PAR2,PARq,PRR,PXR,SAR,SXR" EMSA "Utilizing in vitro EMSA,we found that heterodimer of PXR and the retinoid X receptor binds specifically to DR4-1 and DR4-2 at intensities equal to or exceeding that of the everted-repeat (ER6) element from CYP3A5 promoter." -- -- -- >2KB 21993 E_01_206 25701871 -- hg19 chr5 125345378 125345495 Human HIT DH5A cells (RBC) Low throughput "Luciferase Reporter Assay,4C,PCR" "To test the predicted enhancers experimentally, we perfor-ed dual-luciferase reporter assays with constructs containing regions A, B, C or D, in direct or reverse orientation, upstream of the LMNB1 promoter . Following transfection of the constructs into HEK293T human cell line, regions A and B (in both orientations) signi?cantly increased luciferase expression (A forward = 1.97 ¡À 0.2; A reverse = 1.8 ¡À 1.6; B forward = 2.52 ¡À 0.25; B reverse = 1.85 ¡À 0.10; mean ¡À standard error; P < 0.01; Fig. 3C). In contrast, constructs containing regions C and D did not signi?cantly affect luciferase expression (Fig. 3C). Similar results were obtained when the same plasmids were transfected into NIH3T3 cell line (data not shown), suggesting that the enhancer activity of regions A and B could function across species, at least in mouse ?broblasts. Following these results, regions A and B were subsequently referred to as enhancer region A (Enh-A) and enhancer region B (Enh-B)." Enhancer LMNB1 4C "Luciferase Reporter Assay,PCR" "To test the predicted enhancers experimentally, we perfor-ed dual-luciferase reporter assays with constructs containing regions A, B, C or D, in direct or reverse orientation, upstream of the LMNB1 promoter . Following transfection of the constructs into HEK293T human cell line, regions A and B (in both orientations) signi?cantly increased luciferase expression (A forward = 1.97 ¡À 0.2; A reverse = 1.8 ¡À 1.6; B forward = 2.52 ¡À 0.25; B reverse = 1.85 ¡À 0.10; mean ¡À standard error; P < 0.01; Fig. 3C). In contrast, constructs containing regions C and D did not signi?cantly affect luciferase expression (Fig. 3C). Similar results were obtained when the same plasmids were transfected into NIH3T3 cell line (data not shown), suggesting that the enhancer activity of regions A and B could function across species, at least in mouse ?broblasts. Following these results, regions A and B were subsequently referred to as enhancer region A (Enh-A) and enhancer region B (Enh-B)." "ADLD,LMN,LMN2,LMNB" Adult-onset Autosomal Dominant Demyelinating Leukodystrophy DOID:0060785 C566813 -- -- -- -- -- -- -- -- -- -- >2KB 766877 E_01_207 25701871 -- hg19 chr5 125995862 125995972 Human HIT DH5A cells (RBC) Low throughput "Luciferase Reporter Assay,4C,PCR" "To test the predicted enhancers experimentally, we perfor-ed dual-luciferase reporter assays with constructs containing regions A, B, C or D, in direct or reverse orientation, upstream of the LMNB1 promoter . Following transfection of the constructs into HEK293T human cell line, regions A and B (in both orientations) signi?cantly increased luciferase expression (A forward = 1.97 ¡À 0.2; A reverse = 1.8 ¡À 1.6; B forward = 2.52 ¡À 0.25; B reverse = 1.85 ¡À 0.10; mean ¡À standard error; P < 0.01; Fig. 3C). In contrast, constructs containing regions C and D did not signi?cantly affect luciferase expression (Fig. 3C). Similar results were obtained when the same plasmids were transfected into NIH3T3 cell line (data not shown), suggesting that the enhancer activity of regions A and B could function across species, at least in mouse ?broblasts. Following these results, regions A and B were subsequently referred to as enhancer region A (Enh-A) and enhancer region B (Enh-B). To validate and better characterize Enh-A and -B interactions with the LMNB1 promoter, we performed nested-PCR on circularized DNA which con?rmed that region A interacted in both patient and control, whereas the interaction of region B was patient-speci?c (SupplementaryMaterial). " Enhancer LMNB1 4C "Luciferase Reporter Assay,PCR" "To test the predicted enhancers experimentally, we perfor-ed dual-luciferase reporter assays with constructs containing regions A, B, C or D, in direct or reverse orientation, upstream of the LMNB1 promoter . Following transfection of the constructs into HEK293T human cell line, regions A and B (in both orientations) signi?cantly increased luciferase expression (A forward = 1.97 ¡À 0.2; A reverse = 1.8 ¡À 1.6; B forward = 2.52 ¡À 0.25; B reverse = 1.85 ¡À 0.10; mean ¡À standard error; P < 0.01; Fig. 3C). In contrast, constructs containing regions C and D did not signi?cantly affect luciferase expression (Fig. 3C). Similar results were obtained when the same plasmids were transfected into NIH3T3 cell line (data not shown), suggesting that the enhancer activity of regions A and B could function across species, at least in mouse ?broblasts. Following these results, regions A and B were subsequently referred to as enhancer region A (Enh-A) and enhancer region B (Enh-B). To validate and better characterize Enh-A and -B interactions with the LMNB1 promoter, we performed nested-PCR on circularized DNA which con?rmed that region A interacted in both patient and control, whereas the interaction of region B was patient-speci?c (SupplementaryMaterial). " "ADLD,LMN,LMN2,LMNB" Adult-onset Autosomal Dominant Demyelinating Leukodystrophy DOID:0060785 C566813 -- -- -- -- -- -- -- -- -- -- >2KB 116397 E_01_208 25704602 hg19 chr10 123099588 123100426 Human GMSM-K (human embry-onic oral epithelial cells) Low throughput "Luciferase Reporter Assay,PCR" "The same FGFR2 +254 construct used in the zebra?sh studies (GRCh37, chr10: 123,099,588¨C123,100,426) was modi?ed by PCR-mediated mutagenesis to test the de novo mutation at GRCh37, chr10: 123,099,960. In transient transgenic reporter studies in zebra?sh embryos, we demonstrated that the reference allele of the human +254 kb element has enhancer activity in the neural keel (Figure 3C), brain (Figure 3D), and delaminating neural crest (Figure S18), consistent with expression of Fgfr2 in brain and cranial neural folds in mice and zebra?sh.In parallel experiments, the de novo mutation revealed enhancer activity in fewer embryos (3/83) than the reference allele (41/82; p ? 1.70 3 10 12 )(Figures 3E and S18)." Enhancer FGFR2 -- "Luciferase Reporter Assay,PCR" "The same FGFR2 +254 construct used in the zebra?sh studies (GRCh37, chr10: 123,099,588¨C123,100,426) was modi?ed by PCR-mediated mutagenesis to test the de novo mutation at GRCh37, chr10: 123,099,960. In transient transgenic reporter studies in zebra?sh embryos, we demonstrated that the reference allele of the human +254 kb element has enhancer activity in the neural keel (Figure 3C), brain (Figure 3D), and delaminating neural crest (Figure S18), consistent with expression of Fgfr2 in brain and cranial neural folds in mice and zebra?sh.In parallel experiments, the de novo mutation revealed enhancer activity in fewer embryos (3/83) than the reference allele (41/82; p ? 1.70 3 10 12 )(Figures 3E and S18)." "BBDS, BEK, BFR-1, CD332, CEK3, CFD1, ECT1, JWS, K-SAM, KGFR, TK14, TK25" Cleft Palate -- -- -- -- -- -- -- -- -- -- -- -- >2KB 104142508 E_01_209 25704602 hg19 chr17 54754755 54758235 Human GMSM-K (human embry-onic oral epithelial cells) Low throughput "Luciferase Reporter Assay,PCR" "One element, located +87 kb from the NOG translation start site, is an epithelial enhancer in zebra?sh (Figures 5E and 5F). The second element, at +105 kb, lacked consistent enhancer activity in transient transgenic zebra?sh assays. However, this element had low-level enhancer activity in vitro in human fetal oral epithelial cells (GMSM-K) (Figure 5G) and murine osteoblastic cells (MC3T3) (Figure S19). Interestingly, the enhancer activity of the +105 kb element in both cell types was signi?cantly lower with theNSCL/P-associated allele of rs227727 (i.e., T) than with the unassociated allele (i.e., A) (Figures 5G and S18)." Enhancer NOG -- "Luciferase Reporter Assay,PCR" "One element, located +87 kb from the NOG translation start site, is an epithelial enhancer in zebra?sh (Figures 5E and 5F). The second element, at +105 kb, lacked consistent enhancer activity in transient transgenic zebra?sh assays. However, this element had low-level enhancer activity in vitro in human fetal oral epithelial cells (GMSM-K) (Figure 5G) and murine osteoblastic cells (MC3T3) (Figure S19). Interestingly, the enhancer activity of the +105 kb element in both cell types was signi?cantly lower with theNSCL/P-associated allele of rs227727 (i.e., T) than with the unassociated allele (i.e., A) (Figures 5G and S18)." "SYM1, SYNS1, SYNS1A" Cleft Palate -- -- -- -- -- -- -- -- -- -- -- -- >2KB 85435 E_01_210 25704602 hg19 chr17 54775600 54778000 Human GMSM-K (human embry-onic oral epithelial cells) Low throughput "Luciferase Reporter Assay,PCR" "One element, located +87 kb from the NOG translation start site, is an epithelial enhancer in zebra?sh (Figures 5E and 5F). The second element, at +105 kb, lacked consistent enhancer activity in transient transgenic zebra?sh assays. However, this element had low-level enhancer activity in vitro in human fetal oral epithelial cells (GMSM-K) (Figure 5G) and murine osteoblastic cells (MC3T3) (Figure S19). Interestingly, the enhancer activity of the +105 kb element in both cell types was signi?cantly lower with theNSCL/P-associated allele of rs227727 (i.e., T) than with the unassociated allele (i.e., A) (Figures 5G and S18)." Enhancer NOG -- "Luciferase Reporter Assay,PCR" "One element, located +87 kb from the NOG translation start site, is an epithelial enhancer in zebra?sh (Figures 5E and 5F). The second element, at +105 kb, lacked consistent enhancer activity in transient transgenic zebra?sh assays. However, this element had low-level enhancer activity in vitro in human fetal oral epithelial cells (GMSM-K) (Figure 5G) and murine osteoblastic cells (MC3T3) (Figure S19). Interestingly, the enhancer activity of the +105 kb element in both cell types was signi?cantly lower with theNSCL/P-associated allele of rs227727 (i.e., T) than with the unassociated allele (i.e., A) (Figures 5G and S18)." "SYM1, SYNS1, SYNS1A" Cleft Palate -- -- -- -- -- -- -- -- -- -- -- -- >2KB 105740 E_01_211 25769725 ENH3 Enhancer hg19 chr6 45410618 45417584 Human "B-CPAP,TPC1" Low throughput "Luciferase Reporter Assay,RT-PCR,ChIP" "Next, the DNA fragments corresponding to ENH1, 2, and 3 were cloned into the pGL3-P2 vector downstream of the luciferase reporter gene and their ability to activate the P2 promoter wasassessed by luciferase assay (Fig. 2F). Only ENH3 determined a strong induction of the reporter gene, suggesting a potent activity of this element on the P2 promoter. Noticeably, the activating effect of ENH3 on the P2 promoter was stronger in TPC1 than in BCPAP cells, in line with the higher level of RUNX2 expression detected in TPC1 as compared with BCPAP cells (Fig. 2B; ref. 16).Furthermore, ENH3 maintained the ability of activating the reporter gene when cloned in the inverted orientation and when cloned in the pGL3 vector containing the sP2 promoter.Finally, we have shown by chromatin immunoprecipitation (ChIP) experiments that the binding of RNA Polymerase II (RNA PolII) on the ENH3 was signi?cantly enriched as compared with two downstream RUNX2 exons (exons 5 and 7; Fig.3A and B)." Enhancer RUNX2 -- "Luciferase Reporter Assay,RT-PCR,ChIP" "Next, the DNA fragments corresponding to ENH1, 2, and 3 were cloned into the pGL3-P2 vector downstream of the luciferase reporter gene and their ability to activate the P2 promoter wasassessed by luciferase assay (Fig. 2F). Only ENH3 determined a strong induction of the reporter gene, suggesting a potent activity of this element on the P2 promoter. Noticeably, the activating effect of ENH3 on the P2 promoter was stronger in TPC1 than in BCPAP cells, in line with the higher level of RUNX2 expression detected in TPC1 as compared with BCPAP cells (Fig. 2B; ref. 16).Furthermore, ENH3 maintained the ability of activating the reporter gene when cloned in the inverted orientation and when cloned in the pGL3 vector containing the sP2 promoter.Finally, we have shown by chromatin immunoprecipitation (ChIP) experiments that the binding of RNA Polymerase II (RNA PolII) on the ENH3 was signi?cantly enriched as compared with two downstream RUNX2 exons (exons 5 and 7; Fig.3A and B)." "AML3,CBF-alpha-1,CBFA1,CCD,CCD1,CLCD,OSF-2,OSF2,PEA2aA,PEBP2aA" -- -- -- Inhibition of ENH3 activity is a generalmechanismof action of HDACi in different types of cancer Luciferase Reporter Assay "Finally, we tested whether the TSA-repressive effect was mediated by repression of ENH3 as we have shown in thyroid cancer cells. In all cell lines tested, the presence of ENH3 down-stream of the sP2 promoter determined the activation of the reporter gene as already observed in TPC1 and BCPAP cells (Fig.7C). Similarly, upon TSA treatment, the ENH3 activity was repressed in all cell lines, suggesting the possibility to extend to other cancer types the model of ENH3 functioning that we proposed in thyroid cancer." JUND AP-1 ChIP "We performed supershift assay using anti-c-JUN antibodies to con-firm that AP1 binds to site 2,ChIP experiments using anti-c-JUN antibodies showed that c-JUN actively binds to ENH3 in a context of structured Chromatin." -- -- -- >2KB 118048 E_01_212 25782671 -- hg19 chr7 155845627 155847652 Human Caco-2 Low throughput Luciferase Reporter Assay "The 2026 bp sequence was subcloned into the expression vector pLS_Prom_SHH. As the human colon adenocarcinoma Caco2 cell line expresses SHH,we therefore performed transfections in these cells with the construct containing the 2026 bp sequence in forward (pLS_Prom_SHH_2026F) and reverse orientation (pLS_Prom_SHH_2026R). Both constructs show an approximately twofold decrease of the relative reporter activity, as compared with the control vector without regulatory element pLS_Prom_SHH (Figure 4). Thus, the 2026 bp sequence contains an element capable of repressing the transcriptional activity of the SHH promoter in cis, consistent with a silencer activity." Enhancer SHH -- Luciferase Reporter Assay "The 2026 bp sequence was subcloned into the expression vector pLS_Prom_SHH. As the human colon adenocarcinoma Caco2 cell line expresses SHH,we therefore performed transfections in these cells with the construct containing the 2026 bp sequence in forward (pLS_Prom_SHH_2026F) and reverse orientation (pLS_Prom_SHH_2026R). Both constructs show an approximately twofold decrease of the relative reporter activity, as compared with the control vector without regulatory element pLS_Prom_SHH (Figure 4). Thus, the 2026 bp sequence contains an element capable of repressing the transcriptional activity of the SHH promoter in cis, consistent with a silencer activity." "HHG1,HLP3,HPE3,MCOPCB5,SMMCI,ShhNC,TPT,TPTPS" -- -- -- -- -- -- -- -- -- -- -- -- -- >2KB 251707 E_01_213 25786345 CXCR5 Enhancer hg19 chr11 118767466 118769582 Human MCF-7 Low throughput "Luciferase Reporter Assay,PCR" "Genomic fragment containing cxcr5 promoter was subcloned upstream of luciferase gene in pGL3-Basic reporter vector. Another genomic fragment containing the potential cxcr5 enhancer was subcloned downstream of the luciferase gene. In order to account for lower transfection efficiency of larger plasmids, the second construct included a control genomic fragment from cxcr5 locus which was similar in length to the enhancer fragment but had no characteristic features of a regulatory sequence. Functional comparison of these two constructs in MCF-7 and MCF-7-2si cells (Figure 3A) revealed a marginal modulating effect by the enhancer (15%) towards cxcr5 promoter activity. This effect, however, was stable in both cell lines, indicating that activity of the cxcr5 intronic enhancer in breast cancer cells was independent of the p53 status. " Enhancer CXCR5 -- "Luciferase Reporter Assay,PCR" "Genomic fragment containing cxcr5 promoter was subcloned upstream of luciferase gene in pGL3-Basic reporter vector. Another genomic fragment containing the potential cxcr5 enhancer was subcloned downstream of the luciferase gene. In order to account for lower transfection efficiency of larger plasmids, the second construct included a control genomic fragment from cxcr5 locus which was similar in length to the enhancer fragment but had no characteristic features of a regulatory sequence. Functional comparison of these two constructs in MCF-7 and MCF-7-2si cells (Figure 3A) revealed a marginal modulating effect by the enhancer (15%) towards cxcr5 promoter activity. This effect, however, was stable in both cell lines, indicating that activity of the cxcr5 intronic enhancer in breast cancer cells was independent of the p53 status. " "BLR1,CD185,MDR15" -- -- -- -- -- -- NFASC "NEDCPMD,NF,NRCAML" "ChIP,PCR" "Map of cxcr5 promoter,predicted NF -binding sites and PCR products amplified in ChIP assay." -- -- -- >2KB 14050 E_01_214 26211970 PRE3 Enhancer hg19 chr8 81286250 81293977 Human Lymphoblastoid Cell Lines Low+High throughput "ChIP-seq,Luciferase Reporter Assay,PCR,3C" PRE2 (A) or PRE3 (B) was cloned upstream of a PAG1 promoter driven luciferase reporter with and without the candidate causal SNPs.Our 3C studies provided evidence for allele--specific chromatin looping between PRE2 and PAG1. These results demonstrate that PRE3 acts as a transcriptional enhancer in the presence of the rs2370615:C allergy predisposing allele. Enhancer PAG1 3C Luciferase Reporter Assay PRE2 (A) or PRE3 (B) was cloned upstream of a PAG1 promoter driven luciferase reporter with and without the candidate causal SNPs.Our 3C studies provided evidence for allele--specific chromatin looping between PRE2 and PAG1. These results demonstrate that PRE3 acts as a transcriptional enhancer in the presence of the rs2370615:C allergy predisposing allele. "CBP,PAG" National Institute of Allergy and Infectious Diseases -- D054579 -- -- -- NFKB1 "CVID12,EBP-1,KBF1,NF-kB,NF-kB1,NF-kappa-B1,NF-kappaB,NFKB-p105,NFKB-p50,NFkappaB,p105,p50" ChIP-seq "Based on ENCODE ChIP-seq data for LCLs,17 two transcription factors (TFs) bind to PRE3 and overlap the putative functional variant rs2370615: the RelA (or p65[MIM: 164014]) subunit of the nuclear factor (NF-)TF,which is critical for innate and adaptive immune re_x0002_sponses,22 and the POU domain class 2 transcription factor2 (POU2F2 or Oct-2 [MIM: 164176]),which regulates B-cell-specific genes." rs7009110 81291879 "ChIP-seq,Luciferase Reporter Assay,3C" >2KB 589930 E_01_215 26214525 PROX1-11 Enhancer hg19 chr1 214150511 214151337 Human K-562 Low+High throughput "ChIP-seq,Luciferase Reporter Assay" "The ¨C11 kb element was also cloned distally into the enhancer site of pGL4.12, together with a subcloned region of the Prox1 +4.5 kb element (containing GATA sites 4 and 5, see Methods for details) acting as a promoter. This addition resulted in a significant increase in luciferase activity, demonstrating that the Prox1 ¨C11 kb element is capable of acting as an enhancer (Figure 1D). " Enhancer PROX1 -- Luciferase Reporter Assay "The ¨C11 kb element was also cloned distally into the enhancer site of pGL4.12, together with a subcloned region of the Prox1 +4.5 kb element (containing GATA sites 4 and 5, see Methods for details) acting as a promoter. This addition resulted in a significant increase in luciferase activity, demonstrating that the Prox1 ¨C11 kb element is capable of acting as an enhancer (Figure 1D). " PROX1 Lymphedema DOID:4977 D008209 -- -- -- "GATA2,FOXC2,NFATC1" "DCML,IMD21,MONOMAC,NFE1B,FKHL14,LD,MFH-1,MFH1,NF-ATC,NF-ATc1.2,NFAT2,NFATc" ChIP "ChIP demonstrates that GATA2, FOXC2, and NFATC1 ChIP at the PROX1 ¨C11 kb enhancer in LECs and BECs, but not K-562 cells." -- -- -- >2KB 10353 E_01_216 26214589 -- hg19 chr10 64737032 64737909 Human Mouse Embryonic Stem Cell Low+High throughput "ChIP-seq,Luciferase Reporter Assay" "We observed activating chromatin marks, signals for formaldehyde-assisted isolation of regulatory elements (FAIRE) and/or DNaseI hypersensitivity at five main loci: two loci corresponding to known EGR2 regulatory elements (MSE (myelinating Schwann cell enhancer) 30 and BoneE (bone enhancer) (unpublished data); Supplementary Fig. 8), one to the ADO promoter, and two to GGAA microsatellites (mSat1 and mSat2) that overlapped with EWSR1-FLI1 ChIP-Seq signals (Fig. 3a). Luciferase reporter assays indicated that BoneE and MSE had no or weak activity in Ewing sarcoma, respectively." Enhancer EGR2 -- "ChIP-seq,Luciferase Reporter Assay" "We observed activating chromatin marks, signals for formaldehyde-assisted isolation of regulatory elements (FAIRE) and/or DNaseI hypersensitivity at five main loci: two loci corresponding to known EGR2 regulatory elements (MSE (myelinating Schwann cell enhancer) 30 and BoneE (bone enhancer) (unpublished data); Supplementary Fig. 8), one to the ADO promoter, and two to GGAA microsatellites (mSat1 and mSat2) that overlapped with EWSR1-FLI1 ChIP-Seq signals (Fig. 3a). Luciferase reporter assays indicated that BoneE and MSE had no or weak activity in Ewing sarcoma, respectively." "AT591,CHN1,CMT1D,CMT4E,KROX20" Ewing Sarcoma DOID:3369 D012512 -- -- -- EWSR1 "EWS,EWS-FLI1,bK984G1.4" qRT-PCR ChIP efficacy was validated by qRT-PCR using a CCND1 EWSR1-FLI1 binding site. -- -- -- >2KB 165715 E_01_217 26216945 EBAs Enhancer hg19 chr2 98362566 98364423 Human HEK-293 Low+High throughput "ChIP-seq,Hi-C,Luciferase Reporter Assay" "Similarly, we integrated topologically associated domain (TAD) boundaries inferred from Hi-C chromatin contact maps (34) and found that the MIR insulators are also closer to TAD boundaries (P < 5.4E-8, Mann¨CWhitney test) (Fig. 1 C and D).These findings suggest the predicted MIR insulators have domain barrier function. In addition, we used the ChIA¨CPET interaction data from human CD4+ T cells (35) to test whether the predicted MIR insulators can potentially block enhancer¨Cpromoter interactions. We focused on ChIA¨CPET interactions between enhancers and promoters that are proximal (<500 kb) to MIR-derived insulators and classified them into one-side interactions, i.e., the interaction¡¯s anchors are restricted to one-side of a MIR insulator, and cross-interactions, i.e., the interaction¡¯s anchors are separated by a MIR insulator (Fig. 1E).For the human EBA, a luciferase reporter construct transfected in human HEK 293 cells (Materials and Methods) was used to evaluate three predicted MIR insulators (SI Appendix, Table S2). All three MIR insulators tested here showed enhancer-blocking activity comparable to the 5¡ä HS4 positive control (Fig. 2A) and much larger insulator activity than the second positive control, i.e., the minimal insulator sequence motif of 5¡ä HS4 (II/III). " Enhancer ZAP70 -- Luciferase Reporter Assay "ll three MIR insulators tested here showed enhancer-blocking activity comparable to the 5¡ä HS4 positive control (Fig. 2A) and much larger insulator activity than the second positive control, i.e., the minimal insulator sequence motif of 5¡ä HS4 (II/III).The blocking of the repressive chromatin domain also appears to be functionally important to CD4+ T cells because one of the proximal genes in the adjacent active chromatin domain, i.e., gene ZAP70, is part of the T-cell receptor pathway and its promoter is involved with multiple active ChIA¨CPET interactions (Fig. 2D)." "ADMIO2,IMD48,SRK,STCD,STD,TZK,ZAP-70" -- -- -- -- -- -- -- -- -- -- -- -- -- >2KB 33465 E_01_218 26229090 -- hg19 chr3 186938165 186940165 Human Lymphoma Cell Low+High throughput "ChIP-seq,3C,qRT-PCR" "This highlighted a known BCL6-interacting Enhancer region (6,16,17) 150-250 upstream of BCL6 (SE1),which overlaps the alternate breakpoint region,a recurrent site of BCL6 locus rearrangements (15). It also revealed a second Super-Enhancer overlapping the HGB-04 duplication at ?350 (SE2),and a third such region at ?500 (SE3).We used chromosome conformation capture (3C) analysis of GCB-DLBCL lymphoma cell lines (Supplementary Fig. S3A) to confirm looping of elements from super-enhancer regions to the BCL6 promoter, consistent with roles in activating this oncogene. Intriguingly, our PMBL case, HGB-04, showed a distinct acetylation pattern across these regions, with only the duplicated enhancer region, SE2, showing strong acetylation. In both cases, we detected the expected interactions between the BCL6 and MYC promoters and candidate enhancers in their native locus (Fig. 5C-D). We then used the same enhancer primers to measure interactions with the opposite gene promoter. We detected strong interactions between the MYC promoter and elements within the BCL6 SE1 and SE2 regions in HGB-07, but no such interactions in the non-rearranged tumor, HGB-06." Super-Enhancer BCL6 3C qRT-PCR "This highlighted a known BCL6-interacting Enhancer region (6,16,17) 150-250 upstream of BCL6 (SE1),which overlaps the alternate breakpoint region,a recurrent site of BCL6 locus rearrangements (15). It also revealed a second Super-Enhancer overlapping the HGB-04 duplication at ?350 (SE2),and a third such region at ?500 (SE3).We used chromosome conformation capture (3C) analysis of GCB-DLBCL lymphoma cell lines (Supplementary Fig. S3A) to confirm looping of elements from super-enhancer regions to the BCL6 promoter, consistent with roles in activating this oncogene. Intriguingly, our PMBL case, HGB-04, showed a distinct acetylation pattern across these regions, with only the duplicated enhancer region, SE2, showing strong acetylation. In both cases, we detected the expected interactions between the BCL6 and MYC promoters and candidate enhancers in their native locus (Fig. 5C-D). We then used the same enhancer primers to measure interactions with the opposite gene promoter. We detected strong interactions between the MYC promoter and elements within the BCL6 SE1 and SE2 regions in HGB-07, but no such interactions in the non-rearranged tumor, HGB-06." "BCL5A,BCL6,LAZ3,ZBTB27,ZNF51" B-Cell Lymphoma DOID:707 D016393 -- -- -- MEF2B RSRFR2 ChIP-Seq "Rather, we observed strong, focal MEF2B binding at acetylated elements within BCL6 super-enhancer regions, including four sites in SE1, one in SE2, and two in SE3 (Fig. 3C). ChIP-Seq analysis in the Jeko-1 MCL line revealed a specific increase in H3K27ac at MEF2B binding sites in MEF2B transgene-expressing cells, but not at intervening elements within the BCL6 super-enhancers (Fig. 3E).These data support a direct role for MEF2B in the activation of enhancers that drive BCL6 expression in human lymphomas. " -- -- -- >2KB 499999 E_01_219 26229090 -- hg19 chr3 187088165 187909165 Human Lymphoma Cell Low+High throughput "ChIP-seq,3C,qRT-PCR" "This High throughputlighted a known BCL6-interacting Enhancer region (6,16,17) 150-250 upstream of BCL6 (SE1),which overlaps the alternate breakpoint region,a recurrent site of BCL6 locus rearrangements (15). It also revealed a second Super-Enhancer overlapping the HGB-04 duplication at ?350 (SE2),and a third such region at ?500 (SE3).We used chromosome conformation capture (3C) analysis of GCB-DLBCL lymphoma cell lines (Supplementary Fig. S3A) to confirm looping of elements from super-enhancer regions to the BCL6 promoter, consistent with roles in activating this oncogene. Intriguingly, our PMBL case, HGB-04, showed a distinct acetylation pattern across these regions, with only the duplicated enhancer region, SE2, showing strong acetylation." Super-Enhancer BCL6 3C qRT-PCR "This High throughputlighted a known BCL6-interacting Enhancer region (6,16,17) 150-250 upstream of BCL6 (SE1),which overlaps the alternate breakpoint region,a recurrent site of BCL6 locus rearrangements (15). It also revealed a second Super-Enhancer overlapping the HGB-04 duplication at ?350 (SE2),and a third such region at ?500 (SE3).We used chromosome conformation capture (3C) analysis of GCB-DLBCL lymphoma cell lines (Supplementary Fig. S3A) to confirm looping of elements from super-enhancer regions to the BCL6 promoter, consistent with roles in activating this oncogene. Intriguingly, our PMBL case, HGB-04, showed a distinct acetylation pattern across these regions, with only the duplicated enhancer region, SE2, showing strong acetylation." "BCL5A,BCL6,LAZ3,ZBTB27,ZNF51" B-Cell Lymphoma DOID:707 D016393 -- -- -- MEF2B RSRFR2 ChIP-Seq "Rather, we observed strong, focal MEF2B binding at acetylated elements within BCL6 super-enhancer regions, including four sites in SE1, one in SE2, and two in SE3 (Fig. 3C). ChIP-Seq analysis in the Jeko-1 MCL line revealed a specific increase in H3K27ac at MEF2B binding sites in MEF2B transgene-expressing cells, but not at intervening elements within the BCL6 super-enhancers (Fig. 3E).These data support a direct role for MEF2B in the activation of enhancers that drive BCL6 expression in human lymphomas. " -- -- -- >2KB 59501 E_01_220 26229090 -- hg19 chr3 187559165 187589165 Human Lymphoma Cell Low+High throughput "ChIP-seq,3C,qRT-PCR" "This High throughputlighted a known BCL6-interacting Enhancer region (6,16,17) 150-250 upstream of BCL6 (SE1),which overlaps the alternate breakpoint region,a recurrent site of BCL6 locus rearrangements (15). It also revealed a second Super-Enhancer overlapping the HGB-04 duplication at ?350 (SE2),and a third such region at ?500 (SE3).We used chromosome conformation capture (3C) analysis of GCB-DLBCL lymphoma cell lines (Supplementary Fig. S3A) to confirm looping of elements from super-enhancer regions to the BCL6 promoter, consistent with roles in activating this oncogene. Intriguingly, our PMBL case, HGB-04, showed a distinct acetylation pattern across these regions, with only the duplicated enhancer region, SE2, showing strong acetylation." Super-Enhancer BCL6 3C qRT-PCR "This High throughputlighted a known BCL6-interacting Enhancer region (6,16,17) 150-250 upstream of BCL6 (SE1),which overlaps the alternate breakpoint region,a recurrent site of BCL6 locus rearrangements (15). It also revealed a second Super-Enhancer overlapping the HGB-04 duplication at ?350 (SE2),and a third such region at ?500 (SE3).We used chromosome conformation capture (3C) analysis of GCB-DLBCL lymphoma cell lines (Supplementary Fig. S3A) to confirm looping of elements from super-enhancer regions to the BCL6 promoter, consistent with roles in activating this oncogene. Intriguingly, our PMBL case, HGB-04, showed a distinct acetylation pattern across these regions, with only the duplicated enhancer region, SE2, showing strong acetylation." "BCL5A,BCL6,LAZ3,ZBTB27,ZNF51" B-Cell Lymphoma DOID:707 D016393 -- -- -- MEF2B RSRFR2 ChIP-Seq "Rather, we observed strong, focal MEF2B binding at acetylated elements within BCL6 super-enhancer regions, including four sites in SE1, one in SE2, and two in SE3 (Fig. 3C). ChIP-Seq analysis in the Jeko-1 MCL line revealed a specific increase in H3K27ac at MEF2B binding sites in MEF2B transgene-expressing cells, but not at intervening elements within the BCL6 super-enhancers (Fig. 3E).These data support a direct role for MEF2B in the activation of enhancers that drive BCL6 expression in human lymphomas. " -- -- -- >2KB 135001 E_01_221 26232425 -- hg19 chr20 42977441 42978040 Human Hep G2 Low throughput Luciferase Reporter Assay "The HNF4a P1 promoter consists of the distal enhancer region extending around 26.5 kb fromthe transcription starting site and the proximal promoter region (Ladias et al., 1992; Parviz et al., 2003; Hatzis et al.,2006). Next, we performed cell-based Luc reporter assays using a 7 kb DNA fragment of the HNF4a P1 promoter (Hatzis and Talianidis, 2001) (Fig. 3A). RIF treatment repressed the activity of 7-kb HNF4a P1 promoter constructs by approximately 60% in ShP51 cells, but not in parental HepG2 cells. A deletion of the distal enhancer region (¦¤27/26.4 kb) decreased the activity of the 7-kb construct in both parental HepG2 and ShP51 cells and abrogated the response to RIF treatment in ShP51 cells." Enhancer HNF4A -- "ChIP,Luciferase Reporter Assay" Cell-based reporter and ChIP assays showed that PXR targeted the distal Enhancer of the HNF4a P1 promoter and stimulated dissociation of HNF3b from the distal Enhancer "FRTS4,HNF4,HNF4a7,HNF4a8,HNF4a9,HNF4alpha,MODY,MODY1,NR2A1,NR2A21,TCF,TCF14" Hepatocellular Carcinoma DOID:684 D006528 -- -- -- "FOXM1,DR1,HNF1A" "FKHL16,FOXM1A,FOXM1B,FOXM1C,HFH-11,HFH11,HNF-3,INS-1,MPHOSPH2,MPP-2,MPP2,PIG29,TRIDENT,NC2,NC2-BETA,NC2B,NCB2,HNF-1A,HNF1,HNF4A,IDDM20,LFB1,MODY3,TCF-1,TCF1" "Luciferase Reporter Assay,PCR,ChIP" "Twenty-four hours after transfection, cells were treated with DMSOor RIF for another 24 hours in FBS-free MEM. pRL-TK was included in all transfections as a control. Relative Luc activity was calculated by taking the activity of the cells transfected with pGL3-Basic and treated with DMSO as one." -- -- -- >2KB 6699 E_01_222 26257179 -- hg19 chr4 56895793 56962351 Human Human Embryonic Kidney Cell Low+High throughput "ChIP-seq,5' RACE" "As a representative example, we highlight cheRNA1345, which is produced from a locus ~15 kb downstream of Cep135 and overlaps with an experimentally validated tissue-specific Enhancer element (Figure 4E) (Visel et al., 2007). Peaks of H3K4me3, H3K27ac, and RNAPII decorate the TSS, which we validated by 5' RACE, and also bears a number of validated transcription factor binding sites (Gerstein et al., 2012). Similar patterns are observed with other cheRNA loci (Figure S3A¨CC)." Enhancer CEP135 -- "ChIP-seq,5' RACE" "As a representative example, we highlight cheRNA1345, which is produced from a locus ~15 kb downstream of Cep135 and overlaps with an experimentally validated tissue-specific Enhancer element (Figure 4E) (Visel et al., 2007). Peaks of H3K4me3, H3K27ac, and RNAPII decorate the TSS, which we validated by 5' RACE, and also bears a number of validated transcription factor binding sites (Gerstein et al., 2012). Similar patterns are observed with other cheRNA loci (Figure S3A¨CC)." "CEP4,KIAA0635,MCPH8" -- -- -- -- -- -- -- -- -- -- -- -- -- >2KB 114099 E_01_223 26300977 -- hg19 chr7 114541370 114542201 Human "HEK-293,SK-N-MC" Low throughput Luciferase Reporter Assay "To test whether these regions could act as Enhancer elements we cloned them into a reporter construct in front of a minimal promoter and luciferase reporter gene.Element 1, which had multiple chromatin signatures characteristic of an enhancer, was able to act as a functional enhancer in both cell-lines (Fig. 1d).In HEK293 cells we observed a 3 fold increase of luciferase expression in comparison to the empty vector control.In SK-N-MC cells the luciferase expression increased nearly 7 fold as compared to the control. " Enhancer FOXP2 -- Luciferase Reporter Assay "To test whether these regions could act as Enhancer elements we cloned them into a reporter construct in front of a minimal promoter and luciferase reporter gene.Element 1, which had multiple chromatin signatures characteristic of an enhancer, was able to act as a functional enhancer in both cell-lines (Fig. 1d).In HEK293 cells we observed a 3 fold increase of luciferase expression in comparison to the empty vector control.In SK-N-MC cells the luciferase expression increased nearly 7 fold as compared to the control. " "CAGH44,SPCH1,TNRC10" -- -- -- -- -- -- -- -- -- -- -- -- -- >2KB 815422 E_01_224 26382291 -- hg19 chr6 1614188 1617051 Human Embryonic Stem Cell Low+High throughput Enhancer Assay We experimentally validate four novel Enhancers in this locus and demonstrate that these Enhancers show consistent activity during embryonic development in domains that overlap with the expression of FOXC1 and GMDS. Enhancer "FOXC1,GMDS" -- Enhancer Assay We experimentally validate four novel Enhancers in this locus and demonstrate that these Enhancers show consistent activity during embryonic development in domains that overlap with the expression of FOXC1 and GMDS. "ARA,ASGD3,FKHL7,FREAC-3,FREAC3,IGDA,IHG1,IRID1,RIEG3,GMD,SDR3E1" -- -- -- -- -- -- ZIC1 "CRS6,ZIC,ZNF201" X-Gal Staining Assay "An image from the UCSC Genome Browser showing the enhancer landscape near FOXC1 and GMDS.The lower panel is zoomed in to the regions immediately surrounding the enhancers, to highlight the ZIC1 binding sites. b E11.5 whole-mount images of ZIC mutants CE1¨C3. CE4 only yielded two X gal- positive embryos and was thus not used in the analysis. Tissues that have lacZ expression in at least half of the X-gal-positive embryos in WT but not in ZIC enhancers are noted in red. Arrowheads mark regions of interest." -- -- -- >2KB 4940 E_01_225 26382291 -- hg19 chr6 1615843 1617834 Human Embryonic Stem Cell Low+High throughput Enhancer Assay We experimentally validate four novel Enhancers in this locus and demonstrate that these Enhancers show consistent activity during embryonic development in domains that overlap with the expression of FOXC1 and GMDS. Enhancer "FOXC1,GMDS" -- Enhancer Assay We experimentally validate four novel Enhancers in this locus and demonstrate that these Enhancers show consistent activity during embryonic development in domains that overlap with the expression of FOXC1 and GMDS. "ARA,ASGD3,FKHL7,FREAC-3,FREAC3,IGDA,IHG1,IRID1,RIEG3,GMD,SDR3E1" -- -- -- -- -- -- ZIC1 "CRS6,ZIC,ZNF201" X-Gal Staining Assay "An image from the UCSC Genome Browser showing the enhancer landscape near FOXC1 and GMDS.The lower panel is zoomed in to the regions immediately surrounding the enhancers, to highlight the ZIC1 binding sites. b E11.5 whole-mount images of ZIC mutants CE1¨C3. CE4 only yielded two X gal- positive embryos and was thus not used in the analysis. Tissues that have lacZ expression in at least half of the X-gal-positive embryos in WT but not in ZIC enhancers are noted in red. Arrowheads mark regions of interest." -- -- -- >2KB 6159 E_01_226 26382291 -- hg19 chr6 1619348 1621343 Human Embryonic Stem Cell Low+High throughput Enhancer Assay We experimentally validate four novel Enhancers in this locus and demonstrate that these Enhancers show consistent activity during embryonic development in domains that overlap with the expression of FOXC1 and GMDS. Enhancer "FOXC1,GMDS" -- Enhancer Assay We experimentally validate four novel Enhancers in this locus and demonstrate that these Enhancers show consistent activity during embryonic development in domains that overlap with the expression of FOXC1 and GMDS. "ARA,ASGD3,FKHL7,FREAC-3,FREAC3,IGDA,IHG1,IRID1,RIEG3,GMD,SDR3E1" -- -- -- -- -- -- ZIC1 "CRS6,ZIC,ZNF201" X-Gal Staining Assay "An image from the UCSC Genome Browser showing the enhancer landscape near FOXC1 and GMDS.The lower panel is zoomed in to the regions immediately surrounding the enhancers, to highlight the ZIC1 binding sites. b E11.5 whole-mount images of ZIC mutants CE1¨C3. CE4 only yielded two X gal- positive embryos and was thus not used in the analysis. Tissues that have lacZ expression in at least half of the X-gal-positive embryos in WT but not in ZIC enhancers are noted in red. Arrowheads mark regions of interest." -- -- -- >2KB 9666 E_01_227 26382291 -- hg19 chr6 1701730 1704441 Human Embryonic Stem Cell Low+High throughput Enhancer Assay We experimentally validate four novel Enhancers in this locus and demonstrate that these Enhancers show consistent activity during embryonic development in domains that overlap with the expression of FOXC1 and GMDS. Enhancer "FOXC1,GMDS" -- Enhancer Assay We experimentally validate four novel Enhancers in this locus and demonstrate that these Enhancers show consistent activity during embryonic development in domains that overlap with the expression of FOXC1 and GMDS. "ARA,ASGD3,FKHL7,FREAC-3,FREAC3,IGDA,IHG1,IRID1,RIEG3,GMD,SDR3E1" -- -- -- -- -- -- ZIC1 "CRS6,ZIC,ZNF201" X-Gal Staining Assay "An image from the UCSC Genome Browser showing the enhancer landscape near FOXC1 and GMDS.The lower panel is zoomed in to the regions immediately surrounding the enhancers, to highlight the ZIC1 binding sites. b E11.5 whole-mount images of ZIC mutants CE1¨C3. CE4 only yielded two X gal- positive embryos and was thus not used in the analysis. Tissues that have lacZ expression in at least half of the X-gal-positive embryos in WT but not in ZIC enhancers are noted in red. Arrowheads mark regions of interest." -- -- -- Intron 92406 E_01_228 26422229 -- hg19 chr12 56728500 56735000 Human THP-1 Low+High throughput "ChIP-seq,ChIP" "Our ChIP-seq analysis of markers for transcriptional enhancers in macrophages revealed the presence, in both uninfected and infected THP-1 cells, of an extended region encompassing the IL23A locus of elevated H3K4me1 (Fig. 7A). We screened between the PAN2 and IL23A loci for sequences with homology to extended core AHRE motifs (Fig. 7A). This revealed three potential elements lying 3675, 2025, and 1375 bp upstream of the IL23A transcription start site, one of which (22025) corresponds to an extended consensus sequence. ChIP experiments in THP-1 cells revealed that binding of AHR and its heterodimeric partners ARNT and RELB to these regions is markedly enhanced by infection (Fig. 7B¨CD), with, as expected, the strongest association of all three proteins observed at the extended consensus motif (22025 bp, Fig. 7C)." Enhancer IL23A -- "ChIP-seq,ChIP" "Our ChIP-seq analysis of markers for transcriptional enhancers in macrophages revealed the presence, in both uninfected and infected THP-1 cells, of an extended region encompassing the IL23A locus of elevated H3K4me1 (Fig. 7A). We screened between the PAN2 and IL23A loci for sequences with homology to extended core AHRE motifs (Fig. 7A). This revealed three potential elements lying 3675, 2025, and 1375 bp upstream of the IL23A transcription start site, one of which (22025) corresponds to an extended consensus sequence. ChIP experiments in THP-1 cells revealed that binding of AHR and its heterodimeric partners ARNT and RELB to these regions is markedly enhanced by infection (Fig. 7B¨CD), with, as expected, the strongest association of all three proteins observed at the extended consensus motif (22025 bp, Fig. 7C)." "IL-23,IL-23A,IL23P19,P19,SGRF" Mycobacterium Tuberculosis -- D009169 -- -- -- AHR "RP85, bHLHe76" ChIP ChIP analysis revealed that infection enhanced binding of AHR and ARNT to AHRE (31) in the IL10 promoter. -- -- -- <2KB 912 E_01_229 26813977 -- hg19 chr6 30904342 30904788 Human Embryonic Stem Cell Low throughput "CRISPR/Cas9,Luciferase Reporter Assay,4C-seq" "We designed 1964 sgRNA sequences (Supplemental Table S2) targeting these putative cis-regulatory elements with an average of 11 sgRNA per element (Supplemental Fig. S1A). To eliminate these false positives,we required that a positive cis-regulatory element should have at least two distinct sgRNAs enriched by twofold or more in the eGFP? population in at least three out of the four independent experiments (Supplemental Table S3; Supplemental Fig. S2A). By use of this criterion, no negative control sgRNA passed the filter, while six cis-regulatory elements were identified as positives (Fig. 2A). Next, we tested whether the identified elements could function as enhancers in the classical reporter assays (DHS_37,CTCF_38, DHS_65, DHS_108, and DHS_115).We cloned genomic fragments corresponding to each element into a reporter plasmid containing the 360-bp POU5F1 core promoter region and a luciferase reporter gene. All elements, with the exception of DHS_65, exhibited significant enhancer activities in the H1 cells compared with the control plasmid containing only the POU5F1 core promoter sequence (Fig. 2C). We found that all Temp Enhancers, namely, DHS_37, DHS_65, and DHS_108, are located near regions that show high levels of chromatin interactions with the POU5F1 promoter. To confirm this observation, we also performed 4C-seq experiments in H1 POU5F1-eGFP cells. Again, we found these elements near regions displaying high and consistent interaction frequencies with the POU5F1 promoter (Fig. 4D)." Enhancer POU5F1 4C-seq Luciferase Reporter Assay "Next, we tested whether the identified elements could function as enhancers in the classical reporter assays (DHS_37,CTCF_38, DHS_65, DHS_108, and DHS_115).We cloned genomic fragments corresponding to each element into a reporter plasmid containing the 360-bp POU5F1 core promoter region and a luciferase reporter gene. All elements, with the exception of DHS_65, exhibited significant enhancer activities in the H1 cells compared with the control plasmid containing only the POU5F1 core promoter sequence (Fig. 2C). We found that all Temp Enhancers, namely, DHS_37, DHS_65, and DHS_108, are located near regions that show high levels of chromatin interactions with the POU5F1 promoter. To confirm this observation, we also performed 4C-seq experiments in H1 POU5F1-eGFP cells. Again, we found these elements near regions displaying high and consistent interaction frequencies with the POU5F1 promoter (Fig. 4D)." "OCT3, OCT4, OTF-3, OTF3, OTF4, Oct-3, Oct-4" -- -- -- DHS_65 and DHS_108 act in cis to regulate POU5F1 expression CRISPR/Cas9 Those results support our model that DHS_65 and DHS_108 act in cis to regulate POU5F1 transcription. -- -- -- -- -- -- -- >2KB 3219820 E_01_230 26423156 CARDIAC Enhancer hg19 chr5 148785000 148815000 Human Cardiac Progenitor Cell Low+High throughput "ChIP-seq,Immunohistochemistry,Luciferase Reporter Assay,RIP" "Secondly, ChIP-analysis demonstrates that these transcripts mapped to a highly active cardiac enhancer in both fetal and adult human hearts (Fig. 3B). Notably, this region was shown to harbor a car_x0002_diac SRF/NKX2.5-bound enhancer required for cardiac-specific expres_x0002_sion of miR-143 and -145 during cardiac development.Furthermore, this enhancer has been previously demonstrated to be a Notch-responsive enhancer, capable of modulating miR-143 and -145 expressions in various developmental contexts.Accordingly, the enhancer was able to drive robust cardiac-specific activity in a mouse transgenic reporter assays at E11.5 p.c. (Fig. 3C)." Super-Enhancer NKX2-5 -- "ChIP-seq,Immunohistochemistry,Luciferase Reporter Assay,RIP" "These results indicated that the promoter of NOXA physically interacted with the mbr Enhancer over 3.4Mb and might form a promoter-promoter-mbr complex with the BCL2 promoter and the mbr Enhancer in nucleus through High throughputer-order Chroma_x0002_tin structures. " "CHNG5,CSX,CSX1,HLHS2,NKX2.5,NKX2E,NKX4-1,VSD3" -- -- -- -- -- -- -- -- -- -- -- -- -- >2KB 23859106 E_01_231 26445536 -- hg19 chr22 35686840 35687280 Human Vascular Endothelial Cell Low throughput "ChIP,RT-PCR,qPCR" "To investigate whether the translocated Nrf2 interacted with the HO-1 promoter region, the primer sets for the enhancer region (E2) and promoter region near transcription start site were synthesized for ChIP assays (Figure 5A, upper panel schematic). The ChIP analyses demonstrated that AuNP treatment significantly induced Nrf2 binding to the human HO-1 E2 enhancer region, but not to the promoter region near the transcription start site. The binding to the enhancer region was concentration dependent, as determined by RT-PCR and real-time PCR (Figure 5B and C)." Enhancer HMOX1 -- "ChIP,RT-PCR,qPCR" "To investigate whether the translocated Nrf2 interacted with the HO-1 promoter region, the primer sets for the enhancer region (E2) and promoter region near transcription start site were synthesized for ChIP assays (Figure 5A, upper panel schematic). The ChIP analyses demonstrated that AuNP treatment significantly induced Nrf2 binding to the human HO-1 E2 enhancer region, but not to the promoter region near the transcription start site. The binding to the enhancer region was concentration dependent, as determined by RT-PCR and real-time PCR (Figure 5B and C)." "HMOX1D,HO-1,HSP32,bK286B10" -- -- -- -- -- -- -- -- -- -- -- -- -- >2KB 89999 E_01_232 26501517 HS2 enhancer hg19 chr11 5301600 5302400 Human K-562 Low+High throughput "CRISPR/Cas9,qRT-PCR" "To find optimal sgRNAs for repression of the HS2 enhancer by dCas9-KRAB, we designed a panel of 21 sgRNAs (Cr1¨CCr21) to cover the 400-bp core of the HS2 enhancer (Fig. 1a and Supplementary Table 1). Each sgRNA was transiently transfected into human K562 erythroid leukemia cells that were modified to stably express dCas9 or dCas9-KRAB (Supplementary Fig. 1a). The activity of each sgRNA was screened 3 d after transfection by quantitative real-time PCR (qRT-PCR) for expression of HBE1, HBG1 and HBG2 (HBG1/2), and HBB mRNA (Supplementary Fig. 1b¨Cd). When transfected into K562 cells expressing dCas9-KRAB, most of the 21 sgRNAs caused decreased expression of HBE1 and HBG1/2. " Enhancer "HBE1,HBG1,HBG2" CRISPR/Cas9 qRT-PCR "To find optimal sgRNAs for repression of the HS2 enhancer by dCas9-KRAB, we designed a panel of 21 sgRNAs (Cr1¨CCr21) to cover the 400-bp core of the HS2 enhancer (Fig. 1a and Supplementary Table 1). Each sgRNA was transiently transfected into human K562 erythroid leukemia cells that were modified to stably express dCas9 or dCas9-KRAB (Supplementary Fig. 1a). The activity of each sgRNA was screened 3 d after transfection by quantitative real-time PCR (qRT-PCR) for expression of HBE1, HBG1 and HBG2 (HBG1/2), and HBB mRNA (Supplementary Fig. 1b¨Cd). When transfected into K562 cells expressing dCas9-KRAB, most of the 21 sgRNAs caused decreased expression of HBE1 and HBG1/2. " "HBE,HBG-T2,HBGA,HBGR,HSGGL1,PRO2979,HBG-T1,TNCY" -- -- -- -- -- -- TRIM28 " KAP1, PPP1R157, RNF96, TF1B, TIF1B" ChIP-qPCR We used ChIP-qPCR to evaluate disruption of the interactions of GATA2 and the AP-1 subunit FOSL1 with HS2 by dCas9-KRAB or dCas9. -- -- -- >2KB 12421 E_01_233 26560027 -- hg19 chr11 8271697 8279492 Human Neuroblastoma Cell Lines Low+High throughput "ChIP-seq,Luciferase Reporter Assay" "Acetylation of histone H3 at lysine 27 (H3K27ac) is a hallmark of active enhancers, and ChIP-seq analysis of SHSY5Y (G/G; not MYCN amplified), KELLY (G/?; MYCN amplified), BE2 (G/T; MYCN amplified) and NGP (G/T; MYCN amplified) neuroblastoma cells showed extensive H3K27 acetylation in the first intron of LMO1 across rs2168101, which was not observed in BE2C (T/? ; MYCN amplified; Fig. 3c). This region is classified as a super-enhancer in G-allele-containing lines SHSY5Y, KELLY and BE2 based on enhancer clustering and especially high H3K27ac signal (NGP was just below the threshold; see Methods), a pattern also observed for other known oncogenes and tumour suppressor genes in this disease12 (Fig. 3d and Extended Data Fig. 6a). We next performed luciferase reporter assays to measure the effect of rs2168101 alleles on enhancer activity. HEK293T cells transfected with constructs containing the risk G allele demonstrated 30¨C300-fold higher normalized luminescence compared to the T allele (t-test P = 0.002, Fig. 3e), whereas luciferase activity of the T allele was not significantly different from empty vector, indicating that the intact GATA motif is required for robust enhancer activity. " Super-Enhancer LMO1 -- "ChIP-seq,Luciferase Reporter Assay" "Acetylation of histone H3 at lysine 27 (H3K27ac) is a hallmark of active enhancers, and ChIP-seq analysis of SHSY5Y (G/G; not MYCN amplified), KELLY (G/?; MYCN amplified), BE2 (G/T; MYCN amplified) and NGP (G/T; MYCN amplified) neuroblastoma cells showed extensive H3K27 acetylation in the first intron of LMO1 across rs2168101, which was not observed in BE2C (T/? ; MYCN amplified; Fig. 3c). This region is classified as a super-enhancer in G-allele-containing lines SHSY5Y, KELLY and BE2 based on enhancer clustering and especially high H3K27ac signal (NGP was just below the threshold; see Methods), a pattern also observed for other known oncogenes and tumour suppressor genes in this disease12 (Fig. 3d and Extended Data Fig. 6a). We next performed luciferase reporter assays to measure the effect of rs2168101 alleles on enhancer activity. HEK293T cells transfected with constructs containing the risk G allele demonstrated 30¨C300-fold higher normalized luminescence compared to the T allele (t-test P = 0.002, Fig. 3e), whereas luciferase activity of the T allele was not significantly different from empty vector, indicating that the intact GATA motif is required for robust enhancer activity. " "RBTN1,RHOM1,TTG1" Neuroblastoma DOID:769 D009447 -- -- -- GATA3 "HDR, HDRS" "Luciferase Reporter Assay,RNAi" "HEK293T cells transfected with constructs containing the risk G allele demonstrated 30¨C300-fold higher normalized luminescence compared to the T allele (t-test P = 0.002, Fig. 3e), whereas luciferase activity of the T allele was not significantly different from empty vector, indicating that the intact GATA motif is required for robust enhancer activity. Finally, knockdown of GATA3 in SHSY5Y and KELLY cells resulted in both decreased LMO1 protein levels and suppression of cell growth that was rescued by LMO1 overexpression (Fig. 3f and Extended Data Fig. 7), indicating the central role of GATA3 in regulating LMO1 expression levels in neuroblastoma.Finally, knockdown of GATA3 in SHSY5Y and KELLY cells resulted in both decreased LMO1 protein levels and suppression of cell growth that was rescued by LMO1 overexpression (Fig. 3f and Extended Data Fig. 7), indicating the central role of GATA3 in regulating LMO1 expression levels in neuroblastoma." -- -- -- >2KB 29745 E_01_234 26604133 -- hg19 chr4 26075179 26135897 Human CD4+ T Cell Low+High throughput "ChIP-seq,PCR,ELISA,qPCR" "Genome-wide association studies of rheumatoid arthritis (RA) identi?ed a susceptibility locus, rs874040CC, which implicated the RBPJ gene. Here, chromatin state mapping generated using the chromHMM algorithm reveals strong enhancer regions containing DNase I hypersensitive sites overlapping the rs874040 linkage disequilibrium block in human memory, but not in na?ve CD4+ T cells. The rs874040 overlapping this chromatin state was associated with increased RBPJ expression in stimulated memory CD4+ T cells from healthy subjects homozygous for the risk allele (CC) compared with memory CD4+ T cells bearing the protective allele (GG). In order to understand whether rs874040 allele status can impact gene regulation,we utilized chromatin states annotation of the human reference genome predicted using the chromHMM algorithm (26) with 15-state model parameters that are provided by the NIH Roadmap Epigenomics Consortium in public domain (27).Our analysis reveals (i) enhancer regions containing DNase I hypersensitive sites, and (ii)weak transcriptio nmarks within the rs874040 LD block and upstream of the RBPJ transcription start site (TSS) in memory but not in na?ve T cells (Fig. 1A and B). We found that healthy donors homozygous for the rs874040CC risk allele have increased RBPJ mRNA expression (P = 0.0017) in CD4+ CD45RO+ memory T cells (Fig. 2C). This was con?rmed at the protein level by ELISA, showing signi?cant elevated RBPJ expression in CD4+ CD45RO+ memory T cells (P =0.04) (Fig. 2D). All RBPJ transcripts contain a putative nuclear localization signal, an important component of RBPJ nuclear translocation to activate target gene transcription (32).Therefore,we carried out an isoform speci?c quantitative PCR (qPCR) assay in TCR-stimulated memory T cells. We found a rapid increase in Isoform2 (NM_015874) (P = 0.042), Isoform3 (NM_202283) (P = 0.0083)and Isoform 4 (NM_202284) (P =8.29¡Á10?09 )3h after stimulation of memory T cells from subjects with the rs874040CC risk allele, whilethe expression of Isoform 1 (NM_005349) was unchanged (Supple-mentary Material, Fig. S1B)." Enhancer RBPJ -- "PCR,ELISA,qPCR" "In order to understand whether rs874040 allele status can impact gene regulation,we utilized chromatin states annotation of the human reference genome predicted using the chromHMM algorithm (26) with 15-state model parameters that are provided by the NIH Roadmap Epigenomics Consortium in public domain (27).Our analysis reveals (i) enhancer regions containing DNase I hypersensitive sites, and (ii)weak transcriptio nmarks within the rs874040 LD block and upstream of the RBPJ transcription start site (TSS) in memory but not in na?ve T cells (Fig. 1A and B). We found that healthy donors homozygous for the rs874040CC risk allele have increased RBPJ mRNA expression (P = 0.0017) in CD4+ CD45RO+ memory T cells (Fig. 2C). This was con?rmed at the protein level by ELISA, showing signi?cant elevated RBPJ expression in CD4+ CD45RO+ memory T cells (P =0.04) (Fig. 2D). All RBPJ transcripts contain a putative nuclear localization signal, an important component of RBPJ nuclear translocation to activate target gene transcription (32).Therefore,we carried out an isoform speci?c quantitative PCR (qPCR) assay in TCR-stimulated memory T cells. We found a rapid increase in Isoform2 (NM_015874) (P = 0.042), Isoform3 (NM_202283) (P = 0.0083)and Isoform 4 (NM_202284) (P =8.29¡Á10?09 )3h after stimulation of memory T cells from subjects with the rs874040CC risk allele, whilethe expression of Isoform 1 (NM_005349) was unchanged (Supple-mentary Material, Fig. S1B)." "AOS3,CBF1,IGKJRB,IGKJRB1,KBF2,RBP-J,RBPJK,RBPSUH,SUH,csl" Rheumatoid Arthritis DOID:7148 D001172 -- -- -- -- -- -- -- "rs932036,rs874040" "2609086, 26108197" "ChIP,PCR" >2KB 215793 E_01_235 26610392 -- hg19 chr11 118540532 118575295 Human Normal Human Astrocyte Low throughput "Luciferase Reporter Assay,3C" "After testing candidate SNPs for allele-specific activity in a luciferase-based enhancer scanning assay, we established a subset of 10 functional SNPs in the promoters of PHLDB1 and DDX6, and in a putative enhancer element. Chromatin conformation capture (3C) identified a physical interaction between the enhancer element containing a functional SNP (rs73001406) and the promoter of the DDX6 gene." Enhancer DDX6 3C Luciferase Reporter Assay "After testing candidate SNPs for allele-specific activity in a luciferase-based enhancer scanning assay, we established a subset of 10 functional SNPs in the promoters of PHLDB1 and DDX6, and in a putative enhancer element. Chromatin conformation capture (3C) identified a physical interaction between the enhancer element containing a functional SNP (rs73001406) and the promoter of the DDX6 gene." "LL5A,HLR2,P54,RCK" Glioma DOID:3070 D005910 -- -- -- -- -- -- -- -- -- -- >2KB 80702 E_01_236 26615198 -- hg19 chr7 117011495 117063767 Human "Human Nasal Epithelial Cell,Skin Fibroblasts Cell" Low+High throughput "Hi-C,5C,Luciferase Reporter Assay,PCR" "We tested whether any of the newly identiied looping regions displayed enhancer activity in a reporter assay. As shown in Figure 4A, two of our interacting regions significantly enhanced CFTR promoter activity.The fragment encompassing a DHS in the region E displayed a strong Enhancer function with almost 6-fold effect on the CFTR promoter." Enhancer CFTR -- Luciferase Reporter Assay "As shown in Figure 4A, two of our interacting regions significantly enhanced CFTR promoter activity.The fragment encompassing a DHS in the region E displayed a strong Enhancer function with almost 6-fold effect on the CFTR promoter." "ABC35,ABCC7,CF,CFTR/MRP,MRP7,TNR-CFTR,dJ760C5.1" Cystic Fibrosis DOID:1485 D003550 -- -- -- -- -- -- -- -- -- -- >2KB 82385 E_01_237 26615198 -- hg19 chr7 117282955 117335227 Human "Human Nasal Epithelial Cell,Skin Fibroblasts Cell" Low+High throughput "Hi-C,5C,Luciferase Reporter Assay,PCR" "We tested whether any of the newly identiied looping regions displayed enhancer activity in a reporter assay. As shown in Figure 4A, two of our interacting regions significantly enhanced CFTR promoter activity.The fragment encompassing a DHS in the region E displayed a strong Enhancer function with almost 6-fold effect on the CFTR promoter." Enhancer CFTR -- Luciferase Reporter Assay "As shown in Figure 4A, two of our interacting regions significantly enhanced CFTR promoter activity. The G region containing a DHS and a CTCF binding site also acted as an Enhancer." "ABC35,ABCC7,CF,CFTR/MRP,MRP7,TNR-CFTR,dJ760C5.1" Epithelial Cancer -- -- -- -- -- -- -- -- -- -- -- -- >2KB 189075 E_01_238 26626481 MYC Super-Enhancer hg19 chr8 130386715 130591515 Human Acute Myeloid Leukemia Cell Low throughput "ChIP,ChIP-qPCR" "BRD4 has been shown previously to regulate Myc expression in AML cell via a super-Enhancer (with individual Enhancer constituents E1-E5) located 1.7 Mb downstream of the Myc promoter. ChIP analysis revealed that H3K36 dimethylation, but not H3K36 mono- and tri-methylation, broadly correlated with NSD3-short occupancy at the Myc E1¨CE5 super-enhancer (Figures S5B and S5C and data not shown). Using the ectopically expressed FLAG-tagged NSD3-short, we con?rmed the association of this isoform with the Myc E1¨CE5 super-enhancer using ChIP with anti-FLAG antibodies (Figure 6A)." Super-Enhancer MYC -- "ChIP,ChIP-qPCR" "BRD4 has been shown previously to regulate Myc expression in AML cell via a super-Enhancer (with individual Enhancer constituents E1-E5) located 1.7 Mb downstream of the Myc promoter. ChIP analysis revealed that H3K36 dimethylation, but not H3K36 mono- and tri-methylation, broadly correlated with NSD3-short occupancy at the Myc E1¨CE5 super-enhancer (Figures S5B and S5C and data not shown). Using the ectopically expressed FLAG-tagged NSD3-short, we con?rmed the association of this isoform with the Myc E1¨CE5 super-enhancer using ChIP with anti-FLAG antibodies (Figure 6A)." "MRTL,MYCC,bHLHe39,c-Myc" -- -- -- -- -- -- -- -- -- -- -- -- -- >2KB 1740801 E_01_239 26754925 -- hg19 chr1 181117806 181122451 Human HCT 116 Low+High throughput "ChIP-seq,Luciferase Reporter Assay,4C-seq" "ChIP-seq analysis identified two p53 bind-ing sites 11 kb (RE1) and 46 kb (RE2) downstream of the IER5 gene (Fig.?1F).p53 binding to RE2 was strong,while binding to RE1 was relatively weak. In addition, H3K27 acetylation was detected at RE2, and was strongly increased by 5-FU treatment, showing that RE2 is an active enhancer that is highly activated upon DNA damage.We found sequences highly similar to the p53 consensus binding sequence at the center of RE2 (TRANSFAC match score; 0.94) but not in RE1 (TRANSFAC match score; 0.74, Fig.?1G). We generated DNA oligonucleotides representing RE1 and RE2, and created a version of RE2 mutated in the p53 consensus sequence (RE2 mut; sequences shown in Fig.?1G), and cloned each upstream of a luciferase reporter gene containing a minimal promoter. As shown in Fig.?1H, p53 strongly activated a reporter containing RE2, but not RE2 mut or RE1. We further performed 4C-seq analysis to identify chromatin regions that might interact with RE2. As shown in Fig.1F, we detected chromatin interaction between RE2 and RE1, and between RE2 and the IER5 gene promoter region, in both HCT116 p53 + /+ and ?/? cells. The interactions between the RE2 and the IER5 promoter region seem to be stabilized by 5-FU treatment in HCT116 p53 +/+ cells but not in HCT116 p53 ?/? cells. Collectively, these results show that RE2 is localized in the vicinity of the IER5 promoter when a higher order chromatin structure is formed, and identifies RE2 as the p53-responsive element of the IER5 gene. " Enhancer IER5 4C-seq Luciferase Reporter Assay "We generated DNA oligonucleotides representing RE1 and RE2, and created a version of RE2 mutated in the p53 consensus sequence (RE2 mut; sequences shown in Fig.?1G), and cloned each upstream of a luciferase reporter gene containing a minimal promoter. As shown in Fig.?1H, p53 strongly activated a reporter containing RE2, but not RE2 mut or RE1. We further performed 4C-seq analysis to identify chromatin regions that might interact with RE2. As shown in Fig.1F, we detected chromatin interaction between RE2 and RE1, and between RE2 and the IER5 gene promoter region, in both HCT116 p53 + /+ and ?/? cells. The interactions between the RE2 and the IER5 promoter region seem to be stabilized by 5-FU treatment in HCT116 p53 +/+ cells but not in HCT116 p53 ?/? cells. Collectively, these results show that RE2 is localized in the vicinity of the IER5 promoter when a higher order chromatin structure is formed, and identifies RE2 as the p53-responsive element of the IER5 gene. " SBBI48 Cervical Cancer DOID:4362 D002583 -- -- -- TP53 "BCC7,BMFS5,LFS1,P53,TRP53" Luciferase Reporter Assay "ChIP-seq analyses were performed using antibodies against p53, H3K27ac, H3K4me1, H3K4me3 and phospho-RNAP II. Two p53 binding sites RE1 (11 kb downstream) and RE2 (46 kb downstream) were identified." -- -- -- >2KB 62492 E_01_240 26813977 -- hg19 chr6 31139599 31139921 Human Embryonic Stem Cell Low throughput "CRISPR/Cas9,Luciferase Reporter Assay" "We designed 1964 sgRNA sequences (Supplemental Table S2) targeting these putative cis-regulatory elements with an average of 11 sgRNA per element (Supplemental Fig. S1A). To eliminate these false positives,we required that a positive cis-regulatory element should have at least two distinct sgRNAs enriched by twofold or more in the eGFP? population in at least three out of the four independent experiments (Supplemental Table S3; Supplemental Fig. S2A). By use of this criterion, no negative control sgRNA passed the filter, while six cis-regulatory elements were identified as positives (Fig. 2A). Next, we tested whether the identified elements could function as enhancers in the classical reporter assays (DHS_37,CTCF_38, DHS_65, DHS_108, and DHS_115).We cloned genomic fragments corresponding to each element into a reporter plasmid containing the 360-bp POU5F1 core promoter region and a luciferase reporter gene. All elements, with the exception of DHS_65, exhibited significant enhancer activities in the H1 cells compared with the control plasmid containing only the POU5F1 core promoter sequence (Fig. 2C). Cells with mutations at this proximal Enhancer could not be expanded as they quickly differentiated (Fig. 2E), suggesting that DHS_115 is a major regulator of POU5F1 expression in hESCs. Of note, this region is within a previously defined hESC-specific proximal Enhancer region (Chr 6: 31,247,052¨C31,248,218) that controls POU5F1 expression in primed hESCs (Gafni et al. 2013)." Enhancer POU5F1 4C-seq Luciferase Reporter Assay "Next, we tested whether the identified elements could function as enhancers in the classical reporter assays (DHS_37,CTCF_38, DHS_65, DHS_108, and DHS_115).We cloned genomic fragments corresponding to each element into a reporter plasmid containing the 360-bp POU5F1 core promoter region and a luciferase reporter gene. All elements, with the exception of DHS_65, exhibited significant enhancer activities in the H1 cells compared with the control plasmid containing only the POU5F1 core promoter sequence (Fig. 2C). Consistent with the results from the reporter assay, a cell clone with mutations at DHS_115 (one allele with 13-bp deletion and the other allelewith 4-bp substitution of the 17-bp original sequences) exhibited reduced eGFP levels, which decreased further in additional passages. We found that all Temp Enhancers, namely, DHS_37, DHS_65, and DHS_108, are located near regions that show high levels of chromatin interactions with the POU5F1 promoter. To confirm this observation, we also performed 4C-seq experiments in H1 POU5F1-eGFP cells. Again, we found these elements near regions displaying high and consistent interaction frequencies with the POU5F1 promoter (Fig. 4D)." "OCT3, OCT4, OTF-3, OTF3, OTF4, Oct-3, Oct-4" -- -- -- -- -- -- -- -- -- -- -- -- -- >2KB 7647 E_01_241 26813977 -- hg19 chr6 30646843 30647217 Human Embryonic Stem Cell Low throughput "CRISPR/Cas9,Luciferase Reporter Assay,4C-seq" "We designed 1964 sgRNA sequences (Supplemental Table S2) targeting these putative cis-regulatory elements with an average of 11 sgRNA per element (Supplemental Fig. S1A). To eliminate these false positives,we required that a positive cis-regulatory element should have at least two distinct sgRNAs enriched by twofold or more in the eGFP? population in at least three out of the four independent experiments (Supplemental Table S3; Supplemental Fig. S2A). By use of this criterion, no negative control sgRNA passed the filter, while six cis-regulatory elements were identified as positives (Fig. 2A). Next, we tested whether the identified elements could function as enhancers in the classical reporter assays (DHS_37,CTCF_38, DHS_65, DHS_108, and DHS_115).We cloned genomic fragments corresponding to each element into a reporter plasmid containing the 360-bp POU5F1 core promoter region and a luciferase reporter gene. All elements, with the exception of DHS_65, exhibited significant enhancer activities in the H1 cells compared with the control plasmid containing only the POU5F1 core promoter sequence (Fig. 2C). We found that all Temp Enhancers, namely, DHS_37, DHS_65, and DHS_108, are located near regions that show high levels of chromatin interactions with the POU5F1 promoter. To confirm this observation, we also performed 4C-seq experiments in H1 POU5F1-eGFP cells. Again, we found these elements near regions displaying high and consistent interaction frequencies with the POU5F1 promoter (Fig. 4D)." Enhancer POU5F1 4C-seq Luciferase Reporter Assay "Next, we tested whether the identified elements could function as enhancers in the classical reporter assays (DHS_37,CTCF_38, DHS_65, DHS_108, and DHS_115).We cloned genomic fragments corresponding to each element into a reporter plasmid containing the 360-bp POU5F1 core promoter region and a luciferase reporter gene. All elements, with the exception of DHS_65, exhibited significant enhancer activities in the H1 cells compared with the control plasmid containing only the POU5F1 core promoter sequence (Fig. 2C). We found that all Temp Enhancers, namely, DHS_37, DHS_65, and DHS_108, are located near regions that show high levels of chromatin interactions with the POU5F1 promoter. To confirm this observation, we also performed 4C-seq experiments in H1 POU5F1-eGFP cells. Again, we found these elements near regions displaying high and consistent interaction frequencies with the POU5F1 promoter (Fig. 4D)." "OCT3, OCT4, OTF-3, OTF3, OTF4, Oct-3, Oct-4" -- -- -- -- -- -- -- -- -- -- -- -- -- >2KB 485083 E_01_242 26422397 -- hg19 chr18 60786011 60788011 Human Human T Lymphoid Cell Low throughput "3C,ChIP,RT-PCR" "In the present study, we used chromosome conformation capture (3C) assays and molecular analyses to further investigate the function of the SATB1-mediated higher-order chromatin structure in co-regulation of the anti-apoptotic BCL2 gene and the pro-apoptotic NOXA gene located 3.4Mb downstream on Chromosome 18.We emonstrated that the mbr enhancer spatially juxtaposed the promoters of BCL2 and NOXA genes through SATB1-mediated chromatin-loop in Jurkat cells. Decreased SATB1 levels switched the mbr-BCL2 loop to mbr-NOXA loop, and thus changed expression of these two genes. The PCR products derived from the 3C library showed that the NOXA gene promoter juxtaposed with the mbr enhancer and BCL2 gene promoter (Fig 2). The specificity of the PCR products was validated by DNA sequencing (Supporting information: S1 and S2 Figs). These results indicated that the promoter of NOXA physically interacted with the mbr enhancer over 3.4Mb and might form a promoter-promoter-mbr complex with the BCL2 promoter and the mbr enhancer in nucleus through higher-order chroma- tin structures." Enhancer "NOXA,BCL2" 3C "ChIP,RT-PCR" " Our further analyses with 3C assays revealed that the promoter of the NOXA gene that is 3.4Mb downstream of the BCL2 gene on Chromosome 18 also physically interacted with the mbr enhancer, suggesting that the NOXA gene is targeted by the mbr enhancer." "APR,NOXA,Bcl-2,PPP1R50" -- -- -- -- -- -- SATB1 SATB1 "ChIP,3C" We used ChIP assays with the anti-SATB1 antibody to test SATB1 binding on the NOXA promoter. NOXA-SBS1 sequence located 2.4kb relative to the translational start site were found to be specifically immunoprecipitated with anti-SATB1.3C assays to detect physical interactions of the mbr-NOXA and NOXA-BCL2. -- -- -- >2KB 227548 E_01_243 26813977 -- hg19 chr6 31126537 31126685 Human Embryonic Stem Cell Low throughput "CRISPR/Cas9,Luciferase Reporter Assay,4C-seq" "We designed 1964 sgRNA sequences (Supplemental Table S2) targeting these putative cis-regulatory elements with an average of 11 sgRNA per element (Supplemental Fig. S1A). To eliminate these false positives,we required that a positive cis-regulatory element should have at least two distinct sgRNAs enriched by twofold or more in the eGFP? population in at least three out of the four independent experiments (Supplemental Table S3; Supplemental Fig. S2A). By use of this criterion, no negative control sgRNA passed the filter, while six cis-regulatory elements were identified as positives (Fig. 2A). Next, we tested whether the identified elements could function as enhancers in the classical reporter assays (DHS_37,CTCF_38, DHS_65, DHS_108, and DHS_115).We cloned genomic fragments corresponding to each element into a reporter plasmid containing the 360-bp POU5F1 core promoter region and a luciferase reporter gene. All elements, with the exception of DHS_65, exhibited significant enhancer activities in the H1 cells compared with the control plasmid containing only the POU5F1 core promoter sequence (Fig. 2C). We found that all Temp Enhancers, namely, DHS_37, DHS_65, and DHS_108, are located near regions that show high levels of chromatin interactions with the POU5F1 promoter. To confirm this observation, we also performed 4C-seq experiments in H1 POU5F1-eGFP cells. Again, we found these elements near regions displaying high and consistent interaction frequencies with the POU5F1 promoter (Fig. 4D)." Enhancer POU5F1 4C-seq Luciferase Reporter Assay "Next, we tested whether the identified elements could function as enhancers in the classical reporter assays (DHS_37,CTCF_38, DHS_65, DHS_108, and DHS_115).We cloned genomic fragments corresponding to each element into a reporter plasmid containing the 360-bp POU5F1 core promoter region and a luciferase reporter gene. All elements, with the exception of DHS_65, exhibited significant enhancer activities in the H1 cells compared with the control plasmid containing only the POU5F1 core promoter sequence (Fig. 2C). We found that all Temp Enhancers, namely, DHS_37, DHS_65, and DHS_108, are located near regions that show high levels of chromatin interactions with the POU5F1 promoter. To confirm this observation, we also performed 4C-seq experiments in H1 POU5F1-eGFP cells. Again, we found these elements near regions displaying high and consistent interaction frequencies with the POU5F1 promoter (Fig. 4D)." "OCT3, OCT4, OTF-3, OTF3, OTF4, Oct-3, Oct-4" -- -- -- DHS_65 and DHS_108 act in cis to regulate POU5F1 expression CRISPR/Cas9 Those results support our model that DHS_65 and DHS_108 act in cis to regulate POU5F1 transcription. -- -- -- -- -- -- -- >2KB 5502 E_01_244 26934861 ACP10 Enhancer hg19 chr8 128412386 128415392 Human "LNCaP,C4-2B" Low throughput 4C-seq "We examined the enrichment of H3K4 mono-,di-or tri-methylation,H3K36 tri-methylation and H3K27Ac marks around 4C interactome,and found that H3K4me1,H3K4me2,and H3K27Ac were enriched around identified 4C sites (+/? 500 kb) in both LNCaP and C4-2B cells." Enhancer POU5F1B 4C -- "We examined the enrichment of H3K4 mono-,di-or tri-methylation,H3K36 tri-methylation and H3K27Ac marks around 4C interactome,and found that H3K4me1,H3K4me2,and H3K27Ac were enriched around identified 4C sites (+/? 500 kb) in both LNCaP and C4-2B cells. " "OCT4-PG1,OCT4PG1,OTF3C,OTF3P1,POU5F1P1,POU5F1P4,POU5FLC20,POU5FLC8" Prostate Cancer DOID:10283 D011471 -- -- -- "NKX3-1,FOXA1,AR" "BAPX2,NKX3,NKX3.1,NKX3A,HNF3A,TCF3A,AIS,AR8,DHTR,HUMARA,HYSP1,KD,NR3C4,SBMA,SMAX1,TFM" "4C,ChIP-seq" "We thus compared the ChIP-seq profiles of the three factors and examined their peaks enrichment around our 4C sites (+ /? 500 kb), revealing that they were significantly enriched around the 4C loci compared to random sites in both LNCaP (NKX3.1: p = 7.214e-7, FOXA1: p = 7.871e-15, AR: p = 5.707e-12; Wilcoxon rank sum test, Fig.?5B) and C4-2B cells (NKX3.1: p = 4.593e-6, FOXA1: p = 6.989e-10, AR: p = 1.786e-11; Wilcoxon rank sum test, Fig.?5B). Therefore, we think these transcription factors can bind to accessible 4C regions and play a critical role in prostate cancer cell function." -- -- -- >2KB 13967 E_01_245 26957309 -- hg19 chr13 40839693 40853195 Human CD4+ T Cell Low+High throughput "ChIP-seq,Luciferase Reporter Assay" Distinct chromatin signatures of active Enhancers and promoters. Examples of validated CD4+T cell Enhancers (highlighted in yellow) marked by H2BK20ac but not by H3K27ac Enhancer LINC00548 -- ChIP-seq Distinct chromatin signatures of active Enhancers and promoters. Examples of validated CD4+T cell Enhancers (highlighted in yellow) marked by H2BK20ac but not by H3K27ac LINC00548 -- -- -- -- -- -- -- -- -- -- -- -- -- >2KB 77799 E_01_246 26957309 -- hg19 chr9 4600913 4608373 Human CD4+ T Cell Low+High throughput "ChIP-seq,Luciferase Reporter Assay" Distinct chromatin signatures of active Enhancers and promoters. Examples of validated CD4+T cell Enhancers (highlighted in yellow) marked by H2BK20ac but not by H3K27ac Enhancer SLC1A1 -- ChIP-seq Distinct chromatin signatures of active Enhancers and promoters. Examples of validated CD4+T cell Enhancers (highlighted in yellow) marked by H2BK20ac but not by H3K27ac "DCBXA,EAAC1,EAAT3,SCZD18" -- -- -- -- -- -- -- -- -- -- -- -- -- >2KB 114217 E_01_247 26960733 C11ORF82 Enhancer hg19 chr11 82608774 82612886 Human IMR-90 Low+High throughput "3C-qPCR,Hi-C" "To further assess the performance of EpiTensor, we performed chromosome conformation capture coupled with quantitative PCR (3C-qPCR) on 14 randomly selected pairs from EpiTensor. As shown in Fig. 4, eigenvector 1 from tensor decomposition has three peaks, that is, loci i, ii and iii. Locii and iii correspond to the C11orf82 and RAB30-AS1 promoters,respectively, while locus ii corresponds to an active enhancer in IMR90 with H3K4me1/H3K27ac enrichment. " Enhancer DDIAS 3C-qPCR Hi-C "To further assess the performance of EpiTensor, we performed chromosome conformation capture coupled with quantitative PCR (3C-qPCR) on 14 randomly selected pairs from EpiTensor. As shown in Fig. 4, eigenvector 1 from tensor decomposition has three peaks, that is, loci i, ii and iii. Locii and iii correspond to the C11orf82 and RAB30-AS1 promoters,respectively, while locus ii corresponds to an active enhancer in IMR90 with H3K4me1/H3K27ac enrichment. " "C11orf82,noxin" -- -- -- -- -- -- -- -- -- -- -- -- -- <2KB 1906 E_01_248 26968549 -- hg19 chr20 54937784 54938182 Human G1E-ER4 Cells Low+High throughput "ChIP-seq,ChIP,Luciferase Reporter Assay" "ChIP-seq analysis identiied two GATA-1 peaks at intronic region neighboring FAM210B in K562 cells (+1.9 and +4.0, relative to transcription start site) (Fig. 1c, upper) [1]. Quantitative ChIP analysis veriied GATA-1 chromatin occupancy at these loci, based on analyzes of both K562 cells and cord blood-derived primary erythroblasts (Fig. 1c, lower). To evaluate if the GATA-1-dependent transcriptional activation of FAM210B is dependent on the intronic enhancer region, we conducted luciferase assay. In K562 cells, addition of either FAM210B +1.9 or +4.0 kb region to its promoter signiicantly increased luciferase activity, which was signiicantly decreased by the deletion of the GATA sequence within +4.0 kb, but not +1.9 kb (Fig. 1e). These results demonstrated that FAM210B is a bona ide GATA-1 target gene, presumably through acting on the +4.0 kb intronic enhancer region." Enhancer FAM210B -- Luciferase Reporter Assay "To evaluate if the GATA-1-dependent transcriptional activation of FAM210B is dependent on the intronic enhancer region, we conducted luciferase assay. In K562 cells, addition of either FAM210B +1.9 or +4.0 kb region to its promoter signiicantly increased luciferase activity, which was signiicantly decreased by the deletion of the GATA sequence within +4.0 kb, but not +1.9 kb (Fig. 1e). These results demonstrated that FAM210B is a bona ide GATA-1 target gene, presumably through acting on the +4.0 kb intronic enhancer region." "5A3,C20orf108,dJ1167H4.1" -- -- -- -- -- -- GATA1 "ERYF1,GATA-1,GF-1,GF1,NF-E1,NFE1,XLANP,XLTDA,XLTT" "siRNA,ChIP-seq,ChIP,Luciferase Reporter Assay" "Furthermore, siRNA-mediated knockdown of GATA-1 in HiDEP cells resulted in signiicant down regula tion of FAM210B expression (Fig. 1b). ChIP-seq analysis identiied two GATA-1 peaks at intronic region neighboring FAM210B in K562 cells (+1.9 and +4.0, relative to transcription start site) (Fig. 1c, upper) [1]. Quantitative ChIP analysis veriied GATA-1 chromatin occupancy at these loci, based on analyzes of both K562 cells and cord blood-derived primary erythroblasts (Fig. 1c, lower). To evaluate if the GATA-1-dependent transcriptional activation of FAM210B is dependent on the intronic enhancer region, we conducted luciferase assay. In K562 cells, addition of either FAM210B +1.9 or +4.0 kb region to its promoter signiicantly increased luciferase activity, which was signiicantly decreased by the deletion of the GATA sequence within +4.0 kb, but not +1.9 kb (Fig. 1e). These results demonstrated that FAM210B is a bona ide GATA-1 target gene, presumably through acting on the +4.0 kb intronic enhancer region." -- -- -- Intron 4001 E_01_249 27333824 LAMC2 Enhancer hg19 chr1 183149939 183150336 Human Intestinal Epithelial Cell Low throughput Luciferase Reporter Assay "To determine whether CDX2 regulates the basal LAMC2 gene activity, a luciferase reporter gene driven by the LAMC2 promoter with and without the upstream LAMC2 enhancer identi?ed previously [Boyd et al., 2014] was transfected into Caco-2 cells without or with concomitant CDX2 expression plasmid. Over-expression of CDX2 increased the luciferase activity driven by the enhancer-less construct of LAMC2 by 2.5-fold (P?0.028) relative to cells transfected with the LAMC2 reporter vector alone, whereas the LAMC2 promoter with enhancer construct gave a fourfold increase (P?0.006) in reporter signal when co-transfected with CDX2 expression plasmid relative to cells transfected with the LAMC2 promoter/enhancer reporter vector alone (Fig. 1A). The LAMC2 luciferase reporter constructs were: pGL3-LAMC2 containing the 1.2 kb human LAMC2 promoter region upstream of transcription start site [Olsen et al., 2000], and pGL3-LAMC2+ Enhancer containing the 1.2 kb LAMC2 promoter and the enhancer region at chr1:183,149,939¨C183,150,336 (hg19) [Boyd et al., 2014]." Enhancer LAMC2 -- Luciferase Reporter Assay "To determine whether CDX2 regulates the basal LAMC2 gene activity, a luciferase reporter gene driven by the LAMC2 promoter with and without the upstream LAMC2 enhancer identi?ed previously [Boyd et al., 2014] was transfected into Caco-2 cells without or with concomitant CDX2 expression plasmid. Over-expression of CDX2 increased the luciferase activity driven by the enhancer-less construct of LAMC2 by 2.5-fold (P?0.028) relative to cells transfected with the LAMC2 reporter vector alone, whereas the LAMC2 promoter with enhancer construct gave a fourfold increase (P?0.006) in reporter signal when co-transfected with CDX2 expression plasmid relative to cells transfected with the LAMC2 promoter/enhancer reporter vector alone (Fig. 1A)." "B2T,BM600,CSF,EBR2,EBR2A,LAMB2T,LAMNB2" -- -- -- -- -- -- CDX2 "CDX-3/AS, CDX3, CDX2" ChIP-seq "Thus, to further analyze,whether LAMC2 is a direct target of CDX2 also in the native chromatin environment, we performed ChIP experiments with Caco-2 and with cells purified from human colonic tissue samples." -- -- -- >2KB 5035 E_01_250 27602360 NgR13' Enhancer hg19 chr22 20224442 20228467 Human NT2/D1 Low throughput "Luciferase Reporter Assay,PCR" "The presence of overlapping H3K4me1, H3K9ac, and DNase I hypersensitivity peaks suggests the presence of an enhancer in the NgR1 3 ¡ä region. To investigate if this region has potential enhancer activity, chr22: 20224442¨C20228467 was PCR amplified and cloned into a luciferase reporter gene. Substantial increases in luciferase activity were observed for both constructs in the presence of the sodium channel blocker TTX and the NMDA receptor antagonist D-AP5, which blocks neuronal activity ( fig. 4 b)." Enhancer RTN4R -- "Luciferase Reporter Assay,PCR" "The presence of overlapping H3K4me1, H3K9ac, and DNase I hypersensitivity peaks suggests the presence of an enhancer in the NgR1 3 ¡ä region. To investigate if this region has potential enhancer activity, chr22: 20224442¨C20228467 was PCR amplified and cloned into a luciferase reporter gene. Substantial increases in luciferase activity were observed for both constructs in the presence of the sodium channel blocker TTX and the NMDA receptor antagonist D-AP5, which blocks neuronal activity ( fig. 4 b)." "NGR,NOGOR" -- -- -- "The NgR1 3' region, which contains rs701428, is a neuronal activity-dependent enhancer, and that the minor A allele of rs701428 is susceptible to regulation of enhancer activity by MYBL2." "Luciferase Reporter Assay,PCR" "The NgR1 3 ¡ä Enhancer is downregulated by neuronal activation induced by KCl, and enhanced by blockade of neuronal activation by TTX and D-AP5." MYBL2 BMYB; B-MYB EMSA "To examine if the MYB transcription factor family binds to the region with the schizophrenia risk rs701428-A allele,we performed an electrophoretic mobility shift assay (EMSA). The labeled schizophrenia risk rs701428-A allele probe, but not the labeled reference-G allele probe, formed a binding complex with c-MYB, MYBL1, and MYBL2 ( fig.?5 a, arrow). Furthermore, MYBL2 binding to the labeled rs701428-A allele probe was diminished in the presence of a 200-fold excess of unlabeled rs701428-A allele probe but not unlabeled reference-G allele probe ( fig.?5 b, arrow)." rs701428 20241019 "Luciferase Reporter Assay,PCR" >2KB 2482 E_01_251 27651453 NGB Enhancer hg19 chr14 77635927 77639784 Human SH-SY5Y low throughput "3C,Luciferase Reporter Assay,ChIP,CRISPR/Cas9" "By chromosome conformation capture we identi?ed two novel DREs located ?70 kb upstream and +100 kb downstream from the NGB gene. ENCODE database showed the presence of DNaseI hypersensitive and transcription factors binding sites in these regions. Further analyses using luciferase reporters and chromatin immunoprecipitation suggested that the ?70 kb region upstream of the NGB gene contained a neuronal-speci?c enhancer and GATA transcription factor binding sites. To further illustrate the importance of Element I in inducing expression of the NGB gene, Element I was deleted from SH-SY5Y cells by the CRISPR/Cas9 system. We found that deletion of Element I signiicantly decreased NGB expression levels to 13¨C25%of those observed in control SH-SY5Y cells (Figure 4C)." Enhancer NGB 3C "Luciferase Reporter Assay,ChIP" By chromosome conformation capture we identi?ed two novel DREs located ?70 kb upstream and +100 kb downstream from the NGB gene. ENCODE database showed the presence of DNaseI hypersensitive and transcription factors binding sites in these regions. Further analyses using luciferase reporters and chromatin immunoprecipitation suggested that the ?70 kb region upstream of the NGB gene contained a neuronal-speci?c enhancer and GATA transcription factor binding sites. NGB Alzheimer's Disease DOID:10652 D000544 Deletion of element I reduces NGB gene expression "Luciferase Reporter Assay,CRISPR/Cas9" "The regulatory roles of Element I and GATA-2 were demonstrated by the luciferase reporter and knockdown experiments respectively. To further illustrate the importance of Element I in inducing expression of the NGB gene, Element I was deleted from SH-SY5Y cells by the CRISPR/Cas9 system. We found that deletion of Element I signiicantly decreased NGB expression levels to 13¨C25%of those observed in control SH-SY5Y cells (Figure 4C)." GATA2 "DCML,IMD21,MONOMAC,NFE1B" "Luciferase Reporter Assay,ChIP,shRNA" "Further analyses using luciferase reporters and chromatin immunoprecipitation suggested that the ?70 kb region upstream of the NGB gene contained a neuronal- speci?c enhancer and GATA transcription factor binding sites. Knockdown of GATA-2 caused NGB expression to drop dramatically, indicating GATA-2 as an essential transcription factor for the activation of NGB expression. " -- -- -- >2KB 93977 E_01_252 27869826 IGF2 Enhancer hg19 chr11 2219531 2239062 Human HCC-15 Low+High throughput "ChIP-seq,Luciferase Reporter Assay" "We next performed ChIP-Seq at the IGF2 locus, detecting an accumulation of the active chromatin mark H3K27ac at a previously identified IGF2 enhancer66 herein referred to as IGF2 cognate enhancer (Fig. 5a, Supplementary Fig. 11). An even more pronounced H3K27ac peak, however, intersected with an element previously inferred to represent a lineage-specific super-enhancer in CRC cell lines (VACO-400 and VACO-9M)46.To verify enhancer function, we performed luciferase assays revealing enhancer activity of cloned fragments of this previously inferred super-enhancer46 in the HCT116 colon cancer but not in a control (HeLa) cancer cell line (Supplementary Fig. 12 and Supplementary Table 6). " Super-Enhancer IGF2 -- Luciferase Reporter Assay "To verify enhancer function, we performed luciferase assays revealing enhancer activity of cloned fragments of this previously inferred super-enhancer46 in the HCT116 colon cancer but not in a control (HeLa) cancer cell line (Supplementary Fig. 12 and Supplementary Table 6)." "C11orf43,GRDF,IGF-II,PP9974" Lung Cancer DOID:1324 D008175 this IGF2 region indeed interacts with and hence represents a candidate IGF2 Enhancer. 4C-seq we additionally performed 4C-Seq (chromosome conformation capture sequencing63) experiments employing the putative CRE downstream of IRS4 as a viewpoint -- -- -- -- -- -- -- >2KB 78956 E_01_253 27932455 P2RY2 Enhancer hg19 chr11 72905735 72906735 Human MCF-7 Low throughput "smFISH,ChIP,3C" "Here we monitored the expression and localization of eRNAs derived from the enhancers of the FOXC1 and P2RY2 loci, as well as the expression of their cognate mRNAs over a time course of estrogen induction in individual MCF-7 cells by smFISH. In the uninduced state,FOXC1 and P2RY2 eRNAs were expressed at low levels, independently of MLL1 and ER . Estrogen treatment induced the transcription of eRNAs and target mRNAs with similar kinetics, and required both chromatin modiication by MLL1 and ER recruitment. Chromosome conformation capture (3C) analyses conducted in the presence or absence of enhancer-derived tran-scripts proposed that eRNAs mediate enhancer¨Cpromoter interactions (15,21,25).At this time point, 29% of antisense and 19% of sense FOXC1 eRNA¨CmRNA co-expressing alleles showed a separation of 100 nm or less (Figure 5B). These data show that the small percentage of active transcription sites that co-express eRNAs are infrequently transcribed within a closed enhancer¨Cpromoter loop. Furthermore, these observations suggest that looping interactions are either transient or that eRNAs are preferentially transcribed before enhancer¨Cpromoter loops are established (Figure 5C)." Enhancer P2RY2 3C "smFISH,ChIP" "Here we monitored the expression and localization of eRNAs derived from the enhancers of the FOXC1 and P2RY2 loci, as well as the expression of their cognate mRNAs over a time course of estrogen induction in individual MCF-7 cells by smFISH. In the uninduced state,FOXC1 and P2RY2 eRNAs were expressed at low levels, independently of MLL1 and ER . Estrogen treatment induced the transcription of eRNAs and target mRNAs with similar kinetics, and required both chromatin modiication by MLL1 and ER recruitment. Chromosome conformation capture (3C) analyses conducted in the presence or absence of enhancer-derived tran-scripts proposed that eRNAs mediate enhancer¨Cpromoter interactions (15,21,25).At this time point, 29% of antisense and 19% of sense FOXC1 eRNA¨CmRNA co-expressing alleles showed a separation of 100 nm or less (Figure 5B). These data show that the small percentage of active transcription sites that co-express eRNAs are infrequently transcribed within a closed enhancer¨Cpromoter loop. Furthermore, these observations suggest that looping interactions are either transient or that eRNAs are preferentially transcribed before enhancer¨Cpromoter loops are established (Figure 5C)." "HP2U,P2RU1,P2U,P2U1,P2UR,P2Y2,P2Y2R" -- -- -- eRNA accumulation at enhancer¨Cpromoter loops is not required to sustain target gene transcription. 3C Chromosome conformation capture (3C) analyses conducted in the presence or absence of Enhancer-derived transcripts proposed that eRNAs mediate Enhancer¨Cpromoter interactions . -- -- -- -- -- -- -- >2KB 22500 E_01_254 27932455 FOXC1 Enhancer hg19 chr6 1636681 1642781 Human MCF-7 Low throughput "smFISH,ChIP" "Here we monitored the expression and localization of eRNAs derived from the enhancers of the FOXC1 and P2RY2 loci, as well as the expression of their cognate mRNAs over a time course of estrogen induction in individual MCF-7 cells by smFISH. In the uninduced state,FOXC1 and P2RY2 eRNAs were expressed at low levels, independently of MLL1 and ER. Estrogen treatment induced the transcription of eRNAs and target mRNAs with similar kinetics, and required both chromatin modiication by MLL1 and ER recruitment." Enhancer FOXC1 -- "smFISH,ChIP" "Here we monitored the expression and localization of eRNAs derived from the enhancers of the FOXC1 and P2RY2 loci, as well as the expression of their cognate mRNAs over a time course of estrogen induction in individual MCF-7 cells by smFISH. In the uninduced state,FOXC1 and P2RY2 eRNAs were expressed at low levels, independently of MLL1 and ER. Estrogen treatment induced the transcription of eRNAs and target mRNAs with similar kinetics, and required both chromatin modiication by MLL1 and ER recruitment." "ARA,ASGD3,FKHL7,FREAC-3,FREAC3,IGDA,IHG1,IRID1,RIEG3" -- -- -- eRNA accumulation at enhancer¨Cpromoter loops is not required to sustain target gene transcription. 3C Chromosome conformation capture (3C) analyses conducted in the presence or absence of Enhancer-derived transcripts proposed that eRNAs mediate Enhancer¨Cpromoter interactions . -- -- -- -- -- -- -- >2KB 29051 E_01_255 27980063 hg19 ch3 189109942 189219942 Human Mammary Epithelial Cell MCF10A and MCF12A low throughput "qPCR,ChIP" "ChIP analyses of BRD4 and RNAPII occupancy revealed that decreased BRD4 occupanc on enhancers in response to JQ1 or OTX015 treatment leads to a signiicant concomitant reduction in RNAPII occupancy on these regions (Figure 4H, I and K, L). While BRD4 occupancy was not significantly affected around the Np63 TSS, the potential enhancers displayed a substantial reduction in the occupancy of both BRD4 and RNAPII (Figure 4HandI). Most importantly, BRD4 inhibition resulted in the down regulation of the putative TP63- and GRHL3-associated eRNAs (Figure 4J and N)." Enhancer TP63 -- ChIP "ChIP analyses of BRD4 and RNAPII occupancy revealed that decreased BRD4 occupanc on enhancers in response to JQ1 or OTX015 treatment leads to a significant concomitant reduction in RNAPII occupancy on these regions (Figure 4H, I and K, L).Altogether, these studies demonstrate an enhancer--associated function of BRD4 in modulating the expression of TP63 and GRHL3." "AIS, B(p51A), B(p51B), EEC3, KET, LMS, NBP, OFC8, RHS, SHFM4, TP53CP, TP53L, TP73L, p40, p51, p53CP, p63, p73H, p73L" Breast Cancer DOID:1612 D001943 -- -- -- "BRD4,EGF,AK,FOXO" "CAP,HUNK1,HUNKI,MCAP,HOMG4,URG,AK£¬FOXO" ChIP "F) and GRHL3 (G). Active transcriptional start sites and the direction of transcription are indicated by black arrows. Scale bar is denoted in kilobases. (H, I, K, L) ChIP analyses of BRD4 (H, K) and RNAPII (I, L) occupancy on TP63 (H and I) and GRHL3 (K and L) enhancer and TSS regions after JQ1/0TX015 treatment. Immunoprecipitated DNA was normalized to the input and denoted in percentage. Black dotted line denotes the background DNA precipitated by a negative control IgG." -- -- -- >2KB 184000 E_01_256 27980063 hg19 ch3 189203942 189313942 Human Mammary Epithelial Cell MCF10A and MCF12A low throughput "qPCR,ChIP" "ChIP analyses of BRD4 and RNAPII occupancy revealed that decreased BRD4 occupanc on enhancers in response to JQ1 or OTX015 treatment leads to a signiicant concomitant reduction in RNAPII occupancy on these regions (Figure 4H, I and K, L). While BRD4 occupancy was not significantly affected around the Np63 TSS, the potential enhancers displayed a substantial reduction in the occupancy of both BRD4 and RNAPII (Figure 4HandI). Most importantly, BRD4 inhibition resulted in the down regulation of the putative TP63- and GRHL3-associated eRNAs (Figure 4J and N)." Enhancer TP63 -- ChIP "ChIP analyses of BRD4 and RNAPII occupancy revealed that decreased BRD4 occupanc on enhancers in response to JQ1 or OTX015 treatment leads to a significant concomitant reduction in RNAPII occupancy on these regions (Figure 4H, I and K, L).Altogether, these studies demonstrate an enhancer--associated function of BRD4 in modulating the expression of TP63 and GRHL3." "AIS, B(p51A), B(p51B), EEC3, KET, LMS, NBP, OFC8, RHS, SHFM4, TP53CP, TP53L, TP73L, p40, p51, p53CP, p63, p73H, p73L" Breast Cancer DOID:1612 D001943 -- -- -- "BRD4,EGF,AK,FOXO" "CAP,HUNK1,HUNKI,MCAP,HOMG4,URG,AK£¬FOXO" ChIP "F) and GRHL3 (G). Active transcriptional start sites and the direction of transcription are indicated by black arrows. Scale bar is denoted in kilobases. (H, I, K, L) ChIP analyses of BRD4 (H, K) and RNAPII (I, L) occupancy on TP63 (H and I) and GRHL3 (K and L) enhancer and TSS regions after JQ1/0TX015 treatment. Immunoprecipitated DNA was normalized to the input and denoted in percentage. Black dotted line denotes the background DNA precipitated by a negative control IgG." -- -- -- >2KB 90000 E_01_257 27980063 hg19 ch1 24589812 24645812 Human Mammary Epithelial Cell MCF10A and MCF12A low throughput "qPCR,ChIP" "ChIP analyses of BRD4 and RNAPII occupancy revealed that decreased BRD4 occupanc on enhancers in response to JQ1 or OTX015 treatment leads to a signiicant concomitant reduction in RNAPII occupancy on these regions (Figure 4H, I and K, L). While BRD4 occupancy was not significantly affected around the Np63 TSS, the potential enhancers displayed a substantial reduction in the occupancy of both BRD4 and RNAPII (Figure 4HandI). Most importantly, BRD4 inhibition resulted in the down regulation of the putative TP63- and GRHL3-associated eRNAs (Figure 4J and N)." Enhancer GRHL3 -- ChIP "ChIP analyses of BRD4 and RNAPII occupancy revealed that decreased BRD4 occupanc on enhancers in response to JQ1 or OTX015 treatment leads to a significant concomitant reduction in RNAPII occupancy on these regions (Figure 4H, I and K, L).Altogether, these studies demonstrate an enhancer--associated function of BRD4 in modulating the expression of TP63 and GRHL3." "SOM, TFCP2L4, VWS2" Breast Cancer DOID:1612 D001943 -- -- -- "BRD4,EGF,AK,FOXO" "CAP,HUNK1,HUNKI,MCAP,HOMG4,URG,AK£¬FOXO" ChIP "F) and GRHL3 (G). Active transcriptional start sites and the direction of transcription are indicated by black arrows. Scale bar is denoted in kilobases. (H, I, K, L) ChIP analyses of BRD4 (H, K) and RNAPII (I, L) occupancy on TP63 (H and I) and GRHL3 (K and L) enhancer and TSS regions after JQ1/0TX015 treatment. Immunoprecipitated DNA was normalized to the input and denoted in percentage. Black dotted line denotes the background DNA precipitated by a negative control IgG." -- -- -- >2KB 28243 E_01_258 27989796 SR4 Enhancer hg19 chr17 69541883 69542989 Human TM4 Low throughput "Luciferase Reporter Assay,PCR,Transgenic mice,Immunofluorescence" "Our previous work involving the genomic analysis of isolated DSD patients revealed a 78 kb minimal sex determining region (RevSex) far upstream of SOX9 that was duplicated in 46,XX and deleted in 46,XY DSDs. It was postulated that RevSex contains a gonadal enhancer. However, the most highly conserved sub-region within RevSex, called SR4, was neither responsive to sex determining factors in vitro nor active in the gonads of transgenic mice, suggesting that SR4 may not be functioning as a testicular enhancer. Interestingly, SR4 transgenic mice showed reporter activity in the genital tubercle, the primordium of the penis and clitoris, a previously unreported domain of Sox9 expression. SOX9 protein was detected in the genital tubercle, notably in the urethral plate epithelium, preputial glands, ventral surface ectoderm and corpus cavernosa. SR4 may therefore function as a Sox9 genital tubercle enhancer, mutations of which could possibly lead to hypospadias, a birth defect seen in the DSD patients in the RevSex study. SR4 activity and the observed SOX9 expression pattern suggest that SR4 may function as a Sox9 genital tubercle enhancer. However, conditional ablation of Sox9 in the genital tubercle using Shh-Cre/+;Sox9flox/flox mice revealed no genital tubercle abnormalities, possibly due to compensation by similar Sox factors. To conclude, we have identified a novel regulatory enhancer driving Sox9 expression during external genitalia development. " Enhancer SOX9 -- "Transgenic mice,Immunofluorescence" "The transcriptional activity of the SR4 enhancer in the genital tubercle led us to investigate whether SOX9 protein was also expressed there. Immunofluorescence using a SOX9 antibody on genital tubercle sections from E15.5 mouse embryos was performed (Fig. 5). SOX9 was detected in the genital tubercle of both sexes, specifically in the preputial glands, the urethral plate epithelium, ventral surface ectoderm and the corpus cavernosa. " "CMD1,CMPD1,SRA1,SRXX2,SRXY10" Disorders Of Sexual Development -- D012734 -- -- -- SOX9 "CMD1,CMPD1,SRA1,SRXX2,SRXY10" Luciferase Reporter Assay "SR4 and TESC luciferase constructs containing an E1b minimal promoter, as well as the empty E1b-Luc vector, were transiently co-transfected into TM4 cells with expression plasmids bearing SF-1 and SRY, or SF-1 and SOX9. While activation of the TESCO enhancer was observed in luciferase assays, SR4 was not activated by the transcription factors tested. Data are represented in the form of relative luciferase activity." -- -- -- >2KB 574724 E_01_259 28028313 GIMAP Enhancer hg19 chr7 150360500 150366500 Human T-acute lymphoblastic leukemia (T-ALL) Low+High throughput "ChIP-seq,CRISPR/Cas9,PCR" "DNA binding of the TAL1 complex was observed between the GIMAP2 and GIMAP6 genes across different TAL1-positive T-ALL cell lines (CCRF-CEM, RPMI-8402 and Jurkat) (Figures 1A and 1B, orange box). Notably, this region was also occupied by NOTCH1, which is another prevalent oncogene in T-ALL, with its binding partner RBPJ/CSL. .Importantly, there was substantial binding of RNA polymerase II (RNAP2) and mediator 1 (MED1) as well as extensive acetylation of histone H3 lysine 27 (H3K27Ac), constituting the formation of a super-enhancer in this region (Figure 1A, red box; Supplementary Figure 1C). We next examined whether the region bound by the TAL1 complex (Figures 1 and 2A, orange box) can control the expression of GIMAP genes in T-ALL cells.We designed guide RNAs (gRNAs) targeting either this locus or the whole GIMAP gene cluster (Figure 2B, bottom) and transduced a pair of gRNAs with the Cas9 endonuclease to enable removal of these genomic regions using CRISPR/Cas9 technology.PCR analysis showed successful deletions of the target locus (Supplementary Figure 2C). The chromatogram demonstrated a successful recombination after the cleavage (Supplementary Figure 2D). Importantly, compared to cells transduced with control gRNAs, gene expression of the GIMAP genes was concomitantly downregulated in samples where either the TAL1-bound region or the whole cluster was deleted (Figure 2B). These results demonstrate that the locus bound by the TAL1 complex possesses enhancer activity to induce the expression of the GIMAP genes (GIMAP enhancer). Of note, an additional TAL1 binding site was identified between the GIMAP4 and GIMAP7 genes (Figures 1A and 2A, arrowhead)." Super-Enhancer "GIMAP4,GIMAP7" CRISPR/Cas9 PCR "We next examined whether the region bound by the TAL1 complex (Figures 1 and 2A, orange box) can control the expression of GIMAP genes in T-ALL cells.We designed guide RNAs (gRNAs) targeting either this locus or the whole GIMAP gene cluster (Figure 2B, bottom) and transduced a pair of gRNAs with the Cas9 endonuclease to enable removal of these genomic regions using CRISPR/Cas9 technology.PCR analysis showed successful deletions of the target locus (Supplementary Figure 2C). The chromatogram demonstrated a successful recombination after the cleavage (Supplementary Figure 2D). Importantly, compared to cells transduced with control gRNAs, gene expression of the GIMAP genes was concomitantly downregulated in samples where either the TAL1-bound region or the whole cluster was deleted (Figure 2B). These results demonstrate that the locus bound by the TAL1 complex possesses enhancer activity to induce the expression of the GIMAP genes (GIMAP enhancer). Of note, an additional TAL1 binding site was identified between the GIMAP4 and GIMAP7 genes (Figures 1A and 2A, arrowhead)." "IAN-1,IAN1,IMAP4,MSTP062,IAN7,hIAN7" T-Cell Acute Lymphoblastic Leukemia DOID:5602 D054218 -- -- -- TAL1 "SCL,TCL5,bHLHa17,tal-1" ChIP-seq "We previously identified transcriptional targets directly regulated by TAL1 and its regulatory partners by chromatin immunoprecipitation-sequencing (ChIP-Seq) and microarray analysis in TAL1 -positive T-ALL cell samples. " -- -- -- >2KB 99043 E_01_260 28082341 NBEAL2 Enhancer hg19 chr3 46988575 46990242 Human K-562 Low throughput "Luciferase Reporter Assay,ChIP,RT-PCR" "Chromatin immunoprecipitation sequencing revealed 5 GATA binding sites in a regulatory region 31 kb upstream of NBEAL2 covered by a H3K4Me1 mark indicative of an Enhancer locus.Luciferase reporter constructs containing this region confirmed its Enhancer activity in K562 cells, and mutagenesis of the GATA1 binding sites resulted in significantly reduced Enhancer activity. " Enhancer NBEAL2 -- "Luciferase Reporter Assay,ChIP,RT-PCR" "Chromatin immunoprecipitation sequencing revealed 5 GATA binding sites in a regulatory region 31 kb upstream of NBEAL2 covered by a H3K4Me1 mark indicative of an Enhancer locus.Luciferase reporter constructs containing this region confirmed its Enhancer activity in K562 cells, and mutagenesis of the GATA1 binding sites resulted in significantly reduced Enhancer activity. " "BDPLT4,GPS" -- -- -- -- -- -- GATA1 "ERYF1,GATA-1,GF-1,GF1,NF-E1,NFE1,XLANP,XLTDA,XLTT" Immunoblot Immunoblot analysis for NBEAL2 with a rabbit polyclonal antibody and integrin ¦Â3 were performed (left) using total protein MK lysates at days 8 and 11 for the control and day 11 for GATA1 D218Y (right) using total protein MK lysates at day 13 for the control and GATA1 D218G. -- -- -- >2KB 31763 E_01_261 28086087 YY1 Enhancer hg19 chr12 109986000 109986710 Human WT Mouse Embryonic Fibroblasts Low+High throughput "ChIP-seq,ChIP-seq,RT-qPCR" "Our data demonstrate that CBP binds to RNAs transcribed close to CBP binding sites such as at the YY1 Enhancer (Figure 6A);recruitment of CBP to active Enhancers results in binding to eRNAs, and that eRNA binding to the HAT domain of CBP stimulates its acetyltransferase activity " Enhancer YY1 -- "EMSA,ChIP,RT-qPCR" "Our data demonstrate that CBP binds to RNAs transcribed close to CBP binding sites such as at the YY1 Enhancer (Figure 6A);recruitment of CBP to active Enhancers results in binding to eRNAs, and that eRNA binding to the HAT domain of CBP stimulates its acetyltransferase activity " "DELTA,GADEVS,INO80S,NF-E1,UCRBP,YIN-YANG-1" -- -- -- "By interacting directly with CBP, eRNAs contribute to the unique chromatin structure at active enhancers, which in turn is required for regulation of target genes." ChIP "By interacting directly with CBP, eRNAs contribute to the unique chromatin structure at active Enhancers, which in turn is required for regulation of target genes." -- -- -- -- -- -- -- >2KB 9281254 E_01_262 28123038 -- hg19 chrX 55054584 55054678 Human K-562 Low throughput "3C,Luciferase Reporter Assay,RT-qPCR,CRISPR/Cas9" "A luciferase reporter assay conirmed the enhancer activity of this GATA site in vitro (Figure 1E). Furthermore, the physical proximity between the int-1-GATA and int-8-GATA sites and between the promoter and the int-8-GATA site were veriied by 3C assays (Figure 4H and I), suggesting that the promoter, the int-1-GATA site and the int-8-GATA site could form a potential chromatin enhancer loop. To further interrogate the speciic role of proximal int-1-GATA and distal int-8-GATA enhancer elements in ALAS2 regulation, we employed CRISPR/Cas9 technol-ogy to separately delete the GATA1 binding sites from the intron 1 or 8 enhancer regions in K562 cells.As expected,GATA site deletion in int1 6 and int8 4 cells entirely disrupted GATA1 occupancy at the intron 1 and 8 enhancer regions, respectively, whereas each deletion did not affect GATA1 occupancy at the other intronic GATA site and the promoter region (Figure 7C). We detected signiicant reductions in ALAS2 expression in both int1 6 and int8 4 cells during cytosine arabinoside (AraC)-inducedK562 erythroid differentiation (Fig-Nucleic Acids Research, 2017, Vol. 45, No. 2 667 ure 7D and E)." Enhancer ALAS2 "3C,CRISPR/Cas9" "Luciferase Reporter Assay,RT-qPCR" "A luciferase reporter assay conirmed the enhancer activity of this GATA site in vitro (Figure 1E). Furthermore, the physical proximity between the int-1-GATA and int-8-GATA sites and between the promoter and the int-8-GATA site were veriied by 3C assays (Figure 4H and I), suggesting that the promoter, the int-1-GATA site and the int-8-GATA site could form a potential chromatin enhancer loop. To further interrogate the speciic role of proximal int-1-GATA and distal int-8-GATA enhancer elements in ALAS2 regulation, we employed CRISPR/Cas9 technol-ogy to separately delete the GATA1 binding sites from the intron 1 or 8 enhancer regions in K562 cells.As expected,GATA site deletion in int1 6 and int8 4 cells entirely disrupted GATA1 occupancy at the intron 1 and 8 enhancer regions, respectively, whereas each deletion did not affect GATA1 occupancy at the other intronic GATA site and the promoter region (Figure 7C). We detected signiicant reductions in ALAS2 expression in both int1 6 and int8 4 cells during cytosine arabinoside (AraC)-inducedK562 erythroid differentiation (Fig-Nucleic Acids Research, 2017, Vol. 45, No. 2 667 ure 7D and E)." "ALAS-E,ALASE,ANH1,ASB,SIDBA1,XLDPP,XLEPP,XLSA" -- -- -- -- -- -- GATA1 "ERYF1,GATA-1,GF-1,GF1,NF-E1,NFE1,XLANP,XLTDA,XLTT" ChIP-qPCR "The ChIP-qPCR assay showed the binding of GATA1 to the promoter and the intron 1 and 8 enhancer regions in int1_x0002_6, int8_x0002_4 and WT K562 cells. The DNA sequence at the exon 4-intron 4 junction (exon 4/intron 4) acts as a negative control region. Normal rabbit IgG was employed as the control in all ChIP assays." -- -- -- Intron 19144 E_01_263 28123038 -- hg19 chrX 55041578 55041679 Human K-562 Low throughput "3C,Luciferase Reporter Assay,RT-qPCR,CRISPR/Cas9" "In addition to this int-1-GATA site,another GATA site in ALAS2 intron 8 (int-8-GATA) has also been reported to function as an enhancer through luciferase assays (35). Furthermore, the physical proximity between the int-1-GATA and int-8-GATA sites and between the promoter and the int-8-GATA site were veriied by 3C assays (Figure 4H and I), suggesting that the promoter, the int-1-GATA site and the int-8-GATA site could form a potential chromatin enhancer loop. To further interrogate the speciic role of proximal int-1-GATA and distal int-8-GATA enhancer elements in ALAS2 regulation, we employed CRISPR/Cas9 technol-ogy to separately delete the GATA1 binding sites from the intron 1 or 8 enhancer regions in K562 cells.As expected,GATA site deletion in int1 6 and int8 4 cells entirely disrupted GATA1 occupancy at the intron 1 and 8 enhancer regions, respectively, whereas each deletion did not affect GATA1 occupancy at the other intronic GATA site and the promoter region (Figure 7C). We detected signiicant reductions in ALAS2 expression in both int1 6 and int8 4 cells during cytosine arabinoside (AraC)-inducedK562 erythroid differentiation (Fig-Nucleic Acids Research, 2017, Vol. 45, No. 2 667 ure 7D and E)." Enhancer ALAS2 "3C,CRISPR/Cas9" "Luciferase Reporter Assay,RT-qPCR" "In addition to this int-1-GATA site,another GATA site in ALAS2 intron 8 (int-8-GATA) has also been reported to function as an enhancer through luciferase assays (35). Furthermore, the physical proximity between the int-1-GATA and int-8-GATA sites and between the promoter and the int-8-GATA site were veriied by 3C assays (Figure 4H and I), suggesting that the promoter, the int-1-GATA site and the int-8-GATA site could form a potential chromatin enhancer loop. To further interrogate the speciic role of proximal int-1-GATA and distal int-8-GATA enhancer elements in ALAS2 regulation, we employed CRISPR/Cas9 technol-ogy to separately delete the GATA1 binding sites from the intron 1 or 8 enhancer regions in K562 cells.As expected,GATA site deletion in int1 6 and int8 4 cells entirely disrupted GATA1 occupancy at the intron 1 and 8 enhancer regions, respectively, whereas each deletion did not affect GATA1 occupancy at the other intronic GATA site and the promoter region (Figure 7C). We detected signiicant reductions in ALAS2 expression in both int1 6 and int8 4 cells during cytosine arabinoside (AraC)-inducedK562 erythroid differentiation (Fig-Nucleic Acids Research, 2017, Vol. 45, No. 2 667 ure 7D and E)." "ALAS-E,ALASE,ANH1,ASB,SIDBA1,XLDPP,XLEPP,XLSA" -- -- -- -- -- -- GATA1 "ERYF1,GATA-1,GF-1,GF1,NF-E1,NFE1,XLANP,XLTDA,XLTT" ChIP-qPCR "The ChIP-qPCR assay showed the binding of GATA1 to the promoter and the intron 1 and 8 enhancer regions in int1_x0002_6, int8_x0002_4 and WT K562 cells. The DNA sequence at the exon 4-intron 4 junction (exon 4/intron 4) acts as a negative control region. Normal rabbit IgG was employed as the control in all ChIP assays." -- -- -- Intron 6142 E_01_264 28210006 -- hg19 chr1 92921568 92937818 Human HEL Low+High throughput "ChIP-seq,ChIP-qPCR" "Lysine-speci?c demethylase 1 (LSD1, also known as KDM1A) is the ?rst discovered histone lysine (K) demethylase and has a FAD-dependent activity to demethylase H3K4me1 and H3K4me2 but not H3K4me3.NCD25 and NCD38, small-molecule inhibitors of LSD1. In one of the LSD1 signature, the GFI1 locus, we could identify three regulatory elements de?ned as H3K4me2 peaks (Figure 3a). One of these elements (element 1) was thought to be a promoter because of coincidence of an H3K4me3 peak around the transcription starting sites, whereas the other elements (element 2 and element 3) were thought to be putative enhancers because of location far from the gene body and no H3K4me3 peaks. More notably, H3K27ac peaks, which are a marker of active enhancers, were elevated by NCD38 treatment in element 2. ChIP¨Cquantitative PCR (qPCR) showed that H3K27ac level in the GFI1-SE (element 2) was elevated by twofold after 3 h of NCD38 treatment and up to sixfold after 12 h of treatment. The transcript level of GFI1 hardly increased until after 6 h of treatment but clearly increased after 12 h of treatment. " Super-Enhancer GFI1 -- "ChIP-seq,ChIP-qPCR" "In one of the LSD1 signature, the GFI1 locus, we could identify three regulatory elements de?ned as H3K4me2 peaks (Figure 3a). One of these elements (element 1) was thought to be a promoter because of coincidence of an H3K4me3 peak around the transcription starting sites, whereas the other elements (element 2 and element 3) were thought to be putative enhancers because of location far from the gene body and no H3K4me3 peaks. More notably, H3K27ac peaks, which are a marker of active enhancers, were elevated by NCD38 treatment in element 2. ChIP¨Cquantitative PCR (qPCR) showed that H3K27ac level in the GFI1-SE (element 2) was elevated by twofold after 3 h of NCD38 treatment and up to sixfold after 12 h of treatment. The transcript level of GFI1 hardly increased until after 6 h of treatment but clearly increased after 12 h of treatment. " "GFI-1A, SCN2, ZNF163, GFI1" Leukemia DOID:1240 D007938 -- -- -- -- -- -- -- -- -- -- >2KB 10624 E_01_265 28244015 pCREB Enhancer hg19 chr3 181572712 181574712 Human MCF-7 Low throughput "ChIP,qPCR" "To determine whether BPA-activated pCREB directly regulates SOX2 , ChIP was performed after a 5-minute treatment with either E2 or BPA. pCREB was enriched at an Enhancer region located 143 kilobases downstream of the SOX2 coding region only after BPA treatment (Figure 6), which correlates with gene expression" Enhancer SOX2 -- "ChIP,qPCR" "To determine whether BPA-activated pCREB directly regulates SOX2 , ChIP was performed after a 5-minute treatment with either E2 or BPA. pCREB was enriched at an Enhancer region located 143 kilobases downstream of the SOX2 coding region only after BPA treatment (Figure 6), which correlates with gene expression" "ANOP3, MCOPS3" -- -- -- -- -- -- DST "BP240,BPA,BPAG1,CATX-15,CATX15,D6S1101,DMH,DT,EBSB2,HSAN6,MACF2" ChIP "To determine whether BPA-activated pCREB directly regulates SOX2, ChIP was performed after a 5-minute treatment with either E2 or BPA. pCREB was enriched at an Enhancer region located 143 kilobases downstream of the SOX2 coding region only after BPA treatment (Figure 6), which correlates with gene expression." -- -- -- >2KB 144001 E_01_266 28260788 LMO1 Enhancer hg19 chr11 8240851 8242851 Human "T-acute lymphoblastic leukemia (T-ALL),RPMI-8402 and Jurkat" Low+High throughput "ChIP-seq,Luciferase Reporter Assay,ChIA-PET" "To identify mutations in the T-ALL genome that might signi?cantly alter oncogene expression, we focused our search on aberrant,sample-speci?c enhancers in 10 different human T-ALL cell lines as identi?ed by H3K27ac ChIP-seq. The aberrant active enhancer we identi?ed was present upstream of the LMO1 gene in RPMI-8402 and Jurkat T-ALL cell lines. To ascertain whether this single base-pair substitution can activate LMO1 gene expression, we cloned a 585-bp genomic DNA fragment from either the C allele or T allele upstream of luciferase and tested the enhancer activity of this fragment in a reporter assay.When introduced into Jurkat cells, the construct containing the T allele exhibited robust reporter activity, which was four-fold greater than that of the fragment containing the C allele (Figure 3f).Taken together, we have shown that the somatically acquired C-to-T mutation that creates a MYB binding motif ~ 4 kb upstream of the proximal transcription start site of LMO1 in T-ALL can generate an active transcriptional enhancer that drives monoallelic overexpression of the LMO1 oncogene. We also used chromatin interaction analysis with paired-end tag sequencing (ChIA-PET) 24 in Jurkat cells to demonstrate that the stream of the proximal LMO1 transcription start site used by Jurkat cells (Figure 5d and Supplementary Figure S1), indicating that the active enhancer mediated by the acquired MYB binding motif loops to the LMO1 gene promoter region to regulate the transcription of LMO1 (Figure 5e). " Enhancer LMO1 -- Luciferase Reporter Assay "To ascertain whether this single base-pair substitution can activate LMO1 gene expression, we cloned a 585-bp genomic DNA fragment from either the C allele or T allele upstream of luciferase and tested the enhancer activity of this fragment in a reporter assay.When introduced into Jurkat cells, the construct containing the T allele exhibited robust reporter activity, which was four-fold greater than that of the fragment containing the C allele (Figure 3f).Taken together, we have shown that the somatically acquired C-to-T mutation that creates a MYB binding motif ~ 4 kb upstream of the proximal transcription start site of LMO1 in T-ALL can generate an active transcriptional enhancer that drives monoallelic overexpression of the LMO1 oncogene." "RBTN1,RHOM1,TTG1" -- -- -- -- -- -- MYB "Cmyb, c-myb, c-myb_CDS, efg" ChIP-seq "Analysis of the genomic sequences of both C and T alleles, using UniPROBE and HOCOMOCO databases, identi?ed a de novo-binding motif for the MYB transcription factor (Figure 3a and Supplementary Table S3), while analysis of the MYB and H3K27ac ChIP-seq DNA sequence reads aligned with this site demon-strated that MYB and H3K27ac were bound almost exclusively by the T allele (Figure 3b).Knockdown of MYB expression using lentivirus-transduced shRNA decreased the expression of LMO1 signi?cantly in Jurkat cells (Figure 3d), indicating that MYB binding to the somatically acquired heterozygous MYB binding motif leads to enhanced expression of LMO1 in T-ALL from the same allele." -- -- -- >2KB 3999 E_01_267 28262837 FBP1 Enhancer hg19 chr9 97396388 97401553 Human Hepatocellular Carcinoma Low+High throughput "ChIP-seq,ChIP,RT-PCR" "We noticed that there is a putative H3K4me1-positive enhancer region in the first intron of the FBP1 gene (Fig.?4E).Importantly, we found that at the same region there is a peak of histone H3 lysine-27 acetylation (H3K27Ac) (Fig.?4E), the level of which often correlates with transcriptional activity of the gene. We first examined whether treatment of the HDAC inhibitor NaBu affects H3K27Ac levels in this region. To this end, we erformed chromatin immunoprecipitation (ChIP) assays using H3K27Ac-specific antibody. As demonstrated in Fig.?4F, the H3K27Ac level was readily detectable in the enhancer region of FBP1 gene in both HepG2 and SK-Hep1 cell lines.However, consistent with the finding that NaBu treatment markedly increased expression of FBP1 mRNA (Fig.?2A), it also significantly increased H3K27Ac level in the FBP1 enhancer (Fig.?4F). Similar to the finding that HDAC1 and HDAC2 knockdown induced FBP1 expression, depletion of these proteins alone or together also largely increased H3K27Ac level at this locus (Fig.?4G). This data indicate that the FBP1 gene locus indeed harbors a regulatory region where H3K27Ac level can be modulated by HDAC proteins. Thus, HDAC inhibitor-induced upregulation of H3K27 acetylation in the enhancer of the FBP1 gene correlates with increased expression of FBP1 induced by HDAC inhibitors." Enhancer FBP1 -- "ChIP-seq,ChIP,RT-PCR" "We noticed that there is a putative H3K4me1-positive enhancer region in the first intron of the FBP1 gene (Fig.?4E).Importantly, we found that at the same region there is a peak of histone H3 lysine-27 acetylation (H3K27Ac) (Fig.?4E), the level of which often correlates with transcriptional activity of the gene. We first examined whether treatment of the HDAC inhibitor NaBu affects H3K27Ac levels in this region. To this end, we erformed chromatin immunoprecipitation (ChIP) assays using H3K27Ac-specific antibody. As demonstrated in Fig.?4F, the H3K27Ac level was readily detectable in the enhancer region of FBP1 gene in both HepG2 and SK-Hep1 cell lines.However, consistent with the finding that NaBu treatment markedly increased expression of FBP1 mRNA (Fig.?2A), it also significantly increased H3K27Ac level in the FBP1 enhancer (Fig.?4F). Similar to the finding that HDAC1 and HDAC2 knockdown induced FBP1 expression, depletion of these proteins alone or together also largely increased H3K27Ac level at this locus (Fig.?4G). This data indicate that the FBP1 gene locus indeed harbors a regulatory region where H3K27Ac level can be modulated by HDAC proteins. Thus, HDAC inhibitor-induced upregulation of H3K27 acetylation in the enhancer of the FBP1 gene correlates with increased expression of FBP1 induced by HDAC inhibitors." FBP Hepatocellular carcinoma DOID:684 D006528 HDAC-mediated suppression of FBP1 expression correlated with decreased histone H3K27Ac in the FBP1 enhancer. "ChIP,RT-PCR" Real-time PCR analysis of DNA mmunoprecipitated by control IgG or H3K27Ac antibody from HepG2 and SK-Hep1 cells transfected with control or HDAC1- and/or HDAC2-specific siRNAs. Cells harvested for ChIP assay at 48 hours after transfectio. -- -- -- -- -- -- -- Intron 33557 E_01_268 29379199 -- hg19 chr4 174441057 174442400 Human Neuroblastoma Cell Lines Low+High throughput "ChIP-seq,Western blot" "We observe that two hours after shutdown, MYCN is significantly depleted from promoters and enhancers (Fig. 1f; Supplementary Fig. 2c,e,f) - a result revealed by ChIP-Rx cell-count normalization.Although both traditional ChIP-seq and ChIP-Rx reveal global loss of MYCN at promoters and enhancers, we do observe global loss of additional marks (H3K4me3, CTCF, H3K27ac, and RNA Pol II) at 24 hours post MYCN shutdown only in ChIP-Rx data. In contrast, induction of MYCN in parental SHEP neuroblastoma cells (Fig. 1c,d) results in loading of MYCN at active promoters and enhancers, and is coincident with increases in global H3K27ac. " Enhancer HAND2 -- ChIP-seq "ChIP-seq tracks of MYCN and H3K27ac at 0, 2 and 6hrs post MYCN induction at RPL22, HAND2 and the upstream ID2 enhancer.Visual inspection of MYCN load at highly occupied genes revealed a diversity of binding profiles with some genes exhibiting promoter MYCN binding (RPL22) and others a more mixed array of promoter and enhancer binding (HAND2 & ID2). " "DHAND2,Hed,Thing2,bHLHa26,dHand" Neuroblastoma DOID:769 D009447 -- -- -- MYCN "MODED,N-myc,NMYC,ODED,bHLHe37" "Western blot,ChIP-seq" "Across MYCN-amplified neuroblastoma lines, we used chromatin immunoprecipitation coupled to high-throughput sequencing (ChIP-seq) to generate a consensus genome-wide map of ~10,000 regions that exhibit strong and consistent MYCN occupancy." -- -- -- >2KB 5922 E_01_269 28378740 CD47 SE E5 hg19 chr3 107702615 107714185 Human MCF-7 Low+High throughput "ChIP-seq,Luciferase Reporter Assay" "By rank-ordering of enhancer regions based on H3K27ac enrichment, we discovered that T-cell acute lymphoblastic leukemia (T-ALL (RPMI18402, Jurkat and MOLT3)) diffuse large B-cell lymphoma (DLBCL (LY4)) and breast cancer (MCF7 and HCC1954) cell lines have SEs within B200 kb of CD47 (Fig. 1a). To validate their function experimentally, we cloned each candidate CD47 enhancer (E1¨C9) into an EGFP reporter lentiviral construct containing the minimal (basal) promoter for the thymidine kinase (TK) gene7. We found that two of the CD47 enhancers (E5 and E3.2) had MCF7- and Jurkat-speci?c regulatory activity (Fig. 2a¨Cc).First, E5, in the downstream CD47 SE seen in breast cancers (Fig. 1a,b), showed increased reporter activity speci?cally in the MCF7 breast cancer cell line (Fig. 2a). Further analysis of publicly available Paired-End Tag (ChIA-PET) data22,23 con?rmed that E5 and the downstream CD47 SE in MCF7 are connected by a DNA loop containing RNA Polymerase II.Second, we found that E3.2, located within the upstream CD47 SE (Fig. 1a), had increased reporter expression speci?cally in the Jurkat cell line (Fig. 2b). We also found that a third functional enhancer, E7, located within the upstream CD47 SE (Fig. 1a), drove reporter expression in all of the cancer cell lines tested (Fig. 2d)." Enhancer CD47 -- "ChIP-seq,Luciferase Reporter Assay" "To validate their function experimentally, we cloned each candidate CD47 enhancer (E1¨C9) into an EGFP reporter lentiviral construct containing the minimal (basal) promoter for the thymidine kinase (TK) gene7. We found that two of the CD47 enhancers (E5 and E3.2) had MCF7- and Jurkat-speci?c regulatory activity (Fig. 2a¨Cc).First, E5, in the downstream CD47 SE seen in breast cancers (Fig. 1a,b), showed increased reporter activity speci?cally in the MCF7 breast cancer cell line (Fig. 2a). Further analysis of publicly available Paired-End Tag (ChIA-PET) data22,23 con?rmed that E5 and the downstream CD47 SE in MCF7 are connected by a DNA loop containing RNA Polymerase II.Second, we found that E3.2, located within the upstream CD47 SE (Fig. 1a), had increased reporter expression speci?cally in the Jurkat cell line (Fig. 2b). We also found that a third functional enhancer, E7, located within the upstream CD47 SE (Fig. 1a), drove reporter expression in all of the cancer cell lines tested (Fig. 2d)." "IAP,MER6,OA3" Breast Cancer DOID:1612 D001943 -- -- -- NFKB1 "CVID12,EBP-1,KBF1,NF-kB,NF-kB1,NF-kappa-B1,NF-kappaB,NFKB-p105,NFKB-p50,NFkappaB,p105,p50" shRNA "We?observed?that?72?h?after?shRNA?transduction,?expression?of?CD47?transcript?and?protein?(measured?by?flow?cytometry)?was?significantly?reduced?by?NFKB1?(Fig.?3b,c)?and?PPAR¦Á?(Fig.?3b,?Supplementary?Fig.?3d,e)?shRNAs,?when?compared?to?control?shRNA.?On?the?other?hand,?shRNAs?against?STATs?3,?5?and?6?did?not?reduce?CD47?expression?significantly?(Fig.?3b).?This?information?demonstrates?that?NFKB?and?PPAR?are?involved?in?the?regulation?of?CD47?in?MCF7?cells?and?since?knocking?down?NFKB1?had?the?strongest?effect?on?CD47?gene?expression,?we?focus?this?study?on?the?regulatory?role?of?this?transcription?factor." -- -- -- >2KB 53540 E_01_270 28378740 CD47 SE E3.2 hg19 chr3 107842838 107845592 Human MCF-7 Low+High throughput "ChIP-seq,Luciferase Reporter Assay" "By rank-ordering of enhancer regions based on H3K27ac enrichment, we discovered that T-cell acute lymphoblastic leukemia (T-ALL (RPMI18402, Jurkat and MOLT3)) diffuse large B-cell lymphoma (DLBCL (LY4)) and breast cancer (MCF7 and HCC1954) cell lines have SEs within B200 kb of CD47 (Fig. 1a). To validate their function experimentally, we cloned each candidate CD47 enhancer (E1¨C9) into an EGFP reporter lentiviral construct containing the minimal (basal) promoter for the thymidine kinase (TK) gene7. We found that two of the CD47 enhancers (E5 and E3.2) had MCF7- and Jurkat-speci?c regulatory activity (Fig. 2a¨Cc).First, E5, in the downstream CD47 SE seen in breast cancers (Fig. 1a,b), showed increased reporter activity speci?cally in the MCF7 breast cancer cell line (Fig. 2a). Further analysis of publicly available Paired-End Tag (ChIA-PET) data22,23 con?rmed that E5 and the downstream CD47 SE in MCF7 are connected by a DNA loop containing RNA Polymerase II.Second, we found that E3.2, located within the upstream CD47 SE (Fig. 1a), had increased reporter expression speci?cally in the Jurkat cell line (Fig. 2b). We also found that a third functional enhancer, E7, located within the upstream CD47 SE (Fig. 1a), drove reporter expression in all of the cancer cell lines tested (Fig. 2d)." Enhancer CD47 -- "ChIP-seq,Luciferase Reporter Assay" "To validate their function experimentally, we cloned each candidate CD47 enhancer (E1¨C9) into an EGFP reporter lentiviral construct containing the minimal (basal) promoter for the thymidine kinase (TK) gene7. We found that two of the CD47 enhancers (E5 and E3.2) had MCF7- and Jurkat-speci?c regulatory activity (Fig. 2a¨Cc).First, E5, in the downstream CD47 SE seen in breast cancers (Fig. 1a,b), showed increased reporter activity speci?cally in the MCF7 breast cancer cell line (Fig. 2a). Further analysis of publicly available Paired-End Tag (ChIA-PET) data22,23 con?rmed that E5 and the downstream CD47 SE in MCF7 are connected by a DNA loop containing RNA Polymerase II.Second, we found that E3.2, located within the upstream CD47 SE (Fig. 1a), had increased reporter expression speci?cally in the Jurkat cell line (Fig. 2b). We also found that a third functional enhancer, E7, located within the upstream CD47 SE (Fig. 1a), drove reporter expression in all of the cancer cell lines tested (Fig. 2d)." "IAP,MER6,OA3" Breast Cancer DOID:1612 D001943 -- -- -- NFKB1 "CVID12,EBP-1,KBF1,NF-kB,NF-kB1,NF-kappa-B1,NF-kappaB,NFKB-p105,NFKB-p50,NFkappaB,p105,p50" shRNA "We?observed?that?72?h?after?shRNA?transduction,?expression?of?CD47?transcript?and?protein?(measured?by?flow?cytometry)?was?significantly?reduced?by?NFKB1?(Fig.?3b,c)?and?PPAR¦Á?(Fig.?3b,?Supplementary?Fig.?3d,e)?shRNAs,?when?compared?to?control?shRNA.?On?the?other?hand,?shRNAs?against?STATs?3,?5?and?6?did?not?reduce?CD47?expression?significantly?(Fig.?3b).?This?information?demonstrates?that?NFKB?and?PPAR?are?involved?in?the?regulation?of?CD47?in?MCF7?cells?and?since?knocking?down?NFKB1?had?the?strongest?effect?on?CD47?gene?expression,?we?focus?this?study?on?the?regulatory?role?of?this?transcription?factor." -- -- -- >2KB 82275 E_01_271 28378740 CD47 SE E7 hg19 chr3 107846969 107854683 Human MCF-7 Low+High throughput "ChIP-seq,Luciferase Reporter Assay" "By rank-ordering of enhancer regions based on H3K27ac enrichment, we discovered that T-cell acute lymphoblastic leukemia (T-ALL (RPMI18402, Jurkat and MOLT3)) diffuse large B-cell lymphoma (DLBCL (LY4)) and breast cancer (MCF7 and HCC1954) cell lines have SEs within B200 kb of CD47 (Fig. 1a). To validate their function experimentally, we cloned each candidate CD47 enhancer (E1¨C9) into an EGFP reporter lentiviral construct containing the minimal (basal) promoter for the thymidine kinase (TK) gene7. We found that two of the CD47 enhancers (E5 and E3.2) had MCF7- and Jurkat-speci?c regulatory activity (Fig. 2a¨Cc).First, E5, in the downstream CD47 SE seen in breast cancers (Fig. 1a,b), showed increased reporter activity speci?cally in the MCF7 breast cancer cell line (Fig. 2a). Further analysis of publicly available Paired-End Tag (ChIA-PET) data22,23 con?rmed that E5 and the downstream CD47 SE in MCF7 are connected by a DNA loop containing RNA Polymerase II.Second, we found that E3.2, located within the upstream CD47 SE (Fig. 1a), had increased reporter expression speci?cally in the Jurkat cell line (Fig. 2b). We also found that a third functional enhancer, E7, located within the upstream CD47 SE (Fig. 1a), drove reporter expression in all of the cancer cell lines tested (Fig. 2d)." Enhancer CD47 -- "ChIP-seq,Luciferase Reporter Assay" "To validate their function experimentally, we cloned each candidate CD47 enhancer (E1¨C9) into an EGFP reporter lentiviral construct containing the minimal (basal) promoter for the thymidine kinase (TK) gene7. We found that two of the CD47 enhancers (E5 and E3.2) had MCF7- and Jurkat-speci?c regulatory activity (Fig. 2a¨Cc).First, E5, in the downstream CD47 SE seen in breast cancers (Fig. 1a,b), showed increased reporter activity speci?cally in the MCF7 breast cancer cell line (Fig. 2a). Further analysis of publicly available Paired-End Tag (ChIA-PET) data22,23 con?rmed that E5 and the downstream CD47 SE in MCF7 are connected by a DNA loop containing RNA Polymerase II.Second, we found that E3.2, located within the upstream CD47 SE (Fig. 1a), had increased reporter expression speci?cally in the Jurkat cell line (Fig. 2b). We also found that a third functional enhancer, E7, located within the upstream CD47 SE (Fig. 1a), drove reporter expression in all of the cancer cell lines tested (Fig. 2d)." "IAP,MER6,OA3" Breast Cancer DOID:1612 D001943 -- -- -- NFKB1 "CVID12,EBP-1,KBF1,NF-kB,NF-kB1,NF-kappa-B1,NF-kappaB,NFKB-p105,NFKB-p50,NFkappaB,p105,p50" shRNA "We?observed?that?72?h?after?shRNA?transduction,?expression?of?CD47?transcript?and?protein?(measured?by?flow?cytometry)?was?significantly?reduced?by?NFKB1?(Fig.?3b,c)?and?PPAR¦Á?(Fig.?3b,?Supplementary?Fig.?3d,e)?shRNAs,?when?compared?to?control?shRNA.?On?the?other?hand,?shRNAs?against?STATs?3,?5?and?6?did?not?reduce?CD47?expression?significantly?(Fig.?3b).?This?information?demonstrates?that?NFKB?and?PPAR?are?involved?in?the?regulation?of?CD47?in?MCF7?cells?and?since?knocking?down?NFKB1?had?the?strongest?effect?on?CD47?gene?expression,?we?focus?this?study?on?the?regulatory?role?of?this?transcription?factor." -- -- -- >2KB 88886 E_01_272 28446439 -- hg19 chr11 17666830 17677080 Human Human Fibroblast Cell Lines Low+High throughput "ChIP-seq,4C,Luciferase Reporter Assay" "We found PAX3-FOXO1 most frequently occupied the strong Enhancer chromatin state , exemplified by known PAX3-FOXO1 target FGFR4 and oncogenes MYC, ALK and MET" Super-Enhancer MYOD1 4C "ChIP-seq,Luciferase Reporter Assay" "We found PAX3-FOXO1 most frequently occupied the strong Enhancer chromatin state , exemplified by known PAX3-FOXO1 target FGFR4 and oncogenes MYC, ALK and MET" "MYF3,MYOD,PUM,bHLHc1" Alveolar Rhabdomyosarcoma DOID:4051 D018232 -- -- -- "PAX3,FOXO1" "CDHS,HUP2,WS1,WS3,FKH1,FKHR,FOXO1A" "ChIP-qPCR,ChIP-seq" "To gain insight into the epigenetic consequences of PAX3-FOXO1, we mapped the landscape of active and repressive histone marks by sequencing DNA enriched by chromatin immunoprecipitation (ChIP-seq) from a patient derived fusion positive FP-RMS cell line RH4. " -- -- -- >2KB 69154 E_01_273 28511927 -- hg19 chr11 355447 358949 Human "A549,HEK-293" Low throughput "Luciferase Reporter Assay,CRISPR/Cas9,ChIP,EMSA,3C" "Here, through public data mining, episomal luciferase reporter assay and in vivo CRISPR-Cas9 genome editing, we identified an IFN-responsive enhancer located 35 kb upstream of IFITM3 gene promoter upregulating the IFN-induced expression of IFITM1, 2 and 3 genes. Chromatin immunoprecipitation (ChIP), electrophoretic mobility shift assay (EMSA) and luciferase reporter assay demonstrated that signal transducers and activators of transcription (STAT) 1 bound to the enhancer with the treatment of IFN and was indispensable for the enhancer activity. Furthermore, using chromosome conformation capture technique, we revealed that the IFITM1, 2 and 3 genes physically clustered together and constitutively looped to the distal enhancer through long-range interactions in both HEK293 and A549 cells, providing structural basis for coordinated regulation of IFITM1, 2 and 3 by the enhancer. Finally, we showed that in vivo truncation of the enhancer impaired IFN-induced resistance to influenza A virus (IAV) infection. " Enhancer "IFITM1,IFITM2,IFITM3" "CRISPR/Cas9,3C" "Luciferase Reporter Assay,EMSA" "Here, through public data mining, episomal luciferase reporter assay and in vivo CRISPR-Cas9 genome editing, we identified an IFN-responsive enhancer located 35 kb upstream of IFITM3 gene promoter upregulating the IFN-induced expression of IFITM1, 2 and 3 genes. Chromatin immunoprecipitation (ChIP), electrophoretic mobility shift assay (EMSA) and luciferase reporter assay demonstrated that signal transducers and activators of transcription (STAT) 1 bound to the enhancer with the treatment of IFN and was indispensable for the enhancer activity. Furthermore, using chromosome conformation capture technique, we revealed that the IFITM1, 2 and 3 genes physically clustered together and constitutively looped to the distal enhancer through long-range interactions in both HEK293 and A549 cells, providing structural basis for coordinated regulation of IFITM1, 2 and 3 by the enhancer." "927,CD225,DSPA2a,IFI17,LEU13,1-8D,DSPA2c,1-8U,DSPA2b,IP15" -- -- -- In vivo truncation of the enhancer impaired IFN-induced resistance to influenza A virus (IAV) infection. "RT-PCR,Western blot" "As IFITM1, 2 and 3 are very important for host to resist infection of IAV [4], [8], we infected wild-type and E2-3-truncated HEK293 cells with IAV Puerto Rico/8/1934 (PR8) at a MOI of 0.1 for 24 h. Although IFITM1, 2 and 3 were induced by PR8 virus in both wild-type and E2-3-truncated HEK293 cells, the inductions were greatly impaired upon truncation of E2-3 (Fig. 5A and B), further confirming the regulation of IFITM1, 2 and 3 expression by the enhancer E2-3. Surprisingly, we did not observe a significant effect on the replication of IAV when enhancer E2-3 was truncated, as revealed by the mRNA and protein levels of virus NP and progeny virus titers determined by TCID50 assay (Fig. S9). Nonetheless, when we treated wild-type and E2-3-truncated HEK293 cells with IFN¦Â respectively for 24 h before infection of PR8, NP mRNA and protein levels were found to be much higher in mutant HEK293 cells (Fig. 5C and D). Moreover, consistent with virus NP expression, the viral titers were also increased in E2-3-truncated cells (Fig. 5E), indicating that enhancer E2-3 was responsible for IFN¦Â-induced resistance to IAV infection." STAT1 "CANDF7,IMD31A,IMD31B,IMD31C,ISGF-3,STAT91" "ChIP,EMSA,3C" "Chromatin immunoprecipitation (ChIP), electrophoretic mobility shift assay (EMSA) and luciferase reporter assay demonstrated that signal transducers and activators of transcription (STAT) 1 bound to the enhancer with the treatment of IFN and was indispensable for the enhancer activity. Furthermore, using chromosome conformation capture technique, we revealed that the IFITM1, 2 and 3 genes physically clustered together and constitutively looped to the distal enhancer through long-range interactions in both HEK293 and A549 cells, providing structural basis for coordinated regulation of IFITM1, 2 and 3 by the enhancer. " -- -- -- >2KB 43208 E_01_274 28536097 -- hg19 chr1 221049243 221051243 Human HUVEC Low+High throughput "ChIP-seq,Luciferase Reporter Assay,ChIP" "We also performed H3K27ac ChIP-seq and found that 94% of conserved ERG peaks overlapped H3K27ac-enriched regions, supporting their association with active enhancers. We also identified an ERG-bound enhancer, conserved in cows and humans, ~3.0 kb upstream of the gene H2.0-Like Homeobox (HLX) (Fig. 8B; Fig. S8). We cloned this conserved H3K27ac- and ERG-enriched -3 kb 5¡¯ putative regulatory region (HLX-3a, 1565 bp fragment) upstream of a minimal promoter (SV40) driving a luciferase reporter, and found that it was VEGF-responsive, and that the basal and VEGF-induced activity of this enhancer required ETS DNA binding sequences (Fig. 9D)." Enhancer HLX -- "ChIP-seq,Luciferase Reporter Assay" "We also performed H3K27ac ChIP--seq and found that 94% of conserved ERG peaks overlapped H3K27ac--enriched regions, supporting their association with active enhancers. We also identified an ERG--bound enhancer, conserved in cows and humans, ~3.0 kb upstream of the gene H2.0--Like Homeobox (HLX) (Fig. 8B; Fig. S8). We cloned this conserved H3K27ac-- and ERG--enriched --3 kb 5¡¯ putative regulatory region (HLX--3a, 1565 bp fragment) upstream of a minimal promoter (SV40) driving a luciferase reporter, and found that it was VEGF--responsive, and that the basal and VEGF--induced activity of this enhancer required ETS DNA binding sequences (Fig. 9D)." "HB241, HLX" -- -- -- "A highly-conserved ERG-bound regulatory element was required in the VEGF responsiveness of the angiogenic gene, HLX." "CRISPR/Cas9,PCR" "To further test the functional importance of this enhancer, we utilized CRISPR genome editing to delete a portion (1201 bp; see Fig. 8B for schematic) of the H3K27Ac-enriched, ERG-bound region upstream of HLX in TeloHAECs, an immortalized aortic EC line. Several clonal lines (¦¤HLX15, ¦¤HLX17 and ¦¤HLX21) heterozygous for deletion of this region were generated and confirmed by PCR and DNA sequencing (data not shown). Comparison was made to a clonal line generated following transfection of scrambled control gRNAs (Scr3). While the basal expression of HLX appeared to be unaffected in the deletion lines, the VEGF-dependent induction of HLX was attenuated (Fig. 9G). In contrast, DLL4 induction was unaffected. Furthermore, knock-down of ERG appeared to attenuate the induction of HLX to a greater extent in the control line compared to the deletion lines, implying that ERG acts through the deleted enhancer region. Collectively, these findings demonstrate the requirement of a highly-conserved ERG-bound regulatory element in the VEGF responsiveness of the angiogenic gene, HLX. " "VEGF,ERG" "MVCD1,VEGF,VPF,erg-3,p55" Luciferase Reporter Assay "We cloned this conserved H3K27ac- and ERG-enriched -3 kb 5¡¯ putative regulatory region (HLX-3a, 1565 bp fragment) upstream of a minimal promoter (SV40) driving a luciferase reporter, and found that it was VEGF-responsive, and that the basal and VEGF-induced activity of this enhancer required ETS DNA binding sequences (Fig. 9D). " -- -- -- >2KB 2499 E_01_275 28578223 -- hg19 chr18 61533339 61545796 Human "MonoMac6,K-562" Low+High throughput "ChIP-seq,Luciferase Reporter Assay,qRT-PCR" "A cluster of RNAs, not previously annotated, was found approximately 21 kb upstream of the SERPINB2 gene in SLE monocytes (Shi et al.,2014). The RNAs (772, 774, 775) formed a cluster near an enhancer, de?ned by H3K27ac in UCSC Genome Browser. 774 is centered on the H3K27ac peak, 775 is at the proximal edge of the H3K27ac peak and 772 is at the distal shores of the H3K27ac peak. We will refer to this region as Enhancer 1. We tested the DNA corresponding to the 775 ncRNA in an enhancer assay by sub-cloning it downstream of a minimal thymidine kinase (TK) promoter. The DNA corresponding to the 775 ncRNA location was capable of augmenting luciferase production in this transient transfection assay in two di?erent cell types (K562 and HEK293), thereby assigning it as a probable enhancer (Fig. 2A and B). For this reason, we will refer to the ncRNAs subsequently as eRNAs. Enhancer RNAs are predicted to have chromatin localization. To validate the remaining eRNAs, we used qRT-PCR from three di?erent cell types to demonstrate tissue speci?city and analyzed eRNA sub-cellular localization by fractionating cells. The eRNAs were found predominantly in monocytes and in the chromatin and nucleoplasm fractions predominantly (Fig. 2C and D). This demonstration of enhancer activity of the DNA and chromatin localization support the assignment of the ncRNAs as eRNAs." Enhancer SERPINB2 -- "ChIP-seq,Luciferase Reporter Assay,qRT-PCR" "A cluster of RNAs, not previously annotated, was found approximately 21 kb upstream of the SERPINB2 gene in SLE monocytes (Shi et al.,2014). The RNAs (772, 774, 775) formed a cluster near an enhancer, de?ned by H3K27ac in UCSC Genome Browser. 774 is centered on the H3K27ac peak, 775 is at the proximal edge of the H3K27ac peak and 772 is at the distal shores of the H3K27ac peak. We will refer to this region as Enhancer 1. We tested the DNA corresponding to the 775 ncRNA in an enhancer assay by sub-cloning it downstream of a minimal thymidine kinase (TK) promoter. The DNA corresponding to the 775 ncRNA location was capable of augmenting luciferase production in this transient transfection assay in two di?erent cell types (K562 and HEK293), thereby assigning it as a probable enhancer (Fig. 2A and B). For this reason, we will refer to the ncRNAs subsequently as eRNAs. Enhancer RNAs are predicted to have chromatin localization. To validate the remaining eRNAs, we used qRT-PCR from three di?erent cell types to demonstrate tissue speci?city and analyzed eRNA sub-cellular localization by fractionating cells. The eRNAs were found predominantly in monocytes and in the chromatin and nucleoplasm fractions predominantly (Fig. 2C and D). This demonstration of enhancer activity of the DNA and chromatin localization support the assignment of the ncRNAs as eRNAs." "HsT1201,PAI,PAI-2,PAI2,PLANH2" -- -- -- "Knock-down of the enhancer RNAs compromised stimulus induction of promoter and enhancer chromatin changes. Conversely,over-expression was associated with enhanced recruitment of c-JUN and increased expression of SERPINB2 mRNA expression." qRT-PCR "We knocked down 774 and 775 as SERPINB2-speci?c eRNAs. Knock-down was validated by qRT-PCR of the target eRNAs. Knock-down of 774 and 775 led to inhibition of SERPINB2 mRNAs but not SERPINB10, which is not co-regulated with SERPINB2 (Fig. 3B). We then over-expressed 774 and 775 to determine whether they could regulate SERPIN gene expression when provided in trans. Each over-expression construct was validated as expressing the correct RNA species (Fig. 3C). Over-expression of both 774 and 775 led to higher SERPINB2 RNA but not the distinctly regulated SERPINB10.Ano?-target, control RNA, 477, had no e?ect. Enhancer RNAs have been hypothesized to regulate gene expression via diverse potential mechanisms. We tested whether they regulated histone modi?cations. To de?ne e?ects related to eRNAs, we used anti-sense oligonucleotides to knock-down 774 and 775.We examined chromatin marks of gene activation in MonoMac6 cells at baseline and after treatment with LPS (Fig. 4C¨CF). We compared the e?ects of 774 and 775 knock-down with GFP knock-down. Non-transfected cells were included as assay controls for each experiment but are not shown to streamline the ?gures.The GAPDH promoter was una?ected by LPS stimulation and serves as a control. The anti-sense oligonucleotides to 774 and 775 were associated with diminished acquisition of the chromatin marks seen with LPS stimulation at Enhancer 1. The e?ect was limited to LPS-stimulated cells and was not observed in resting cells. Knock-down of 774 and 775 primarily a?ected H3K4me3 and H3K27ac at the promoter. These data demonstrate that eRNAs regulate histone modi?cations not just at the enhancer but also at their target gene promoter. " "NSMF,CDK9" "HH9,NELF,C-2k,CDC2L4,CTK1,PITALRE,TAK" ChIP "To examine whether NELF and its major kinase are localized to the promoter and Enhancer of SERPINB2, we performed ChIP studies for NELF and CDK9. " -- -- -- >2KB 15370 E_01_276 28637769 -- hg19 Chr11 45846943 45846959 Human U-2 OS Low throughput Luciferase Reporter Assay "To identify components necessary for cell-autonomous circadian transcription of the hCry2 gene, a 3-kbp 5'-flanking region upstream from the hCry2 transcription start site was isolated and subcloned into the upstream of the luciferase gene, and the hCry2-driven luciferase activity was monitored in real time using a cell-based system (Figure 1A).To exclude systemic factors such as blood-borne factors and body temperature, it was necessary to examine cell-autonomous transcriptional regulatory mechanisms under an in vitro culture condition. The data clearly showed a robust circadian oscillation of the bioluminescence in U2OS human steosarcoma cells (Figure 1B), indicating that the cloned genomic region contained elements essential for circadian transcription.The 60-bp region is functionally divided into two; a 43-bp region (from -158 to -116) contains elements which enhance oscillation amplitude, while the 17-bp region (from -175 to -159) is indispensable for the generation of transcriptional oscillation. After narrowing down the transcriptional regulatory region required for the cell-autonomous circadian transcription of hCry2,and further narrowing down the genomic region essential for cell-autonomous circadian transcription,the results strongly suggested that the essential regulatory elements are contained in the region from -175 to -116 (60-bp length) (Figure 1D). To identify consensus sequences for transcription factor binding within the latter region, we performed an in silico search by sequence alignment, and found interspecies-conserved E-boxes within the 17-bp region (E1 and E2, Figure 1C). To investigate whether each of the E-boxes is functional for circadian transcription of the hCry2 gene, we produced constructs carrying hCry2 lacking either one of the E-boxes (Figure 2A). To confirm whether the BMAL1 and CLOCK complex actually activates hCry2 transcription via each E-box, we performed dual luciferase assays with these constructs (Figures 2B). The data showed that overexpression of BMAL1 and CLOCK activated hCry2 transcription via the region spanning from -249 to +41, and that deletion of one of the E-boxes produced severe attenuation of transcription, while deletion of both produced an almost complete loss of activation. " Enhancer CRY2 -- Luciferase Reporter Assay "To identify components necessary for cell-autonomous circadian transcription of the hCry2 gene, a 3-kbp 5'-flanking region upstream from the hCry2 transcription start site was isolated and subcloned into the upstream of the luciferase gene, and the hCry2-driven luciferase activity was monitored in real time using a cell-based system (Figure 1A).To exclude systemic factors such as blood-borne factors and body temperature, it was necessary to examine cell-autonomous transcriptional regulatory mechanisms under an in vitro culture condition. The data clearly showed a robust circadian oscillation of the bioluminescence in U2OS human steosarcoma cells (Figure 1B), indicating that the cloned genomic region contained elements essential for circadian transcription.The 60-bp region is functionally divided into two; a 43-bp region (from -158 to -116) contains elements which enhance oscillation amplitude, while the 17-bp region (from -175 to -159) is indispensable for the generation of transcriptional oscillation. After narrowing down the transcriptional regulatory region required for the cell-autonomous circadian transcription of hCry2,and further narrowing down the genomic region essential for cell-autonomous circadian transcription,the results strongly suggested that the essential regulatory elements are contained in the region from -175 to -116 (60-bp length) (Figure 1D). To identify consensus sequences for transcription factor binding within the latter region, we performed an in silico search by sequence alignment, and found interspecies-conserved E-boxes within the 17-bp region (E1 and E2, Figure 1C). To investigate whether each of the E-boxes is functional for circadian transcription of the hCry2 gene, we produced constructs carrying hCry2 lacking either one of the E-boxes (Figure 2A). To confirm whether the BMAL1 and CLOCK complex actually activates hCry2 transcription via each E-box, we performed dual luciferase assays with these constructs (Figures 2B). The data showed that overexpression of BMAL1 and CLOCK activated hCry2 transcription via the region spanning from -249 to +41, and that deletion of one of the E-boxes produced severe attenuation of transcription, while deletion of both produced an almost complete loss of activation. " "HCRY2,PHLL2" -- -- -- the interdependent behavior of enhancer elements including not only E-boxboxes but also ROR response elements (ROREs) might contribute to limit cycle oscillations by increasing transcriptional nonlinearity. EMSA "Immunoprecipitation was performed with antibody-conjugated beads, and the immunoprecipitates were eluted with an excess amount of tag-peptide.A mutation of E1 resulted in a severe attenuation of the affinity of the BMAL1 and CLOCK complex to the probe, and the position of the shifted band was slightly changed, probably because of the reduction of the mole ratio between the binding proteins and probe. A mutation of E2 led to almost complete loss of the physical interaction between them. Taken together, the present EMSA data indicate that each of the hCry2 tandem E-boxes physically interacts directly with the BMAL1 and CLOCK complex in an interdependent and not independent manner. " "ARNTL,CLOCK" "BMAL1,BMAL1c,JAP3,MOP3,PASD3,TIC,bHLHe5,KAT13D, bHLHe8" "EMSA,Pull-down assay" "We therefore performed electrophoretic mobility shift assays (EMSAs) to investigate the physical interaction of purified BMAL1 and CLOCK proteins to double-stranded DNA fragments (Figure 3A).We confirmed that the electrophoretic mobility of the probe was shifted by the physical interaction with BMAL1¨CCLOCK. A mutation of E1 resulted in a severe attenuation of the affinity of the BMAL1 and CLOCK complex to the probe, and the position of the shifted band was slightly changed, probably because of the reduction of the mole ratio between the binding proteins and probe. A mutation of E2 led to almost complete loss of the physical interaction between them. Taken together, the present EMSA data indicate that each of the hCry2 tandem E-boxes physically interacts directly with the BMAL1 and CLOCK complex in an interdependent and not independent manner. Next, we performed hCry2-oligodeoxynucleotide (ODN) pull-down assays and confirmed that endogenous BMAL1 and CLOCK expressed in the liver bound to a fragment containing the hCry2 tandem E-boxes (Figure 3B). " -- -- -- >2KB 21717 E_01_277 28717659 SIX6 Enhancer hg19 chr14 60973427 60975430 Human Eye low throughput "Transgenic zebrafish,Luciferase Reporter Assay" "A evolutionarily conserved enhancer has been shown to control Six6 expression in the vertebrate retina (Conte et al. 2010).We ?rst inserted the entire control human enhancer sequence in the ZED transgenic vector (Bessa et al. 2009) and investigated if the plasmid could drive GFP reporter expression in the retina of zebra?sh embryos, similarly to what has been previously observed for the medaka orthologous region (Conte et al. 2010). We generated 38 F0 transgenic embryos, as determined by RFP expression in the somites (Fig. 4C) that acts as a positive control (Bessa et al.2009). About half of them (18) showed a clear GFP expression driven by the human enhancer element in the eye and occasionally in other CNS regions." Enhancer SIX6 -- Transgenic zebrafish "A evolutionarily conserved enhancer has been shown to control Six6 expression in the vertebrate retina (Conte et al. 2010).We ?rst inserted the entire control human enhancer sequence in the ZED transgenic vector (Bessa et al. 2009) and investigated if the plasmid could drive GFP reporter expression in the retina of zebra?sh embryos, similarly to what has been previously observed for the medaka orthologous region (Conte et al. 2010). We generated 38 F0 transgenic embryos, as determined by RFP expression in the somites (Fig. 4C) that acts as a positive control (Bessa et al.2009). About half of them (18) showed a clear GFP expression driven by the human enhancer element in the eye and occasionally in other CNS regions." "MCOPCT2,ODRMD,OPTX2,Six9" Primary Open-Angle Glaucoma DOID:13550 D005902 -- -- -- -- -- -- -- rs33912345 60976537 Luciferase Reporter Assay <2KB 1509 E_01_278 28737489 -- hg19 chr3 181432912 181433312 Human Lung Adenocarcinoma low throughput "Luciferase Reporter Assay,ChIP-qPCR" "To further delineate the molecular mechanism of SOX2 regulation by NFATc2, we screened, in silico, the genomic sequences spanning 5 kb up- and downstream of the SOX2 transcription start site (TSS), which identified 4 regions encompassing multiple conserved NFAT binding sequences (Figure 6-figure supplement 1A). Luciferase assays confirmed these regions are active transcriptional regulatory regions (Figure 6-figure supplement 1B). Further evaluation with respective SOX2 luciferase reporter revealed transcriptional activities were mediated by sites 1, 2, 4, and 5 (Figure 6B). Using H441 lung cancer cell line with NFATc2 transient overexpression, we observed only sites 1, 4 and 5 showed statistically significant increased reporter activities while those of sites 4 and 5 were reciprocally abolished by CSA treatment (Figure 6C)." Enhancer SOX2 -- "Luciferase Reporter Assay,ChIP-qPCR" "To further delineate the molecular mechanism of SOX2 regulation by NFATc2, we screened, in silico, the genomic sequences spanning 5 kb up- and downstream of the SOX2 transcription start site (TSS), which identified 4 regions encompassing multiple conserved NFAT binding sequences (Figure 6-figure supplement 1A). Luciferase assays confirmed these regions are active transcriptional regulatory regions (Figure 6-figure supplement 1B). Further evaluation with respective SOX2 luciferase reporter revealed transcriptional activities were mediated by sites 1, 2, 4, and 5 (Figure 6B). Using H441 lung cancer cell line with NFATc2 transient overexpression, we observed only sites 1, 4 and 5 showed statistically significant increased reporter activities while those of sites 4 and 5 were reciprocally abolished by CSA treatment (Figure 6C). " "ANOP3,MCOPS3" Lung Cancer DOID:1324 D008175 -- -- -- NFATC2 "NFAT1,NFATP" "Luciferase Reporter Assay,ChIP-qPCR" "Finally, site directed mutagenesis of NFAT motifs (GGAAA to GACTA) prevented reporter activities of sites 4 and 5 only (Figure 6D), and the findings were supported by data from A549 and H1299 cells ectopically expressing NFATc2, respectively (Figure 6-figure supplement 1C,D). Thus, the data suggested NFATc2 was highly likely to regulate SOX2 expression through binding to 3¡¯ enhancers at sites 4 and 5. For validation, NFATc2 ChIP-qPCR assays were performed using A549 with NFATc2 upregulation, which showed statistically significant enrichment of sites 4 and 5 sequences compared to vector control (Figure 6F). In HCC827 cells, sites 4 and 5 sequences were significantly enriched by anti-NFATc2 antibody compared to IgG control. Conversely, these sequences were significantly reduced upon NFATc2 knockout in HCC827, compared to their endogenous levels in control cells, indicating de novo physical binding of NFATc2 to SOX2 at sites 4 and 5 (Figure 6G). Together, the data showed NFATc2 upregulates SOX2 by binding to its 3¡¯ enhancer region at around 3.2 kb (site 4) and 3.6 kb (site 5) from the TSS, respectively " -- -- -- >2KB 3400 E_01_279 28751304 -- hg19 chr7 129854050 129895632 Human Adipose Progenitor Cell Low+High throughput "ChIP-seq,FISH" "We also searched for putative MIR335 short- and long-range Enhancers:to this end, we combined chromatin state modeling data from ENC ODE ChIP-seq experiments showing enrichment in H3K4me1 and/or H3K27ac, histone modifications found on inactive and active Enhancers, respectively (Fig. S5 A), with ENCODE genome-wide chromosome conformation capture of Hi-C data (Rao et al., 2014). The Hi-C data reveal multiple pairwise interactions of these predicted enhancers with the MIR335 promoter (Fig. S5, B¨CD), providing six MIR335-predicted distal enhancer sites (regions r6¨Cr11; Fig. S5 D). Acetylation of H3K27 on MIR335 enhancers detected in ASCLMNA(R482W) after adipogenic induction suggests interac-tions with the MIR335 promoter by chromatin looping (Zhang et al., 2013). We tested this hypothesis by two-color FISH using distinctly labeled promoter and enhancer probes. Probes were generated to cover the MIR335 gene and promoter as well as the distal enhancer element r11 located ?200 kb upstream of MIR335. In both undifferentiated and differentiated ASCs that were either native or expressing WT LMNA, these promoter and enhancer elements displayed a relatively low incidence of colocalization on the majority of alleles (?80%; Fig.?6, C and D). After induction of differentiation, however, this proportion markedly increased to 56% of alleles in the LMNA(R482W) mutants, reflecting interaction of a high proportion of MIR335 promoter and enhancer sites (Fig. 6, C and D)." Enhancer MIR335 -- "ChIP-seq,FISH" "We also searched for putative MIR335 short- and long-range Enhancers:to this end, we combined chromatin state modeling data from ENC ODE ChIP-seq experiments showing enrichment in H3K4me1 and/or H3K27ac, histone modifications found on inactive and active Enhancers, respectively (Fig. S5 A), with ENCODE genome-wide chromosome conformation capture of Hi-C data (Rao et al., 2014). The Hi-C data reveal multiple pairwise interactions of these predicted enhancers with the MIR335 promoter (Fig. S5, B¨CD), providing six MIR335-predicted distal enhancer sites (regions r6¨Cr11; Fig. S5 D). Acetylation of H3K27 on MIR335 enhancers detected in ASCLMNA(R482W) after adipogenic induction suggests interac-tions with the MIR335 promoter by chromatin looping (Zhang et al., 2013). We tested this hypothesis by two-color FISH using distinctly labeled promoter and enhancer probes. Probes were generated to cover the MIR335 gene and promoter as well as the distal enhancer element r11 located ?200 kb upstream of MIR335. In both undifferentiated and differentiated ASCs that were either native or expressing WT LMNA, these promoter and enhancer elements displayed a relatively low incidence of colocalization on the majority of alleles (?80%; Fig.?6, C and D). After induction of differentiation, however, this proportion markedly increased to 56% of alleles in the LMNA(R482W) mutants, reflecting interaction of a high proportion of MIR335 promoter and enhancer sites (Fig. 6, C and D)." "MIRN335,hsa-mir-335,miRNA335,mir-335" -- -- -- -- -- -- -- -- -- -- -- -- -- >2KB 261110 E_01_280 28753427 -- hg19 chr6 12490000 12590000 Human HUVEC Low throughput "4C-seq,Luciferase Reporter Assay" "The newly released ENCODE phase 3 data, however, includes four aortic artery samples. In these aortic samples, a strong enhancer signal overlies rs9349379 (Figure 2B), spanning 1 kb. 4C-seq from the rs9349379 site demonstrated a smaller contact region that extended from chr6:12,690,000¨C13,350,000. There was a possible area of over-lap intergenic to EDN1 and PHACTR1 (Figure 5A). Notably, this region featured high levels of H3K27Ac signal in ECs, consistent with a super enhancer (Figure 5B). The common contact region contains four distinct peaks of H3K27Ac signal. Reporter assays with each of these four sites cloned into an SV40-driven luciferase plasmid demonstrated that each cis element possessed strong enhancer activity in 293T cells (2- to 12-fold luciferase induction) comparedwith SV40-promoter sequence alone (Figure 5C)." Super-Enhancer EDN1 4C-seq Luciferase Reporter Assay "The newly released ENCODE phase 3 data, however, includes four aortic artery samples. In these aortic samples, a strong enhancer signal overlies rs9349379 (Figure 2B), spanning 1 kb. 4C-seq from the rs9349379 site demonstrated a smaller contact region that extended from chr6:12,690,000¨C13,350,000. There was a possible area of over-lap intergenic to EDN1 and PHACTR1 (Figure 5A). Notably, this region featured high levels of H3K27Ac signal in ECs, consistent with a super enhancer (Figure 5B). The common contact region contains four distinct peaks of H3K27Ac signal. Reporter assays with each of these four sites cloned into an SV40-driven luciferase plasmid demonstrated that each cis element possessed strong enhancer activity in 293T cells (2- to 12-fold luciferase induction) comparedwith SV40-promoter sequence alone (Figure 5C)." "ARCND3,ET1,HDLCQ7,PPET1,QME" "Coronary Artery Disease,Migraine Headache,Cervical Artery Dissection,Fibromuscular Dysplasia,Hypertension" DOID:10763 D006973 -- -- -- -- -- -- -- rs9349379 12803957 RNA-seq >2KB 249472 E_01_281 28881808 -- hg19 chr4 77511788 77605824 Human "LNCaP,CWR22Rv1" Low+High throughput "ChIP-seq,Dual-Luciferase Reporter Assay,ChIP,PCR" "By analyzing AR ChIP-seq data in LNCaP cells, we found that there are two obvious AR binding peaks located within CXCL13 intron I.Corresponding to these two peaks of CHIP-seq results, there were two AR-binding sites (called as ARBS-1 and ARBS-2) in CXCL13 intron I. To investigate the effects of these two ARBS in androgen responsiveness, we constructed two luciferase reporter plasmids (named CXCL13-Luc1 and CXCL13-Luc2) separately containing the two ARBS sequences and then transfected the plasmids into LNCaP cells for luciferase assay. The results of dual-luciferase assay in LNCaP cells showed that ARBS-1 (CXCL13-Luc1) was found to have approximately 3.3-folds increased in luciferase activity by comparing with pGL3-promoter (empty vector), while no obvious luciferase activity changes were observed in ARBS-2 (CXCL13-Luc2; Figure 4D). Furthermore, we generated luciferase reporter plasmids containing key nucleotides mutations within the four ARE sequences of ARBS-1 (called as ARE1-mut, ARE2-mut, ARE3-mut and ARE4-mut).Upon transfection of reporter plasmids into LNCaP cells, the luciferase activities of ARE3-mut and ARE4-mut were obviously decreased by comparing with the luciferase activity of ARBS-1 (CXCL13-Luc1). Moreover, mutant ARE3-mut (mutated at the ARE3 sequence) almost completely abrogated the increased luciferase activity of ARBS-1 (CXCL13-Luc1; Figure 4E). To further verify the ARBS-1 of CXCL13 gene is the androgen responsive element (ARE), CHIP assays were carried out next.Shown as in Figure 4G, PCR analysis using specific primers designed to amplify the ARBS-1 region revealed a significant increment of AR binding in response to Mib stimulation, suggesting this region is the bona fide AR-binding region within CXCL13 enhancer." Enhancer CXCL13 -- "ChIP-seq,Dual-Luciferase Reporter Assay,ChIP,PCR" "By analyzing AR ChIP-seq data in LNCaP cells, we found that there are two obvious AR binding peaks located within CXCL13 intron I.Corresponding to these two peaks of CHIP-seq results, there were two AR-binding sites (called as ARBS-1 and ARBS-2) in CXCL13 intron I. To investigate the effects of these two ARBS in androgen responsiveness, we constructed two luciferase reporter plasmids (named CXCL13-Luc1 and CXCL13-Luc2) separately containing the two ARBS sequences and then transfected the plasmids into LNCaP cells for luciferase assay. The results of dual-luciferase assay in LNCaP cells showed that ARBS-1 (CXCL13-Luc1) was found to have approximately 3.3-folds increased in luciferase activity by comparing with pGL3-promoter (empty vector), while no obvious luciferase activity changes were observed in ARBS-2 (CXCL13-Luc2; Figure 4D). Furthermore, we generated luciferase reporter plasmids containing key nucleotides mutations within the four ARE sequences of ARBS-1 (called as ARE1-mut, ARE2-mut, ARE3-mut and ARE4-mut).Upon transfection of reporter plasmids into LNCaP cells, the luciferase activities of ARE3-mut and ARE4-mut were obviously decreased by comparing with the luciferase activity of ARBS-1 (CXCL13-Luc1). Moreover, mutant ARE3-mut (mutated at the ARE3 sequence) almost completely abrogated the increased luciferase activity of ARBS-1 (CXCL13-Luc1; Figure 4E). To further verify the ARBS-1 of CXCL13 gene is the androgen responsive element (ARE), CHIP assays were carried out next.Shown as in Figure 4G, PCR analysis using specific primers designed to amplify the ARBS-1 region revealed a significant increment of AR binding in response to Mib stimulation, suggesting this region is the bona fide AR-binding region within CXCL13 enhancer." "ANGIE,ANGIE2,BCA-1,BCA1,BLC,BLR1L,SCYB13" Prostate Cancer DOID:10283 D011471 AR bound the enhancer of CXCL13 gene to up-regulate its expression. "siRNA,Western blot,PCR" "AR is known to be the primarily function as a transcription factor by binding the enhancers of its target genes.Shown as in PCR analysis using specific primers designed to amplify the ARBS-1 region revealed a significant increment of AR binding in response to Mib stimulation, suggesting this region is the bona fide ARbinding region within CXCL13 enhancer.CXCL13 is an AR target gene and involved in AR-mediated cell migration and invasion in primary PCa." AR "AIS,AR8,DHTR,HUMARA,HYSP1,KD,NR3C4,SBMA,SMAX1,TFM" PCR "AR is known to be the primarily function as a transcription factor by binding the enhancers of its target genes.Shown as in PCR analysis using specific primers designed to amplify the ARBS-1 region revealed a significant increment of AR binding in response to Mib stimulation, suggesting this region is the bona fide AR binding region within CXCL13 enhancer." -- -- -- Intron 874100 E_01_282 28887413 -- hg19 chr4 94419116 94437803 Human "231/ER¦ÁWt Cells,231/ER¦ÁLQ Cells" Low+High throughput "3C-qPCR,ChIP-seq,ChIP-qPCR" 3C-qPCR assays showing chromatin looping from a distal ER¦Á-binding site to the OTUB2 promoter in 231/ER¦ÁWt and 231/ER¦ÁLQ cells with 45 min of E2 treatment. Enhancer OTUB2 3C-qPCR "ChIP-seq,ChIP-qPCR" 3C-qPCR assays showing chromatin looping from a distal ER¦Á-binding site to the OTUB2 promoter in 231/ER¦ÁWt and 231/ER¦ÁLQ cells with 45 min of E2 treatment. "C14orf137,OTB2,OTU2" Breast Cancer DOID:1612 D001943 -- -- -- -- -- -- -- -- -- -- >2KB 64263 E_01_283 28916223 DGKA Enhancer hg19 chr12 56330413 56330909 Human A9 Low+High throughput "ChIP-seq,PCR,ChIP" "Analysis of publicly available data from the ENCODE project [18] (Fig. 1A), as well as our own ChIP experiments [8] revealed that the DGKA DMR carries H3K27ac. ChIP showed that JQ1 reduced the deposition of H3K27ac levels specifically at the DGKA enhancer site(DMR), but did not affect the upstream DGKA promoter sites 1 and 2 (Fig. 2C). " Enhancer DGKA -- "ChIP,PCR" "From various NHDFs,we selected three fibroblast lines (data not shown) with significant differences in DGKA DNA methylation at the DGKA enhancer site(Fig. 1A) and inducible DGKA mRNA expression (Fig. 1B). Increased DNA methylation at the DGKA enhancer was associated with a lack of DGKA induction after exposure to bleomycin (Fig. 1B, Fibroblast3), confirming our previous findings in numerous patient-derived NHDF.EGR1 was significantly induced in two NHDF showing high DGKA induction (Fig. 1C)." "DAGK,DAGK1,DGK-alpha" Fibrotic Disease -- -- -- -- -- EGR1 "AT225,G0S30,KROX-24,NGFI-A,TIS8,ZIF-268,ZNF225" qPCR "Relative DGKA (B) and EGR1 (C) mRNA expression levels in three individual NHDF exposed to bleomycin (40 mM) or vehicle control (DMSO) for 48 h. *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.0001, n.s. not significant." -- -- -- >2KB 5716 E_01_284 28916725 "rs6758183 enhancer" hg19 chr2 226998914 227004586 Human Human Corneal Epithelial Cells Low+High throughput "Luciferase Reporter Assay,ChIP-seq" "Because of their importance in controlling cell type¨Cspecific gene expression, we also defined SEs, using the strength of the H3K27ac mark as described previously (5, 6). We identified 1154 SEs and 12,424 TEs in corneal epithelial cells as well as 76,205 distal regulatory regions, marked by H3K4me1 alone (Fig. 1A). As expected, the majority of enhancers was found at distal sites in the genome rather than in proximal promoters (Fig. 1B). Although TEs showed a trend toward being localized near genes, SEs were even more strongly enriched near genes; most SEs were found within 50 kb of a transcriptional start site.Together, these data indicate that most epithelial distal regulatory regions are common to multiple epithelial cell types, differing only in the type of enhancer and/or the level of enhancer activity, as indicated by the activation mark H3K27Ac (Fig. 2C)." Enhancer IRS1 -- ChIA-PET "As insulin signaling is important for growth and development of both the corneal epithelium and the stroma, IRS1 is a candidate target for the enhancer containing rs6758183. Consistent with this possibility, in previously published siRNA data for EHF in corneal epithelial cells, we found that knockdown of EHF caused a small but significant increase in IRS1 gene expression (Figure 7G) (18). Together, these data suggest EHF regulates IRS1 through binding to the rs6758183 enhancer and that disruption of the EHF motif causes reduced affinity of EHF for this site, resulting in aberrant IRS1 expression during corneal development. " HIRS-1 -- -- -- -- -- -- EHF "ESE3, ESE3B, ESEJ" "ChIP,siRNA" "We hypothesized that these SNP variants could disrupt transcription factor binding, altering enhancer activity for each allele. At rs6758183, the disease associated A allele increased the strength of a SMAD motif, and decreased the strength of an ETS motif (Figure 7D). Using ChIP, we found that EHF bound to the WT allele of rs6758183, suggesting disruption of EHF binding by the SNP could result in the observed differences in enhancer activity (Figure 7F). Intriguingly, the WT version of the SNP showed less enhancer activity than the mutant, suggesting EHF binding acts to repress the enhancer region. When this binding is lost, the enhancer becomes overactive, a change that could result in growth imbalances that cause alterations in the curvature and refractive power of the cornea. Consistent with this possibility, in previously published siRNA data for EHF in corneal epithelial cells, we found that knockdown of EHF caused a small but significant increase in IRS1 gene expression (Figure 7G) (18). Together, these data suggest EHF regulates IRS1 through binding to the rs6758183 enhancer and that disruption of the EHF motif causes reduced affinity of EHF for this site, resulting in aberrant IRS1 expression during corneal development. " rs6758183 226136270 Luciferase Reporter Assay >2KB 594283 E_01_285 28916725 hg19 chr6 31092587 31094587 Human Human Corneal Epithelial Cells Low+High throughput "Luciferase Reporter Assay,ChIP-seq" "Because of their importance in controlling cell type¨Cspecific gene expression, we also defined SEs, using the strength of the H3K27ac mark as described previously (5, 6). We identified 1154 SEs and 12,424 TEs in corneal epithelial cells as well as 76,205 distal regulatory regions, marked by H3K4me1 alone (Fig. 1A). As expected, the majority of enhancers was found at distal sites in the genome rather than in proximal promoters (Fig. 1B). Although TEs showed a trend toward being localized near genes, SEs were even more strongly enriched near genes; most SEs were found within 50 kb of a transcriptional start site.Together, these data indicate that most epithelial distal regulatory regions are common to multiple epithelial cell types, differing only in the type of enhancer and/or the level of enhancer activity, as indicated by the activation mark H3K27Ac (Fig. 2C)." Enhancer PSORS1C1 -- "Luciferase Reporter Assay,ChIP" "SNP rs3815087 is located in an exon of PSORS1C (Figure 7A), a gene identified as a psoriasis susceptibility locus, although evidence points to this SNP being non-coding as the exon in which it is located is not translated in any of PSORS1C¡¯s known isoforms. SNP rs6758183 is located in a gene desert, approximately 500kb from the nearest coding gene (Figure 7A, 7B).When transfected into HCE cells, the genomic region of rs3815087 showed enhancer activity, and rs6758183 showed a trend toward increased activity compared with the negative control (Fig. 7C).In contrast, the region containing the disease-linked variant of rs6758183 showed significantly increased enhancer activity compared with the WT allele and significantly increased enhancer activity above the negative control (Fig. 7C). " "C6orf16,SEEK1" -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- E_01_286 28973302 -- hg19 chr8 27455903 27456103 Human HEK-293 Low throughput "Luciferase Reporter Assay,CRISPR/Cas9,EMSA,ChIP" "We have previously shown that the alleles at rs2279590 differentially regulate clusterin (CLU) gene expression in lens capsule tissues. This polymorphism resides in an active regulatory region marked by H3K27Ac and DNase I hypersensitive site and is an eQTL for CLU expression. Here, we report the presence of an enhancer element in surrounding region of rs2279590. Deletion of a 115 base pair intronic region flanking the rs2279590 variant through CRISPRB Cas9 genome editing in HEK293 cells demonstrated a decreased clusterin gene expression. Electrophoretic mobility shift and chromatin immunoprecipitation assays show that rs2279590 with allele ¡°A¡± constitutes a transcription factor binding site for heat shock factorB1 (HSF1) but not with allele ¡°G¡±. By binding to allele ¡°A¡±, HSF1 abrogates the enhancer effect of the locus as validated by reporter assays. Interestingly, rs2279590 locus have a widespread enhancer effect on two nearby genes, Protein tyrosine kinase 2 beta (PTK2B) and epoxide hydrolaseB2 (EPHX2); both of which have been previously associated with AD as risk factors." Enhancer "CLU,EPHX2,PTK2B" CRISPR/Cas9 "Luciferase Reporter Assay,EMSA,ChIP" "We have previously shown that the alleles at rs2279590 differentially regulate clusterin (CLU) gene expression in lens capsule tissues. This polymorphism resides in an active regulatory region marked by H3K27Ac and DNase I hypersensitive site and is an eQTL for CLU expression. Here, we report the presence of an enhancer element in surrounding region of rs2279590. Deletion of a 115 base pair intronic region flanking the rs2279590 variant through CRISPRB Cas9 genome editing in HEK293 cells demonstrated a decreased clusterin gene expression. Electrophoretic mobility shift and chromatin immunoprecipitation assays show that rs2279590 with allele ¡°A¡± constitutes a transcription factor binding site for heat shock factorB1 (HSF1) but not with allele ¡°G¡±. By binding to allele ¡°A¡±, HSF1 abrogates the enhancer effect of the locus as validated by reporter assays. Interestingly, rs2279590 locus have a widespread enhancer effect on two nearby genes, Protein tyrosine kinase 2 beta (PTK2B) and epoxide hydrolaseB2 (EPHX2); both of which have been previously associated with AD as risk factors." "AAG4,APO-J,APOJ,CLI,CLU1,CLU2,KUB1,NA1/NA2,SGP-2,SGP2,SP-40,TRPM-2,TRPM2,CADTK,CAKB,FADK2,FAK2,PKB,PTK,PYK2,RAFTK" Alzheimer's Disease DOID:10652 D000544 -- -- -- HSF1 HSTF1 "EMSA,ChIP" "Electrophoretic mobility shift and chromatin immunoprecipitation assays show that rs2279590 with allele ""A""constitutes a transcription factor binding site for heat shock factor-1 (HSF1) but not with allele ¡°G¡±.By binding to allele ¡°A¡±, HSF1 abrogates the Enhancer effect of the locus as validated by reporter assays." rs2279590 27456253 "EMSA,ChIP" Intron 1570 E_01_287 28991225 PGC-1¦Á super-Enhancer hg19 chr4 23811733 23994829 Human CHL-1 Low+High throughput ChIP-seq We performed chromatin immunoprecipitation followed by DNA sequencing (ChIP-seq) using antibodies against H3K27ac and BRD4. Super-Enhancer PPARGC1A -- ChIP-seq We performed chromatin immunoprecipitation followed by DNA sequencing (ChIP-seq) using antibodies against H3K27ac and BRD4. "LEM6,PGC-1(alpha),PGC-1alpha,PGC-1v,PGC1,PGC1A,PPARGC1" Melanoma DOID:1909 D008545 BRD4 binding at super-enhancers that drive the expression of PGC-1¦Á and SOX10 "ChIP-seq,ChIP-qPCR" "We provide evidence for a direct role of BRD4 binding at super-enhancers that drive the expression of PGC-1¦Á and SOX10, a transcription factor involved in melanocyte development. " -- -- -- -- -- -- -- >2KB 109638 E_01_288 29035388 HGE(HER2 gene body Enhancer ) hg19 chr17 37879571 37880263 Human K-562 Low throughput "DNaseI-seq,ChIP-PCR,ChIP,Luciferase Reporter Assay" "With the ENCODE Regulation Super-track Settings, DNase I hypersensitivity sites, H3K4me1 and H3K27ac were found in the previously identified intron 1 enhancer.Chromatin immunoprecipitation (ChIP)-PCR amplicons are shown. NC: sequences to which nonspecific controls were generated." Enhancer ERBB2 -- "ChIP,Luciferase Reporter Assay" "Chromatin immunoprecipitation of TFAP2C, H3K4me1 and H3K27ac were performed in SKBR3 cells and TFAP2C occupancies were confirmed at promoters 1 and 2, the intron 1 enhancer and HE.We tested the ability of the HGE to regulate the transcriptional activities of the HER2 promoters by luciferase reporter assay in 293T, SKBR3 and BT474 cells (Figure 2a). " "CD340,HER-2,HER-2/neu,HER2,MLN19,NEU,NGL,TKR1" Breast Cancer DOID:1612 D001943 -- -- -- TFAP2C "AP2-GAMMA,ERF1,TFAP2G,hAP-2g" CRISPR/Cas9 The transcriptional activities of these constructs were analyzed using the luciferase assay in 293T and SKBR3 cells.We next carried out genomic editing using CRISPR-Cas9 system to determine the role of the TFAP2C-binding sites at the HGE in the regulation of HER2 expression. -- -- -- Intron 35581 E_01_289 29091765 -- hg19 chr3 180728712 180730712 Human Neural Stem Cells Low+High throughput "ChIP-seq,ChIP-qPCR,4C-seq,Hi-C" "To understand if loss of CTCF binding affects chromatin contacts at the SOX2 locus, we performed circularized chromosome conformation capture (4C-Seq) using the SOX2 promoter as bait to identify genomic interactions that may regulate its expression (Figure 6B, S6B)." Enhancer SOX2 4C-seq Hi-C "To understand if loss of CTCF binding affects chromatin contacts at the SOX2 locus, we performed circularized chromosome conformation capture (4C-Seq) using the SOX2 promoter as bait to identify genomic interactions that may regulate its expression (Figure 6B, S6B).To see if this same pattern of chromatin contacts is maintained in other cell types, we examined the HiC profiles of hESCs, which express high levels of SOX2 , and IMR90 lung fibroblasts, which are low for SOX2These findings suggest that in LGA SOX2 downregulation is due to aberrant chromatin looping that results in disassociation of the SOX2 promoter from a downstream enhancer." "ANOP3,MCOPS" Low-grade astrocytomas -- -- -- -- -- CTCF MRD21 ChIP-qPCR "To assess whether CTCF occupancy at these motifs had changed, we performed ChIP-qPCR for CTCF. Every site assayed had an 2-fold decrease in CTCF occupancy, supporting the hypothesis that DNA hypermethylation in 3-hit NSCs leads to reduced CTCF occupancy around. the SOX2 locus." -- -- -- >2KB 700000 E_01_290 29129929 BACH2 Enhancer hg19 chr6 91004796 91006944 Human B Cell low throughput "RT-PCR,ChIP-qPCR,Luciferase Reporter Assay" "DNA immunoprecipitated by total ELK1 antibody or immunoglobulin G (IgG CTL) was amplified by real-time PCR using primers flanking the putative ELK1 binding site in BACH2 intron 1 (predicted BACH2 enhancer position), known ELK1 binding regions in MCL1, MYD88 and FOS genes, and negative control primers as a reference (negative site). ChIP analysis of histone modifications and p300 binding in IL-2-primed D3 B cells. We demonstrated an in vivo binding of ELK1 to this putative enhancer by chromatin immunoprecipitation followed by QPCR (ChIP-QPCR) in IL2-primed B cells collected at D3." Enhancer BACH2 -- Luciferase Reporter Assay "We decided to detail the functional DNA regulatory sequence by luciferase reporter assay in the context of primary human naive B cell activation. The BACH2 proximal promoter41 (minPBACH2, ?725; +146) ligated upstream from the nano-luciferase coding sequence (NanoLuc) demonstrated a 4.2?¡À?0.2 (n?=?15) fold more transcriptional activity than the promoterless NanoLuc vector (pNL1.1), whatever the time point of B-cell electroporation (from D0 to D3). The enhancer region (+1265; +1493) was inserted in the native and reverse orientation downstream from the minPBACH2. " "BTBD25,IMD60" -- -- -- -- -- -- ELK1 ELK1 ChIP-qPCR "ChIP assay of the in vivo binding of ELK1 in IL-2-primed D3 B cells. DNA immunoprecipitated by total ELK1 antibody or immunoglobulin G (IgG CTL) was amplified by real-time PCR using primers flanking the putative ELK1 binding site in BACH2 intron 1 (predicted BACH2 enhancer position), known ELK1 binding regions in MCL1, MYD88 and FOS genes, and negative control primers as a reference (negative site). Means ¡À s.e.m. of four independent experiments." -- -- -- Intron 369624 E_01_291 29142074 -- hg19 chr7 94002773 94005073 Human Lung Myofibroblast Low throughput "Luciferase Reporter Assay,ChIP" The structure of the COL1A2 far-upstream enhancer and the HS4 region is shown in Figure 1A.Enhancer activation was determined by measuring luciferase expression. Enhancer COL1A2 -- "Luciferase Reporter Assay,ChIP" "We created two different luciferase reporter gene constructs under the control of the COL1A2 enhancer and proximal promoter.When the enhancer is engaged, it contacts the RNA polymerase complex forming at the collagen promoter. Thus, an RNA polymerase ChIP signal is a direct measure of enhancer engagement. " "EDSARTH2,EDSCV,OI4" -- -- -- -- -- -- STAT3 "ADMIO,ADMIO1,APRF,HIES" ChIP-qPCR "ChIP analysis using qPCR for the binding of STAT3, JunB, and RNA polymerase II, in the HS4 region of SSc fibroblasts after treatment with SD1029, STAT3 siRNA (siSTAT3), or nontargeting siRNA (siNT). * = p < 0.05." -- -- -- >2KB 19949 E_01_292 29149598 -- hg19 chr12 47471429 47487762 Human SKmel147 Low+High throughput "ATAC-seq,ChIP-seq,Luciferase Reporter Assay,Transfection,CRISPR/Cas9" "Using Assay for Transposase-Accessible Chromatin (ATAC)-seq (Buenrostro et al., 2013) and ChIP-seq for H3K4me1 and H3K27ac (Creyghton et al., 2010), we identified ~1,400 constituent enhancers within the SEs of melanoma cells (Table S5) and found them largely devoid of open chromatin, H3K27ac, and H3K4me1 in NHMs (Figures 5F and ?and5G).To test the functionality of the predicted enhancers (Figures 6B and ?and7A), we cloned E1¨CE5 upstream of a minimal promoter driving luciferase expression (Prescott et al., 2015) and trans-fected these constructs into SKmel147 cells. We next used CRISPR/Cas9 editing to individually delete the genomic regions containing E2¨CE3 and E4¨CE5." Super-Enhancer AMIGO2 CRISPR/Cas9 Luciferase Reporter Assay "Schematic of AMIGO2 SE showing enhancers E1¨CE5 (B), deleted sequences for CRISPR-Cas9 editing (C), and DNA segments used for E3 luciferase assay (D). Luciferase reporter assays performed in SKmel147 cells for AMIGO2 enhancer elements E3A¨CC and E3A deleted for TF motifs (E3A-Del) " "ALI1,AMIGO-2,DEGA" -- -- -- -- -- -- "FOSL2,TEAD4" "FRA2£¬EFTR-2, RTEF1, TCF13L1, TEF-3, TEF3, TEFR-1, hRTEF-1B" Immunoblot "FOSL2 (left), TEAD4 (right), and AMIGO2 immunoblots of SKmel147 cells 96 hr post-transduction with shSCR or shFOSL2 (shF2 #1 and #2) and shSCR or shTEAD4+TEAD1 (shT4 #1+shT1 #1 and shT4 #2+shT1 #1). HSP90 and GAPDH were used as loading controls." -- -- -- >2KB 10107 E_01_293 29151363 -- hg19 chr19 34845032 34847032 Human Prostate Cancer Cell Lines Low+High throughput "WHG-STARR-seq,ChIP-seq,Luciferase Reporter Assay,ATAC-seq,RNA-seq" "In the current study, we use a human prostate cancer cell line, LNCaP as a model to perform whole human genome STARR-seq (WHG-STARR-seq) to reliably obtain an assessment of enhancer activity.Furthermore, these datasets can only identify putative enhancers based on TF and histone chromatin immunoprecipitation (ChIP-seq) data and therefore do not necessarily exhaustively define functional enhancers, leading to false negatives. In order to validate the function of the active enhancers determined by WHG-STARR-seq, we selected 15 regions for all activity measurements and measured their activity using traditional Renilla luciferase reporter assays." Enhancer GPI -- "ATAC-seq,mRNA-seq,WHG-STARR-seq" "Following treatment with TSA, we performed assay for transposase accessible chromatin sequencing (ATAC-seq) and messenger RNA-sequencing (mRNA-seq) experiments to measure the genome-wide chromatic accessibility and gene expression levels, respectively.Genomic snapshot displaying the GPI locus region as detected by WHG-STARR-seq. There is a strong enhancer region approximately 10 kb upstream of GPI transcriptional start site and another weaker enhancer region in the 3¡¯UTR of GPI. " "AMF,GNPI,NLK,PGI,PHI,SA-36,SA36" -- -- -- -- -- -- -- -- -- -- -- -- -- >2KB 9612 E_01_294 29155818 -- hg19 chr13 28545205 28547205 Human Colorectal Cancer Low+High throughput "ChIP-seq,ChIP,EMSA,Luciferase Reporter Assay" "To identify candidate cis-regulatory elements targeted by FOXA factors, we performed literature search, interrogated the ECR browser for evolutionary conserved regions (ECRs), and integrated this information with the results of the FOXA1 and FOXA2 ChIP-seq analysis.Moreover, the ENCODE data base lists several FOXA1 ChIP-seq peaks at the CDH1 locus in different cell lines, especially in the second intron. Notably, at the +7.8 kb enhancer region a FOXA1 ChIP-seq signal overlapped with an ECR (Fig 5A). To find out if FOXA factors could be involved in the function of the intronic CDH1 enhancer in CRC cells, we performed ChIP analyses." Enhancer CDX2 -- "ChIP,Luciferase Reporter Assay" "To test for binding of FOXA proteins at this site of the CDX2 locus in CRC cells we performed ChIP analyses. Indeed, this region was occupied by FOXA1 in LS174T and HT29 cells. Furthermore, FOXA3 was bound at the CDX2 +10.0 kb region in LS174T, SW480 and HT29 cells (Fig 5D). Even though binding did not correlate with CDX2 expression in the CRC cells (S10 Fig, panel B), this result suggests that FOXA proteins act upon a transcriptional enhancer at the CDX2 locus in CRC cells.To test the importance of the FOXA binding sites for CDH1, CDX2 and EPHB3 enhancer activity, we performed luciferase reporter assays in LS174T cells." "CDX-3,CDX2/AS,CDX3" -- -- -- -- -- -- FOXA1 "HNF3A,TCF3A" "EMSA,Luciferase Reporter Assay" "EMSA to test for in vitro binding of FOXA1 and FOXA3 to the CDX2 +10.0 kb enhancer. (C) (Top) Scheme of the EPHB3 ?2.3 kb enhancer with its known transcription factor binding sites. TBE: TCF/LEF-binding element. (Bottom) EMSA to test for in vitro binding of FOXA1 and FOXA3 to the EPHB3 ?2.3 kb enhancer. (D, E, F) Luciferase reporter assay in LS174T cells with constructs covering the CDH1 (D), the CDX2 (E), and the EPHB3 (F) enhancer. Mutations of the respective FOX binding motifs are indicated by red crosses. Shown is the mean and SEM; n_x0015_3. RLA: relative luciferase activity. Statistical significance was calculated relative to the wild-type luciferase reporter constructs. " -- -- -- >2KB 10001 E_01_295 29155818 -- hg19 chr16 68777928 68779928 Human Colorectal Cancer Low+High throughput "ChIP-seq,ChIP,EMSA,Luciferase Reporter Assay" "To identify candidate cis-regulatory elements targeted by FOXA factors, we performed literature search, interrogated the ECR browser for evolutionary conserved regions (ECRs), and integrated this information with the results of the FOXA1 and FOXA2 ChIP-seq analysis.Moreover, the ENCODE data base lists several FOXA1 ChIP-seq peaks at the CDH1 locus in different cell lines, especially in the second intron. Notably, at the +7.8 kb enhancer region a FOXA1 ChIP-seq signal overlapped with an ECR (Fig 5A). To find out if FOXA factors could be involved in the function of the intronic CDH1 enhancer in CRC cells, we performed ChIP analyses." Enhancer CDH1 -- "EMSA,Luciferase Reporter Assay" "Since occupancy of the CDH1 +7.8 kb and EPHB3 ?2.3 kb enhancers by FOXA factors had not yet been described, we validated binding of FOXA1 and FOXA3 to the predicted FOXA binding motifs at these enhancers in vitro by EMSAs.To test the importance of the FOXA binding sites for CDH1, CDX2 and EPHB3 enhancer activity, we performed luciferase reporter assays in LS174T cells." "Arc-1,BCDS1,CD324,CDHE,ECAD,LCAM,UVO" -- -- -- -- -- -- FOXA1 "HNF3A,TCF3A" "EMSA,Luciferase Reporter Assay" "EMSA to test for in vitro binding of FOXA1 and FOXA3 to the CDX2 +10.0 kb enhancer. (C) (Top) Scheme of the EPHB3 ?2.3 kb enhancer with its known transcription factor binding sites. TBE: TCF/LEF-binding element. (Bottom) EMSA to test for in vitro binding of FOXA1 and FOXA3 to the EPHB3 ?2.3 kb enhancer. (D, E, F) Luciferase reporter assay in LS174T cells with constructs covering the CDH1 (D), the CDX2 (E), and the EPHB3 (F) enhancer. Mutations of the respective FOX binding motifs are indicated by red crosses. Shown is the mean and SEM; n_x0015_3. RLA: relative luciferase activity. Statistical significance was calculated relative to the wild-type luciferase reporter constructs. " -- -- -- >2KB 7736 E_01_296 29155818 -- hg19 chr3 184276287 184278287 Human Colorectal Cancer Low+High throughput "ChIP-seq,ChIP,EMSA,Luciferase Reporter Assay" "To identify candidate cis-regulatory elements targeted by FOXA factors, we performed literature search, interrogated the ECR browser for evolutionary conserved regions (ECRs), and integrated this information with the results of the FOXA1 and FOXA2 ChIP-seq analysis.Moreover, the ENCODE data base lists several FOXA1 ChIP-seq peaks at the CDH1 locus in different cell lines, especially in the second intron. Notably, at the +7.8 kb enhancer region a FOXA1 ChIP-seq signal overlapped with an ECR (Fig 5A). To find out if FOXA factors could be involved in the function of the intronic CDH1 enhancer in CRC cells, we performed ChIP analyses." Enhancer EPHB3 -- "ChIP,Luciferase Reporter Assay" "In line with this, we detected by ChIP FOXA1 occupancy at the EPHB3 ?2.3 kb enhancer in LS174T and HT29 cells (Fig 5F). Furthermore, FOXA3 was found to be present at the EPHB3 enhancer at -2.3 kb in all CRC cells except for HCT116 cells.To test the importance of the FOXA binding sites for CDH1, CDX2 and EPHB3 enhancer activity, we performed luciferase reporter assays in LS174T cells." "EK2,ETK2,HEK2,TYRO6" -- -- -- -- -- -- FOXA1 "HNF3A,TCF3A" "EMSA,Luciferase Reporter Assay" "EMSA to test for in vitro binding of FOXA1 and FOXA3 to the CDX2 +10.0 kb enhancer. (C) (Top) Scheme of the EPHB3 ?2.3 kb enhancer with its known transcription factor binding sites. TBE: TCF/LEF-binding element. (Bottom) EMSA to test for in vitro binding of FOXA1 and FOXA3 to the EPHB3 ?2.3 kb enhancer. (D, E, F) Luciferase reporter assay in LS174T cells with constructs covering the CDH1 (D), the CDX2 (E), and the EPHB3 (F) enhancer. Mutations of the respective FOX binding motifs are indicated by red crosses. Shown is the mean and SEM; n_x0015_3. RLA: relative luciferase activity. Statistical significance was calculated relative to the wild-type luciferase reporter constructs. " -- -- -- >2KB 2299 E_01_297 29236325 -- hg19 chr4 55859107 55860311 Human "Kasumi-1,U937-AE" Low throughput "PCR,Luciferase Reporter Assay,ChIP-qPCR,ChIP-3C" "Intriguingly, we also identified a DHS site located at 30 kb downstream of TSS, which was also enriched by p300 (GSE6284728) and thus defined as the intronic enhancer (Figure 2a).To verify whether these co-factors binding to the promoter and intron regions of cKIT, we performed ChIP-qPCR assays with a series of primers for these regions using t(8;21) positive Kasumi-1 cells (Figure 2c)." Enhancer KIT ChIP-3C "Luciferase Reporter Assay,ChIP-qPCR" "We firstly detected the luciferase activity of c-KIT promoter or promoter plus intronic enhancer in Kasumi-1 cells.As shown in Figure 4c, the activity of c-KIT promoter plus intronic enhancer (pGL3-c-KIT-P+I) was significantly higher than that of the c-KIT promoter (pGL3-c-KIT-P).The motif analysis (Figure 2b) as well as the finding that the intronic enhance region increased the activity of c-KIT promoter prompt us to ask whether there exists an interaction between the promoter and intronic enhancer region of c-KIT. To demonstrate our speculation, we firstly performed ChIPqPCR experiments using antibodies of CTCF and a cohesion complex member, RAD21, in Kasumi-1 cell line.Secondly, to provide further evidence that looping occurs in vivo between promoter and enhancer, we performed ChIP combined chromosome conformation capture (ChIP-3C) assays in Kasumi-1 cells. " "C-Kit,CD117,MASTC,PBT,SCFR" -- -- -- The intronic Enhancer region of c-KIT prompts the transactivation of AML1/ETO to c-KIT. "Luciferase Reporter Assay,siRNA" "We firstly detected the luciferase activity of c-KIT promoter or promoter plus intronic Enhancer in Kasumi-1 cells. As shown in Figure 4c, the activity of c-KIT promoter plus intronic Enhancer (pGL3-c-KIT-P+I) was significantly higher than that of the c-KIT promoter (pGL3-c-KIT-P). Further knockdown of AML1/ETO (Figure 4c) led to a dramatic decrease of the activity of both reporter plasmids (Figure 4b). Secondly, we induced AML1/ETO expression using PA in U937-AE cell line (Figure 4d) and measured the luciferase activity of the aforementioned plasmids before and after AML1/ETO induction. As shown in Figure 4e, the luciferase activity of all the reporter plasmids was increased after 48 hours of AML1/ETO induction. The activity of reporter plasmid that contained both c-KIT promoter and intronic Enhancer regions was significantly higher than that contain only c-KIT promoter region." RUNX1 "AML1,AML1-EVI-1,AMLCR1,CBF2alpha,CBFA2,EVI-1,PEBP2aB,PEBP2alpha" ChIP-3C "Knockdown of AML1/ETO led to a dramatic decrease of the activity of both reporter plasmids.Secondly, to provide further evidence that looping occurs in vivo between promoter and Enhancer, we performed ChIP combined chromosome conformation capture (ChIP-3C) assays in Kasumi-1 cells. Collectively, these findings suggest that AML1/ETO prompts the formation of DNA looping between the c-KIT promoter and intronic Enhancer. " -- -- -- >2KB 335615 E_01_298 29326336 -- hg19 chr10 63525013 63527013 Human T-acute lymphoblastic leukemia (T-ALL) Low+High throughput "ChIA-PET,CRISPR/Cas9" "A ChIP-seq analysis of the ARID5B protein showed that ARID5B binds directly to the NOTCH-driven MYC enhancer (N-Me), which has been reported previously to be activated by NOTCH1 in T-ALL cells.Analysis of transcriptional activity using a luciferase reporter assay with the N-Me sequence also demonstrated that ARID5B knockdown significantly inhibited the enhancer activity.Hence, these results indicated that ARID5B directly activates the expression of MYC in T-ALL." Enhancer ARID5B CRISPR/Cas9 ChIA-PET "Importantly, a chromatin¨Cchromatin interaction analysis by paired-end tag sequencing (ChIA-PET) for the cohesin protein demonstrated a cis-regulatory interaction between the ?135-kb element and the ARID5B transcriptional start site (TSS).In fact, genetic knockout of the ?135-kb element by the CRISPR/Cas9 method resulted in a marked down-regulation of ARID5B gene expression.Thus, these results indicate that the TAL1-bound region at ?135 kb is the enhancer controlling ARID5B expression in T-ALL cells." "DESRT, MRF-2, MRF2" T-Cell Acute Lymphoblastic Leukemia DOID:5602 D054218 The TAL1-bound region at ?135 kb is the Enhancer controlling ARID5B expression in T-ALL cells. CRISPR/Cas9 "In fact, genetic knockout of the -135-kb element by the CRISPR/Cas9 method resulted in a marked down-regulation of ARID5B gene expression" TAL1 "SCL,TCL5,bHLHa17,tal-1" CRISPR/Cas9 "In fact, genetic knockout of the ?135- kb element by the CRISPR/Cas9 method resulted in a marked down-regulation of ARID5B gene expression (Fig. 1G; Supplemental Fig. S1B¨CE). Thus, these results indicate that the TAL1-bound region at ?135 kb is the enhancer controlling ARID5B expression in T-ALL cells. " -- -- -- >2KB 282956 E_01_299 29348663 LEENE Enhancer hg19 chr14 56245000 56285000 Human Endothelial Cells (ECs) Low+High throughput "ChIP-seq,qPCR,Hi-C" "To test this possibility, we first attempted to identify the promoter/enhancer region of LEENE. In evaluating the genomic region surrounding LEENE, i.e., 20?kb up- and down-stream of the transcription start site (TSS) on chr14: 56,245,000¨C56,285,000, we observed the enrichments of H3K27ac and H3K4me1 in the HUVEC chromatin immunoprecipitation (ChIP)-seq data available in the ENCODE Database, indicating an ¡°enhancer¡± state of this DNA region.We did find that PS led to significant increase in the H3K27ac in the LEENE region as measured by ChIP-qPCR, indicating the activation of LEENE as an enhancer in ECs subjected to PS vs. " Enhancer LINC00520 -- "ChIP-qPCR,qPCR,Hi-C" "As shown in Fig. 1f, quantitative PCR (qPCR) with LEENE RNA-specific primers also revealed the significantly higher level of LEENE in ECs subjected to PS than to OS. To confirm the flow regulation of LEENE in ECs and explore its relevance to endothelial function, we tested whether LEENE is differentially regulated by tumor necrosis factor alpha (TNF¦Á), which exerts pro-inflammatory effects similar to OS, and atorvastatin (ATV), which confers endothelial protective effects similar to PS. Resembling the opposite effects of OS and PS, TNF¦Á decreased, while ATV increased the level of LEENE.These findings are in line with the differential levels of KLF2/KLF4-eNOS signaling." C14orf34 -- -- -- LEENE-associated Enhancer plays a role in positive regulation of eNOS transcription CRISPR/Cas9 "As a result of the Enhancer ablation, the transcription of LEENE and eNOS was significantly suppressed, in both DMSO (a control vehicle) and ATV-treated ECs" "KLF2,KLF4" "LKLF,EZF,GKLF" ChIP-qPCR "To confirm the association of such TFs on the promoter of LEENE, we performed ChIP-qPCR, which detected a robust binding between KLF4 and multiple regions within the promoter region of LEENE (marked by H3K4me3 peaks, Fig. 2a); these interactions were significantly increased by Ad-KLF4, which mimics the effect of PS and ATV." -- -- -- >2KB 17148 E_01_300 29378668 -- hg19 chr2 11633299 11641134 Human MCF-7 Low+High throughput "ChIP-seq,3C,ChIP-qPCR" "Additionally, overlap with datasets containing histone ChIP-Seq data reveals the enrichment of histone marks corresponding to active/poised enhancers and depleted of those markings found at repressed/silenced enhancers.Validations were performed using conventional ChIP-qPCR.Compared to genomic background, global analysis of the high-confidence TDG peaks revealed that E2-dependent TDG binding was enriched at promoters as well as distal to promoters with approximately 60% occurring intergenically.However, overlapping TDG peaks with sites of E2-dependent ER¦Á localization revealed that 45% of TDG peaks occur at the same sites where ER¦Á localizes in response to E2." Enhancer GREB1 3C -- "ChIP using ER in the presence and absence of TDG as well as ? or + E2, showing that ER binding is unaltered during depletion of TDG. e Loss of TDG prevents enhancer¨Cpromoter looping at the GREB1 locus. MCF7 cells were treated with siControl or siTDG, and then treated with 100 nM E2 for 1 h. 3C, semiquantitative method of measuring the looping between the GREB1 enhancer and promoter, revealed that E2-driven looping of the enhancer and promoter is disrupted upon TDG knockdown." GREB1 Breast Cancer DOID:1612 D001943 -- -- -- TDG hTDG CRISPR/Cas9 "Remarkably, at a subset of enhancers that E2 targets, we found that TDG depletion abrogates E2-mediated eRNA, disrupts 3-dimensional reorganization at ER-targets such as GREB1 and disrupts E2-mediated transcription of corresponding ER-target genes. To investigate whether TDG plays a functional role in E2 signaling in breast cancer, we engineered an MCF7 TDG knockout cell line using CRISPR technology and found that TDG knockout and depletion leads to defects in E2-mediated proliferation and sensitizes MCF7 cells to the anti-estrogen. " -- -- -- >2KB 37024 E_01_301 29379199 -- hg19 chr2 8818311 8820522 Human Neuroblastoma Cell Lines Low+High throughput "ChIP-seq,Western blot" "We observe that two hours after shutdown, MYCN is significantly depleted from promoters and enhancers (Fig. 1f; Supplementary Fig. 2c,e,f) - a result revealed by ChIP-Rx cell-count normalization.Although both traditional ChIP-seq and ChIP-Rx reveal global loss of MYCN at promoters and enhancers, we do observe global loss of additional marks (H3K4me3, CTCF, H3K27ac, and RNA Pol II) at 24 hours post MYCN shutdown only in ChIP-Rx data. In contrast, induction of MYCN in parental SHEP neuroblastoma cells (Fig. 1c,d) results in loading of MYCN at active promoters and enhancers, and is coincident with increases in global H3K27ac. " Enhancer ID2 -- ChIP-seq "ChIP-seq tracks of MYCN and H3K27ac at 0, 2 and 24hrs post MYCN shutdown at RPL22, HAND2 and the upstream ID2 enhancer.Profiling of the TH-MYCN genetically engineered neuroblastoma mouse model28 revealed MYCN binding at the promoters of classic MYC target genes (Npm1/Rpl22), as well as at enhancers for key neuroblastoma and/or neural crest associated genes (ID2 and GATA2)." "GIG8,ID2A,ID2H,bHLHb26" Neuroblastoma DOID:769 D009447 -- -- -- MYCN "MODED,N-myc,NMYC,ODED,bHLHe37" "Western blot,ChIP-seq" "Across MYCN-amplified neuroblastoma lines, we used chromatin immunoprecipitation coupled to high-throughput sequencing (ChIP-seq) to generate a consensus genome-wide map of ~10,000 regions that exhibit strong and consistent MYCN occupancy." -- -- -- >2KB 2695 E_01_302 29379199 -- hg19 chr4 174435828 174438057 Human Neuroblastoma Cell Lines Low+High throughput "ChIP-seq,Western blot" "We observe that two hours after shutdown, MYCN is significantly depleted from promoters and enhancers (Fig. 1f; Supplementary Fig. 2c,e,f) - a result revealed by ChIP-Rx cell-count normalization.Although both traditional ChIP-seq and ChIP-Rx reveal global loss of MYCN at promoters and enhancers, we do observe global loss of additional marks (H3K4me3, CTCF, H3K27ac, and RNA Pol II) at 24 hours post MYCN shutdown only in ChIP-Rx data. In contrast, induction of MYCN in parental SHEP neuroblastoma cells (Fig. 1c,d) results in loading of MYCN at active promoters and enhancers, and is coincident with increases in global H3K27ac. " Enhancer HAND2 -- ChIP-seq "ChIP-seq tracks of MYCN and H3K27ac at 0, 2 and 6hrs post MYCN induction at RPL22, HAND2 and the upstream ID2 enhancer.Visual inspection of MYCN load at highly occupied genes revealed a diversity of binding profiles with some genes exhibiting promoter MYCN binding (RPL22) and others a more mixed array of promoter and enhancer binding (HAND2 & ID2). " "DHAND2,Hed,Thing2,bHLHa26,dHand" Neuroblastoma DOID:769 D009447 -- -- -- MYCN "MODED,N-myc,NMYC,ODED,bHLHe37" "Western blot,ChIP-seq" "Across MYCN-amplified neuroblastoma lines, we used chromatin immunoprecipitation coupled to high-throughput sequencing (ChIP-seq) to generate a consensus genome-wide map of ~10,000 regions that exhibit strong and consistent MYCN occupancy." -- -- -- >2KB 10708 E_01_303 28378740 -- hg19 chr3 107662655 107762655 Human MCF-7 Low+High throughput "ChIP-seq,Luciferase Reporter Assay,ChIA-PET" "By rank-ordering of enhancer regions based on H3K27ac enrichment, we discovered that T-cell acute lymphoblastic leukemia (T-ALL (RPMI18402, Jurkat and MOLT3)) diffuse large B-cell lymphoma (DLBCL (LY4)) and breast cancer (MCF7 and HCC1954) cell lines have SEs within ~200 kb of CD47 (Fig. 1a). To validate their function experimentally, we cloned each candidate CD47 enhancer (E1¨C9) into an EGFP reporter lentiviral construct containing the minimal (basal) promoter for the thymidine kinase (TK) gene7. We found that two of the CD47 enhancers (E5 and E3.2) had MCF7- and Jurkat-speci?c regulatory activity (Fig. 2a¨Cc).First, E5, in the downstream CD47 SE seen in breast cancers (Fig. 1a,b), showed increased reporter activity speci?cally in the MCF7 breast cancer cell line (Fig. 2a). Further analysis of publicly available Paired-End Tag (ChIA-PET) data22,23 con?rmed that E5 and the downstream CD47 SE in MCF7 are connected by a DNA loop containing RNA Polymerase II.Second, we found that E3.2, located within the upstream CD47 SE (Fig. 1a), had increased reporter expression speci?cally in the Jurkat cell line (Fig. 2b). We also found that a third functional enhancer, E7, located within the upstream CD47 SE (Fig. 1a), drove reporter expression in all of the cancer cell lines tested (Fig. 2d)." Super-Enhancer CD47 -- "ChIP-seq,Luciferase Reporter Assay,ChIA-PET" "By rank-ordering of enhancer regions based on H3K27ac enrichment, we discovered that T-cell acute lymphoblastic leukemia (T-ALL (RPMI18402, Jurkat and MOLT3)) diffuse large B-cell lymphoma (DLBCL (LY4)) and breast cancer (MCF7 and HCC1954) cell lines have SEs within ~200 kb of CD47 (Fig. 1a). To validate their function experimentally, we cloned each candidate CD47 enhancer (E1¨C9) into an EGFP reporter lentiviral construct containing the minimal (basal) promoter for the thymidine kinase (TK) gene7. We found that two of the CD47 enhancers (E5 and E3.2) had MCF7- and Jurkat-speci?c regulatory activity (Fig. 2a¨Cc).First, E5, in the downstream CD47 SE seen in breast cancers (Fig. 1a,b), showed increased reporter activity speci?cally in the MCF7 breast cancer cell line (Fig. 2a). Further analysis of publicly available Paired-End Tag (ChIA-PET) data22,23 con?rmed that E5 and the downstream CD47 SE in MCF7 are connected by a DNA loop containing RNA Polymerase II.Second, we found that E3.2, located within the upstream CD47 SE (Fig. 1a), had increased reporter expression speci?cally in the Jurkat cell line (Fig. 2b). We also found that a third functional enhancer, E7, located within the upstream CD47 SE (Fig. 1a), drove reporter expression in all of the cancer cell lines tested (Fig. 2d)." "IAP,MER6,OA3" Breast Cancer DOID:1612 D001943 -- -- -- NFKB1 "CVID12,EBP-1,KBF1,NF-kB,NF-kB1,NF-kappa-B1,NF-kappaB,NFKB-p105,NFKB-p50,NFkappaB,p105,p50" shRNA "We?observed?that?72?h?after?shRNA?transduction,?expression?of?CD47?transcript?and?protein?(measured?by?flow?cytometry)?was?significantly?reduced?by?NFKB1?(Fig.?3b,c)?and?PPAR¦Á?(Fig.?3b,?Supplementary?Fig.?3d,e)?shRNAs,?when?compared?to?control?shRNA.?On?the?other?hand,?shRNAs?against?STATs?3,?5?and?6?did?not?reduce?CD47?expression?significantly?(Fig.?3b).?This?information?demonstrates?that?NFKB?and?PPAR?are?involved?in?the?regulation?of?CD47?in?MCF7?cells?and?since?knocking?down?NFKB1?had?the?strongest?effect?on?CD47?gene?expression,?we?focus?this?study?on?the?regulatory?role?of?this?transcription?factor." -- -- -- >2KB 49285 E_01_304 29385519 -- hg19 chrx 106959618 106962917 Human U-2 OS Low+High throughput "ChIP-qPCR,4C" "However, in contrast to the effects on the hormone-induced levels, these effects do not consistently correlate with deletions at the GILZ GBS1¨C4 enhancer and might reflect difficulties to quantify these transcripts by qPCR due to low expression levels in the absence of hormone treatment.To test if deletion of an individual GBS influences GR occupancy at the edited enhancer, we analyzed GR binding by ChIP-qPCR in a representative clonal cell line in which we deleted the GILZ GBS1. " Enhancer TSC22D3 CRISPR/Cas9 -- "To experimentally test the contribution of the smallest regulatory units of gene regulation, individual GR binding sequences (GBSs), we used the CRISPR/Cas9-system (25) to delete selected GBSs in their endogenous genomic context. We chose a GBS located 1.5 kb upstream of the GR target gene GILZ (glucocorticoid induced leucine zipper, alias TSC22D3) and one GBS 1.5 kb upstream of the target gene DUSP1." "SOM,TFCP2L4,VWS2" -- -- -- -- -- -- NR3C1 "GCCR,GCR,GCRST,GR,GRL" ChIP-seq "To study the global connection between GR binding and GR-dependent gene regulation, we combined data from genome-wide GR binding experiments (Chromatin Immunoprecipitation followed by sequencing (ChIP-seq)) with RNA-seq data regarding gene expression changes upon GR-activation in A549 cells (3)." -- -- -- >2KB 4818 E_01_305 29408204 -- hg19 chr3 69863244 70187654 Human Melanoma Low+High throughput "ChIP-seq,Western blot,siRNA,qPCR" "H3K27ac chromatin immunoprecipitation sequencing (ChIP-seq) profiling, which marks active enhancers, indicated the presence of SEs at the MITF gene locus in MITF-hi cells and substantially less enhancer signal at the same location in an MITF-lo cell line (LOXIMVI) and a normal human dermal fibroblast line." Super-Enhancer MITF -- "qPCR,ChIP-seq" "To identify a set of MITF high- and low-expressing cells, we performed quantitative real-time reverse transcriptase-PCR analysis on 18 melanoma lines and found 8 lines with high MITF (MITF-hi) (>2-fold mean normalized MITF levels) and 5 lines with low MITF (MITF-lo) (2KB 236864 E_01_306 29416716 -- hg19 chr14 99770698 99782638 Human A673/TR/shEF1 Cells Low throughput "Luciferase Reporter Assay,qRT-PCR" " Expression of these genes appeared to be induced by EWSR1-FLI1-bound super-enhancers, which showed high activity in reporter assays.In fact, EWSR1-FLI1 is known to convert non-functional GGAA-microsatellites into potent enhancers to steer a large proportion of its target genes [24¨C26]. Strong EWSR1-FLI1-dependent enhancer activity of these GGAA-microsatellites in luciferase reporter assays was consistently observed. " Super-Enhancer BCL11B -- "qRT-PCR,ChIP-seq" "These data in cell lines suggested that ATP1A1, BCL11B, and GLG1 may be direct EWSR1-FLI1 target genes. Testing this hypothesis involved analyzing available ChIP-Seq and DNase-Seq data generated in Ewing sarcoma cell lines, which showed strong EWSR1-FLI1-binding to GGAA-microsatellites close to these genes." "ATL1,ATL1-alpha,ATL1-beta,ATL1-delta,ATL1-gamma,CTIP-2,CTIP2,IDDFSTA,IMD49,RIT1,ZNF856B,hRIT1-alpha" Ewing Sarcoma DOID:3369 D012512 -- -- -- -- -- -- -- -- -- -- >2KB 141045 E_01_307 29416716 -- hg19 chr1 116896120 116904325 Human A673/TR/shEF1 Cells Low throughput "Luciferase Reporter Assay,qRT-PCR" " Expression of these genes appeared to be induced by EWSR1-FLI1-bound super-enhancers, which showed high activity in reporter assays.In fact, EWSR1-FLI1 is known to convert non-functional GGAA-microsatellites into potent enhancers to steer a large proportion of its target genes [24¨C26]. Strong EWSR1-FLI1-dependent enhancer activity of these GGAA-microsatellites in luciferase reporter assays was consistently observed. " Super-Enhancer ATP1A1 -- "qRT-PCR,ChIP-seq" "These data in cell lines suggested that ATP1A1, BCL11B, and GLG1 may be direct EWSR1-FLI1 target genes. Testing this hypothesis involved analyzing available ChIP-Seq and DNase-Seq data generated in Ewing sarcoma cell lines, which showed strong EWSR1-FLI1-binding to GGAA-microsatellites close to these genes." "CMT2DD,HOMGSMR2" Ewing Sarcoma DOID:3369 D012512 -- -- -- -- -- -- -- -- -- -- >2KB 15571 E_01_308 29416716 -- hg19 chr16 74568579 74583481 Human A673/TR/shEF1 Cells Low throughput "Luciferase Reporter Assay,qRT-PCR" " Expression of these genes appeared to be induced by EWSR1-FLI1-bound super-enhancers, which showed high activity in reporter assays.In fact, EWSR1-FLI1 is known to convert non-functional GGAA-microsatellites into potent enhancers to steer a large proportion of its target genes [24¨C26]. Strong EWSR1-FLI1-dependent enhancer activity of these GGAA-microsatellites in luciferase reporter assays was consistently observed. " Super-Enhancer GLG1 -- "qRT-PCR,ChIP-seq" "These data in cell lines suggested that ATP1A1, BCL11B, and GLG1 may be direct EWSR1-FLI1 target genes. Testing this hypothesis involved analyzing available ChIP-Seq and DNase-Seq data generated in Ewing sarcoma cell lines, which showed strong EWSR1-FLI1-binding to GGAA-microsatellites close to these genes." "CFR-1,ESL-1,MG-160,MG160" Ewing Sarcoma DOID:3369 D012512 -- -- -- -- -- -- -- -- -- -- >2KB 94706 E_01_309 29440643 -- hg19 chr6 138226720 138233280 Human "B Cell,Monocytes" Low+High throughput "CRISPR/Cas9,Hi-C,ChIP-seq" "Recent studies have begun to address mammalian enhancer function using CRISPR-mediated genome editing, including in vivo analysis of mouse genes.To identify enhancers activated by inflammatory stimuli,we analyzed ATAC-seq and ChIP-seq data sets generated in our laboratory (GSE43036, GSE9836926, and GSE104638) for chromatin accessibility, binding of PU.1 and C/EBP (which bind to regulatory elements and help define enhancers in myeloid cells), and induction of acetylation of histone 3 lysine 27 (H3K27-Ac), which indicates enhancer activation, at enhancer peaks after LPS stimulation of primary monocytes." Enhancer TNFAIP3 -- "Hi-C,ChIP-seq" "To guide selection of BACs for generation of humanized transgenic mice, we analyzed chromatin conformation, accessibility, and enhancer-related histone marks at the TNFAIP3 locus using our laboratory¡¯s (GSE4303620, GSE10038321 and previously unpublished data, GSE104628) and ENCODE and NIH Roadmap ChIP-seq data (available at genome.ucsc.edu and roadmapepigenomics.org) and Hi-C data.Analysis of pre-existing high-resolution Hi-C (chromatin conformation) data in GM12878 B cells identified a 305?kb topologically associating domain (TAD) flanked by CTCF sites roughly centered around the TNFAIP3 gene body." "A20,AISBL,OTUD7C,TNFA1P2" Autoimmune Diseases -- D001327 These findings provide insights into enhancers that regulate human A20 expression to prevent inflammatory pathology and autoimmunity. "PCR,CRISPR/Cas9" "Collectively, the results show that deletion of the TT>A Enhancer at the TNFAIP3 locus results in an autoimmune/inflammatory phenotype most clearly evident as athritis." -- -- -- -- -- -- -- >2KB 41676 E_01_310 29453285 -- hg19 chr7 45920458 45923258 Human Hep G2 Low throughput "FAIRE-qPCR,Luciferase Reporter Assay" "The effect of cAMP on the changes of chromatin structure in the IGFBP-1 enhancer region were examined by FAIRE-qPCR. cAMP significantly increased the relative ratio of FAIRE enrichment in the IGFBP-1 enhancer region,indicating that the chromatin structure of the IGFBP-1 enhancer region becomes looser during decidualization." Enhancer IGFBP1 CRISPR/Cas9 -- we used HepG2 cells to examine the endogenous function of the IGFBP-1 enhancer region by a genome editing approach.The enhancer-deleted clones showed significantly lower expression of IGFBP-1 mRNA than the wild-type clones . These results demonstrate that the enhancer region is responsible for IGFBP-1 mRNA expression. "AFBP,IBP1,IGF-BP25,PP12,hIGFBP-1" -- -- -- -- -- -- FOXO1 "FKH1, FKHRA, FOXO1" ChIP "A ChIP assay revealed that cAMP increased the recruitment of the transcriptional regulators CCAAT Enhancer-binding protein ¦Â (C/EBP¦Â), forkhead box O1 (FOXO1), and p300 to the IGFBP-1 Enhancer in ESCs." -- -- -- >2KB 6100 E_01_311 29507293 -- hg19 chr15 66074477 66098025 Human "Erythroleukemia Cell,B lymphoblastoid Cell" Low+High throughput "Hi-C,ChIP-seq" "Finally, if an enhancer element within hierarchical SEs overlaps with a bin associated with a z-score greater than the threshold H-score, the element is referred to a hub enhancer, whereas the remaining enhancers at the same SE are termed non-hub enhancers" Super-Enhancer "MYO1D,SMYD3 " CRISPR/Cas9 -- "we employed CRISPR-Cas9-mediated genome engineering to delete individual hub or non-hub enhancers with paired sgRNAs flanking the enhancer elements at the MYO1D SE.We observed that 3 of the 5 genes within the SE-containing TAD domain MYO1D,TMEM98 and SPACA3)displayed significant downregulation in mRNA expression, whereas the other two genes (PSMD11 and CDK5R1) remained unaffected , suggesting that the MYO1D SE may regulate only a subset of genes within the same TAD domain." "PPP1R108,myr4,KMT3E,ZMYND1,ZNFN3A1,bA74P14.1" -- -- -- Hub enhancers are the major constituents responsible for SE functional and structural organization. "ChIP-seq,CRISPR/Cas9" The signals for H3K27ac and DNase I hypersensitivity are slightly higher at hub than other types of Enhancers. "GATA1,TAL1,PAX5,EBF1" "ERYF1,GATA-1,GF-1,GF1,NF-E1,NFE1,XLANP,XLTDA,XLTT,SCL,ALL3,BSAPTCL5,bHLHa17,tal-1,ALL3,BSAP,COE1,EBF,O/E-1,OLF1" ChIP-seq "Hub Enhancers contain significantly higher ChIP-seq binding signals for lineage-regulating master regulators than non-hub Enhancers, such as GATA1 and TAL1 in K562 cells, and PAX5 and EBF1 in GM12878 cells." -- -- -- >2KB 35266712 E_01_312 29507293 -- hg19 chr17 31209116 31240452 Human "Erythroleukemia Cell,B lymphoblastoid Cell" Low+High throughput "Hi-C,ChIP-seq,CRISPR/Cas9" "Finally, if an enhancer element within hierarchical SEs overlaps with a bin associated with a z-score greater than the threshold H-score, the element is referred to a hub enhancer, whereas the remaining enhancers at the same SE are termed non-hub enhancers" Super-Enhancer "MYO1D,SMYD3 " CRISPR/Cas9 -- "we employed CRISPR-Cas9-mediated genome engineering to delete individual hub or non-hub enhancers with paired sgRNAs flanking the enhancer elements at the MYO1D SE.We observed that 3 of the 5 genes within the SE-containing TAD domain MYO1D,TMEM98 and SPACA3)displayed significant downregulation in mRNA expression, whereas the other two genes (PSMD11 and CDK5R1) remained unaffected , suggesting that the MYO1D SE may regulate only a subset of genes within the same TAD domain." "PPP1R108,myr4,KMT3E,ZMYND1,ZNFN3A1,bA74P14.1" -- -- -- Hub enhancers are the major constituents responsible for SE functional and structural organization. "ChIP-seq,CRISPR/Cas9" The signals for H3K27ac and DNase I hypersensitivity are slightly higher at hub than other types of Enhancers. "GATA1,TAL1,PAX5,EBF1" "ERYF1,GATA-1,GF-1,GF1,NF-E1,NFE1,XLANP,XLTDA,XLTT,SCL,ALL3,BSAPTCL5,bHLHa17,tal-1,ALL3,BSAP,COE1,EBF,O/E-1,OLF1" ChIP-seq "Hub Enhancers contain significantly higher ChIP-seq binding signals for lineage-regulating master regulators than non-hub Enhancers, such as GATA1 and TAL1 in K562 cells, and PAX5 and EBF1 in GM12878 cells." -- -- -- >2KB 405245 E_01_313 29511351 HPSE eRNA hg19 chr4 84277627 84281723 Human AGS Low+High throughput "3C,ChIP-seq,qPCR" "Overlapping analysis of FANTOM5 expression atlas and chromatin interaction dataset derived from 4DGenome revealed one potential enhancer region (chr4:84277627¨C84281723) adjacent to human HPSE promoter(chr4: 84255915¨C84256935). Chromosome conformation capture (3C) and nucleic acid sequencing revealed the endogenous chromatin looping between HPSE promoter and super enhancer in AGS cells" Super-Enhancer HPSE -- "Luciferase Reporter Assay,qPCR" "Dual-luciferase assay revealed that this super enhancer increased the activity of HPSE promoter in gastric cancer cell lines SGC-7901 and AGS, independent of the orientation or location." "HPA,HPA1,HPR1,HPSE1,HSE1" Gastric Cancer DOID:10534 D013274 HPSE eRNA promotes cancer progression through driving chromatin looping and regulating hnRNPU/p300/EGR1/HPSE axis. "ChIP,qPCR" ChIP and qPCR assay revealed that knockdown or ectopic expression of hnRNPU prevented the increased or decreased binding of hnRNPU or p300 to super Enhancer region following stable ectopic expression or knockdown of HPSE eRNA EGR1 "AT225,G0S30,KROX-24,NGFI-A,TIS8,ZIF-268,ZNF225" "ChIP-seq,qPCR" "ChIP and qPCR assay revealed that ectopic expression or knockdown of HPSE eRNA increased and decreased the enrichment of EGR1 on HPSE promoter,which was attenuated by knockdown or overexpression of hnRNPU or p300, respectively." -- -- -- >2KB 66062 E_01_314 29628309 -- hg19 chr3 150450617 150456921 Human MCF-7 Low+High throughput "ChIP-seq,ChIP-qPCR" "We next assessed whether those estrogen-induced JMJD6 binding sites shared enhancer characteristics, including highly enriched H3K4me1/2 but low levels of H3K4me3. Heatmap and tag density plots confirmed they were, indeed, ERa-bound enhancers . Furthermore, it was reported recently that a group of enhancers, namely active enhancers, were essential for the transcriptional activation of estrogeninduced coding target genes" Enhancer SIAH2 -- "ChIP-qPCR,qRT-PCR" "To validate JMJD6 effects on eRNA production, total RNA extracted from MCF7 cells transfected with control siRNA or two independent siRNAs pecifically against JMJD6 in the presence or absence of strogen treatment were subjected to qRT-PCR analysis using primers specifically targeting several ERa and JMJD6 co-bound active enhancers nearby estrogen-induced coding genes, such as FOXC1, SIAH2, GREB1, NRIP1, and SMAD7." hSiah2 Breast Cancer DOID:1612 D001943 -- -- -- "JMJD6,ESR1" "PSR,PTDSR,PTDSR1,ER,ESR,ESRA,ESTRR,Era,NR3A1" "siRNA,RT-qPCR" "To validate JMJD6 effects on eRNA production, total RNA extracted from MCF7 cells transfected with control siRNA or two independent siRNAs specifically against JMJD6 in the presence or absence of estrogen treatment were subjected to RT-qPCR analysis using primers specifically targeting several ER¦Á and JMJD6 co-bound active enhancers nearby estrogen-induced coding genes." -- -- -- >2KB 5140 E_01_315 29628309 -- hg19 chr6 1679010 1683521 Human MCF-7 Low+High throughput "ChIP-seq,ChIP-qPCR" "We next assessed whether those estrogen-induced JMJD6 binding sites shared enhancer characteristics, including highly enriched H3K4me1/2 but low levels of H3K4me3. Heatmap and tag density plots confirmed they were, indeed, ERa-bound enhancers . Furthermore, it was reported recently that a group of enhancers, namely active enhancers, were essential for the transcriptional activation of estrogeninduced coding target genes" Enhancer FOXC1 -- "ChIP-qPCR,qRT-PCR" "To validate JMJD6 effects on eRNA production, total RNA extracted from MCF7 cells transfected with control siRNA or two independent siRNAs pecifically against JMJD6 in the presence or absence of strogen treatment were subjected to qRT-PCR analysis using primers specifically targeting several ERa and JMJD6 co-bound active enhancers nearby estrogen-induced coding genes, such as FOXC1, SIAH2, GREB1, NRIP1, and SMAD7." "ARA,ASGD3,FKHL7,FREAC-3,FREAC3,IGDA,IHG1,IRID1,RIEG3" Breast Cancer DOID:1612 D001943 -- -- -- "JMJD6,ESR1" "PSR,PTDSR,PTDSR1,ER,ESR,ESRA,ESTRR,Era,NR3A1" "siRNA,RT-qPCR" "To validate JMJD6 effects on eRNA production, total RNA extracted from MCF7 cells transfected with control siRNA or two independent siRNAs specifically against JMJD6 in the presence or absence of estrogen treatment were subjected to RT-qPCR analysis using primers specifically targeting several ER¦Á and JMJD6 co-bound active enhancers nearby estrogen-induced coding genes." -- -- -- >2KB 70586 E_01_316 29641996 -- hg19 chr8 128206162 128314310 Human HCT 116 Low+High throughput "4C,ChIP-seq" "Among the DNA loop structures observed in these datasets, a large 2.8 Mb DNA loop was evident in all four cell types. This loop connects CTCF sites encompassing the MYC gene and qualifies as an insulated neighborhood. The DNA anchor sites of this 2.8 Mb DNA loop occur at the boundaries of a TAD found in all cells." Super-Enhancer MYC CRISPR/Cas9 "ChIA-PET,ChIP-qPCR" CTCF chromatin immunoprecipitation quantative polymerase chain reaction (ChIP-qPCR) showed complete loss of CTCF binding to this site in K562 and HCT-116 cells and a 60%¨C70% reduction in CTCF binding at this site in Jurkat and MCF7 cells. RNA analysis revealed a 70%¨C80% reduction of endogenous MYC mRNA in the absence of the enhancer-docking site in all of these cell types. "MRTL,MYCC,bHLHe39,c-Myc" -- -- -- The CTCF site in the promoter-proximal region of MYC is important for optimal interaction with distal Enhancers and supports the idea that this CTCF site functions as an Enhancer-docking site. "4C,CRISPR/Cas9,ChIP-qPCR" "Similar results were obtained in HCT-116 cells, where the viewpoint was centered on the super-Enhancer located -0.4 Mb upstream of the MYC gene" CTCF MRD21 CRISPR/Cas9 "An inducible CRISPR/Cas9 perturbation model showed reduced proliferation for these four cell types upon induction of CTCF-site deletions .These results indicate that the CTCF motif in the MYC Enhancer-docking site is necessary for CTCF binding, for high levels of MYC expression and for cellular proliferation." -- -- -- >2KB 488078 E_01_317 29641996 -- hg19 chr8 129046628 129096067 Human K-562 Low+High throughput "4C,ChIP-seq" "Among the DNA loop structures observed in these datasets, a large 2.8 Mb DNA loop was evident in all four cell types. This loop connects CTCF sites encompassing the MYC gene and qualifies as an insulated neighborhood. The DNA anchor sites of this 2.9 Mb D?" Super-Enhancer MYC CRISPR/Cas9 "ChIA-PET,ChIP-qPCR" CTCF chromatin immunoprecipitation quantative polymerase chain reaction (ChIP-qPCR) showed complete loss of CTCF binding to this site in K562 and HCT-116 cells and a 60%¨C70% reduction in CTCF binding at this site in Jurkat and MCF8 cells. RNA analysis rev? "MRTL,MYCC,bHLHe39,c-Myc" -- -- -- The CTCF site in the promoter-proximal region of MYC is important for optimal interaction with distal Enhancers and supports the idea that this CTCF site functions as an Enhancer-docking site. "4C,CRISPR/Cas9,ChIP-qPCR" The 4C-seq data in K562 cells indicated that the MYC Enhancer docking site interacts predominantly with distal super-Enhancers located -0.3 Mb and -2 Mb downstream of the MYC gene and that the majority of these interactions were significantly reduced when the putative CTCF motif was perturbed . CTCF MRD21 CRISPR/Cas9 "An inducible CRISPR/Cas9 perturbation model showed reduced proliferation for these four cell types upon induction of CTCF-site deletions .These results indicate that the CTCF motif in the MYC Enhancer-docking site is necessary for CTCF binding, for high levels of MYC expression and for cellular proliferation." -- -- -- >2KB 323034 E_01_318 29641996 -- hg19 chr8 130569971 130616320 Human K-562 Low+High throughput "4C,ChIP-seq" "Among the DNA loop structures observed in these datasets, a large 2.8 Mb DNA loop was evident in all four cell types. This loop connects CTCF sites encompassing the MYC gene and qualifies as an insulated neighborhood. The DNA anchor sites of this 210 Mb D?" Super-Enhancer MYC CRISPR/Cas9 "ChIA-PET,ChIP-qPCR" CTCF chromatin immunoprecipitation quantative polymerase chain reaction (ChIP-qPCR) showed complete loss of CTCF binding to this site in K562 and HCT-116 cells and a 60%¨C70% reduction in CTCF binding at this site in Jurkat and MCF9 cells. RNA analysis rev? "MRTL,MYCC,bHLHe39,c-Myc" -- -- -- The CTCF site in the promoter-proximal region of MYC is important for optimal interaction with distal Enhancers and supports the idea that this CTCF site functions as an Enhancer-docking site. "4C,CRISPR/Cas9,ChIP-qPCR" The 4C-seq data in K562 cells indicated that the MYC Enhancer docking site interacts predominantly with distal super-Enhancers located -0.3 Mb and -2 Mb downstream of the MYC gene and that the majority of these interactions were significantly reduced when the putative CTCF motif was perturbed . CTCF MRD21 CRISPR/Cas9 "An inducible CRISPR/Cas9 perturbation model showed reduced proliferation for these four cell types upon induction of CTCF-site deletions .These results indicate that the CTCF motif in the MYC Enhancer-docking site is necessary for CTCF binding, for high levels of MYC expression and for cellular proliferation." -- -- -- >2KB 1844832 E_01_319 29641996 -- hg19 chr8 130693569 130739918 Human K-562 Low+High throughput "4C,ChIP-seq" "Among the DNA loop structures observed in these datasets, a large 2.8 Mb DNA loop was evident in all four cell types. This loop connects CTCF sites encompassing the MYC gene and qualifies as an insulated neighborhood. The DNA anchor sites of this 211 Mb D?" Super-Enhancer MYC CRISPR/Cas9 "ChIA-PET,ChIP-qPCR" CTCF chromatin immunoprecipitation quantative polymerase chain reaction (ChIP-qPCR) showed complete loss of CTCF binding to this site in K562 and HCT-116 cells and a 60%¨C70% reduction in CTCF binding at this site in Jurkat and MC10 cells. RNA analysis rev "MRTL,MYCC,bHLHe39,c-Myc" -- -- -- The CTCF site in the promoter-proximal region of MYC is important for optimal interaction with distal Enhancers and supports the idea that this CTCF site functions as an Enhancer-docking site. "4C,CRISPR/Cas9,ChIP-qPCR" The 4C-seq data in K562 cells indicated that the MYC Enhancer docking site interacts predominantly with distal super-Enhancers located -0.3 Mb and -2 Mb downstream of the MYC gene and that the majority of these interactions were significantly reduced when the putative CTCF motif was perturbed . CTCF MRD21 CRISPR/Cas9 "An inducible CRISPR/Cas9 perturbation model showed reduced proliferation for these four cell types upon induction of CTCF-site deletions .These results indicate that the CTCF motif in the MYC Enhancer-docking site is necessary for CTCF binding, for high levels of MYC expression and for cellular proliferation." -- -- -- >2KB 1968430 E_01_320 29695788 OCT4 Intronic Enhancer hg19 chr6 31137269 31137697 Human Embryonic Stem Cell Low+High throughput "ATAC-seq,Luciferase Reporter Assay,ChIP-seq" "In human pre-implantation blastocyst, however, neither enhancer appears open, whereas two putative enhancers appear downstream of the POU5F1 TSS. Each of these peaks contains a cluster of AP2 sites and a KLF site, indicating that they could be opened by the combinatorial activity of these transcription factors during preimplantation development." Enhancer POU5F1 CRISPR/Cas9 -- "We found normal expression of OCT4 and self-renewal in the primed state, but a dramatic loss of OCT4 expression accompanied by differentiation upon reversion to the naive state. This indicates a potential direct role for TFAP2C in regulating the pluripotency master-regulator OCT4 by binding to a previously unknown enhancer, which in turn is likely to be important for preimplantation OCT4 expression." "OCT3, OCT4, OTF-3, OTF3, OTF4, Oct-3, Oct-4" -- -- -- TFAP2C functions to maintain pluripotency and repress neuroectodermal differentiation during the transition from primed to naive by facilitating the opening of enhancers proximal to pluripotency factors. "Luciferase Reporter Assay,CRISPR/Cas9" "This indicates a potential direct role for TFAP2C in regulating the pluripotency master-regulator OCT4 by binding to a previously unknown Enhancer, which in turn is likely to be important for preimplantation OCT4 expression." TFAP2C "AP2-GAMMA,ERF1,TFAP2G,hAP-2g" Luciferase Reporter Assay "Luciferase activity from a pGL3 construct in which WT or mutant Intron Element 1 had been cloned, normalized to signal from a pGL3 construct with no enhancer. Results are shown from two independent experiment, except for the ¦¤AP2 sample, for which there are n=3 replicates from two experiments. All signals were first normalized for Renilla signal." -- -- -- Intron 5370 E_01_321 29706059 AHRR Enhancer hg19 chr5 372500 376000 Human Circulating Monocytes Cell Low+High throughput "ChIP-seq,RNA-seq" "We tested total RNA samples from a group of CRU-mRNA with available RNA (n=11smokers/10 nonsmokers) and observed three ofthe SM-DMR locations(AHRR, C5orf55-EXOC-AS, and SASH1) showedup-regulation of correspondinge RNA expression as well as mRNA." Enhancer AHRR -- RT-PCR The Figure6C bar graph displays the significants moking-associated fold-change results for each RT-qPCR relative to their genome position in the AHRR region. "AHH,AHHR,bHLHe77" Cardiovascular Cancer DOID:176 -- -- -- -- -- -- -- -- -- -- -- >2KB 69960 E_01_322 29765516 ERG Super-Enhancer hg19 chr21 40003867 40015913 Human HEL Low+High throughput "ChIP-qPCR,ChIP-seq" "Chromatin-immunoprecipitation followed by quantitative PCR (ChIP-qPCR) analysis for GFI1B and RUNX1 was performed at highly conserved noncoding areas including upstream, promotor, 3¡¯ UTR, and SE regions." Super-Enhancer ERG -- ChIP-qPCR "Furthermore,ChIP-qPCR analysis for H3K27ac revealed that the level of H3K27ac was elevated only at the SE region after the NCD38 treatment." "erg-3,p55" Leukemia DOID:1240 D007938 -- -- -- "GFI1B,RUNX1,KDM1A" "BDPLT17,ZNF163B,AML1, AML1-EVI-1, AMLCR1, CBF2alpha, CBFA2, EVI-1, PEBP2aB, PEBP2alpha,AOF2, BHC110, CPRF, KDM1, LSD1" ChIP-qPCR "ChIP-qPCR analysis for GFI1B, RUNX1, or control IgG in the conserved non-coding regions at the ERG locus in HEL cells.ChIP-qPCR analysis for GFI1B (C), LSD1 (D), and CoREST (E) at the ERG-SE locus in HEL cells treated with DMSO or NCD38 for 24 hours. All ChIP-qPCR assays were performed independently twice and the means are displayed." -- -- -- >2KB 270708 E_01_323 29898989 MLH1 Enhancer hg19 chr3 36999364 37001216 Human "SW620,RKO" Low throughput "ChIP-qPCR,3C,Luciferase Reporter Assay,CRISPR/Cas9" "We provide the first description of an enhancer for the MLH1 gene. This -35kb enhancer was CTCF-bound, and disruption of the consensus binding site within the enhancer significantly impairs MLH1 expression in SW620 colorectal carcinoma cells." Enhancer MLH1 "3C,CRISPR/Cas9" "ChIP-qPCR,Luciferase Reporter Assay" "We deleted a DNA fragment corresponding to the -35kb region from SW620 cells,showed that the -35kb region functions as an MLH1 Enhancer in SW620 colorectal cells." "COCA2,FCC2,HNPCC,HNPCC2,hMLH1" Lynch Syndrome DOID:3883 D003123 -- -- -- CTCF MRD21 CRISPR/Cas9 "To evaluate the impact on endogenous MLH1 expression, we next deleted a region containing the CTCF binding motif in SW620 and K562 cells using CRISPR-Cas9 (Fig 3A, ¦¤CTCF). Similar to deletion of the entire Enhancer, we noted significant reduction in MLH1 expression in SW620 but not in K562 cells." -- -- -- >2KB 34550 E_01_324 29903739 COUP-TFII Enhancer hg19 chr15 96725740 96737960 Human E11.5 Heart Low+High throughput "ChIP-seq,Hi-C" "we made use of our TBX20 ChIP-Seq analysis in E11.5 hearts and attributed TBX20 bound sites to the nearest expressed gene in E11.5 control or Tbx20 cKO cardiomyocytes. Next, we overlaid our RNA-Seq data with that of our TBX20 ChIP-Seq analysis in E11.5 hearts and identified 548 genes that were differentially expressed in Tbx20 cKO cardiomyocytes and were marked by one or more TBX20 binding events in the vicinity of the gene." Enhancer NR2F2 -- Hi-C "We next established that this Enhancer was functionally connected with COUP-TFII. We performed a promoter-based Capture Hi-C in iPSC-derived human cardiomyocytes to identify long-range physical interactions between genes and Enhancers. We observed that this Enhancer directly loops and contacts the COUP-TFII promoter,140-Kb away,confirming that this is a COUP-TFII Enhancer." "ARP-1,ARP1,CHTD4,COUPTF2,COUPTFB,COUPTFII,NF-E3,SVP40,TFCOUP2" Congenital Heart Disease DOID:1682 D006330 This Enhancer interacted with the NR2F2 promoter in human cardiomyocytes and conferred atrial specific gene expression in a transgenic mouse in a TBX20 dependent manner. Hi-C "Taken together, these results linked TBX20 binding to an evolutionary conserved Enhancer that regulates COUP-TFII expression in developing atrial cardiomyocytes, uncovering a mechanism by which COUP-TFII expression is TBX20 dependent." TBX20 ASD4 X-Gal Staining Assay Section analysis of COUP-TFII enh1::lacZ transgenic embryo demonstrates X-gal staining in developing atrial myocardium (arrows) and caval vein (arrowheads). -- -- -- >2KB 137306 E_01_325 29912188 -- hg19 chr12 132285987 132290767 Human Ishikawa Cell Low+High throughput ChIP-seq Ensure that each regulatory element to be targeted with Enhancer-i has at least 4 unique oligonucleotides designed for it. Order these sequences along with the ¡°U6_internal¡± primers Enhancer MMP17 CRISPR/Cas9 -- "To determine the contributions of ER-bound Enhancers near the estrogen_x0002_regulated gene MMP17,which has 3 binding sites nearby as defined by ChIP-seq.Figure 2D shows results from an Enhancer dissection experiment, where multiple Enhancers nearby MMP17 are targeted alone and in combination using Enhancer-i." "MMP-17,MT4-MMP,MT4MMP,MTMMP4" -- -- -- -- -- -- -- -- -- -- -- -- -- >2KB 24560 E_01_326 29912188 -- hg19 chr12 132281970 132287430 Human Ishikawa Cell Low+High throughput ChIP-seq Ensure that each regulatory element to be targeted with Enhancer-i has at least 4 unique oligonucleotides designed for it. Order these sequences along with the ¡°U6_internal¡± primers Enhancer MMP17 CRISPR/Cas9 -- "To determine the contributions of ER-bound Enhancers near the estrogen_x0002_regulated gene MMP17,which has 3 binding sites nearby as defined by ChIP-seq.Figure 2D shows results from an Enhancer dissection experiment, where multiple Enhancers nearby MMP18 are t." "MMP-17,MT4-MMP,MT4MMP,MTMMP4" -- -- -- -- -- -- -- -- -- -- -- -- -- >2KB 28237 E_01_327 29912188 -- hg19 chr12 132342570 132347690 Human Ishikawa Cell Low+High throughput ChIP-seq Ensure that each regulatory element to be targeted with Enhancer-i has at least 4 unique oligonucleotides designed for it. Order these sequences along with the ¡°U6_internal¡± primers Enhancer MMP17 CRISPR/Cas9 -- "To determine the contributions of ER-bound Enhancers near the estrogen_x0002_regulated gene MMP17,which has 3 binding sites nearby as defined by ChIP-seq.Figure 2D shows results from an Enhancer dissection experiment, where multiple Enhancers nearby MMP19 are t." "MMP-17,MT4-MMP,MT4MMP,MTMMP4" -- -- -- -- -- -- -- -- -- -- -- -- -- >2KB 32193 E_01_328 30008316 -- hg19 chr1 160536893 160537651 Human "HL-60,293T" Low+High throughput "ChIP-seq,ChIP-qPCR" "Notably, INTS11-gained regions were largely enriched for proximal promoters(62.2%), mirroring the known RNAPII-associated activity of the complex, and displayed robust binding of INTS13 as well. Conversely, INTS13-gained regions were largely found distal from the TSS of protein-coding genes (82.5%) and only partially overlapped with INTS11." Enhancer CD84 "CRISPR/Cas9,3C" qRT-PCR "We performed chromosome conformation capture (3C) on the enhancer of CSF1R to infer the consequences of INTS13 depletion on genome architecture. During differentiation of HL-60 cells, we detected a robust interaction between the intronic enhancer of CSF1R and the proximal promoter, as compared to other regions within the CSF1R gene or with the promoter of neighboring genes." "LY9B,SLAMF5,hCD84,mCD84" -- -- -- -- -- -- "INTS13,EGR1,NAB2" "ASUN,C12orf11,GCT1,Mat89Bb,NET48,SPATA30,AT225,G0S30,KROX-24,NGFI-A,TIS8,ZIF-268,ZNF225,MADER" "Immunoprecipitation,Colony-forming Unit (CFU) Assay" Immunoprecipitation of INTS13 in undifferentiated (CTRL) and differentiated (PMA) HL-60 cells confirms the interaction between INTS13 and NAB2 after PMA treatment.Colony-forming unit (CFU) assay of cord blood derived CD34+ cells infected with shRNAs against NAB2 and EGR1 shows that the number of monocytic/macrophagic colonies is significantly reduced in both NAB2- and EGR1-depleted cells when compared to control. -- -- -- >2KB 26389 E_01_329 30008316 -- hg19 chr1 160542651 160543257 Human "HL-60,293T" Low+High throughput "ChIP-seq,ChIP-qPCR" "Notably, INTS11-gained regions were largely enriched for proximal promoters(62.2%), mirroring the known RNAPII-associated activity of the complex, and displayed robust binding of INTS13 as well. Conversely, INTS14-gained regions were largely found distal" Enhancer CD84 "CRISPR/Cas9,3C" qRT-PCR "We performed chromosome conformation capture (3C) on the enhancer of CSF1R to infer the consequences of INTS13 depletion on genome architecture. During differentiation of HL-60 cells, we detected a robust interaction between the intronic enhancer of CSF1R and the proximal promoter, as compared to other regions within the CSF1R gene or with the promoter of neighboring genes." "LY9B,SLAMF5,hCD84,mCD84" -- -- -- -- -- -- "INTS13,EGR1,NAB2" "ASUN,C12orf11,GCT1,Mat89Bb,NET48,SPATA30,AT225,G0S30,KROX-24,NGFI-A,TIS8,ZIF-268,ZNF225,MADER" "Immunoprecipitation,Colony-forming Unit (CFU) Assay" Immunoprecipitation of INTS13 in undifferentiated (CTRL) and differentiated (PMA) HL-60 cells confirms the interaction between INTS13 and NAB2 after PMA treatment.Colony-forming unit (CFU) assay of cord blood derived CD34+ cells infected with shRNAs against NAB2 and EGR1 shows that the number of monocytic/macrophagic colonies is significantly reduced in both NAB2- and EGR1-depleted cells when compared to control. -- -- -- >2KB 32071 E_01_330 30008316 -- hg19 chr5 149457542 149458807 Human "HL-60,293T" Low+High throughput "ChIP-seq,ChIP-qPCR" "Notably, INTS11-gained regions were largely enriched for proximal promoters(62.2%), mirroring the known RNAPII-associated activity of the complex, and displayed robust binding of INTS13 as well. Conversely, INTS15-gained regions were largely found distal" Enhancer CSF1R "CRISPR/Cas9,3C" qRT-PCR "We performed chromosome conformation capture (3C) on the enhancer of CSF1R to infer the consequences of INTS13 depletion on genome architecture. During differentiation of HL-60 cells, we detected a robust interaction between the intronic enhancer of CSF1R and the proximal promoter, as compared to other regions within the CSF1R gene or with the promoter of neighboring genes." "BANDDOS,C-FMS,CD115,CSF-1R,CSFR,FIM2,FMS,HDLS,M-CSF-R" -- -- -- -- -- -- "INTS13,EGR1,NAB2" "ASUN,C12orf11,GCT1,Mat89Bb,NET48,SPATA30,AT225,G0S30,KROX-24,NGFI-A,TIS8,ZIF-268,ZNF225,MADER" 3C We performed Chromosome Conformation Capture (3C) on the enhancer of CSF1R to infer the consequences of INTS13 depletion on genome architecture. -- -- -- >2KB 25322 E_01_331 30012865 HBG-4kb HS site hg19 chr11 5279732 5280272 Human K-562 Low+High throughput ChIP-seq We generated a synthetic zinc finger DNA-binding domain (ZF) targeting the HBG-4kb HS. The HBG-4kb ZF interacted with the target site in vitro and in the context of cells with high affinity and specificity. Enhancer HBG2 -- PCR "The regions that we analyzed were the HBG-4kb HS,a 327 region located 4 kb upstream (HBG-8kb) or 1 and 2 kb downstream(HBG-3kb and HBG-2kb) of 328 the HBG-4kb HS,the G¦Ã-(HBG2) and A¦Ã-(HBG1) globin gene promoter regions." "HBG-T1,TNCY" Erythrocytic Leukemia -- -- the HBG-4kb HS site is important for fetal globin production and suggest that it may act by opening chromatin in a directional manner. CRISPR/Cas9 "Expression of the adult ¦Â-globin, the embryonic -globin, and the GATA1 genes was not affected by the HBG-4kb ZF. Delivery of the Control ZF did not cause any change in expression of the ¦Ã-globin, ¦Â-globin, -globin, or GATA1 genes." "USF1,USF2,EGR1,MAFK,NFE2" "FCHL,FCHL1,HYPLIP1,MLTF,MLTFI,UEF,bHLHb11,FIP,bHLHb12,AT225,G0S30,KROX-24,NGFI-A,TIS8,ZIF-268,ZNF225,NFE2U,P18,NF-E2,p45" ChIP "Reduced binding of transcription factors with the HBG-4kb HS in K562 cells expressing the HBG-4kb ZF. K562 cells (Untreated) or K562 cells exposed to 500 nM HBG-4kb ZF for 12 h were subjected to ChIP using antibodies specific for EGR1, USF1, USF2, NF-E2 (p45), MafK, or the negative control, IgG." -- -- -- >2KB 5582 E_01_332 30033119 -- hg19 chr8 38291071 38304326 Human Embryonic Stem Cell Low+High throughput ChIP-STARR-seq "Visual inspection of selected genomic regions illustrates the broad spectrum of enhancer activity measured by ChIP-STARR-seq. For instance, ChIP-seq for NANOG indicates two strong binding sites up- and downstream of SOX2, but only the downstream binding site resulted in ChIP-STARR-seq RNA in excess of plasmid abundance." Super-Enhancer FGFR1 CRISPR/Cas9 "Luciferase Reporter Assay,qRT-PCR" Luciferase assays confirmed spatially restricted Enhancer activity of DNA in the neighborhood of the central active region of the FGFR1 SE. "BFGFR,CD331,CEK,ECCL,FGFBR,FGFR-1,FLG,FLT-2,FLT2,HBGFR,HH2,HRTFDS,KAL2,N-SAM,OGD,bFGF-R-1" -- -- -- "Although transposable elements associate with putative enhancers, only some exhibit activity." "Luciferase Reporter Assay,CRISPR/Cas9" Luciferase assays confirmed spatially restricted Enhancer activity of DNA in the neighborhood of the central active region of the FGFR1 SE POU5F1 "OCT3,OCT4,OTF-3,OTF3,OTF4,Oct-3,Oct-4,ANOP3,MCOPS3,NANOG" ChIP-qPCR "To investigate the functional potential of enhancers in ESCs, we first focused on primed H9 ESCs (Figures S1A and S1B) and performed ChIP for NANOG, OCT4, H3K4me1 and H3K27ac.ChIP-qPCR and ChIP-seq were similar to previous results (Figures S1C and 1D).Applying this threshold to regions bound by NANOG, OCT4, H3K4me1, H3K27ac, or combinations of these factors indicates that only a minority of ChIP-seq peaks showed enhancer activity (Figure 2D and S3C), with regions bound by OCT4 having the highest proportion of high activity enhancers." -- -- -- >2KB 29044 E_01_333 30119690 -- hg19 chr16 86422446 86426834 Human HEK-293 Low+High throughput "ChIP-seq,Luciferase Reporter Assay,CRISPR/Cas9" "Notably, two of these five sgRNAs,sgRNAs-AP169 and sgRNA-AP171, are independent sgRNAs that target the same enhancer region,hence increasing the confidence that these are true-positive hits. ENCODE ChIP-seq data confirmed a strong binding of both FOS and JUN to this region." Enhancer FOXF1 CRISPR/Cas9 qRT-PCR "Furthermore,we identified FOXF1 as the tar_x0002_get gene of this Enhancer and demonstrated that it regu_x0002_lates the senescence phenotype." "ACDMPV,FKHL5,FREAC1" -- -- -- We identify FOXF1 as the gene regulated by this enhancer and demonstrate that FOXF1 mediates EnhAP1-OIS1 effect on the senescence phenotype. "qRT-PCR,Western blot" "the physical interactions between EnhAP1-OIS1 and the promoter region of FOXF1 were significantly stronger , suggesting a robust transcriptional regulation of EnhAP1-OIS1." JUN "AP-1,AP1,c-Jun,cJUN,p39" "CRISPR/Cas9,Luciferase Reporter Assay" "We thus constructed a CRISPR-Cas9 sgRNA library designed to target senescence-induced Enhancers that are putatively regulated by AP-1 and used it in a functional screen.That screen was confined to regions that are directly bound by p53 as detected by p53 chromatin immunoprecipitation-sequencing (ChIP-seq) analysis and therefore missed Enhancers that are critical for OIS enforcement and are regulated by other TFs, in either a p53-dependent or -independent manner." -- -- -- >2KB 119492 E_01_334 30139998 -- hg19 chr20 30244800 30316534 Human MDA-MB-231 Low+High throughput "ChIP-qPCR,ChIP-seq,Luciferase Reporter Assay" "In contrast to the broad RING1B ChIP-seq signals in pluripotent cells, RING1B peaks in the breast cell lines were narrow, resembling ChIP-seq signals of transcription factors. Therefore, we assessed whether RING1B is recruited to specific transcription factor-binding sites at SEs." Super-Enhancer BCL2L1 -- qRT-PCR "Indeed,we observed RING1B recruitment at SE regions near BCL2L1 in MDA-MB-231 and ESR1 in T47D23." "BCL-XL/S,BCL2L,BCLX,Bcl-X,PPP1R52" Breast Cancer DOID:1612 D001943 RING1B regulates Enhancer activity and gene transcription not only by promoting the expression of oncogenes but also by regulating chromatin accessibility. ATAC-seq "RING1B was recruited to regions targeted by transcription factors and its depletion deregulated breast cancer signaling pathways as well as FOXA1 and ER¦Á localization to chromatin, we next hypothesized that RING1B regulates transcriptional programs in breast cancer by orchestrating chromatin accessibility. To test this, we performed transposase-accessible chromatin sequencing (ATAC-seq)35 in RING1B-depleted cells.These results indicate that RING1B depletion affects chromatin accessibility at enhancer regions." RNF2 "BAP-1,BAP1,DING,HIPI3,RING1B,RING2" ChIP-qPCR "RING1B, H3K27me3 and H3K27ac ChIP-qPCR of RIN1GB-containing enhancers in control and RING1B-depleted T47D cells. IgG antibody was used as a negative control. As additional control, RING1B ChIP-qPCR were performed using a different RING1B antibody from the one used for ChIP-seq." -- -- -- >2KB 28407 E_01_335 30139998 -- hg19 chr6 37997692 38001538 Human T-47D Low+High throughput "ChIP-qPCR,ChIP-seq,Luciferase Reporter Assay" "In contrast to the broad RING1B ChIP-seq signals in pluripotent cells, RING1B peaks in the breast cell lines were narrow, resembling ChIP-seq signals of transcription factors. Therefore, we assessed whether RING1B is recruited to specific transcription factor-binding sites at SEs." Super-Enhancer FOXA1 -- qRT-PCR "In T47D cells, RING1B bound to a putative SE downstream of FOXA1, suggesting that it plays an activating role in regulating FOXA1 expression." "HNF3A,TCF3A" Breast Cancer DOID:1612 D001943 RING1B regulates Enhancer activity and gene transcription not only by promoting the expression of oncogenes but also by regulating chromatin accessibility. ATAC-seq "RING1B was recruited to regions targeted by transcription factors and its depletion deregulated breast cancer signaling pathways as well as FOXA1 and ER¦Á localization to chromatin, we next hypothesized that RING1B regulates transcriptional programs in breast cancer by orchestrating chromatin accessibility. To test this, we performed transposase-accessible chromatin sequencing (ATAC-seq)35 in RING1B-depleted cells.These results indicate that RING1B depletion affects chromatin accessibility at enhancer regions." RNF2 "BAP-1,BAP1,DING,HIPI3,RING1B,RING2,ER,ESR,ESRA,ESTRR,Era,NR3A1" ChIP-qPCR "RING1B, H3K27me3 and H3K27ac ChIP-qPCR of RIN1GB-containing enhancers in control and RING1B-depleted T47D cells. IgG antibody was used as a negative control. As additional control, RING1B ChIP-qPCR were performed using a different RING1B antibody from the one used for ChIP-seq." -- -- -- >2KB 59141 E_01_336 30139998 -- hg19 chr6 38010769 38019538 Human T-47D Low+High throughput "ChIP-qPCR,ChIP-seq,Luciferase Reporter Assay" "In contrast to the broad RING1B ChIP-seq signals in pluripotent cells, RING1B peaks in the breast cell lines were narrow, resembling ChIP-seq signals of transcription factors. Therefore, we assessed whether RING1B is recruited to specific transcription factor-binding sites at SEs." Super-Enhancer FOXA1 -- qRT-PCR "In T47D cells, RING1B bound to a putative SE downstream of FOXA1, suggesting that it plays an activating role in regulating FOXA1 expression." "HNF3A,TCF3A" Breast Cancer DOID:1612 D001943 RING1B regulates Enhancer activity and gene transcription not only by promoting the expression of oncogenes but also by regulating chromatin accessibility. ATAC-seq "RING1B was recruited to regions targeted by transcription factors and its depletion deregulated breast cancer signaling pathways as well as FOXA1 and ER¦Á localization to chromatin, we next hypothesized that RING1B regulates transcriptional programs in breast cancer by orchestrating chromatin accessibility. To test this, we performed transposase-accessible chromatin sequencing (ATAC-seq)35 in RING1B-depleted cells.These results indicate that RING1B depletion affects chromatin accessibility at enhancer regions." RNF2 "BAP-1,BAP1,DING,HIPI3,RING1B,RING2,ER,ESR,ESRA,ESTRR,Era,NR3A1" ChIP-qPCR "RING1B, H3K27me3 and H3K27ac ChIP-qPCR of RIN1GB-containing enhancers in control and RING1B-depleted T47D cells. IgG antibody was used as a negative control. As additional control, RING1B ChIP-qPCR were performed using a different RING1B antibody from the one used for ChIP-seq." -- -- -- >2KB 43602 E_01_337 30139998 -- hg19 chr6 38032307 38035230 Human T-47D Low+High throughput "ChIP-qPCR,ChIP-seq,Luciferase Reporter Assay" "In contrast to the broad RING1B ChIP-seq signals in pluripotent cells, RING1B peaks in the breast cell lines were narrow, resembling ChIP-seq signals of transcription factors. Therefore, we assessed whether RING1B is recruited to specific transcription factor-binding sites at SEs." Super-Enhancer FOXA1 -- qRT-PCR "In T47D cells, RING1B bound to a putative SE downstream of FOXA1, suggesting that it plays an activating role in regulating FOXA1 expression." "HNF3A,TCF3A" Breast Cancer DOID:1612 D001943 RING1B regulates Enhancer activity and gene transcription not only by promoting the expression of oncogenes but also by regulating chromatin accessibility. ATAC-seq "RING1B was recruited to regions targeted by transcription factors and its depletion deregulated breast cancer signaling pathways as well as FOXA1 and ER¦Á localization to chromatin, we next hypothesized that RING1B regulates transcriptional programs in breast cancer by orchestrating chromatin accessibility. To test this, we performed transposase-accessible chromatin sequencing (ATAC-seq)35 in RING1B-depleted cells.These results indicate that RING1B depletion affects chromatin accessibility at enhancer regions." RNF2 "BAP-1,BAP1,DING,HIPI3,RING1B,RING2,ER,ESR,ESRA,ESTRR,Era,NR3A1" ChIP-qPCR "RING1B, H3K27me3 and H3K27ac ChIP-qPCR of RIN1GB-containing enhancers in control and RING1B-depleted T47D cells. IgG antibody was used as a negative control. As additional control, RING1B ChIP-qPCR were performed using a different RING1B antibody from the one used for ChIP-seq." -- -- -- >2KB 24987 E_01_338 30139998 -- hg19 chr6 151917241 152028736 Human T-47D Low+High throughput "ChIP-qPCR,ChIP-seq,Luciferase Reporter Assay" "In contrast to the broad RING1B ChIP-seq signals in pluripotent cells, RING1B peaks in the breast cell lines were narrow, resembling ChIP-seq signals of transcription factors. Therefore, we assessed whether RING1B is recruited to specific transcription factor-binding sites at SEs." Super-Enhancer ESR1 -- qRT-PCR "Indeed,we observed RING1B recruitment at SE regions near BCL2L1 in MDA-MB-231 and ESR1 in T47D23." "ER,ESR,ESRA,ESTRR,Era,NR3A1" Breast Cancer DOID:1612 D001943 RING1B regulates Enhancer activity and gene transcription not only by promoting the expression of oncogenes but also by regulating chromatin accessibility. ATAC-seq "RING1B was recruited to regions targeted by transcription factors and its depletion deregulated breast cancer signaling pathways as well as FOXA1 and ER¦Á localization to chromatin, we next hypothesized that RING1B regulates transcriptional programs in breast cancer by orchestrating chromatin accessibility. To test this, we performed transposase-accessible chromatin sequencing (ATAC-seq)35 in RING1B-depleted cells.These results indicate that RING1B depletion affects chromatin accessibility at enhancer regions." RNF2 "BAP-1,BAP1,DING,HIPI3,RING1B,RING2" ChIP-qPCR "RING1B, H3K27me3 and H3K27ac ChIP-qPCR of RIN1GB-containing enhancers in control and RING1B-depleted T47D cells. IgG antibody was used as a negative control. As additional control, RING1B ChIP-qPCR were performed using a different RING1B antibody from the one used for ChIP-seq." -- -- -- >2KB 38641 E_01_339 30397001 gtPBREM Enhancer hg19 chr2 234665349 234665739 Human Hep G2 Low throughput "Luciferase Reporter Assay,qRT-PCR" "These observations suggested that a specific enhancer region, such as neighboring transcriptional factors, is required for CAR/CCAR1 interaction." Enhancer UGT1A1 -- ChIP The phenobarbital-responsive Enhancer module of UGT1A1 (gtPBREM)-driven Luciferaseplasmid (gtPBREM-Luc.) containing a 290-bp distal Enhancer module (23570/23180) was constructed based on pGL3-promoter (Promega). "BILIQTL1,GNT1,HUG-BR1,UDPGT,UDPGT1-1,UGT1,UGT1A" Hepatocellular Carcinoma DOID:684 D006528 CCAR1 enhances transcriptional activity of CAR and acts as an Enhancer-dependent selective cofactor of CAR for UGT1A1 expression. Luciferase Reporter Assay The phenobarbital-responsive Enhancer module of UGT1A1 (gtPBREM)-driven luciferase reporter plasmid (gtPBREM-Luc.) containing a 290-bp distal Enhancer module (-23570/-23180) was constructed based on pGL3-promoter (Promega). CCAR1 CCAR1 ChIP "CCAR1 regulates CAR-mediated mRNA expression (Figs. 4 and 5) and reporter activity (Figs. 2 and 3) of UGT1A1 but not CYP2B6.We speculated that CCAR1 is differentially recruited to CAR-responsive Enhancer regions. To investigate this hypothesis,we carried out ChIP experiments in HepTR/CAR cells." -- -- -- >2KB 3374 E_01_340 30511964 -- hg19 chr14 76419740 76421740 Human LNCaP Low+High throughput "ChIP-seq,ChIP-qPCR" Analysis of FOXA1 ChIP-seq previously performed in LNCaP cells identified strong FOXA1 binding at an enhancer of approximately 3.7 kb upstream of the TGFB3 gene promoter. Enhancer TGFB3 -- ChIP-qPCR "Analysis of FOXA1 ChIP-seq previously performed in LNCaP cells identified strong FOXA1 binding at an Enhancer of approximately 3.7 kb upstream of the TGFB3 gene promoter.Importantly, qRT-PCR analysis demonstrated that CRISPR-mediated Enhancer deletion in only one-fourth of the cell population was able to substantially restore the transcription of TGFB3 gene, comparing cells transfected with TGFB3-targeting gRNAs to those with control gRNAs." "ARVD,ARVD1,LDS5,RNHF,TGF-beta3" Prostate Cancer DOID:10283 D011471 -- -- -- FOXA1 "HNF3A,TCF3A" CRISPR/Cas9 "To demonstrate that FOXA1 binding at the upstream Enhancer indeed inhibits TGFB3 gene transcription in vivo, we used a lentiviral CRISPR/Cas9 editing system to delete the Enhancer elements with small guide RNAs.Taken together, these results strongly support that FOXA1 binds to an upstream Enhancer to directly repress TGFB3 transcription." -- -- -- >2KB 3663 E_01_341 30518632 -- hg19 chr1 47704791 47705107 Human T-acute lymphoblastic leukemia (T-ALL) Low throughput "ChIP-qPCR,qRT-PCR,ChIP,Transgenic mice" "We found robust cytotoxicity against T-ALL cells, but not normal bone marrow progenitors, for two N-alkylated TCP derivatives, S2116 and S2157. The two compounds induced apoptosis in TCP-resistant T-ALL cells in vitro and in vivo by repressing transcription of the NOTCH3 and TAL1 genes through increased H3K9 methylation and reciprocal H3K27 deacetylation at super-enhancer regions." Super-Enhancer TAL1 -- "qRT-PCR,ChIP" "we found that both S2116 and S2157 readily increased the methylation level of H3K9 and reciprocally reduced the acetylation level of H3K27 at Super-Enhancer regions of the NOTCH3 and TAL1 genes (GRCh38/hg38: 15,198,031-15,197,862 and GRCh38/hg38: 47,239,435-47,239,119,respectively) using ChIP assays.In addition,global ChIP-seq analyses revealed that the acetylation level of H3K27 was readily decreased by LSD1 inhibition through the entire range of NOTCH3 and TAL1 Enhancers." "SCL,TCL5,bHLHa17,tal-1" Central Nervous System Leukemia DOID:12969 -- -- -- -- ZEB2 "HSPC082,SIP-1,SIP1,SMADIP1,ZFHX1B" RT-PCR "We used the Expression Assays(Hs01097987 for TAL1, Hs01128537 for NOTCH3, Hs00207691 for ZEB2, and Hs01922876 forGAPDH) and TaqMan Universal PCR Master Mix (Applied Biosystems, Waltham, MA) for real-time quantitative RT-PCR (RQ-PCR)." -- -- -- >2KB 22988 E_01_342 30518632 -- hg19 chr19 15308673 15308842 Human T-acute lymphoblastic leukemia (T-ALL) Low throughput "ChIP-qPCR,qRT-PCR,ChIP,Transgenic mice" "We found robust cytotoxicity against T-ALL cells, but not normal bone marrow progenitors, for two N-alkylated TCP derivatives, S2116 and S2157. The two compounds induced apoptosis in TCP-resistant T-ALL cells in vitro and in vivo by repressing transcription of the NOTCH3 and TAL1 genes through increased H3K9 methylation and reciprocal H3K27 deacetylation at super-enhancer regions." Super-Enhancer NOTCH3 -- "qRT-PCR,ChIP" "we found that both S2116 and S2157 readily increased the methylation level of H3K9 and reciprocally reduced the acetylation level of H3K27 at Super-Enhancer regions of the NOTCH3 and TAL1 genes (GRCh38/hg38: 15,198,031-15,197,862 and GRCh38/hg38: 47,239,435-47,239,119,respectively) using ChIP assays.In addition,global ChIP-seq analyses revealed that the acetylation level of H3K27 was readily decreased by LSD1 inhibition through the entire range of NOTCH3 and TAL1 Enhancers." "CADASIL,CADASIL1,CASIL,IMF2,LMNS" Central Nervous System Leukemia DOID:12969 -- -- -- -- ZEB2 "HSPC082,SIP-1,SIP1,SMADIP1,ZFHX1B" RT-PCR "We used the Expression Assays(Hs01097987 for TAL1, Hs01128537 for NOTCH3, Hs00207691 for ZEB2, and Hs01922876 forGAPDH) and TaqMan Universal PCR Master Mix (Applied Biosystems, Waltham, MA) for real-time quantitative RT-PCR (RQ-PCR)." -- -- -- >2KB 38315 E_01_343 30566872 -- hg19 chr10 33492690 33519530 Human Normal Epidermal Cell Low+High throughput "siRNA,ChIP-seq" "Two clusters of active enhancers were bound by p63 . Regions in C3 showed higher p63-binding signals.nearby genes were involved in apoptosis and epidermis development. Regions in C4 had relatively lower p63-binding signals, and nearby genes were involved in keratinocyte differentiation." Enhancer NRP1 -- siRNA "Indeed, we observed a clear decrease of H3K27ac signals at Enhancers that had higher H3K27ac signals and bound by RUNX1 in p63 mutant keratinocytes (Figure 6B; n = 6,035) in two biological replicas, such as Enhancers near NRP1, which is involved in angiogenesis ." "BDCA4,CD304,NP1,NRP,VEGF165R" EEC syndrome DOID:0060782 C536189 -- -- -- "TP63,RUNX1" "AIS,B(p51A),B(p51B),EEC3,KET,LMS,NBP,OFC8,RHS,SHFM4,TP53CP,TP53L,TP73L,p40,p51,p53CP,p63,p73H,p73L,AML1, AML1-EVI-1, AMLCR1, CBF2alpha, CBFA2, EVI-1, PEBP2aB, PEBP2alpha" "siRNA,qRT-PCR" Gene expression analysis by qRT-PCR of TP63 and RUNX1 expression in TP63 knockdown (siTP63) compared to non-targeting siRNA (siNT) in control keratinocytes. -- -- -- >2KB 39692 E_01_344 30566872 -- hg19 chr10 33512793 33539633 Human Normal Epidermal Cell Low+High throughput "siRNA,ChIP-seq" "Two clusters of active enhancers were bound by p63 . Regions in C3 showed higher p63-binding signals.nearby genes were involved in apoptosis and epidermis development. Regions in C4 had relatively lower p63-binding signals, and nearby genes were involved in keratinocyte differentiation." Enhancer NRP1 -- siRNA "Indeed, we observed a clear decrease of H3K27ac signals at Enhancers that had higher H3K27ac signals and bound by RUNX1 in p63 mutant keratinocytes (Figure 6B; n = 6,035) in two biological replicas (Figures 6H and S7E), such as Enhancers near NRP1, which is involved in angiogenesis (Figure 6I)." "BDCA4,CD304,NP1,NRP,VEGF165R" EEC syndrome DOID:0060782 C536189 -- -- -- "TP63,RUNX1" "AIS,B(p51A),B(p51B),EEC3,KET,LMS,NBP,OFC8,RHS,SHFM4,TP53CP,TP53L,TP73L,p40,p51,p53CP,p63,p73H,p73L,AML1, AML1-EVI-1, AMLCR1, CBF2alpha, CBFA2, EVI-1, PEBP2aB, PEBP2alpha" "siRNA,qRT-PCR" Gene expression analysis by qRT-PCR of TP63 and RUNX1 expression in TP63 knockdown (siTP63) compared to non-targeting siRNA (siNT) in control keratinocytes. -- -- -- >2KB 59795 E_01_345 30566872 -- hg19 chr10 33637535 33664375 Human Normal Epidermal Cell Low+High throughput "siRNA,ChIP-seq" "Two clusters of active enhancers were bound by p63 . Regions in C3 showed higher p63-binding signals.nearby genes were involved in apoptosis and epidermis development. Regions in C4 had relatively lower p63-binding signals, and nearby genes were involved in keratinocyte differentiation." Enhancer NRP1 -- siRNA "Indeed, we observed a clear decrease of H3K27ac signals at Enhancers that had higher H3K27ac signals and bound by RUNX1 in p63 mutant keratinocytes (Figure 6B; n = 6,035) in two biological replicas (Figures 6H and S7E), such as Enhancers near NRP1, which is involved in angiogenesis (Figure 6I)." "BDCA4,CD304,NP1,NRP,VEGF165R" EEC syndrome DOID:0060782 C536189 -- -- -- "TP63,RUNX1" "AIS,B(p51A),B(p51B),EEC3,KET,LMS,NBP,OFC8,RHS,SHFM4,TP53CP,TP53L,TP73L,p40,p51,p53CP,p63,p73H,p73L,AML1, AML1-EVI-1, AMLCR1, CBF2alpha, CBFA2, EVI-1, PEBP2aB, PEBP2alpha" "siRNA,qRT-PCR" Gene expression analysis by qRT-PCR of TP63 and RUNX1 expression in TP63 knockdown (siTP63) compared to non-targeting siRNA (siNT) in control keratinocytes. -- -- -- >2KB 184537 E_01_346 30568279 -- hg19 chr14 106327635 106328903 Human GM12878 Low+High throughput "Luciferase Reporter Assay,ChIP-seq" "Surprisingly, this enrichment stayed constant thereafter for promoter regions, and increased modestly for enhancer regions, ultimately surpassing the enrichment seen for promoters. In fact, even though promoter chromatin states were more enriched at intermediate HiDRA activity levels,enhancer chromatin states were the most enriched at the highest HiDRA activity levels , suggesting that enhancer elements have a greater dynamic range of regulatory activity potential, which has implications for the regulatory architecture of genes." Enhancer IGHE -- Luciferase Reporter Assay We observed that the peak of HiDRA activity is centered precisely within the region previously identi?ed as driving Enhancer activity in low-throughput luciferase assays (Fig. 2c). IgE -- -- -- -- -- -- -- -- -- -- -- -- -- >2KB 261866 E_01_347 30568279 -- hg19 chr9 126900000 126900625 Human GM12878 Low+High throughput "Luciferase Reporter Assay,ChIP-seq" "Surprisingly, this enrichment stayed constant thereafter for promoter regions, and increased modestly for enhancer regions, ultimately surpassing the enrichment seen for promoters. In fact, even though promoter chromatin states were more enriched at intermediate HiDRA activity levels,enhancer chromatin states were the most enriched at the highest HiDRA activity levels , suggesting that enhancer elements have a greater dynamic range of regulatory activity potential, which has implications for the regulatory architecture of genes." Enhancer NEK6 -- Luciferase Reporter Assay A visualization of 14 luciferase-tested Enhancers in the serine/threonine kinase NEK6 locus shows a strong correspondence between luciferase assay results and HiDRA active regions. SID6-1512 -- -- -- -- -- -- -- -- -- -- -- -- -- >2KB 119571 E_01_348 30568279 -- hg19 chr9 126964375 126965000 Human GM12878 Low+High throughput "Luciferase Reporter Assay,ChIP-seq" "Surprisingly, this enrichment stayed constant thereafter for promoter regions, and increased modestly for enhancer regions, ultimately surpassing the enrichment seen for promoters. In fact, even though promoter chromatin states were more enriched at intermediate HiDRA activity levels,enhancer chromatin states were the most enriched at the highest HiDRA activity levels , suggesting that enhancer elements have a greater dynamic range of regulatory activity potential, which has implications for the regulatory architecture of genes." Enhancer NEK6 -- Luciferase Reporter Assay A visualization of 14 luciferase-tested Enhancers in the serine/threonine kinase NEK7 locus shows a strong correspondence between luciferase assay results and HiDRA active regions. SID6-1512 -- -- -- -- -- -- -- -- -- -- -- -- -- >2KB 55196 E_01_349 30568279 -- hg19 chr9 126980625 126981125 Human GM12878 Low+High throughput "Luciferase Reporter Assay,ChIP-seq" "Surprisingly, this enrichment stayed constant thereafter for promoter regions, and increased modestly for enhancer regions, ultimately surpassing the enrichment seen for promoters. In fact, even though promoter chromatin states were more enriched at intermediate HiDRA activity levels,enhancer chromatin states were the most enriched at the highest HiDRA activity levels , suggesting that enhancer elements have a greater dynamic range of regulatory activity potential, which has implications for the regulatory architecture of genes." Enhancer NEK6 -- Luciferase Reporter Assay A visualization of 14 luciferase-tested Enhancers in the serine/threonine kinase NEK8 locus shows a strong correspondence between luciferase assay results and HiDRA active regions. SID6-1512 -- -- -- -- -- -- -- -- -- -- -- -- -- >2KB 39009 E_01_350 30568279 -- hg19 chr9 127001250 127001875 Human GM12878 Low+High throughput "Luciferase Reporter Assay,ChIP-seq" "Surprisingly, this enrichment stayed constant thereafter for promoter regions, and increased modestly for enhancer regions, ultimately surpassing the enrichment seen for promoters. In fact, even though promoter chromatin states were more enriched at intermediate HiDRA activity levels,enhancer chromatin states were the most enriched at the highest HiDRA activity levels , suggesting that enhancer elements have a greater dynamic range of regulatory activity potential, which has implications for the regulatory architecture of genes." Enhancer NEK6 -- Luciferase Reporter Assay A visualization of 14 luciferase-tested Enhancers in the serine/threonine kinase NEK9 locus shows a strong correspondence between luciferase assay results and HiDRA active regions. SID6-1512 -- -- -- -- -- -- -- -- -- -- -- -- -- >2KB 18321 E_01_351 30568279 -- hg19 chr9 127047500 127048375 Human GM12878 Low+High throughput "Luciferase Reporter Assay,ChIP-seq" "Surprisingly, this enrichment stayed constant thereafter for promoter regions, and increased modestly for enhancer regions, ultimately surpassing the enrichment seen for promoters. In fact, even though promoter chromatin states were more enriched at intermediate HiDRA activity levels,enhancer chromatin states were the most enriched at the highest HiDRA activity levels , suggesting that enhancer elements have a greater dynamic range of regulatory activity potential, which has implications for the regulatory architecture of genes." Enhancer NEK6 -- Luciferase Reporter Assay A visualization of 14 luciferase-tested Enhancers in the serine/threonine kinase NEK10 locus shows a strong correspondence between luciferase assay results and HiDRA active regions. SID6-1512 -- -- -- -- -- -- -- -- -- -- -- -- -- >2KB 28054 E_01_352 30590035 POU5F1 proximal Enhancer hg19 chr6 31139519 31140167 Human Human Primordial Germ Cell Low+High throughput "ATAC-seq,ChIP-seq" "These data show that the PE, DE, and NE are all open in hPGCLCs at day 4 of aggregate differentiation, with the NE being more open in hPGCs than the DE and PE. This observation raises the possibility that restriction of OCT4 expression between days 2 and 3 of aggregate differentiation may be due to NE and/or the DE enhancer activation at the OCT4 locus. Given that the NE is bound by TFAP2C in groundstate naive pluripotent stem cells, whereas the DE is not , we next confirmed that the OCT4 NE is also a target of TFAP2C during hPGCLC differentiation." Enhancer POU5F1 -- ChIP-qPCR "Screenshot of ATAC-seq and TFAP2C ChIP-seq signals showing three Enhancers at the POU5F1 locus (encoding OCT4). Shaded boxes highlight the naive Enhancer(NE),proximalEnhancer(PE),anddistalEnhancer(DE)atthePOU5F1locus.DEdeletion andNEdeletion indicategenomicregionsthatweredeleted by CRISPR/Cas9-mediated genome editing. Primers for ChIP-qPCR (P1-2,P3-4,P5-6) of NE and control regions are shown." "OCT3, OCT4, OTF-3, OTF3, OTF4, Oct-3, Oct-4" -- -- -- "TFAP2C regulates human germline cell formation is opening naive-specific Enhancers, with one of these Enhancers corresponding to the NE at the OCT4 locus." CRISPR/Cas9 "Using CRISPR/Cas9, we show that deleting the TFAP2C-bound naive enhancer at the OCT4 locus (also called POU5F1) results in impaired OCT4 expression and a negative effect on hPGCLC identity." TFAP2C "AP2-GAMMA,ERF1,TFAP2G,hAP-2g" CRISPR/Cas9 "Using CRISPR/Cas9, we show that deleting the TFAP2C-bound naive Enhancer at the OCT4 locus (also called POU5F1) results in impaired OCT4 expression and a negative effect on hPGCLC identity." -- -- -- >2KB 7730 E_01_353 30590035 POU5F1 naive Enhancer hg19 chr6 31137056 31138028 Human Human Primordial Germ Cell Low+High throughput "ATAC-seq,ChIP-seq" "These data show that the PE, DE, and NE are all open in hPGCLCs at day 4 of aggregate differentiation, with the NE being more open in hPGCs than the DE and PE. This observation raises the possibility that restriction of OCT4 expression between days 3 and" Enhancer POU5F1 -- ChIP-qPCR "Screenshot of ATAC-seq and TFAP2C ChIP-seq signals showing three Enhancers at the POU5F1 locus (encoding OCT4). Shaded boxes highlight the naive Enhancer(NE),proximalEnhancer(PE),anddistalEnhancer(DE)atthePOU5F1locus.DEdeletion andNEdeletion indicategenomicregionsthatweredeleted by CRISPR/Cas9-mediated genome editing. Primers for ChIP-qPCR (P1-2,P3-4,P5-6) of NE and control regions are shown." "OCT3, OCT4, OTF-3, OTF3, OTF4, Oct-3, Oct-4" -- -- -- "The PE, DE, and NE are all open in hPGCLCs at day 4 of aggregate differentiation." ATAC-seq "First, to determine whether the NE is also open in human germline cells, we compared ATAC-seq peaks for the NE in hPGCLCs and hPGCs to this region in naive and primed hESCs (Figure 5C). These data show that the PE, DE, and NE are all open in hPGCLCs at day 4 of aggregate differentiation, with the NE being more open in hPGCs than the DE and PE." TFAP2C "AP2-GAMMA,ERF1,TFAP2G,hAP-2g" CRISPR/Cas9 "Using CRISPR/Cas9, we show that deleting the TFAP2C-bound naive Enhancer at the OCT4 locus (also called POU5F1) results in impaired OCT4 expression and a negative effect on hPGCLC identity." -- -- -- >2KB 5429 E_01_354 30590035 POU5F1 distal Enhancer hg19 chr6 31140556 31141204 Human Human Primordial Germ Cell Low+High throughput "ATAC-seq,ChIP-seq,ChIP-qPCR" "Using chromatin immunoprecipitation sequencing (ChIP-seq) for TFAP2C in 5i/L/FA cultured naive hPSCs (Pastor et al., 2018), we found that the 1,892 hPGC and hPGCLC overlapping peaks are also bound by TFAP2C in 5i/L/FA cultured cells (Figure 3C).Analysis of the 1,892 peaks enriched in the naive-specific and germline cell-specific intersect group revealed a significant enrichment of AP2 motifs, as expected (Figure S3B).This observation raises the possibility that restriction of OCT4 expression between days 2 and 3 of aggregate differentiation may be due to NE and/or the DE enhancer activation at the OCT4 locus. Given that the NE is bound by TFAP2C in ground-state naive pluripotent stem cells, whereas the DE is not (Figure 5C), we next confirmed that the OCT4 NE is also a target of TFAP2C during hPGCLC differentiation. To do this, we performed ChIP-qPCR on day 4 aggregates containing hPGCLCs and discovered that TFAP2C is bound to the NE, whereas it is not bound to a genomic region that does not contain AP2 sites (Figure 5D). Taken together, these experiments suggest that OCT4 regulation during hPGCLC differentiation may involve enhancer activation at the DE as well as TFAP2C-bound enhancer activation at the NE." Enhancer POU5F1 -- ChIP-qPCR "Screenshot of ATAC-seq and TFAP2C ChIP-seq signals showing three Enhancers at the POU5F1 locus (encoding OCT4). Shaded boxes highlight the naive Enhancer(NE),proximalEnhancer(PE),anddistalEnhancer(DE)atthePOU5F1locus.DEdeletion andNEdeletion indicategenomicregionsthatweredeleted by CRISPR/Cas9-mediated genome editing. Primers for ChIP-qPCR (P1-2,P3-4,P5-6) of NE and control regions are shown." "OCT3, OCT4, OTF-3, OTF3, OTF4, Oct-3, Oct-4" -- -- -- OCT4 regulation during hPGCLC differentiation may involve enhancer activation at the DE as well as TFAP2C-bound enhancer activation at the NE. "ATAC-seq,ChIP-qPCR" "Taken together, these experiments suggest that OCT4 regulation during hPGCLC differentiation may involve enhancer activation at the DE as well as TFAP2C-bound enhancer activation at the NE." TFAP2C "AP2-GAMMA,ERF1,TFAP2G,hAP-2g" CRISPR/Cas9 "Using CRISPR/Cas9, we show that deleting the TFAP2C-bound naive Enhancer at the OCT4 locus (also called POU5F1) results in impaired OCT4 expression and a negative effect on hPGCLC identity." -- -- -- >2KB 8767 E_01_355 18695675 IgH 3' Enhancer hg19 chr14 105051517 105351516 Human "DHL-4,DHL-9" Low+High throughput "3C,PCR,ChIP" "To determine whether the IgH 3' enhancers interact with the bcl-2 promoter region, 3C analysis was performed with primers near the bcl-2 ranscriptional start sites on chromosome 18 and primers on chromosome 14 (105,051,517-105,351,516). The region of chromosome 14 is centered approximately between the two IgH 3' enhancer regions downstream of C¦Á1 and C¦Á2." Enhancer BCL2 3C PCR "To determine whether the IgH 3' Enhancers interact with the bcl-2 promoter region, 3C analysis was performed with primers near the bcl-2 transcriptional start sites on chromosome 18 and primers on chromosome 14." "Bcl-2,PPP1R50" Lymphoma DOID:0060058 D008223 "The IgH 3' enhancers physically interact with the bcl-2 promoter region over a 350 kb genomic region in t(14;18) lymphoma cells. The physical Interactions of the IgH enhancers with the bcl-2 5' region are functionally involved in the transcriptional contr" 3C "Using the chromosome conformation capture (3C) technique, we found that the IgH 3' Enhancers physically interact with the bcl-2 promoter region over a 350 kb genomic region in t(14;18) lymphoma cells." POU2F2 "OCT2,OTF2,Oct-2" ChIP We showed by chromatin immunoprecipitation assay (ChIP) and siRNA transfection studies that the POU2 family transcription factor Oct-2 and its cofactor Bob-1 play a critical role in mediating the IgH Enhancer-bcl-2 promoter region interactions. -- -- -- >2KB 44410939 E_01_356 20093418 FKBP51 Enhancer hg19 chr6 35628362 35641362 Human A549 Low throughput "ChIP,Transgenic mice" Our quantitative chromatin immunoprecipitation scans and enhancer activity analyses indicate that activation of the FKBP51 locus by glucocorticoids in vivo is triggered by the loading of GR to enhancers at about 34 kb 5 and about 87 kb 3 of the transcription start site. Enhancer FKBP4 -- "qRT-PCR,Western blot" Schematic locations of the GREs and regions analyzed in reporter and ChIP assays. "FKBP51,FKBP52,FKBP59,HBI,Hsp56,PPIase,p52" -- -- -- -- -- -- CTCF MRD21 ChIP We used the genome-wide CTCF-binding information from the CTCF-binding site database for choosing the regions to be ChIP scanned with anti-CTCF antibody. -- -- -- >2KB 32730755 E_01_357 20952403 RET Enhancer hg19 chr10 43569045 43569456 Human SK-N-BE Low throughput ChIP "We then performed chromatin immunoprecipitation (ChIP) experiments to verify the presence of RARa, in RA-stimulated and unstimulated cells, at three different regions of RET locus:the enhancer region located about 3.4-kb upstream from the TSS, containing functional SOX10 and Pax3 binding sites and displaying a putative complete site for RARa binding;" Enhancer RET -- "Luciferase Reporter Assay,qRT-PCR" "The general structure of human RET gene and the main regulatory elements have been partially investigated and it has been reported that a conserved Enhancer region, located about 3000-bp upstream from a promoter region, contains binding sites for Sox10 and Pax3 transcription factors." "CDHF12,CDHR16,HSCR1,MEN2A,MEN2B,MTC1,PTC,RET-ELE1" -- -- -- The primary epigenetic determinants of RA-induced RET activation differ between Enhancer and promoter regions. "Luciferase Reporter Assay,qRT-PCR,ChIP" "Then we performed luciferase transcriptional assays in order to investigate whether a 3.4-kb RET gene regulatory region,located upstream from TSS and including both promoter and Enhancer,was sufficient for RA responsiveness.The results,shown in Figure 2A,demonstrated that plasmid constructs containing the 3.4-kb RET region upstream the luciferase gene were more active than empty plasmids(pGL3-basic);however, no significant increase of luciferase activity was observed when trans_x0002_fected cells were treated with RA." "SOX10,PAX3" "DOM,PCWH,WS2E,WS4,WS4C,CDHS,HUP2,WS1,WS3" ChIP "We then performed chromatin immunoprecipitation (ChIP) experiments to verify the presence of RARa, in RA-stimulated and un_x0002_stimulated cells, at three different regions of RET locus (Figure 1A):(i) the Enhancer region located about 3.4-kb upstream from the TSS, containing functional SOX10 and Pax3 binding sites." -- -- -- >2KB 3265 E_01_358 22543974 -- hg19 chr12 114463712 114464080 Human "HeLa,293T" Low+High throughput "EMSA,PCR,ChIP-seq" "This strategy considered multiple genome-wide ChIP-seq data sets including the enhancer associated transcriptional co-activator p300, cardiac transcription factors Mef2a, TBX5, Gata4, Nkx2.5, SRF and computationally predicted enhancers, as well as the enhancer-associated histonemodificationH3K4me1and evolutionary conservation to identify and prioritize likely functional non-coding regions." Enhancer TBX5 -- "EMSA,PCR" "We identified three elements (CREs 2, 9, 16) with patterns of Enhancer activity overlapping the endogenous expression pattern of TBX5 and the Enhancer-trap BACs in which they were contained." HOS Congenital Heart Disease DOID:1682 D006330 "A significant number of CHD associated with TBX5 dysfunction might arise from non-coding mutations in TBX5 heart enhancers, effectively decoupling the heart and hand phenotypes of the Holt-Oram syndrome." "EMSA,ChIP-seq" "Notably, all lines exhibited weak expression in the left atrium and almost no ex_x0002_pression in the right atrium, suggesting that the bulk of TBX5 atrial expression is regulated by Enhancers outside the genomic region covered by this BAC. " TBX5 HOS EMSA "We hypothesized that the variant nucleotide disrupts transcrip_x0002_tion factor binding to Enhancer 9. To test this, we performed an electrophoretic mobility shift assay using short DNA probes spanning either the wild-type (G) or variant (T) allele." -- -- -- >2KB 327838 E_01_359 22543974 -- hg19 chr12 114701207 114704691 Human "HeLa,293T" Low+High throughput "EMSA,PCR,ChIP-seq" "This strategy considered multiple genome-wide ChIP-seq data sets including the enhancer associated transcriptional co-activator p300, cardiac transcription factors Mef2a, TBX5, Gata4, Nkx2.6, SRF and computationally predicted enhancers, as well as the enh?" Enhancer TBX5 -- "EMSA,PCR" "We identified three elements (CREs 2, 9, 16) with patterns of Enhancer activity overlapping the endogenous expression pattern of TBX5 and the Enhancer-trap BACs in which they were contained." HOS Congenital Heart Disease DOID:1682 D006330 "Asignificant number of CHD associated with TBX5 dysfunction might arise from non-coding mutations in TBX5 heart enhancers, effectively decoupling the heart and hand phenotypes of the Holt-Oram syndrome." "EMSA,ChIP-seq" "Notably, all lines exhibited weak expression in the left atrium and almost no ex_x0002_pression in the right atrium, suggesting that the bulk of TBX5 atrial expression is regulated by Enhancers outside the genomic region covered by this BAC. " TBX5 HOS EMSA "We hypothesized that the variant nucleotide disrupts transcrip_x0002_tion factor binding to Enhancer 9. To test this, we performed an electrophoretic mobility shift assay using short DNA probes spanning either the wild-type (G) or variant (T) allele." -- -- -- >2KB 88785 E_01_360 22543974 -- hg19 chr12 114853271 114858238 Human "HeLa,293T" Low+High throughput "EMSA,PCR,ChIP-seq" "This strategy considered multiple genome-wide ChIP-seq data sets including the enhancer associated transcriptional co-activator p300, cardiac transcription factors Mef2a, TBX5, Gata4, Nkx2.7, SRF and computationally predicted enhancers, as well as the enh?" Enhancer TBX5 -- "EMSA,PCR" "We identified three elements (CREs 2, 9, 16) with patterns of Enhancer activity overlapping the endogenous expression pattern of TBX5 and the Enhancer-trap BACs in which they were contained." HOS Congenital Heart Disease DOID:1682 D006330 "A significant number of CHD associated with TBX5 dysfunction might arise from non-coding mutations in TBX5 heart enhancers, effectively decoupling the heart and hand phenotypes of the Holt-Oram syndrome." "EMSA,ChIP-seq" "Notably, all lines exhibited weak expression in the left atrium and almost no ex_x0002_pression in the right atrium, suggesting that the bulk of TBX5 atrial expression is regulated by Enhancers outside the genomic region covered by this BAC. " TBX5 HOS EMSA "We hypothesized that the variant nucleotide disrupts transcrip_x0002_tion factor binding to Enhancer 9. To test this, we performed an electrophoretic mobility shift assay using short DNA probes spanning either the wild-type (G) or variant (T) allele." -- -- -- >2KB 64021 E_01_361 23108396 -- hg19 chr11 69468761 69470761 Human "MDA-MB-23,MDA-MB-436" Low+High throughput "3C,ChIP,qPCR" "ChIP confirmed that H2A.Z siRNA treatment lead to markedly reduced binding of H2A.Z to the CCND1 TSS. Concomitant to increased mRNA levels, polymerase II (pol II) recruitment to the CCND1 TSS slightly increased." Enhancer CCND1 3C qPCR "We conclude that enh2 has an active role in ER-independent CCND1 activation.To investigate whether the CCND1 TSS interacts with enh2, we used a chromatin conformation capture (3C) assay,which detects physical proximity between distal DNA sites by ligation of cross-linked restricted DNA fragments." "BCL1,D11S287E,PRAD1,U21B31" -- -- -- The enh2 has an active role in ER-independent CCND1 activation. ChIP "In addition, CCND1 activation in siH2AZ-transfected MDA-MB231 cells was accompanied by a strong recruitment of pol II to enh2 (Figure 1c, right panel), as described previously for E2 stimulated MCF-7 cells. We conclude that enh2 has an active role in ER-independent CCND1 activation." -- -- -- -- -- -- -- >2KB 13889 E_01_362 23637611 -- hg19 chr11 69468873 69470873 Human MCF-7 Low throughput "ChIP,PCR,3C" "Long-range chromatin interactions between ERa recognition sequences and enhancers have been proposed to regulate ERa target genes in breast cancer cells. The main enhancer regulating CCND1 is located at the 39 end of the gene, 14 kb distant from the promoter." Enhancer CCND1 3C PCR "The main Enhancer regulating CCND1 is located at the 3' end of the gene,14 kb distant from the promoter." "BCL1,D11S287E,PRAD1,U21B31" Breast Tumor DOID:3459 D001943 -- -- -- KAT5 "ESA1,HTATIP,HTATIP1,PLIP,TIP,TIP60,ZC2HC5,cPLA2" ChIP TIP60 binding to the CCND1 promoter was analyzed by ChIP and shown as percent of input (n = 2). -- -- -- >2KB 14001 E_01_363 24443471 -- hg19 chr7 116374406 116376406 Human COLO 829 Low throughput "3C,ChIP" "Chromosome conformation capture (3C)was performed in COLO829 cells in the presence or absence of PLX4032 using the MET +63-kb enhancer as an anchor region. We observed that BRAF inhibition strikingly increased the interaction between the MET +63-kb enhancer and the MET TSS, demonstrating that this lineage-specific enhancer undergoes inducible chromatin looping in response to oncogene withdrawal in melanoma cells." Enhancer MET 3C ChIP Chromosome conformation capture (3C)was performed in COLO829 cells in the presence or absence of PLX4032 using the MET +63-kb Enhancer as an anchor region.We observed that BRAF inhibition strikingly increased the interaction between the MET +63-kb Enhancer and the MET TSS. "AUTS9,DFNB97,HGFR,RCCP2,c-Met" Melanoma DOID:1909 D008545 This enhancer displays inducible chromatin looping with the MET promoter to up-regulate MET expression upon BRAF inhibition. "ChIP,3C" "ChIP and 3C analysis were performed on wild-type or Enhancer-edited cells treated with PLX4032.A significant decrease in both H3K27 acetylation,a histone modification associated with active Enhancer state,and Enhancer-promoter chromatin looping was observed in Enhancer-edited cells. " MITF "CMM8,COMMAD,MI,WS2,WS2A,bHLHe32" "ChIP-seq,3C" "To test the necessity of MITF for inducible chromatin looping between the MET +63-kb Enhancer and MET TSS in response to BRAF inhibition, 3C was performed in COLO829 cells in the presence or absence of PLX4032 and MITF depletion." -- -- -- >2KB 62963 E_01_364 24498324 -- hg19 chr11 47363009 47364009 Human Human Myeloid Cells Low throughput ChIP "Using ChIP we focused on loci with more than 2-fold occupancy relative to control antibody. We dentified diffuse occupancy of CTCF with peaks involving amplicons -16.6 (downstream URE), -14.4 (Enhancer), and -11 (Element) as well as in the neighboring mplicons: -15.6, -13.7, -13.4, -13.3, -12.4 and also close to the promoter at -0.15." Enhancer SPI1 -- "RT-PCR,Western blot,Luciferase Reporter Assay" "To conclude this part,CTCF and SMARCA5 co-occupy its newly validated target SPI1 using ChIP assay at the -14.4 Enhancer upon AZA-mediated DNA demethylation." "OF,PU.1,SFPI1,SPI-1,SPI-A" Acute Myeloid Leukemia DOID:9119 D015470 -- -- -- -- -- -- -- -- -- -- >2KB 12899 E_01_365 25453903 -- hg19 chr20 21452815 21462458 Human "HeLa,293T" Low+High throughput "ChIP-seq,PCR,3C" "WDR5 occupancy at activated EWS-FLI1 sites in SKNMC and A673 cells was also detected by ChIP-seq. Accordingly, ChIP-seq profiling of MSCs before and after fusion gene induction demonstrated that EWS-FLI1 recruits WDR5 to activated enhancers." Enhancer NKX2-2 3C PCR "Genes proximal to EWS-FLI1 bound Enhancers include known regulators with critical functions in Ewing sarcoma,such as CCND1 and NKX2-2,as well as many novel targets." "NKX2.2,NKX2B" Ewing Sarcoma DOID:3369 D012512 -- -- -- ELF1 "EFTUD1,RIA1" ChIP-seq "We focused on the ETS-related transcription factor ELF1,which has similar sequence preference to FLI1 and is expressed in A673 and SKNMC cells. We surveyed ELF1 binding in SKNMC cells by ChIP-seq before and after EWS-FLI1 knock-down." -- -- -- >2KB 34017 E_01_366 26166704 -- hg19 chr21 42668000 42671000 Human Breast Cancer Cell Low+High throughput "ChIP-seq,ChIP,GRO-seq" "Previous work from our lab and others established that a subgroup of ER-¦Á/H3K27Ac co-bound enhancers (n=1,248,), exhibiting E2-upregulated eRNAs, high intensity of ER-¦Á binding and close proximity to estrogen target genes, constitute the major E2-activated functional enhancers in MCF-7 cells, referred to as E2-induced ""active enhancers"" or ¡°eRNA+ enhancers¡±. " Enhancer TFF1 3C ChIP-qPCR Genome browser image showing the results of GRO-Seq and Pol II ChIP-Seq at TFF1 locus in the presence of condensin knockdown ( siNCAPG or siNCAPD3 ) vs siCTL transfected cell. "BCEI,D21S21,HP1.A,HPS2,pNR-2,pS2" -- -- -- -- -- -- -- -- -- -- -- -- -- >2KB 1112890 E_01_367 26445536 -- hg19 chr22 35736840 35737280 Human Endothelial Cells (ECs) Low+High throughput "ChIP,PCR" "The ChIP analyses demonstrated that AuNP treatment significantly induced Nrf2 binding to the human HO-1 E2 enhancer region, but not to the promoter region near the transcription start site. " Enhancer HMOX1 -- "Western blot,Immunofluorescence" The Chromatin immunoprecipitation assay revealed that the translocated Nrf2 bound to the antioxidant-response element located in the E2 Enhancer region of the HO-1 gene promoter and acted as a transcrip- tion factor. "HMOX1D,HO-1,HSP32,bK286B10" -- -- -- -- -- -- -- -- -- -- -- -- -- >2KB 39999 E_01_368 26829750 -- hg19 chr9 14538685 14540685 Human MCF-7 Low+High throughput "Luciferase Reporter Assay,RT-PCR,ChIP,ChIP-seq" Here we identify super-enhancer translocations that drive overexpression of the oncogenic transcription factor MYB as a recurrent theme in adenoid cystic carcinoma (ACC). Whole-genome sequencing data and chromatin maps highlight distinct chromosomal rearrangements that juxtapose super-enhancers to the MYB locus. Chromosome conformation capture confirms that the translocated enhancers interact with the MYB promoter. Enhancer NFIB 3C "ChIP,ChIP-seq" "To test whether specific enhancers within the translocated super_x0002_enhancers might activate the MYB promoter, we examined their physical proximity to the promoter using chromosome conforma_x0002_tion capture (3C). First, we examined an ACC with a translocation involving MYB and the NFIB locus. " "CTF,HMGIC/NFIB,MACID,NF-I/B,NF1-B,NFI-B,NFI-RED2,NFIB3,NFIB" Adenoid Cystic Carcinoma DOID:0080202 D003528 Here we identify the juxtaposition of super-enhancer regions to the MYB locus as the unifying feature of ACC translocations.Detailed genomic and epigenomic analyses of ACCs identify alternate rearrangements that translocate super-enhancers in the NFIB and 3C "To test whether specific enhancers within the translocated super_x0002_enhancers might activate the MYB promoter, we examined their physical proximity to the promoter using chromosome conforma_x0002_tion capture (3C).These data suggest that the translocations reposition super-enhancers that subsequently loop to the MYB promoter and sustain high-level MYB expression." TP63 "AIS, B(p51A), B(p51B), EEC3, KET, LMS, NBP, OFC8, RHS, SHFM4, TP53CP, TP53L, TP73L, p40, p51, p53CP, p63, p73H, p73L" ChIP-seq "To directly test whether TP63 co-binds with MYB, we mapped binding of this transcription factor by ChIP-seq.Remarkably, we found that 81% of TP63-binding sites in ACC were co-bound by MYB.These data suggest that MYB,TP63 and Notch signaling may coordinately orchestrate the diverse expression programs in ACC." -- -- -- >2KB 457843 E_01_369 26829750 -- hg19 chr9 14923979 14925979 Human MCF-7 Low+High throughput "Luciferase Reporter Assay,RT-PCR,ChIP,ChIP-seq" Here we identify super-enhancer translocations that drive overexpression of the oncogenic transcription factor MYB as a recurrent theme in adenoid cystic carcinoma (ACC). Whole-genome sequencing data and chromatin maps highlight distinct chromosomal rearrangements that juxtapose super-enhancers to the MYB locus. Chromosome conformation capture confirms that the translocated enhancers interact with the MYB promoter. Enhancer NFIB 3C "ChIP,ChIP-seq" "To test whether specific enhancers within the translocated super_x0002_enhancers might activate the MYB promoter, we examined their physical proximity to the promoter using chromosome conforma_x0002_tion capture (3C). First, we examined an ACC with a translocation involving MYB and the NFIB locus. " "CTF,HMGIC/NFIB,MACID,NF-I/B,NF1-B,NFI-B,NFI-RED2,NFIB3,NFIB" Adenoid Cystic Carcinoma DOID:0080202 D003528 Here we identify the juxtaposition of super-enhancer regions to the MYB locus as the unifying feature of ACC translocations.Detailed genomic and epigenomic analyses of ACCs identify alternate rearrangements that translocate super-enhancers in the NFIB and 3C "To test whether specific enhancers within the translocated super_x0002_enhancers might activate the MYB promoter, we examined their physical proximity to the promoter using chromosome conforma_x0002_tion capture (3C).These data suggest that the translocations reposition super-enhancers that subsequently loop to the MYB promoter and sustain high-level MYB expression." TP63 "AIS, B(p51A), B(p51B), EEC3, KET, LMS, NBP, OFC8, RHS, SHFM4, TP53CP, TP53L, TP73L, p40, p51, p53CP, p63, p73H, p73L" ChIP-seq "To directly test whether TP63 co-binds with MYB, we mapped binding of this transcription factor by ChIP-seq.Remarkably, we found that 81% of TP63-binding sites in ACC were co-bound by MYB.These data suggest that MYB,TP64 and Notch signaling may coordinately orchestrate the diverse expression programs in ACC." -- -- -- >2KB 843137 E_01_370 26829750 -- hg19 chr9 15066136 15068136 Human MCF-7 Low+High throughput "Luciferase Reporter Assay,RT-PCR,ChIP,ChIP-seq" Here we identify super-enhancer translocations that drive overexpression of the oncogenic transcription factor MYB as a recurrent theme in adenoid cystic carcinoma (ACC). Whole-genome sequencing data and chromatin maps highlight distinct chromosomal rearrangements that juxtapose super-enhancers to the MYB locus. Chromosome conformation capture confirms that the translocated enhancers interact with the MYB promoter. Enhancer NFIB 3C "ChIP,ChIP-seq" "To test whether specific enhancers within the translocated super_x0002_enhancers might activate the MYB promoter, we examined their physical proximity to the promoter using chromosome conforma_x0002_tion capture (3C). First, we examined an ACC with a translocation involving MYB and the NFIB locus. " "CTF,HMGIC/NFIB,MACID,NF-I/B,NF1-B,NFI-B,NFI-RED2,NFIB3,NFIB" Adenoid Cystic Carcinoma DOID:0080202 D003528 Here we identify the juxtaposition of super-enhancer regions to the MYB locus as the unifying feature of ACC translocations.Detailed genomic and epigenomic analyses of ACCs identify alternate rearrangements that translocate super-enhancers in the NFIB and 3C "To test whether specific enhancers within the translocated super_x0002_enhancers might activate the MYB promoter, we examined their physical proximity to the promoter using chromosome conforma_x0002_tion capture (3C).These data suggest that the translocations reposition super-enhancers that subsequently loop to the MYB promoter and sustain high-level MYB expression." TP63 "AIS, B(p51A), B(p51B), EEC3, KET, LMS, NBP, OFC8, RHS, SHFM4, TP53CP, TP53L, TP73L, p40, p51, p53CP, p63, p73H, p73L" ChIP-seq "To directly test whether TP63 co-binds with MYB, we mapped binding of this transcription factor by ChIP-seq.Remarkably, we found that 81% of TP63-binding sites in ACC were co-bound by MYB.These data suggest that MYB,TP65 and Notch signaling may coordinately orchestrate the diverse expression programs in ACC." -- -- -- >2KB 985294 E_01_371 26829750 -- hg19 chr1 92170642 92172642 Human MCF-7 Low+High throughput "Luciferase Reporter Assay,RT-PCR,ChIP,ChIP-seq" Here we identify super-enhancer translocations that drive overexpression of the oncogenic transcription factor MYB as a recurrent theme in adenoid cystic carcinoma (ACC). Whole-genome sequencing data and chromatin maps highlight distinct chromosomal rearrangements that juxtapose super-enhancers to the MYB locus. Chromosome conformation capture confirms that the translocated enhancers interact with the MYB promoter. Enhancer TGFBR3 3C "ChIP,ChIP-seq" "To test whether specific enhancers within the translocated super_x0002_enhancers might activate the MYB promoter, we examined their physical proximity to the promoter using chromosome conforma_x0002_tion capture (3C).We also examined a second ACC with a MYB-TGFBR3 translocation. In this case, seven of the nine H3K27ac peaks tested interacted with the MYB promoter." "BGCAN, betaglycan" Adenoid Cystic Carcinoma DOID:0080202 D003528 Here we identify the juxtaposition of super-enhancer regions to the MYB locus as the unifying feature of ACC translocations.Detailed genomic and epigenomic analyses of ACCs identify alternate rearrangements that translocate super-enhancers in the NFIB and 3C "To test whether specific enhancers within the translocated super_x0002_enhancers might activate the MYB promoter, we examined their physical proximity to the promoter using chromosome conforma_x0002_tion capture (3C).These data suggest that the translocations reposition super-enhancers that subsequently loop to the MYB promoter and sustain high-level MYB expression." TP63 "AIS, B(p51A), B(p51B), EEC3, KET, LMS, NBP, OFC8, RHS, SHFM4, TP53CP, TP53L, TP73L, p40, p51, p53CP, p63, p73H, p73L" ChIP-seq "To directly test whether TP63 co-binds with MYB, we mapped binding of this transcription factor by ChIP-seq.Remarkably, we found that 81% of TP63-binding sites in ACC were co-bound by MYB.These data suggest that MYB,TP66 and Notch signaling may coordinately orchestrate the diverse expression programs in ACC." -- -- -- >2KB 25742 E_01_372 26829750 -- hg19 chr1 92254801 92256801 Human MCF-7 Low+High throughput "Luciferase Reporter Assay,RT-PCR,ChIP,ChIP-seq" Here we identify super-enhancer translocations that drive overexpression of the oncogenic transcription factor MYB as a recurrent theme in adenoid cystic carcinoma (ACC). Whole-genome sequencing data and chromatin maps highlight distinct chromosomal rearrangements that juxtapose super-enhancers to the MYB locus. Chromosome conformation capture confirms that the translocated enhancers interact with the MYB promoter. Enhancer TGFBR3 3C "ChIP,ChIP-seq" "To test whether specific enhancers within the translocated super_x0002_enhancers might activate the MYB promoter, we examined their physical proximity to the promoter using chromosome conforma_x0002_tion capture (3C).We also examined a second ACC with a MYB-TGFBR3 translocation. In this case, seven of the nine H3K27ac peaks tested interacted with the MYB promoter." "BGCAN, betaglycan" Adenoid Cystic Carcinoma DOID:0080202 D003528 Here we identify the juxtaposition of super-enhancer regions to the MYB locus as the unifying feature of ACC translocations.Detailed genomic and epigenomic analyses of ACCs identify alternate rearrangements that translocate super-enhancers in the NFIB and 3C "To test whether specific enhancers within the translocated super_x0002_enhancers might activate the MYB promoter, we examined their physical proximity to the promoter using chromosome conforma_x0002_tion capture (3C).These data suggest that the translocations reposition super-enhancers that subsequently loop to the MYB promoter and sustain high-level MYB expression." TP63 "AIS, B(p51A), B(p51B), EEC3, KET, LMS, NBP, OFC8, RHS, SHFM4, TP53CP, TP53L, TP73L, p40, p51, p53CP, p63, p73H, p73L" ChIP-seq "To directly test whether TP63 co-binds with MYB, we mapped binding of this transcription factor by ChIP-seq.Remarkably, we found that 81% of TP63-binding sites in ACC were co-bound by MYB.These data suggest that MYB,TP67 and Notch signaling may coordinately orchestrate the diverse expression programs in ACC." -- -- -- >2KB 109901 E_01_373 26829750 -- hg19 chr1 92331533 92333533 Human MCF-7 Low+High throughput "Luciferase Reporter Assay,RT-PCR,ChIP,ChIP-seq" Here we identify super-enhancer translocations that drive overexpression of the oncogenic transcription factor MYB as a recurrent theme in adenoid cystic carcinoma (ACC). Whole-genome sequencing data and chromatin maps highlight distinct chromosomal rearrangements that juxtapose super-enhancers to the MYB locus. Chromosome conformation capture confirms that the translocated enhancers interact with the MYB promoter. Enhancer TGFBR3 3C "ChIP,ChIP-seq" "To test whether specific enhancers within the translocated super_x0002_enhancers might activate the MYB promoter, we examined their physical proximity to the promoter using chromosome conforma_x0002_tion capture (3C).We also examined a second ACC with a MYB-TGFBR3 translocation. In this case, seven of the nine H3K27ac peaks tested interacted with the MYB promoter." "BGCAN, betaglycan" Adenoid Cystic Carcinoma DOID:0080202 D003528 Here we identify the juxtaposition of super-enhancer regions to the MYB locus as the unifying feature of ACC translocations.Detailed genomic and epigenomic analyses of ACCs identify alternate rearrangements that translocate super-enhancers in the NFIB and 3C "To test whether specific enhancers within the translocated super_x0002_enhancers might activate the MYB promoter, we examined their physical proximity to the promoter using chromosome conforma_x0002_tion capture (3C).These data suggest that the translocations reposition super-enhancers that subsequently loop to the MYB promoter and sustain high-level MYB expression." TP63 "AIS, B(p51A), B(p51B), EEC3, KET, LMS, NBP, OFC8, RHS, SHFM4, TP53CP, TP53L, TP73L, p40, p51, p53CP, p63, p73H, p73L" ChIP-seq "To directly test whether TP63 co-binds with MYB, we mapped binding of this transcription factor by ChIP-seq.Remarkably, we found that 81% of TP63-binding sites in ACC were co-bound by MYB.These data suggest that MYB,TP68 and Notch signaling may coordinately orchestrate the diverse expression programs in ACC." -- -- -- >2KB 186633 E_01_374 26829750 -- hg19 chr1 92359751 92361751 Human MCF-7 Low+High throughput "Luciferase Reporter Assay,RT-PCR,ChIP,ChIP-seq" Here we identify super-enhancer translocations that drive overexpression of the oncogenic transcription factor MYB as a recurrent theme in adenoid cystic carcinoma (ACC). Whole-genome sequencing data and chromatin maps highlight distinct chromosomal rearrangements that juxtapose super-enhancers to the MYB locus. Chromosome conformation capture confirms that the translocated enhancers interact with the MYB promoter. Enhancer TGFBR3 3C "ChIP,ChIP-seq" "To test whether specific enhancers within the translocated super_x0002_enhancers might activate the MYB promoter, we examined their physical proximity to the promoter using chromosome conforma_x0002_tion capture (3C).We also examined a second ACC with a MYB-TGFBR3 translocation. In this case, seven of the nine H3K27ac peaks tested interacted with the MYB promoter." "BGCAN, betaglycan" Adenoid Cystic Carcinoma DOID:0080202 D003528 Here we identify the juxtaposition of super-enhancer regions to the MYB locus as the unifying feature of ACC translocations.Detailed genomic and epigenomic analyses of ACCs identify alternate rearrangements that translocate super-enhancers in the NFIB and 3C "To test whether specific enhancers within the translocated super_x0002_enhancers might activate the MYB promoter, we examined their physical proximity to the promoter using chromosome conforma_x0002_tion capture (3C).These data suggest that the translocations reposition super-enhancers that subsequently loop to the MYB promoter and sustain high-level MYB expression." TP63 "AIS, B(p51A), B(p51B), EEC3, KET, LMS, NBP, OFC8, RHS, SHFM4, TP53CP, TP53L, TP73L, p40, p51, p53CP, p63, p73H, p73L" ChIP-seq "To directly test whether TP63 co-binds with MYB, we mapped binding of this transcription factor by ChIP-seq.Remarkably, we found that 81% of TP63-binding sites in ACC were co-bound by MYB.These data suggest that MYB,TP69 and Notch signaling may coordinately orchestrate the diverse expression programs in ACC." -- -- -- >2KB 214851 E_01_375 26829750 -- hg19 chr1 92392919 92394919 Human MCF-7 Low+High throughput "Luciferase Reporter Assay,RT-PCR,ChIP,ChIP-seq" Here we identify super-enhancer translocations that drive overexpression of the oncogenic transcription factor MYB as a recurrent theme in adenoid cystic carcinoma (ACC). Whole-genome sequencing data and chromatin maps highlight distinct chromosomal rearrangements that juxtapose super-enhancers to the MYB locus. Chromosome conformation capture confirms that the translocated enhancers interact with the MYB promoter. Enhancer TGFBR3 3C "ChIP,ChIP-seq" "To test whether specific enhancers within the translocated super_x0002_enhancers might activate the MYB promoter, we examined their physical proximity to the promoter using chromosome conforma_x0002_tion capture (3C).We also examined a second ACC with a MYB-TGFBR3 translocation. In this case, seven of the nine H3K27ac peaks tested interacted with the MYB promoter." "BGCAN, betaglycan" Adenoid Cystic Carcinoma DOID:0080202 D003528 Here we identify the juxtaposition of super-enhancer regions to the MYB locus as the unifying feature of ACC translocations.Detailed genomic and epigenomic analyses of ACCs identify alternate rearrangements that translocate super-enhancers in the NFIB and 3C "To test whether specific enhancers within the translocated super_x0002_enhancers might activate the MYB promoter, we examined their physical proximity to the promoter using chromosome conforma_x0002_tion capture (3C).These data suggest that the translocations reposition super-enhancers that subsequently loop to the MYB promoter and sustain high-level MYB expression." TP63 "AIS, B(p51A), B(p51B), EEC3, KET, LMS, NBP, OFC8, RHS, SHFM4, TP53CP, TP53L, TP73L, p40, p51, p53CP, p63, p73H, p73L" ChIP-seq "To directly test whether TP63 co-binds with MYB, we mapped binding of this transcription factor by ChIP-seq.Remarkably, we found that 81% of TP63-binding sites in ACC were co-bound by MYB.These data suggest that MYB,TP70 and Notch signaling may coordinately orchestrate the diverse expression programs in ACC." -- -- -- >2KB 248019 E_01_376 26829750 -- hg19 chr1 92567177 92569177 Human MCF-7 Low+High throughput "Luciferase Reporter Assay,RT-PCR,ChIP,ChIP-seq" Here we identify super-enhancer translocations that drive overexpression of the oncogenic transcription factor MYB as a recurrent theme in adenoid cystic carcinoma (ACC). Whole-genome sequencing data and chromatin maps highlight distinct chromosomal rearrangements that juxtapose super-enhancers to the MYB locus. Chromosome conformation capture confirms that the translocated enhancers interact with the MYB promoter. Enhancer TGFBR3 3C "ChIP,ChIP-seq" "To test whether specific enhancers within the translocated super_x0002_enhancers might activate the MYB promoter, we examined their physical proximity to the promoter using chromosome conforma_x0002_tion capture (3C).We also examined a second ACC with a MYB-TGFBR3 translocation. In this case, seven of the nine H3K27ac peaks tested interacted with the MYB promoter." "BGCAN, betaglycan" Adenoid Cystic Carcinoma DOID:0080202 D003528 Here we identify the juxtaposition of super-enhancer regions to the MYB locus as the unifying feature of ACC translocations.Detailed genomic and epigenomic analyses of ACCs identify alternate rearrangements that translocate super-enhancers in the NFIB and 3C "To test whether specific enhancers within the translocated super_x0002_enhancers might activate the MYB promoter, we examined their physical proximity to the promoter using chromosome conforma_x0002_tion capture (3C).These data suggest that the translocations reposition super-enhancers that subsequently loop to the MYB promoter and sustain high-level MYB expression." TP63 "AIS, B(p51A), B(p51B), EEC3, KET, LMS, NBP, OFC8, RHS, SHFM4, TP53CP, TP53L, TP73L, p40, p51, p53CP, p63, p73H, p73L" ChIP-seq "To directly test whether TP63 co-binds with MYB, we mapped binding of this transcription factor by ChIP-seq.Remarkably, we found that 81% of TP63-binding sites in ACC were co-bound by MYB.These data suggest that MYB,TP71 and Notch signaling may coordinately orchestrate the diverse expression programs in ACC." -- -- -- >2KB 422277 E_01_377 26886256 ES-specific enhancers (ESSEs) hg19 chr6 43891400 43898360 Human K-562 Low+High throughput ChIP-seq "We termed these promoter-distal elements, which carry the H3K4me1 mark in ES but not in hematopoietic cells,the ES-specific enhancers (ESSEs) of the hematopoietic cell lineage." enhancer VEGFA "4C-seq,CRISPR/Cas9" Luciferase Reporter Assay "To determine whether this ESSE is able to enhance transcription, a 1,500 bp fragment surrounding the ESSE methylation site was cloned upstream to a minimal SV40 promoter in a luciferase reporter plasmid and transfected to K562 cells.Indeed, the ESSE segment significantly enhanced expression when placed upstream or downstream to the minimal promoter. Thus,the DNA element surrounding the ESSE site is a transcriptional enhancer in leukemia cells.The Circularized Chromosome Conformation Capture (4C-seqencing) assay allowed mapping the interactions between the VEGFA promoter and DNA sequences across the K562 cell genome.To determine whether VEGFA expression depends upon the ESSE, we mutated the ESSE site in K562 cells by utilization of the CRISPR/Cas9 genome-editing system." "MVCD1, VEGF, VPF" -- -- -- -- -- -- STAT1/2 "CANDF7,IMD31A,IMD31B,IMD31C,ISGF-3,STAT91,IMD44,ISGF-3,P113,STAT113" ChIP-seq Focused (<600 bp) cluster of transcription factor binding sites (Chip-seq data) within the predicted enhancer. The factors bound in K562 cells are listed. This is the only cluster within 20 kb around the ESSE. -- -- -- >2kb 156934 E_01_378 26916345 PRE hg19 chr1 67739940 67741075 Human Lymphoblastoid Cell Lines low throughput DNase I Hypersensitivity Assay "We identified a PRE (Chr1:67739940¨C67741075) located 14.2 kb downstream of IL23R and 33.1 kb upstream of IL12RB2.This region is DNase I hypersensitive (in Th1-cells, but not Th17-cells) and exhibits TF binding (lymphoblastoid cell line; GM12878) and enhancer-associated H3K4me1 methylation (CD4+ CD25? IL-17A? T-cells; PMA and ionomycin-stimulated)." enhancer IL23R -- DNase I Hypersensitivity Assay "We identified a PRE (Chr1:67739940¨C67741075) located 14.2 kb downstream of IL23R and 33.1 kb upstream of IL12RB2.This region is DNase I hypersensitive (in Th1-cells, but not Th17-cells) and exhibits TF binding (lymphoblastoid cell line; GM12878) and enhancer-associated H3K4me1 methylation (CD4+ CD25? IL-17A? T-cells; PMA and ionomycin-stimulated)." IL23R Ankylosing Spondylitis DOID:7147 D013167 -- -- -- -- -- -- -- rs11209026 67705958 "DNase I Hypersensitivity Assay,Luciferase Reporter Assay" >2kb 135918 E_01_379 27149122 hg19 chr5 110175800 110176600 Human 293T low throughput Luciferase Reporter Assay "To experimentally test the effect of rs9885413 on enhancer activity, the 100 bp region flanking the SNP (50 bp on either side) was cloned into a reporter vector and transfected into HEK293 cells expressing NHLH1 (S1 Text). Luciferase activity measured after 24 hours was 4-fold higher with a construct corresponding to the risk allele as compared to the wild-type allele (S4 Fig, P < 0.001), indicating that the risk allele of rs9885413 substantially increases enhancer activity." enhancer TSLP -- Luciferase Reporter Assay "To experimentally test the effect of rs9885413 on enhancer activity, the 100 bp region flanking the SNP (50 bp on either side) was cloned into a reporter vector and transfected into HEK293 cells expressing NHLH1 (S1 Text). Luciferase activity measured after 24 hours was 4-fold higher with a construct corresponding to the risk allele as compared to the wild-type allele (S4 Fig, P < 0.001), indicating that the risk allele of rs9885413 substantially increases enhancer activity." TSLP Heart Failure -- D006333 -- -- -- -- -- -- -- rs9885413 110176128 "Transfection,Luciferase Reporter Assay" >2kb 229578 E_01_380 26581162 -- hg19 chr3 108015219 108015290 Human 293T Low throughput "Luciferase Reporter Assay,Transient Transfection Assay,RNA-seq,PCR" "In this study, we identify a targeted hotspot for CRISPR acti_x0002_vation within the long terminal repeat (LTR) enhancer region,at the junction between two nuclear factor (NF)-¦ÊB transcrip_x0002_tion factor¨Cbinding sites.Twenty-three possible NGG Streptococcus pyogenes(Sp) Cas9 photospacer adjacent motif (PAM) sites for targeting of sgRNAs were identified in the U3 region of the LTR, upstream of the HIV transcriptional start site(TSS) (?450 to 0 bp; HIV genome HXB2) within the enhancer and modulatory region of the promoter. " Enhancer HHLA2 CRISPR/Cas9 Luciferase Reporter Assay All sgRNAs were named according to the 3¡ä adjacent sense strand nucleotide cleavage site catalyzed by nuclease-active Cas9 (Figure? 1a) and were screened together with dCas9-VP64 or dCas9-VP160 for their activation properties by transient transfection with the reporter NL4-3.Luc.R_x005f_x0002_E-£¬a full-length HIV molec_x0002_ular clone where luciferase is driven by the viral LTR. "B7-H5,B7-H7,B7H7,B7y" -- -- -- -- -- -- NFKB1 "CVID12,EBP-1,KBF1,NF-kB,NF-kB1,NF-kappa-B1,NF-kappaB,NFKB-p105,NFKB-p50,NFkappaB,p105,p50" CRISPR/Cas9 "Since sg362F fully occupies the sequence across both NF-¦ÊB¨Cbinding sites, we next determined if CRISPR activation via the sg362F target site was coupled to NF-¦ÊB function. these data strongly support a conclusion where sg362F/dCas9-VP64 functions independently of NF-¦ÊB as a stand-alone transactivator of LTR activation. " -- -- -- <2KB 98 E_01_381 28905448 -- hg19 chr11 109985003 109985222 Human Osteosarcoma Stem Cells Low throughput "ChIP-PCR,Luciferase Reporter Assay" "Interestingly, mutation of the Sox2 binding site at mt 11, whose sequence is not con©\ served, had no effect. Mutations at positions m8, m14,and m15 strongly affected the luciferase activity of thereporter plasmid in both cell types. Overexpression of Sox2 did not significantly stimulate the m8, m14, and m15 mutant plasmid activity in 293 cells, indicating that these motifs are critical for enhancer activity." Enhancer YAP1 -- ChIP-PCR "To further confirm the binding of MZF1 to the YAP1 enhancer region ChIP¨CPCR was performed.The results showed binding of MZF1 to specific DNA sequences within the YAP1 enhancer region (Fig. 2C)." "COB1,YAP,YAP2,YAP65,YKI" Osteosarcoma DOID:3347 D012516 Osteogenic Differentiation "CRISPR/Cas9,Alkaline Phosphatase Staining" MZF1 binding to the YAP enhancer is essential for the endogenous expression of YAP;Osteogenic differentiation was detected using alkaline phosphatase staining "MZF1,SOX2" "MZF-1B,ZFP98,ZNF42,ZSCAN6,MZF1,Sox-2,lcc,ysb" "EMSA,Luciferase Reporter Assay" "EMSA analysis of the binding of GABP, and MZF1 transcription factor to the YAP enhancer region;A reporter plasmid expressing luciferase under the control of the FGF4 minimal promoter and the YAP enhancer was transfected into 293 cells in combination with different plasmids expressing MZF1, Sox2 and GABP cDNA." -- -- -- Intron 102053115 E_01_382 29348663 BY707159.1 Enhancer hg19 chr7 150700000 150705000 Human Endothelial Cells (ECs) Low+High throughput "ChIP-seq,qPCR,4C-seq,Hi-C" "Combining RNA-sequencing and chromatin conformation capture methods, we demonstrate that LEENE is co-regulated with eNOS and that its enhancer resides in proximity to eNOS promoter in endothelial cells (ECs)." Enhancer NOS3 CRISPR/Cas9 "ChIP-seq,qPCR,ChIP-qPCR" "Consistent with the Hi-C data, 4C-seq also revealed the chromosomal proximity between the LEENE enhancer and eNOS promoter and this interaction is substantially increased in ECs subjected to PS as compared with OS(Fig. 3g)." "ECNOS,eNOS" -- -- -- LEENE-associated Enhancer plays a role in positive regulation of eNOS transcription CRISPR/Cas9 "As shown in Fig. 7e, LNA inhibiting of LEENE homolog indeed significantly decreased the mRNA level of eNOS in the mouse lung ECs. " "KLF2,KLF4" "LKLF,EZF,GKLF" ChIP-seq "Similar to the human LEENE, BY707159.1 is also transcribed from the negative strand, and the surrounding DNA region contains multiple KLF2/KLF4 binding sites." -- -- -- >2KB 103053401 E_01_383 26745862 hg19 chr6 36640937 36642937 Human "MCF-7,A549,HeLa,293T" Low throughput "ChIP,qPCR" "Our ChIP experiments showed that prior to activation p53 was exclusively found at the upstream enhancer that harbors a high-affinity, near-consen_x0002_sus binding site for p53 (Fig 5A). Occupancy of this site increased incrementally after 1.5 and 4 hours of CDK9 inhibition (Fig 5B). In contrast to this pre-loaded ""primary"" enhancer, treat_x0002_ment with 067 was followed by de novo recruitment of p53 to additional, secondary binding sites, most notably to an intronic region that maps to position +4 kb downstream of the p21 TSS and contains another near-consensus p53 motif (Fig 5A). After four hours of treatment,p53 occupancy of this site reached similar levels as found at the upstream enhancer (Fig 5B)." Enhancer CDKN1A -- ChIP-qPCR "Our ChIP experiments showed that prior to activation p53 was exclusively found at the upstream enhancer that harbors a high-affinity, near-consen_x0002_sus binding site for p53 (Fig 5A). Occupancy of this site increased incrementally after 1.5 and 4 hours of CDK9 inhibition (Fig 5B). In contrast to this pre-loaded ""primary"" enhancer, treat_x0002_ment with 067 was followed by de novo recruitment of p53 to additional, secondary binding sites, most notably to an intronic region that maps to position +4 kb downstream of the p21 TSS and contains another near-consensus p53 motif (Fig 5A). After four hours of treatment,p53 occupancy of this site reached similar levels as found at the upstream enhancer (Fig 5B)." "CAP20, CDKN1, CIP1, MDA-6,?P21, SDI1, WAF1, p21CIP1" Tumour -- -- -- -- -- TP53 "BCC7, BMFS5, LFS1,?P53, TRP53" qPCR "MCF7 cells, which harbour functional wild-type p53, were treated for 20 minutes to 4 hours with 10 ¦ÌM 067 and both nascent unspliced precursor (pre) and mature spliced mRNA was analyzed by quantitative PCR (qPCR). Inhibitor treatment led to significant(2.5-fold) repression of nascent RNA synthesis of the p53 target gene p21 within 20 minutes (Fig1A)." -- -- -- Intron 2299 E_01_384 26745862 hg19 chr6 36647237 36649237 Human "MCF-7,A549,HeLa,293T" Low throughput "ChIP,qPCR" "Our ChIP experiments showed that prior to activation p53 was exclusively found at the upstream enhancer that harbors a high-affinity, near-consen_x0002_sus binding site for p53 (Fig 5A). Occupancy of this site increased incrementally after 1.5 and 4 hours of CDK9 inhibition (Fig 5B). In contrast to this pre-loaded ""primary"" enhancer, treat_x0002_ment with 067 was followed by de novo recruitment of p53 to additional, secondary binding sites, most notably to an intronic region that maps to position +4 kb downstream of the p21 TSS and contains another near-consensus p53 motif (Fig 5A). After four hours of treatment,p53 occupancy of this site reached similar levels as found at the upstream enhancer (Fig 5B)." Enhancer CDKN1A -- ChIP-qPCR "Our ChIP experiments showed that prior to activation p53 was exclusively found at the upstream enhancer that harbors a high-affinity, near-consen_x0002_sus binding site for p53 (Fig 5A). Occupancy of this site increased incrementally after 1.5 and 4 hours of CDK9 inhibition (Fig 5B). In contrast to this pre-loaded ""primary"" enhancer, treat_x0002_ment with 067 was followed by de novo recruitment of p53 to additional, secondary binding sites, most notably to an intronic region that maps to position +4 kb downstream of the p21 TSS and contains another near-consensus p53 motif (Fig 5A). After four hours of treatment,p53 occupancy of this site reached similar levels as found at the upstream enhancer (Fig 5B)." "CAP20, CDKN1, CIP1, MDA-6,?P21, SDI1, WAF1, p21CIP1" Tumour -- -- -- -- -- TP53 "BCC7, BMFS5, LFS1,?P53, TRP53" qPCR "MCF7 cells, which harbour functional wild-type p53, were treated for 20 minutes to 4 hours with 10 ¦ÌM 067 and both nascent unspliced precursor (pre) and mature spliced mRNA was analyzed by quantitative PCR (qPCR). Inhibitor treatment led to significant(2.5-fold) repression of nascent RNA synthesis of the p53 target gene p21 within 20 minutes (Fig1A)." -- -- -- Intron 4001 E_01_385 2116990 hg19 chr11 5288664 5288684 Human "K-562,HeLa" low throughput "Transient Assay,DNaseI-seq,Luciferase Reporter Assay" "When the same constructs were transfected into HeLa cells, the HS II enhancer had modest activity (relative activity: no HS II, 1.0 + 0.1;HS II (1455 bp), 2.8 + 0.8; HS II (46 bp), 3.9 + 0.1; HS IIenh-, 0.7 _ 0.1. Consistent with the marked increase in luciferase activity following induction, correctly initiated luciferase transcripts were seen in the uninduced state only after long exposures. These data suggest that the HS II enhancer element may have a role in the increase in globin gene expression that occurs during erythroid maturation." enhancer HBE1 -- Luciferase Reporter Assay "The enhancer consists of tandem AP-l-binding sites, phased 10 bp apart, which are both required for full activity. DNA-protein binding assays with nuclear extracts from induced cells demonstrate a high molecular weight complex on the enhancer. The formation of this complex also requires both AP-1 sites and correlates with maximal enhancer activity. Induction of the enhancer may have a role in the increase in globin gene transcription that characterizes erythroid maturation." HBE -- -- -- -- -- -- SPl SPl EMSA "To investigate the basis for the increase in enhancer activity with hemin induction, we performed gel mobility-shift assays with the enhancer using nuclear extracts from uninduced and induced K562 cells (Fig. 7A).In addition to having protein concentrations determined, uninduced and induced nuclear extracts were standardized by use of a probe to the widely distributed transcription factor Spl (lanes 1 and 2)." -- -- -- <2kb 905 E_01_386 2339058 hg19 chr11 5268575 5268621 Human K-562 low throughput DNase I Hypersensitivity Assay A 46 bp fragment was made that contained this sequence (46bpE) and a 1.5 kb fragment was made (HS II-E) that was identical to the wildtype HS II fragment except that a portion of the minimal enhancer was replaced by a plasmid polylinker ablating both API sites (fig.7). This data clearly shows that the 46 bp minimal enhancer is both necessary and sufficient for gamma-globin gene induction with hemin induced erythroid maturation in K562 cells. enhancer HBG1 -- "DNase I Hypersensitivity Assay,Southern blot,PCR" "To determine the effect of fragments containing LAR sequences on globin gene expression, mRNA from a marked gamma_x0002_globin gene linked to LAR fragments was assayed in stably transfected K562 erythroleukemia cells. DNasel hypersensitive site 11 (HS 11), located 10.9 kb upstream of the epsilon-globin gene, was required for high level gamma-globin gene expression." "HBG-T2, HBGA, HBGR, HSGGL1, PRO2979" -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- E_01_387 10523648 -- hg19 chr11 5266021 5266708 Human K-562 Low throughput "Transfection,RNase Protection Assay,DNase I Hypersensitivity Assay" "We investigated the requirements for enhancer-promoter communication by using the human b-globin locus control region (LCR) DNase I-hypersensitive site 2 (HS2) enhancer and the ?epsilon-globin gene in chromatinized minichromosomes in erythroid cells.Activation of globin genes during development is accompanied by local_x0002_ized alterations of chromatin structure, and CACCC binding factors and GATA-1, which interact with both globin promoters and the LCR, are believed to be critical for globin gene transcription activation." Enhancer HBE1 -- DNase I Hypersensitivity Assay "We found that an HS2 element mutated in its GATA motif failed to remodel the ?-globin promoter or activate tran_x0002_scription yet HS2 nuclease accessibility did not change. Accessibility and transcription were reduced at promoters with mutated GATA-1 or CACCC sites. Strikingly, these mutations also resulted in reduced accessibility at HS2." HBE -- -- -- "Further, at least in this instance, transcription activation and promoter remodeling by a distant enhancer are not separable." DNase I Hypersensitivity Assay "To determine whether the HS2 mutations affected transcription, ¦Å-globin RNA levels were measured by RNase protection as illustrated in Fig. 4A. The minichromosomal ¦Å-globin gene is not transcribed in the absence of an enhancer, while inclusion of HS2 results in transcription of the linked gene.The results suggest that in chromatin,multiple interactions contribute to enhancer-promoter communication." "GATA1,NFE2" "ERYF1,GATA-1,GF-1,GF1,NF-E1,NFE1,XLANP,XLTDA,XLTT,NF-E2,p45" DNase I Hypersensitivity Assay "Because of the reduced DNase I sensitivity of HS2 in the absence of a globin gene, cleavage at sites close to the HS2 GATA-1 and NF-E2 binding motifs could be visualized at the lowest enzyme concentration.However, DNase I sensi_x0002_tivity at HS2 was reduced when either the promoter 2165 GATA site or the CACCC site was mutated.Aquantitative assessment of these changes by MscI digestion of nuclei revealed that accessibility at HS2 for these promoter mutants was reduced to about half and differed significantly from the wild-type level (P,0.05). Thus, the struc_x0002_tural effect on HS2 of each of these mutations is equivalent to the effect of removing the globin gene entirely." -- -- -- >2KB 23214 E_01_388 10688646 -- hg19 chr16 80617 80837 Human K-562 Low throughput "ChIP,PCR,Transfection" "In this study,we have further investigated the molecular basis of this model. First, human erythroid K562 cells stablyintegrated with various HS-40 mutants cis linked to a human a-globin promoter-growth hormone hybrid gene were analyzed by genomic footprinting and expression analysis. By the assay, we demonstrate that factors bound at different motifs of HS-40 indeed act in concert to build a fully functional enhanceosome.Thus,modification of factor binding at a single motif could drastically change the configuration and function of the HS-40 enhanceosome." Enhancer GATA1 -- "ChIP,PCR" "Substitution of 3 bp in the factor-binding cores of 59-NA and GATA-1(c) motifs, as well as the 1-bp mutation of 39-NA, had no apparent effects on the presence of the HS-40 genomic footprints (Fig. 2C, E, and G, respectively).On the other hand, 3-bp mutation of either 39-NA or the GT motif greatly affected the genomic footprints. In particular, the HS-40 enhancer carrying the GT mutation becomes empty(Fig. 2F), while factor binding at both the GT and GATA-1(c)motifs of HS-40 with the 39-NA(I) mutation was abolished(Fig. 2D)." "ERYF1,GATA-1,GF-1,GF1,NF-E1,NFE1,XLANP,XLTDA,XLTT" -- -- -- "The HS-40 enhancer is the major cis-acting regulatory element responsible for the developmental stage- and erythroid lineage-specific expression of the human a-like globin genes, the embryonic z and the adult a2/a/1." "Footprinting,ChIP" "A model has been proposed in which competitive factor binding at one of the HS-40 motifs, 3¡ä-NA, modulates the capability of HS-40 to activate the embryonic ¦Æ-globin promoter. Furthermore, this modulation was thought to be mediated through configurational changes of the HS-40 enhanceosome during development." NFE2 "NF-E2, p45" "ChIP,EMSA" "Finally, we show by chromatin immunoprecipitation experiments that only binding of NF-E2, not AP1,could be detected in vivo in K562 cells around the HS-40 region. These data exclude a role for AP1 in the developmental regulation of the human a-globin locus via the 3*-NA motif of HS-40 in embryonic/fetal erythroid cells. " -- -- -- >2kb 48564255 E_01_389 2304460 -- hg19 chr11 5248824 5249485 Human Embryonic Blood Cells Low throughput DNase I Hypersensitivity Assay "We found that sequences between -201 and -136 are essential for expression of the G¦Ã-globin gene,whereas those upstream of -201 have little effect on the level or tissue or stage specificity of G¦Ã-globin expression. The G¦Ã-globin upstream sequences from -201 to -136 were, furthermore, capable of activating a linked ¦Â-globin gene in embryonic blood cells; however, a G¦Ã-globin fragment from -383 to -206 was similarly active in this assay, and the complete fragment from -383 to -136 was considerably more active than either of the smaller fragments, suggesting the presence of multiple cis-acting elements for embryonic blood cells. Our data also suggested the possibility of a negative regulatory element between -201 and -136. " Enhancer "HBG1,HBB" -- DNase I Hypersensitivity Assay "Finally,we observed that removal of the ,¦Â-globin3'-flanking sequences,including the 3' enhancer,from the G¦Ã-globin upstream-¦Â-globin hybrid gene resulted in a 25-fold reduction in expression in embryonic blood cells. This suggests that the ¦Â-globin 3'enhancer is potentially active at the embryonic stage and thus cannot be solely responsible for the fetal or adult specificity of the ¦Â-globin gene. " "HBG-T2,HBGA,HBGR,HSGGL1,PRO2979£»CD113t-C,ECYT6,beta-globin" -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- E_01_390 10891470 hg19 chr11 5255062 5258441 Human K-562 Low throughput "Luciferase Reporter Assay,ChIP,PCR,Southern blot" "To determine whether NF-E2 interacts directly with HS2 of the endogenous ¦Â-globin LCR , we used a ChIP assay.The immunoprecipi_x0002_tated DNA was analyzed by PCR with primers specific for HS2, the Ag-globin promoter, and the HS12 enhancer element of the IgH locus.These results provide strong evidence that NF-E2 associates with HS2 in intact cells, and the controls illustrate the specificity of the interaction." Enhancer HBB -- "ChIP,PCR" "To determine whether the tandem MAREs were required for the interaction of NF-E2 with HS2, K562 cells were stably transfected with the HS2(Gal4)5.1gluciferase reporter construct in which a single Gal4 binding site was substituted for the tandem MAREs while maintaining the correct spacing between adjacent cis-acting elements. These results support a direct role for NF-E2 in the regulation of b-glo_x0002_bin gene expression through activation of the LCR." "CD113t-C,ECYT6,beta-globin" Erythrocytic Leukemia -- -- -- -- -- NFE2 "NF-E2, p45" "ChIP,Luciferase Reporter Assay" "Using a chromatin immunoprecipitation assay, we provide evidence for NF-E2 binding directly and specifically to HS2 in living erythroleukemia cells and in mouse fetal liver. " -- -- -- >2kb 10056 E_01_391 9878258 hg19 chr3 108296550 108297750 Human K-562 Low throughput "CAT Assay,PCR,RT-PCR" "Functional tests with the CAT reporter gene assays demonstrate that the human 59 HS5 LTR activates the cis-linked CAT gene and possesses enhancer and pro_x0002_moter activities in erythroid cells.We found an LTR retrotranspo_x0002_son belonging to the ERV-9 family of human endoge_x0002_nous retroviruses in the apparent 5* boundary area of the LCR. This ERV-9 LTR contains an unusual U3 en_x0002_hancer region composed of 14 tandem repeats with recurrent GATA, CACCC, and CCAAT motifs." Enhancer HBB -- "CAT Assay,RT-PCR" We next determined whether the endogenous 59 HS5 LTR also exhibits enhancer and promoter activities and can activate the transcription of the downstream R region and the flanking genomic DNA in the b-globin LCR.The above RT-PCR results indicate that the endog_x0002_enous 59 HS5 LTR possesses apparent enhancer and promoter activities and is capable of promoting the transcription of the R and U5 regions in the LTR and of further downstream genomic DNA in the LCR. "CD113t-C,ECYT6,beta-globin" -- -- -- "In both recombinant constructs and the endog_x0002_enous human genome, the LTR enhancer and promoter activate the transcription of cis-linked DNA preferentially in erythroid cells." CAT Assay Functional tests with the CAT reporter gene assays demonstrate that the human 59 HS5 LTR activates the cis-linked CAT gene and possesses enhancer and pro_x0002_moter activities in erythroid cells. -- -- -- -- -- -- -- >2kb 103050454 E_01_392 10595394 hg19 chr11 5250264 5250295 Human "K-562,HEL" Low throughput "Luciferase Reporter Assay,EMSA" "DNA footprinting analyses and electrophoretic mobility shift assays (EMSAs) indicate that two sites for which ¦ÃPE has a high affinity, 3' ¦ÃPE1 and 3' ¦ÃPE2, span from +1992 to + 2023 and +2263 to +2288, respectively, and contain the consensus sequence ATTANNNGGAANNCT(N)NNNNTAATGG. Three low-affinity sites,3' ¦ÃPE1 (+2245 to +2225),3' ¦ÃPE3 (+2432 to +2447), and 3' ¦ÃPE4 (+2479 to +2505),contain the consensus sequences 59 -AAAAN(T/A)A(A/T)TT-3'. " Enhancer HBG1 -- Luciferase Reporter Assay "A large nuclear protein complex, termed ¦ÃPE (for ¦Ã-globin promoter and enhancer binding factor), binds to five sites located 5' and 3' of the human ¦Ã-globin gene. Transient cotransfections of a HOXB2 cDNA and a c -globin¨Cluciferase reporter gene con_x0002_struct into cells expressing SATB1 suggested that SATB1 has a positive and HOXB2 a negative regulatory effect on transcription." "HBG-T2,HBGA,HBGR,HSGGL1,PRO2979" Chronic myelogenous leukemia DOID:8552 D015464 -- -- -- "SATB1,HOXB2" "CLEVER-1,FEEL-1,FELE-1,FEX1,SCARH2,STAB-1,HOX2,HOX2H,Hox-2.8,K8" EMSA "The present work showed that antisera directed against either SATB1 or HOXB2 reacted specifically with the entire ¦ÃPE complex in electrophoretic mobility shift assays (EMSAs), suggesting that the two pro_x0002_teins can bind to the ¦ÃPE binding site simultaneously. " -- -- -- >2kb 19222 E_01_393 10595394 hg19 chr11 5250535 5250560 Human "K-562,HEL" Low throughput "Luciferase Reporter Assay,EMSA" "DNA footprinting analyses and electrophoretic mobility shift assays (EMSAs) indicate that two sites for which g PE has a high affinity, 3' ¦ÃPE1 and 3' ¦ÃPE2, span from +1992 to + 2023 and +2263 to +2288, respectively, and contain the consensus sequence ATTANNNGGAANNCT(N)NNNNTAATGG. Three low-affinity sites,3' ¦ÃPE1 (+2245 to +2225),3' ¦ÃPE3 (+2432 to +2447), and 3' ¦ÃPE4 (+2479 to +2505),contain the consensus sequences 59 -AAAAN(T/A)A(A/T)TT-3'. " Enhancer HBG1 -- Luciferase Reporter Assay "A large nuclear protein complex, termed ¦ÃPE (for ¦Ã-globin promoter and enhancer binding factor), binds to five sites located 5' and 3' of the human ¦Ã-globin gene. Transient cotransfections of a HOXB2 cDNA and a c -globin¨Cluciferase reporter gene con_x0002_struct into cells expressing SATB1 suggested that SATB1 has a positive and HOXB2 a negative regulatory effect on transcription. " "HBG-T2,HBGA,HBGR,HSGGL1,PRO2979" Chronic myelogenous leukemia DOID:8552 D015464 -- -- -- "SATB1,HOXB2" "CLEVER-1,FEEL-1,FELE-1,FEX1,SCARH2,STAB-1,HOX2,HOX2H,Hox-2.8,K8" EMSA "The present work showed that antisera directed against either SATB1 or HOXB2 reacted specifically with the entire ¦ÃPE complex in electrophoretic mobility shift assays (EMSAs), suggesting that the two pro_x0002_teins can bind to the ¦ÃPE binding site simultaneously. " -- -- -- >2kb 18954 E_01_394 10595394 hg19 chr11 5248027 5248047 Human "K-562,HEL" Low throughput "Luciferase Reporter Assay,EMSA" "DNA footprinting analyses and electrophoretic mobility shift assays (EMSAs) indicate that two sites for which g PE has a high affinity, 3' ¦ÃPE1 and 3' ¦ÃPE2, span from +1992 to + 2023 and +2263 to +2288, respectively, and contain the consensus sequence ATTANNNGGAANNCT(N)NNNNTAATGG. Three low-affinity sites,3' ¦ÃPE1 (+2245 to +2225),3' ¦ÃPE3 (+2432 to +2447), and 3' ¦ÃPE4 (+2479 to +2505),contain the consensus sequences 59 -AAAAN(T/A)A(A/T)TT-3'. " Enhancer HBG1 -- Luciferase Reporter Assay "A large nuclear protein complex, termed ¦ÃPE (for ¦Ã-globin promoter and enhancer binding factor), binds to five sites located 5' and 3' of the human ¦Ã-globin gene. Transient cotransfections of a HOXB2 cDNA and a c -globin¨Cluciferase reporter gene con_x0002_struct into cells expressing SATB1 suggested that SATB1 has a positive and HOXB2 a negative regulatory effect on transcription." "HBG-T2,HBGA,HBGR,HSGGL1,PRO2979" Chronic myelogenous leukemia DOID:8552 D015464 -- -- -- "SATB1,HOXB2" "CLEVER-1,FEEL-1,FELE-1,FEX1,SCARH2,STAB-1,HOX2,HOX2H,Hox-2.8,K8" EMSA "The present work showed that antisera directed against either SATB1 or HOXB2 reacted specifically with the entire ¦ÃPE complex in electrophoretic mobility shift assays (EMSAs), suggesting that the two pro_x0002_teins can bind to the ¦ÃPE binding site simultaneously. " -- -- -- >2kb 21465 E_01_395 10595394 hg19 chr11 5250704 5250719 Human "K-562,HEL" Low throughput "Luciferase Reporter Assay,EMSA" "DNA footprinting analyses and electrophoretic mobility shift assays (EMSAs) indicate that two sites for which g PE has a high affinity, 3' ¦ÃPE1 and 3' ¦ÃPE2, span from +1992 to + 2023 and +2263 to +2288, respectively, and contain the consensus sequence ATTANNNGGAANNCT(N)NNNNTAATGG. Three low-affinity sites,3' ¦ÃPE1 (+2245 to +2225),3' ¦ÃPE3 (+2432 to +2447), and 3' ¦ÃPE4 (+2479 to +2505),contain the consensus sequences 59 -AAAAN(T/A)A(A/T)TT-3'. " Enhancer HBG1 -- Luciferase Reporter Assay "A large nuclear protein complex, termed ¦ÃPE (for ¦Ã-globin promoter and enhancer binding factor), binds to five sites located 5' and 3' of the human ¦Ã-globin gene. Transient cotransfections of a HOXB2 cDNA and a c -globin¨Cluciferase reporter gene con_x0002_struct into cells expressing SATB1 suggested that SATB1 has a positive and HOXB2 a negative regulatory effect on transcription." "HBG-T2,HBGA,HBGR,HSGGL1,PRO2979" Chronic myelogenous leukemia DOID:8552 D015464 -- -- -- "SATB1,HOXB2" "CLEVER-1,FEEL-1,FELE-1,FEX1,SCARH2,STAB-1,HOX2,HOX2H,Hox-2.8,K8" EMSA "The present work showed that antisera directed against either SATB1 or HOXB2 reacted specifically with the entire ¦ÃPE complex in electrophoretic mobility shift assays (EMSAs), suggesting that the two pro_x0002_teins can bind to the ¦ÃPE binding site simultaneously. " -- -- -- >2kb 18790 E_01_396 10595394 hg19 chr11 5250751 5250777 Human "K-562,HEL" Low throughput "Luciferase Reporter Assay,EMSA" "DNA footprinting analyses and electrophoretic mobility shift assays (EMSAs) indicate that two sites for which g PE has a high affinity, 3' ¦ÃPE1 and 3' ¦ÃPE2, span from +1992 to + 2023 and +2263 to +2288, respectively, and contain the consensus sequence ATTANNNGGAANNCT(N)NNNNTAATGG. Three low-affinity sites,3' ¦ÃPE1 (+2245 to +2225),3' ¦ÃPE3 (+2432 to +2447), and 3' ¦ÃPE4 (+2479 to +2505),contain the consensus sequences 59 -AAAAN(T/A)A(A/T)TT-3'. " Enhancer HBG1 -- Luciferase Reporter Assay "A large nuclear protein complex, termed ¦ÃPE (for ¦Ã-globin promoter and enhancer binding factor), binds to five sites located 5' and 3' of the human ¦Ã-globin gene.Transient cotransfections of a HOXB2 cDNA and a c -globin¨Cluciferase reporter gene con_x0002_struct into cells expressing SATB1 suggested that SATB1 has a positive and HOXB2 a negative regulatory effect on transcription. " "HBG-T2,HBGA,HBGR,HSGGL1,PRO2979" Chronic myelogenous leukemia DOID:8552 D015464 -- -- -- "SATB1,HOXB2" "CLEVER-1,FEEL-1,FELE-1,FEX1,SCARH2,STAB-1,HOX2,HOX2H,Hox-2.8,K8" EMSA "The present work showed that antisera directed against either SATB1 or HOXB2 reacted specifically with the entire ¦ÃPE complex in electrophoretic mobility shift assays (EMSAs), suggesting that the two pro_x0002_teins can bind to the ¦ÃPE binding site simultaneously. " -- -- -- >2kb 18738 E_01_397 12861010 hg19 chr11 5293686 5294250 Human K-562 Low throughput "PCR,DNaseI-seq" "We have also found DNase I HSs at kb -85.5 (HS-85.5) and -84.5 in mouse, but there is no corresponding se_x0002_quence or structure in human for these HSs.By comparison,neither the active _x0002_maj-globin gene promoter nor a region located ~1 kb from HS5 shows significant enrichment. HS-85.5 exhibits a modest (twofold) enrichment." Enhancer LOC110006319 -- "DNaseI-seq,Southern blot,PCR" "In ery_x0002_throid cells, the active _x0002_-globin locus contains several DNase I HSs, which have been shown to map to sequences with regu_x0002_latory functionIn addition, histone hyperacetylation anddimethylation of histone H3 K4 are not uniform features of the nuclease-sensitive mouse _x0001_-globin domain but rather define distinct subdomains within it. Our results reveal a complex chromatin landscape for the active _x0001_-globin locus and illustrate the complexity of broad structural changes that accompany gene activation." LOC110006319 -- -- -- -- -- -- CTCF MRD21 ChIP "To determine whether CTCF binds to these sequences in vivo, we performed ChIP analysis using antibodies to CTCF." -- -- -- >2kb 70188 E_01_398 17637499 hg19 chr11 5211019 5213983 Human K-562 Low throughput Luciferase Reporter Assay "Enhancer activity of reporter constructs was determined with the dual luciferase system in transient transfection assays. Notch signaling transactivated a reporter construct harboring a con_x0002_served RBP-J (CBF1) binding site in the hypersensitive site 2 (HS2) of human b-globin. Transactivation by activated Notch was completely abolished when this RBP-J site was mutated to prevent RBP-J binding." Enhancer HBB -- "RT-PCR,Northern blot" Expression was analyzed by Northern blotting and real-time polymerase chain reac_x0002_tion.Notch signaling transactivated a reporter construct harboring a con_x0002_served RBP-J (CBF1) binding site in the hypersensitive site 2 (HS2) of human b-globin. "CD113t-C, ECYT6, beta-globin" -- -- -- -- -- -- RBPJ "AOS3, CBF1, IGKJRB, IGKJRB1, KBF2, RBP-JK, RBPSUH, SUH, csl, RBPJ " Luciferase Reporter Assay "Luciferase reporter constructs containing either the functional RBP-J binding site of the HS2 [38] or a mutated sequence (Fig. 5A) that does not bind RBP-J [53] or showed reduced RBP-J binding [38] were transiently transfected into rNERTneo and rneo FDCP-mix cells and luciferase activity was determined in the presence or absence of OHT." -- -- -- >2kb 34195 E_01_399 17283048 hg19 chr11 5264845 5266345 Human K-562 Low throughput "FISH,Transfection" "Minichromosomes, maintained at stable, moderate copy num_x0002_bers in K562 cells, contained the strong HS2 enhancer of the -globin LCR and the ¦Å-globin gene in the form of a 5.2-kb minilocus in which the enhancer and gene reside about 2.5 kb apart." Enhancer HBE1 -- "ChIP,PCR,FISH" "In K562 cells, three copies of chromosome 11, which is the loca_x0002_tion of the _x0002_-globin locus, were detected in most nuclei by hybridizing a DNA probe corresponding to the centromeric _x0008_-satellite repeat region. RNA FISH performed on the same slides revealed that only two chromosomes actively transcribe ¦Å-globin." HBE -- -- -- ¦Å-Globin Intergenic Transcription and Histone Acetylation Dependent on an Enhancer. "FISH,ChIP,PCR" "To ascertain whether this is the case,we employed DNA/RNA FISH.Here, we studied the establishment and function of histone acetylation and transcription in noncoding sequences by using a model locus linking the ¦Å-globin HS2 enhancer and the embryonic ¦Å-globin gene in chromatin. An intact HS2 enhancer that recruits RNA Pol II is required for intergenic transcription and histone H3 acetylation and K4 methylation between the enhancer and target gene. " NFE2 "NF-E2, p45" ChIP "Chromatin was prepared from K562 cells carrying NF-E2 mutant minichromosomes, and ChIP was car_x0002_ried out as described for panel B.Within HS2, Pol II was maximal at the core NF-E2 sites, where it is recruited by transcription activa_x0002_tors, but levels above background were detected 5_x0006_ of the core and between HS2 and ¦Å-globin. When the NF-E2 motifs in HS2 were eliminated, Pol II was almost completely lost from the gene, consistent with the loss of HS2 enhancer activity and loss of gene transcription." -- -- -- >2kb 3907 E_01_400 15371553 HS2 enhancer hg19 chr11 5271421 5273421 Human K-562 Low throughput RNase Protection Assay The minichromosomes were stably maintained in human erythroid K562 cells in which the endogenous e-globin gene is actively transcribed. RNase protection assays were performed to test the influence of the insulators on enhancer activation of the e-globin gene. enhancer HBE1 -- Luciferase Reporter Assay "The minichromosomes were stably maintained in human erythroid K562 cells in which the endogenous e-globin gene is actively transcribed. RNase pro_x0002_tection assays were performed to test the influence of the insulators on enhancer activation of the ¦Å-globin gene.Representative assays of RNA from different cHS4 insula_x0002_tor-containing clones are illustrated in Figure 1B, and the results for least 12 individual clones of each type are summar_x0002_ized in Figure 1C. Abundant transcription of the e-globin RNA occurred when the gene was linked to HS2. " HBE -- -- -- -- -- -- NFE2 "NF-E2, p45" ChIP "Therefore, we used antibodies to the HS2 enhancer factor NF-E2 and the insulator factor CTCF in ChIP assays of the cHS4in and cHS4out insulated loci." -- -- -- >2kb 2919 E_01_401 14585970 HS2 enhancer hg19 chr11 5286080 5288080 Human K-562 Low throughput "DNase I Hypersensitivity Assay,RT-PCR,ChIP" "To investigate the chromatin anatomy of a more complete enhancer-gene locus, we created minichromosomes linking ¦Å-globin to a longer 1.46-kb KpnI-to-BglII LCR HS2 enhancer fragment. Formation of HS2 and the ¦Å-globin promoter DNase I hypersensitive site are enhancer dependent.We compared the transcriptionally active locus to one in which HS2 was inactivated by mutations in the core NF-E2 sites. In contrast to inactive templates, nucleosomes were mobilized in discrete areas of the active locus, including the HS2 core and the proximal promoter." enhancer HBE1 -- DNase I Hypersensitivity Assay "We studied nucleosome remodeling and covalent histone modification mediated by the¦Å-globin locus control region HS2 enhancer at nucleosome-level resolution throughout a 5.5-kb globin gene model locus in vivo in K562 cells.We compared the transcriptionally active locus to one in which HS2 was inactivated by mutations in the core NF-E2 sites. In contrast to inactive templates, nucleosomes were mobilized in discrete areas of the active locus, including the HS2 core and the proximal promoter. Large differences in restriction enzyme accessibility between the active and inactive templates were limited to the regions of nucleosome mobilization, which subsumed the DNase I hypersensitive sites." HBE -- -- -- -- -- -- NFE2 "NF-E2, p45" RNase Protection Assay "To investigate the effect on transcription activation of the NF-E2 mutation in the context of an extended HS2, we performed RNase protection assays on RNA isolated from clones with the wild-type (HS2L¦Å) or mutated [HS2L(mut)¦Å] enhancer.A representative experiment is de_x0002_picted in Fig. 2A, and the results of multiple determinations for six different clones of HS2L¦Å and HS2L(mut)¦Å are summa_x0002_rized in Fig. 2B. Abundant transcription of ¦Å-globin RNA is seen when the gene is linked to the longer 1.46-kb HS2 se_x0002_quence (HS2L¦Å). Destruction of the tandem NF-E2 binding sites in HS2 abolished transcription of the ¦Å-globin gene [HS2L(mut)¦Å]." -- -- -- >2kb 17578 E_01_402 27149122 -- hg19 chr5 110175120 110177136 Human Blood Low+High throughput "Luciferase Reporter Assay,ChIP-seq" "To experimentally test the effect of rs9885413 on enhancer activity, the 100 bp region flank_x0002_ing the SNP (50 bp on either side) was cloned into a reporter vector and transfected intoHEK293 cells expressing NHLH1 (S1 Text). Luciferase activity measured after 24 hours was 4-fold higher with a construct corresponding to the risk allele as compared to the wild-type allele (S4 Fig, P < 0.001), indicating that the risk allele of rs9885413 substantially increases enhancer activity." Enhancer TSLP -- "Luciferase Reporter Assay,qPCR,ChIP-seq" We further observed association of the genetic variant with a DNA methylation signature in blood that in turn is associated with allergy and expression of the gene TSLP (Thymic stromal lymphoprotein) in blood. Knockdown of the transcription factor predicted to bind the enhancer region also resulted in lower TSLP expression. TSLP Heart Failure -- D006333 -- -- -- NHLH1 "HEN1,NSCL,NSCL1,bHLHa35" siRNA "To examine whether the transcription factor NHLH1 affects the expression of any of the five genes in the locus (Fig 1), we knocked down NHLH1 in HEK293 cells using siRNAs. " rs9885413 110176128 "Luciferase Reporter Assay,qPCR,ChIP-seq" >2kb 229650 E_01_403 27207652 -- hg19 chr11 95817801 95818824 Human "MCF10CA1h,MCF10CA1a" Low+High throughput "ChIP,qPCR,Transfection,Western blot" "The difference in DNA meth_x0002_ylation within MAML2 in RSV treated versus control cells was determined as 0.37 in MCF10CA1h and 0.1 in MCF10CA1a cells based on the array data. These elevated levels of methylation were identified in a fragment corresponding to the predic_x0002_tive enhancer within MAML2 gene body. It has been demonstrated that active enhancers are enriched with H3K27ac.We confirmed by ChIP assay that this histone mark is present at MAML2 in cancer cells and its occupancy is significantly reduced by RSV.These data strongly support the role of this region as a gene enhancer and the role of RSV in decreasing the activ_x0002_ity of this enhancer." Enhancer MAML2 -- "ChIP,Transfection,Western blot" "MAML2, a coactivator of NOTCH targets, is methylated at the enhancer region and transcriptionally silenced in response to stilbenoids, possibly explaining the downregulation of NOTCH target genes.It has been demonstrated that active enhancers are enriched with H3K27ac.We confirmed by ChIP assay that this histone mark is present at MAML2 in cancer cells and its occupancy is significantly reduced by RSV." "MAM-3,MAM2,MAM3,MLL-MAML2" Breast Cancer DOID:1612 D001943 -- -- -- POU2F1 "OCT1,OTF1,oct-1B" "ChIP,qPCR" "Using ChIP, we confirmed OCT1 binding to MAML2 enhancer (Figure?6C, control cells). The binding dramati_x0002_cally decreases upon treatment of MCF10CA1a with 15?¦ÌM RSV.The lack of OCT1 binding may be directly linked to MAML2 transcriptional silencing. To test this hypothesis, we depleted OCT1 in MCF10CA1a cells using SmartPool siRNA mixture and four siRNA sequences separately, as confirmed by QPCR and western blot and tested the effect of OCT1 absence on MAML2 expression." -- -- -- >2kb 106873 E_01_404 26919345 PRE hg19 chr1 67739940 67741075 Human "GM12878,Th17,Th1" Low throughput DNase I Hypersensitivity Assay "We identified a PRE (Chr1:67739940¨C67741075) located 14.2 kb downstream of IL23R and 33.1 kb upstream of IL12RB2.This region is DNase I hypersensitive (in Th1--cells, but not Th17--cells) and exhibits TF binding (lymphoblastoid cell--line; GM12878) and enhancer--associated H3K4me1 methylation (CD4+ CD25? IL--17A? T--cells; PMA and ionomycin--stimulated). " enhancer IL23R-IL12RB2 -- DNase I Hypersensitivity Assay "We identified a PRE (Chr1:67739940¨C67741075) located 14.2 kb downstream of IL23R and 33.1 kb upstream of IL12RB2.This region is DNase I hypersensitive (in Th1-cells, but not Th17-cells) and exhibits TF binding (lymphoblastoid cell-line; GM12878) and enhancer-associated H3K4me1 methylation (CD4+ CD25? IL-17A? T-cells; PMA and ionomycin-stimulated). " IL23R-IL12RB2 Ankylosing Spondylitis DOID:7147 D013167 -- -- -- -- -- -- -- rs11209032 67740092 "DNase I Hypersensitivity Assay,ChIP-qPCR,Luciferase Reporter Assay" -- -- E_01_405 26427657 -- hg19 chr3 78597238 79767998 Human "Colorectal Cancer,Breast Cancer Cell" Low+High throughput "qPCR,ChIP-seq" "We identified an identical germline deletion of the ROBO1 gene, which was confirmed by an independent microarray platform and qPCR analysis in three unrelated patients with BC and/or CRC. The ROBO1 deletion identified comprises an intronic region (intron 4) of four protein-coding splice variants of ROBO1" Enhancer ROBO1 -- "qPCR,ChIP-seq" "We identified an identical germline deletion of the ROBO1 gene, which was confirmed by an independent microarray platform and qPCR analysis in three unrelated patients with BC and/or CRC. The ROBO1 deletion identified comprises an intronic region (intron 4) of four protein-coding splice variants of ROBO1" "AW494633, AW742721, DUTT1, Gm310" "Colorectal Cancer,Breast Cancer" "DOID:9256,DOID:1612" D001943 -- -- -- -- -- -- -- -- -- -- >2kb 536230 E_01_406 26776159 -- hg19 chr9 91737604 92057238 Human Primary Human BJ Fibroblasts Low+High throughput "Luciferase Reporter Assay,ChIP-qPCR,GRO-seq,ChIP-seq,4C" "Thus,we assessed whether p53BERs can induce lincRNAs with essential roles within the p53 pathway. We first genome-wide mapped p53BERs using both publically available p53 chromatin immuno_x0002_precipitation sequencing (ChIP-seq) data sets10 and enhancer domains epigenetically defined by the Broad Institute segmentation." Enhancer lincRNA00475 4C -- "we identified and characterized a novel p53-bound intronic enhancer that controls the expression of its host, the lincRNA00475(linc-475). We demonstrate the requirement of linc-475 for the proper induction of a p53-dependent cell cycle inhibitory response." -- -- -- -- -- -- -- "CTCF,TP53" "MRD21,BCC7,BMFS5,LFS1,P53,TRP53" ChIP-qPCR "In addition,we assessed whether CTCF could influence the p53-dependent regulation of linc-475. To tackle this question, we first evaluated and confirmed the binding of CTCF upstream of linc-475 using ChIP¨Cquantitative PCR.Next, we tested whether knocking down CTCF could interfere with the expression levels of linc-475. " -- -- -- -- -- E_01_407 26780995 -- hg19 chr19 33299481 33300688 Human Breast Cancer Cell Low+High throughput "DNase I Hypersensitivity Assay,ATAC-seq,ChIP-seq" "ChIP-seq of TFs predicts a subset of putative enhancers (reviewed in [13]) whereas ChIP_x0002_seq of p300 covers enhancers more ubiquitously [28]. High-throughput profiling of DNase I hypersensitive sites (DHSs) allows identification of enhancers [15], although DHSs also include other regulatory DNA regions such as promoters, insulators, and silencers." Enhancer CEP89 -- "GWAS,ChIP-seq" "It was found that rs10411210, which is associated with colorectal cancer [76], is located on an enhancer linked to the centrosomal protein 89kDa(CEP89) gene, which is listed in the COSMIC database. Although rs10411210 was mapped to the rhophilin, Rho GTPase binding protein 2 (RHPN2) gene based on the genomic distance,FANTOM5 CAGE data indicated that an enhancer covering rs10411210 targeted the CEP89 gene in addition to the RHPN2 gene based on the correlation of the transcription of the enhancer and that of target genes." "CCDC123, CEP123" Colorectal Cancer DOID:9256 -- -- -- -- EP300 "KAT3B,MKHK2,RETS2,P300" ChIP-seq "ChIP-seq of TFs predicts a subset of putative enhancers (reviewed in [13]) whereas ChIP_x0002_seq of p300 covers enhancers more ubiquitously [28]. High-throughput profiling of DNase I hypersensitive sites (DHSs) allows identification of enhancers [15], although DHSs also include other regulatory DNA regions such as promoters, insulators, and silencers." rs10411210 33532300 "DNase I Hypersensitivity Assay,ATAC-seq,ChIP-seq" >2KB 69819 E_01_408 26795348 -- hg19 chr2 202120390 202125986 Human Human Mammary Epithelial Normal Breast Cell Line Low+High throughput "PCR,Luciferase Reporter Assay,ChIP-seq" "Variants rs3769823, rs3769821, and rs10197246 were chosen to investigate for ability to affect enhancer activity. To create the enhancer constructs, we performed PCR using genomic DNA from T-47D, which is homozygous for risk alleles at all three SNPs, and MCF10A, which is homozygous for neutral alleles, using the following primer pairs." Enhancer CASP8 -- DNase I Hypersensitivity Assay "To prioritize further, we used expression-quantitative trait locus analysis of RNA sequencing from breast tissues, gene regulation annota_x0002_tions from the ENCODE consortium, and functional assays for differential enhancer activities. Notably, we implicate three regulatory variants at 2q33 that target CASP8 (rs3769823, rs3769821 in CASP8, and rs10197246 in ALS2CR12) as func_x0002_tionally relevant." "CASP-8, FLICE, MACH, Mch5" Breast Cancer DOID:1612 D001943 -- -- -- "STAT3,MYC,CTCF" "1110034C02Rik, AW109958, Aprf,MRTLC,bHLHe39,c-Myc,MYC,MRD21" "DNase I Hypersensitivity Assay,ChIP-seq" "The first two reside 435 bp apart in a promoter proximal region that exhibits DNAse I hypersensitivity in mammary epithelial and breast cancer cell lines, and binds multiple transcription factors(STAT3, MYC, Pol2, and CTCF) based on ENCODE data (22).Also notable are results from an eQTL study in blood (23),indicating highly significant decreased CASP8 expression associ_x0002_ated with these cFSVs and suggesting the expression signature is also readily observed in blood." -- -- -- >2KB 25022 E_01_409 26809031 -- hg19 chr12 131949637 131949801 Human Normal Human Bronchial Epithelial Cells Low throughput "RT-qPCR,ChIP,EMSA" DNA sequence alignment of the E1A enhancer regions of divergent Ad serotypes revealed that the E2F binding site located between nt 270¨C290 is highly conserved. Enhancer EP400 -- qPCR "This finding indicated that an IFN-induced repressor binding site is located in the downstream half of the E1A enhancer region, corresponding to Ad5 nt 270¨C358." "1700020J09Rik, AU023439, mDomino, mKIAA1498, p400" -- -- -- -- -- -- "E2F1,SP1" "E2F-1,RBAP1,RBBP3,RBP3,SP1" "ChIP,EMSA" The cellular transcription factor Sp1 binds to several sites in the Ad5 inverted terminal repeat (ITR)[26] that are adjacent to the E1A enhancer region. We analyzed if Sp1 binding to the ITR was altered by IFN signaling in vivo. Sp1 bound to the Ad5 ITR at similar levels in the presence and absence of IFNs (Fig 3B). This result suggests that reduced binding of GABP to the E1A enhancer following IFN treatment is target specific and not due to reduced global accessibility of the left-end of the Ad5 genome. -- -- -- >2KB 484746 E_01_410 26814967 -- hg19 chr8 128310315 128312315 Human Medulloblastoma Cell Lines Low+High throughput "qPCR,ChIP-seq,RNA-seq,4C-seq" "To deeply characterize the active cis-regulatory circuitry of a single disease entity, here medul_x0002_loblastoma, we performed high-resolution chromatin immuno_x0002_precipitation with sequencing (ChIP-seq) for active enhancers (H3K27ac) in 28 primary tumour specimens and three established cell lines. " Enhancer MYC -- ChIP-seq "Given the apparent limitations of using cell lines to faithfully study the tumour epigenome, and the recognized subgroup-dependent hetero_x0002_geneity of medulloblastoma, we collected a series of 28 treatment_x0002_naive, fresh-frozen medulloblastoma specimens and profiled the active enhancer landscape by H3K27ac ChIP-seq." "AU0167572, Niard, Nird, bHLHe39, Myc" Medulloblastoma DOID:0050902 D008527 -- -- -- "POU5F1,SOX2,NANOG" "OCT3,OCT4,OTF-3,OTF3,OTF4,Oct-3,Oct-4,ANOP3,MCOPS3,NANOG" ChIP-seq "Computational reconstruction of core regulatory circuitry identified a master set of transcription factors, validated by ChIP-seq, that is responsible for subgroup divergence, and implicates candidate cells of origin for Group 4. " -- -- -- >2KB 436999 E_01_411 26814967 -- hg19 chr2 29511793 29765640 Human Medulloblastoma Cell Lines Low+High throughput "qPCR,ChIP-seq,RNA-seq,4C-seq" "To deeply characterize the active cis-regulatory circuitry of a single disease entity, here medul_x0002_loblastoma, we performed high-resolution chromatin immuno_x0002_precipitation with sequencing (ChIP-seq) for active enhancers (H3K27ac) in 29 primary tumour specimens and three established cell lines. " Enhancer ALK -- ChIP-seq "To validate the robustness of our methods, we used 4C-seq19 to query Group 3-specific enhancer¨Cpromoter interactions for enhancers showing conserved activity in both primary Group 3 tumours and cell lines. This approach con_x0002_firmed enhancer¨Cpromoter interactions for both TGFBR1 and SMAD9in the Group 3 cell line HD-MB03, a low-passage line more faithful to primary Group 3 tumours than older models." "CD246, NBLST3" Medulloblastoma DOID:0050902 D008527 -- -- -- "POU5F1,SOX2,NANOG" "OCT3,OCT4,OTF-3,OTF3,OTF4,Oct-3,Oct-4,ANOP3,MCOPS3,NANOG" ChIP-seq "Computational reconstruction of core regulatory circuitry identified a master set of transcription factors, validated by ChIP-seq, that is responsible for subgroup divergence, and implicates candidate cells of origin for Group 4. " -- -- -- >2KB 223077 E_01_412 26814967 -- hg19 chr5 893433 1083433 Human Medulloblastoma Cell Lines Low+High throughput "qPCR,ChIP-seq,RNA-seq,4C-seq" "To deeply characterize the active cis-regulatory circuitry of a single disease entity, here medul_x0002_loblastoma, we performed high-resolution chromatin immuno_x0002_precipitation with sequencing (ChIP-seq) for active enhancers (H3K27ac) in 30 primary tumour specimens and three established cell lines. " Super-Enhancer NKD2 -- ChIP-seq "Compared to typ_x0002_ical enhancers, SEs showed higher occupancy of BRD4 and greater enhancer signal dynamic range between subgroups.Targets of differential enhancers contained within SEs included a large fraction of established medullo_x0002_blastoma signature genes, as well as novel candidates.Medulloblastoma SEs were inferred to regulate known Cancer Gene Census genes, including the aforemen_x0002_tioned ALK in WNT, SMO and NTRK3 in SHH, LMO1, LMO2, and MYC in Group 3, and ETV4 and PAX5 in Group 4, among others. " Naked2 Medulloblastoma DOID:0050902 D008527 -- -- -- "POU5F1,SOX2,NANOG" "OCT3,OCT4,OTF-3,OTF3,OTF4,Oct-3,Oct-4,ANOP3,MCOPS3,NANOG" ChIP-seq "Computational reconstruction of core regulatory circuitry identified a master set of transcription factors, validated by ChIP-seq, that is responsible for subgroup divergence, and implicates candidate cells of origin for Group 4. " -- -- -- >2KB 20508 E_01_413 26814967 -- hg19 chr21 47716490 47892045 Human Medulloblastoma Cell Lines Low+High throughput "qPCR,ChIP-seq,RNA-seq,4C-seq" "To deeply characterize the active cis-regulatory circuitry of a single disease entity, here medul_x0002_loblastoma, we performed high-resolution chromatin immuno_x0002_precipitation with sequencing (ChIP-seq) for active enhancers (H3K27ac) in 31 primary tumour specimens and three established cell lines. " Super-Enhancer PCNT -- ChIP-seq "Compared to typ_x0002_ical enhancers, SEs showed higher occupancy of BRD4 and greater enhancer signal dynamic range between subgroups.Targets of differential enhancers contained within SEs included a large fraction of established medullo_x0002_blastoma signature genes, as well as novel candidates.Medulloblastoma SEs were inferred to regulate known Cancer Gene Census genes, including the aforemen_x0002_tioned ALK in WNT, SMO and NTRK3 in SHH, LMO1, LMO2, and MYC in Group 3, and ETV4 and PAX5 in Group 4, among others. " "KEN, MOPD2, PCN2, PCNTB, PCTN2, SCKL4, PCNT" Medulloblastoma DOID:0050902 D008527 -- -- -- "POU5F1,SOX2,NANOG" "OCT3,OCT4,OTF-3,OTF3,OTF4,Oct-3,Oct-4,ANOP3,MCOPS3,NANOG" ChIP-seq "Computational reconstruction of core regulatory circuitry identified a master set of transcription factors, validated by ChIP-seq, that is responsible for subgroup divergence, and implicates candidate cells of origin for Group4. " -- -- -- >2KB 60292 E_01_414 26814967 -- hg19 chr2 221040922 221069549 Human Medulloblastoma Cell Lines Low+High throughput "qPCR,ChIP-seq,RNA-seq,4C-seq" "To deeply characterize the active cis-regulatory circuitry of a single disease entity, here medul_x0002_loblastoma, we performed high-resolution chromatin immuno_x0002_precipitation with sequencing (ChIP-seq) for active enhancers (H3K27ac) in 32 primary tumour specimens and three established cell lines. " Super-Enhancer HLX -- ChIP-seq "Compared to typ_x0002_ical enhancers, SEs showed higher occupancy of BRD4 and greater enhancer signal dynamic range between subgroups.Targets of differential enhancers contained within SEs included a large fraction of established medullo_x0002_blastoma signature genes, as well as novel candidates.Medulloblastoma SEs were inferred to regulate known Cancer Gene Census genes, including the aforemen_x0002_tioned ALK in WNT, SMO and NTRK3 in SHH, LMO1, LMO2, and MYC in Group 3, and ETV4 and PAX5 in Group 4, among others. " "HB241, HLX" Medulloblastoma DOID:0050902 D008527 -- -- -- "POU5F1,SOX2,NANOG" "OCT3,OCT4,OTF-3,OTF3,OTF4,Oct-3,Oct-4,ANOP3,MCOPS3,NANOG" ChIP-seq "Computational reconstruction of core regulatory circuitry identified a master set of transcription factors, validated by ChIP-seq, that is responsible for subgroup divergence, and implicates candidate cells of origin for Group 4. " -- -- -- >2KB 2493 E_01_415 26814967 -- hg19 chr5 121568382 121839811 Human Medulloblastoma Cell Lines Low+High throughput "qPCR,ChIP-seq,RNA-seq,4C-seq" "To deeply characterize the active cis-regulatory circuitry of a single disease entity, here medul_x0002_loblastoma, we performed high-resolution chromatin immuno_x0002_precipitation with sequencing (ChIP-seq) for active enhancers (H3K27ac) in 33 primary tumour specimens and three established cell lines. " Super-Enhancer SNCAIP -- ChIP-seq "Compared to typ_x0002_ical enhancers, SEs showed higher occupancy of BRD4 and greater enhancer signal dynamic range between subgroups.Targets of differential enhancers contained within SEs included a large fraction of established medullo_x0002_blastoma signature genes, as well as novel candidates.Medulloblastoma SEs were inferred to regulate known Cancer Gene Census genes, including the aforemen_x0002_tioned ALK in WNT, SMO and NTRK3 in SHH, LMO1, LMO2, and MYC in Group 3, and ETV4 and PAX5 in Group 4, among others. " "SYPH1, Sph1" Medulloblastoma DOID:0050902 D008527 -- -- -- "POU5F1,SOX2,NANOG" "OCT3,OCT4,OTF-3,OTF3,OTF4,Oct-3,Oct-4,ANOP3,MCOPS3,NANOG" ChIP-seq "Computational reconstruction of core regulatory circuitry identified a master set of transcription factors, validated by ChIP-seq, that is responsible for subgroup divergence, and implicates candidate cells of origin for Group 4. " -- -- -- >2KB 56642 E_01_416 26817450 -- hg19 chr1 230293530 230305966 Human Hep G2 Low+High throughput "ChIP-seq,Luciferase Reporter Assay,EMSA" EMSA and luciferase assays were used to validate the allele specific binding and to test the enhancer activity of the regulatory element harboring the AS-SNP rs4846913 as well as the neighboring rs2144300 which are in high LD with rs4846914. Enhancer GALNT2 -- GWAS Using luciferase assays we found that rs4846913 and the neighboring rs2144300 displayed allele specific enhancer activity. We propose that an inhibitor binds preferentially to the rs4846913-C allele with an inhibitory boost from the synergistic binding of other TFs at the neighboring SNP rs2144300. These events influence the transcription level of GALNT2. GalNAc-T2 -- -- -- -- -- -- ZBTB3 ZBTB3 "EMSA,Luciferase Reporter Assay" Using luciferase assays we found that rs4846913 and the neighboring rs2144300 displayed allele specific enhancer activity. We propose that an inhibitor binds preferentially to the rs4846913-C allele with an inhibitory boost from the synergistic binding of other TFs at the neighboring SNP rs2144300. "rs4846913,rs2144300" "230294715,230294916" "ChIP-seq,Luciferase Reporter Assay,EMSA" >2KB 96792 E_01_417 26818267 -- hg19 chr22 18897006 18899006 Human DLBCL Cells Low+High throughput "Luciferase Reporter Assay,PCR,ChIP-seq" "Deletion of the LTR as well as insertion of the chimpanzee orthologous regulatory sequence imparts the activity of the human PRODH pro_x0002_moter in luciferase assays, showing that the LTR of hsERV_PRODH is critical for maintaining human_x0002_specific and high expression of PRODH." Enhancer PRODH -- Luciferase Reporter Assay 2.2 kb upstream of the PRODH promoter the LTR of a hsERV (hsERV_PRODH) functions as a neuron-specific enhancer in hippocampus where it regulates PRODH expression in synergy with a CpG island that overlaps PRODH exon 2. "HSPOX2, PIG6, POX1, PRODH2, TP53I6, PRODH" Diffuse Large B-Cell Lymphoma DOID:0050745 D016403 Several studies have shown that human endogenous retroviruses and endogenous retrovirus-like repeats (here collec_x0002_tively HERVs) impose direct regulation on human genes through enhancer and promoter motifs present in their long terminal repeats (LTRs Luciferase Reporter Assay "Deletion of the LTR as well as insertion of the chimpanzee orthologous regulatory sequence imparts the activity of the human PRODH pro_x0002_moter in luciferase assays, showing that the LTR of hsERV_PRODH is critical for maintaining human_x0002_specific and high expression of PRODH." -- -- -- -- -- -- -- >2KB 2200 E_01_418 26859151 -- hg19 chr5 172196326 172199378 Human "MCF-7,MDA-MB-231" Low+High throughput ChIP-seq "Similar co-localisation studies were conducted in MDA-MB-231 cells to further understand the potential contribution of DUSP family members in the mesenchymal state. Similar to above, DUSP1 showed high levels of co-localization with the active promoter marks H3K9me1 (PCC = 0.65) and H3K4me3 (PCC = 0.72) (Fig 2E), while DUSP4 showed definite co-localisa_x0002_tion with the enhancer marks H3K27ac (PCC = 0.56) and strong co-localisation with H3K4me1 (PCC = 0.78) (Fig 2F) but not H3K4me3 or H3K9me3 (Fig C in S2 Fig)." Enhancer DUSP1 -- "ChIP,RT-qPCR" "Interestingly, we found that siRNA-mediated knockdown of DUSP1 and DUSP4 does not affect the expression of these genes (data not shown). Taken together,these data indicate a novel chromatin-anchored role for DUSP1 and DUSP4 in mesenchymal breast cancer cells." "CL100,HVH1,MKP-1,MKP1,PTPN10" Breast Cancer DOID:1612 D001943 -- -- -- -- -- -- -- -- -- -- >2KB 2759 E_01_419 26859151 -- hg19 chr8 29203575 29214287 Human "MCF-7,MDA-MB-231" Low+High throughput ChIP-seq "DUSP4 co-localised with the enhancer mark H3K27ac [40] in MCF-7/PMA (PCC = 0.40) but not in non-stimulated MCF-7 cells (PCC = -0.30). Moreover, DUSP4 co-localised with the enhancer mark H3K4me1 in MCF-7 cells (PCC = 0.35), which increased in MCF-7/PMA cells (PCC = 0.44) (Fig 2D). In comparison, there was absent or minimal co-localisation of DUSP4 with H3K4me3, H3K4me1, and H3K9me3 in MCF-7 and MCF-7/PMA cells (Fig B in S2 Fig). Conversely, DUSP6 was expressed at very low levels or did not co-localise with any of the tested histone modifications in MCF-7 or MCF-7/PMA cells (Fig A in S3 Fig)." Enhancer DUSP4 -- "ChIP,RT-qPCR" "Similarly, DUSP4 was enriched at the promoters of these genes in MCF-7 cells and dis_x0002_played reduced enrichment in MCF-7/PMA cells at PLAUR and IL6 but not FN1 gene promot_x0002_ers (Fig 3B).Furthermore, DUSP4 half_x0002_ChIP on MCF-7 and MCF-7/PMAAD nuclear extracts showed that DUSP4 associates with both key phosphorylation residues (Ser2 and Ser5) of a key indicator of active chromatin, RNA Polymerase II (Fig 3C)." "HVH2,MKP-2,MKP2,TYP" Breast Cancer DOID:1612 D001943 -- -- -- EP300 "KAT3B,MKHK2,RSTS2,p300" "Immunofluorescence,Half-ChIP,RT-qPCR" "Immunofluoresence anal_x0002_ysis showed that the nuclear fluorescence intensity of p300 increased in the mesenchymal state,in MCF-7/PMASUS and MDA-MB-231 cells compared to MCF-7 cells (Fig 4A). Moreover,DUSP4 and p300 significantly co-localise in MCF-7/PMASUS (PCC = 0.42) and MDA-MB-231 cells (PCC = 0.50) and minimally co-localised in MCF-7 cells (PCC = 0.15) (Fig 4A). Consis_x0002_tent with these findings, DUSP4 half-ChIP on MCF-7 and MCF-7/PMAAD nuclear extracts showed that DUSP4 increased its association with p300 in the mesenchymal state (S4 Fig).Next, we investi_x0002_gated the impact of DUSP4 knockdown on p300 phosphorylation by immunofluorescence analysis of either MOCK or validated DUSP4 siRNA-treated MCF-7 cells prior and subsequent to stimulation with PMA. DUSP4 knockdown was first confirmed by real-time PCR and immunoblotting (Figs A, B, C in S5 Fig)." -- -- -- >2KB 18352 E_01_420 26864944 -- hg19 chr14 24435744 24437744 Human "Hep G2,HL-7702" Low throughput "Luciferase Reporter Assay,ChIP" "we searched for and identified a putative enhancer region located 13.8 kb downstream from the TSS of DHRS4-AS117,18. We performed chromatin immunoprecipitation (ChIP) to confirm the enrichment of histone modifications in hepatocellular carcinoma cells (HepG2) and normal hepatocytes (HL7702) (Fig.?1D). We then examined the potential enhancement of luciferase reporter activity by using luciferase reporter assays in HepG2 and HL7702 cells (Fig.?1E). " Enhancer DHRS4-AS1 3C "Luciferase Reporter Assay,ChIP,qPCR" "In a search of the DHRS4-AS1 region for nearby enhancers, we identified an enhancer located 13.8 kb downstream of the DHRS4-AS1 transcriptional start site. We further showed, by using a chromosome conformation capture (3C) assay, that this enhancer is capable of physically interacting with the DHRS4-AS1 promoter through chromosomal looping. " "AS1DHRS4,C14orf167,C14orf67,DHRS4AS1,PRO1488" -- -- -- -- -- -- EP300 "KAT3B,MKHK2,RSTS2,p300" ChIP "RNA pol II and the transcriptional coactivator p300/CBP occupy enhancer regions and target gene promoters to maintain chromatin loops29. We next characterized the effect of AS1eRNA knockdown on the binding levels of RNA pol II and p300 at the AS1 enhancer and the DHRS4-AS1 promoter (Fig.?4A,B)." -- -- -- >2KB 28804 E_01_421 26884396 -- hg19 chr14 61121917 61123917 Human Nephron Progenitors Low+High throughput ChIP-seq "ChIP-seq was performed on 17 week fetal kidneys and E16.5 mouse kidneys to assess chromatin marks associated with active genes and enhancers (H3K27ac) and transcriptionally silenced chromatin (H3K27me3). Similarly, the human SIX1 locus was bound by SIX2 at multiple conserved elements and displayed prominent H3K27ac throughout the gene body and the SIX2-bound regions (Fig. 3A)." Enhancer SIX1 -- RNA-seq "Using RNA-seq data from nephron progenitors, we compared expression of genes between human and mouse and identified genes that were >5-fold enriched in either species and were also a species-specific target (Fig. 2E, right panel,highlighted genes). SIX1 is expressed in the human ITGA8+progenitors, but not in mouse nephron progenitors, identifying SIX1 as a human specific target on the basis of both cis-interactions around the SIX1 gene and active SIX1 expression (right panelFig. 2E; Table S3)." "BOS3,DFNA23,TIP39" -- -- -- -- -- -- SIX2 SIX2 ChIP The differing number of peaks was due to lower levels of SIX2 ChIP enrichment in the second replicate (Fig. S1C). Examination of SIX2 binding near MEOX1 and WT1 highlighted the similar binding profiles for SIX2 replicates(Fig. 1B). -- -- -- >2KB 11500 E_01_422 26884396 -- hg19 chr14 61106417 61108417 Human Nephron Progenitors Low+High throughput ChIP-seq "ChIP-seq was performed on 17 week fetal kidneys and E16.5 mouse kidneys to assess chromatin marks associated with active genes and enhancers (H3K27ac) and transcriptionally silenced chromatin (H3K27me3). Similarly, the human SIX1 locus was bound by SIX2 at multiple conserved elements and displayed prominent H3K27ac throughout the gene body and the SIX2-bound regions (Fig. 3A)." Enhancer SIX1 -- RNA-seq "Using RNA-seq data from nephron progenitors, we compared expression of genes between human and mouse and identified genes that were >5-fold enriched in either species and were also a species-specific target (Fig. 2E, right panel,highlighted genes). SIX1 is expressed in the human ITGA8+progenitors, but not in mouse nephron progenitors, identifying SIX1 as a human specific target on the basis of both cis-interactions around the SIX1 gene and active SIX1 expression (right panelFig. 2E; Table S3)." "BOS3,DFNA23,TIP39" -- -- -- -- -- -- SIX2 SIX2 ChIP The differing number of peaks was due to lower levels of SIX2 ChIP enrichment in the second replicate (Fig. S1C). Examination of SIX2 binding near MEOX1 and WT1 highlighted the similar binding profiles for SIX2 replicates(Fig. 1B). -- -- -- >2KB 4000 E_01_423 26679052 -- hg19 chr8 140657900 141002079 Human 5637 Low throughput "Luciferase Reporter Assay,ChIP,RT-PCR" "Realtime PCR analysis showed that antrocin downregulated the expression of mRNA of several MMPs, including MMP-2. Moreover, the phosphorylation of ERK and c-Fos were also attenuated by antrocin" Enhancer FAK -- "Luciferase Reporter Assay,ChIP,RT-PCR" "Realtime PCR analysis showed that antrocin downregulated the expression of mRNA of several MMPs, including MMP-2. Moreover, the phosphorylation of ERK and c-Fos were also attenuated by antrocin" "FADK, FAK, FAK1, FRNK, PPP1R71, p125FAK, pp125FAK" Bladder Cancer DOID:11054 D001749 -- -- -- -- -- -- -- -- -- -- >2kb 838491 E_01_424 26505625 -- hg19 chr7 95210132 95260132 Human ccRCC Cells Low+High throughput "ChIP,ChIP-seq" "rs11762213 alone is found in a regulatory element of MET likely in an enhancer domain, and this has implications for its interaction both with MET itself and with potentially other cis-acting target genes.To further characterize the functional significance of rs11762213 we used our previously generated genome-wide chromatin annotation maps using cultured human proximal tubular epithelial cells (HKC8) and overlaid them with previously generated gene regulatory annotation maps from a panel of ChIP-seq data using the hidden Markov model-based ChromHMM chromatin segmentation program (Figure 4). Notably, rs11762213 maps to an H3K4me1 histone modification mark, which serves as an enhancer marker and is therefore consistent with the hypothesis that the SNP has a regulatory function.Mapping of rs11762213 to regulatory regions within the genome suggests that it may impact a DNA enhancer region." Enhancer MET -- "ChIP,RNA-seq" "Next, we assessed the impact of rs11762213 on MET steady-state mRNA and protein tumor expression using available RNA seq data and reverse phase protein array (RPPA) data from the TCGA. We found no difference in tumor MET expression by SNP status (p=0.47 Mann Whitney) including all detected MET isoforms (n=18) (Supplemental figure 2A). Since rs11762213 is located in the coding region of exon 2 we further explored exon level expression differences by genotype and again did not find any difference by genotype for exon 2 MET expression (p=0.29) of any other exon (Supplemental figure 3). Additionally, we did not see differences in tumor MET protein (p=0.88) or MET phospho-protein expression (Y1235) (Supplemental figure 2B and 2C).rs11762213 was associated with higher normal tissue MET expression (p=0.019), however, in an independent normal kidney Affymetrix mRNA array data set this finding did not validate (n=95) (data not shown)." "AI838057,HGF,HGFR,Par4,c-Met" Clear Cell Renal Cell Carcinoma DOID:4467 D002292 -- -- -- -- -- -- -- -- -- -- -- -- E_01_425 26443750 -- hg19 chr1 153628434 153632039 Human HEKa Low throughput "RT-PCR,Western blot" "Methylation analysis was also performed for three other EDC genes of known expression pattern (involucrin, loricrin, and NICE-1) and a recently identified evolutionary conserved region with defined enhancer properties." Enhancer LCE1A -- "RT-PCR,Western blot" "Methylation analysis was also performed for three other EDC genes of known expression pattern (involucrin, loricrin, and NICE-1) and a recently identified evolutionary conserved region with defined enhancer properties." LEP1 Skin Barrier -- -- -- -- -- -- -- -- -- -- -- -- -- -- E_02_001 25209250 -- mm10 chr10 52388022 52388224 Mouse P19 Low throughput Luciferase Reporter Assay Luciferase constructs containing the Nepn TSS and upstream promoter regions were assayed in P19 cells treated with DMSO or RA (0.1 ¦ÌM). The length of the upstream region for each construct is indicated in the graphic. Relative luciferase activity represents the fold increase observed in the presence of RA relative to DMSO. Enhancer Nepn CRISPR/Cas9 "Luciferase Reporter Assay,RT-PCR" Luciferase assays indicated that promoter activity of the construct (2.3 kb) that contains both Sox17-binding motif and RARE was dramatically enhanced with ectopic expression of Sox17 in the presence of RA. "Npn,periolin,5730521E12Rik" -- -- -- -- -- -- "Sox17,Rara" "Sox17,Nr1b1,RAR,RARalpha1" "Luciferase Reporter Assay,EMSA" "The luciferase activity of these constructs was evaluated in P19 cells in the presence of ectopically expressed Sox17. A significant drop in Sox17-inducible activity occurred when the sequence between ?842?bp and ?640?bp upstream of the Nepn TSS was deleted, suggesting that an enhancer region that is dependent on Sox17 lies in this region.EMSA assay of nuclear protein binding to the proposed RARE from the Nepn promoter. Myc-tagge RAR¦Á was ectopically expressed in P19 cells treated with RA (0.1 ¦ÌM) and nuclear extracts were incubated with 32P-labelled RARE from the Nepn promoter. Competition with a 100-fold excess of unlabeled wild-type or mutated RARE is indicated. Anti-Myc antibodies were included in the binding assay as indicated. Sindicates the RAR¦Á-DNA complex; SS indicates the super shifted complex in the presence of anti-Myc antibody. **P<0.01. Mut, mutated; NE, nuclear extracts; Wt, wild type." -- -- -- <2KB 742 E_02_002 18552207 -- mm10 chr7 126406352 126406616 Mouse "B Cell,T Cell" Low throughput "Luciferase Reporter Assay,ChIP" "Transient transfection assays in different mouse cell lines. A mouse Cd19 promoter reporter construct (_x005f_x005f_x005f_x005f_x005f_x005f_x005F_x005f_x005F_x005f_x005F_x005f_x005F_x005f_x005F_x005f_x005F_x005f_x005F_x005f_x005F_x005f_x005F_x005f_x005F_x005f_x005F_x005f_x005F_x005f_x005F_x005f_x005F_x005f_x005F_x005f_x005F_x005f_x005F_x005f_x005F_x005f_x005F_x005f_x005F_x005f_x005F_x005f_x005F_x0007_7 to _x005F_x0007_200 bp; e) or a construct containing the _x0007_2 kb DHS (_x0007_1832 to _x0007_2096 bp) combined with the Cd19 promoter (f) was transiently transfected into various cell lines. The relative activity was determined as the luciferase activity of each construct over control vector pXPG. Data represent the mean of 2 to 4 experiments performed in triplicate" Enhancer Cd19 -- ChIP "As previously reported in human cells, the promoter was active in all analyzed cell types (Figure 1C white bars). In contrast, the _x005F_x0007_2 kb DHS had enhancer activity only in B cells (Figure 1C black bars), providing evidence that this enhancer is involved in the regulation of tissue specific expression of Cd19." AW495831 -- -- -- Enhancer is involved in the regulation of tissue specific expression of Cd19 Transfection "As previously reported in human cells, the promoter was active in all analyzed cell types (Figure 1C white bars). In contrast, the -2 kb DHS had Enhancer activity only in B cells (Figure 1C black bars), providing evidence that this Enhancer is involved in the regulation of tissue specific expression of Cd19." "Tcf3,Ebf1,Pax5" "A1,AA408400,ALF2,AW209082,E12,E12/E47,E2A,E47,KA1,ME2,Pan1,Pan2,TCF-3,Tcfe2a,VDIR,bHLHb21,Ebf,O/E-1,OE-1,Olf-1,Olf1,BSAP,EBB-1,KLP,Pax-5" "ChIP,DMS Footprinting Assay" "We performed an in vivo DMS footprinting assay to answer the question of which transcription factors bind to the Cd19 promoter and enhancer.The specific binding of these proteins was confirmed by ChIP analysis, demonstrating that EBF and PAX5 bound to the enhancer in B cells and that E2A bound in B and T cells." -- -- -- <2KB 1963 E_02_003 18984737 ¦Â-cell specific Enhancer mm10 chr3 51508734 51512187 Mouse "¦ÂTC-3,INS-1,¦ÁTC1.6,NIH3T3" Low throughput Luciferase Reporter Assay "To directly test whether this region of the Setd7 gene might be regulated in ¦Â-cells, a DNA fragment containing this conserved region was placed upstream of the prolactin minimal promoter driving luciferase and used in reporter gene analysis studies.¦Â-cell lines displayed enhancement of luciferase activity with a DNA fragment containing _x0003_6,584 bp upstream of the transcriptional start site. This enhancement was significantly attenuated when a fragment containing _x0003_3,131 bp from the transcriptional start site was used, suggesting the potential for a ¦Â-cell specific enhancer located between _x0003_3,131 and _x0003_6,584 bp." Enhancer Setd7 -- Luciferase Reporter Assay "¦Â-cell lines displayed enhancement of luciferase activity with a DNA fragment containing _x0003_6,584 bp upstream of the transcriptional start site. This enhancement was significantly attenuated when a fragment containing _x0003_3,131 bp from the transcriptional start site was used, suggesting the potential for a ¦Â-cell specific enhancer located between _x0003_3,131 and _x0003_6,584 bp." "1600028F23Rik,H3K4MT,KMT7,Set7,Set7/9,mKIAA1717" Insulin Deficiency -- -- -- -- -- Pdx1 "IDX-1,IPF-1,Ipf1,Mody4,STF-1,pdx-1" "RT-PCR,ChIP,Luciferase Reporter Assay" "To directly test whether this region of the Setd7 gene might be regulated in ¦Â-cells, a DNA fragment containing this conserved region was placed upstream of the prolactin minimal promoter driving luciferase and used in reporter gene analysis studies. As shown in Fig. 2B, this fragment displayed relative enhancement of luciferase activity in _x0001_TC3 and INS-1,¦Â-cell lines,but not in NIH3T3 cells. Upon cotransfection with a plasmid encoding Pdx1, this fragment displayed an _x0008_25-fold enhancement in the ¦Â-cell lines INS-1 and_x0001_TC3." -- -- -- >2KB 4856 E_02_004 19041414 -- mm10 chr15 97977739 97977942 Mouse HeLa Low throughput "Luciferase Reporter Assay,EMSA" "Transient transfections of Sox9, Smad3, and T_x0002_R-I(TD) did not increase luciferase activities of pGL3-B plasmids in SW1353 cells (pGL3-B). In pGL3-585E systems, Sox9 enhanced a relative luciferase activity to a level as high as 2.2-fold over the control. Cotransfection of Smad3 augmented a luciferase activity up to 2.3-fold higher level of Sox9-transfected cells. The additional transfection of constitutively active form of T_x0002_R-I(TD) induced an approximately 36% increase of the activity in Sox9- and Smad3-transfected SW1353 cells. Luciferase activities of pGL3-585E were not increased in the absence of Sox9. Note that Smad3 and T_x0002_R-I(TD) synergistically activated the native Col2a1 reporter-mediated transcription in a Sox9-dependent manner. " Enhancer Col2a1 -- "Luciferase Reporter Assay,EMSA" Purified Sox9 associated with the Col2a1 enhancer probe in EMSA. "Col2,Col2a,Col2a-1,Del1,Dmm,Lpk,M100413,Rgsc413,Rgsc856" -- -- -- -- -- -- Sox9 "2010306G03Rik,AV220920,mKIAA4243" EMSA Purified Sox9 associated with the Col2a1 enhancer probe in EMSA. The unlabeled competitor decreased the signal of Sox9¨CDNA complex. Supershifted band was observed in the presence of anti-Sox9 antibody. -- -- -- Intron 2240 E_02_005 19285986 -- mm10 chr8 105528423 105529045 Mouse N38 Low throughput "Transgenic mice,Luciferase Reporter Assay" "Specifically, Diiib had as much activity as subregion Diii on its own but only in the h295R cells, while subregion Diiia had the same activity as the parent Diii region but only in the N38 cells." Enhancer Agrp -- "Transgenic mice,Luciferase Reporter Assay,qPCR" qPCR shows robust expression of AgRP in the tongue but at lower levels than in the hypothalamus. (c) Immunohistochemistry of mouse tongue sections shows AgRP expression in epithelial cells of wild-type (AgRP+/+) but not AgRP-deficient (AgRP?/?) mice "Agrt,Art" -- -- -- -- -- -- -- -- -- -- -- -- -- >2KB 37960 E_02_006 19345119 MHB Enhancer mm10 chr4 44520195 44520234 Mouse Lymphopoiesis B Cell Low throughput "Transfection,ChIP,EMSA" "By using transgenic analysis, we have previously identified a 435 bp enhancer (located 5.6 kb upstream of exon 1A), which is sufficient to drive reporter gene expression in the developing MHB region.To confirm these footprinting data, we performed ChIP analysis with unstimulated cells of the mature B cell line WEHI-279 (Figure 7C; Figure S14A) and with in vitro cultured pro-B cells (Figure S14B) to screen all HS sites of the Pax5 locus for binding of PU.1, IRF4, IRF8, NF-kB, and Myc. Electrophoretic mobility shift assays(EMSAs)with an oligonucleotide probe containing the EBF1 recognition sequence of HS-7 revealed efficient protein binding to this site with a pro-B cell nuclear extract." Enhancer Pax5 -- Transgenic mice "A DNA fragment containing the entire Pax5 promoter region from the MHB enhancer (at position -6,074) to exon 2 (at position +13,368) was fused in-frame in exon 2 to a Gfp reporter gene to generate transgene PromGFP.GFP expression was determined by flow cytometry in different B cell types of a PromGFP transgenic mouse. PromGFP transgenic mice failed to express GFP in pro-B and mature B cells, but gave rise to low GFP expression in pre-B and immature B cells(Figure 4A) similar to the BAC1-DIn5 transgene ." "BSAP,EBB-1,KLP,Pax-5" -- -- -- -- -- -- "Spi1,Irf4,Irf8,Nfkb1" "Dis-1,Dis1,PU.1,Sfpi-1,Sfpi1,Spi-1,Tcfpu1,Tfpu.1,AI385587,IRF-4,LSIRF,NF-EM5,Spip,AI893568,ICSBP,IRF-8,Icsbp1,Myls,NF-KB1,NF-kappaB,NF-kappaB1,p105,p50,p50/p105" "ChIP,DMS Footprinting Assay" "PU.1, IRF4, IRF8, and NF-kB Control the Activity of the Pax5 Enhancer.To confirm these footprinting data, we performed ChIP analysis with unstimulated cells of the mature B cell line WEHI-279 and with in vitro cultured pro-B cells to screen all HS sites of the Pax5 locus for binding of PU.1, IRF4, IRF8, NF-kB, and Myc. " -- -- -- >2KB 6053 E_02_007 20042469 CNS-2 Enhancer mm10 chr3 37225520 37228865 Mouse CD4+ T Cell Low throughput "Luciferase Reporter Assay,ChIP-qPCR,ChIP,PCR" "To determine the function of IL-21P and CNS-2 in the transcriptional control of IL-21 expression by c-Maf, we performed a reporter assay. IL-21P-Luci containing a fragment of IL-21P (positions ¨C261 to 17), IL-21P-Luci-CNS-2 containing IL-21P and CNS-2 (position +2931 to +3443), or control pGL3 was transfected into EL4 cells, together with pcDNA3 c-Maf or control pcDNA3, and the luciferase activity of the reporter construct was evaluated with or without PMA+I stimulation. As shown in Figure3B, IL-21P-Luci responded to PMA+I stimulation, and c-Maf enhanced the activity of IL-21P-Luci (n=3; P<0.05) further. IL-21P-Luci-CNS-2 responded much greater to c-Maf in the presence of PMA+I stimulation (Fig. 3B). These results suggest that CNS-2 functions as an enhancer of the IL-21 gene and that c-Maf activates IL-21P as well as CNS-2.After cells were stimulated with or without PMA+I for 1 h, ChIP-qPCR assay for IL-21P, CNS-2, and CNS-4 was performed with anti-c-Maf anti-body or control rabbit IgG. Results are expressed as the percent input for each ChIP fraction." Enhancer Il21 -- ChIP "To determine whether c-Maf actually binds to IL-21P and the CNS-2 enhancer in CD4 + T cells, we performed ChIP analysis.These results suggest that CNS-2 functions as an Enhancer of the IL-21 gene and that c-Maf acti_x005F_x0002_vates IL-21P as well as CNS-2." IL-21 -- -- -- c-Maf induces IL-21 production directly in CD4_x005f_x0002_ T cells by activating IL-21P and the CNS-2 Enhancer and that TGF-¦Â suppresses c-Maf-induced IL-21 production in CD4_x0002_ T cells. "Luciferase Reporter Assay,PCR" "Taken together with the data shown in Figure 3, these results suggest that c-Maf binds di_x005f_x005f_x005f_x005F_x005f_x005F_x005f_x005F_x0002_rectly to IL-21P and the CNS-2 Enhancer in CD4_x005F_x0001_ T cells after IL-6 stimulation and that TGF-_x0001_ enhances IL-6-induced c-Maf binding to IL-21P and the Enhancer." Maf "2810401A20Rik,A230108G15Rik,AW047063,c-maf" ChIP "To determine whether c-Maf actually binds to IL-21P and the CNS-2 Enhancer in CD4 T cells, we performed ChIP analysis." -- -- -- >2KB 4435 E_02_008 20864515 -- mm10 chr6 124718594 124718929 Mouse "Hep G2,NMuLi" Low throughput "ChIP,Luciferase Reporter Assay" "By ChIP, two of these sites bound Prep1 (Fig. 7A; positions nucleotide [nt] ?2,489 to ?2,139, nt ?2,113 to ?1,778), but re-ChIP assays revealed that Pbx1 was simultaneously present only at position nt ?2,113 to ?1,778.To investigate whether this last region features enhancer activity, we subsequently cloned this fragment in the pgl3 basic construct upstream the luciferase gene (pgl3LUC). The construct was then cotransfected in HeLa cells together with the Prep1, Pbx1 cDNAs or both and luciferase activity was measured (Fig. 7B).Prep1 and Pbx1 increased the SHP1 reporter activity, respectively, by 7.1-fold and 6-fold. Simultaneous cotransfection of the two plasmids caused an almost 30-fold induction, indicating SHP1 transcriptional regulation by the Prep1/Pbx1 complex." Enhancer Nr0b2 -- Luciferase Reporter Assay "lin-dependent IR phosphorylation and glycogen accumulation. Both Prep1 and Pbx1 bind SHP1 promoter at a site located between nucleotides -2,113 and -1,778. This fragment features Enhancer activity and induces luciferase function by 7-, 6-, and 30-fold, respectively,in response to Prep1, Pbx1, or both." "SHP,SHP-1,Shp1" Diabetes Mellitus DOID:9351 D003920 "Both Prep1 and Pbx1 bind SHP1 promoter at a site located between nucleotides -2,113 and -1,778. This fragment features Enhancer activity and induces luciferase function by 7-, 6-, and 30-fold, respectively, in response to Prep1, Pbx1, or both." "Luciferase Reporter Assay,ChIP" "In these assays, a fragment containing single Prep1/Pbx1 binding site displays a powerful Enhancer function. In addition, ChIp experiments with Prep1 and Pbx1 antibod_x005f_x0002_ies showed that these proteins bind the SHP1 regulatory region, suggesting that SHP1 gene is a target of the Prep1/Pbx1 complex and not just Prep1." "Pknox1,Pbx1" "D17Wsu76e,PREP1,2310056B04Rik,D230003C07Rik,Pbx-1" "Luciferase Reporter Assay,ChIP" "In these assays, a fragment containing single Prep1/Pbx1 binding site displays a powerful Enhancer function. The construct was then cotransfected in HeLa cells together with the Prep1, Pbx1 cDNAs or both and luciferase activity was measured In addition, ChIp experiments with Prep1 and Pbx1 antibodies showed that these proteins bind the SHP1 regulatory region, suggesting that SHP1 gene is a target of the prep1/Pbx1 complex and not just Prep1." -- -- -- >2KB 8834613 E_02_009 20878775 -- mm10 chr13 53463266 53463541 Mouse "C2C12,Primary Bone Marrow¨CMesenchymal Stem Cell" Low throughput "PCR,Luciferase Reporter Assay" The murine Dlx5 promoter from -1036 to +30 was obtained by polymerase chain reaction (PCR) and cloned in the pGL2basic vector. Murine Msx2 and Runx2 enhancers (from -3340 to -3615 for Msx2 and from -1170 to -1388 for Runx2) also were obtained by PCR and subcloned in the minimal promoter of c-fos promoter. Enhancer Msx2 -- Luciferase Reporter Assay "The Msx2 gene shows a conserved region 3.5 kb upstream of the transcriptional start site, which also includes a combination of a TCF/LEF1 box and both GC-rich and CAGA SBEs. This region has been shown to be necessary and sufficient to confer BMP responsiveness in vitro and in vivo.Finally, a similar enhancer is located 1 kb upstream of the P1 osteoblast-specific promoter of the Runx2 gene (Supplemental Fig. S3). To test whether these regions have the ability to render responsiveness to Wnt3a and/or BMP-2, we generated the corresponding luciferase constructs and assayed them in C2C12 cells. " "BB122635,Hox-8,Hox8,Hox8.1" -- -- -- -- -- -- "Msx2,Dlx3,Dlx5,Atf4,Runx2,Sp7" "BB122635,Hox-8,Hox8,Hox8.1,AV237891,Dlx-3,AI385752,Atf-4,C/ATF,CREB2,TAXREB67,AML3,Cbf,Cbfa-1,Cbfa1,LS3,Osf2,PEBP2aA,Pebp2a1,Pebpa2a,6430578P22Rik,C22,Osx" "qRT-PCR,Luciferase Reporter Assay,ChIP" "To confirm that cooperative transcriptional interactions between canonical Wnt signaling and the Smad pathway on osteogenic genes occur in vivo, ChIP assays were performed in C2C12 cells.As shown in Fig.7D,TCF4,b-catenin,and Smad1 and Smad4 interacted with the responsive regions of Dlx5, Msx2, and Runx2 genes in vivo." -- -- -- >2KB 3476 E_02_010 20878775 -- mm10 chr17 44494486 44495486 Mouse "C2C12,Primary Bone Marrow¨CMesenchymal Stem Cell" Low throughput "PCR,Luciferase Reporter Assay" The murine Dlx5 promoter from -1036 to +30 was obtained by polymerase chain reaction (PCR) and cloned in the pGL2basic vector. Murine Msx2 and Runx2 enhancers (from -3340 to -3615 for Msx2 and from -1170 to -1388 for Runx2) also were obtained by PCR and subcloned in the minimal promoter of c-fos promoter. Enhancer Runx2 -- Luciferase Reporter Assay "The Msx2 gene shows a conserved region 3.5 kb upstream of the transcriptional start site, which also includes a combination of a TCF/LEF1 box and both GC-rich and CAGA SBEs. This region has been shown to be necessary and sufficient to confer BMP responsiveness in vitro and in vivo.Finally, a similar enhancer is located 1 kb upstream of the P1 osteoblast-specific promoter of the Runx2 gene (Supplemental Fig. S3). To test whether these regions have the ability to render responsiveness to Wnt3a and/or BMP-2, we generated the corresponding luciferase constructs and assayed them in C2C12 cells. " "AML3,Cbf,Cbfa-1,Cbfa1,LS3,Osf2,PEBP2aA,Pebp2a1,Pebpa2a" -- -- -- -- -- -- "Msx2,Dlx3,Dlx5,Atf4,Runx2,Sp7" "BB122635,Hox-8,Hox8,Hox8.1,AV237891,Dlx-3,AI385752,Atf-4,C/ATF,CREB2,TAXREB67,AML3,Cbf,Cbfa-1,Cbfa1,LS3,Osf2,PEBP2aA,Pebp2a1,Pebpa2a,6430578P22Rik,C22,Osx" "qRT-PCR,Luciferase Reporter Assay,ChIP" "To confirm that cooperative transcriptional interactions between canonical Wnt signaling and the Smad pathway on osteogenic genes occur in vivo, ChIP assays were performed in C2C12 cells.As shown in Fig.7D,TCF4,b-catenin,and Smad1 and Smad4 interacted with the responsive regions of Dlx5, Msx2, and Runx2 genes in vivo." -- -- -- <2KB 1000 E_02_011 20878775 -- mm10 chr17 44494598 44494816 Mouse "C2C12,Primary Bone Marrow¨CMesenchymal Stem Cell" Low throughput "PCR,Luciferase Reporter Assay" The murine Dlx5 promoter from -1036 to +30 was obtained by polymerase chain reaction (PCR) and cloned in the pGL2basic vector. Murine Msx2 and Runx2 enhancers (from -3340 to -3615 for Msx2 and from -1170 to -1388 for Runx2) also were obtained by PCR and subcloned in the minimal promoter of c-fos promoter. Enhancer Runx2 -- Luciferase Reporter Assay "The Msx2 gene shows a conserved region 3.5 kb upstream of the transcriptional start site, which also includes a combination of a TCF/LEF1 box and both GC-rich and CAGA SBEs. This region has been shown to be necessary and sufficient to confer BMP responsiveness in vitro and in vivo.Finally, a similar enhancer is located 1 kb upstream of the P1 osteoblast-specific promoter of the Runx2 gene (Supplemental Fig. S3). To test whether these regions have the ability to render responsiveness to Wnt3a and/or BMP-2, we generated the corresponding luciferase constructs and assayed them in C2C12 cells. " "AML3,Cbf,Cbfa-1,Cbfa1,LS3,Osf2,PEBP2aA,Pebp2a1,Pebpa2a" -- -- -- -- -- -- "Msx2,Dlx3,Dlx5,Atf4,Runx2,Sp7" "BB122635,Hox-8,Hox8,Hox8.1,AV237891,Dlx-3,AI385752,Atf-4,C/ATF,CREB2,TAXREB67,AML3,Cbf,Cbfa-1,Cbfa1,LS3,Osf2,PEBP2aA,Pebp2a1,Pebpa2a,6430578P22Rik,C22,Osx" "qRT-PCR,Luciferase Reporter Assay,ChIP" "To confirm that cooperative transcriptional interactions between canonical Wnt signaling and the Smad pathway on osteogenic genes occur in vivo, ChIP assays were performed in C2C12 cells.As shown in Fig.7D,TCF4,b-catenin,and Smad1 and Smad4 interacted with the responsive regions of Dlx5, Msx2, and Runx2 genes in vivo." -- -- -- <2KB 1279 E_02_012 20942803 Slc30a8 Enhancer mm10 chr15 52315678 52316298 Mouse Islet B-Cell Low throughput "Luciferase Reporter Assay,PCR" "To address this hypothesis these regions, designated intronic enhancers A and B, respectively, were isolated using PCR and ligated 5¡ä of a heterologous thymidine kinase (TK)-luciferase fusion gene containing TK genomic sequence between ?105 and +51, relative to the transcription start site.Luciferase expression directed by these fusion genes was then analyzed by transient transfection of ¦ÂTC-3 cells, an islet beta cell-derived line [31], ¦ÁTC-6 cells, an islet alpha cell-derived line [32, 33] and HeLa cells, a cervix-derived cell line." Enhancer Slc30a8 -- "PCR,Luciferase Reporter Assay" "Luciferase expression directed by these fusion genes was then analyzed by transient transfection of ¦ÂTC-3 cells, an islet beta cell-derived line [31], ¦ÁTC-6 cells, an islet alpha cell-derived line [32, 33] and HeLa cells, a cervix-derived cell line. Figure 2 shows that in ¦ÂTC-3 cells, but not ¦ÁTC-6 or HeLa cells, intronic enhancer A elevated reporter gene expression beyond that driven by the TK-luciferase fusion gene alone, indicating that this region is an islet beta cell-specific enhancer. In contrast, intronic enhancer B had no effect on fusion gene expression in any of the cell lines." "C820002P14Rik,ZnT-8,ZnT8" -- -- -- -- -- -- Pbx1 "2310056B04Rik,D230003C07Rik,Pbx-1" "ChIP,EMSA" "Gel retardation and chromatin immunoprecipitation (ChIP) assays revealed that the islet-enriched transcription factor Pdx-1 binds Enhancer A in vitro and in situ, respectively." -- -- -- Intron 20436 E_02_013 21056086 -- mm10 chr4 136140539 136140546 Mouse "Immortalized Murine Gonadotrope L??T2 Cell,Ovarian Cancer Cell" Low throughput "Transfection,EMSA,Luciferase Reporter Assay,ChIP" "In silico analyses suggested that the upstream enhancer in the human ID3 gene might be conserved in murine Id3. In that report, an enhancer was described upstream of the promoter region we investigated here in mouse. We obtained the reporter constructs used in Shepherd et al. (2008) and examined their BMP2 induction in L¦ÂT2cells(Fig.6(A)). The data reflect the mean (+SEM) of three independent experiments and are presented relative to untreated cells transfected with the empty vector, pGL3-Basic. EMSAs were performed with the indicated radio-labeled (*) probes corresponding to ?568/?502 of the murine Id3 promoter" Enhancer Id3 -- ChIP The analysis of the human gene revealed a BMP response element (BRE) within a distal Enhancer (?2632/?2625) that when mutated (TGGCGCC¡úTGGTGCT) greatly reduced the fold BMP4 response.We identified the same sequence in the murine gene (?3283/?3276) and observed through ChIP analysis that BMP2 stimulated recruitment of SMAD1 to this region of the endogenous Id3 gene in L¦ÂT2 cells. "Hlh462,Idb3,bHLHb25" -- -- -- A more distal Enhancer was shown to mediate BMP4-induction of the human ID3 gene in ovarian cancer cells. ChIP "We identified the same sequence in the murine gene (?3283/?3276) and observed through ChIP analysis that BMP2 stimulated recruitment of SMAD1 to this region of the endogenous Id3 gene in L_x005f_x0002_T2 cells. Mutation of the distal BRE in the longer murine Id3 reporter greatly reduced, but did not abolish, BMP2 induction." Smad1 "AI528653,Mad1,Madh1,Madr1,Mlp1,MusMLP,dwf-A,mMad1" ChIP "We identified the same sequence in the murine gene (?3283/?3276) and observed through ChIP analysis that BMP2 stimulated recruitment of SMAD1 to this region of the endogenous Id3 gene in L¦ÂT2 cells. Mutation of the distal BRE in the longer murine Id3 reporter greatly reduced, but did not abolish, BMP2 induction." -- -- -- >2KB 3278 E_02_014 21331042 -- mm10 chr3 34563695 34565695 Mouse Axial Stem Cell Low throughput "EMSA,Immunostaining,Transgenic mice" "Our earlier studies have indicated that among a number of enhancers regulating Sox2, enhancer N1 is responsible for Sox2 activation in the caudally extendin neural plate5,6 (Fig.1a-c; Supplementary Fig.1)." Enhancer Sox2 -- "EMSA,Immunostaining" "Finally, we asked whether the suppression of Sox2 enhancer N1 activation by Tbx6 involves direct interaction of the Tbx6 protein with the enhancer N1 DNA sequence. Various overlapping fragments of the N1 sequence were tested for Tbx6 binding using electrophoretic mobility shift assays (EMSA). Exogenous Sox2-HA was successfully expressed in the caudal-most mesodermal compartment (Fig.4b), as indicated by HA-tag immunostaining and the mesodermal Sox2 immunostaining (Fig.4c-e). This result demonstrates that Sox2 expression is sufficient for the initiation of neural tube development from the primitive paraxial mesoderm, even in wild type embryos." "Sox-2,lcc,ysb" -- -- -- -- -- -- Tbx6 rv EMSA Various overlapping fragments of the N1 sequence were tested for Tbx6 binding using electrophoretic mobility shift assays (EMSA). -- -- -- >2KB 85299 E_02_015 21368045 -- mm10 chr14 67742375 67743103 Mouse "Epithelial Cell,Granulosa Cells,GT1-7,GN11,Embryonic Stem Cell" Low throughput "RT-PCR,Luciferase Reporter Assay" "Furthermore, studies in mice bearing either ?3446/+23 or ?2078/+23 bp of the mGnRH promoter fused to the LUC reporter defined a critical enhancer region for the in vivo expression of hypothalamic mGnRH (Kim et al., 2002).The quantitative RT-PCR primer sequences are available on request." Enhancer Gnrh1 -- Luciferase Reporter Assay "In our previous study, using the luciferase (LUC) reporter gene, we found that the proximal ?1005 bp of the mouse GnRH (mGnRH) promoter contains the critical elements for appropriate neuronal and ovarian expression of mGnRH (Kim et al., 2002). Furthermore, studies in mice bearing either ?3446/+23 or ?2078/+23 bp of the mGnRH promoter fused to the LUC reporter defined a critical enhancer region for the in vivo expression of hypothalamic mGnRH (Kim et al., 2002)." "Gnrh,Gnrh2,LHRH,Lhrh1,Lnrh,hpg" -- -- -- This work supports the hypothesisthatthe region ofthe GnRH promoter contained between -2806 and -2078 bp acts as a cell-specific Enhancer in the GnRH neuron and as a repressor in the ovary. Luciferase Reporter Assay "In this work, to further characterize the mGnRH promoter,we have constructed an additional transgenic mouse bearing a -2806/+23 bp fragment fused to the LUC reporter gene. This model demonstrates that critical hypothalamic Enhancer se_x005f_x0002_quences and ovarian repressor elements for the in vivo expression of mGnRH are located between -2806 and -2078 bp on the mGnRH promoter." "Oct1,Gata4" "2810482H01Rik,NF-A1,Oct-1,Oct1,Otf-1,Otf1,Gata-4" Luciferase Reporter Assay Oct-1 and GATA-4 were identified as important transcription factors that bind to the neuron-specific Enhancer and regulate GnRH expression in vitro. -- -- -- >2KB 2441 E_02_016 21454523 LCR mm10 chr7 103823354 103846754 Mouse 745A Low throughput "ChIP,qRT-PCR,3C" "The presence of the cohesin holo-complex was confirmed by ChIP using antibodies specific for SMC1 and SA1.Most notably, HS2 functions as an enhancer, and the outer HS5 functions as an insulator. B, adult ¦Â-globin gene induction following DMSO treatment. End point RT-PCR of ¦Â-major gene induction and q-RT-PCR analysis of ¦Â-major and ¦Â-minor genes in comparison to GAPDH were examined over 4 days of DMSO treatment. Q-RT-PCR values were normalized to ribonuclease/angiogenin inhibitor 1 (rnh1). " Enhancer Hbb-y 3C ChIP 3C and ChIP-loop analysis of the HS2 and ¦Â-major promoter interaction at 4 days after DMSO treatment. The interactions of a region containing HS4 and HS5 (HS4/5) with either 3¡äHS1 or HS-62 are compared. "Ey,Hby" Haploinsufficiency -- D057895 Nipped-B-like (Nipbl) bind to the locus control region (LCR) at the CTCF insulator and distal enhancer regions as well as at the specific target globin gene that undergoes activation upon differentiation "ChIP,3C,PCR" "The results indicate that cohesin binds to these regions when they interact. Taken together, cohesin and Nipbl are recruited not only to the HS5 insulator, but also to the Enhancer and the active _x005f_x0001_-globin gene regions. This correlates with the induction of the interaction of these domains and ¦Â-globin gene activation." "Nipbl,Ctcf,Nfe2" "Idn3,AW108038,NF-E2,NF-E2/P45,p45,p45NFE2,p45nf-e2" ChIP "Differentiation-induced Cohesin and Nipbl Binding at the ¦Â-globin Locus Is Associated with LCR Enhancer-globin Gene Interactions.,ChIP analysis of cohesin (Rad21),Nipbl,CTCF, and NF-E2 binding to the ¦Â-globin locus." -- -- -- >2KB 16699 E_02_017 21464046 -- mm10 chrx 7954360 7956360 Mouse Embryonic Stem Cell Low throughput "Luciferase Reporter Assay,RT-PCR" The 3.9 kb Gata1 hematopoietic enhancer and minigene was previously described.We have previously generated a transgenic mouse model using an Nkx2-5 cardiac specific enhancer to direct EYFP reporter expression to the developing heart.Transcriptional assay in K562 cells reveal a dose-dependent repression of luciferase activity by Nkx2-5 using the 3.9 kb erythroid enhancer. Enhancer Gata1 -- Transfection "Utilizing transgenic technologies to isolate Nkx2-5 expressing cells, we observed an induction of the erythroid molecular program, including Gata1, in the Nkx2-5 null embryos. We demonstrated that Nkx2-5 binds to the Gata1 gene enhancer and represses the transcriptional activity of the Gata1 gene." "Gata-1,Gf-1,eryf1" -- -- -- -- -- -- Nkx2-5 "MyoR,bHLHa22,Tbx5,Csx,Nkx-2.5,Nkx2.5,tinman" Luciferase Reporter Assay "Using transcriptional assays,we showed that one of the targets of Nkx2-5 is the Gata1 gene,an essential transcription factor in erythroid differentiation." -- -- -- >2KB 3899 E_02_018 21486496 -- mm10 chr5 115358083 115360083 Mouse Embryonic Stem Cell Low+High throughput "Luciferase Reporter Assay,ChIP-seq" "The deleted BAC constructs were introduced into ESCs, and stable transformants were established for each BAC reporter construct. In a similar manner to the RA treatment and Luciferase reporter assay conducted with EBs (shown in Figure ?Figure2A),the Msi1 transcriptional activities were quantified using the deletion-containing reporters D1-D5 and the full-length BAC reporter D0. These results suggested that the 10-kb region from 55 kb to 65 kb upstream of the TSS might contain Msi1 transcriptional enhancers, and we named this region the 'upstream 10-kb enhancer.'" Enhancer Msi1 -- Luciferase Reporter Assay "We then used homologous recombination techniques to insert the ffLuc reporter gene into the Msi1 translational initiation site of the RP24-132L16 BAC (Figure1A).The ffLuc reporter gene encodes a fusion protein of the fluorescent protein Venus and firefly Luciferase.This reporter gene both visualized Msi1 transcriptional activity in vivo, and allowed us to use Luciferase bioluminescence to quantify the level of transcriptional activity." "Msi1h,Musahi1,m-Msi-1" -- -- -- The intronic Enhancer can transactivate Msi1 gene expression with cell-type specificity markedly similar to the endogenous Msi1 expression patterns. Luciferase Reporter Assay "Furthermore, when the region after the transcription site (exons and introns coding region) was deleted, the Msi1 transcriptional activity diminished even in the presence of the¡®upstream 10-kb Enhancer¡¯ (Figure 3C). These results indicated two regions responsible for Msi2 transcription." "Qsox1,Tfap2a" "1300003H02Rik,QSOX,Qscn6,SOx,b2b2673Clo,AP-2,AP2alpha,Ap-2(a),Ap2,Ap2tf,Tcfap2a" Luciferase Reporter Assay "Through these investigations, we have identified a new Msi1 transcription Enhancer element in NS/PCs. This region is located in the 595-bp region within the sixth intron of the Msi1 gene, and contains SOX- and AP-2-binding sites." -- -- -- <2KB 1 E_02_019 21487009 TCCR Enhancer mm10 chr14 78384835 78446843 Mouse T Cell Low throughput "ChIP-chip,3C,ChIP,Luciferase Reporter Assay,PCR" "We used ChIP-chip analysis in mouse primary T cells and a T cell hybridoma to define the regulatory enhancers responsible for this up-regulation and to characterize their properties.Our previous ChIP-chip analyses of the mouse Rankl locus suggested that the long distance Rankl enhancers were routinely marked in osteoblasts by both basal and 1,25(OH)2D3-, PTH-, or cytokine-inducible levels of H4ac as well as increased RNA pol II density. " Enhancer Tnfsf11 3C -- "To determine whether looping might provide a mechanism for Rankl activation via the mRL-D5 and the TCCR regions, we treated 2b4.11 cells with either vehicle or PI and used 3C analysis to assess potential interactions between the promoter and enhancers as described under ¡°Experimental Procedures.¡± " "Ly109l,ODF,OPGL,RANKL,Trance" -- -- -- mRL-D5 and segments of the TCCR were located in proximity to the Rankl gene promoter and thus potentially able to influence directly Rankl gene promoter activity. "3C,ChIP,Luciferase Reporter Assay,PCR" "These data also suggest that osteoblasts and T cell both may utilize a common Enhancer such as mRL-D5 and cell type-specific Enhancers such as the TCCR to regulate Rankl gene expression. " "Fos,Rela" "D12Rfj1,c-fos,cFos,p65,p65 NF-kappa B,p65 NFkB" "3C,ChIP" "They also indicate that despite our inability to detect its presence in the ChIP-ChIP analysis,Nfat or related transcription factor activation may be involved in transcriptional regulation through the putative mRL-T1A Enhancer." -- -- -- >2KB 138394 E_02_020 21487009 mRL-D5 Enhancer mm10 chr14 78351421 78354313 Mouse T Cell Low throughput "ChIP-chip,3C,ChIP,Luciferase Reporter Assay,PCR" "We used ChIP-chip analysis in mouse primary T cells and a T cell hybridoma to define the regulatory enhancers responsible for this up-regulation and to characterize their properties.Our previous ChIP-chip analyses of the mouse Rankl locus suggested that the long distance Rankl enhancers were routinely marked in osteoblasts by both basal and 1,25(OH)2D3-, PTH-, or cytokine-inducible levels of H4ac as well as increased RNA pol II density. " Enhancer Tnfsf11 3C -- "To determine whether looping might provide a mechanism for Rankl activation via the mRL-D5 and the TCCR regions, we treated 2b4.11 cells with either vehicle or PI and used 3C analysis to assess potential interactions between the promoter and enhancers as described under ¡°Experimental Procedures.¡± " "Ly109l,ODF,OPGL,RANKL,Trance" -- -- -- mRL-D5 and segments of the TCCR were located in proximity to the Rankl gene promoter and thus potentially able to influence directly Rankl gene promoter activity. "3C,ChIP,Luciferase Reporter Assay,PCR" " These data also suggest that osteoblasts and T cell both may utilize a common Enhancer such as mRL-D5 and cell type-specific Enhancers such as the TCCR to regulate Rankl gene expression. " "Fos,Rela" "D12Rfj1,c-fos,cFos,p65,p65 NF-kappa B,p65 NFkB" "3C,ChIP" "They also indicate that despite our inability to detect its presence in the ChIP-ChIP analysis,Nfat or related transcription factor activation may be involved in transcriptional regulation through the putative mRL-T1A Enhancer." -- -- -- >2KB 75422 E_02_021 21527504 -- mm10 chr14 67745496 67746000 Mouse GT1-1 Low throughput "Luciferase Reporter Assay,Transfection,ChIP,qRT-PCR,EMSA" "To examine the enhancer activity of intron A, we constructed reporter vectors containing the full-length mouse intron A sequence (1003 bp) in either the forward or reversed direction followed by the simian vacuolating virus 40 (SV40) minimal promoter (SV40p) driving luciferase expression.These constructs were transfected into GnRH-expressing GT1-1 cells, and luciferase activity was then measured. Cells harboring the vectors with the full-length intron A exhibited luciferase activity significantly increased by 2- to 3-fold compared with those harboring only the SV40p (the ¡°empty vector¡± in this study) (Fig. 1B). These findings strongly suggest that GnRH intron A contains a putative transcriptional enhancer that is likely involved in GnRH gene regulation." Enhancer Gnrh1 -- Luciferase Reporter Assay "Schematic diagrams of the reporter constructs and their relative luciferase activities in comparison with those of SV40p-Ex1-Luc and SV40p-Ex1IA-315-Luc are shown in Fig. 4. Thus, these results strongly suggest that the region between 12 and 25 nt in the mouse GnRH intron A harbors a key cis-element for the enhancer function." "Gnrh,Gnrh2,LHRH,Lhrh1,Lnrh,hpg" -- -- -- Class-C SOX Transcription Factors Control GnRH Gene Expression via the Intronic Transcriptional Enhancer. "Luciferase Reporter Assay,ChIP,EMSA" "Chromatin immunoprecipitation shows that endogenous SOX-C factors recognize and bind to the intronic Enhancer in GT1-1 cells and the hypothalamus. Taken together,these findings demonstrate that SOX-C transcription factors function as important transcriptional regulators of cell type-specific GnRH gene expression by acting on the intronic transcriptional Enhancer." "Sox4,Sox11,Sox12" "AA682046,Sox-4,1110038H03Rik,6230403H02Rik,AI836553,end1,2010205A02Rik" "ChIP,EMSA" "Chromatin immunoprecipitation (ChIP) assay re vealed that SOX11 binds to the Ex1-proximal Enhancer region of intron A, and overexpression of SOX11 in creases this binding in GT1-1 cells.In accor_x005f_x0002_dance with the result in the reporter assay, EMSA revealed that in vitro translated SOX4 and SOX11 proteins can recognize both SOX-binding sites in mouse intronA; the binding was completely abrogated when both sites were mutated but partially diminished in the probes with a deletion of one of them." -- -- -- <2KB 568 E_02_022 21549805 -- mm10 chr18 61099404 61099740 Mouse B Cell Low throughput "Luciferase Reporter Assay,EMSA,ChIP" "luciferase expression was driven by FIRE sequences, and tested these constructs for their responsiveness to PAX5 expression in the RAW264 macrophage cell line and the 38B9 B-cell line using transient transfection assays.When testing the binding of PAX5 across Csf1r by ChIPassays in primary pro-B cells, we noticed that PAX5 did not just bind to the promoter, but also associated with the intronic enhancer (FIRE).This observation was confirmed by EMSA experiments, demonstrating that none of the DNA sequences present on FIRE were capable of competing for the binding of PAX5 to DNA containing a PAX5 consensus sequence." Enhancer Csf1r -- "Immunoprecipitation,Transfection,ChIP" "We performed PAX5-specific chromatin immunoprecipitation analyses across the Csfr1 locus. We investigated the role of PAX5 in regulating Csf1r sense and antisense promoter activity by transient transfections and by employing a Pax5/ proLBcell line expressing an inducible PAX5 protein. When testing the binding of PAX5 across Csf1r by ChIPassays in primary pro-B cells, we noticed that PAX5 did not just bind to the promoter, but also associated with the intronic enhancer (FIRE). " "AI323359,CD115,CSF-1R,Csfmr,Fim-2,Fim2,Fms,M-CSF-R,M-CSFR" -- -- -- -- -- -- Pax5 "BSAP,EBB-1,KLP,Pax-5 " "ChIP,EMSA" "PAX5 binds to the intronic enhancer of Csf1r (FIRE).When testing the binding of PAX5 across Csf1r by ChIP assays in primary pro-B cells, we noticed that PAX5 did not just bind to the promoter, but also associated with the intronic Enhancer (FIRE).This observation was confirmed by EMSA experiments." -- -- -- >2KB 5999 E_02_023 21566115 Il12b Enhancer mm10 chr11 44390342 44392251 Mouse "Myeloid Cells,T Cell" Low throughput "Luciferase Reporter Assay,qRT-PCR" We focused on a conserved enhancer about 10kb upstream of the Il12b start site that is targeted by TLR signaling to increase Il12b transcription in response to LPS (33). We made luciferase reporter constructs that fused the Il12b promoter to the upstream enhancer. Enhancer Il12b -- "Luciferase Reporter Assay,qRT-PCR" "To search for transcription factors that could be involved in the suppression of Il12b transcription, we used qRT-PCR to measure the expression of transcription factor mRNAs induced by IL-10 in the presence of an inflammatory costimulus.We made luciferase reporter constructs that fused the Il12b promoter to the upstream enhancer." "Il-12b,Il-12p40,Il12p40,p40" Autoimmune and Inflammatory Diseases -- -- -- -- -- Nfil3 "AV225605,E4BP4" "qRT-PCR,Luciferase Reporter Assay" A NFIL3 binding site is identified in the enhancer region of Il12b. -- -- -- >2KB 8765 E_02_024 21652712 -- mm10 chr5 147267614 147267639 Mouse "HIT,Hep G2,INS-1" Low throughput "Transfection,Luciferase Reporter Assay,EMSA,ChIP,qPCR" "Previous studies have identified several transcriptional factors for the Pdx-1 promoter that consists of two important regulatory regions as follows: the proximal and distal enhancer regions.To delineate the sites responsible for SREBP-lc suppression o¨ª Pdx-1, we generated a series of 5'-deletion constructs and analyzed these reporters by transfection into HIT cells (Fig.2C). SREBP-lc-mediated inhibition was completely abolished In PDX-1 (-93)-Luc. " Enhancer Pdx1 -- "Luciferase Reporter Assay,EMSA" "SREBP-lc-mediated inhibition was completely abolished In PDX-1 (-93)-Luc. In the distal enhancer region, EMSA analysis showed that potent binding of PDX-1 to the probe containing a PDX-1 -binding site in area I (-2517 to - 2492 bp) was dose-dependently inhibited by SREBP-lc (Fig. 6£©" "IDX-1,IPF-1,Ipf1,Mody4,STF-1,pdx-1" Diabetes Mellitus DOID:9351 D003920 -- -- -- Pdx1 "IDX-1,IPF-1,Ipf1,Mody4,STF-1,pdx-1" EMSA "In the distal Enhancer region, EMSA analysis showed that potent binding of PDX-1 to the probe containing a PDX-1-binding site in area I (-2517 to - 2492 bp) was dose-dependently inhibited by SREBP-lc." -- -- -- >2KB 2503 E_02_025 21652712 -- mm10 chr5 147267670 147267721 Mouse "HIT,Hep G2,INS-1" Low throughput "Transfection,Luciferase Reporter Assay,EMSA,ChIP,qPCR" "Previous studies have identified several transcriptional factors for the Pdx-1 promoter that consists of two important regulatory regions as follows: the proximal and distal enhancer regions.To delineate the sites responsible for SREBP-lc suppression o¨ª Pdx-1, we generated a series of 5'-deletion constructs and analyzed these reporters by transfection into HIT cells (Fig.2C). SREBP-lc-mediated inhibition was completely abolished In PDX-1 (-93)-Luc. " Enhancer Pdx1 -- "Luciferase Reporter Assay,EMSA" "SREBP-lc-mediated inhibition was completely abolished In PDX-1 (-93)-Luc. In the distal enhancer region, EMSA analysis showed that potent binding of PDX-1 to the probe containing a PDX-1 -binding site in area I (-2517 to - 2492 bp) was dose-dependently inhibited by SREBP-lc (Fig. 6£©" "IDX-1,IPF-1,Ipf1,Mody4,STF-1,pdx-1" Diabetes Mellitus DOID:9351 D003920 -- -- -- Pdx1 "IDX-1,IPF-1,Ipf1,Mody4,STF-1,pdx-1" EMSA "In the distal Enhancer region,EMSA analysis showed that potent binding of PDX-1 to the probe containing a PDX-1 -binding site in area I (-2517 to - 2492 ) was dose-dependently inhibited by SRE -lc." -- -- -- >2KB 2434 E_02_026 21652712 -- mm10 chr5 147267701 147267751 Mouse "HIT,Hep G2,INS-1" Low throughput "Transfection,Luciferase Reporter Assay,EMSA,ChIP,qPCR" "Previous studies have identified several transcriptional factors for the Pdx-1 promoter that consists of two important regulatory regions as follows: the proximal and distal enhancer regions.To delineate the sites responsible for SREBP-lc suppression o¨ª Pdx-1, we generated a series of 5'-deletion constructs and analyzed these reporters by transfection into HIT cells (Fig.2C). SREBP-lc-mediated inhibition was completely abolished In PDX-1 (-93)-Luc. " Enhancer Pdx1 -- "Luciferase Reporter Assay,EMSA" "SREBP-lc-mediated inhibition was completely abolished In PDX-1 (-93)-Luc. In the distal enhancer region, EMSA analysis showed that potent binding of PDX-1 to the probe containing a PDX-1 -binding site in area I (-2517 to - 2492 bp) was dose-dependently inhibited by SREBP-lc (Fig. 6£©" "IDX-1,IPF-1,Ipf1,Mody4,STF-1,pdx-1" Diabetes Mellitus DOID:9351 D003920 -- -- -- Pdx1 "IDX-1,IPF-1,Ipf1,Mody4,STF-1,pdx-1" EMSA "In the distal Enhancer region,EMSA analysis showed that potent binding of PDX-1 to the probe containing a PDX-1 -binding site in area I (-2517 to - 2492 ) was dose-dependently inhibited by SRE -lc." -- -- -- >2KB 2404 E_02_027 21673114 -- mm10 chr14 55518040 55518674 Mouse HEK-293 Low throughput "Transfection,EMSA,PCR" "To refine the cis-regulatory elements involved in controlling Nrl expression further, we tested the sequences within the three conserved clusters by in vivo transfection." Enhancer Nrl -- EMSA "In silico analysis using the TRANSFAC database (46) revealed a number of conserved putative transcription factor binding sites within B2(transcriptionally active sequence in clusterB)and clusterA(Fig.3,A and B, respectively). To identify the involvement of specific transcription factors, we performed EMSA using several different oligonucleotides spanning the conserved sequence elements(Fig. 4)." D14H14S46E -- -- -- -- -- -- "Rorb,Crx,,Nrl,Nr2e3" "Nr1f2,RZR-beta,RZRBeta,hstp,Rorb,Crx1,D14H14S46E,A930035N01Rik,PNR,RNR,rd7" EMSA "Electrophoretic mobility shift assays using mouse retinal nuclear extracts, in combination with specific antibodies,demonstrate the binding of retinoid-related orphan nuclear receptor(ROR¦Â),cone rod homeobox,orthodenticle homolog 2,and cyclic AMP response element-binding protein to predicted consensus elements within clusters A and B.Our studies demonstrate Nrl as a direct transcriptional target of ROR¦Â and suggest that combinatorial action of multiple regulatory factors modulates the expression of Nrl in developing and mature retina." -- -- -- <2KB 620 E_02_028 21797989 -- mm10 chr7 19409838 19410044 Mouse MM14 Low throughput "EMSA,Immunofluorescence,ChIP" Of particular interest was a 95-bp region (+901 to +995) that was subsequently proven to exhibit the properties of a transcriptional enhancer (Figure 1). Enhancer Ckm -- ChIP The chromatin immunoprecipitation (ChIP) primer pairs (black lines) that span the 5¡¯-enhancer sequence were used as positive controls for MyoD and myogenin binding to functional E-boxes. "CPK-Mm,M-CK,MCK,?Ckm" -- -- -- -- -- -- Mef2 "5430401D19Rik,9930028G15Rik,AV011172,Mef2" ChIP-seq MEF2 ChIP-seq occupancy at the 6.5- MCK regulatory region in differentiated C2 C12 cell shows that MEF2 is present at all three control regions. -- -- -- <2KB 1152 E_02_029 21798992 -- mm10 chr15 52315678 52316298 Mouse "¦ÂTC-3,¦ÁTC-6" Low throughput "Luciferase Reporter Assay,PCR" "The analysisof SLC30A8-luciferase expression in bTC-3 cells revealed that the proximal promoter region, located between K6154 and K1, relativeto the translation startsite, was onlyactivein stablebut not transient transfections.To assess whether chromatin structure was essential for human SLC30A8 and mouse Slc30a8 promoter function, luciferase fusion genes containing either the human SLC30A8 sequence from K6154 to K1 or the mouse Slc30a8 sequence from K1803 to K1 were analyzed in the context of the pGL4 vector." Enhancer Slc30a8 -- "PCR,Luciferase Reporter Assay" "To determine whether the C16 579 and C16 954 region of intron 2 in the human SLC30A8 gene also represents a transcriptional enhancer, this region was isolated using PCR and ligated 5 0 of the K150/C3 G6PC2-luciferase fusion gene described above." "C820002P14Rik,ZnT-8,ZnT8" Type 2 Diabetes Mellitus DOID:9352 D003924 -- -- -- -- -- -- -- -- -- -- >2KB 20436 E_02_030 21798992 -- mm10 chr15 5249678 5250298 Mouse ¦ÂTC-3 Low throughput Luciferase Reporter Assay "The analysisof SLC30A8-luciferase expression in bTC-3 cells revealed that the proximal promoter region, located between K6154 and K1, relativeto the translation startsite, was onlyactivein stablebut not transient transfections.To assess whether chromatin structure was essential for human SLC30A8 and mouse Slc30a8 promoter function, luciferase fusion genes containing either the human SLC30A8 sequence from K6154 to K1 or the mouse Slc30a8 sequence from K1803 to K1 were analyzed in the context of the pGL4 vector." Enhancer Slc30a8 -- "PCR,Luciferase Reporter Assay" "To determine whether the C16 579 and C16 954 region of intron 2 in the human SLC30A8 gene also represents a transcriptional enhancer, this region was isolated using PCR and ligated 5 0 of the K150/C3 G6PC2-luciferase fusion gene described above." "C820002P14Rik,ZnT-8,ZnT8" -- -- -- -- -- -- -- -- -- -- -- -- -- >2KB 47045564 E_02_031 21822303 -- mm10 chr3 34646162 34646551 Mouse MCF-7 Low throughput Luciferase Reporter Assay We transfected MCF7 and T47D cells with luciferase reporter vectors for the upstream distal enhancer and the core promoter to test whether induction of Sox2 protein was achieved through transcriptional activation of Sox2 promoter. Enhancer Sox2 -- Luciferase Reporter Assay We transfected MCF7 and T47D cells with luciferase reporter vectors for the upstream distal enhancer and the core promoter to test whether induction of Sox2 protein was achieved through transcriptional activation of Sox2 promoter. "Sox-2,lcc,ysb" -- -- -- -- -- -- -- -- -- -- -- -- -- >2KB 3637 E_02_032 21937508 u-PAR Enhancer mm10 chr7 24461573 24462573 Mouse Colon Cancer Cell Low throughput "RT-PCR,ChIP" "In order to gain insight into the epigenetic regulation of u-PAR expression, ChIP assays were employed in an unbiased screen to identify histone modifications or exchange of histone variants at the u-PAR promoter and enhancer under conditions of gene activation.Quantitative real-time PCR (qPCR) was performed using chromatin generated from either unstimulated or PMA-stimulated GEO cells using primer sets corresponding to the promoter, enhancer and intron 3 (Figure 1A and C). " Enhancer Plaur -- "RT-PCR,ChIP" "In order to gain insight into the epigenetic regulation of u-PAR expression, ChIP assays were employed in an unbiased screen to identify histone modifications or exchange of histone variants at the u-PAR promoter and enhancer under conditions of gene activation.Quantitative real-time PCR (qPCR) was performed using chromatin generated from either unstimulated or PMA-stimulated GEO cells using primer sets corresponding to the promoter, enhancer and intron 3 (Figure 1A and C). " "Cd87,u-PAR,uPAR" -- -- -- The role of an AP-1-harboring intragenic Enhancer in the regulation of both constitutive and inducible u-PAR expression. ChIP "In order to gain insight into the epigenetic regulation of u-PAR expression, ChIP assays were employed in an unbiased screen to identify histone modifications or exchange of histone variants at the u-PAR promoter and Enhancer under conditions of gene activation." Jun "AP-1c,c-jun,Jun" ChIP AP-1 binding to the u-PAR gene precedes H2A.Z eviction. ChIP assay showing the kinetics of (A) AP-1 (using a mixture of pan antibodies directed against Fos and Jun family members) (B) H2A.Z (C) RNAP II (using an antibody against RNAP II phosphorylated at Serine 5) deposition at the u-PAR promoter and enhancer in response to 100?nm PMA for the indicated times. -- -- -- <2kb 1137 E_02_033 22072553 -- mm10 chr1 38469166 38512500 Mouse BM Cell Low throughput "Transfection,ChIP-chip,Luciferase Reporter Assay" "Furthermore, a subset of these sites shows enhancer activity in transient transfection assays. ChIP-chip data were analyzed with MA2C 36 and a customized R package.These studies showed that H3 and H4 acetylation and p300/CBP binding is centered on the H/M peaks and is flanked by regions of histone H3K4me1 in a bimodal distribution characteristic of enhancer sequences (Figure 3B). 3To test for potential enhancer (or repressor activity), 22 Hoxa9/Meis1 binding sites as well as 2 control regions randomly selected in the genome where there is no Hoxa9/Meis1 binding were cloned as ~1000-bp fragments. These were inserted into the multiple cloning site of the pTAL-Lucvector,electroporated into myeloblastic K562 cells and were analyzed by Dual Luciferase Reporter Assays (Promega; supplemental Figure 3)." Enhancer Aff3 -- Luciferase Reporter Assay "To test for potential enhancer (or repressor activity), 22 Hoxa9/Meis1 binding sites as well as 2 control regions randomly selected in the genome where there is no Hoxa9/Meis1 binding were cloned as ~1000-bp fragments. These were inserted into the multiple cloning site of the pTAL-Lucvector,electroporated into myeloblas- tic K562 cells and were analyzed by Dual Luciferase Reporter Assays (Promega; supplemental Figure 3)." "3222402O04Rik,A730046J16,LAF-4,Laf4" Acute Leukemia DOID:12603 -- -- -- -- "Hoxa9,Meis1" "D6a9,Hox-1.7,C530044H18Rik,Evi8" ChIP-qPCR Confirmation of selected Hoxa9 and Meis1 binding sites by ChIP and quantitative PCR. -- -- -- >2KB 315441 E_02_034 22264824 -- mm10 chr11 32244715 32246895 Mouse Primary Erythroid Cell Low+High throughput "RT-PCR,ChIP,ChIP-seq" "Beyond this AFE the isoform structure appears identical to the full-length transcript. Interestingly, the AFE coincides with one of the known intragenic enhancers.First, we tested wild-type mouse tissues by RT-PCR to verify the existence of this alternative transcript. " Enhancer Nprl3 -- "RT-PCR,ChIP,ChIP-seq" "Beyond this AFE the isoform structure appears identical to the full-length transcript. Interestingly, the AFE coincides with one of the known intragenic enhancers.First, we tested wild-type mouse tissues by RT-PCR to verify the existence of this alternative transcript. " "Aag,CGTHBA,HS-26,HS-40,Mare,Phg,Prox1,m(alpha)RE" -- -- -- "Intragenic Enhancers frequently act as alternative tissue_x005f_x0002_specific promoters producing a class of abundant,spliced, multiexonic poly(A)+ RNAs (meRNAs) which reflect the host gene¡¯s structure." "ChIP,RT-PCR,ChIP-seq" "To address this problem, we analyzed transcrip_x005f_x0002_tion of the Nprl3 gene after deleting its constitutive promoter. This revealed that intragenic Enhancers act as highly active, alternative tissue-specific promoters for the gene containing them. " -- -- -- -- -- -- -- >2KB 13843 E_02_035 22264824 -- mm10 chr11 32249976 32252156 Mouse Primary Erythroid Cell Low+High throughput "RT-PCR,ChIP,ChIP-seq" "Beyond this AFE the isoform structure appears identical to the full-length transcript. Interestingly, the AFE coincides with one of the known intragenic enhancers.First, we tested wild-type mouse tissues by RT-PCR to verify the existence of this alternative transcript. " Enhancer Nprl3 -- "RT-PCR,ChIP,ChIP-seq" "Beyond this AFE the isoform structure appears identical to the full-length transcript. Interestingly, the AFE coincides with one of the known intragenic enhancers.First, we tested wild-type mouse tissues by RT-PCR to verify the existence of this alternative transcript. " "Aag,CGTHBA,HS-26,HS-40,Mare,Phg,Prox1,m(alpha)RE" -- -- -- "Intragenic Enhancers frequently act as alternative tissue_x005f_x0002_specific promoters producing a class of abundant,spliced, multiexonic poly(A)+ RNAs (meRNAs) which reflect the host gene¡¯s structure." "ChIP,RT-PCR,ChIP-seq" "To address this problem, we analyzed transcrip_x005f_x0002_tion of the Nprl3 gene after deleting its constitutive promoter. This revealed that intragenic Enhancers act as highly active, alternative tissue-specific promoters for the gene containing them. " -- -- -- -- -- -- -- >2KB 19104 E_02_036 22264824 -- mm10 chr11 32255189 32257369 Mouse Primary Erythroid Cell Low+High throughput "RT-PCR,ChIP,ChIP-seq" "Beyond this AFE the isoform structure appears identical to the full-length transcript. Interestingly, the AFE coincides with one of the known intragenic enhancers.First, we tested wild-type mouse tissues by RT-PCR to verify the existence of this alternative transcript. " Enhancer Nprl3 -- "RT-PCR,ChIP,ChIP-seq" "Beyond this AFE the isoform structure appears identical to the full-length transcript. Interestingly, the AFE coincides with one of the known intragenic enhancers.First, we tested wild-type mouse tissues by RT-PCR to verify the existence of this alternative transcript. " "Aag,CGTHBA,HS-26,HS-40,Mare,Phg,Prox1,m(alpha)RE" -- -- -- "Intragenic Enhancers frequently act as alternative tissue_x005f_x0002_specific promoters producing a class of abundant,spliced, multiexonic poly(A)+ RNAs (meRNAs) which reflect the host gene¡¯s structure." "ChIP,RT-PCR,ChIP-seq" "To address this problem, we analyzed transcrip_x005f_x0002_tion of the Nprl3 gene after deleting its constitutive promoter. This revealed that intragenic Enhancers act as highly active, alternative tissue-specific promoters for the gene containing them. " -- -- -- -- -- -- -- >2KB 24317 E_02_037 22264824 -- mm10 chr11 32268128 32270308 Mouse Primary Erythroid Cell Low+High throughput "RT-PCR,ChIP,ChIP-seq" "Beyond this AFE the isoform structure appears identical to the full-length transcript. Interestingly, the AFE coincides with one of the known intragenic enhancers.First, we tested wild-type mouse tissues by RT-PCR to verify the existence of this alternative transcript. " Enhancer Nprl3 -- "RT-PCR,ChIP,ChIP-seq" "Beyond this AFE the isoform structure appears identical to the full-length transcript. Interestingly, the AFE coincides with one of the known intragenic enhancers.First, we tested wild-type mouse tissues by RT-PCR to verify the existence of this alternative transcript. " "Aag,CGTHBA,HS-26,HS-40,Mare,Phg,Prox1,m(alpha)RE" -- -- -- "Intragenic Enhancers frequently act as alternative tissue_x005f_x0002_specific promoters producing a class of abundant,spliced, multiexonic poly(A)+ RNAs (meRNAs) which reflect the host gene¡¯s structure." "ChIP,RT-PCR,ChIP-seq" "To address this problem, we analyzed transcrip_x005f_x0002_tion of the Nprl3 gene after deleting its constitutive promoter. This revealed that intragenic Enhancers act as highly active, alternative tissue-specific promoters for the gene containing them. " -- -- -- -- -- -- -- >2KB 37256 E_02_038 22264824 DHS-12 Enhancer mm10 chr11 32264052 32266232 Mouse Primary Erythroid Cell Low+High throughput "RT-PCR,ChIP,ChIP-seq" "Beyond this AFE the isoform structure appears identical to the full-length transcript. Interestingly, the AFE coincides with one of the known intragenic enhancers.First, we tested wild-type mouse tissues by RT-PCR to verify the existence of this alternative transcript. " Enhancer Nprl3 -- "RT-PCR,ChIP,ChIP-seq" "Beyond this AFE the isoform structure appears identical to the full-length transcript. Interestingly, the AFE coincides with one of the known intragenic enhancers.First, we tested wild-type mouse tissues by RT-PCR to verify the existence of this alternative transcript. " "Aag,CGTHBA,HS-26,HS-40,Mare,Phg,Prox1,m(alpha)RE" -- -- -- "Intragenic Enhancers frequently act as alternative tissue_x005f_x0002_specific promoters producing a class of abundant,spliced, multiexonic poly(A)+ RNAs (meRNAs) which reflect the host gene¡¯s structure." "ChIP,RT-PCR,ChIP-seq" "To address this problem, we analyzed transcrip_x005f_x0002_tion of the Nprl3 gene after deleting its constitutive promoter. This revealed that intragenic Enhancers act as highly active, alternative tissue-specific promoters for the gene containing them. " -- -- -- -- -- -- -- >2KB 33180 E_02_039 22323542 -- mm10 chr6 70763764 70765090 Mouse "S194,MPC-11" Low throughput "DNase I Hypersensitivity Assay,Luciferase Reporter Assay,Transfection" "We identified a DNA sequence that exhibited B cell-specific, longrange interactions with Ei and E39 using chromosome conformation-capture technology.Mapping and sequence analysis of DNase I-hypersensitive site HS10.To determine whether HS10 corresponded to a new transcriptional enhancer in the locus, we performed transient-transfection assays with a luciferase reporter gene containing a Vk gene promoter (Fig. 2A, construct 1)." Enhancer Igk -- RT-PCR "Finally,to confirm the above results we performed real-timePCR assays to quantitate Igk gene transcript levels in plasma cell populations before and 5 d after immunization." kappa -- -- -- -- -- -- -- -- -- -- -- -- -- >2KB 3208791 E_02_040 22323542 Ei Enhancer mm10 chr6 70725576 70726090 Mouse "S194,MPC-11" Low throughput "DNaseI-seq,Luciferase Reporter Assay,PCR" "We identified a DNA sequence that exhibited B cell-specific, longrange interactions with Ei and E39 using chromosome conformation-capture technology.Mapping and sequence analysis of DNase I-hypersensitive site HS10.To determine whether HS10 corresponded to a new transcriptional enhancer in the locus, we performed transient-transfection assays with a luciferase reporter gene containing a Vk gene promoter (Fig. 2A, construct 1)." Enhancer Igk -- RT-PCR "Finally,to confirm the above results we performed real-timePCR assays to quantitate Igk gene transcript levels in plasma cell populations before and 5 d after immunization." kappa -- -- -- -- -- -- -- -- -- -- -- -- -- >2KB 3170197 E_02_041 22323542 Ed Enhancer mm10 chr6 70743710 70744950 Mouse "S194,MPC-11" Low throughput "DNaseI-seq,Luciferase Reporter Assay,PCR" "We identified a DNA sequence that exhibited B cell-specific, longrange interactions with Ei and E39 using chromosome conformation-capture technology.Mapping and sequence analysis of DNase I-hypersensitive site HS10.To determine whether HS10 corresponded to a new transcriptional enhancer in the locus, we performed transient-transfection assays with a luciferase reporter gene containing a Vk gene promoter (Fig. 2A, construct 1)." Enhancer Igk -- RT-PCR "Finally,to confirm the above results we performed real-timePCR assays to quantitate Igk gene transcript levels in plasma cell populations before and 5 d after immunization." kappa -- -- -- -- -- -- -- -- -- -- -- -- -- >2KB 3188694 E_02_042 22323542 E3' Enhancer mm10 chr6 70735256 70736064 Mouse "S194,MPC-11" Low throughput "DNaseI-seq,Luciferase Reporter Assay,PCR" "We identified a DNA sequence that exhibited B cell-specific, longrange interactions with Ei and E39 using chromosome conformation-capture technology.Mapping and sequence analysis of DNase I-hypersensitive site HS10.To determine whether HS10 corresponded to a new transcriptional enhancer in the locus, we performed transient-transfection assays with a luciferase reporter gene containing a Vk gene promoter (Fig. 2A, construct 1)." Enhancer Igk -- RT-PCR "Finally,to confirm the above results we performed real-timePCR assays to quantitate Igk gene transcript levels in plasma cell populations before and 5 d after immunization." kappa -- -- -- -- -- -- -- -- -- -- -- -- -- >2KB 3180024 E_02_043 22378757 -- mm10 chr10 62128342 62130342 Mouse Germ Cell Low throughput "ChIP,PCR,Luciferase Reporter Assay" "To investigate this further, we used a ChIP method to determine whether STAT3 binds Neurog3 regulatory elements in THY1+ spermatogonia. PCR analysis with primers designed to span a 200-bp segment of the Neurog3 distal promoter/enhancer region including the STAT3 binding site produced an amplicon of expected size;" Enhancer Neurog3 -- "Luciferase Reporter Assay,Transfection" "Next, we determined whether STAT3 directly regulates Neurog3 promoter/enhancer activity. To investigate this, we used a reporter construct in which the Neurog3 promoter/enhancer sequence was cloned upstream of the firefly luciferase gene, termed pNeurog3-Luc.The transfection efficiency of cultured SSC/progenitor spermatogonia with plasmid DNA vectors is low, thus HeLa cells were used with the reporter vector to determine whether STAT3 is capable of stimulating Neurog3 promoter/enhancer activity." "Atoh5,Math4B,bHLHa7,ngn3" -- -- -- -- -- -- Stat3 "1110034C02Rik,AW109958,Aprf" ChIP "Moreover,using a ChIP approach we found that STAT3 physically interacts with the distal Neurog3 promoter/Enhancer in THY1 undifferentiated spermatogonia and using a reporter construct show that STAT3 regulates transcription through this promoter." -- -- -- >2KB 3747 E_02_044 22396734 -- mm10 chr4 115100426 115102426 Mouse 416B Low throughput "Luciferase Reporter Assay,ChIP,EMSA" "To investigate whether the +45 region functions as a transcriptional enhancer, luciferase reporter constructs were generated using a 1.76 kb genomic fragment encompassing the complete +45 element and their activity was assessed in 416B cells hematopoietic progenitor cells (Figure 1B)." Enhancer Tal1 -- "DNaseI-seq,EMSA,ChIP" "Here we report identification of a DNaseI hypersensitive site at the 39 end of the Scl/Map17 domain and 45 kb downstream of the Scl transcription start site.To investigate whether CTCF binding is responsible for the footprinting results observed in Figure 3A, we performed electrophoretic mobility shift assay (EMSA) and ChIP-on-chip experiments." "Hpt,SCL/tal-1,Scl,bHLHa17,tal-1" -- -- -- -- -- -- Ctcf AW108038 ChIP-seq Analysis of publicly available CTCF ChIP-seq data from the ENCODE consortium clearly demonstrates CTCF binding to the +45 element across a wide range of mouse tissues consistent with the presence of a ubiquitous CTCF site. -- -- -- >2KB 45001 E_02_045 22401818 Scl +19 Enhancer mm10 chr4 115074426 115076426 Mouse "Hematopoietic Stem Cell,Progenitor Cell" Low throughput "RT-PCR,qPCR,ChIP" "Real-time semi-quantitative PCR performed with primers specific for the Scl gene [16] showed that expression levels of Scl in erythroid (Ter119 ? ), myeloid (Mac-1 ? ), megakaryocytic (CD41 ? ), and T-cell (CD4 ? ) lineages were similar for WTand Scl D19/D19 mice (Fig. 1D). To determine whether deletion of the Scl +19 enhancer affects embryonic/fetal hematopoiesis, we quantified the number of progenitors in the fetal liver using methylcellulose-based colony assay." Enhancer Tal1 -- ChIP "To identify possible redundancy between the ?19 enhancer and other Scl regulatory elements, we performed chromatin immunoprecipitation (ChIP) assays on E14.5 fetal liver cells with markers for repressive (H3K9me3 and H3K9me2) and active (H3K4me3 and H3K9Ac) chromatin." "Hpt,SCL/tal-1,Scl,bHLHa17,tal-1" Acute Leukemia DOID:12603 -- "The Scl +19 Enhancer plays a key role in the development of hematopoietic stem/progen_x005f_x0002_itor cells,but is not necessary for mature hematopoietic lineages." ChIP "Interestingly,both active euchromatin marks are reduced in SclD19/D19 fetal liver cells,indicating that the Scl t19 element is required for enhanced activity of the Scl promoter." "Spi1,Erg" "Dis-1,Dis1,PU.1,Sfpi-1,Sfpi1,Spi-1,Tcfpu1,Tfpu.1,D030036I24Rik" ChIP-seq "Using whole-genome transcription factor binding mapping by ChIP-Seq,we have recently identified other important hematopoietic regulators that also bind to the Scl t19 Enhancer,including the Ets factors Spi1 and Erg." -- -- -- >2KB 19001 E_02_046 22701669 CONS3.8 Enhancer mm10 chr14 47056226 47056692 Mouse LS-8 Cells Low throughput "EMSA,Phylogenetic Footprinting" In vitro electrophoretic mobility shift assays (EMSA) and in vivo transgenic reporter mutational analyses revealed that this site supports Pitx binding and that the site is necessary to recapitulate aspects of endogenous Bmp4 expression in developing craniofacial and limb tissues. Enhancer Bmp4 -- "ChIP,PCR" "Pitx2 chromatin immunoprecipitation (ChIP) demonstrate direct binding of Pitx2 to this Bmp4 enhancer site in a dental epithelial cell line.Using primers that target the Pitx1/2 binding motif in the 396 bp Bmp4 enhancer sequence (CONS3.8), DNA purified from crosslinked LS8 chromatin after immunoprecipitation with an anti-Pitx2 antibody yielded a 4.7-fold increase in amplicon abundance, relative to an IgG control, by PCR and qPCR (Figure 6A, B)." "Bmp-4,Bmp2b,Bmp2b-1,Bmp2b1" -- -- -- Pitx2 ChIP demonstrated direct binding of Pitx2 to this Bmp4 enhancer site in a dental epithelial cell line. "ChIP,PCR,qPCR" "Using primers that target the Pitx1/2 binding motif in the 396 bp Bmp4 Enhancer sequence (CONS3.8), DNA purified from crosslinked LS8 chromatin after immunoprecipitation with an anti-Pitx2 antibody yielded a 4.7-fold increase in amplicon abundance, relative to an IgG control, by PCR and qPCR." Pitx2 "9430085M16Rik,Brx1,Brx1b,Munc30,Otlx2,Ptx2,Rieg" "EMSA,ChIP" "Electrophoretic Mobility Shift Assay (EMSA) exhibits robust binding of Pitx1 protein to both a positive control bicoid DNA sequence and to the consensus Pitx1/2 binding site in with a 25 probe sequence in the CONS3.8 sequenceTo determine whether Pitx2 physically binds the CONS3.8 Bmp4 Enhancer in living cells,we performed Chromatin Immunoprecipitation (ChIP) assays in LS8 mouse dental epithelial cells." -- -- -- >2KB 672935 E_02_047 22728936 Scn5a Enhancer mm10 chr9 119469800 119469811 Mouse HL-1 Low+High throughput "Luciferase Reporter Assay,ChIP-seq" "We tested the ability of this putative enhancer to activate transcription in a TBX5-dependent manner. TBX5 dramatically upregulated enhancer-dependent luciferase reporter expression from this genomic region (chr9:119,378,051¨C119,379,479;NCBI build 37/mm9) in vitro (Figure 6B). " Enhancer Scn5a -- Luciferase Reporter Assay "We tested the ability of this putative enhancer to activate transcription in a TBX5-dependent manner. TBX5 dramatically upregulated enhancer-dependent luciferase reporter expression from this genomic region (chr9:119,378,051¨C119,379,479;NCBI build 37/mm9) in vitro (Figure 6B). " "Nav1.5,Nav1.5c,SkM1,SkM2,mH1" Cardiac Conduction System Disease DOID:10273 D000075224 "TBX5-responsive Enhancer downstream of Scn5a sufficient to drive VCS expression in vivo, dependent on canonical T-box binding sites. " ChIP-seq "Here, we found a direct molecular link between TBX5 and Scn5a via a TBX5-respon_x005f_x0002_sive downstream Enhancer that was sufficient to direct VCS-specif-ic gene expression. Our results identified a TBX5-Scn5a molecular network essential for function of the mature VCS. " Tbx5 Tbx5 ChIP-seq We bioinformatically interrogated the Scn5a locus to identify potential TBX5-responsive Enhancers using the overlap of 4 independent data sets: (a) evolutionary conservation (21);(b) ChIP-seq studies identifying TBX5 binding sites in the atrial cardiomyocyte HL-1 cell line. -- -- -- >2KB 13597 E_02_048 22728936 Scn5a Enhancer mm10 chr9 119469879 119469890 Mouse HL-1 Low+High throughput "Luciferase Reporter Assay,ChIP-seq" "We tested the ability of this putative enhancer to activate transcription in a TBX5-dependent manner. TBX5 dramatically upregulated enhancer-dependent luciferase reporter expression from this genomic region (chr9:119,378,051¨C119,379,479;NCBI build 37/mm9) in vitro (Figure 6B). " Enhancer Scn5a -- Luciferase Reporter Assay "We tested the ability of this putative enhancer to activate transcription in a TBX5-dependent manner. TBX5 dramatically upregulated enhancer-dependent luciferase reporter expression from this genomic region (chr9:119,378,051¨C119,379,479;NCBI build 37/mm9) in vitro (Figure 6B). " "Nav1.5,Nav1.5c,SkM1,SkM2,mH1" Cardiac Conduction System Disease DOID:10273 D000075224 "TBX5-responsive Enhancer downstream of Scn5a sufficient to drive VCS expression in vivo, dependent on canonical T-box binding sites. " ChIP-seq "Here, we found a direct molecular link between TBX5 and Scn5a via a TBX5-respon_x005f_x0002_sive downstream Enhancer that was sufficient to direct VCS-specif-ic gene expression. Our results identified a TBX5-Scn5a molecular network essential for function of the mature VCS. " Tbx5 Tbx5 ChIP-seq We bioinformatically interrogated the Scn5a locus to identify potential TBX5-responsive Enhancers using the overlap of 4 independent data sets: (a) evolutionary conservation (21);(b) ChIP-seq studies identifying TBX5 binding sites in the atrial cardiomyocyte HL-1 cell line. -- -- -- >2KB 13518 E_02_049 22728936 Scn5a Enhancer mm10 chr9 119469902 119469913 Mouse HL-1 Low+High throughput "Luciferase Reporter Assay,ChIP-seq" "We tested the ability of this putative enhancer to activate transcription in a TBX5-dependent manner. TBX5 dramatically upregulated enhancer-dependent luciferase reporter expression from this genomic region (chr9:119,378,051¨C119,379,479;NCBI build 37/mm9) in vitro (Figure 6B). " Enhancer Scn5a -- Luciferase Reporter Assay "We tested the ability of this putative enhancer to activate transcription in a TBX5-dependent manner. TBX5 dramatically upregulated enhancer-dependent luciferase reporter expression from this genomic region (chr9:119,378,051¨C119,379,479;NCBI build 37/mm9) in vitro (Figure 6B). " "Nav1.5,Nav1.5c,SkM1,SkM2,mH1" Cardiac Conduction System Disease DOID:10273 D000075224 "TBX5-responsive Enhancer downstream of Scn5a sufficient to drive VCS expression in vivo, dependent on canonical T-box binding sites. " ChIP-seq "Here, we found a direct molecular link between TBX5 and Scn5a via a TBX5-respon_x005f_x0002_sive downstream Enhancer that was sufficient to direct VCS-specif-ic gene expression. Our results identified a TBX5-Scn5a molecular network essential for function of the mature VCS. " Tbx5 Tbx5 ChIP-seq We bioinformatically interrogated the Scn5a locus to identify potential TBX5-responsive Enhancers using the overlap of 4 independent data sets: (a) evolutionary conservation (21);(b) ChIP-seq studies identifying TBX5 binding sites in the atrial cardiomyocyte HL-1 cell line. -- -- -- >2KB 13495 E_02_050 22761896 -- mm10 chr14 67741735 67744661 Mouse GT1-7 Low throughput "Luciferase Reporter Assay,ChIP,PCR" "the luciferase activities in GT1¨C7 cells were noticeably decreased without HDACIs treatments, suggesting that potential enhancer elements located in the regions (-3446,-2087 bp and -1134,-520 bp). " Enhancer Gnrh1 -- "Western blot,Immunofluorescence,ChIP,EMSA" "The Otx2 protein levels were also obviously reduced in GT1¨C7 cells showed by western blot analysis (Figure 5B) and immunofluorescence staining (Figure 5C).The results of ChIP assay showed that the binding ability of Otx2 to neuron-specific elements (2356,2249 bp) in Gnrh1 promoter obviously decreased after HDACIs treatments (Figure 6B).To further analyze the affinities of Otx2 to the two conserved binding sites in mouse Gnrh1 promoter respectively, EMSA was performed with probes containing the 2268,2239 bp and 2330,2301 bp sequences [20](Figure 6C, above)." "Gnrh,Gnrh2,LHRH,Lhrh1,Lnrh,hpg" -- -- -- -- -- -- Otx2 E130306E05Rik "ChIP,EMSA" Chromatin immunoprecipitation and electrophoretic mobility shift assays demonstrated that the DNA-binding activity of Otx2 to Gnrh1 gene was suppressed by TSA and VPA. -- -- -- <2KB 1982 E_02_051 22877652 GnRH-E1 Enhancer mm10 chr14 67743318 67743610 Mouse GT1-7 Low throughput "ChIP,PCR,Luciferase Reporter Assay,EMSA" "To determine whether AR interacts with endogenous chromatin in the GnRH-E1 region in GT1-7 cells, ChIP assays were performed. GT1-7 cells were transiently transfected with luciferase reporter constructs containing the GnRH-P (P), GnRH-E1 (E1), and/or GnRH-E2 (E2) regions, and/or the RSV promoter (RSVp) or enhancer (RSVe), as indicated, along with the AR expression vector. " Enhancer Gnrh1 -- Luciferase Reporter Assay "AR associates with the enhancer 1 region of the GnRH regulatory region and represses transcription.GT1-7 cells were transiently transfected with luciferase reporter constructs containing the GnRH-P (P), GnRH-E1 (E1),and/or GnRH-E2 (E2) regions, and/or the RSV promoter (RSVp) or enhancer (RSVe), as indicated, along with the AR expression vector. " "Gnrh,Gnrh2,LHRH,Lhrh1,Lnrh,hpg" Polycystic Ovarian Syndrome DOID:11612 D011085 AR represses GnRH transcription via the major Enhancer (GnRH-E1). ChIP "For the rest of this study, we chose to evaluate the mechanism of repression by AR using GnRH-E1 upstream of RSVp to avoid com_x005f_x0002_pensation by the androgen-responsive GnRH promoter.To determine whether AR interacts with endogenous chromatin in the GnRH-E1 region in GT1-7 cells,ChIP assays were performed." "Oct1,Pbx,Nkx2-1" "2810482H01Rik,NF-A1,Oct-1,Oct1,Otf-1,Otf1,Gata-4,D17Wsu76e,PREP1,AV026640,Nkx2.1,T/EBP,Titf1,Ttf-1" "EMSA,PCR" "AR bound to the -1796/-1791 sequence in electrophoretic mobility shift assays, indicating a direct interaction with DNA or other transcription factors in this region." -- -- -- <2KB 1716 E_02_052 22877652 GnRH-E2 Enhancer mm10 chr14 67742046 67742550 Mouse GT1-7 Low throughput "ChIP,PCR,Luciferase Reporter Assay,EMSA" "To determine whether AR interacts with endogenous chromatin in the GnRH-E1 region in GT1-7 cells, ChIP assays were performed. GT1-7 cells were transiently transfected with luciferase reporter constructs containing the GnRH-P (P), GnRH-E1 (E1), and/or GnRH-E2 (E2) regions, and/or the RSV promoter (RSVp) or enhancer (RSVe), as indicated, along with the AR expression vector. " Enhancer Gnrh1 -- Luciferase Reporter Assay "AR associates with the enhancer 1 region of the GnRH regulatory region and represses transcription.GT1-7 cells were transiently transfected with luciferase reporter constructs containing the GnRH-P (P), GnRH-E1 (E1),and/or GnRH-E2 (E2) regions, and/or the RSV promoter (RSVp) or enhancer (RSVe), as indicated, along with the AR expression vector. " "Gnrh,Gnrh2,LHRH,Lhrh1,Lnrh,hpg" Polycystic Ovarian Syndrome DOID:11612 D011085 AR represses GnRH transcription via the major Enhancer (GnRH-E1). ChIP "For the rest of this study, we chose to evaluate the mechanism of repression by AR using GnRH-E1 upstream of RSVp to avoid com_x005f_x0002_pensation by the androgen-responsive GnRH promoter.To determine whether AR interacts with endogenous chromatin in the GnRH-E1 region in GT1-7 cells,ChIP assays were performed." "Oct1,Pbx,Nkx2-1" "2810482H01Rik,NF-A1,Oct-1,Oct1,Otf-1,Otf1,Gata-4,D17Wsu76e,PREP1,AV026640,Nkx2.1,T/EBP,Titf1,Ttf-1" "EMSA,PCR" "AR bound to the -1796/-1791 sequence in electrophoretic mobility shift assays, indicating a direct interaction with DNA or other transcription factors in this region." -- -- -- >2KB 2882 E_02_053 22877652 GnRH-E3 Enhancer mm10 chr14 67740982 67741286 Mouse GT1-7 Low throughput "ChIP,PCR,Luciferase Reporter Assay,EMSA" "To determine whether AR interacts with endogenous chromatin in the GnRH-E1 region in GT1-7 cells, ChIP assays were performed. GT1-7 cells were transiently transfected with luciferase reporter constructs containing the GnRH-P (P), GnRH-E1 (E1), and/or GnRH-E2 (E2) regions, and/or the RSV promoter (RSVp) or enhancer (RSVe), as indicated, along with the AR expression vector. " Enhancer Gnrh1 -- Luciferase Reporter Assay "AR associates with the enhancer 1 region of the GnRH regulatory region and represses transcription.GT1-7 cells were transiently transfected with luciferase reporter constructs containing the GnRH-P (P), GnRH-E1 (E1),and/or GnRH-E2 (E2) regions, and/or the RSV promoter (RSVp) or enhancer (RSVe), as indicated, along with the AR expression vector. " "Gnrh,Gnrh2,LHRH,Lhrh1,Lnrh,hpg" Polycystic Ovarian Syndrome DOID:11612 D011085 AR represses GnRH transcription via the major Enhancer (GnRH-E1). ChIP "For the rest of this study, we chose to evaluate the mechanism of repression by AR using GnRH-E1 upstream of RSVp to avoid com_x005f_x0002_pensation by the androgen-responsive GnRH promoter.To determine whether AR interacts with endogenous chromatin in the GnRH-E1 region in GT1-7 cells,ChIP assays were performed." "Oct1,Pbx,Nkx2-1" "2810482H01Rik,NF-A1,Oct-1,Oct1,Otf-1,Otf1,Gata-4,D17Wsu76e,PREP1,AV026640,Nkx2.1,T/EBP,Titf1,Ttf-1" "EMSA,PCR" "AR bound to the -1796/-1791 sequence in electrophoretic mobility shift assays, indicating a direct interaction with DNA or other transcription factors in this region." -- -- -- >2KB 4046 E_02_054 23024062 -- mm10 chr1 60908875 60908895 Mouse T Cell Low throughput Luciferase Reporter Assay "T cells were stimulated with CPT after transfection of CTLA4 luciferase reporter constructs (including -1221, -343, -238, -167, and-63 bp relative to the CTLA4 transcription start site). suggesting the presence of an enhancer binding site in this region. " Enhancer Ctla4 -- ChIP "The promoter region from 2150 to 2130 bp contains a putative CREB binding site, a well known downstream transcription factor of cAMP signaling. To define whether the putative CREB binding site (2150 to 2143 bp) is responsive to cAMP, we performed ChIP assay with CREB antibody in T cells, with or without CPT treatment." "Cd152,Ctla-4,Ly-56" -- -- -- -- -- -- Creb1 "2310001E10Rik,3526402H21Rik,AV083133,Creb,Creb-1" ChIP "The promoter region from -150 to -130 bp contains a putative CREB binding site, a well known downstream transcription factor of cAMP signaling." -- -- -- <2KB 139 E_02_055 23154183 Il12b Enhancer mm10 chr11 44390063 44390123 Mouse HEK-293 Low throughput "ChIP,Luciferase Reporter Assay" "We then examined by ChIP assay the binding of IRFs and other transcription factors to the enhancer region in mouse peritoneal macrophages following stimulation with either LPS for TLR4 activation or poly(I:C) for RLR activation. Interestingly,TLR4 stimulation resulted in the recruitment of IRF5, C/EBPb and Oct-1/-2 transcription factors to the enhancer." Enhancer Il12b -- Transfection "The above observations suggest that, similar to the Il12b promoter, TLR-activated IRF5 also activates the enhancer through binding to the ISREs, whereas the binding of the RLR-activated IRF3 suppresses the IRF5-mediated activation. To test this, we carried out a transient reporter assay." "Il-12b,Il-12p40,Il12p40,p40" -- -- -- -- -- -- Irf4 "LSIRF,MUM1,NF-EM5,SHEP8" ChIP "We then examined by ChIP assay the bindingof IRFs and other transcription factors to the Enhancer region in mouse peritoneal macrophages following stimulation with either LPS for TLR4 activation or poly(I:C) for RLR activation. Interestingly,TLR4 stimulation resulted in the recruitment of IRF5, C/EBPb and Oct-1/- transcription factors to the Enhancer." -- -- -- >2KB 9969 E_02_056 23307821 -- mm10 chr17 26829229 26831311 Mouse Cardiac Progenitor Cell Low throughput "ChIP,PCR,Luciferase Reporter Assay,EMSA" "To examine in detail the potential binding of YY1 to Nkx2.5 enhancer, we performed an electromobility shift assay (EMSA) using the 2.1 kb cardiac enhancer located within -9435 and -7353 bases upstream from the murine NKX2.5 transcriptional start site.Binding of site #1 to YY1 was further confirmed using a DNA supershift assay (Figure 2C). To corroborate these in vitro data with their binding to the Nkx2.5 cardiac enhancer in vivo , we performed chromatin immunoprecipitation (ChIP) and PCR analysis using anti-YY1 antibodies on day 6 differentiated NK ESCs (Figure 2D), FACS-purified eGFP+ CPCs and eGFP- cells from day 6 differentiated NK ESCs (Figure 2E), and from embryonic day 9.5 hearts and body. " Enhancer Nkx2-5 -- "EMSA,ChIP,PCR" "To examine in detail the potential binding of YY1 to Nkx2.5 enhancer, we performed an electromobility shift assay (EMSA) using the 2.1 kb cardiac enhancer located within -9435 and -7353 bases upstream from the murine NKX2.5 transcriptional start site.Binding of site #1 to YY1 was further confirmed using a DNA supershift assay (Figure 2C). To corroborate these in vitro data with their binding to the Nkx2.5 cardiac enhancer in vivo , we performed chromatin immunoprecipitation (ChIP) and PCR analysis using anti-YY1 antibodies on day 6 differentiated NK ESCs (Figure 2D), FACS-purified eGFP+ CPCs and eGFP- cells from day 6 differentiated NK ESCs (Figure 2E), and from embryonic day 9.5 hearts and body. " "Csx,Nkx-2.5,Nkx2.5,tinman" -- -- -- YY1 regulates Nkx2.5 expression via a 2.1-kb cardiac-specific enhancer "EMSA,ChIP,PCR" "To examine in detail the potential binding of YY1 to Nkx2.5 Enhancer, we performed an electromobility shift assay (EMSA) using the 2.1 kb cardiac Enhancer located within -9435 and -7353 bases upstream from the murine NKX2.5 transcriptional start site." Yy1 "NF-E1,YY-1" "ChIP,Luciferase Reporter Assay,EMSA" "YY1 regulates Nkx2.5 expression via a 2.1 kb cardiac-specific enhancer as demonstrated by in vitro luciferase-based assays and in vivo chromatin immunoprecipitation (ChIP) and genome-wide sequencing analysis.To examine in detail the potential binding of YY1 to Nkx2.5 enhancer, we performed an electromobility shift assay (EMSA) using the 2.1 kb cardiac enhancer located within -9435 and -7353 bases upstream from the murine NKX2.5 transcriptional start site." -- -- -- >2KB 8394 E_02_057 23321477 -- mm10 chr6 122726952 122727236 Mouse N2a Low throughput "Transfection,Luciferase Reporter Assay,PCR,EMSA,ChIP" Transient transfection assays in N2A neuroblasts using murine glut3-luciferase reporter constructs mapped the hypoxia-induced enhancer activities to -857- to -573-bp and -203- to -177-bp regions. Enhancer Slc2a3 -- ChIP "Hypoxia-exposed N2A nuclear extracts demonstrated an increase in HIF-1a and p-Creb binding to HRE (-828 to -824 bp) and AP-1 (-187 to -180 bp) cis-elements, respectively, in electromobility shift and supershift assays, which was confirmed by chromatin immunoprecipitation assays." "AA408729,AL023014,AL024341,AU040424,C78366,Glut-3,Glut3" -- -- -- -- -- -- "Hif1a,Creb1" "AA959795,HIF-1-alpha,HIF1-alpha,HIF1alpha,MOP1,bHLHe78,2310001E10Rik,3526402H21Rik,AV083133,Creb,Creb-1" "ChIP,EMSA" "Hypoxia-exposed N2A nuclear extracts demonstrated an increase in HIF-1¦Á and p-Creb binding to HRE (-828 to -824 bp) and AP-1 (-187 to -180 bp) cis-elements, respectively, in electromobility shift and supershift assays, which was confirmed by chromatin immunoprecipitation assays." -- -- -- <2KB 714 E_02_058 23321477 -- mm10 chr6 122727606 122727632 Mouse N2a Low throughput "Transfection,Luciferase Reporter Assay,PCR,EMSA,ChIP" Transient transfection assays in N2A neuroblasts using murine glut3-luciferase reporter constructs mapped the hypoxia-induced enhancer activities to -857- to -573-bp and -203- to -177-bp regions. Enhancer Slc2a3 -- ChIP "Hypoxia-exposed N2A nuclear extracts demonstrated an increase in HIF-1a and p-Creb binding to HRE (-828 to -824 bp) and AP-1 (-187 to -180 bp) cis-elements, respectively, in electromobility shift and supershift assays, which was confirmed by chromatin immunoprecipitation assays." "AA408729,AL023014,AL024341,AU040424,C78366,Glut-3,Glut3" -- -- -- -- -- -- "Hif1a,Creb1" "AA959795,HIF-1-alpha,HIF1-alpha,HIF1alpha,MOP1,bHLHe78,2310001E10Rik,3526402H21Rik,AV083133,Creb,Creb-1" "ChIP,EMSA" "Hypoxia-exposed N2A nuclear extracts demonstrated an increase in HIF-1¦Á and p-Creb binding to HRE (-828 to -824 bp) and AP-1 (-187 to -180 bp) cis-elements, respectively, in electromobility shift and supershift assays, which was confirmed by chromatin immunoprecipitation assays." -- -- -- <2KB 189 E_02_059 23642367 Tal1 +40k Enhancer mm10 chr4 115095426 115097426 Mouse Embryonic Stem Cell Low throughput "ChIP,EMSA" "Several Tal1 enhancers have been identified, but only the +40k enhancer region contains E-box motifs (Ogilvy et al., 2007), potential binding sites for Mesp1 (Figure 3B, left panel)." Enhancer Tal1 -- "ChIP,EMSA" "We performed ChIP in day 3 EBs after a prior 24 hr Mesp1 pulse and found that Mesp1 was enriched at this cis -regulatory element (Figure 3B, right panel) with a concurrent upregulation of Tal1 expression (Supplementary Figure S3B).We next performed electrophoretic mobility shift assays to confirm the direct interaction between Mesp1 and the Tal1 +40k Enhancer." "Hpt,SCL/tal-1,Scl,bHLHa17,tal-1" -- -- -- -- -- -- Nkx2-5 "Csx,Nkx-2.5,Nkx2.5,tinman" RT-PCR "At the protein level,the late pulse of Mesp1 elevated the expression of Nkx2.5,a cardiogenic transcription factor,and downstream markers,cTnT,cTnI and sarcomeric ¦Á-actinin." -- -- -- >2KB 40001 E_02_060 23993744 CE Enhancer mm10 chr7 46352720 46353957 Mouse C2C12 Low+High throughput "siRNA,ChIP-qPCR,qRT-PCR,ChIP-seq,DNaseI-seq" "To this end, we devised a screening approach where ten different small interfering RNA duplexes (siRNA) were designed to target various regions upstream of MYOD1 with proximity to MyoD+/MyoG+/H3K4me1+ peaks (Figure 3A, #1¨C10). As controls, siRNAs were designed to target four eRNA+ regions on other chromosomes (Figure S3) and another against green fluorescent protein (GFPi). Transfected C2C12 cells were placed in differentiation media (DM) for 4¨C6hrs and harvested thereafter for analysis. " Enhancer Myod -- "ChIP-seq,RNA-seq" "Within this region, the concerted activity of two DNA Enhancer elements, CE and DRR, specifies the level of MyoD expression in the myogenic lineage.In this context, we surveyed for the enhancer signature (i.e. H3K4me1) within ~1¨C2kb of MyoG+ sites. We obtained and analyzed ChIP-Seq data for H3K4me1 (Table S1) and observed the majority (~80%) of the extragenic MyoG+ peaks within H3K4me1+ domains (Figure 1B¨CD).To ascertain whether these regions were transcriptionally active, we performed paired-end RNA-Seq (PE-Seq)from ribosome-depleted RNA fraction from MT (76bp/end, see Table S1 for number of reads). " "AI503393,MYF3,MyoD,?Myod-1,bHLHc1" -- -- -- The eRNAs regulate genomic access of the transcriptional complex to defined regulatory regions. ChIP "The above findings prompted us to evaluate whether RNA-assisted PolII recruitment could also explain the effect of DRRRNA on MyoG expression (Figure 4A,B). Indeed, ChIP experiments revealed that DRRi resulted in the reduction of PolII at MYOG, but not at MYOD1. " "Mef2a,Foxo1,Myod1,Myog" "A430079H05Rik,AI876417,Afxh,FKHR,Fkhr1a,Foxo1,MYF4,bHLHc3,myo,AI503393,MYF3,MyoD,Myod-1,bHLHc1" "qRT-PCR,ChIP-seq" "Similar to other nuclear eRNAs examined (FigureS1G¨CI), DRRRNA levels were significantly reduced following MyoDi and to a lesser extent by MyoGi (Figure 2D), suggesting that its transcriptional regulation is under the concerted control of these transcription factors." -- -- -- >2KB 23134 E_02_061 23993744 DRR Enhancer mm10 chr7 46370375 46374302 Mouse C2C12 Low+High throughput "siRNA,ChIP-qPCR,qRT-PCR,ChIP-seq,DNaseI-seq" "To this end, we devised a screening approach where ten different small interfering RNA duplexes (siRNA) were designed to target various regions upstream of MYOD1 with proximity to MyoD+/MyoG+/H3K4me1+ peaks (Figure 3A, #1¨C10). As controls, siRNAs were designed to target four eRNA+ regions on other chromosomes (Figure S3) and another against green fluorescent protein (GFPi). Transfected C2C12 cells were placed in differentiation media (DM) for 4¨C6hrs and harvested thereafter for analysis. " Enhancer Myod -- "ChIP-seq,RNA-seq" "Within this region, the concerted activity of two DNA Enhancer elements, CE and DRR, specifies the level of MyoD expression in the myogenic lineage.In this context, we surveyed for the enhancer signature (i.e. H3K4me1) within ~1¨C2kb of MyoG+ sites. We obtained and analyzed ChIP-Seq data for H3K4me1 (Table S1) and observed the majority (~80%) of the extragenic MyoG+ peaks within H3K4me1+ domains (Figure 1B¨CD).To ascertain whether these regions were transcriptionally active, we performed paired-end RNA-Seq (PE-Seq)from ribosome-depleted RNA fraction from MT (76bp/end, see Table S1 for number of reads). " "AI503393,MYF3,MyoD,?Myod-1,bHLHc1" -- -- -- The eRNAs regulate genomic access of the transcriptional complex to defined regulatory regions. ChIP "The above findings prompted us to evaluate whether RNA-assisted PolII recruitment could also explain the effect of DRRRNA on MyoG expression (Figure 4A,B). Indeed, ChIP experiments revealed that DRRi resulted in the reduction of PolII at MYOG, but not at MYOD1. " "Mef2a,Foxo1,Myod1,Myog" "A430079H05Rik,AI876417,Afxh,FKHR,Fkhr1a,Foxo1,MYF4,bHLHc3,myo,AI503393,MYF3,MyoD,Myod-1,bHLHc1" "qRT-PCR,ChIP-seq" "Similar to other nuclear eRNAs examined (FigureS1G¨CI), DRRRNA levels were significantly reduced following MyoDi and to a lesser extent by MyoGi (Figure 2D), suggesting that its transcriptional regulation is under the concerted control of these transcription factors." -- -- -- >2KB 4134 E_02_062 24000349 -- mm10 chr17 40206839 40206900 Mouse "Somatic Spleen Cells,Spermatogenic Germ Cells" Low throughput "ChIP,qRT-PCR" "This reflects the fact that both the human and mouse Pgk2 gene promoters contain highly conserved regulatory elements including a GC-box, a CAAT-box and an enhancer region just upstream from the CAAT-box." Enhancer Pgk2 -- "ChIP,qRT-PCR" "Our results indicate there is an ordered series of molecular events that lead to activation of transcription of the Pgk2 gene in spermatogenic cells, and that these events are signaled by distinct elements in the Pgk2 promoter/Enhancer region." "Pgk-2,Tcp-2" -- -- -- Activation of Pgk2 transcription is regulated by testis-specific demethylation of DNA and binding of testis-specific transcription factors to Enhancer and core promoter elements. "ChIP,qRT-PCR" "Our results indicate there is an ordered series of molecular events that lead to activation of transcription of the Pgk2 gene in spermatogenic cells, and that these events are signaled by distinct elements in the Pgk2 promoter/Enhancer region." -- -- -- -- -- -- -- <2KB 147 E_02_063 24014243 -- mm10 chr5 107680655 107682655 Mouse "Hematopoietic Cell,B Cell,T Cell" Low throughput "ChIP,PCR,Luciferase Reporter Assay" "To investigate whether Mysm1 regulates Gfi1 expression by binding to the regulatory elements of Gfi1 in HSC, we performed chromatin immunoprecipitation (ChIP) assays along the Gfi1 regulatory elements. A panel of PCR primers to encompass the 23.4-kb promoter and 235-kb enhancer of the Gfi1 locus was designed (Figure 5A)." Enhancer Gfi1 -- "ChIP,PCR" "To investigate whether Mysm1 regulates Gfi1 expression by binding to the regulatory elements of Gfi1 in HSC, we performed chromatin immunoprecipitation (ChIP) assays along the Gfi1 regulatory elements. A panel of PCR primers to encompass the 23.4-kb promoter and 235-kb enhancer of the Gfi1 locus was designed (Figure 5A)." "AW495828,Gfi-1,Pal-1,Pal1" -- -- -- -- -- -- "Gata2,Runx1" "Gata-2,AML1,CBF-alpha-2,Cbfa2,Pebp2a2,Pebpa2b" "Luciferase Reporter Assay,ChIP,PCR" Mechanistic studies revealed that Mysm1 modulates histone modifications and directs the recruitment of key transcriptional factors such as Gata2 and Runx1 to the Gfi1 locus in HSCs. We found that Mysm1 directly associates with the Gfi1 Enhancer element and promotes its transcription through Gata2 and Runx1 transactivation. -- -- -- >2KB 35000 E_02_064 24040411 ECR18 Enhancer mm10 chr7 6502892 6504892 Mouse "HeLa,HEK-293" Low+High throughput "ChIP-seq,3C,Luciferase Reporter Assay,PCR,ChIP" "In vitro assays also reveal ECR18 as a potential enhancer or repressor for the promoter of Peg3.The identified ECRs were also examined in terms of their histone modifications using the ChIP-seq data set of the ENCODE project.To further characterize the predicted functions of the ECRs, we performed the Chromatin Conformation Capture (3C) approach with the tissues derived from mouse. " Enhancer Vegfa 3C ChIP "In the current study, we performed a series of Chromatin Conformation Capture (3C) and Chromatin Immunoprecipitation (ChIP) analyses to further confirm this prediction. Overall, the current study identifies one region,ECR18, as a key regulatory region for the transcription and imprinting of the Peg3 domain." "Vegf,Vpf" -- -- -- -- -- -- -- -- -- -- -- -- -- >2KB 39513100 E_02_065 24157522 -- mm10 chr5 28476034 28479024 Mouse Colon Adenocarcinoma Cell Lines Low+High throughput "RT-PCR,ChIP-seq,Transient Transfection Assay,Luciferase Reporter Assay,3C,DNaseI-seq" "The ChIP-seq data of the ENCODE project show that H3K4me1, but not H3K4me3, marks all four DNaseI HS sites in the adult lung.To examine whether each of the DNase I HS sites is responsible for upregulating the expression of Shh, we employed a transgenic reporter assay using BAC clones (Figs. 1A and 3A)." Enhancer Shh 3C Luciferase Reporter Assay "To determine whether SLGE physically interacts with the Shh promoter in association with transcriptional activity, we conducted a 3C assay, which has been used to measure associations between two genomic regions. Assessment of the functional properties of SLGE by 3C and luciferase assays." "9530036O11Rik,Dsh,Hhg1,Hx,Hxl3,M100081NC,Shh" -- -- -- "Shh expression in the endodermal epithelial lining, which is enabled by the coordinated actions of the Enhancers, is necessary for proper endodermal development." "RT-PCR,Luciferase Reporter Assay,3C" "We identified a novel, less-conserved cis-regulatory element for Shh expres_x005f_x0002_sion in the esophageal epithelium, as well as in the lung and intestinal epithelium, in a region 100 Kb upstream of the Shh CDS. The new element compensates for the Enhancer activi_x0002_ties of the three previously identified Enhancers, and the combined activities of all four Enhancers permit the seamless Shh expression in the epithelial linings of the whole endoder_x0002_mal tissues in the mouse embryo." "Rbfox3,Socs1" "Fox-3,Hrnbp3,NeuN,Neuna60,Cish1,Cish3,JAB,SOCS-1,SSI-1" DNaseI-seq "Putative binding sites for Fox transcription factors and STAT are found in the SLGE.In human cancer cell lines, including Caco-2, putative Fox- and STAT-binding sites are found in some of the DNase I HS sites around the SHH CDS." -- -- -- >2KB 20690 E_02_066 24157522 -- mm10 chr5 28495262 28497421 Mouse Colon Adenocarcinoma Cell Lines Low+High throughput "RT-PCR,ChIP-seq,Transient Transfection Assay,Luciferase Reporter Assay,3C,DNaseI-seq" "The ChIP-seq data of the ENCODE project show that H3K4me1, but not H3K4me3, marks all four DNaseI HS sites in the adult lung.To examine whether each of the DNase I HS sites is responsible for upregulating the expression of Shh, we employed a transgenic reporter assay using BAC clones (Figs. 1A and 3A)." Enhancer Shh 3C Luciferase Reporter Assay "To determine whether SLGE physically interacts with the Shh promoter in association with transcriptional activity, we conducted a 3C assay, which has been used to measure associations between two genomic regions. Assessment of the functional properties of SLGE by 3C and luciferase assays." "9530036O11Rik,Dsh,Hhg1,Hx,Hxl3,M100081NC,Shh" -- -- -- "Shh expression in the endodermal epithelial lining, which is enabled by the coordinated actions of the Enhancers, is necessary for proper endodermal development." "RT-PCR,Luciferase Reporter Assay,3C" "We identified a novel, less-conserved cis-regulatory element for Shh expres_x005f_x0002_sion in the esophageal epithelium, as well as in the lung and intestinal epithelium, in a region 100 Kb upstream of the Shh CDS. The new element compensates for the Enhancer activi_x0002_ties of the three previously identified Enhancers, and the combined activities of all four Enhancers permit the seamless Shh expression in the epithelial linings of the whole endoder_x0002_mal tissues in the mouse embryo." "Rbfox3,Socs1" "Fox-3,Hrnbp3,NeuN,Neuna60,Cish1,Cish3,JAB,SOCS-1,SSI-1" DNaseI-seq "Putative binding sites for Fox transcription factors and STAT are found in the SLGE.In human cancer cell lines, including Caco-2, putative Fox- and STAT-binding sites are found in some of the DNase I HS sites around the SHH CDS." -- -- -- >2KB 39503 E_02_067 24157522 -- mm10 chr5 28500370 28502363 Mouse Colon Adenocarcinoma Cell Lines Low+High throughput "RT-PCR,ChIP-seq,Transient Transfection Assay,Luciferase Reporter Assay,3C,DNaseI-seq" "The ChIP-seq data of the ENCODE project show that H3K4me1, but not H3K4me3, marks all four DNaseI HS sites in the adult lung.To examine whether each of the DNase I HS sites is responsible for upregulating the expression of Shh, we employed a transgenic reporter assay using BAC clones (Figs. 1A and 3A)." Enhancer Shh 3C Luciferase Reporter Assay "To determine whether SLGE physically interacts with the Shh promoter in association with transcriptional activity, we conducted a 3C assay, which has been used to measure associations between two genomic regions. Assessment of the functional properties of SLGE by 3C and luciferase assays." "9530036O11Rik,Dsh,Hhg1,Hx,Hxl3,M100081NC,Shh" -- -- -- "Shh expression in the endodermal epithelial lining, which is enabled by the coordinated actions of the Enhancers, is necessary for proper endodermal development." "RT-PCR,Luciferase Reporter Assay,3C" "We identified a novel, less-conserved cis-regulatory element for Shh expres_x005f_x0002_sion in the esophageal epithelium, as well as in the lung and intestinal epithelium, in a region 100 Kb upstream of the Shh CDS. The new element compensates for the Enhancer activi_x0002_ties of the three previously identified Enhancers, and the combined activities of all four Enhancers permit the seamless Shh expression in the epithelial linings of the whole endoder_x0002_mal tissues in the mouse embryo." "Rbfox3,Socs1" "Fox-3,Hrnbp3,NeuN,Neuna60,Cish1,Cish3,JAB,SOCS-1,SSI-1" DNaseI-seq "Putative binding sites for Fox transcription factors and STAT are found in the SLGE.In human cancer cell lines, including Caco-2, putative Fox- and STAT-binding sites are found in some of the DNase I HS sites around the SHH CDS." -- -- -- >2KB 44528 E_02_068 24157522 -- mm10 chr5 28561915 28564905 Mouse Colon Adenocarcinoma Cell Lines Low+High throughput "RT-PCR,ChIP-seq,Transient Transfection Assay,Luciferase Reporter Assay,3C,DNaseI-seq" "The ChIP-seq data of the ENCODE project show that H3K4me1, but not H3K4me3, marks all four DNaseI HS sites in the adult lung.To examine whether each of the DNase I HS sites is responsible for upregulating the expression of Shh, we employed a transgenic reporter assay using BAC clones (Figs. 1A and 3A)." Enhancer Shh 3C Luciferase Reporter Assay "To determine whether SLGE physically interacts with the Shh promoter in association with transcriptional activity, we conducted a 3C assay, which has been used to measure associations between two genomic regions. Assessment of the functional properties of SLGE by 3C and luciferase assays." "9530036O11Rik,Dsh,Hhg1,Hx,Hxl3,M100081NC,Shh" -- -- -- "Shh expression in the endodermal epithelial lining, which is enabled by the coordinated actions of the Enhancers, is necessary for proper endodermal development." "RT-PCR,Luciferase Reporter Assay,3C" "We identified a novel, less-conserved cis-regulatory element for Shh expres_x005f_x0002_sion in the esophageal epithelium, as well as in the lung and intestinal epithelium, in a region 100 Kb upstream of the Shh CDS. The new element compensates for the Enhancer activi_x0002_ties of the three previously identified Enhancers, and the combined activities of all four Enhancers permit the seamless Shh expression in the epithelial linings of the whole endoder_x0002_mal tissues in the mouse embryo." "Rbfox3,Socs1" "Fox-3,Hrnbp3,NeuN,Neuna60,Cish1,Cish3,JAB,SOCS-1,SSI-1" DNaseI-seq "Putative binding sites for Fox transcription factors and STAT are found in the SLGE.In human cancer cell lines, including Caco-2, putative Fox- and STAT-binding sites are found in some of the DNase I HS sites around the SHH CDS." -- -- -- >2KB 106571 E_02_069 24204311 ECR9 Enhancer mm10 chr19 10215891 10216703 Mouse Oligodendroglial Cell Low throughput "ChIP,Luciferase Reporter Assay,EMSA,qPCR" "Considering the large number of potential Sox binding sites and the low predictive power of their presence, we decided to assess regulatory potential and Sox10-responsiveness of the ECR in luciferase assays.To verify that Sox10 is bound to ECR9 in OL we next performed chromatin immunoprecipitation (ChIP) experiments. Some were clustered so that eight oligonucleotides were sufficient to cover all sites in electrophoretic mobility shift assays (EMSA)." Enhancer Myrf -- Immunoprecipitation To address this issue we performed immunoprecipitation experiments with anti-Sox10 antibodies on extracts of the OL-like OLN93 cell line. "6030439E18,Gm1804,Gm98,Mrf" -- -- -- -- -- -- Sox10 "Dom,Sox21,gt" ChIP To verify that Sox10 is bound to ECR9 in OL we next performed chromatin immunoprecipitation (ChIP) experiments. -- -- -- >2KB 8027 E_02_070 24257627 -- mm10 chr2 119321593 119322529 Mouse "293T,Progenitor Cell" Low throughput "ChIP,PCR,EMSA" "In addition, we carried out chromatin immunoprecipitation (ChIP) assays to show that both Ascl1 and Neurog1 were able to occupy in vivo the critical E-box region of the CR1 Enhancer in cell culture and mouse embryonic neural tubes, whereas no enrichment was shown for control DNA in Dll4 3¡ä UTR." Enhancer Dll4 -- EMSA "We performed an electrophoretic mobility shift assay (EMSA) to test whether Ascl1 and Neurog proteins were able to directly bind to the minimal enhancer region using an oligo probe containing the critical E-box (Probe 2) and a control upstream probe that lacks an E-box (Probe 1) " Delta4 -- -- -- "Foxn4, Ascl1 and Neurog1 were able to activate gene expression through an evolutionarily conserved Dll4 Enhancer CR1 found to be active in the retina. " "ChIP,EMSA" "In addition, we carried out chromatin immunoprecipitation (ChIP) assays to show that both Ascl1 and Neurog1 were able to occupy in vivo the critical E-box region of the CR1 Enhancer in cell culture and mouse embryonic neural tubes, whereas no enrichment was shown for control DNA in Dll4 3¡ä UTR.Thus, Ascl1 and Neurog factors may bind to the same E-box in the Dll4 Enhancer but have differential effects on Dll4 gene expression." "Foxn4,Ascl1" "AI225900,ASH1,Mash1,bHLHa46" "EMSA,ChIP" We performed an electrophoretic mobility shift assay (EMSA) to test whether Ascl1 and Neurog proteins were able to directly bind to the minimal Enhancer region using an oligo probe containing the critical E-box (Probe 2) and a control upstream probe that lacks an E-box. -- -- -- >2KB 3722 E_02_071 24257627 -- mm10 chr2 119321593 119322529 Mouse "293T,Progenitor Cell" Low throughput "ChIP,PCR,EMSA" "we tested whether Foxn4, Ascl1 and Neurog1 were able to activate gene expression through an evolutionarily conserved Dll4 enhancer CR1 found to be active in the retina. In the chick spinal cord, CR1 was able to drive DsRed reporter expression in a pattern that mimics that of the mouse Dll4 along the dorsoventral axis, predominantly in the V2 domain" Enhancer Dll4 -- "ChIP,PCR,EMSA" "In addition, we carried out chromatin immunoprecipitation (ChIP) assays to show that both Ascl1 and Neurog1 were able to occupy in vivo the critical E-box region of the CR1 Enhancer in cell culture and mouse embryonic neural tubes, whereas no enrichment was shown for control DNA in Dll4 3¡ä UTR." Delta4 -- -- -- "Foxn4, Ascl1 and Neurog1 were able to activate gene expression through an evolutionarily conserved Dll4 Enhancer CR1 found to be active in the retina. " "ChIP,EMSA" "In addition, we carried out chromatin immunoprecipitation (ChIP) assays to show that both Ascl1 and Neurog1 were able to occupy in vivo the critical E-box region of the CR1 Enhancer in cell culture and mouse embryonic neural tubes, whereas no enrichment was shown for control DNA in Dll4 3¡ä UTR.Thus, Ascl1 and Neurog factors may bind to the same E-box in the Dll4 Enhancer but have differential effects on Dll4 gene expression." "Foxn4,Ascl1" "AI225900,ASH1,Mash1,bHLHa46" "EMSA,ChIP" We performed an electrophoretic mobility shift assay (EMSA) to test whether Ascl1 and Neurog proteins were able to directly bind to the minimal Enhancer region using an oligo probe containing the critical E-box (Probe 2) and a control upstream probe that lacks an E-box. -- -- -- >2KB 3722 E_02_072 30146478 -- mm10 chr11 111838889 111839334 Mouse Cartilage Low+High throughput ChIP-qPCR "We next performed the ChIP-qPCR using anti-H3K4me1 and H3K27ac antibody in all five regions and found that RCSE regions were significantly enriched in both H3K4me1 and H3K27ac ChIP, suggesting the potential enhancer." Enhancer Sox9 3C PCR These data suggest that there is a long-range interaction between the Sox9 promoter and RCSE region in primary chondrocytes. "2010306G03Rik,AV220920,mKIAA4243" -- -- -- TG mice with the Sox9 promoter and 43 RCSE region.These data suggest that the RCSE region functions as a costal- and sternum cartilage-specific Enhancer. Transgenic mice TG mice with the Sox9 promoter and 43 RCSE region.These data suggest that the RCSE region functions as a costal- and sternum cartilage-specific Enhancer. Stat3 "1110034C02Rik,AW109958,Aprf" "Luciferase Reporter Assay,Immunostaining" "Furthermore, transcription factors that regulate Sox9 expres sion were screened using a CRISPR/Cas9-chromatin immuno precipitation (ChIP)-mass spectrometry (MS) system targeting the RCSE region in chondrocytes; signal transducer and acti vator of transcription 3 (Stat3) was identified as a transacting fac tor for the Sox9 Enhancer." -- -- -- >2KB 943097 E_02_073 24324617 -- mm10 chr16 59471850 59472124 Mouse EL4 Low+High throughput "ChIP,Luciferase Reporter Assay" These results suggested: (1) that region (264/+19) contains an element that mediates strong reporter activity suggestive of an enhancer; and (2) that fragment(+19/+354) contains a Mina promoter comparable in activity to the SV40 promoter. Enhancer Riox2 -- qRT-PCR "In summary, two promoters were identified in a ,2 kb Mina TSS-spanning genomic fragment: P1 in region (264/+19) and P2 in region (+150/+280). Further, we inferred the existence of a P1-specific Enhancer E1 located in region (+80/+354)." "1810047J07Rik,2410057H13Rik,3830408E23Rik,AI449204,Mina" Pulmonary Inflammation -- D011014 -- -- -- "Sp1,Sp3" "1110003E12Rik,AA450830,AI845540-1,Sp1,D130027J01Rik" "EMSA,RT-PCR,ChIP,Luciferase Reporter Assay" EMSA supershift analysis of Sp1/3 association with the four Sp1/3 binding sites comprising the Mina P1 promoter. -- -- -- <2KB 234 E_02_074 24368734 -- mm10 chr6 115409226 115436116 Mouse Embryonic Stem Cell Low+High throughput "ChIP-seq,qPCR,RNA-seq" "To characterize the genomic features of MLL4 binding regions during differentiation, we performed ChIP-Seq of H3K4me1/2/3, H3K27ac and Pol II during adipogenesis. Then, we used histone marks to define four types of gene regulatory elements: active enhancer, silent enhancer, active promoter, and silent promoter. Average profile plots revealed that at day 2 of adipogenesis, MLL4 and adipogenic TFs C/EBP¦Á, C/EBP¦Â and PPAR¦Ã were enriched on active enhancers." Enhancer Pparg -- qPCR "Importantly, deletion of Mll4 markedly decreased H3K4me1 and H3K27ac on over 90% of the 591 recovered C/EBP¦Â+ MLL4+ adipogenic Enhancers (Figure 8C¨CD). MLL4-dependent de novo H3K4me1 and H3K27ac were also observed on the C/EBP¦Â+ adipogenic Enhancers located on Pparg gene locus." "Nr1c3,PPAR-gamma,PPAR-gamma2,PPARgamma,PPARgamma2" -- -- -- -- -- -- "Ppargc1a,Cebpa,Cebpb" "A830037N07Rik,Gm11133,PGC-1,PPARGC-1-alpha,Pgc-1alpha,Pgc1,Pgco1,Ppargc1,C/EBPbeta,CRP2,IL-6DBP,LAP,LIP,NF-IL6,NF-M,Nfil6,C/ebpalpha,CBF-A,Cebp" ChIP-seq "To experimentally validate the predicted motifs, we performed ChIP-Seq of C/EBP¦Á, C/EBP¦Â and PPAR¦Ã at day 2 of adipogenesis and of MyoD in myocytes. By comparing the genomic localizations of MLL4 with those of C/EBP¦Á, C/EBP¦Â and PPAR¦Ã, we found that consistent with the motif analysis, ¡«64% of MLL4 binding regions overlapped with those of C/EBP¦Á/¦Â or PPAR¦Ã at day 2 of adipogenesis." -- -- -- >2KB 61795 E_02_075 24368734 -- mm10 chr6 115507545 115533315 Mouse Embryonic Stem Cell Low+High throughput "ChIP-seq,PCR,RNA-seq" "To characterize the genomic features of MLL4 binding regions during differentiation, we performed ChIP-Seq of H3K4me1/2/3, H3K28ac and Pol II during adipogenesis. Then, we used histone marks to define four types of gene regulatory elements: active enhance" Enhancer Pparg -- qPCR "Importantly, deletion of Mll4 markedly decreased H3K4me1 and H3K27ac on over 90% of the 591 recovered C/EBP¦Â+ MLL4+ adipogenic Enhancers (Figure 8C¨CD). MLL4-dependent de novo H3K4me1 and H3K27ac were also observed on the C/EBP¦Â+ adipogenic Enhancers located on Pparg gene locus." "Nr1c3,PPAR-gamma,PPAR-gamma2,PPARgamma,PPARgamma2" -- -- -- -- -- -- "Ppargc1a,Cebpa,Cebpb" "A830037N07Rik,Gm11133,PGC-1,PPARGC-1-alpha,Pgc-1alpha,Pgc1,Pgco1,Ppargc1,C/EBPbeta,CRP2,IL-6DBP,LAP,LIP,NF-IL6,NF-M,Nfil6,C/ebpalpha,CBF-A,Cebp" ChIP-seq "To experimentally validate the predicted motifs, we performed ChIP-Seq of C/EBP¦Á, C/EBP¦Â and PPAR¦Ã at day 2 of adipogenesis and of MyoD in myocytes. By comparing the genomic localizations of MLL4 with those of C/EBP¦Á, C/EBP¦Â and PPAR¦Ã, we found that consistent with the motif analysis, ¡«64% of MLL4 binding regions overlapped with those of C/EBP¦Á/¦Â or PPAR¦Ã at day 2 of adipogenesis." -- -- -- >2KB 159554 E_02_076 24374176 -- mm10 chr2 115794340 115800495 Mouse Embryonic Stem Cell Low+High throughput "ChIP-qPCR,ChIP-seq,ChIP,3C" "Based on these results, we identified sequence d as a MB Meis2 enhancer, and hereafter designate it as MBE. We went on to look for accumulation of histone H3K27 acetylation (H3K27ac) and H3K4 monomethylation (H3K4me1), which demarcate active and potential enhancers, respectively, at MBE by ChIP-seq." Enhancer Meis2 3C Immuno-FISH "Since Meis2 and its upstream region are highly homologous between mouse and human, we used two human BAC clones that contained mainly the upstream or gene body region of Meis2 to test for inclusion of an MB-specific Enhancer." "A430109D20Rik,Mrg1,Stra10" -- -- -- -- -- -- Rnf2 "AI326319,AI450156,AU019207,Ring1B,dinG" "ChIP-qPCR,ChIP-seq" "RING1B ChIP-seq data (GSE48464) over 10 Mb of the mouse genome (mm9) around Meis2 in FB,MB, and LM at 11.5 dpcBinding of H3K27ac and RING1B in 11.5 dpc MB of WT and Ring1 mutant (Ring1 mut) revealed by ChIP-quantitative PCR (ChIP-qPCR) analysis." -- -- -- >2KB 63845 E_02_077 24385922 -- mm10 chr6 52296135 52334522 Mouse Limb Cells Low+High throughput "ChIP,PCR,3C,5C,ChIP-seq" "Sequences distinct from proximal promoters (RefSeq) that were bound by RNAP2 and at least one other mark, or by both p300 and H3K27Ac were retained as candidate enhancers. Using these criteria, 19 putative enhancers were identified within 850 kb upstream of Hoxa13." Enhancer Hoxa 3C PCR "Here,we report on the first identification of bona fide transcriptional Enhancers controlling HoxA genes in developing limbs and show that these Enhancers are grouped into distinct topological domains at the sub-megabase scale." Hox-1 -- -- -- -- -- -- Ctcf AW108038 5C "Interestingly,loci bound either by CTCF or cohesin in limb buds have been recently identified and comparison with our data shows that almost all loci interacting with 59 HoxA genes overlap with either CTCF o cohesin binding." -- -- -- -- -- E_02_078 24385922 -- mm10 chr6 52585919 52613016 Mouse Limb Cells Low+High throughput "ChIP,PCR,3C,5C,ChIP-seq" "Sequences distinct from proximal promoters (RefSeq) that were bound by RNAP2 and at least one other mark, or by both p300 and H3K27Ac were retained as candidate enhancers. Using these criteria, 19 putative enhancers were identified within 851 kb upstream true???" Enhancer Hoxa 3C PCR "Here,we report on the first identification of bona fide transcriptional Enhancers controlling HoxA genes in developing limbs and show that these Enhancers are grouped into distinct topological domains at the sub-megabase scale." Hox-1 -- -- -- -- -- -- Ctcf AW108038 5C "Interestingly,loci bound either by CTCF or cohesin in limb buds have been recently identified and comparison with our data shows that almost all loci interacting with 59 HoxA genes overlap with either CTCF o cohesin binding." -- -- -- -- -- E_02_079 24385922 -- mm10 chr6 52765812 52891511 Mouse Limb Cells Low+High throughput "ChIP,PCR,3C,5C,ChIP-seq" "Sequences distinct from proximal promoters (RefSeq) that were bound by RNAP2 and at least one other mark, or by both p300 and H3K27Ac were retained as candidate enhancers. Using these criteria, 19 putative enhancers were identified within 852 kb upstream true???" Enhancer Hoxa 3C PCR "Here,we report on the first identification of bona fide transcriptional Enhancers controlling HoxA genes in developing limbs and show that these Enhancers are grouped into distinct topological domains at the sub-megabase scale." Hox-1 -- -- -- -- -- -- Ctcf AW108038 5C "Interestingly,loci bound either by CTCF or cohesin in limb buds have been recently identified and comparison with our data shows that almost all loci interacting with 59 HoxA genes overlap with either CTCF o cohesin binding." -- -- -- -- -- E_02_080 24385922 -- mm10 chr6 52908070 53037532 Mouse Limb Cells Low+High throughput "ChIP,PCR,3C,5C,ChIP-seq" "Sequences distinct from proximal promoters (RefSeq) that were bound by RNAP2 and at least one other mark, or by both p300 and H3K27Ac were retained as candidate enhancers. Using these criteria, 19 putative enhancers were identified within 853 kb upstream true???" Enhancer Hoxa 3C PCR "Here,we report on the first identification of bona fide transcriptional Enhancers controlling HoxA genes in developing limbs and show that these Enhancers are grouped into distinct topological domains at the sub-megabase scale." Hox-1 -- -- -- -- -- -- Ctcf AW108038 5C "Interestingly,loci bound either by CTCF or cohesin in limb buds have been recently identified and comparison with our data shows that almost all loci interacting with 59 HoxA genes overlap with either CTCF or cohesin binding." -- -- -- -- -- E_02_081 24391132 -- mm10 chr4 147962887 147964479 Mouse 293T Low+High throughput "RT-PCR,3C,ChIP,Luciferase Reporter Assay" "Among the 11 CRs identified, only CR9 coincided with the binding sites of RNA polymerase II and p300, and overlapped with the gene areas modified by H3K4me1, and filled all criteria for the enhancer." Enhancer Nppb 3C "RT-PCR,Transgenic mice" "The Enhancer is a 650-bp fragment within 50 kb of the Nppa and Nppb loci.To identify the organs in which CR9 functioned as a stress-responsive Enhancer, we examined the luciferase reporter expression in each organ by quantitative PCR." "AA408272,BNF,BNP" Heart Failure -- D006333 -- -- -- "Nkx2-5,Ctcf" "CHNG5,CSX,CSX1,HLHS2,NKX2.5,NKX2E,NKX4-1,VSD3,AW108038" ChIP-seq "ChIP-seq data for H3K4me1, p300, and CTCF were obtained from an open database of the adult mouse heart. Some CRs coincided with the peaks for H3K4me1, RNA polymerase II, and the transcriptional coactivator protein p300." -- -- -- >2KB 22104 E_02_082 24391766 -- mm10 chr7 100474191 100474841 Mouse Platinum E cells Low+High throughput "Luciferase Reporter Assay,PCR,EMSA,ChIP-seq" "The first intron of the hamster is shorter compared to mouse and rat corresponding to the first half of the introns in these species.Generally, sequence conservation was low across the first intron,except a region of high conservation ranging from IVS(intervening sequence)1+1200 to IVS1+1850 with the IVS1+1505G/A baseexchange in the center of this region." Enhancer Ucp3 -- "Luciferase Reporter Assay,PCR,EMSA" "Generally, sequence conservation was low across the first intron,except a region of high conservation ranging from IVS(intervening sequence)1+1200 to IVS1+1850 with the IVS1+1505G/A base exchange in the center of this region." "AI645527,Slc25a9,UCP-3" -- -- -- This intronic region is the main Enhancer driving UCP3 expression with SP1/3 and PPARc as the core factors required for expression. "EMSA,ChIP-seq" "While we understand that simple EMSA experiments are not sufficient to validate presence of a complex transcription factor binding module conserved across the whole mammalian class, our data provide good evidence that it an intronic Enhancer in the first intron of the UCP3 gene is of importance in non-rodent species as well." "Sp1,Sp3" "1110003E12Rik,AA450830,AI845540-1,Sp1,D130027J01Rik" "EMSA,ChIP-seq" "We discovered that the transcription factors SP1 and SP3 were binding to the IVS1+1505G element, whereas binding to the mutant allele was strongly diminished. Direct binding of PPAR¦Ã and RXRa to the IVS1+1505G element could be ruled out." -- -- -- Intron 1526 E_02_083 24692107 -- mm10 chr17 44464986 44466986 Mouse Osteoblast-Specific Cell Low throughput "Luciferase Reporter Assay,ChIP,EMSA" and found that a 1.3©\kb region located about 30 kb upstream of the transcription start site of type II Runx2 was highly conserved among all of the species Enhancer Runx2 -- "Transgenic mice,RT-PCR" "We searched for regulatory elements of the Runx2 gene,and identified a 343bp Enhancer sequence,which specifically directed the reporter gene expression to osteoblasts." "AML3,Cbf,Cbfa-1,Cbfa1,LS3,Osf2,PEBP2aA,Pebp2a1,Pebpa2a" -- -- -- -- -- -- "Dlx5,Mef2c,Tcf7,Ctnnb1,Sp7,Smad1,Sox6" "AI385752,5430401D19Rik,9930028G15Rik,AV011172,Mef2,AI465550,TCF-1,Tcf1,Bfc,Catnb,Mesc,6430578P22Rik,C22,Osx,AI528653,Mad1,Madh1,Madr1,Mlp1,MusMLP,dwf-A,mMad1,AI987981,SOX-LZ" "EMSA,ChIP" "In EMSA, Mef2c, Dlx5, and Msx2 but not Tcf7, Ctnnb1, Smad1, Sox6, and Sp7 directly bound to the 89©\bp core sequence.In ChIP analysis, the bindings of Dlx5, Dlx6, Msx2, Mef2, Tcf7, Ctnnb1, Smad1, Sox5, Sox6, and Sp7 to the 343 enhancer region were detected using each antibody, whereas the binding of Foxa2 was not detected in primary osteoblasts despite the presence of a conserved Foxa2 binding motif in the core 89©\bp sequence." -- -- -- >2KB 30000 E_02_084 24705708 -- mm10 chr6 52199038 52200503 Mouse "Lung,Stomach" Low+High throughput "EMSA,ChIP" A 2.1-kb mesodermal (MES) enhancer important for Hoxa5 paraxial and lateral plate mesoderm expression in the cervico-upper thoracic region of the A-P axis is positioned 39 of the Hoxa5 gene. Enhancer Vegfa -- "Transgenic mice,EMSA,qRT-PCR" A 1.5-kb XbaI-XbaI DNA fragment located in Hoxa4-Hoxa5 intergenic sequences at 3.0-kb upstream the Hoxa4 gene (positions +9351-bp to +10816-bp) was able to target Hoxa5 expression in lung and stomach. "Vegf,Vpf" -- -- -- -- -- -- Yy1 "NF-E1,YY-1" "ChIP,qPCR" "In summary, YY1 acts as a positive regulator of Hoxa5 lung, stomach and intestine expression during embryogenesis." -- -- -- >2KB 6182779 E_02_085 24734888 -- mm10 chr1 133347808 133348049 Mouse Kidney Low throughput "RT-PCR,Southern blot" "The mdE, defined by 240 bp DNA sequence, was identified by transient transfection of 50 deletion constructs of the mouse Ren-1c gene into As4.1 cells. This tissue-specific enhancer is located 2.6 kb upstream (_x0002_2866 to _x0002_2625 bp) of the transcriptional start site of the mouse renin gene and its homologous sequence is present about 12 kb upstream of the human renin start site of transcription." Enhancer Ren1 -- "Transgenic mice,RT-PCR" "Structural analysis of the transgenic mdE region. In vivo Cre-loxP recombination shown in the left was confirmed by Southern blot analysis of tail DNA from parental,wt,and mut TgM lines,in which the DNA was digested with EcoRI (E),separated on agarosegels,transferred to a nylon membrane,and hybridized to the probe (shaded rectangles in the left panel,from nt 2635 to 2214 relative to transcription start site). The sizes of the expected bands are indicated (in kb) in the right panel and those expected from the endogenous locus are in parentheses." "D19352,Ren,Ren-1,Ren-Ac,Ren1d,Rn-1,Rnr,?Ren1" -- -- -- -- -- -- -- -- -- -- -- -- -- >2KB 2744 E_02_086 24812424 -- mm10 chr16 23143036 23145036 Mouse Offspring Adipocytes Low throughput "ChIP,qRT-PCR,Western blot" "Active epigenetic marks were significantly decreased in the promoter and enhancer regions of the AdipoQ gene in 24-week-old offspring from SF-exposed mothers. In contrast,the inactive epigenetic marks in the AdipoQ gene (5-mC and histone H3K9m2) were significantly higher in SF offspring adipocytes" Enhancer Adipoq -- "qRT-PCR,Western blot" "SF exposures during late gestation alter epigenetic markers in the AdipoQ gene promoter and Enhancer regions in adipocytes from VWAT. Active (5-hmC and H3K4me3) and inactive (5-mC and H3K9me2) marks were mapped to the Enhancer (22,500 bp) and promoter (2500 bp) regions and downstream to the transcription start site (+500 bp) region, respectively, using ChIP assays." "30kDa,APN,Acdc,Acrp30,Ad,Adid,GBP28,adipo,apM1" Sleep Fragmentation -- D012892 -- -- -- -- -- -- -- -- -- -- >2KB 2499 E_02_087 24905168 -- mm10 chr4 124638991 124641160 Mouse Mouse Embryonic Stem Cell Low+High throughput "ChIP-seq,RNA-seq" "To address whether changes in enhancer utilization patterns occur in the majority of cells within the differentiating population, we isolated dual transgenic enhancer reporter lines, in which ESC-specific or EpiLC-specific enhancers of interest were cloned upstream of a minimal promoter and one of two distinct fluorescent reporters and integrated into the ESC genome using a PiggyBac transposon system." Enhancer Pou3f1 -- "qPCR,IP-Western" "Upon differentiation, Otx2?/? cells underwent morphological changes indistinguishable from those seen in wt EpiLCs, but showed defects in expression of certain epiblast-associated genes, such as Fgf5 and Oct6. RNA-seq analysis of Otx2?/? EpiLCs identified 260 and 141 genes significantly downregulated or upregulated, respectively, as compared to the genetically-matched wt EpiLCs" "Oct-6,Oct6,Otf-6,Otf6,Scip,Test1,Tst-1,Tst1" -- -- -- -- -- -- "Otx2,Oct4" "E130306E05Rik,2810482H01Rik,NF-A1,Oct-1,Oct1,Otf-1,Otf1" ChIP-seq Our results illuminate regulatory mechanisms underlying pluripotency and suggest that capacity of transcription factors such as Otx2 and Oct4 to pioneer new Enhancer sites is highly context dependent. -- -- -- >2KB 17569 E_02_088 24905168 -- mm10 chr4 124645999 124647927 Mouse Mouse Embryonic Stem Cell Low+High throughput "ChIP-seq,RNA-seq" "To address whether changes in enhancer utilization patterns occur in the majority of cells within the differentiating population, we isolated dual transgenic enhancer reporter lines, in which ESC-specific or EpiLC-specific enhancers of interest were cloned upstream of a minimal promoter and one of two distinct fluorescent reporters and integrated into the ESC genome using a PiggyBac transposon system." Enhancer Pou3f1 -- "qPCR,IP-Western" "Upon differentiation, Otx2?/? cells underwent morphological changes indistinguishable from those seen in wt EpiLCs, but showed defects in expression of certain epiblast-associated genes, such as Fgf5 and Oct6. RNA-seq analysis of Otx2?/? EpiLCs identified 260 and 141 genes significantly downregulated or upregulated, respectively, as compared to the genetically-matched wt EpiLCs" "Oct-6,Oct6,Otf-6,Otf6,Scip,Test1,Tst-1,Tst1" -- -- -- -- -- -- "Otx2,Oct4" "E130306E05Rik,2810482H01Rik,NF-A1,Oct-1,Oct1,Otf-1,Otf1" ChIP-seq Our results illuminate regulatory mechanisms underlying pluripotency and suggest that capacity of transcription factors such as Otx2 and Oct4 to pioneer new Enhancer sites is highly context dependent. -- -- -- >2KB 10682 E_02_089 24905168 -- mm10 chr5 119677099 119681977 Mouse Mouse Embryonic Stem Cell Low+High throughput "ChIP-seq,RNA-seq" "To address whether changes in enhancer utilization patterns occur in the majority of cells within the differentiating population, we isolated dual transgenic enhancer reporter lines, in which ESC-specific or EpiLC-specific enhancers of interest were cloned upstream of a minimal promoter and one of two distinct fluorescent reporters and integrated into the ESC genome using a PiggyBac transposon system." Enhancer Tbx3 -- "qPCR,IP-Western" "However, comparison between ESC and EpiLC showed substantial reorganization of Oct4 occupancy during differentiation with gain of binding near genes associated with post-implantation epiblast (e.g. Fgf5, Oct6, Wnt8a) and loss near genes associated with na?ve pluripotency (e.g. Klf4, Tbx3, Prdm14)" D5Ertd189e -- -- -- -- -- -- "Otx2,Oct4" "E130306E05Rik,2810482H01Rik,NF-A1,Oct-1,Oct1,Otf-1,Otf1" ChIP-seq Our results illuminate regulatory mechanisms underlying pluripotency and suggest that capacity of transcription factors such as Otx2 and Oct4 to pioneer new Enhancer sites is highly context dependent. -- -- -- >2KB 9220 E_02_090 24905168 -- mm10 chr5 119704124 119707831 Mouse Mouse Embryonic Stem Cell Low+High throughput "ChIP-seq,RNA-seq" "To address whether changes in enhancer utilization patterns occur in the majority of cells within the differentiating population, we isolated dual transgenic enhancer reporter lines, in which ESC-specific or EpiLC-specific enhancers of interest were cloned upstream of a minimal promoter and one of two distinct fluorescent reporters and integrated into the ESC genome using a PiggyBac transposon system." Enhancer Tbx3 -- "qPCR,IP-Western" "However, comparison between ESC and EpiLC showed substantial reorganization of Oct4 occupancy during differentiation with gain of binding near genes associated with post-implantation epiblast (e.g. Fgf5, Oct6, Wnt8a) and loss near genes associated with na?ve pluripotency (e.g. Klf4, Tbx3, Prdm14)" D5Ertd189e -- -- -- -- -- -- "Otx2,Oct4" "E130306E05Rik,2810482H01Rik,NF-A1,Oct-1,Oct1,Otf-1,Otf1" ChIP-seq Our results illuminate regulatory mechanisms underlying pluripotency and suggest that capacity of transcription factors such as Otx2 and Oct4 to pioneer new Enhancer sites is highly context dependent. -- -- -- >2KB 35660 E_02_091 24905168 -- mm10 chr5 119707931 119710758 Mouse Mouse Embryonic Stem Cell Low+High throughput "ChIP-seq,RNA-seq" "To address whether changes in enhancer utilization patterns occur in the majority of cells within the differentiating population, we isolated dual transgenic enhancer reporter lines, in which ESC-specific or EpiLC-specific enhancers of interest were cloned upstream of a minimal promoter and one of two distinct fluorescent reporters and integrated into the ESC genome using a PiggyBac transposon system." Enhancer Tbx3 -- "qPCR,IP-Western" "However, comparison between ESC and EpiLC showed substantial reorganization of Oct4 occupancy during differentiation with gain of binding near genes associated with post-implantation epiblast (e.g. Fgf5, Oct6, Wnt8a) and loss near genes associated with na?ve pluripotency (e.g. Klf4, Tbx3, Prdm14)" D5Ertd189e -- -- -- -- -- -- "Otx2,Oct4" "E130306E05Rik,2810482H01Rik,NF-A1,Oct-1,Oct1,Otf-1,Otf1" ChIP-seq Our results illuminate regulatory mechanisms underlying pluripotency and suggest that capacity of transcription factors such as Otx2 and Oct4 to pioneer new Enhancer sites is highly context dependent. -- -- -- >2KB 39027 E_02_092 24905168 -- mm10 chr5 119710660 119714758 Mouse Mouse Embryonic Stem Cell Low+High throughput "ChIP-seq,RNA-seq" "To address whether changes in enhancer utilization patterns occur in the majority of cells within the differentiating population, we isolated dual transgenic enhancer reporter lines, in which ESC-specific or EpiLC-specific enhancers of interest were cloned upstream of a minimal promoter and one of two distinct fluorescent reporters and integrated into the ESC genome using a PiggyBac transposon system." Enhancer Tbx3 -- "qPCR,IP-Western" "However, comparison between ESC and EpiLC showed substantial reorganization of Oct4 occupancy during differentiation with gain of binding near genes associated with post-implantation epiblast (e.g. Fgf5, Oct6, Wnt8a) and loss near genes associated with na?ve pluripotency (e.g. Klf4, Tbx3, Prdm14)" D5Ertd189e -- -- -- -- -- -- "Otx2,Oct4" "E130306E05Rik,2810482H01Rik,NF-A1,Oct-1,Oct1,Otf-1,Otf1" ChIP-seq Our results illuminate regulatory mechanisms underlying pluripotency and suggest that capacity of transcription factors such as Otx2 and Oct4 to pioneer new Enhancer sites is highly context dependent. -- -- -- >2KB 42391 E_02_093 24905168 -- mm10 chr5 98290964 98295647 Mouse Mouse Embryonic Stem Cell Low+High throughput "ChIP-seq,RNA-seq" "To address whether changes in enhancer utilization patterns occur in the majority of cells within the differentiating population, we isolated dual transgenic enhancer reporter lines, in which ESC-specific or EpiLC-specific enhancers of interest were cloned upstream of a minimal promoter and one of two distinct fluorescent reporters and integrated into the ESC genome using a PiggyBac transposon system." Enhancer Fgf5 -- "qPCR,IP-Western" "Interrogation of our ChIP-seq datasets revealed presence of a proximal Enhancer (PE) at the Fgf5 locus, which exists in a poised state in ESCs, as well as a cluster of four Enhancers (depicted as E1-E4) located within 25¨C60 kb downstream from the Fgf5 TSS that are unmarked in ESCs, but activated de novo during the transition." "Fgf-5,HBGF-5,angora,go" -- -- -- -- -- -- "Otx2,Oct4" "E130306E05Rik,2810482H01Rik,NF-A1,Oct-1,Oct1,Otf-1,Otf1" ChIP-seq Our results illuminate regulatory mechanisms underlying pluripotency and suggest that capacity of transcription factors such as Otx2 and Oct4 to pioneer new Enhancer sites is highly context dependent. -- -- -- >2KB 39123 E_02_094 24905168 -- mm10 chr5 98307793 98312281 Mouse Mouse Embryonic Stem Cell Low+High throughput "ChIP-seq,RNA-seq" "To address whether changes in enhancer utilization patterns occur in the majority of cells within the differentiating population, we isolated dual transgenic enhancer reporter lines, in which ESC-specific or EpiLC-specific enhancers of interest were cloned upstream of a minimal promoter and one of two distinct fluorescent reporters and integrated into the ESC genome using a PiggyBac transposon system." Enhancer Fgf5 -- "qPCR,IP-Western" "Interrogation of our ChIP-seq datasets revealed presence of a proximal Enhancer (PE) at the Fgf5 locus, which exists in a poised state in ESCs, as well as a cluster of four Enhancers (depicted as E1-E4) located within 25¨C60 kb downstream from the Fgf5 TSS that are unmarked in ESCs, but activated de novo during the transition." "Fgf-5,HBGF-5,angora,go" -- -- -- -- -- -- "Otx2,Oct4" "E130306E05Rik,2810482H01Rik,NF-A1,Oct-1,Oct1,Otf-1,Otf1" ChIP-seq Our results illuminate regulatory mechanisms underlying pluripotency and suggest that capacity of transcription factors such as Otx2 and Oct4 to pioneer new Enhancer sites is highly context dependent. -- -- -- >2KB 55854 E_02_095 24905168 -- mm10 chr5 98310330 98314427 Mouse Mouse Embryonic Stem Cell Low+High throughput "ChIP-seq,RNA-seq" "To address whether changes in enhancer utilization patterns occur in the majority of cells within the differentiating population, we isolated dual transgenic enhancer reporter lines, in which ESC-specific or EpiLC-specific enhancers of interest were cloned upstream of a minimal promoter and one of two distinct fluorescent reporters and integrated into the ESC genome using a PiggyBac transposon system." Enhancer Fgf5 -- "qPCR,IP-Western" "Interrogation of our ChIP-seq datasets revealed presence of a proximal Enhancer (PE) at the Fgf5 locus, which exists in a poised state in ESCs, as well as a cluster of four Enhancers (depicted as E1-E4) located within 25¨C60 kb downstream from the Fgf5 TSS that are unmarked in ESCs, but activated de novo during the transition." "Fgf-5,HBGF-5,angora,go" -- -- -- -- -- -- "Otx2,Oct4" "E130306E05Rik,2810482H01Rik,NF-A1,Oct-1,Oct1,Otf-1,Otf1" ChIP-seq Our results illuminate regulatory mechanisms underlying pluripotency and suggest that capacity of transcription factors such as Otx2 and Oct4 to pioneer new Enhancer sites is highly context dependent. -- -- -- >2KB 58196 E_02_096 24905168 -- mm10 chr5 98253530 98257713 Mouse Mouse Embryonic Stem Cell Low+High throughput "ChIP-seq,RNA-seq" "To address whether changes in enhancer utilization patterns occur in the majority of cells within the differentiating population, we isolated dual transgenic enhancer reporter lines, in which ESC-specific or EpiLC-specific enhancers of interest were cloned upstream of a minimal promoter and one of two distinct fluorescent reporters and integrated into the ESC genome using a PiggyBac transposon system." Enhancer Fgf5 -- "qPCR,IP-Western" "Interrogation of our ChIP-seq datasets revealed presence of a proximal Enhancer (PE) at the Fgf5 locus, which exists in a poised state in ESCs, as well as a cluster of four Enhancers (depicted as E1-E4) located within 25¨C60 kb downstream from the Fgf5 TSS that are unmarked in ESCs, but activated de novo during the transition." "Fgf-5,HBGF-5,angora,go" -- -- -- -- -- -- "Otx2,Oct4" "E130306E05Rik,2810482H01Rik,NF-A1,Oct-1,Oct1,Otf-1,Otf1" ChIP-seq Our results illuminate regulatory mechanisms underlying pluripotency and suggest that capacity of transcription factors such as Otx2 and Oct4 to pioneer new Enhancer sites is highly context dependent. -- -- -- <2KB 1439 E_02_097 24915132 -- mm10 chr2 25423079 25423874 Mouse Th1 Low throughput ChIP "Incontrast, lung fibroblasts, which are of mesenchymal origin andwhich lack Fut7 mRNA and selectin ligand expression, carried adifferential histone modification pattern with an enrichment ofH3K27me3 at the CNS and the Fut7 gene locus and absenceof H3K4me2 marks. This suggests that the pattern of histone mod-ifications might control tissue-specific expression of Fut7, ratherthan the differentiation and activation dependent expression inT cells" Enhancer Fut7 -- "qRT-PCR,Luciferase Reporter Assay" The CNS was cloned in different orientations in the Enhancer position of the pGL3 vector carrying the +728/+1523 construct in promoter position and transfected into Th1 cells. "AI853193,FTVII,Fuc-TVII,FucT-VII" -- -- -- -- -- -- Ep300 "A430090G16,A730011L11,KAT3B,p300,p300 HAT" ChIP "As our histone ChIP data as well as analysis of genome-wide p300 binding data published by Vahedi et al.(2012) revealed distribution ofthe active histone mark as well as enrichment of p300 binding (data not shown) beyond the homology region, i.e. the CNS, we extended the CNS by about 300 bp upstream and 150 bp downstream of the original construct." -- -- -- <2KB 1127 E_02_098 25030696 -- mm10 chr17 31172998 31173656 Mouse COS-1 Low throughput "3C,ChIP,RNA-seq,GRO-seq,Luciferase Reporter Assay" "Interestingly, primarily RXR-induced eRNA production could be detected on an enhancer assigned to Vegfa or Tgm2, while an enhancer of Abcg1 also showed robust LXR ligand activation, as expected. These data suggested that the eRNAs can be easily validated and show ligand induction similar to the regulated genes and therefore most likely are linked." Enhancer Abcg1 3C qRT-PCR "Box plot representation of the distribution of interaction frequency of Abcg1 B1 Enhancer (chr17: 31,172,998¨C31,173,656) and Vegfa B1 Enhancer (chr17: 45,890,060¨C45,890,829) determined by 3C-seq." "AW413978,Abc8,White" -- -- -- "A set of angiogenic genes, including Vegfa, has liganded RXR-controlled enhancers and provides the macrophage with a novel inducible program" "3C,3C-seq" "Using chromosome conformation capture (3C) and 3C combined with sequencing (3C-seq), and we show that selected long-range Enhancers communicate with promoters via stable or RXR-induced loops and that some of the Enhancers interact with each other, forming an interchromosomal network." Rxra "9530071D11Rik,Nr2b1,RXRalpha1" "RNA-seq,ChIP-seq,GRO-seq,3C-seq" "In our studies, we tried to solve these issues by combining RNA-seq, ChIP-seq, GRO-seq (global run-on sequencing), and 3C-seq (chromosome conformation capture [3C] combined with sequencing) in a highly integrated way to unravel the mechanism of RXR-induced transcriptional events in mouse bone marrow-derived macrophages (BMDMs) and, as the result of the process,discovered and validated a novel biological activity promoted by the receptor." -- -- -- >2KB 115634 E_02_099 25030696 -- mm10 chr17 45890060 45890829 Mouse COS-1 Low throughput "3C,ChIP,RNA-seq,GRO-seq,Luciferase Reporter Assay" "Interestingly, primarily RXR-induced eRNA production could be detected on an enhancer assigned to Vegfa or Tgm2, while an enhancer of Abcg1 also showed robust LXR ligand activation, as expected. These data suggested that the eRNAs can be easily validated and show ligand induction similar to the regulated genes and therefore most likely are linked." Enhancer Vegfa 3C qRT-PCR "Box plot representation of the distribution of interaction frequency of Abcg1 B1 Enhancer (chr17: 31,172,998¨C31,173,656) and Vegfa B1 Enhancer (chr17: 45,890,060¨C45,890,829) determined by 3C-seq." "Vegf,Vpf" -- -- -- "A set of angiogenic genes, including Vegfa, has liganded RXR-controlled enhancers and provides the macrophage with a novel inducible program" "3C,3C-seq" "Using chromosome conformation capture (3C) and 3C combined with sequencing (3C-seq), and we show that selected long-range Enhancers communicate with promoters via stable or RXR-induced loops and that some of the Enhancers interact with each other, forming an interchromosomal network." Rxra "9530071D11Rik,Nr2b1,RXRalpha1" "RNA-seq,ChIP-seq,GRO-seq,3C-seq" "In our studies, we tried to solve these issues by combining RNA-seq, ChIP-seq, GRO-seq (global run-on sequencing), and 3C-seq (chromosome conformation capture [3C] combined with sequencing) in a highly integrated way to unravel the mechanism of RXR-induced transcriptional events in mouse bone marrow-derived macrophages (BMDMs) and, as the result of the process,discovered and validated a novel biological activity promoted by the receptor." -- -- -- >2KB 126547 E_02_100 25153150 -- mm10 chrx 140806807 140808327 Mouse F9 Low throughput "Luciferase Reporter Assay,ChIP" "This suggests that the -65 to +20 bp region is a minimal promoter region for Tex13 gene expression, and various additional repressive and Enhancer regions are present in the -1500 to +20 bp region." Enhancer Tex13b -- "PCR,Transfection" "This suggests that the -65 to +20 bp region is a minimal promoter region for Tex13 gene expression, and various additional repressive and Enhancer regions are present in the -1500 to +20 bp region." "Tex13,4933403J23Rik" -- -- -- -- -- -- -- -- -- -- -- -- -- <2KB 740 E_02_101 25185713 -- mm10 chr2 91067374 91069374 Mouse "Bone Marrow Cell,Leukemic Stem Cell,Hematopoietic Stem Cell,416B" Low throughput "ChIP,3C,Transgenic mice" "Mechanistically, by using a knockin mouse model in which all three Runx binding sites in the214kb enhancer of PU.1 are disrupted, we observed failure to form chromosomal interactions between the PU.1 enhancer and its proximal promoter. Consequently, decreased PU.1 levels resulted in diminished long-term HSC function through HSC exhaustion, which could be rescued by reintroducing a PU.1 transgene." Enhancer Spi1 3C "RT-PCR,PCR,Transgenic mice" "Mechanistically, by using a knockin mouse model in which all three Runx binding sites in the 214kb Enhancer of PU.1 are disrupted, we observed failure to form chromosomal interactions between the PU.1 Enhancer and its proximal promoter.Runx-dependent PU.1 chromatin interaction and transcription of PU.1 are essential for both normal and leukemia stem cells." "Dis-1,Dis1,PU.1,Sfpi-1,Sfpi1,Spi-1,Tcfpu1,Tfpu.1" Leukemia DOID:1240 D007938 -- -- -- "Runx1,Runx2,Runx3" "AML1,CBF-alpha-2,Cbfa2,Pebp2a2,Pebpa2b,AML3,Cbf,Cbfa-1,Cbfa1,LS3,Osf2,PEBP2aA,Pebp2a1,Pebpa2a,AML2,Cbfa3,Pebp2a3,Rx3" ChIP "ChIP analyses of total bone marrow cells con?rmed the loss of Runx binding to the 214kb URE in PU.1-URE-mRunx mice (supplemental Figure 1C). Importantly, PU.1 mRNA levels in HSCs of PU.1-URE-mRunxmicewere reduced by 72%in comparisonwith controls (WT), greater than the average reduction of 63% observed in Runx1 knockout mice (Figure 1D). These results revealed the major role of the URE Runx sites for PU.1 transcription in HSCs." -- -- -- >2KB 14007 E_02_102 25223790 -- mm10 chr17 35502632 35504632 Mouse Embryonic Stem Cell Low throughput "Luciferase Reporter Assay,ChIP,ChIP-qPCR" We first investigated activation of the Oct4 enhancer by luciferase reporter assay 48 h after transfection of TALE-A and dCas9-A/gRNA in MEFs. These luciferase constructs contain the 2.4 kb region covering all three upstream regulatory elements of the Oct4 locus Enhancer Oct4 CRISPR/Cas9 "qRT-PCR,Luciferase Reporter Assay" The Oct4 luciferase assay reporter constructs carried the genomic DNA 2.4 kb upstream of the Oct4 transcrip_x0002_tion start site (TSS). The region encompasses the 1.7 kb distal and proximal Enhancers and the 0.2 kb promoter. "NF-A3,Oct-3,Oct-3/4,Oct-4,Oct3,Oct3/4,?Oct4,Otf-3,Otf-4,Otf3,Otf3-rs7,Otf3g,Otf4" -- -- -- -- -- -- "Klf4,Oct4,Nanog,Sox2" "EZF,Gklf,Zie,NF-A3,Oct-3,Oct-3/4,Oct-4,Oct3,Oct3/4,Oct4,Otf-3,Otf-4,Otf3,Otf3-rs7,Otf3g,Otf4,2410002E02Rik,ENK,ecat4,Sox-2,lcc,ysb" ChIP-seq "To address this possibility, we reviewed the ChIP-seq information of several pluripotency transcription factors, including KLF4,OCT4,NANOG and SOX2 at the Nanog 5kb upstream Enhancer region(42) and found that the Site 2 (targeted by both TALE and dCas9) was surrounded by the predicted KLF4 and NANOG binding sites." -- -- -- >2KB 2400 E_02_103 25223790 -- mm10 chr6 122700483 122702483 Mouse Embryonic Stem Cell Low throughput "Luciferase Reporter Assay,ChIP,ChIP-qPCR" "We also designed gRNA constructs and TALE-As to target the Nanog 5 kb upstream enhancer. Similar to the Oct4 locus, dCas9-As could bind their targeted regions and effectively activate the luciferase reporter carrying the 5 kb upstream enhancer " Enhancer Nanog CRISPR/Cas9 "qRT-PCR,Luciferase Reporter Assay" "For Nanog luciferase assay reporter, the ¡«1.0 kb DNA frag_x0002_ment of the Nanog 5 kb Enhancer (?5145 to ?4154) was cloned into a mini promoter luciferase vector." "2410002E02Rik,ENK,ecat4" -- -- -- -- -- -- "Klf4,Oct4,Nanog,Sox2" "EZF,Gklf,Zie,NF-A3,Oct-3,Oct-3/4,Oct-4,Oct3,Oct3/4,Oct4,Otf-3,Otf-4,Otf3,Otf3-rs7,Otf3g,Otf4,2410002E02Rik,ENK,ecat4,Sox-2,lcc,ysb" ChIP-seq "To address this possibility, we reviewed the ChIP-seq information of several pluripotency transcription factors, including KLF4,OCT4,NANOG and SOX2 at the Nanog 5kb upstream Enhancer region(42) and found that the Site 2 (targeted by both TALE and dCas9) was surrounded by the predicted KLF4 and NANOG binding sites." -- -- -- >2KB 6005 E_02_104 25249570 -- mm10 chr9 99131669 99132862 Mouse Ventricular Cardiomyocytes (NRVMs) Low+High throughput "Luciferase Reporter Assay,ChIP-seq" "To measure the transcriptional activity of the YAP-bound region of Pik3cb, which we refer to as the Pik3cb enhancer, we cloned a 552 bp genomic DNA fragment containing the conserved element and potential TEAD binding sites into a minimal promoter luciferase reporter construct. Co-transfection with Yap in NRVMs showed that Yap stimulates activity of the enhancer by ~5-fold." Enhancer Pik3cb -- "qRT-PCR,ChIP-qPCR" "To measure the transcriptional activity of the YAP-bound region of Pik3cb, which we refer to as the Pik3cb Enhancer, we cloned a 552 bp genomic DNA fragment containing the conserved element and potential TEAD binding sites into a minimal promoter luciferase reporter construct." "1110001J02Rik,AI447572,p110beta" Heart Injuries -- D006335 "YAP and TEAD occupied a conserved Enhancer within the first intron of Pik3cb, and this Enhancer drove YAP_x0002_dependent reporter gene expression." "ChIP-seq,ChIP-qPCR,Luciferase Reporter Assay" "To measure the transcriptional activity of the YAP-bound region of Pik3cb, which we refer to as the Pik3cb Enhancer, we cloned a 552 bp genomic DNA fragment containing the conserved element and potential TEAD binding sites into a minimal promoter luciferase reporter construct. Co-transfection with Yap in NRVMs showed that Yap stimulates activity of the Enhancer by ~5-fold. " Yap1 "AI325207,Yap,Yap65,Yki,Yorkie" "ChIP-seq,ChIP-qPCR,Luciferase Reporter Assay" "The HL1 ChIP-seq data revealed a YAP-bound sequence residing in the first intron of Pik3cb (Fig. 2A). We validated YAP binding to the identified sequence by ChIP-qPCR,using a pair of primers spanning the YAP bound sequence and a control pair recognizing a sequence 1.3 kb away." -- -- -- >2KB 93864 E_02_105 25263596 -- mm10 chr1 182851627 182852853 Mouse Tet2-/- Low+High throughput "ChIP-seq,ChIP" "We therefore hypothesized that loss of Tet2 could lead to reduced oxidation and increased 5mC at enhancers. Indeed, hypermethylated DMRs (hyper-DMRs) in Tet2?/? cells exhibit several hallmarks of enhancers including: evolutionary sequence conservation,enrichment for enhancer chromatin marks H3K4me1 and H3K27ac, and significant overlap with co-activator p300 binding sites (o/e = 5.9, p < 1E-200), DNase I hypersensitive sites(o/e = 4.5, p < 1E-200), and predicted enhancers(o/e = 7.1, p < 1E-200)" Enhancer Lefty1 -- "PCR,Transgenic mice" "Indeed,an Enhancer that physically interacts with the developmental gene Left-Right Determination Factor 1 (Lefty1) is hypermethylated and hypoacetylated in Tet6?/? cells,potentially explaining the significantly decreased expression of this gene." "AI450052,Ebaf,Leftb,Stra3,Tgfb4,lefty,lefty-1" -- -- -- -- -- -- "Oct4,Sox4,Nanog" "EZF,Gklf,Zie,NF-A3,Oct-3,Oct-3/4,Oct-4,Oct3,Oct3/4,Oct4,Otf-3,Otf-4,Otf3,Otf3-rs7,Otf3g,Otf4,2410002E02Rik,ENK,ecat4,Sox-2,lcc,ysb" ChIP-seq "Weaker occupancy of the ES-cell core transcription factors (TFs) OCT4,SOX6,and NANOG than those that do not change DNA methylation status." -- -- -- >2KB 1917202 E_02_106 25263596 -- mm10 chr1 182852133 182854320 Mouse Tet2-/- Low+High throughput "ChIP-seq,ChIP" "We therefore hypothesized that loss of Tet2 could lead to reduced oxidation and increased 5mC at enhancers. Indeed, hypermethylated DMRs (hyper-DMRs) in Tet3?/? cells exhibit several hallmarks of enhancers including: evolutionary sequence conservation,enr" Enhancer Lefty1 -- "PCR,Transgenic mice" "Indeed,an Enhancer that physically interacts with the developmental gene Left-Right Determination Factor 1 (Lefty1) is hypermethylated and hypoacetylated in Tet7?/? cells,potentially explaining the significantly decreased expression of this gene." "AI450052,Ebaf,Leftb,Stra3,Tgfb4,lefty,lefty-1" -- -- -- -- -- -- "Oct4,Sox4,Nanog" "EZF,Gklf,Zie,NF-A3,Oct-3,Oct-3/4,Oct-4,Oct3,Oct3/4,Oct4,Otf-3,Otf-4,Otf3,Otf3-rs7,Otf3g,Otf4,2410002E02Rik,ENK,ecat4,Sox-2,lcc,ysb" ChIP-seq "Weaker occupancy of the ES-cell core transcription factors (TFs) OCT4,SOX7,and NANOG than those that do not change DNA methylation status." -- -- -- >2KB 1918189 E_02_107 25263596 mm10 chr1 182856227 182857187 Mouse Tet2-/- Low+High throughput "ChIP-seq,ChIP" "We therefore hypothesized that loss of Tet2 could lead to reduced oxidation and increased 5mC at enhancers. Indeed, hypermethylated DMRs (hyper-DMRs) in Tet4?/? cells exhibit several hallmarks of enhancers including: evolutionary sequence conservation,enr" Enhancer Lefty1 -- "PCR,Transgenic mice" "Indeed,an Enhancer that physically interacts with the developmental gene Left-Right Determination Factor 1 (Lefty1) is hypermethylated and hypoacetylated in Tet8?/? cells,potentially explaining the significantly decreased expression of this gene." "AI450052,Ebaf,Leftb,Stra3,Tgfb4,lefty,lefty-1" -- -- -- -- -- -- "Oct4,Sox4,Nanog" "EZF,Gklf,Zie,NF-A3,Oct-3,Oct-3/4,Oct-4,Oct3,Oct3/4,Oct4,Otf-3,Otf-4,Otf3,Otf3-rs7,Otf3g,Otf4,2410002E02Rik,ENK,ecat4,Sox-2,lcc,ysb" ChIP-seq "Weaker occupancy of the ES-cell core transcription factors (TFs) OCT4,SOX5,and NANOG than those that do not change DNA methylation status." -- -- -- >2KB 1921669 E_02_108 25263596 -- mm10 chr11 35178343 35180368 Mouse Tet2-/- Low+High throughput "ChIP-seq,ChIP" "We therefore hypothesized that loss of Tet2 could lead to reduced oxidation and increased 5mC at enhancers. Indeed, hypermethylated DMRs (hyper-DMRs) in Tet5?/? cells exhibit several hallmarks of enhancers including: evolutionary sequence conservation,enrInstance"": 858, ""TopActivityInstance"": 856, ""Success"": true}" Enhancer Slit3 -- "PCR,Transgenic mice" "we also differentiated a previously reported Tet2 knockdown mESC line and performed quantitative real-time PCR analysis. Consistent with our observations in TET2?/? mESCs, we also find delayed induction of the three marker genes slit3, lmo4, and irx3" "Slil2,Slit1,b2b2362.1Clo" -- -- -- -- -- -- "Oct4,Sox2,Nanog" "2810482H01Rik,NF-A1,Oct-1,Oct1,Otf-1,Otf1,Sox-2,lcc,ysb,2410002E02Rik,ENK,ecat4" ChIP-seq "Weaker occupancy of the ES-cell core transcription factors (TFs) OCT4,SOX2,and NANOG than those that do not change DNA methylation status." -- -- -- >2KB 57901 E_02_109 25263596 -- mm10 chr11 35813868 35814943 Mouse Tet2-/- Low+High throughput "ChIP-seq,ChIP" "We therefore hypothesized that loss of Tet2 could lead to reduced oxidation and increased 5mC at enhancers. Indeed, hypermethylated DMRs (hyper-DMRs) in Tet6?/? cells exhibit several hallmarks of enhancers including: evolutionary sequence conservation,enr" Enhancer Slit3 -- "PCR,Transgenic mice" "we also differentiated a previously reported Tet2 knockdown mESC line and performed quantitative real-time PCR analysis. Consistent with our observations in TET2?/? mESCs, we also find delayed induction of the three marker genes slit3, lmo4, and irx3" "Slil2,Slit1,b2b2362.1Clo" -- -- -- -- -- -- "Oct4,Sox4,Nanog" "EZF,Gklf,Zie,NF-A3,Oct-3,Oct-3/4,Oct-4,Oct3,Oct3/4,Oct4,Otf-3,Otf-4,Otf3,Otf3-rs7,Otf3g,Otf4,2410002E02Rik,ENK,ecat4,Sox-2,lcc,ysb" ChIP-seq "Weaker occupancy of the ES-cell core transcription factors (TFs) OCT4,SOX4,and NANOG than those that do not change DNA methylation status." -- -- -- >2KB 692951 E_02_110 25271055 -- mm10 chr11 102006458 102008458 Mouse Osteocyte Cell Lines Low throughput "Luciferase Reporter Assay,ChIP" "Three lines of evidence support a direct role for HDAC5 in regulating MEF2C activity at the +45 kB SOST enhancer over the course of osteocyte differentiation. First, HDAC5 shRNA causes increased activity of this element, but not of the proximal SOST promoter, in luciferase assays. MEF2C shRNA reduces the activity of this enhancer. Second, HDAC5 overexpression dose-dependently inhibits the activity of a MEF2-driven reporter from the desmin and the SOST¡¯9¡¯ (+45kB) enhancer. Third, endogenous HDAC5 association with this region in control (but not HDAC5 shRNA) Ocy454 cells can be detected by ChIP." Enhancer Sost -- "ChIP,qRT-PCR,Western blot" "Using Chromatin immunoprecipitation,we have mapped endogenous MEF2C binding in the SOST gene to a distal intergenic Enhancer 45 downstream from the transcription start site." 5430411E23Rik -- -- -- -- -- -- Mef2c "5430401D19Rik,9930028G15Rik,AV011172,Mef2" "ChIP,PCR" We performed Chromatin immunoprecipitation (ChIP) to determine MEF2C occupancy in Ocy454 cell cultured at 37¡ãC for 14 days (a time point in which SOST expression is High throughput). -- -- -- >2KB 45001 E_02_111 25321476 -- mm10 chr10 34385685 34385834 Mouse MCT Cells Low throughput "Transgenic mice,ChIP,qRT-PCR,EMSA" "These results show that multiple copies of the enhancer (150-bp Col10a1 distal promoter) mediates higher level and cellspecific reporter activity, whereas the 10-kb Col10a1 promoter/intronic element contains nonspecific regulatory elements,in addition to the 150-bp enhancer." Enhancer Col10a1 -- "ChIP,qRT-PCR,EMSA" Top panel displays Col10a1 gene structure and the 10 -kb promoter and intronic element.Positions of the 150-bp (?4296 to ?4147 bp) Col10a1 cis-Enhancer (purple bar) and its 330-bp (?220 to +110 bp) basal promoter (red bar) are shown. "Col10,Col10a-1" Skeletal Diseases -- -- -- -- -- Runx2 "AML3,Cbf,Cbfa-1,Cbfa1,LS3,Osf2,PEBP2aA,Pebp2a1,Pebpa2a" "qRT-PCR,EMSA" "The qRT-PCR was performed to examine the mRNA transcript level of marker genes Col10a1 and Runx2 in MCT cells. As illustrated, both Col10a1 and Runx2 are significantly upregulated in hypertrophic MCT cells compared with that in proliferative MCT cells." -- -- -- >2KB 4220 E_02_112 25369933 -- mm10 chr15 63254341 63256341 Mouse T-acute lymphoblastic leukemia (T-ALL) Low+High throughput "ChIP-seq,EMSA,3C" "Notch1 is depleted from the NDME site by short-term gammasecretase inhibitor (GSI) treatment, and loading and unloading of Notch1 is associated with rapid changes in H3K27ac, features that characterize genomic Notch1 binding sites that dynamically regulate gene expression.Notch1 also bound the NDME in primary murine T-ALL cells and in DN3 thymocytes, a stage of T-cell development marked by high levels of Notch1 activation" Enhancer Myc 3C "ChIP,qRT-PCR,Luciferase Reporter Assay" "Schematic of the Luciferasegene constructs including the NDME WT and mutant sequences.To test for chromatin looping between the NDME and the Myc promoter, chromatin conformation capture (3C) assays were carried out in T6E cells." "AU0167572,Niard,Nird,bHLHe39,?Myc" Acute Lymphoblastic Leukemia DOID:9952 D054198 Altered long-range enhancer activity can mediate resistance to targeted therapies and provide a mechanistic rationale for combined targeting of Notch and Brd4 in leukemia. 3C "To determine whether Notch signaling was required to maintain the interaction between the Myc promoter and the NDME,3C analysis was performed in T6E cells cultured in the presence or absence of GSI." Notch "9930111A19Rik,Mis6,N1,Tan1,lin-12" "ChIP-seq,3C" Analysis of the ChIP-seq data revealed an RBPJ/Notch1 binding site associated with high levels of H3K4me1 marks located 1.43 Mb 3¡ä of the Myc promoter (Fig. 4A) that shares sequence homology with the murine NDME. -- -- -- >2KB 1270001 E_02_113 25403490 -- mm10 chr7 53527011 53528648 Mouse C2C12 Low throughput "ChIP,PCR" "To gain insight into the mechanism by which MUNC facilitates muscle differentiation, we examined by ChIP the binding of MyoD and myogenin to the regulatory sites known to bind the two transcription factors, the core enhancer region (CER) at -20kb relative to MyoD TSS and theDRR (at -5kb relative to MyoD TSS), and to the sites in the -0.5kb region of the Myogenin promoter" Enhancer Myod -- "Western blot,qRT-PCR,Immunofluorescence" "RNAs 2 and 3 were of particular interest be_x0002_cause they were located upstream of the TSS of MyoD,a master regulator of skeletal muscle differentiation (12),with ncRNA 2 overlapping a previously known MyoD Enhancer element,known as the distal regulatory region (DRR)." "AI503393,MYF3,MyoD,?Myod-1,bHLHc1" -- -- -- -- -- -- Myod1 "AI503393,MYF3,MyoD,Myod-1,bHLHc1" ChIP "To gain insight into the mechanism by which MUNC facilitates muscle differentiation,we examined by ChIP the binding of MyoD and myogenin to the regulatory sites known to bind the two transcription factors,the core en_x0002_hancer region (CER) at kb _x0006_20 relative to MyoD TSS and the DRR,(at kb _x0006_5 relative to MyoD TSS),and to the sites in the kb _x0006_0.5 region of the Myogenin promoter." -- -- -- >2KB 7151357 E_02_114 25453760 -- mm10 chr2 167687771 167689503 Mouse Monocytes Low+High throughput "ChIP-seq,ATAC-seq" analysis of active enhancers (H3K27ac) revealed the same relationship between H3K27ac intensity and cell type specific genes. ChIP-Seq of in vivo splenic pDCs showed similar patterns to our in vitro pDC model with respect to H3K4me1 and H3K27ac in enhancer regions associated to pDC and moDC specific genes Enhancer Cebpb -- "qPCR,RNA-seq,Western blot" "Taken together,our results suggest that Irf8 and Cebpb form a double negative feedback loop and a positive self-auto-regulatory loop.Such a composite self-reinforcing negative feedback loop confers bi-stability,leading to either expression of one TF or the other;as a consequence, the entire epigenetic landscape is directed towards either a pDC or moDC Enhancer state." "C/EBPbeta,CRP2,IL-6DBP,LAP,LIP,NF-IL6,NF-M,Nfil6" -- -- -- -- -- -- "Tcf4,Spib,Bcl11a" "5730422P05Rik,ASP-I2,E2-2,E2.2,ITF-2,ITF-2b,ITF2,ME2,MITF-2A,MITF-2B,SEF-2,SEF2,SEF2-1,TFE,Tcf-4,bHLHb19,Spi-B,2810047E18Rik,BCL-11A,Ctip1,D930021L15Rik,Evi9,Evi9a,Evi9b,Evi9c,mKIAA1809" "ATAC-seq,ChIP-seq" "To better focus on open chromatin accessible to TF binding within these Enhancers, we narrowed down our search to overlapping peak of ATAC-seq signal." -- -- -- <2KB 277 E_02_115 25453760 -- mm10 chr4 66837166 66852551 Mouse Dendritic Cells Low+High throughput "ChIP-seq,ATAC-seq" analysis of active enhancers (H3K27ac) revealed the same relationship between H3K27ac intensity and cell type specific genes. ChIP-Seq of in vivo splenic pDCs showed similar patterns to our in vitro pDC model with respect to H3K4me1 and H3K28ac in enhance Enhancer Tlr4 -- "qPCR,RNA-seq,Western blot" "Specifically,we profiled genome-wide epigenetic modifications corresponding to candidate Enhancers (monomethylation of histone 3 lysine 4,H3K4me1),active Enhancers (H3K27ac) and promoter regions (H3K4me3) in both moDCs and pDCs." "Lps,Ly87,Ran/M1,Rasl2-8" -- -- -- -- -- -- "Tcf4,Spib,Bcl11a" "5730422P05Rik,ASP-I2,E2-2,E2.2,ITF-2,ITF-2b,ITF2,ME2,MITF-2A,MITF-2B,SEF-2,SEF2,SEF2-1,TFE,Tcf-4,bHLHb19,Spi-B,2810047E18Rik,BCL-11A,Ctip1,D930021L15Rik,Evi9,Evi9a,Evi9b,Evi9c,mKIAA1809" ChIP "Molecularly,pDCs display many features of lymphocytes such as the B cell marker (B220),nucleic acid-sensing TLRs (TLR7,9) and transcription factors (Tcf4,SpiB,Bcl11a),whereas cDCs and moDCs express high levels of receptors that sense bacterial components (TLR 2,4) and inflammatory response genes." -- -- -- >2KB 17309 E_02_116 25453760 -- mm10 chr8 120737580 120746913 Mouse Monocytes Low+High throughput "ChIP-seq,ATAC-seq" analysis of active enhancers (H3K27ac) revealed the same relationship between H3K27ac intensity and cell type specific genes. ChIP-Seq of in vivo splenic pDCs showed similar patterns to our in vitro pDC model with respect to H3K4me1 and H3K29ac in enhance Enhancer Irf8 -- "qPCR,RNA-seq,Western blot" "Taken together,our results suggest that Irf8 and Cebpb form a double negative feedback loop and a positive self-auto-regulatory loop.Such a composite self-reinforcing negative feedback loop confers bi-stability,leading to either expression of one TF or the other;as a consequence, the entire epigenetic landscape is directed towards either a pDC or moDC Enhancer state." "AI893568,ICSBP,IRF-8,Icsbp1,Myls" -- -- -- -- -- -- "Tcf4,Spib,Bcl11a" "5730422P05Rik,ASP-I2,E2-2,E2.2,ITF-2,ITF-2b,ITF2,ME2,MITF-2A,MITF-2B,SEF-2,SEF2,SEF2-1,TFE,Tcf-4,bHLHb19,Spi-B,2810047E18Rik,BCL-11A,Ctip1,D930021L15Rik,Evi9,Evi9a,Evi9b,Evi9c,mKIAA1809" "ATAC-seq,ChIP-seq" "To better focus on open chromatin accessible to TF binding within these Enhancers, we narrowed down our search to overlapping peak of ATAC-seq signal." -- -- -- >2KB 5890 E_02_117 25486239 ARC-specific Enhancer mm10 chr1 133303739 133325138 Mouse Kisspeptin-Immunoreactive Cell Low+High throughput "DNase-seq,Immunofluorescence,Luciferase Reporter Assay,PCR,3C" "Using open Chromatin mapping by DNase-seq in HTE cell,the core of DHS-44 was localized to 600 at hg 19,Chr7:117075400-117076000.This region was marked by enrichment of H3K27Ac in 16HBE14o-cell,consistent with DHS-44 encompassing an active Enhancer element." Enhancer Kiss1 3C Transgenic mice "The present study aimed to determine ARC-specific enhancer(s) of the Kiss1 gene by an in vivo reporter assay.The 3C assay was performed to detect interactions between the Kiss1 promoter region and the 5 -upstream region, the latter of which was identified as an ARCspecific enhancer in the present study" "kisspeptin,metastatin" -- -- -- The 5'-upstream region of Kiss1 locus functions as an enhancer for ARC Kiss1 gene expression in mice. 3C "In addition,the current 3C and ER ChIP assays suggest that the Chromatin loop formation between the ARC-specific Enhancer and the promoter region of Kiss1 gene and unoccupied ER recruitment to the putative Enhancer region are involved in Kiss1 gene expression in the ARC" Eral1 "2610524P08Rik,9130407C09Rik,AU019798,Era,M-ERA,MERA-S,MERA-W" ChIP The binding of ERa in the 5' upstream region (Figure 4) was determined by a ChIP assay with ERa antibody. -- -- -- >2KB 7447 E_02_118 25486255 -- mm10 chr3 34743178 34776096 Mouse Embryonic Stem Cell Low+High throughput "ChIP-seq,RNA-seq" "We examined ENCODE ChIP-seq data from 23 different mouse tissues or cell types focusing on the Sox2 locus. Using H3K27ac, a histone mark for active enhancers, we observed a distal SE that is only present in mouse ESC lines, which is approximately 100kb downstream from the Sox2 locus. This sequence spans a relatively large 13kb region, and corresponds to a recently defined super-enhancer or stretch enhancer." Super-Enhancer Sox2 CRISPR/Cas9 PCR We analyzed epigenomic data within the 1.5 Mb gene-desert regions around the Sox2 gene and identified a 13kb-long super-Enhancer (SE) located 100kb downstream of Sox2 in mouse ESCs. "Sox-2,lcc,ysb" -- -- -- "SE is occupied by Oct4, Sox2, Nanog,and the mediator complex, and physically interacts with the Sox2 locus via DNA looping" "RT-qPCR,ChIP-seq" "To test the in vivo function of Sox2-SEdistal, we sought to delete the entire Enhancer sequence from the endogenous locus. Given that the SE spans a,13kb region, deletion of this region using conventional methods would be very inefficient.Therefore, we explored whether the recently developed CRISPR technology could be utilized to delete this large non-coding sequence in mouse ESCs." "Oct4,Sox2,Nanog" "2810482H01Rik,NF-A1,Oct-1,Oct1,Otf-1,Otf1,Sox-2,lcc,ysb,2410002E02Rik,ENK,ecat4" ChIP-seq "First, we observed strong H3K27ac signal at this SE in two additional mouse ES cell lines (F123 and J1), indicating that usage of this SE is conserved across different mice strains.Previously published ChIP-seq data shows that Sox2-SEdistal is occupied by Oct4,Sox2 and Nanog." -- -- -- >2KB 109643 E_02_119 25487574 -- mm10 chr11 98979280 98979880 Mouse COS-7 Low throughput "Luciferase Reporter Assay,ChIP" "The Cx30.2-Luc reporter construct was generated by subcloning a 4.1-kb Cx30.2 enhancer fragment (-4.1 to 0.0 relative to the translation start site) using KpnI-XhoI into pGL2 E1b LUC, which contains a minimal TATA box, followed by the luciferase coding sequence." Enhancer Gjd3 -- "Western blot,ChIP,qRT-PCR" "Consistent with our in vitro data, MyoR associated more strongly with the Cx30.2 Enhancer in the AVC relative to the atria and ventricles. Furthermore, sequen_x0002_tial ChIP experiments with MyoR and Gata4 antibodies demon_x0002_strated that both MyoR and Gata4 simultaneously occupy the Cx30.2 Enhancer in vivo." "Gja11,Gjc1,cx30.2" -- -- -- -- -- -- "Msc,Tbx5,Nkx2-5" "MyoR,bHLHa22,Tbx5,Csx,Nkx-2.5,Nkx2.5,tinman" "Luciferase Reporter Assay,ChIP,qRT-PCR" Human and mouse genetic studies have demonstrated that the transcription factors Tbx5 and Nkx2.5 function during development to specify lower AV nodal cells and promote their morphogenesis. -- -- -- >2KB 2599 E_02_120 25487574 Cx30.2 Enhancer mm10 chr14 63196014 63196614 Mouse Cx30.2+ Cells Low throughput "Luciferase Reporter Assay,ChIP" "The Cx30.2-Luc reporter construct was generated by subcloning a 4.1-kb Cx30.2 enhancer fragment (-4.1 to 0.0 relative to the translation start site) using KpnI-XhoI into pGL2 E1b LUC, which contains a minimal TATA box, followed by the luciferase coding sequence." Enhancer Gata4 -- "Western blot,ChIP,qRT-PCR" Schematic depiction of the Cx30.2-Luciferase construct used in panel B that contains the Cx30.2 Enhancer fused to a minimal TATA box and Luciferase coding sequence. The 4.1- Enhancer fragment encompasses the _x0004_2.9/_x0004_2.3 region that contains the minimal AVC-specific Cx30.2 Enhancer. Gata-4 -- -- -- -- -- -- Msc "MyoR,bHLHa22" ChIP Coimmunoprecipitation analysis between MyoR and Gata4. Myc-Gata4 and either Flag vector alone or Flag-MyoR were cotransfected into COS7 cell. -- -- -- >2KB 2599 E_02_121 25505291 ECR2 Enhancer mm10 chr4 136148484 136149239 Mouse Hep G2 Low throughput Luciferase Reporter Assay "Because enhancers were previously described for Id3 (32), we searched for evolutionally conserved regions that may function as enhancers and enable Smad2/3-dependent upregulation of Id3 in macrophages. Using ECR browser, we found two conserved regions, ECR1 located between -3177 and -2660 bp upstream of transcription start and ECR2 located between +4517 and 4662 bp downstream of the gene . ECR1 overlaps with the enhancer described by Shepherd et al. whereas ECR2 has not been described to date.Luciferase activity of pGL3-ID3prom ECR1 ECR2 plasmid cotransfected with plasmids containing BMP-specific Smad1 and Smad5." Enhancer Id3 -- "siRNA,RT-PCR" "To assess the effect of chromatin structure on Id3 gene expression, we used one pan¨C-HDAC inhibitor (TSA) and two selective HDAC inhibitors: MS-275, an inhibitor of HDAC1, and Apicidin, an inhibitor of HDAC2 and HDAC3." "Hlh462,Idb3,bHLHb25" -- -- -- -- -- -- Tgfb1 "TGF-beta1,TGFbeta1,Tgfb,Tgfb-1" Western blot "Western blot analysis of Smad phosphorylation upon stimulation of primary Human monocytederived macrophages with TGF-b1,BMPs,and their combinations." -- -- -- >2KB 5041 E_02_122 25505291 ECR1 Enhancer mm10 chr4 136140654 136141162 Mouse Hep G2 Low throughput Luciferase Reporter Assay "Because enhancers were previously described for Id3 (32), we searched for evolutionally conserved regions that may function as enhancers and enable Smad2/3-dependent upregulation of Id3 in macrophages. Using ECR browser, we found two conserved regions, ECR1 located between -3177 and -2660 bp upstream of transcription start and ECR2 located between +4517 and 4662 bp downstream of the gene . ECR1 overlaps with the enhancer described by Shepherd et al. whereas ECR2 has not been described to date.Luciferase activity of pGL3-ID3prom ECR1 ECR2 plasmid cotransfected with plasmids containing BMP-specific Smad1 and Smad5." Enhancer Id3 -- "siRNA,RT-PCR" "To assess the effect of chromatin structure on Id3 gene expression, we used one pan¨C-HDAC inhibitor (TSA) and two selective HDAC inhibitors: MS-275, an inhibitor of HDAC1, and Apicidin, an inhibitor of HDAC2 and HDAC4." "Hlh462,Idb3,bHLHb25" -- -- -- -- -- -- Tgfb1 "TGF-beta1,TGFbeta1,Tgfb,Tgfb-1" Western blot "Western blot analysis of Smad phosphorylation upon stimulation of primary Human monocytederived macrophages with TGF-b1,BMPs,and their combinations." -- -- -- >2KB 2913 E_02_123 25548254 E8VI Enhancer mm10 chr6 71341692 71345212 Mouse CD4+ T Cell Low throughput "ChIP,PCR" "Strikingly, 6 ECRs (ECR-2, -3, -6, -7, -8, and -10) overlapped with previously identified Cd8 enhancers. This indicated a good correlation between the location of already described Cd8 cis-regulatory elements and the presence of ECRs." Enhancer "Cd8a,Cd8b1" -- "Transgenic mice,PCR,ChIP" "ChIP assays revealed that E8VI was bound by Runx/CBFb complexes and Bcl11b,and the analysis of Runx3-null CD8+T cell showed that E8VI activity was,in part,dependent on Runx3." "BB154331,Ly-2,Ly-35,Ly-B,Lyt-2,Cd8b,Ly-3,Ly-C,Lyt-3" -- -- -- -- -- -- Bcl11b "9130430L19Rik, AI604821, B630002E05Rik, BCL-11B, Ctip2, Rit1" ChIP "ChIP assays revealed that E8VI was bound by Runx/CBFb complexes and Bcl11b,and the analysis of Runx3-null CD8+T cell showed that E8VI activity was,in part,dependent on Runx3." -- -- -- >2KB 29974 E_02_124 25582196 -- mm10 chr10 62893206 62923461 Mouse Embryonic Stem Cell Low+High throughput "ChIP-seq,Luciferase Reporter Assay" "To identify cis-regulatory elements in Tet1, we surveyed its epigenome in mouse ESCs using ChIP-seq data sets.The combinatorial binding profiles of pluripotency factors, Mediator,and cohesin units have previously delineated a superenhancer domain in Tet1, a 15.2-kb region that spans from exon 1c at the distal end to a conserved noncoding sequence (CNS) upstream of the CDS ChIP-seq showed strong binding profiles of Oct4 within the CNS,as previously shown, but also several more strong peaks in the distal region between exons 1b and 1c." Super-Enhancer Tet1 -- "PCR,EMSA" "Here we defined the promoter and Enhancer domains in Tet1 and Tet2.Within a 15- ¡°superEnhancer¡± of Tet1,there are two transcription start sites (TSSs) with different activation patterns during development." "2510010B09Rik,AA517754,BB001228,Cxxc6,D10Ertd17e,LCX,mKIAA1676" -- -- -- -- -- -- "Oct4,Sox2" "2810482H01Rik,NF-A1,Oct-1,Oct1,Otf-1,Otf1,Sox-2,lcc,ysb" EMSA Contribution of Oct and Sox motifs in Tet1 and Tet2 enhancer fragments. -- -- -- >2KB 103765 E_02_125 25582196 -- mm10 chr3 133531418 133563926 Mouse Embryonic Stem Cell Low+High throughput "ChIP-seq,Luciferase Reporter Assay" "To identify cis-regulatory elements in Tet1, we surveyed its epigenome in mouse ESCs using ChIP-seq data sets.The combinatorial binding profiles of pluripotency factors, Mediator,and cohesin units have previously delineated a superenhancer domain in Tet2,?" Super-Enhancer Tet2 -- "PCR,EMSA" "A second TSS downstream,associated with a constitutively weak CpG-rich promoter,is activated by a neighboring Enhancer in naive embryonic stem cell (ES cells) and primed epiblast-like cell (EpiLCs)." "Ayu17-449,E130014J05Rik,mKIAA1546" -- -- -- -- -- -- "Oct4,Sox2" "2810482H01Rik,NF-A1,Oct-1,Oct1,Otf-1,Otf1,Sox-2,lcc,ysb" EMSA Contribution of Oct and Sox motifs in Tet1 and Tet2 enhancer fragments. -- -- -- >2KB 83996 E_02_126 25605944 -- mm10 chr12 3944001 3947078 Mouse "AtT-20,Pituitary Corticotrope Cell" Low+High throughput "ChIP-seq,3C,ChIP" "Further, we have defined the subset of the enhancers that have active chromatin markers (H3K27ac and p300).Motif enrichment analysis of H3K27ac+ p300+ enhancers revealed enrichment of binding sites for the CCCTC-binding factor (CTCF), neurofibromin 1 (NF1), bHLH, FORKHEAD, X-BOX,and T-BOX families of transcription factors. Although members of the CTCF and NF1 family are general cellular transcription factors, the factors in the bHLH and other families may be critical for lineage development and enhancer programming in corticotropes." Enhancer Pomc "3C,CRISPR/Cas9" "qPCR,ChIP,GRO-seq" We found that enhancer:promoter interactions between LDB1-bound enhancers and the adjacent promoters of POMC and lung carcinoma myc related oncogene 1 (Lmyc1) genes were greatly diminished following knockdown of Ldb1. "ACTH,BE,Beta-LPH,Clip,Gamma-LPH,Npp-1,Pomc1,alpha-MSH,alphaMSH,beta-MSH,gamma-MSH,?Pomc" -- -- -- LDB1-mediated enhancer:promoter looping appears to be required for both activation and repression of these target genes "qPCR,3C,GRO-seq" "Together,our data reveal that Enhancer-bound LDB1 has a role in mediating the actions of both activating and repressive Enhancers in a cell type_x0002_specific gene-regulation program." -- -- -- -- -- -- -- >2KB 9404 E_02_127 25605944 -- mm10 chr12 3956137 3961266 Mouse "AtT-20,Pituitary Corticotrope Cell" Low+High throughput "ChIP-seq,3C,ChIP" "Further, we have defined the subset of the enhancers that have active chromatin markers (H3K27ac and p300).Motif enrichment analysis of H3K27ac+ p300+ enhancers revealed enrichment of binding sites for the CCCTC-binding factor (CTCF), neurofibromin 1 (NF1), bHLH, FORKHEAD, X-BOX,and T-BOX families of transcription factors. Although members of the CTCF and NF1 family are general cellular transcription factors, the factors in the bHLH and other families may be critical for lineage development and enhancer programming in corticotropes." Enhancer Pomc "3C,CRISPR/Cas9" "qPCR,ChIP,GRO-seq" We found that enhancer:promoter interactions between LDB1-bound enhancers and the adjacent promoters of POMC and lung carcinoma myc related oncogene 1 (Lmyc1) genes were greatly diminished following knockdown of Ldb2. "ACTH,BE,Beta-LPH,Clip,Gamma-LPH,Npp-1,Pomc1,alpha-MSH,alphaMSH,beta-MSH,gamma-MSH,?Pomc" -- -- -- LDB1-mediated enhancer:promoter looping appears to be required for both activation and repression of these target genes "qPCR,3C,GRO-seq" "Together,our data reveal that Enhancer-bound LDB1 has a role in mediating the actions of both activating and repressive Enhancers in a cell type_x0002_specific gene-regulation program." -- -- -- -- -- -- -- >2KB 3758 E_02_128 25605944 -- mm10 chr12 3938522 3942093 Mouse Pituitary Corticotrope Cell Low+High throughput "ChIP-seq,3C,ChIP" "Further, we have defined the subset of the enhancers that have active chromatin markers (H3K27ac and p300).Motif enrichment analysis of H3K27ac+ p300+ enhancers revealed enrichment of binding sites for the CCCTC-binding factor (CTCF), neurofibromin 1 (NF1), bHLH, FORKHEAD, X-BOX,and T-BOX families of transcription factors. Although members of the CTCF and NF1 family are general cellular transcription factors, the factors in the bHLH and other families may be critical for lineage development and enhancer programming in corticotropes." Enhancer Pomc "3C,CRISPR/Cas9" "qPCR,ChIP,GRO-seq" We found that enhancer:promoter interactions between LDB1-bound enhancers and the adjacent promoters of POMC and lung carcinoma myc related oncogene 1 (Lmyc1) genes were greatly diminished following knockdown of Ldb3. "ACTH,BE,Beta-LPH,Clip,Gamma-LPH,Npp-1,Pomc1,alpha-MSH,alphaMSH,beta-MSH,gamma-MSH,?Pomc" -- -- -- LDB1-mediated enhancer:promoter looping appears to be required for both activation and repression of these target genes "ChIP,PCR" "Together,our data reveal that Enhancer-bound LDB1 has a role in mediating the actions of both activating and repressive Enhancers in a cell type_x0002_specific gene-regulation program." -- -- -- -- -- -- -- >2KB 14636 E_02_129 25644605 Ucp1 Enhancer mm10 chr8 83287848 83288068 Mouse Brown Fat Cell Low throughput "ChIP,RT-PCR" we hypothesized that PRDM16 might be recruited to the enhancer region of the Ucp1 gene through the interaction with PGC-1a. This possibility was tested in an immobilized template assay with purified proteins and a beadimmobilized DNA fragment spanning the 220-bp Ucp1 enhancer. Enhancer Ucp1 -- "ChIP,RT-PCR" "Overall, these results indicate that the ZF1 domain is required for the recruitment of PRDM16 to the Ucp1 enhancer through MED1 and PGC-1a." "AI385626,Slc25a7,Ucp" -- -- -- -- -- -- Prdm16 "5730557K01Rik,csp1,mel1" ChIP "To determine whether PRDM16 is recruited to the Enhancer following Ucp1 induction, we treated Med1 +/+MEFs that stably express PPARg and PRDM16 with forskolin and performed a chromatin immunoprecipitation (ChIP) assay with an antibody specific for PRDM16." -- -- -- >2KB 2389 E_02_130 25651906 MyoD distal Enhancer mm10 chr7 46351197 46355693 Mouse C2C12 Low throughput "Luciferase Reporter Assay,ChIP" "C2C12 myoblasts stably carrying the P6P (?5870 to +95) -driven reporter (MyoD-P6P-luc) or another composite reporter,MyoD-PE-luc, containing P6P plus the DE (?25277 to?20780) were treated with Wnt3a medium. We found that Wnt3a marginally, but significantly, activated MyoDP6P-luc activity; however, Wnt3a strongly activated MyoD-PE-luc, suggesting that one or multiple Wnt-response elements(WREs) are located within the DE." Enhancer Myod1 -- "Western blot,qRT-PCR" "A distal Enhancer(DE) centred at ? 20 kb, when combined with a ? 2.5k proximal pro_x0002_moter, can also recapitulate human MyoD expression in vivo,demonstrating its critical role in determining the spatiotemporal expression of human MyoD." "AI503393,MYF3,MyoD,?Myod-1,bHLHc1" -- -- -- -- -- -- Wnt3a "Wnt-3a,vt" Luciferase Reporter Assay "Serial deletion mutants of DE region were generated later to identify the minimal WRE,and we found that a region of 3 kb (fragment 10, ?24 to ?21 kb)in DE was required to retain the full Wnt3a response.Deletion of sequence at either the 5_x0002_ end (fragment 8) or the 3_x0002_ end(fragment 7) significantly reduced its Wnt3a response, comparedwith fragment." -- -- -- >2KB 23028 E_02_131 25652130 Grhl3 Enhancer mm10 chr4 135548688 135550688 Mouse NMuMG Low throughput "Luciferase Reporter Assay,3C" We reported previously that intron 2 of Cdh1 is essential for proper E-cad expression.In order to analyze this large DNA sequence(>40 kb) for putative enhancers we focused on evolutionary conserved sequences. Enhancer Grhl3 3C "ChIP,qPCR" "Weshow thatCdh1 activity during MET isgovernedby 33 two Enhancers at +7.8 and at +11.5 within intron 2 that are activated by binding of Grhl3 and Hnf4¦Á, 34 respectively." "AI561912,Get1,Som,Tfcp2l4,ct" -- -- -- -- -- -- -- -- -- -- -- -- -- >2KB 7801 E_02_132 25652130 -- mm10 chr8 106613868 106615868 Mouse NMuMG Low throughput "Luciferase Reporter Assay,3C" We reported previously that intron 2 of Cdh1 is essential for proper E-cad expression.In order to analyze this large DNA sequence(>41 kb) for putative enhancers we focused on evolutionary conserved sequences. Enhancer Cdh1 3C "ChIP,qPCR" "Weshow that Cdh1 activity during MET is governedby two Enhancers at +7.8 and at +11.5 within intron 2 that are activated by binding of Grhl3 and Hnf4¦Á,respectively." "AA960649,ARC-1,E-cad,Ecad,L-CAM,UVO,Um" -- -- -- -- -- -- Hnf4a "HNF-4,Hnf4lpha,MODY1,Nr2a1,TCF-14,Tcf14,Hnf4a" "Luciferase Reporter Assay,ChIP" "Using luciferase reporter assays, we first tested whether Hnf4¦Á could activate Cl.5 in NMuMG cells.In contrast to Grhl3 enrichment on the Cl.3 intronic sites, Hnf4¦Á recruitment to the chromatin was detectable already in untreated NMuMG cells under steady state conditions, suggesting a distinct function for this Enhancer." -- -- -- Intron 11519 E_02_133 25652130 -- mm10 chr8 109134068 109136068 Mouse NMuMG Low throughput "Luciferase Reporter Assay,3C" We reported previously that intron 2 of Cdh1 is essential for proper E-cad expression.In order to analyze this large DNA sequence(>42 kb) for putative enhancers we focused on evolutionary conserved sequences. Enhancer Cdh1 3C "ChIP,qPCR" "We show that Cdh1 activity during MET is governedby two Enhancers at +7.8 and at +11.5 within intron 2 that are activated by binding of Grhl3 and Hnf4¦Á,respectively." "AA960649,ARC-1,E-cad,Ecad,L-CAM,UVO,Um" -- -- -- Enhancer cooperativity as a novel mechanism underlying the transcriptional regulation of E-cadherin during mesenchymal to epithelial transition. Transgenic mice Enhancer cooperativity as a novel mechanism underlying the transcriptional regulation of E-cadherin during mesenchymal to epithelial transition. Grhl3 "AI561912,Get1,Som,Tfcp2l4,ct" "3C,qPCR,ChIP" A mutation at site 7.8b alone was sufficient to abrogate Grhl3-dependent luciferase reporter activation and also the lacZ-reporter activity in transgenic embryos. -- -- -- Intron 2531719 E_02_134 25652130 -- mm10 chr8 106610150 106612150 Mouse Non-tumorigenic Mouse Mammary Gland Cells Low throughput "Luciferase Reporter Assay,3C" We reported previously that intron 2 of Cdh1 is essential for proper E-cad expression.In order to analyze this large DNA sequence(>43 kb) for putative enhancers we focused on evolutionary conserved sequences. Enhancer Cdh1 3C "ChIP,qPCR" "We show that Cdh1 activity during MET is governed by two Enhancers at +7.8 kb and at +11.5 kb within intron 2 that are activated by binding of Grhl3 and Hnf4¦Á,respectively." "AA960649,ARC-1,E-cad,Ecad,L-CAM,UVO,Um" -- -- -- Enhancer cooperativity as a novel mechanism underlying the transcriptional regulation of E-cadherin during mesenchymal to epithelial transition. ChIP Enhancer cooperativity as a novel mechanism underlying the transcriptional regulation of E-cadherin during mesenchymal to epithelial transition. "Grhl3,Hnf4a" "AI561912,Get1,Som,Tfcp2l4,ct,HNF-4,Hnf4lpha,MODY1,Nr2a1,TCF-14,Tcf14,Hnf4a" "Luciferase Reporter Assay,ChIP" "Using luciferase reporter assays,we first tested whether Hnf4¦Á could activate Cl.5 in NMuMG cells.We found that indeed Hnf4¦Á was able to exert a robust enhancing effect on the E-cad Cl.5 reporter construct.Two different mutations in the putative Hnf4¦Á binding site decreased the basal activity of this construct and inhibited its Hnf4¦Á dependent activation." -- -- -- >2KB 7801 E_02_135 25775043 -- mm10 chr17 35499359 35507923 Mouse Mouse Embryonic Stem Cell Low throughput "CRISPR/Cas9,3C,Luciferase Reporter Assay,ATAC-seq" "We generated mouse embryonic stem cells (mESCs) expressing versions of Neisseria meningitidis (Nm) dCas9 fused with LSD1, a non-effector BirA affinity tag (BAT), or a KRAB repressor and used a viral delivery system for sgRNAs. We first targeted the well-characterized cis-regulatory region of Oct417, a factor critical for the ESC state. Oct4 expression is regulated by a proximal enhancer (OPE) active in epiblast cells, and a distal enhancer (ODE) active in mESCs and cells of the inner cell mass. Targeting of LSD1 to the ODE resulted in loss of Oct4 expression and appearance of OCT4-negative colonies accompanied by phenotypic changes compared to control dCas9-BAT cells targeted to the same enhancer." Enhancer Oct-4 "3C,CRISPR/Cas9" "PCR,ChIP" "For follow-up, we focused on the putative Enhancer with the highest differential score,Enh1.Test of Enh1 in a reporter assay confirmed its ability to enhance expression at comparable levels to an Oct4 DE sequence.." "NF-A3,Oct-3,Oct-3/4,Oct-4,Oct3,Oct3/4,?Oct4,Otf-3,Otf-4,Otf3,Otf3-rs7,Otf3g,Otf4" -- -- -- "We conclude that the dCas9-LSD1 fusion protein allows for an effector dependent definition of functional, native enhancer elements that help to maintain a given cellular state. " 3C "We conclude that the dCas9-LSD1 fusion protein allows for an effector dependent definition of functional,native Enhancer elements that help to maintain a given cellular state.Accordingly,dCas9-LSD1 provides a rapid and powerful approach to understanding distal cis-regulatory regions such as Enhancers without major disruption of the local genomic architecture." Tbx3 D5Ertd189e "PCR,Luciferase Reporter Assay,ChIP,3C" "This previously unannotated ESC_x0002_specific Enhancer is positioned ~10kb upstream of the transcription factor Tbx3, a gene previously implicated in the maintenance of pluripotency20. We therefore hypothesized that Enh1 may function in the ESC network by regulating Tbx3 expression." -- -- -- >2KB 2391 E_02_136 25775043 -- mm10 chr5 119654061 119663588 Mouse Mouse Embryonic Stem Cell Low throughput "CRISPR/Cas9,3C,Luciferase Reporter Assay,ATAC-seq" "We generated mouse embryonic stem cells (mESCs) expressing versions of Neisseria meningitidis (Nm) dCas9 fused with LSD2, a non-effector BirA affinity tag (BAT), or a KRAB repressor and used a viral delivery system for sgRNAs. We first targeted the well-c" Enhancer Tbx3 "3C,CRISPR/Cas9" "PCR,ChIP" "This previously unannotated ESC specific Enhancer is positioned ~10kb upstream of the transcription factor Tbx3 (Fig. 1d), a gene previously implicated in the maintenance of pluripotency.We therefore hypothesized that Enh1 may function in the ESC network by regulating Tbx3 expression." D5Ertd189e -- -- -- "We conclude that the dCas9-LSD1 fusion protein allows for an effector dependent definition of functional, native enhancer elements that help to maintain a given cellular state. " 3C "We conclude that the dCas9-LSD1 fusion protein allows for an effector dependent definition of functional,native Enhancer elements that help to maintain a given cellular state.Accordingly,dCas9-LSD1 provides a rapid and powerful approach to understanding distal cis-regulatory regions such as Enhancers without major disruption of the local genomic architecture." Tbx3 D5Ertd189e "PCR,Luciferase Reporter Assay,ChIP,3C" "This previously unannotated ESC_x0002_specific Enhancer is positioned ~10kb upstream of the transcription factor Tbx3, a gene previously implicated in the maintenance of pluripotency20. We therefore hypothesized that Enh1 may function in the ESC network by regulating Tbx3 expression." -- -- -- >2KB 11493 E_02_137 25801169 -- mm10 chr1 13041306 13127367 Mouse Embryonic Stem Cell Low throughput Luciferase Reporter Assay "The five super-enhancers show physical interactions with their respective associated genes and are located within insulated neighborhoods in the ESC genome , suggesting that these genes represent the bona fide physiological targets of the five SEs." Super-Enhancer Prdm14 CRISPR/Cas9 -- "The results showed that deletion of most (12/14) super-enhancer constituents led to reduced expression of the associated gene, while in one case (Prdm14 E5) the deletion caused a small increase in expression of the associated gene" Prdm14 -- -- -- -- -- -- "Oct4,Sox2,Nanog" "2810482H01Rik,NF-A1,Oct-1,Oct1,Otf-1,Otf1,Sox-2,lcc,ysb,2410002E02Rik,ENK,ecat4" ChIP-seq "ChIP-Seq binding profiles for OCT4,SOX2 and NANOG (merged) and Mediator (MED1) at the miR-290-295 locus in ES cells.Enhancer activity measured in luciferase reporter assays in wild type cells and the change in enhancer activity after OCT4 shutdown is plotted for each constituent enhancer within the super-enhancer. The super-enhancer is depicted as a black bar above the binding profiles. The difference in values after OCT4 shutdown is statistically significant for all constituents, except from miR-290-295 M1, M3 and M5." -- -- -- >2KB 29090 E_02_138 25801169 -- mm10 chr17 31794375 31829875 Mouse Embryonic Stem Cell Low throughput Luciferase Reporter Assay "The five super-enhancers show physical interactions with their respective associated genes and are located within insulated neighborhoods in the ESC genome , suggesting that these genes represent the bona fide physiological targets of the five SEs." Super-Enhancer Sik1 CRISPR/Cas9 -- "These five super-enhancers control transcription of Prdm14, miR-290-295, Sik1, Klf2 and Pou5f1 (Oct5), which play important roles in ESC self-renewal, pluripotency and differentiation." "Hrt-20,Msk,Sik,Sik-1,Snf1lk" -- -- -- -- -- -- -- -- -- -- -- -- -- >2KB 32124 E_02_139 25801169 -- mm10 chr17 35501272 35506872 Mouse Embryonic Stem Cell Low throughput Luciferase Reporter Assay "The five super-enhancers show physical interactions with their respective associated genes and are located within insulated neighborhoods in the ESC genome , suggesting that these genes represent the bona fide physiological targets of the five SEs." Super-Enhancer Pou5f1 CRISPR/Cas9 -- Enhancer activity measured in Luciferaseassays in wild type cell and the change in Enhancer activity after OCT4 shutdown is plotted for each constituent Enhancer within the Super-enahncer. "NF-A3,Oct-3,Oct-3/4,Oct-4,Oct3,Oct3/4,Oct4,Otf-3,Otf-4,Otf3,Otf3-rs7,Otf3g,Otf4" -- -- -- -- -- -- -- -- -- -- -- -- -- >2KB 4734 E_02_140 25801169 -- mm10 chr7 3183157 3229407 Mouse Embryonic Stem Cell Low throughput Luciferase Reporter Assay "The five super-enhancers show physical interactions with their respective associated genes and are located within insulated neighborhoods in the ESC genome , suggesting that these genes represent the bona fide physiological targets of the five SEs." Super-Enhancer Mir290-295 CRISPR/Cas9 -- "These five super-enhancers control transcription of Prdm14, miR-290-295, Sik1, Klf2 and Pou5f1 (Oct5), which play important roles in ESC self-renewal, pluripotency and differentiation." "Mir290, Mirn290, mir-290a, mmu-miR-290, mmu-mir-290a" -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- E_02_141 25804738 -- mm10 chr3 127641624 127642545 Mouse Embryonic Stem Cell Low+High throughput "ChIP-qPCR,ChIP-seq,RNA-seq" "A conserved 447?bp genomic fragment within this?Neurog2?3¡ä Foxa1-bound peak showed enhancer activity specifically in midbrain floor plate and also in the cerebral cortex, when analysed in mouse transient transgenic experiments at E12.5" Enhancer Neurog2 -- "Transgenic mice,qRT-PCR" "By contrast, the identification of a Foxa1-bound peak downstream of Neurog2 coding sequences (Neurog2 3¡ä) raised the possibility that Foxa2 might directly activate Neurog2 expression through this putative Enhancer region in embryonic mDA progenitors." "Atoh4,Math4A,bHLHa8,ngn-2,ngn2" -- -- -- -- -- -- "Otx2,Foxa1,Foxa2" "E130306E05Rik,HNF3-beta,HNF3beta,Hnf-3b,Hnf3b,Tcf-3b,Tcf3b,Hnf-3a,Hnf3a,Tcf-3a,Tcf3a" "ChIP-seq,RNA-seq" Our genome-wide Foxa1-bound cis-regulatory sequences from ChIP-Seq and Foxa1/2 candidate target genes from RNA-Seq analyses of embryonic midbrain dopamine cells also provide an excellent resource for probing mechanistic insights into gene regulatory networks involved in the differentiation of midbrain dopamine neurons. -- -- -- >2KB 8941 E_02_142 25810254 -- mm10 chr4 34888479 34889907 Mouse ¦ÁT3-1 Cells Low throughput "qPCR,ChIP,3C" "Using quantitative PCR (qPCR) on DNase-treated reverse-transcribed RNA from a gonadotrope cell line, we amplified fragments between ?5300 and ?3872 bp upstream of the Cga TSS, which includes most of the reported enhancer." Enhancer Cga 3C "qPCR,ChIP" "qPCR analysis confirmed the interaction of the distal enhancer regions (?6.7 to ?6.58 and ?4.9 to ?4.85 kbp) with the proximal Cga promoter in both the treated and untreated cells and,with greater frequency, in WT and eRNA-knockdown cells after normalization to the relative levels of the Fsh¦Â chimeric fragment" "CG-alpha,FSHA,GPHA1,GPHalpha,HCG,LHA,Tsha,aGSU,alpha-GSU,alphaGSU,alphaSU" -- -- -- -- -- -- -- -- -- -- -- -- -- >2KB 4585 E_02_143 26110280 Ccnb1ip1 Enhancer mm10 chr14 50787449 50787634 Mouse Mitotic Cell Low throughput "Luciferase Reporter Assay,ChIP,RT-PCR" "PCR amplification of JF1 genomic DNA using several primer pairs targeted to differentially methylated regions identified a 1.8 kbp genomic segment that was present in the JF1 strain but absent in the B6 strain . Sequence analysis revealed that this 1.8kbp segment consisted of a 1,579 bp interval deleted in the B6 genome, and the remaining 220 bp interval present in the B6 genome but in an inverted orientation." Enhancer Ccnb1ip1 -- "Luciferase Reporter Assay,ChIP,RT-PCR" "We hypothesized the existence of an insulator-type element that prevents the spreadof DNA methylation within the1.8 k segment,and actually identified a 242- and a 185- fragments that were located adjacent to eachother and showed insulatorand Enhancer activities,respectively,in reporter assays. We designated these genomicregions as the Ccnb1ip1 insulator and the Ccnb1ip1Enhancer. The Ccnb1ip1 insulator showed Enhancer-blocking activity in the Luciferaseiferase assays andbarrieractivity in the colony formation assays. Further examination of the Ccnb1ip1locus in other mammalian speciesrevealed that the insulator and Enhancerare High throughput lyconserve damonga wide variety of species,and are located immediately upstream of the transcriptional start site of Ccnb1ip1." "Gm288,Hei10,mei4" -- -- -- -- -- -- -- -- -- -- -- -- -- <2KB 1706 E_02_144 26163528 Id2 Enhancer mm10 chr12 25090158 25090663 Mouse "HCT 116,SW480" Low throughput "Luciferase Reporter Assay,ChIP-PCR,ChIP" "Expression of Id2 was stimulated by Wnt signaling through the enhancer region of the Id2 promoter at the early stage of tumorigenesis in Apc+/¦¤716 (Apc¦¤716) mice. Genetic depletion of Id2 in Apc¦¤716 mice caused ?80% reduction in the number of ileal polyps, but had little effect on tumor size." Enhancer Id2 -- "Transgenic mice,RT-PCR,Western blot" "Expression of Id2 was stimulated by Wnt signaling through the enhancer region of the Id2 promoter at the early stage of tumorigenesis in Apc+/¦¤716 (Apc¦¤716) mice. Genetic depletion of Id2 in Apc¦¤716 mice caused ?80% reduction in the number of ileal polyps, but had little effect on tumor size." "AI255428,C78922,Idb2,bHLHb26" Colorectal Carcinoma DOID:0080199 D015179 -- -- -- Tcf4 "5730422P05Rik,ASP-I2,E2-2,E2.2,ITF-2,ITF-2b,ITF2,ME2,MITF-2A,MITF-2B,SEF-2,SEF2,SEF2-1,TFE,Tcf-4,bHLHb19" "ChIP-PCR,Luciferase Reporter Assay" "To determine if mouse Id2 is also a target gene of Wnt signaling, we performed ChIP-PCR analysis of the binding of Tcf4 to the promoter region of the Id2 gene.Sequencing analysis revealed ten putative Tcf/Lef-binding consensus sequences in the Id2 promoter region spanning from ?4000 to ?1. As expected, Tcf4 bound to the DNA region spanning from ?3300 to ?3146 of the Id2 distal promoter.To test this Tcf4 binding region for enhancer activity, we conducted luciferase reporter assays using the constructs .The putative Id2-enhancer fragment significantly activated Wnt-signaling dependent transcription from the CMV minimal promoter in two colorectal cancer cell lines." -- -- -- >2KB 3387 E_02_145 26303528 -- mm10 chr8 84899088 84902859 Mouse "Murine erythroleukemia (MEL) line 745A,32DEpo1,293T" Low throughput ChIP "We have shown by cotransfection and in vivo analyses that the small 950-bp region adjacent to the EKLF transcription start site is sufficient for tissue-restricted expression and wished to address whether the epigenetic profile of this region supports its importance. Database perusal of modified histone H3 interactions within the critical 950-bp region of the EKLF promoter/enhancer during hematopoiesis shows that levels of the H3K4me1 active enhancer mark increase exactly in parallel with activation of EKLF expression during hematopoiesis , that is, low within the hematopoietic repopulating cells and in myeloid (CMP and MPP) progenitors but high in MEPs and erythroblasts" Enhancer Klf1 -- shRNA "Collectively, these studies demonstrate that DEK is required for EKLF expression and directly interacts in vitro and in vivo with its cognate site in the EKLF EHS1 Enhancer." "Eklf,Nan" -- -- -- -- -- -- -- -- -- -- -- -- -- <2KB 953 E_02_146 26321200 AHF Enhancer mm10 chr13 83661050 83662862 Mouse Embryonic Stem Cell Low throughput "3C-qPCR,3C-seq" "Multiple promoter-interacting elements were detected in the Mef2c lo_x0002_cus, of which some showed a lower signal in Ldb1-deficientEBs (Figure 5B). Importantly, we observed interactions of the AHF enhancer with the promoter area and with the 30 end of the Mef2c gene, which were decreased in Ldb1-deficient EBs(Figure 5B). 3C-qPCR experiments revealed a similar long-range interaction pattern in WT versus Ldb1-/-EBs during the course of cardiac differentiation and in dissected SHF and hearts of E8¨C10.5 embryos, confirming the 3C-seq results (Figure 5C)." Enhancer Mef2c "3C-qPCR,3C-seq" -- "Multiple promoter-interacting elements were detected in the Mef2c lo_x0002_cus, of which some showed a lower signal in Ldb1-deficientEBs (Figure 5B). Importantly, we observed interactions of the AHF enhancer with the promoter area and with the 30 end of the Mef2c gene, which were decreased in Ldb1-deficient EBs(Figure 5B). 3C-qPCR experiments revealed a similar long-range interaction pattern in WT versus Ldb1-/-EBs during the course of cardiac differentiation and in dissected SHF and hearts of E8¨C10.5 embryos, confirming the 3C-seq results (Figure 5C)." "5430401D19Rik,9930028G15Rik,AV011172,Mef2" Heart Failure -- D006333 -- -- -- -- -- -- -- -- -- -- >2KB 157923 E_02_147 26321200 OFTRV Enhancer mm10 chr8 59795280 59797080 Mouse Embryonic Stem Cell Low throughput 3C-qPCR "Using 3C-qPCR anal_x0002_ysis with the Hand2 promoter as a viewpoint, we observed a specific close proximity of the promoter with the OFTRV enhancer in d5 EBs from WT ESCs, but not in d5 EBs from Ldb1-deficient cells (Figure 5A)." Enhancer Hand2 3C-qPCR -- "Using 3C-qPCR anal_x0002_ysis with the Hand2 promoter as a viewpoint, we observed a specific close proximity of the promoter with the OFTRV enhancer in d5 EBs from WT ESCs, but not in d5 EBs from Ldb1-deficient cells (Figure 5A)." "AI225906,AI661148,Ehand2,Hed,Th2,Thing2,bHLHa26,dHAND" Heart Failure -- D006333 -- -- -- -- -- -- -- -- -- -- >2KB 2475198 E_02_148 26364592 CNS1 Enhancer mm10 chr10 78046488 78046636 Mouse Thymic Medullary Epithelial Cells (mTECs) Low throughput qPCR "To determine the in vivo function of the Aire enhancer region,we generated knockout mice (CNS1-KO) with a deletion of 45 bp within CNS1 including both NF-¦ÊB sites.We first studied thymic expression of Aire in purified thymic epithelial cell subsets-cTEC, mTEClo, and mTEChi-from homozygous CNS1-KO mice by qPCR targeting exons 7¨C9. Strik_x0002_ingly, Aire was not expressed in mTEChi from CNS1-KO mice,whereas the gene was highly expressed in mTEChi from WT mice(Fig. 2A)." Enhancer Aire -- "qPCR,Immunofluorescence,RT-PCR" "To determine whether the upstream CNS1 region regulates these variants, we analyzed the expression levels of the majority of exons in the Aire coding region by qPCR.Aire-KO mouse results are shown in Supporting Information Fig.2B), indicating that CNS1 is required for the thymic expression of the majority of, if not all, Aire splice variants.Deficiency of Aire protein expression was confirmed in situ by immunofluorescence staining, which showed no Aire protein in the thymi of CNS1-KO(Fig. 2D). These results established that the CNS1 region is critical for Aire mRNA and protein expression in the mouse thymus." Aire -- -- -- CNS1 is indispensable for RANK-induced Aire expression and that CNS1 is activated by NF-¦ÊB pathway complexes containing RelA. "3C,Luciferase Reporter Assay,EMSA" "To further examine the role of CNS1 in Aire gene regulation, we evaluated the spa_x0002_tial proximity between the CNS1 Enhancer and Aire promoter by chromosome conformation capture (3C) method.This result suggests that these distant regulatory regions are brought close to each other in intact chromatin." Nfkb1 "NF-KB1,NF-kappaB,NF-kappaB1,p105,p50,p50/p105" "Luciferase Reporter Assay,EMSA" "We next analyzed the CNS1 region for its responsiveness to NF-¦ÊB stimulation in luciferase reporter assays in vitro.We inserted a CNS1 fragment containing the two NF-¦ÊB binding sites upstream of the IFN-¦Â minimal promoter and luciferase coding region and then introduced specific mutations into the NF-¦ÊB binding sites A and B,either individually or in combination." -- -- -- >2KB 16541 E_02_149 26443845 CD47 Enhancer mm10 chr16 49740654 49742654 Mouse G1E Cells Low throughput "RT-qPCR,ChIP" "CD47 gene contacts with its distal enhancer at ?80 kb dur_x0002_ing synchronized transcriptional elongation. (A) Map of the CD47 lo_x0002_cus. Blue bars indicate fragments of BglII, and red bars indicate the regions used for ChIP and RT-qPCR." Enhancer Cd47 3C "RT-qPCR,ChIP" "This locus has a well-character- ized Enhancer residing 114 kb upstream of the transcrip- tion start site (TSS),and its coding region encompasses ¡«82 kb,thus lending itself to finely space-and time-re- solved 3C analysis (Jing et al. 2008) No- tably,additional dynamic contacts of the Enhancer with the coding region were observed at the positions of elon- gating RNAPII. Similar dynamic Enhancer gene body con- tacts were identified at the CD47 locus.CD47 gene contacts with its distal enhancer at ?80 kb dur_x0002_ing synchronized transcriptional elongation. (A) Map of the CD47 lo_x0002_cus. Blue bars indicate fragments of BglII, and red bars indicate the regions used for ChIP and RT-qPCR." "9130415E20Rik,AA407862,AI848868,AW108519,B430305P08Rik,IAP,Itgp" -- -- -- -- -- -- -- -- -- -- -- -- -- >2KB 113598 E_02_150 26494787 Nodal proximal epiblast Enhancer (PEE) mm10 chr10 61403948 61408233 Mouse Embryonic Stem Cell Low+High throughput "ChIP,ChIP-seq" "ChIP (red) and input (blue) wiggle plot overlays showing enrichment of Lhx1 ChIP-seq density at Hesx1,Fzd8,Embigin,Nodal,Otx2,and Foxa3. Purple boxes indicate the positions of previously mapped Nodal Enhancer elements." Enhancer Nodal -- "ChIP,ChIP-seq" "Conditional inactivation of Lhx1 dis_x0002_rupts anterior definitive endoderm development and impedes node and midline morphogenesis in part due to severe disturbances in visceral endoderm displacement. Transcriptional profiling and ChIP-seq (chromatin immunopre_x0002_cipitation [ChIP] followed by high-throughput sequencing) experiments identified Lhx1 target genes, including numerous anterior definitive endoderm markers and components of the Wnt signaling pathway. Interestingly, Lhx1-binding sites were enriched at enhancers, including the Nodal-proximal epiblast enhancer element and enhancer regions controlling Otx2 and Foxa2 expression. " Tg.413d -- -- -- -- -- -- "Lhx1,Eomes" "Lim1,C77258,TBR-2,Tbr2" ChIP "Several putative Lhx1 target genes were represented in our ChIP data set. For example, Lhx1 binding was detected at two distinct regions at the Hesx1 locus, including a regulatory element in the 5¡ä untranslated region (containing two Lhx1-binding motifs) and a 3¡ä distal enhancer. Lhx1 ChIP peaks were also present upstream of Embigin exon 1." -- -- -- >2KB 11880 E_02_151 26494787 Nodal Enhancer (NDE) mm10 chr10 61402781 61405448 Mouse Embryonic Stem Cell Low+High throughput "ChIP,ChIP-seq" "ChIP (red) and input (blue) wiggle plot overlays showing enrichment of Lhx1 ChIP-seq density at Hesx1,Fzd8,Embigin,Nodal,Otx2,and Foxa3. Purple boxes indicate the positions of previously mapped Nodal Enhancer elements." Enhancer Nodal -- "ChIP,ChIP-seq" "Conditional inactivation of Lhx1 dis_x0002_rupts anterior definitive endoderm development and impedes node and midline morphogenesis in part due to severe disturbances in visceral endoderm displacement. Transcriptional profiling and ChIP-seq (chromatin immunopre_x0002_cipitation [ChIP] followed by high-throughput sequencing) experiments identified Lhx1 target genes, including numerous anterior definitive endoderm markers and components of the Wnt signaling pathway. Interestingly, Lhx1-binding sites were enriched at enhancers, including the Nodal-proximal epiblast enhancer element and enhancer regions controlling Otx2 and Foxa2 expression. " Tg.413d -- -- -- -- -- -- "Lhx1,Eomes" "Lim1,C77258,TBR-2,Tbr2" ChIP "Several putative Lhx1 target genes were represented in our ChIP data set. For example, Lhx1 binding was detected at two distinct regions at the Hesx1 locus, including a regulatory element in the 5¡ä untranslated region (containing two Lhx1-binding motifs) and a 3¡ä distal enhancer. Lhx1 ChIP peaks were also present upstream of Embigin exon 1." -- -- -- >2KB 13856 E_02_152 26537192 EphA7 Enhancer mm10 chr4 29115647 29140947 Mouse Embryo Low throughput "PCR,Transgenic mice" "Transgenic LacZ reporter analysis, using overlapping EphA7 BACs, was used to narrow down the dorsal midline-specific enhancer, revealing the 25.3 kb genomic region as the enhancer candidate. Strikingly, this genomic DNA was located far downstream of the EphA7 transcription start site, +302.6 kb to +327.9 kb." Enhancer Epha7 -- "PCR,Transgenic mice" "Further enhancer mapping, using comparative genomic analysis and transgenic methods, showed that the 187 bp genomic DNA alone, approximately 305 kb downstream of the EphA7 transcription start site, was sufficient to act as the dorsal midline-specific enhancer of EphA7." "Cek11,Ebk,Ehk3,Hek11,Mdk1" -- -- -- The 187 bp dorsal midline-specific enhancer is critically regulated by homeobox transcription factors during the development of the dien- and mesencephalon. PCR "Importantly, we found that transgenic embryos carrying ECR3, but not ECR1 or 2, displayed LacZ expression in the dorsal midline of the dien- and mesencephalon" Sbe3 ECR3 PCR "To investigate whether the conserved transcription factor binding sites are functionally important for the 187 bp ECR3 enhancer activity, three putative transcription factor binding sites were selected for deletion. The deleted ECR3 DNA was examined for its effect on the enhancer activity by creating transgenic mouse embryos (Figs. 4A and 4B)." -- -- -- >2KB 315250 E_02_153 26537192 EphA7 Enhancer mm10 chr4 29119847 29120047 Mouse Embryo Low throughput "PCR,Transgenic mice" "Transgenic LacZ reporter analysis, using overlapping EphA7 BACs, was used to narrow down the dorsal midline-specific enhancer, revealing the 25.3 kb genomic region as the enhancer candidate. Strikingly, this genomic DNA was located far downstream of the EphA7 transcription start site, +302.6 kb to +327.9 kb." Enhancer Epha7 -- "PCR,Transgenic mice" "Further enhancer mapping, using comparative genomic analysis and transgenic methods, showed that the 187 bp genomic DNA alone, approximately 305 kb downstream of the EphA7 transcription start site, was sufficient to act as the dorsal midline-specific enhancer of EphA7." "Cek11,Ebk,Ehk3,Hek11,Mdk1" -- -- -- The 187 bp dorsal midline-specific enhancer is critically regulated by homeobox transcription factors during the development of the dien- and mesencephalon. PCR "Importantly, we found that transgenic embryos carrying ECR3, but not ECR1 or 2, displayed LacZ expression in the dorsal midline of the dien- and mesencephalon" Sbe3 ECR3 PCR "To investigate whether the conserved transcription factor binding sites are functionally important for the 187 bp ECR3 enhancer activity, three putative transcription factor binding sites were selected for deletion. The deleted ECR3 DNA was examined for its effect on the enhancer activity by creating transgenic mouse embryos (Figs. 4A and 4B)." -- -- -- >2KB 306900 E_02_154 26550034 -- mm10 chr4 55565908 55608014 Mouse Mouse Embryonic Stem Cell Low+High throughput "ChIP-seq,ChIP" "The genome-wide mapping of Tip60 binding in mESCs by chromatin immunoprecipitation (ChIP) coupled with high-throughput sequencing (ChIP-seq) shows that the Tip60 complex is present at promoter regions of predominantly active genes that are bound by RNA polymerase II (Pol II) and contain the H3K4me3 histone mark. The coactivator HAT complexes, Tip60- and Mof (KAT8)- containing (NSL and MSL), show a global overlap at promoters, whereas distinct binding profiles at enhancers suggest different regulatory functions of each essential HAT complex. Interestingly, Tip60 enrichment peaks at about 200 bp downstream of the transcription start sites suggesting a function for the Tip60 complexes in addition to histone acetylation." Super-Enhancer Klf4 -- ChIP-seq "As two of the so-called ¡®super Enhancer¡¯ regions of the Nanog gene and the Enhancer of the Klf4 gene are bound by Tip60,but not by Nsl1 and Msl1(Fig.?7a, b)." "EZF,Gklf,Zie" -- -- -- -- -- -- Kat5 "AI839539,CPLA2,Htatip,Htatip1,PLIP,Tip55,Tip60" "ChIP-qPCR,ChIP-seq" "The validation of several randomly selected ChIP-seq positive sites by ChIP-qPCR indicated specific Tip60 enrichments at these sites, when compared to control IgG ChIP signals and to background enrichment at an intergenic region negative for Tip60 binding (Fig.?1b).These results, together with the ChIP-qPCR validation (Fig.? 1b), indicate that the obtained anti-Tip60 ChIP-seq signal is specific." -- -- -- >2KB 59825 E_02_155 26550034 -- mm10 chr6 122653828 122675804 Mouse Mouse Embryonic Stem Cell Low+High throughput "ChIP-seq,ChIP" "The genome-wide mapping of Tip60 binding in mESCs by chromatin immunoprecipitation (ChIP) coupled with high-throughput sequencing (ChIP-seq) shows that the Tip60 complex is present at promoter regions of predominantly active genes that are bound by RNA polymerase II (Pol II) and contain the H3K4me3 histone mark. The coactivator HAT complexes, Tip60- and Mof (KAT8)- containing (NSL and MSL), show a global overlap at promoters, whereas distinct binding profiles at enhancers suggest different regulatory functions of each essential HAT complex. Interestingly, Tip60 enrichment peaks at about 200 bp downstream of the transcription start sites suggesting a function for the Tip60 complexes in addition to histone acetylation." Super-Enhancer Nanog -- ChIP-seq "As two of the so-called ¡®super Enhancer¡¯ regions of the Nanog gene and the Enhancer of the Klf4 gene are bound by Tip60,but not by Nsl1 and Msl1(Fig.?7a, b)." "2410002E02Rik,ENK,ecat4" -- -- -- -- -- -- "Myc,Stat3,Nfkb1,E2f1,Trp53" "AU0167572,Niard,Nird,bHLHe39,Myc,1110034C02Rik,E2F-1,Tg(Wnt1cre)2Sor,mKIAA4009,AW109958,Aprf,NF-KB1,NF-kappaB,NF-kappaB1,p105,p50,p50/p105,Tp53,bbl,bfy,bhy,p44,p53" ChIP-seq "Mammalian Tip60 has been described as a transcriptional co-activator complex that is supposed to mediate the action of large variety of transcription factors,including nuclear receptors,c-Myc,STAT3,NF-kappaB,E2F1,p55 and others.To further address the global distribution and function of the Tip60 complex in mESCs, we compared high-confidence Tip60 binding sites with marks, which are either associated with active transcription at promoters (Pol II and H3K4me3) or enriched at enhancer sites (H3K4me1 and H3K27ac)." -- -- -- >2KB 42672 E_02_156 26550034 -- mm10 chr6 122696853 122708211 Mouse Mouse Embryonic Stem Cell Low+High throughput "ChIP-seq,ChIP" "The genome-wide mapping of Tip60 binding in mESCs by chromatin immunoprecipitation (ChIP) coupled with high-throughput sequencing (ChIP-seq) shows that the Tip60 complex is present at promoter regions of predominantly active genes that are bound by RNA polymerase II (Pol II) and contain the H3K4me3 histone mark. The coactivator HAT complexes, Tip60- and Mof (KAT8)- containing (NSL and MSL), show a global overlap at promoters, whereas distinct binding profiles at enhancers suggest different regulatory functions of each essential HAT complex. Interestingly, Tip60 enrichment peaks at about 200 bp downstream of the transcription start sites suggesting a function for the Tip60 complexes in addition to histone acetylation." Super-Enhancer Nanog -- ChIP-seq "As two of the so-called ¡®super Enhancer¡¯ regions of the Nanog gene and the Enhancer of the Klf4 gene are bound by Tip60,but not by Nsl1 and Msl1(Fig.?7a, b)." "2410002E02Rik,ENK,ecat4" -- -- -- -- -- -- "Myc,Stat3,Nfkb1,E2f1,Trp53" "AU0167572,Niard,Nird,bHLHe39,Myc,1110034C02Rik,E2F-1,Tg(Wnt1cre)2Sor,mKIAA4009,AW109958,Aprf,NF-KB1,NF-kappaB,NF-kappaB1,p105,p50,p50/p105,Tp53,bbl,bfy,bhy,p44,p53" ChIP-seq "Mammalian Tip60 has been described as a transcriptional co-activator complex that is supposed to mediate the action of large variety of transcription factors,including nuclear receptors,c-Myc,STAT3,NF-kappaB,E2F1,p55 and others.To further address the global distribution and function of the Tip60 complex in mESCs, we compared high-confidence Tip60 binding sites with marks, which are either associated with active transcription at promoters (Pol II and H3K4me3) or enriched at enhancer sites (H3K4me1 and H3K27ac)." -- -- -- >2KB 4956 E_02_157 26595656 -- mm10 chr12 85433906 85439068 Mouse Mouse Cortical Neurons Low+High throughput "ChIP-seq,Luciferase Reporter Assay" "These enhancers, which we identified in primary cultures of mouse cortical neurons, appear to be active in the intact brain throughout development, as all of them were tightly aligned with the chroma_x0002_tin immunoprecipitation (ChIP)-seq peaks of the active enhancer marker, acetylated H3K27 (H3K27ac)(Supplementary Fig. 1)." Enhancer Fos 3C -- "Characterizing individual c-fos Enhancers by chromosome-conformation capture (3C) analysis and an Enhancer reporter assay, we have corroborated the idea that eRNA is a reliable marker for identifying activated Enhancers." "D12Rfj1,c-fos,cFos" -- -- -- These results demonstrate that the combinatorial activation of the c-fosenhancers enables the c-fos gene to be broadly responsive to various signaling pathways. "CRISPRi,RT-qPCR" "We also evaluated the functionality of individual enhancers in their native context by targeted transcription silencing via CRISPR interference (CRISPRi). Using eRNA as a proxy for enhancer activity, we found that distinc_x0002_tive subsets of the c-fos enhancers are activated in the intact brain following chemically induced seizure or light stimulation." "Creb,srf,Mef2a,Mef2d,Mef2c,Npas4" "CREB,CREB-1,MCM1,ADCAD1,RSRFC4,RSRFC9,mef2,C5DELq14.3,DEL5q14.3,Le-PAS,NXF,PASD10,bHLHe79" ChIP-seq "ChIP-_x0002_seq analysis has revealed that the c-fos promoter and enhancers were bound by different combinations of several activity-regulated TFs6 (Supplementary Fig. 1).Knockdown of CREB, MEF2A and NPAS4 significantly impaired c-fos induction by Cl-mediated membrane depolarization (Fig. 4a and Supplementary Fig. 4). Although knockdown of MEF2 family members MEF2D and MEF2C showed a slight decrease in c-fos expression, it was not significant and had a weaker impact than MEF2A knockdown. The decrease in c-fos expression was paralleled with little effect on eRNA transcription (Supplementary Fig. 5)." -- -- -- >2KB 37413 E_02_158 26595656 -- mm10 chr12 85452549 85458571 Mouse Mouse Cortical Neurons Low+High throughput "ChIP-seq,Luciferase Reporter Assay" "These enhancers, which we identified in primary cultures of mouse cortical neurons, appear to be active in the intact brain throughout development, as all of them were tightly aligned with the chroma_x0002_tin immunoprecipitation (ChIP)-seq peaks of the active enhancer marker, acetylated H3K27 (H3K27ac)(Supplementary Fig. 1)." Enhancer Fos 3C -- "Characterizing individual c-fos Enhancers by chromosome-conformation capture (3C) analysis and an Enhancer reporter assay, we have corroborated the idea that eRNA is a reliable marker for identifying activated Enhancers." "D12Rfj1,c-fos,cFos" -- -- -- These results demonstrate that the combinatorial activation of the c-fosenhancers enables the c-fos gene to be broadly responsive to various signaling pathways. "CRISPRi,RT-qPCR" "We also evaluated the functionality of individual enhancers in their native context by targeted transcription silencing via CRISPR interference (CRISPRi). Using eRNA as a proxy for enhancer activity, we found that distinc_x0002_tive subsets of the c-fos enhancers are activated in the intact brain following chemically induced seizure or light stimulation." "Creb,srf,Mef2a,Mef2d,Mef2c,Npas4" "CREB,CREB-1,MCM1,ADCAD1,RSRFC4,RSRFC9,mef2,C5DELq14.3,DEL5q14.3,Le-PAS,NXF,PASD10,bHLHe79" ChIP-seq "ChIP-_x0002_seq analysis has revealed that the c-fos promoter and enhancers were bound by different combinations of several activity-regulated TFs6 (Supplementary Fig. 1).Knockdown of CREB, MEF2A and NPAS4 significantly impaired c-fos induction by Cl-mediated membrane depolarization (Fig. 4a and Supplementary Fig. 4). Although knockdown of MEF2 family members MEF2D and MEF2C showed a slight decrease in c-fos expression, it was not significant and had a weaker impact than MEF2A knockdown. The decrease in c-fos expression was paralleled with little effect on eRNA transcription (Supplementary Fig. 5)." -- -- -- >2KB 18340 E_02_159 26595656 -- mm10 chr12 85466416 85469570 Mouse Mouse Cortical Neurons Low+High throughput "ChIP-seq,Luciferase Reporter Assay" "These enhancers, which we identified in primary cultures of mouse cortical neurons, appear to be active in the intact brain throughout development, as all of them were tightly aligned with the chroma_x0002_tin immunoprecipitation (ChIP)-seq peaks of the active enhancer marker, acetylated H3K27 (H3K27ac)(Supplementary Fig. 1)." Enhancer Fos 3C -- "Characterizing individual c-fos Enhancers by chromosome-conformation capture (3C) analysis and an Enhancer reporter assay, we have corroborated the idea that eRNA is a reliable marker for identifying activated Enhancers." "D12Rfj1,c-fos,cFos" -- -- -- These results demonstrate that the combinatorial activation of the c-fosenhancers enables the c-fos gene to be broadly responsive to various signaling pathways. "CRISPRi,RT-qPCR" "We also evaluated the functionality of individual enhancers in their native context by targeted transcription silencing via CRISPR interference (CRISPRi). Using eRNA as a proxy for enhancer activity, we found that distinc_x0002_tive subsets of the c-fos enhancers are activated in the intact brain following chemically induced seizure or light stimulation." "Creb,srf,Mef2a,Mef2d,Mef2c,Npas4" "CREB,CREB-1,MCM1,ADCAD1,RSRFC4,RSRFC9,mef2,C5DELq14.3,DEL5q14.3,Le-PAS,NXF,PASD10,bHLHe79" ChIP-seq "ChIP-_x0002_seq analysis has revealed that the c-fos promoter and enhancers were bound by different combinations of several activity-regulated TFs6 (Supplementary Fig. 1).Knockdown of CREB, MEF2A and NPAS4 significantly impaired c-fos induction by Cl-mediated membrane depolarization (Fig. 4a and Supplementary Fig. 4). Although knockdown of MEF2 family members MEF2D and MEF2C showed a slight decrease in c-fos expression, it was not significant and had a weaker impact than MEF2A knockdown. The decrease in c-fos expression was paralleled with little effect on eRNA transcription (Supplementary Fig. 5)." -- -- -- >2KB 5907 E_02_160 26595656 -- mm10 chr12 85470646 85474086 Mouse Mouse Cortical Neurons Low+High throughput "ChIP-seq,Luciferase Reporter Assay" "These enhancers, which we identified in primary cultures of mouse cortical neurons, appear to be active in the intact brain throughout development, as all of them were tightly aligned with the chroma_x0002_tin immunoprecipitation (ChIP)-seq peaks of the active enhancer marker, acetylated H3K27 (H3K27ac)(Supplementary Fig. 1)." Enhancer Fos 3C -- "Characterizing individual c-fos Enhancers by chromosome-conformation capture (3C) analysis and an Enhancer reporter assay, we have corroborated the idea that eRNA is a reliable marker for identifying activated Enhancers." "D12Rfj1,c-fos,cFos" -- -- -- These results demonstrate that the combinatorial activation of the c-fosenhancers enables the c-fos gene to be broadly responsive to various signaling pathways. "CRISPRi,RT-qPCR" "We also evaluated the functionality of individual enhancers in their native context by targeted transcription silencing via CRISPR interference (CRISPRi). Using eRNA as a proxy for enhancer activity, we found that distinc_x0002_tive subsets of the c-fos enhancers are activated in the intact brain following chemically induced seizure or light stimulation." "Creb,srf,Mef2a,Mef2d,Mef2c,Npas4" "CREB,CREB-1,MCM1,ADCAD1,RSRFC4,RSRFC9,mef2,C5DELq14.3,DEL5q14.3,Le-PAS,NXF,PASD10,bHLHe79" ChIP-seq "ChIP-_x0002_seq analysis has revealed that the c-fos promoter and enhancers were bound by different combinations of several activity-regulated TFs6 (Supplementary Fig. 1).Knockdown of CREB, MEF2A and NPAS4 significantly impaired c-fos induction by Cl-mediated membrane depolarization (Fig. 4a and Supplementary Fig. 4). Although knockdown of MEF2 family members MEF2D and MEF2C showed a slight decrease in c-fos expression, it was not significant and had a weaker impact than MEF2A knockdown. The decrease in c-fos expression was paralleled with little effect on eRNA transcription (Supplementary Fig. 5)." -- -- -- <2KB 1534 E_02_161 26595656 -- mm10 chr12 85482048 85490068 Mouse Mouse Cortical Neurons Low+High throughput "ChIP-seq,Luciferase Reporter Assay" "These enhancers, which we identified in primary cultures of mouse cortical neurons, appear to be active in the intact brain throughout development, as all of them were tightly aligned with the chroma_x0002_tin immunoprecipitation (ChIP)-seq peaks of the active enhancer marker, acetylated H3K27 (H3K27ac)(Supplementary Fig. 1)." Enhancer Fos 3C -- "Characterizing individual c-fos Enhancers by chromosome-conformation capture (3C) analysis and an Enhancer reporter assay, we have corroborated the idea that eRNA is a reliable marker for identifying activated Enhancers." "D12Rfj1,c-fos,cFos" -- -- -- These results demonstrate that the combinatorial activation of the c-fosenhancers enables the c-fos gene to be broadly responsive to various signaling pathways. "CRISPRi,RT-qPCR" "We also evaluated the functionality of individual enhancers in their native context by targeted transcription silencing via CRISPR interference (CRISPRi). Using eRNA as a proxy for enhancer activity, we found that distinc_x0002_tive subsets of the c-fos enhancers are activated in the intact brain following chemically induced seizure or light stimulation." "Creb,srf,Mef2a,Mef2d,Mef2c,Npas4" "CREB,CREB-1,MCM1,ADCAD1,RSRFC4,RSRFC9,mef2,C5DELq14.3,DEL5q14.3,Le-PAS,NXF,PASD10,bHLHe79" ChIP-seq "ChIP-_x0002_seq analysis has revealed that the c-fos promoter and enhancers were bound by different combinations of several activity-regulated TFs6 (Supplementary Fig. 1).Knockdown of CREB, MEF2A and NPAS4 significantly impaired c-fos induction by Cl-mediated membrane depolarization (Fig. 4a and Supplementary Fig. 4). Although knockdown of MEF2 family members MEF2D and MEF2C showed a slight decrease in c-fos expression, it was not significant and had a weaker impact than MEF2A knockdown. The decrease in c-fos expression was paralleled with little effect on eRNA transcription (Supplementary Fig. 5)." -- -- -- >2KB 12158 E_02_162 26659182 -- mm10 chr2 168744572 168752225 Mouse Induced Pluripotent Stem Cells Low+High throughput "ChIP-seq,ATAC-seq,CRISPR/Cas9" "To validate these observations with an independent, higher resolution method, we performed Assay of Transposase Accessible Chromatin using sequencing (ATAC-seq), which detects integration of the Tn5 transposase in open chromatin regions27 (Extended Data Fig. 7c). Consistent with the SONO-seq data, ATAC-seq analysis of early reprogramming intermediates showed a more accessible chromatin configuration at regulatory regions including ESC-specific enhancers upon suppression of Chaf1a (Fig. 5a; P value <10?15). Moreover, Chaf1a knockdown caused a significant increase in chromatin accessibility across ESC-specific super-enhancers at day 3 of iPSC formation (Supplementary Table 3 and Extended Data Fig. 7d,e; P value <5.3¡Á10?16). Of note, super-enhancers linked to specialized cell types such as macrophages, lymphocytes and muscle cells were also significantly more accessible in Chaf1a depleted reprogramming intermediates compared to controls (Extended Data Fig. 7f). " Super-Enhancer Sall4 -- "ChIP-seq,ATAC-seq" "Moreover,Chaf1a knockdown caused a significant increase in Chromatin accessibility across ES-cell-specific Super-Enhancers at day 3 of iPS cell formation." "5730441M18Rik,AA407717,AL022809,AW536104,C330011P20Rik,C78083,C78563,Tex20" -- -- -- -- -- -- Sox2 "Sox-2,lcc,ysb" "ChIP-seq,ATAC-seq" "Next,we performed ChIP-seq analysis for Sox2 at day 3 of OKSM expression in order to test our hypothesis that increased Chromatin accessibility at Enhancer lements influences reprogramming factor binding. Of the Sox2-bound super-Enhancers unique to CAF-1 knockdown cells, a subset also showed a more accessible chromatin structure by ATAC-seq analysis. " -- -- -- <2KB 68 E_02_163 26659182 -- mm10 chr8 89041889 89090124 Mouse Induced Pluripotent Stem Cells Low+High throughput "ChIP-seq,ATAC-seq,CRISPR/Cas9" "To validate these observations with an independent, higher resolution method, we performed Assay of Transposase Accessible Chromatin using sequencing (ATAC-seq), which detects integration of the Tn5 transposase in open chromatin regions27 (Extended Data Fig. 7c). Consistent with the SONO-seq data, ATAC-seq analysis of early reprogramming intermediates showed a more accessible chromatin configuration at regulatory regions including ESC-specific enhancers upon suppression of Chaf1a (Fig. 5a; P value <10?15). Moreover, Chaf1a knockdown caused a significant increase in chromatin accessibility across ESC-specific super-enhancers at day 3 of iPSC formation (Supplementary Table 3 and Extended Data Fig. 7d,e; P value <5.3¡Á10?16). Of note, super-enhancers linked to specialized cell types such as macrophages, lymphocytes and muscle cells were also significantly more accessible in Chaf1a depleted reprogramming intermediates compared to controls (Extended Data Fig. 7f). " Super-Enhancer Sall1 -- "ChIP-seq,ATAC-seq" "Next,we performed ChIP-seq analysis for Sox2 at day 3 of OKSM expression in order to test our hypothesis that increased Chromatin accessibility at Enhancer lements influences reprogramming factor binding. Of the Sox2-bound super-Enhancers unique to CAF-1 knockdown cells, a subset also showed a more accessible chromatin structure by ATAC-seq analysis (for example, Sall1; Fig. 5c and Supplementary Table 4)." Msal-3 -- -- -- -- -- -- Sox2 "Sox-2,lcc,ysb" "ChIP-seq,ATAC-seq" "Next,we performed ChIP-seq analysis for Sox2 at day 3 of OKSM expression in order to test our hypothesis that increased Chromatin accessibility at Enhancer lements influences reprogramming factor binding. Of the Sox2-bound super-Enhancers unique to CAF-1 knockdown cells, a subset also showed a more accessible chromatin structure by ATAC-seq analysis." -- -- -- >2KB 38766 E_02_164 26663721 -- mm10 chr12 85515101 85543901 Mouse Macrophage Low+High throughput ChIP-seq "To identify the sites involved in the GC response in the two species,Chromatin immunoprecipitation for GR and sequencing (ChIP-seq) was performed 2 h after dexamethasone treatment in both mBMDM and hMDM. Representative UCSC browser tracks for GR binding in mBMDM and hMDM are shown in Fig. 2A and 2C.ChIP-seq data tracks from the UCSC browser for the Fos ¨C Jdp2 region, for GR binding in mBMDM. Data from ChIP with anti-GR antibodies after treatment with 100nM dexamethasone for 2h (Dex GR IP), input material (Dex input) and immunoprecipitated material from a vehicle treated control (Vehicle GR IP) are shown. Enriched motifs found de novo within GR bound sites in mBMDM and hMDM." Enhancer Fos-Jdp2? -- ChIP-seq "To identify the sites involved in the GC response in the two species,Chromatin immunoprecipitation for GR and sequencing (ChIP-seq) was performed 2 h after dexamethasone treatment in both mBMDM and hMDM. Representative UCSC browser tracks for GR binding in mBMDM and hMDM are shown in Fig. 2A and 2C.ChIP-seq data tracks from the UCSC browser for the Fos ¨C Jdp2 region, for GR binding in mBMDM. Data from ChIP with anti-GR antibodies after treatment with 100nM dexamethasone for 2h (Dex GR IP), input material (Dex input) and immunoprecipitated material from a vehicle treated control (Vehicle GR IP) are shown. Enriched motifs found de novo within GR bound sites in mBMDM and hMDM." "D12Rfj1,c-fos,cFos,Jundm2,Jundp2,TIF" -- -- -- -- -- -- -- -- -- -- -- -- -- >2KB 55601 E_02_165 26673693 TRA Enhancer mm10 chr14 54227135 54228033 Mouse P5424 Low+High throughput "ChIP-seq,ChIP" "We found that eRNAs essentially reca_x0002_pitulated Pol II patterns during DN to DP transition as wellas those of Ets1 in DP only, suggesting highest enhancer ac_x0002_tivity in class 4 at the DN stage and classes 4¨C5 at the DP stage (Figure 3C). Thus, distal Ets1 enhancers broadly re_x0002_cruit Pol II that is able to generate paused transcripts.Significance of gene expression differences by increasing Ets1-fold change. Expression fold changes for all classes, shown as a boxplot. Significance levels for each comparison are indicated above, with 1 star denoting P < 0.05, 2 stars denoting P < 0.01 and 3 stars denoting P < 0.005." Enhancer Dad1 -- "ChIP-seq,Western blot,ChIP-qPCR" "We further validated the dy_x0002_namic recruitment of TCF1 and Runx1 by ChIP-qPCR at the Tcra Enhancer, which is bound by similar levels of Ets1 in both DN and DP thymocytes.UCSC genome browser screenshot of the Tcra (left) and Cd2 loci (right)showing increased H3K4me1 signal at the Ets1 binding site following knockdown in the P5424 cell line." AI323713 -- -- -- -- -- -- "Ets1,Tcf1,Runx1" "AI196000,AI448617,D230050P06,Ets-1,Tpl1,p54,vs,AI465550,TCF-1,Tcf1,AML1,CBF-alpha-2,Cbfa2,Pebp2a2,Pebpa2b" "ChIP-qPCR,Western blot" "We further validated the dynamic recruitment of TCF1 and Runx1 by ChIP-qPCR at the Tcra Enhancer, which is bound by similar levels of Ets1 in both DN and DP thymocytes." -- -- -- >2KB 7900 E_02_166 26673693 E8II Enhancer mm10 chr6 71321750 71323696 Mouse P5424 Low+High throughput "ChIP-seq,ChIP" "We found that eRNAs essentially reca_x0002_pitulated Pol II patterns during DN to DP transition as wellas those of Ets1 in DP only, suggesting highest enhancer ac_x0002_tivity in class 4 at the DN stage and classes 4¨C5 at the DP stage (Figure 3C). Thus, distal Ets1 enhancers broadly re_x0002_cruit Pol II that is able to generate paused transcripts.Significance of gene expression differences by increasing Ets1-fold change. Expression fold changes for all classes, shown as a boxplot. Significance levels for each comparison are indicated above, with 1 star denoting P < 0.05, 2 stars denoting P < 0.01 and 3 stars denoting P < 0.005." Enhancer Cd8b1 -- "ChIP-seq,Western blot,ChIP-qPCR" "As exemplified at the E8II Enhancer of the Cd8 locus, and at the Tcra and Tcrb Enhancers,Ets1 in fact seemed to recruit Pol II as well as promoting High throughputer H3K4me3 in some cases." "Cd8b,Ly-3,Ly-C,Lyt-3" -- -- -- -- -- -- "Ets1,Tcf1,Runx1" "AI196000,AI448617,D230050P06,Ets-1,Tpl1,p54,vs,AI465550,TCF-1,Tcf1,AML1,CBF-alpha-2,Cbfa2,Pebp2a2,Pebpa2b" ChIP-qPCR "We further validated the dynamic recruitment of TCF1 and Runx1 by ChIP-qPCR at the Tcra Enhancer,which is bound by similar levels of Ets1in both DN and DP thymocytes." -- -- -- <2KB 88 E_02_167 26748758 -- mm10 chr2 172513228 172520500 Mouse Mouse Embryonic Stem Cell Low+High throughput "ChIP-seq,RT-qPCR" "To test if Foxd3 was indeed able to silence na?ve pluripotency enhancers, we evaluated the impact of Foxd3 overexpression on some of these regulatory elements. tetOn Foxd3 mESC were treated with Dox and the levels of various histone modifications (i.e. H3K27ac, H3K27me2, H3K4me1, H3K4me2) at selected enhancers were evaluated by ChIP-qPCR. Compared to control cells, tetOn Foxd3 cells displayed lower levels of H3K27ac and H3K4me2 (active enhancers) , increased H3K27me2 (inactive enhancers) and minor changes in H3K4me1 levels (active and inactive enhancers)at most of the analysed na?ve enhancers (Figure 4B-C, Figure S5A-B)." Enhancer Tfap2C -- "ChIP-seq,RT-qPCR" "ChIP-Seq profiles generated in Foxd3-FH mES cells with anti-HA and anti-Flag antibodies around a representative locus (i.e.,Tfap2c)." "AA409384,AP2gamma,Ap-2.2,Stra2,Tcfap2c" -- -- -- -- -- -- Foxd3 "CWH3,Genesis,Hfh2" ChIP-qPCR "To test if Foxd3 was indeed able to silence naive pluripotency enhancers, we evaluated the impact of Foxd3 overexpression on some of these regulatory elements. tetOn Foxd3 mESCs were treated with Dox, and the levels of various histone modifications (i.e., H3K27ac, H3K27me2, H3K4me1, and H3K4me2) at selected enhancers were evaluated by ChIP-qPCR. Compared to control cells, tetOn Foxd3 cells displayed lower levels of H3K27ac and H3K4me2 (active enhancers) , increased H3K27me2 (inactive enhancers) and minor changes in H3K4me1 levels (active and inactive enhancers)at most of the analysed na?ve enhancers (Figure 4B-C, Figure S5A-B).Using RT-qPCR, we found that Foxd3 overexpression significantly reduced eRNA levels at most of the analysed enhancers (Figure S5C). The role of Foxd3 was similarly evaluated using Foxd3?/? mESC, which displayed increased levels of H3K27ac and H3K4me2 (Figure 4D-E) and minor changes in H3K27me2 (Figure S5D) at most of the analysed enhancers." -- -- -- >2KB 28814 E_02_168 26748758 -- mm10 chr2 172541319 172544955 Mouse Mouse Embryonic Stem Cell Low+High throughput "ChIP-seq,RT-qPCR" "To test if Foxd3 was indeed able to silence na?ve pluripotency enhancers, we evaluated the impact of Foxd3 overexpression on some of these regulatory elements. tetOn Foxd3 mESC were treated with Dox and the levels of various histone modifications (i.e. H3K27ac, H3K27me2, H3K4me1, H3K4me2) at selected enhancers were evaluated by ChIP-qPCR. Compared to control cells, tetOn Foxd3 cells displayed lower levels of H3K27ac and H3K4me2 (active enhancers) , increased H3K27me2 (inactive enhancers) and minor changes in H3K4me1 levels (active and inactive enhancers)at most of the analysed na?ve enhancers (Figure 4B-C, Figure S5A-B)." Enhancer Tfap2C -- "ChIP-seq,RT-qPCR" "ChIP-Seq profiles generated in Foxd3-FH mES cells with anti-HA and anti-Flag antibodies around a representative locus (i.e.,Tfap2c)." "AA409384,AP2gamma,Ap-2.2,Stra2,Tcfap2c" -- -- -- -- -- -- Foxd3 "CWH3,Genesis,Hfh2" ChIP-qPCR "To test if Foxd3 was indeed able to silence naive pluripotency enhancers, we evaluated the impact of Foxd3 overexpression on some of these regulatory elements. tetOn Foxd3 mESCs were treated with Dox, and the levels of various histone modifications (i.e., H3K27ac, H3K27me2, H3K4me1, and H3K4me2) at selected enhancers were evaluated by ChIP-qPCR. Compared to control cells, tetOn Foxd3 cells displayed lower levels of H3K27ac and H3K4me2 (active enhancers) , increased H3K27me2 (inactive enhancers) and minor changes in H3K4me1 levels (active and inactive enhancers)at most of the analysed na?ve enhancers (Figure 4B-C, Figure S5A-B).Using RT-qPCR, we found that Foxd3 overexpression significantly reduced eRNA levels at most of the analysed enhancers (Figure S5C). The role of Foxd3 was similarly evaluated using Foxd3?/? mESC, which displayed increased levels of H3K27ac and H3K4me2 (Figure 4D-E) and minor changes in H3K27me2 (Figure S5D) at most of the analysed enhancers." -- -- -- >2KB 2541 E_02_169 26748758 -- mm10 chr2 172574046 172577682 Mouse Mouse Embryonic Stem Cell Low+High throughput "ChIP-seq,RT-qPCR" "To test if Foxd3 was indeed able to silence na?ve pluripotency enhancers, we evaluated the impact of Foxd3 overexpression on some of these regulatory elements. tetOn Foxd3 mESC were treated with Dox and the levels of various histone modifications (i.e. H3K27ac, H3K27me2, H3K4me1, H3K4me2) at selected enhancers were evaluated by ChIP-qPCR. Compared to control cells, tetOn Foxd3 cells displayed lower levels of H3K27ac and H3K4me2 (active enhancers) , increased H3K27me2 (inactive enhancers) and minor changes in H3K4me1 levels (active and inactive enhancers)at most of the analysed na?ve enhancers (Figure 4B-C, Figure S5A-B)." Enhancer Tfap2C -- "ChIP-seq,RT-qPCR" "ChIP-Seq profiles generated in Foxd3-FH mES cells with anti-HA and anti-Flag antibodies around a representative locus (i.e.,Tfap2c)." "AA409384,AP2gamma,Ap-2.2,Stra2,Tcfap2c" -- -- -- -- -- -- Foxd3 "CWH3,Genesis,Hfh2" ChIP-qPCR "To test if Foxd3 was indeed able to silence naive pluripotency enhancers, we evaluated the impact of Foxd3 overexpression on some of these regulatory elements. tetOn Foxd3 mESCs were treated with Dox, and the levels of various histone modifications (i.e., H3K27ac, H3K27me2, H3K4me1, and H3K4me2) at selected enhancers were evaluated by ChIP-qPCR. Compared to control cells, tetOn Foxd3 cells displayed lower levels of H3K27ac and H3K4me2 (active enhancers) , increased H3K27me2 (inactive enhancers) and minor changes in H3K4me1 levels (active and inactive enhancers)at most of the analysed na?ve enhancers (Figure 4B-C, Figure S5A-B).Using RT-qPCR, we found that Foxd3 overexpression significantly reduced eRNA levels at most of the analysed enhancers (Figure S5C). The role of Foxd3 was similarly evaluated using Foxd3?/? mESC, which displayed increased levels of H3K27ac and H3K4me2 (Figure 4D-E) and minor changes in H3K27me2 (Figure S5D) at most of the analysed enhancers." -- -- -- >2KB 30186 E_02_170 26766440 -- mm10 chr14 69196226 69202226 Mouse G1ER Erythroid Cell Low+High throughput "CRISPR/Cas9,ChIP-seq" "Specifically, we analyzed active enhancer-associated histone modifications H3K4me1 and H3K27ac by chromatin immunoprecipitation sequencing (ChIP-seq) and transcriptomic profiles in four distinct populations of human HSPCs or ProEs at fetal or adult stage (Figure 1B-E; Figure S1A).Identification of lineage or developmental stage-specific enhancers. Venn diagram shows the overlap between HSPC and ProE, or fetal and adult enhancers. The numbers of lost, shared, or gained enhancers in each comparison are shown.We designed sequence-specific guide RNAs (or sgRNAs) flanking each constituent enhancer (E1, E2 and E3) or the promoter (P) (Figure 3B). " Enhancer Slc25a37 -- "ChIP-seq,ChIP-qPCR" "Specifically, while deletion of E1 or E2 individually only modestly or slightly impairs Slc25a37 activation during differentiation, respectively, E3 deletion abolishes its activation resulting in a 15-fold decrease in expression (48h after ¦Â-estradiol treatment; Figure 3C).The enhancer cluster upstream of the SLC25A37 gene, consisting of three distinct constituent enhancers as measured by H3K4me1 and H3K27ac ChIP-seq, is defined as an erythroid-specific super-enhancer in both human (A5 ProE) and mouse (G1ER) erythroid cells (Figure 3A; Table S4)." "Mfrn,Mscp,Mfrn1,AI848481,frascati,mitoferrin,1700020E22Rik,4930513O14Rik,4930526G11Rik,C330015G08Rik" -- -- -- GATA2-to-GATA1 switch is prevalent at dynamic enhancers and drives erythroid enhancer commissioning. CRISPR/Cas9 "Importantly, by CRISPR/Cas9-mediated genomic editing, we uncover functional hierarchy of constituent enhancers within the SLC25A37 super-enhancer. Despite indistinguishable chromatin features, we reveal through genomic editing the functional diversity of several GATA switch enhancers in which enhancers with opposing functions cooperate to coordinate transcription." "Gata1,Gata2" "Gata-1,Gf-1,eryf1,Gata-2" "ChIP-seq,CRISPR/Cas9" "To further dissect the role of GATA switch in enhancer turnover, we compared GATA2 and GATA1 occupancy within A0 and A5 enhancers by ChIP-seq. Specifically, we enumerated the distribution of enhancers occupied by GATA2-Only, GATA1-Only, or GATA2-to-GATA1 switch.We then employed genomic editing to dissect the requirement of GATA switch enhancers within the paradigmatic Gata2 locus." -- -- -- >2KB 42621 E_02_171 26766440 -- mm10 chr14 119710660 119714758 Mouse G1ER Erythroid Cell Low+High throughput "CRISPR/Cas9,ChIP-seq" "Specifically, we analyzed active enhancer-associated histone modifications H3K4me1 and H3K27ac by chromatin immunoprecipitation sequencing (ChIP-seq) and transcriptomic profiles in four distinct populations of human HSPCs or ProEs at fetal or adult stage (Figure 1B-E; Figure S1A).Identification of lineage or developmental stage-specific enhancers. Venn diagram shows the overlap between HSPC and ProE, or fetal and adult enhancers. The numbers of lost, shared, or gained enhancers in each comparison are shown." Enhancer Slc25a37 -- "ChIP-seq,ChIP-qPCR" "Specifically, while deletion of E1 or E2 individually only modestly or slightly impairs Slc25a37 activation during differentiation, respectively, E3 deletion abolishes its activation resulting in a 15-fold decrease in expression (48h after ¦Â-estradiol treatment; Figure 3C)." "Mfrn,Mscp,Mfrn1,AI848481,frascati,mitoferrin,1700020E22Rik,4930513O14Rik,4930526G11Rik,C330015G08Rik" -- -- -- GATA2-to-GATA1 switch is prevalent at dynamic enhancers and drives erythroid enhancer commissioning. CRISPR/Cas9 "Importantly, by CRISPR/Cas9-mediated genomic editing, we uncover functional hierarchy of constituent enhancers within the SLC25A37 super-enhancer. Despite indistinguishable chromatin features, we reveal through genomic editing the functional diversity of several GATA switch enhancers in which enhancers with opposing functions cooperate to coordinate transcription." "Gata1,Gata2" "Gata-1,Gf-1,eryf1,Gata-2" "ChIP-seq,CRISPR/Cas9" "To further dissect the role of GATA switch in enhancer turnover, we compared GATA2 and GATA1 occupancy within A0 and A5 enhancers by ChIP-seq. Specifically, we enumerated the distribution of enhancers occupied by GATA2-Only, GATA1-Only, or GATA2-to-GATA1 switch.We then employed genomic editing to dissect the requirement of GATA switch enhancers within the paradigmatic Gata2 locus." -- -- -- >2KB 50470862 E_02_172 26766440 -- mm10 chr14 69221635 69222851 Mouse G1ER Erythroid Cell Low+High throughput "CRISPR/Cas9,ChIP-seq" "Specifically, we analyzed active enhancer-associated histone modifications H3K4me1 and H3K27ac by chromatin immunoprecipitation sequencing (ChIP-seq) and transcriptomic profiles in four distinct populations of human HSPCs or ProEs at fetal or adult stage (Figure 1B-E; Figure S1A).Identification of lineage or developmental stage-specific enhancers. Venn diagram shows the overlap between HSPC and ProE, or fetal and adult enhancers. The numbers of lost, shared, or gained enhancers in each comparison are shown." Enhancer Slc25a37 -- "ChIP-seq,ChIP-qPCR" "Loss of E3 leads to near absence of H3K27ac, GATA1 and TAL1 occupancy at the neighboring E1 and E2 Enhancers, whereas loss of E1 or E2 has minimal impact on H3K27ac or GATA1/TAL1 binding at neighboring Enhancers (Figure 3D-G). These results strongly suggest that, despite the indistinguishable chromatin features and TF occupancy at the Slc25a37 constituent Enhancers, the E3 Enhancer is functionally more potent than its neighboring Enhancers in directing transcriptional activation." "Mfrn,Mscp,Mfrn1,AI848481,frascati,mitoferrin,1700020E22Rik,4930513O14Rik,4930526G11Rik,C330015G08Rik" -- -- -- GATA2-to-GATA1 switch is prevalent at dynamic enhancers and drives erythroid enhancer commissioning. CRISPR/Cas9 "Importantly, by CRISPR/Cas9-mediated genomic editing, we uncover functional hierarchy of constituent enhancers within the SLC25A37 super-enhancer. Despite indistinguishable chromatin features, we reveal through genomic editing the functional diversity of several GATA switch enhancers in which enhancers with opposing functions cooperate to coordinate transcription. " "Gata1,Gata2" "Gata-1,Gf-1,eryf1,Gata-2" "ChIP-seq,CRISPR/Cas9" "To further dissect the role of GATA switch in enhancer turnover, we compared GATA2 and GATA1 occupancy within A0 and A5 enhancers by ChIP-seq. Specifically, we enumerated the distribution of enhancers occupied by GATA2-Only, GATA1-Only, or GATA2-to-GATA1 switch.We then employed genomic editing to dissect the requirement of GATA switch enhancers within the paradigmatic Gata2 locus." -- -- -- >2KB 19604 E_02_173 26808502 Tce1 Enhancer mm10 chr2 10136078 10138078 Mouse T Cell Low throughput "CRISPR/Cas9,ChIP,RT-PCR" "We next asked whether this 1.2-kb Gata3 Tce1 fragment was also necessary for the transcription of a reporter gene in T cells. To answer this question, we prepared another EGFP reporter plasmid in which the 1.2-kb sequence was deleted from full-length Tce1 (Figure 5A) and generated additional F0 Tg mice (Tg¦¤1.2 mice). None of the F0 Tg¦¤1.2 mice expressed EGFP in peripheral CD4 T cells (Table 1 and Supplemental Figure 5C) or in thymocytes (Figure 5, B and C), except possibly at the DN4 stage (Table 2). We also analyzed Tg¦¤1.7, Tg¦¤1.5, and Tg¦¤2.7 mice and found that all of them expressed EGFP in peripheral CD4 T cells (Tables 1 and ?and2).2). Therefore, the EGFP expression in those mice confirmed that these sequences were not required for Tce1 enhancer activity in peripheral CD4 T cells. These data show that the 1.2-kb T cell enhancer fragment within Tce1 is also necessary for reporter gene transcription in T cells and, taken together with the data shown in Figure 1, that this fragment functions as an enhancer core element in vivo for Gata3 T cell¨Cspecific transcription. " Enhancer Gata3 CRISPR/Cas9 "ChIP,PCR" "As anticipated, homozygous loss of Tce1 (Tce1¨C/¨C) led to a reduction in the number of ETPs (62% of that of heterozygous controls), and Gata3 mRNA in the remaining ETPs was only 52% of that of controls (Figure 1B). In contrast, the number of Tce1¨C/¨C DN2 and DN3 stage cells was unaltered compared with that of controls, although Gata3 mRNA levels in DN2 thymocytes were reduced (83% of that of heterozygous controls; Figure 1B). " "Gata-3,jal" -- -- -- "Together, our data show that Tce1 is both necessary and sufficient for critical aspects of Gata3 T cell¨Cspecific transcriptional activity." "CRISPR/Cas9,ChIP,RT-PCR" "Instead, we demonstrated that multiple lineage-affiliated transcription factors bind to Tce1 and that this enhancer confers T lymphocyte¨Cspecific Gata3 activation in vivo, as targeted deletion of Tce1 in a mouse model abrogated critical functions of this T cell¨Cregulatory element." "Tcf7,Tcf12,Rbpj" "AI465550,TCF-1,Tcf1,A130037E08Rik,ALF1,HEB,HEBAlt,HTF-4,HTF4,ME1,REB,bHLHb20,AI843960,CBF1,Igkjrb,Igkrsbp,RBP-J,RBP-Jkappa,RBP-Jkappa,RBPjk,Rbpsuh" ChIP "We therefore examined the association of CSL/RBP-J with predicted binding sites within Tce1. CSL/RBP-J binding to the Cd25 locus, a robust direct Notch target gene, was used as a positive control with an anti¨CRBP-J antibody in ChIP assays.Taken together, these data show that at least 3 critical T cell¨Caffiliated transcription factors, TCF-1, HEB, and CSL/RBP-J, occupy binding sites within Tce1 at different developmental stages.Since all 3 have been shown to vitally affect T cell development, it seems likely that these factors and their associated signaling pathways directly modulate Gata3 expression through their binding to multiple consensus sites within Tce1." -- -- -- >2KB 280001 E_02_174 26937964 -- mm10 chr7 35156353 35161233 Mouse Myeloid Cells Low+High throughput "ChIP-seq,CRISPR/Cas9,Transgenic mice" "To determine the effect of deleting the enhancer in vivo, we now characterize C57BL/6 mice in which loxP sites flank a 688 bp DNA segment containing the enhancer. " Enhancer Cebpa CRISPR/Cas9 ChIP-seq "These findings demonstrate a critical role for the +37 kb Cebpa Enhancer for hematopoietic-specific Cebpa expression,with Enhancer deletion leading to impaired myelopoiesis and potentially preleukemic progenitor expansion." "C/ebpalpha,CBF-A,Cebp" -- -- -- "These findings demonstrate a critical role for the +37 kb Cebpa enhancer for hematopoietic-specific Cebpa expression, with enhancer deletion leading to impaired myelopoiesis and potentially preleukemic progenitor expansion." "CRISPR/Cas9,PCR,Western blot" "Together, these data indicate that reduced Cebpa expression consequent to +37 kb Enhancer deletion impairs hematopoietic cell autonomous granulopoiesis in vitro, leading to preservation of immature myeloid progenitors capable of long-term, IL-3-dependent proliferation without complete terminal maturation,a preleukemic phenotype." Runx1 "AML1,CBF-alpha-2,Cbfa2,Pebp2a2,Pebpa2b" ChIP-seq "Runx1,C/EBP¦Á,Spi1,Erg,Fli-1,GATA2,Scl,Meis1,and Gfi-1b bind chromatin in the region of this Enhancer in hematopoietic cells as determined by ChIP-Seq." -- -- -- >2KB 39501 E_02_175 27881681 -- mm10 chr7 129554284 129564284 Mouse Rat Chondrosarcoma Cells (RCS cells) Low+High throughput "Luciferase Reporter Assay,ChIP-seq" "However, ChIP sequencing revealed that SOX9 does not strongly bind to intron 1, but rather it binds to intron 6 and a site 30 kb upstream of the transcription start site. Here, we aimed to determine the role of the novel SOX9-binding site in intron 6. We prepared reporter constructs that contain a Col2a1 promoter, intron 1 with or without intron 6, and the luciferase gene. " Enhancer Col2a1 CRISPR/Cas9 "Luciferase Reporter Assay,ChIP-seq" "the ChIP-Seq data of SOX9 in the Col2a1 gene using rat chondrosarcoma cells (RCS cells)show that there is a strong SOX9-binding site in intron 6 and a weaker binding site in intron 1.Although the reporter constructs were not activated by SOX9 alone, the construct that contained both introns 1 and 6 was activated 5¨C10-fold by the SOX9/SOX5 or the SOX9/SOX6 combination in transient-transfection assays in 293T cells. This enhancement was also observed in rat chondrosarcoma cells that stably expressed the construct. CRISPR/Cas9-induced deletion of intron 6 in RCS cells revealed that a 10-bp region of intron 6 is necessary both for Col2a1 expression and SOX9 binding. " "Col2,Col2a,Col2a-1,Del1,Dmm,Lpk,M100413,Rgsc413,Rgsc856" Skeletal Diseases -- -- -- -- -- Sox9 "2010306G03Rik,AV220920,mKIAA4243" "ChIP-seq,ChIP-qPCR,EMSA" "SOX9 bind to intron 6 and the binding of SOX5/SOX6 to intron 6 may facilitate the binding of SOX9 to the intron 1.To explore this possibility, we tested whether SOX9 and SOX5 bound to the SB2 fragment(600bp)shownabove or an even shorter fragment by means of an EMSA." -- -- -- Intron 31583683 E_02_176 27895109 -- mm10 chr9 78368337 78376479 Mouse Embryonic Stem Cell Low+High throughput "Luciferase Reporter Assay,RT-qPCR,ChIP-seq" "Reporter assays revealed that each of these regions contained an active enhancer (Fig. 2B). Deletion of the entire EC(DppUp) significantly affects the expression of both Dppa5a and Ooep, although the effect on Ooep is much greater (92% reduction compared to a 36% reduction).Enhancer activity was identified in a reporter assay for the ?1 enhancer and the ?5.8 enhancer within the EC(DppUpA) and EC(DppUpB) regions, respectively." Enhancer "Dppa5a,Ooep" -- "Luciferase Reporter Assay,RT-qPCR,ChIP-seq" "We found that the enhancer cluster EC(DppUp), which lies between the Dppa5a and Ooep genes, regulated both Dppa5a and Ooep in cis. We considered two alternatives for the mechanism underlying this observation: Either this region contained two distinct regulatory modules, one for Dppa5a and one for Ooep, or each of the enhancers within the cluster contributed to the expression of both genes. To differentiate between these two alternatives, we split the EC into two segments and deleted each individually (Fig. 2). The region closest to Ooep, EC(DppUpB), contains one MTL bound by four transcription factors, whereas the region closest to Dppa5a, EC(DppUpA), contains three separate MTLs. Reporter assays revealed that each of these regions contained an active enhancer (Fig. 2B)." "AA536857,Dppa5,Esg1,ecat2,2410146L05Rik,Floped,Moep19,Sddr" -- -- -- "Robust transcriptional output can be achieved by an isolated enhancer,clusters of enhancers acting on a common target gene act in a partially redundant manner to fine tune transcriptional output of their target genes." ChIP-seq This occurs despite the fact that deletion of EC(DppDn) alone has only a modest effect on both Dppa5a (16% reduction) and Ooep (15% reduction) ex_x0002_pression (Table 1). This finding indicates partially redundant control of Dppa5a and Ooep by Enhancers in the upstream and downstream EC. -- -- -- -- -- -- -- >2KB 5357 E_02_177 27920250 -- mm10 chr5 137465620 137479420 Mouse Renal EPO-producing (REP) Cells Low throughput "Luciferase Reporter Assay,Transgenic mice" "17k-GFP contains the 17-kb upstream and 15-kb downstream regions, whereas 17kXM-GFP contains only the 17-kb upstream region as a regulatory element. As in 22k-GFP mice, GFP expression was observed in the kidney and liver in 17k-GFP mice under anemic conditions (Fig. 1C)." Enhancer Epo -- "Luciferase Reporter Assay,qRT-PCR" "we generated a series of transgenic GFP reporter constructs using 60k-GFP and 22k-GFP as the starting materia GFP expression was observed in the kidney but not in the liver in 17kXM-GFP mice.By means of long genomic PCR assays utilizing a primer set recognizing sequences located 18.6 kb upstream (forward primer) and 3.7 kb downstream (reverse primer) from the Epo gene transcription start site, we confirmed that the bulk of the Epo-BAC DNA containing a 17-kb upstream region and a downstream hepatic enhancer region was integrated into the genome of the transgenic mice in the right order (Fig. 4C)." Epo Renal Anemia DOID:784 D051436 -- -- -- -- -- -- -- -- -- -- >2KB 10499 E_02_178 27941798 -- mm10 chr15 63833002 63863200 Mouse HCT 116 Low+High throughput ChIP-seq "To determine if control of enhancer activity by SWI/SNF complexes is coordinated with TFs, we analyzed sequence motifs at enhancers sensitive to ARID1A loss. The CTCF motif was relatively depleted in regions of H3K27ac loss, implying CTCF-bound insulator regions may resist modulation, while the AP1 (JUND/FOSL1) motif was most enriched (Fig. 5a, Supplementary Table 5).Among factors assessed in HCT116 cells, binding of these TFs correlated most strongly with SWI/SNF occupancy (Supplementary Fig. 6b) and was higher at enhancers that lost activity than at enhancers that were unaffected by ARID1A deficiency (Fig. 5c). " Super-Enhancer Arid1a -- "ChIP-seq,ChIP-qPCR" "To determine if ARID1A loss impairs enhancer activity?in vivo, we examined H3K27ac and gene expression in the colonic epithelium of wildtype and Villin-CreER-T2?Arid1afl/fl?mice. Again, whereas promoter activation states were changed little (Fig. 5d), the effects on H3K27ac at enhancers were significant and were correlated with changes in mRNA levels of the nearest genes (Fig. 5e).?" "1110030E03Rik, BAF250a, Osa1, Smarcf1" Colon Cancer DOID:219 D003110 -- -- -- "Jun,Cebpb" "AP-1c,c-jun,Jun,C/EBPbeta,CRP2,IL-6DBP,LAP,LIP,NF-IL6,NF-M,Nfil6" ChIP-seq "ChIP-Seq profiles for HCT116 cells, including those generated by ENCODE21 (Encyclopedia of DNA Elements, Supplementary Table 6), revealed further that H3K27ac loss was most strongly associated with sites bound in WT cells by SWI/SNF complexes and/or TFs including AP1, CEBPB, and TEAD4 (Fig. 5b). Among factors assessed in HCT116 cells, binding of these TFs correlated most strongly with SWI/SNF occupancy (Supplementary Fig. 6b) and was higher at enhancers that lost activity than at enhancers that were unaffected by ARID1A deficiency (Fig. 5c)" -- -- -- >2KB 72131 E_02_179 27941798 -- mm10 chr15 63865799 63896492 Mouse HCT 116 Low+High throughput ChIP-seq "To determine if control of enhancer activity by SWI/SNF complexes is coordinated with TFs, we analyzed sequence motifs at enhancers sensitive to ARID1A loss. The CTCF motif was relatively depleted in regions of H3K27ac loss, implying CTCF-bound insulator regions may resist modulation, while the AP1 (JUND/FOSL1) motif was most enriched (Fig. 5a, Supplementary Table 5).Among factors assessed in HCT116 cells, binding of these TFs correlated most strongly with SWI/SNF occupancy (Supplementary Fig. 6b) and was higher at enhancers that lost activity than at enhancers that were unaffected by ARID1A deficiency (Fig. 5c). " Super-Enhancer Arid1a -- "ChIP-seq,ChIP-qPCR" "To determine if control of enhancer activity by SWI/SNF complexes is coordinated with TFs, we analyzed sequence motifs at enhancers sensitive to ARID1A loss. The CTCF motif was relatively depleted in regions of H3K27ac loss, implying CTCF-bound insulator regions may resist modulation, while the AP1 (JUND/FOSL1) motif was most enriched (Fig. 5a, Supplementary Table 5).Among factors assessed in HCT116 cells, binding of these TFs correlated most strongly with SWI/SNF occupancy (Supplementary Fig. 6b) and was higher at enhancers that lost activity than at enhancers that were unaffected by ARID1A deficiency (Fig. 5c). " "1110030E03Rik, BAF250a, Osa1, Smarcf1" Colon Cancer DOID:219 D003110 -- -- -- "Arid1a,Arid1b" "1110030E03Rik,BAF250a,Osa1,Smarcf1,8030481M12,9330189K18Rik,AI836955,Ardi1b,B230217J03Rik,BAF250B,mKIAA1235" ChIP-seq "ChIP-Seq profiles for HCT116 cells, including those generated by ENCODE21?(Encyclopedia of DNA Elements,?Supplementary Table 6), revealed further that H3K27ac loss was most strongly associated with sites bound in WT cells by SWI/SNF complexes and/or TFs including AP1, CEBPB, and TEAD4 (Fig. 5b). Among factors assessed in HCT116 cells, binding of these TFs correlated most strongly with SWI/SNF occupancy (Supplementary Fig. 6b) and was higher at enhancers that lost activity than at enhancers that were unaffected by ARID1A deficiency (Fig. 5c)" -- -- -- >2KB 105176 E_02_180 27941798 -- mm10 chr15 63894512 63932136 Mouse HCT 116 Low+High throughput ChIP-seq "To determine if control of enhancer activity by SWI/SNF complexes is coordinated with TFs, we analyzed sequence motifs at enhancers sensitive to ARID1A loss. The CTCF motif was relatively depleted in regions of H3K27ac loss, implying CTCF-bound insulator regions may resist modulation, while the AP1 (JUND/FOSL1) motif was most enriched (Fig. 5a, Supplementary Table 5).Among factors assessed in HCT116 cells, binding of these TFs correlated most strongly with SWI/SNF occupancy (Supplementary Fig. 6b) and was higher at enhancers that lost activity than at enhancers that were unaffected by ARID1A deficiency (Fig. 5c). " Super-Enhancer Arid1a -- "ChIP-seq,ChIP-qPCR" "To determine if control of enhancer activity by SWI/SNF complexes is coordinated with TFs, we analyzed sequence motifs at enhancers sensitive to ARID1A loss. The CTCF motif was relatively depleted in regions of H3K27ac loss, implying CTCF-bound insulator regions may resist modulation, while the AP1 (JUND/FOSL1) motif was most enriched (Fig. 5a, Supplementary Table 5).Among factors assessed in HCT116 cells, binding of these TFs correlated most strongly with SWI/SNF occupancy (Supplementary Fig. 6b) and was higher at enhancers that lost activity than at enhancers that were unaffected by ARID1A deficiency (Fig. 5c). " "1110030E03Rik, BAF250a, Osa1, Smarcf1" Colon Cancer DOID:219 D003110 -- -- -- "Arid1a,Arid1b" "1110030E03Rik,BAF250a,Osa1,Smarcf1,8030481M12,9330189K18Rik,AI836955,Ardi1b,B230217J03Rik,BAF250B,mKIAA1235" ChIP-seq "ChIP-Seq profiles for HCT116 cells, including those generated by ENCODE21?(Encyclopedia of DNA Elements,?Supplementary Table 6), revealed further that H3K27ac loss was most strongly associated with sites bound in WT cells by SWI/SNF complexes and/or TFs including AP1, CEBPB, and TEAD4 (Fig. 5b). Among factors assessed in HCT116 cells, binding of these TFs correlated most strongly with SWI/SNF occupancy (Supplementary Fig. 6b) and was higher at enhancers that lost activity than at enhancers that were unaffected by ARID1A deficiency (Fig. 5c)" -- -- -- >2KB 137354 E_02_181 27986456 -- mm10 chr18 69656860 69683465 Mouse HoxB8-FL cells Low+High throughput ATAC-seq "ATAC-Seq analysis of Tcf4 locus in primary DCs. Shown are ATAC-Seq peaks across ~1.5 Mb of the Tcf4 locus; indicated are proximal promoters of Tcf4L (green) and Tcf4S (purple), the 3¡ä enhancer (orange) and the cluster of 5¡ä regulatory elements (yellow). In this and subsequent panels, the scale was adjusted between different samples based on signals from housekeeping genes.We analyzed the 3¡ä region by ChIP during the differentiation of HoxB8-FL cells. Histone modifications associated with active enhancers, including histone 3 lysine 4 monomethylation (H3K4me1) and lysine 27 acetylation (H3K27ac), were induced in this region on day 4, i.e. at the onset of pDC differentiation (Fig. 5D)." Super-Enhancer Tcf4 CRISPR/Cas9 "ChIP-seq,ATAC-seq" ChIP analysis of histone modifications (H3K4me1 and H3K27ac) and of TCF4 binding to the 3¡ä Enhancer in differentiating HoxB8-FL cells. "5730422P05Rik,ASPI2,E2-2,E2.2,ITF-2,ITF-2b,ITF2,ME2,MITF-2A,MITF-2B,SEF-2,SEF2,SEF2-1,TFE,Tcf-4,bHLHb19" -- -- -- "A conserved Enhancer downstream of Tcf4 was required for its upregulation during pDC differentiation, revealing a positive feedback loop" "qRT-PCR,CRISPR/Cas9" "By qRT-PCR, Tcf4S was prominently expressed in pDCs but also present in other cell types including B cells and cDCs" Tcf4 "5730422P05Rik,ASPI2,E2-2,E2.2,ITF-2,ITF-2b,ITF2,ME2,MITF-2A,MITF-2B,SEF-2,SEF2,SEF2-1,TFE,Tcf-4,bHLHb19" qRT-PCR "By qRT-PCR, Tcf4S was prominently expressed in pDCs but also present in other cell types including B cells and cDCs. " -- -- -- >2KB 325676 E_02_182 28045957 TES/TESCO Enhancer mm10 chr11 112769210 112772210 Mouse Testis Low throughput "Transgenic mice,CRISPR/Cas9,qRT-PCR" "During mouse sex determination, transient expression of the Y-linked gene Sry up-regulates its direct target gene Sox9, via a 3.2 kb testis specific enhancer of Sox9 (TES), which includes a core 1.4 kb element, TESCO. SOX9 activity leads to differentiation of Sertoli cells, rather than granulosa cells from the bipotential supporting cell precursor lineage. Here, we present functional analysis of TES/TESCO, using CRISPR/Cas9 genome editing in mice. Deletion of TESCO or TES reduced Sox9 expression levels in XY fetal gonads to 60 or 45% respectively relative to wild type gonads, and reduced expression of the SOX9 target Amh. " Enhancer Sox9 -- qRT-PCR "These results of qRT-PCR clearly indicate that the TESCO Enhancer is an important regulator of Sox9 expression throughout testis development, accounting for around 40% of its expression levels." "2010306G03Rik,AV220920,mKIAA4243" -- -- -- reduced expression of the SOX9 target Amh. CRISPR/Cas9 "Here, we present functional analysis of TES/TESCO, using CRISPR/Cas9 genome editing in mice. Deletion of TESCO or TES reduced Sox9 expression levels in XY fetal gonads to 60 or 45% respectively relative to wild type gonads, and reduced expression of the SOX9 target Amh." -- -- -- -- -- -- -- >2KB 11500 E_02_183 26163528 Id2 Enhancer mm10 chr12 25090158 25096935 Mouse Apoptotic Cell Low throughput Luciferase Reporter Assay "To test this Tcf4 binding region for enhancer activity, we conducted luciferase reporter assays using the constructs shown in Fig. 1F. The putative Id2-enhancer fragment significantly activated Wnt-signaling dependent transcription from the CMV minimal promoter in two colorectal cancer cell lines (HCT116 and SW480) (Fig. 1G). Therefore, these results indicate that Id2 expression is stimulated by Wnt signaling through the enhancer region, in tumor epithelial cells of Apc¦¤716 mice at an early stage of tumorigenesis." Enhancer Id2 -- Luciferase Reporter Assay Schematic representation of theLuciferase (Luciferase) expression constructs containing the Id2 Enhancer with/without a CMV-minimal promoter. "AI255428,C78922,Idb2,bHLHb26" Colorectal Carcinoma DOID:0080199 D015179 -- -- -- -- -- -- -- -- -- -- <2KB 251 E_02_184 28087634 -- mm10 chr2 105072843 105076843 Mouse Sertoli Cells Low+High throughput "ChIP-seq,DNaseI-seq" "H3K27ac ChIP-seq on E13.5 Sertoli cells identified >28,000 H3K27ac peaks, ?70% of which overlapped a DHS in our E13.5 Sertoli cell dataset (Fig. 3A, Fig. S4).H3K27ac-positive DHSs, which are indicative of active enhancers, were significantly enriched specifically in neighboring Sertoli cell-expressed genes (Fig. 3D), as expected." Enhancer Wt1 -- "ChIP-seq,DNaseI-seq" "To demonstrate this, we performed a transient transgenic assay with a putative active enhancer of the Wt1 gene (Fig. 5A,B).We identified a DHS peak located 50?kb upstream of Wt1 that was unique to E13.5 and E15.5 Sertoli cells compared with the other cell types we examined, and marked by H3K27ac, suggesting that it functions as an active enhancer." "D630046I19Rik,Wt-1" Genitourinary Defects -- -- -- -- -- -- -- -- -- -- -- -- >2KB 51686 E_02_185 28104492 -- mm10 chr15 97898854 97902854 Mouse YAMC Cell Low throughput "Luciferase Reporter Assay,Western blot,DNaseI-seq" "Zella et al. [22] found that transcription of the mouse VDR gene is regulated at seven promoter and enhancer regions (Fig. 5A) in the mouse osteoblast cell line MC3T3-E1. In addition, the ENCODE project has identified DNAse I hypersensitive sites in the mouse large intestine that overlap with these regions." Enhancer Vdr -- "Luciferase Reporter Assay,Western blot" "However, chromatin accessibility to the VDR gene at the proximal promoter (_x0001_300 bp), an Enhancer region at _x0001_6 kb, and an Enhancer region located in exon 7 was significantly reduced in RAS transformed YAMC cells (YAMC-RAS)." Nr1i1 Colon Cancer DOID:219 D003110 -- -- -- -- -- -- -- -- -- -- >2KB 46428 E_02_186 28104492 -- mm10 chr15 97911426 97915997 Mouse YAMC Cell Low throughput "Luciferase Reporter Assay,Western blot,DNaseI-seq" "Zella et al. [22] found that transcription of the mouse VDR gene is regulated at seven promoter and enhancer regions (Fig. 5A) in the mouse osteoblast cell line MC3T3-E1. In addition, the ENCODE project has identified DNAse I hypersensitive sites in the mouse large intestine that overlap with these regions." Enhancer Vdr -- "Luciferase Reporter Assay,Western blot" "However, chromatin accessibility to the VDR gene at the proximal promoter (_x0001_300 bp), an Enhancer region at _x0001_6 kb, and an Enhancer region located in exon 8 was significantly reduced in RAS transformed YAMC cells (YAMC-RAS)." Nr1i1 Colon Cancer DOID:219 D003110 -- -- -- -- -- -- -- -- -- -- >2KB 59286 E_02_187 28121289 -- mm10 chr6 88202011 88204330 Mouse Embryonic Endothelial Cell Low+High throughput "Luciferase Reporter Assay,ChIP-seq,Western blot" "Viewing Ep300fb bioChiP-seq signal at genes selectively expressed in heart or brain confirmed robust tissue-specific differences that overlapped enhancers with known tissue-specific activity.To further functionally validate the transcriptional activity of these heterodimer motifs, we measured their enhancer activity using luciferase reporter assays." Enhancer Gata2 -- "Luciferase Reporter Assay,ChIP-seq" "At the skeletal muscle specific gene Myod, Myf5Cre drove strong Ep300fb bioChiP-seq signal at a known distal Enhancer (Goldhamer et al., 1992), as well as a second Ep300 bound region about 12 kb upstream from the transcriptional start site." Gata-2 -- -- -- -- -- -- Ep300 "A430090G16,A730011L11,KAT3B,p300,p300 HAT" ChIP-seq "However, antibodies for Ep300 are marginal for robust ChIP-seq, particularly from tissues, leading to low reproducibility, variation between antibody lots, and inefficient Enhancer identification." -- -- -- >2KB 9735 E_02_188 28213503 -- mm10 chr11 44386118 44393090 Mouse Macrophage Low+High throughput ChIP-seq "However, following treatment with acute IL-10, H3K27ac was significantly lower at peaks associated with genes from Cluster 2 than at peaks associated with genes from Cluster 1 (p-value <0.01 by Mann-Whitney test, Fig. 6A). Examples of Cluster 1 and Cluster 2 enhancers are shown in Fig. 6B and Fig. 6C, respectively. Interestingly this analysis identified a peak inhibited by acute IL-10 that encompasses a DNAse hypersensitivity site 10 kb upstream of Il12b that has previously been demonstrated in a reporter assay to exhibit enhancer activity (Fig. 6C)." Enhancer Cxcl2 -- "ChIP-seq,RT-PCR" "To evaluate this issue, we compared induction of Cxcl2 transcription in IL-10-deficient macrophages stimulated with LPS alone, both LPS and IL-10, or stimulated with IL-10 for 1 hour prior to stimulation with LPS. As predicted, LPS rapidly induced Cxcl2 pre-mRNA within 1h hour of stimulation. Surprisingly, addition of IL-10 at the time of LPS stimulation or addition 1 hour prior to LPS stimulation had little influence on Cxcl2 pre-mRNA at 1h post LPS stimulation, although significant suppression was observed at 2h post LPS stimulation (Fig. 8A). " "CINC-2a,GROb,Gro2,MIP-2,MIP-2a,Mgsa-b,Mip2,Scyb,Scyb2" Inflammatory Disease -- -- -- -- -- Stat3 "1110034C02Rik, AW109958, Aprf" ChIP-seq "To address this possibility, we performed ChIP-seq with an anti-STAT3 antibody to identify STAT3 binding sites.Interestingly, virtually all identified STAT3 binding sites were located within H3K4me1 peaks, suggesting that STAT3 binds to enhancers. As anticipated, we found strong STAT3 binding near Cluster 3 genes induced by IL-10, such as Socs3 (Fig. 7, bottom left), and IL-10 induced an increase in average mean acetylation at STAT3 binding sites associated with genes in Cluster 3, consistent with enhancer activation (Fig. 7, bottom right)." -- -- -- >2KB 46514294 E_02_189 28262751 -- mm10 chr4 97123309 97712957 Mouse Embryonic Fibroblast Low+High throughput ChIP-seq "Following inactivation of either Smarcb1 or Smarca4, we observed little change in H3K27ac levels at promoters, but a marked reduction at many enhancers (Fig. 1b,c). This reduction at enhancers was robust and consistent across replicate experiments performed on MEFs derived from independent mice (Methods, Supplementary Figs 3-7)." Enhancer Nfia -- "ChIP-seq,Western blot" "To investigate whether SWI/SNF is acting directly at active enhancers, we performed ChIP-Seq for the core SWI/SNF subunits SMARCC1 and SMARCA4 in wild-type and Smarcb1-deficient cells. Because the genome-wide binding profiles of these two subunits were very similar (Supplementary Fig. 3), we considered the average of the two experiments to be representative of SWI/SNF binding for each condition. Interestingly, we found that SWI/SNF was bound at the vast majority of enhancers in wild-type cells-over 95% of enhancers showed enrichment (IP>input; Fig. 2a and Supplementary Fig. 9a). Deletion of the Smarcb1 subunit led to a widespread reduction in SWI/SNF binding (Fig. 2b and Supplementary Fig. 9b).Taken together, the targeting of SWI/SNF to enhancers and the association between loss of SWI/SNF binding and the loss of H3K27ac suggests a direct role for the SWI/SNF complex in regulating the enhancer chromatin landscape in MEFs." "1110047K16Rik,9430022M17Rik,CTF,NF1-A,NF1A" Malignancies -- -- -- -- -- Ep300 "A430090G16,A730011L11,KAT3B,p300,p300 HAT" ChIP-seq "To further evaluate how the interaction between SWI/SNF and p300 regulates H3K27ac levels, we immunoprecipitated SWI/SNF complexes using an antibody against the core subunit SMARCC1 and measured histone acetylation activity using recombinant histones as a substrate. With immunoprecipitated SWI/SNF complexes from a RT cell line that lacked SMARCB1, the sample lacked acetyltransferase activity. However, re-expression of SMARCB1 increased H3K27-specific acetyltransferase activity, despite acetylation of H3K9ac being not significantly changed, suggesting the presence of H3K27-specific acetylation activity (Fig. 3d). " -- -- -- >2KB 163582 E_02_190 28335007 -- mm10 chr5 9034150 9036900 Mouse Liver Low+High throughput ChIP-seq "On the other hand, the enhancer regions associated with lncRNAs tended to have much higher intensities of H3K27ac marks than the enhancers not associated with lncRNAs (KS test, P-value < 2.2 ¡Á 10?16) (Figure ?(Figure2E).2E). The enhancers associated with circadian lncRNAs have even higher levels of H3K27ac marks than those associated with lncRNAs (KS test, P-value = 0.0017). Taken together, there is a strong association between circadian lncRNAs and super-enhancers." Super-Enhancer Gm40264 4C "qPCR,RNA-seq,ChIP-seq" "The enhancers defined from mouse liver GRO-seq and 5? CAGE technology were consistent with H3K27ac marks of enhancer at this lnc-Crot region (Supplementary Figure S3C). Using RACE assay, we obtained the full-length sequence of lnc-Crot (Figure ?(Figure4B).4B). Both BMAL1 and REV-ERB¦Á binding sites were found on the promoter and gene body of lnc-Crot (Figure ?(Figure4B)4B) but not on the promoters of nearby circadian genes including Crot, Tmem243 and Dmtf1 (Supplementary Figure S3D).By qPCR, we showed that Crot and lnc-Crot were co-expressed (r = 0.8) across adult mouse tissues with elevated expression in liver and kidney (Supplementary Figure S3F).4C signals from three biological replicates at each circadian time were highly reproducible judging from their cross-sample correlations between 0.89 and 0.95. About 44% of 4C signals fell within chromosome 5 where lnc-Crot is situated and the signal profile around lnc-Crot region was displayed in Supplementary Figure S5A. " lnc-Crot -- -- -- Enhancer-associated circadian lncRNAs mark the genomic loci modulating long-range circadian gene regulation and shed new lights on the evolutionary origin of lncRNAs. "qPCR,CRISPR/Cas9" Our result points to a model that the lnc-Crot locus and nearby genes first form a scaffold upon which histone modifications and TF bindings then take place to confer tissue- and time-dependent expression (Figure ?(Figure5G).5G). The circadian lncRNAs that we examined may be a result of circadian transcription at the enhancer regions while the lncRNAs themselves do not influence the circadian gene regulation. "Arntl,Nr1d2" "Arnt3,BMAL1b,Bmal1,MOP3,bHLHe5,bmal1b',RVR,Rev-erb" "ChIP-seq,RNA-seq,Knockout mice" "We defined BMAL1 and REV-ERB binding sites from two sets of BMAL1 ChIP-seq studies and two sets of REV-ERB ChIP-seq data in mouse liver respectively, resulting in 8001 BMAL1and 17545REV-ERB binding sites.Atotal of 259 (43%) circadian lncRNA clusters contain either BMAL1 or REV-ERB binding sites.For each lncRNA cluster, we quantified its expression in the BMAL1 knockout and WT mouse livers at CT0 and CT12 using our RNA-seq data.This suggested that circadian lncRNAs were regulated by these two master circadian TFs in the similar way as circadian protein coding genes.Circadian expression of lnc-Crot was disrupted when BMAL1 or REV-ERB¦Á was knocked out, resulting in its downregulation at CT0 in BMAL1 knockout mice and upregulation at CT12 in REV-ERB¦Á knockout mice (Figure ?(Figure4B,4B, Supplementary Figure S3E). This demonstrated that lnc-Crot was directly regulated by BMAL1 and REV-ERB¦Á." -- -- -- >2KB 1075054 E_02_191 28335007 -- mm10 chr5 9037806 9041881 Mouse Liver Low+High throughput ChIP-seq "On the other hand, the enhancer regions associated with lncRNAs tended to have much higher intensities of H3K27ac marks than the enhancers not associated with lncRNAs (KS test, P-value < 2.2 ¡Á 10?16) (Figure ?(Figure2E).2E). The enhancers associated with circadian lncRNAs have even higher levels of H3K27ac marks than those associated with lncRNAs (KS test, P-value = 0.0017). Taken together, there is a strong association between circadian lncRNAs and super-enhancers." Super-Enhancer Gm40264 4C "qPCR,RNA-seq,ChIP-seq" "The enhancers defined from mouse liver GRO-seq and 5? CAGE technology were consistent with H3K27ac marks of enhancer at this lnc-Crot region (Supplementary Figure S3C). Using RACE assay, we obtained the full-length sequence of lnc-Crot (Figure ?(Figure4B).4B). Both BMAL1 and REV-ERB¦Á binding sites were found on the promoter and gene body of lnc-Crot (Figure ?(Figure4B)4B) but not on the promoters of nearby circadian genes including Crot, Tmem243 and Dmtf1 (Supplementary Figure S3D).By qPCR, we showed that Crot and lnc-Crot were co-expressed (r = 0.8) across adult mouse tissues with elevated expression in liver and kidney (Supplementary Figure S3F).4C signals from three biological replicates at each circadian time were highly reproducible judging from their cross-sample correlations between 0.89 and 0.95. About 44% of 4C signals fell within chromosome 5 where lnc-Crot is situated and the signal profile around lnc-Crot region was displayed in Supplementary Figure S5A. " lnc-Crot -- -- -- Enhancer-associated circadian lncRNAs mark the genomic loci modulating long-range circadian gene regulation and shed new lights on the evolutionary origin of lncRNAs. "qPCR,CRISPR/Cas9" Our result points to a model that the lnc-Crot locus and nearby genes first form a scaffold upon which histone modifications and TF bindings then take place to confer tissue- and time-dependent expression (Figure ?(Figure5G).5G). The circadian lncRNAs that we examined may be a result of circadian transcription at the enhancer regions while the lncRNAs themselves do not influence the circadian gene regulation. "Arntl,Nr1d2" "Arnt3,BMAL1b,Bmal1,MOP3,bHLHe5,bmal1b',RVR,Rev-erb" "ChIP-seq,RNA-seq,Knockout mice" "We defined BMAL1 and REV-ERB binding sites from two sets of BMAL1 ChIP-seq studies and two sets of REV-ERB ChIP-seq data in mouse liver respectively, resulting in 8001 BMAL1and 17545REV-ERB binding sites.Atotal of 259 (43%) circadian lncRNA clusters contain either BMAL1 or REV-ERB binding sites.For each lncRNA cluster, we quantified its expression in the BMAL1 knockout and WT mouse livers at CT0 and CT12 using our RNA-seq data.This suggested that circadian lncRNAs were regulated by these two master circadian TFs in the similar way as circadian protein coding genes." -- -- -- >2KB 1079373 E_02_192 28335007 -- mm10 chr5 9038118 9038593 Mouse Liver Low+High throughput ChIP-seq "On the other hand, the enhancer regions associated with lncRNAs tended to have much higher intensities of H3K27ac marks than the enhancers not associated with lncRNAs (KS test, P-value < 2.2 ¡Á 10?16) (Figure ?(Figure2E).2E). The enhancers associated with circadian lncRNAs have even higher levels of H3K27ac marks than those associated with lncRNAs (KS test, P-value = 0.0017). Taken together, there is a strong association between circadian lncRNAs and super-enhancers." Super-Enhancer Gm40264 4C "qPCR,RNA-seq,ChIP-seq" "The enhancers defined from mouse liver GRO-seq and 5? CAGE technology were consistent with H3K27ac marks of enhancer at this lnc-Crot region (Supplementary Figure S3C). Using RACE assay, we obtained the full-length sequence of lnc-Crot (Figure ?(Figure4B).4B). Both BMAL1 and REV-ERB¦Á binding sites were found on the promoter and gene body of lnc-Crot (Figure ?(Figure4B)4B) but not on the promoters of nearby circadian genes including Crot, Tmem243 and Dmtf1 (Supplementary Figure S3D).By qPCR, we showed that Crot and lnc-Crot were co-expressed (r = 0.8) across adult mouse tissues with elevated expression in liver and kidney (Supplementary Figure S3F).4C signals from three biological replicates at each circadian time were highly reproducible judging from their cross-sample correlations between 0.89 and 0.95. About 44% of 4C signals fell within chromosome 5 where lnc-Crot is situated and the signal profile around lnc-Crot region was displayed in Supplementary Figure S5A. " lnc-Crot -- -- -- Enhancer-associated circadian lncRNAs mark the genomic loci modulating long-range circadian gene regulation and shed new lights on the evolutionary origin of lncRNAs. "qPCR,CRISPR/Cas9" Our result points to a model that the lnc-Crot locus and nearby genes first form a scaffold upon which histone modifications and TF bindings then take place to confer tissue- and time-dependent expression (Figure ?(Figure5G).5G). The circadian lncRNAs that we examined may be a result of circadian transcription at the enhancer regions while the lncRNAs themselves do not influence the circadian gene regulation. "Arntl,Nr1d2" "Arnt3,BMAL1b,Bmal1,MOP3,bHLHe5,bmal1b',RVR,Rev-erb" "ChIP-seq,RNA-seq,Knockout mice" "We defined BMAL1 and REV-ERB binding sites from two sets of BMAL1 ChIP-seq studies and two sets of REV-ERB ChIP-seq data in mouse liver respectively, resulting in 8001 BMAL1and 17545REV-ERB binding sites.Atotal of 259 (43%) circadian lncRNA clusters contain either BMAL1 or REV-ERB binding sites.For each lncRNA cluster, we quantified its expression in the BMAL1 knockout and WT mouse livers at CT0 and CT12 using our RNA-seq data.This suggested that circadian lncRNAs were regulated by these two master circadian TFs in the similar way as circadian protein coding genes." -- -- -- >2KB 1077885 E_02_193 28575289 -- mm10 chr1 136170878 136192613 Mouse C2C12 Low+High throughput ChIP-seq "To further monitor the progressive remodeling of SE during the MB differentiation into MTs, we generated a time series of H3K27ac ChIP-seq data covering the differentiating course of ?24,0,6,12,24 and 72 hr (Supplementary Data S2). As shown in Figure??Figure1R,1R, progressive temporal changes in enhancer usage were readily observed during the progression of differentiation." Super-Enhancer Myog 3C "qRT-PCR,ChIP-seq" "To further illuminate whether the cooperation is mediated through physical interaction through chromosomal looping, 3C (chromosome confirmation capture) assay was performed followed by PCR to analyze the chromatin loop structure (Figure ?(Figure5L).5L). Detectable looping frequency between H3 and H4 or the promoter but not with the negative control regions upstream of H1 or downstream of Myogenin gene was observed upon early differentiation at 24 h and persisted throughout the differentiation whereas the interaction between H3 and H1 or H2 occurred in a later stage (Figure ?(Figure5L5L¨CN and Supplementary Figure S6F), confirming H3 is the earliest to be activated then probably helped the establishment of H4 and others through their interactions." "MYF4,bHLHc3,myo" -- -- -- -- -- -- "Foxo3,Myod1" "1110048B16Rik,2010203A17Rik,C76856,FKHRL1,Fkhr2a,Foxo3,AI503393,MYF3,MyoD,Myod-1,bHLHc1" "CRISPR/Cas9,Western blot" "Indeed, the deletion of MyoD by CRISPR-Cas9 in the cells (Supplementary Figure S3A¨CD) induced extraordinary changes at SE landscape; 584 SEs in WT were lost while 257 new ones were gained (Supplementary Figure S3E and F). The deletion not only sharply attenuated the enhancer activity at the WT SE hotspots (Figure ?(Figure3A),3A), but also resulted in the activation of genes that acquired de novo SEs in KO cells (Supplementary Figure S3G).HA-tagged MyoD was co-transfected with plasmid expressing Flag-tagged full length FoxO3. Lysates were pulled down by HA antibody and examined by western blotting using Flag antibody. For all the above panels, the representative images from two replicates are shown. " -- -- -- >2KB 1891743 E_02_194 28614915 ephrin-A5 Enhancer mm10 chr17 62627957 62633457 Mouse Mesencephalon Low throughput Luciferase Reporter Assay "Luciferase activity assay showing that Ascl1- E47 fusion protein induces transcriptional activation through the ephrin-A5 E-box DNA.Taken together, our findings suggest that the mesencephalon-specific enhancer of ephrin-A5 resided in a genomic region that is +11.6 kb to +57.7 kb from the TSS and is within the first intron (Fig. 1A)." Enhancer Efna5 -- "Luciferase Reporter Assay,PCR" "To further restrict the possible locations of the mesencephalon-specific enhancer within the first intron, the 375B7 BAC was modified using a different targeting vector. Importantly, transgenic embryos carrying this recombinant BAC did not show significant LacZ expression in the mesencephalon (Fig. 2C, first panel from the left). This suggested that the deleted 30.4 kb region included the mesencephalon-specific enhancer of ephrin-A5.This suggests that the potential enhancer for ephrin-A5 expression in the mesencephalon was in a 5.5 kb genomic region from +25 kb to +30.5 kb of the TSS." "AL-1,AV158822,EFL-5,Ephrin-A5,Epl7,LERK-7,RAGS" -- -- -- -- -- -- Ascl1 "AI225900,ASH1,Mash1,bHLHa46" "EMSA,Luciferase Reporter Assay" "Finally, both electrophoretic mobility shift assay and luciferase-based transactivation assay revealed that the transcription factor Ascl1 bound the bHLH consensus binding site in the mesencephalon-specific ephrin-A5 enhancer in vitro. " -- -- -- Intron 27749 E_02_195 28667437 -- mm10 chr9 88325101 88326201 Mouse Hepatic Myofibroblasts Low throughput Luciferase Reporter Assay "The measured luciferase activity noticeably decreased between F1- and F2-Luc constructs, suggesting that F1-Luc construct contains a potential enhancer sequence, which spans approximately 1000 bp (?2069 to ?996 bp) and is absent in the shorter F2-Luc construct." Enhancer Nt5e -- "Luciferase Reporter Assay,PCR" Luciferase activity as_x0002_says showed that the ENH1074-F5-Luc construct containing the full putative Enhancer (-2069 to -996 bp) sequence has the ability to enhance the luciferase activity of the identified putative mouse Nt5e promoter. "2210401F01Rik,5'-NT,AI447961,CD73,NT,Nt5,eNT" Hepatic Fibrosis -- D008103 -- -- -- Elf2 "2610036A20Rik,A230104O07Rik,AW111824,BB183398,EU32,NERF,NERF-1A,NERF-1B,NERF-2" Luciferase Reporter Assay "We found that mutation of Elf2 responsive element alone was sufficient to significantly increase the F5-Luc construct luciferase activity (F5-Luc, 11.79 ¡À 1.41 vs. mut Elf2-F5-Luc,20.48 ¡À 2.96, **p < 0.01).These data indicate that repression of mouse Nt5e promoter activity is likely mediated by transcription factor(s) acting at the identified Elf2 site in NIH3T3 fibroblasts." -- -- -- <2KB 1957 E_02_196 28671686 -- mm10 chr8 36785613 36985613 Mouse Brain Low+High throughput "ChIP-seq,Hi-C" "We then assessed longer-range chromosomal contacts, spanning >200kb of linear genome. We identified genome-wide 110 long-range loop contacts affected in mutant neurons (DESeq2 P<0.05). Unexpectedly, the large majority, 84/110 or 76%, represented clustered, locus-specific ¡®loop aggregates¡¯ showing massive weakening, or complete loss, after neuronal Setdb1 ablation (Figure 1d, Supplementary Figure 2c). hese deficits were specific, because ChiP-seq profiling for open chromatin-associated acetyl-H3-lysine 27 (H3K27ac) showed that 96.4% of 1,112 differentially tagged sequences were hyperacetylated in Setdb1-deficient neurons (Figure 2a, Supplementary Tables 1,2)." Enhancer Setdb1 -- ChIP-seq "Therefore, we predicted that higher order chromatin organization at these positions will be highly conserved in human brain cells, and specifically in neurons, with long-range loopings radiating from ~200kb R1 towards cPCDH promoter and Enhancers primarily anchored in chromatin at and around the Setdb1 peak. To explore, we surveyed with 40kb resolution the cPCDH -bound chromosomal contacts in our in situ Hi-C datasets generated from human neurons and their isogenic neural precursors cells (NPC) and NPC-differentiated astrocytes." "AU022152,ESET,KMT1E,mKIAA0067" Schizophrenia DOID:5419 D012559 -- -- -- Ctcf AW108038 ChIP-seq ChIP-seq data from stem cells and CD19+ B lymphocytes significant CTCF motif enrichment.CTCF motif (red) enrichment in sequences H3K9me3 hypomethylated in KO. (e) (Left) FACS plots showing separation of crosslinked NeuN+ from NeuN? nuclei (adult cortex) for cell-type specific CTCF ChIP-seq. -- -- -- >2KB 58437911 E_02_197 28749941 -- mm10 chr10 67853705 67855705 Mouse Embryo Low+High throughput "ChIP-seq,4C-seq" "To investigate the correlation between the activity of the Krox20 cis-regulatory elements and their chromatin modifications and conformation, we first performed ChIP-seq experiments to analyse two histone modifications: H3K4me1 (broad peaks covering active enhancers) and H3K27ac (punctuated peaks covering active enhancers and promoters).In E8.5 wild type embryo heads, a number of H3K4me1 peaks were observed, including those that expectedly correspond to the A, B and C elements and to a previously known neural crest element (NCE; Fig 4A). " Enhancer Egr2 4C-seq -- "To better characterize the Krox20 regulatory neighborhood, we used circular chromosome conformation capture (4C-seq) on multiple viewpoints in the locus. In samples prepared from total embryos at embryonic day (E) 9.5, when Krox20 is no more transcribed, the Krox20 gene and its distant regulatory element A (separated by over 200 kb) show highly similar distributions of 4C-seq signal (Fig 3A and S2B and S2C Fig) preferentially located in the Krox20 TAD. " "Egr-2,Krox-20,Krox20,NGF1-B,Zfp-25,Zfp-6" -- -- -- "Enhancer element C possesses a dual activity: besides its classical Enhancer function, it is also permanently required in cis to potentiate the autoregulatory activity of element A, by increasing its chromatin accessibility." ChIP-seq "Here, using both enhancer knock-outs and investigations of chromatin organisation, we show that element C possesses a dual activity: besides its classical enhancer function, it is also permanently required in cis to potentiate the autoregulatory activity of element A, by increasing its chromatin accessibility. " -- -- -- -- -- -- -- >2KB 320253 E_02_198 28749941 -- mm10 chr10 67926705 67928705 Mouse Embryo Low+High throughput "ChIP-seq,4C-seq" "To investigate the correlation between the activity of the Krox20 cis-regulatory elements and their chromatin modifications and conformation, we first performed ChIP-seq experiments to analyse two histone modifications: H3K4me1 (broad peaks covering active enhancers) and H3K27ac (punctuated peaks covering active enhancers and promoters).In E8.5 wild type embryo heads, a number of H3K4me1 peaks were observed, including those that expectedly correspond to the A, B and C elements and to a previously known neural crest element (NCE; Fig 4A). " Enhancer Egr2 4C-seq -- "To better characterize the Krox20 regulatory neighborhood, we used circular chromosome conformation capture (4C-seq) on multiple viewpoints in the locus. In samples prepared from total embryos at embryonic day (E) 9.5, when Krox20 is no more transcribed, the Krox20 gene and its distant regulatory element A (separated by over 200 kb) show highly similar distributions of 4C-seq signal (Fig 3A and S2B and S2C Fig) preferentially located in the Krox20 TAD. " "Egr-2,Krox-20,Krox20,NGF1-B,Zfp-25,Zfp-6" -- -- -- "Enhancer element C possesses a dual activity: besides its classical Enhancer function, it is also permanently required in cis to potentiate the autoregulatory activity of element A, by increasing its chromatin accessibility." ChIP-seq "Here, using both enhancer knock-outs and investigations of chromatin organisation, we show that element C possesses a dual activity: besides its classical enhancer function, it is also permanently required in cis to potentiate the autoregulatory activity of element A, by increasing its chromatin accessibility. " -- -- -- -- -- -- -- >2KB 393253 E_02_199 28836200 Col1a2 Enhancer mm10 chr6 4438697 4440197 Mouse Mesenchymal-Fibroblast Cells Low+High throughput "Luciferase Reporter Assay,DNase I Hypersensitivity Assay,ChIP-seq" "Look for regions that have high conservation and good his_x0002_tone peaks and DNase1 peaks; you can use other tracks as exclusion criteria¡¯s, but, generally, these marks are good indica_x0002_tors for enhancers without needing to conduct ChIP-seq or DNaseI hypersensitivity experiments (Fig. 5)." Enhancer Col1a2 -- "Luciferase Reporter Assay,DNase I Hypersensitivity Assay,ChIP-seq" In vivo imaging of transgenic mice that harbor a luciferase gene can be used as a noninvasive longitudinal monitoring of the gene expression. Make sure to determine plateau of constant luciferase activity rather than peak of activity (see Notes 13). You may need to shade high-expressing areas to see other low levels (see Notes 14 and 15). "AA960264,AI325291,Col1a-2,Cola-2,Cola2,oim" Fibrotic Disease -- -- -- -- -- -- -- -- -- -- -- -- >2KB 66170 E_02_200 28982762 -- mm10 chr4 55449628 55461828 Mouse Mouse Embryonic Stem Cell low throughput "Luciferase Reporter Assay,3C" "DNA fragments containing E1 and E2 specifically exhibited high transcriptional activity in na?ve state mESCs relative to primed EpiSCs as determined by transgene-driven luciferase assays, consistent with the Klf4 expression pattern in these two pluripotent cell types (Fig. 1B,C).To probe whether the putative enhancers E1 and E2 communicate with the Klf4 promoter ?55 kb away, we performed chromosome conformation capture (3C) assays to detect long-range interactions. For both E1 and E2, there was a significantly higher frequency of cross-linking to the Klf4 promoter in mESCs compared with EpiSCs (Fig. 1F), indicating that E1 and E2 spatially juxtapose the promoter in mESCs." Enhancer Klf4 "CRISPR/Cas9,3C" -- "Additionally, biallelic deletion of either E1 or E2 by CRISPR/Cas9-mediated genome editing reduced Klf4 expression by ?70% and 85%, respectively, without significantly affecting the expression of an adjacent gene (Rad23b) or another pluripotency gene (Pou5f1) (Fig. 1E).Nevertheless, for the remainder of this report, we primarily discuss our findings regarding E1 and E2, as these two enhancers clearly represent dominant elements that regulate Klf4 expression in na?ve state mESCs." "EZF,Gklf,Zie" -- -- -- -- -- -- "Oct4,Sox2,Stat3 " "2810482H01Rik,NF-A1,Oct-1,Oct1,Otf-1,Otf1,Sox-2,lcc,ysb,1110034C02Rik,AW109958,Aprf" "ChIP-exo,CRISPR/Cas9,ChIP-qPCR" "To validate the direct binding of these candidate TFs to the Klf4 Enhancers, we performed genome-wide high-resolution ChIP-exo experiments for SOX2, STAT3, and ESRRB in mESCs.Here, we probed whether the binding activities of OCT4, SOX2, ESRRB, and STAT3 at E2 are interdependent through ChIP-qPCR (ChIP combined with quantitative PCR) analysis following endogenous deletion of individual TF-binding sites.Peaks identified by ChIP-exo were consistent with peaks from previous ChIP-seq (ChIP combined with high-throughput sequcing) protocols (Supplemental Fig. S2B,C). As expected, we observed significant enrichment of all three TFs at the Klf4 enhancer cluster, particularly at E2 (Fig. 2D)." -- -- -- >2KB 71408 E_02_201 28982762 -- mm10 chr4 55453628 55465828 Mouse Mouse Embryonic Stem Cell low throughput "Luciferase Reporter Assay,3C" "DNA fragments containing E1 and E2 specifically exhibited high transcriptional activity in na?ve state mESCs relative to primed EpiSCs as determined by transgene-driven luciferase assays, consistent with the Klf4 expression pattern in these two pluripotent cell types (Fig. 1B,C).To probe whether the putative enhancers E1 and E2 communicate with the Klf4 promoter ?55 kb away, we performed chromosome conformation capture (3C) assays to detect long-range interactions. For both E1 and E2, there was a significantly higher frequency of cross-linking to the Klf4 promoter in mESCs compared with EpiSCs (Fig. 1F), indicating that E1 and E2 spatially juxtapose the promoter in mESCs." Enhancer Klf4 "CRISPR/Cas9,3C" -- "Additionally, biallelic deletion of either E1 or E2 by CRISPR/Cas9-mediated genome editing reduced Klf4 expression by ?70% and 85%, respectively, without significantly affecting the expression of an adjacent gene (Rad23b) or another pluripotency gene (Pou5f1) (Fig. 1E).Nevertheless, for the remainder of this report, we primarily discuss our findings regarding E1 and E2, as these two enhancers clearly represent dominant elements that regulate Klf4 expression in na?ve state mESCs." "EZF,Gklf,Zie" -- -- -- -- -- -- "Oct4,Sox2,Stat3 " "2810482H01Rik,NF-A1,Oct-1,Oct1,Otf-1,Otf1,Sox-2,lcc,ysb,1110034C02Rik,AW109958,Aprf" "ChIP-exo,CRISPR/Cas9,ChIP-qPCR" "To validate the direct binding of these candidate TFs to the Klf4 Enhancers, we performed genome-wide high-resolution ChIP-exo experiments for SOX2, STAT3, and ESRRB in mESCs.Here, we probed whether the binding activities of OCT4, SOX2, ESRRB, and STAT3 at E2 are interdependent through ChIP-qPCR (ChIP combined with quantitative PCR) analysis following endogenous deletion of individual TF-binding sites.Peaks identified by ChIP-exo were consistent with peaks from previous ChIP-seq (ChIP combined with high-throughput sequcing) protocols (Supplemental Fig. S2B,C). As expected, we observed significant enrichment of all three TFs at the Klf4 enhancer cluster, particularly at E2 (Fig. 2D)." -- -- -- >2KB 67408 E_02_202 29042628 -- mm10 chr16 92625802 92627802 Mouse HPC-7 Low+High throughput "4C-seq,ChIP" 4C-seq in HPC-7 cells using the P1 and P2 promoters and the +24 enhancer as ¡®baits¡¯ identified genomic interactions at Runx1 (Fig. 1a) Enhancer Runx1 4C-seq -- "Strikingly, the +24 enhancer in particular forms many significant interactions outside the Runx1 domain (Fig. 1b,d). Many of these long-range interactions are with other genes or gene promoters on mouse chromosome 16 (Fig. 2). " "AML1,CBF-alpha-2,Cbfa2,Pebp2a2,Pebpa2b" Leukaemia -- D007938 known haematopoietic enhancers that are likely to be involved in regulating?Runx1?expression in haematopoietic progenitor cells. "DNase I Hypersensitivity Assay,4C-seq,Hi-C" "These regions not only drive haematopoietic expression, but also physically connect with the Runx1-P1 promoter, lending confidence to the concept that they are bona fide regulators of Runx1 transcription." -- -- -- -- -- -- -- >2KB 25337 E_02_203 29078395 Il2ra Enhancer mm10 chr8 11564419 11614821 Mouse T Cell Low+High throughput "ChIP-seq,ChIA-PET" "To clarify how distal enhancer elements within superenhancer loci form enhancer-promoter loops and chromatin interactions, we performed RNA Pol II-based chromatin interaction analysis by paired-end tag (ChIA-PET) sequencing (24, 25) using mouse CD4+ T cells that were preactivated by TCR stimulation (anti-CD3 plus anti-CD28) and then not treated or treated with IL-2 for 4 h.Interestingly, both H3K4me3 (blue peaks in Fig. 3, primarily at the promoter) and H3K27Ac (green peaks in Fig. 3, spanning the Il2ra superenhancer region; also shown in Fig. 1E) were strong after TCR preactivation and only modestly affected by stimulation with IL-2, in marked contrast to the changes in STAT5 binding and chromatin looping. " Super-Enhancer Il2ra -- "ChIP-seq,RNA-seq" "As noted above, STAT5 and H3K27Ac are highly enriched at a subset of superenhancers, including at the Il2ra superenhancer (Fig. 1 A, B, and E), correlating with IL-2¨Cinduced gene expression (Fig. 2B and Fig. S1C). We also found extensive H3K27Ac and STAT5 binding at the human IL2RA locus (Fig. S2), analogous to the mouse Il2ra superenhancer, with significant conservation of some of the enhancer elements (Fig. S2)." "CD25,Il2r,Ly-43" -- -- -- -- -- -- "Stat5a,Stat3" "AA959963,STAT5,1110034C02Rik,AW109958,Aprf" "ChIP-seq,CRISPR/Cas9" "Based on our genome-wide analysis of chromatin immunoprecipitation coupled to DNA sequencing (ChIP-Seq) profiles, we observed that STAT5-enriched Super-Enhancers spanned large genomic regions (average size of ¡«19 kb) and indeed were associated with high levels of H3K27Ac." -- -- -- >2KB 53171 E_02_204 29113912 -- mm10 chr14 18175266 18186392 Mouse Hepatocellular Carcinoma Low+High throughput ChIP-seq "Notably, histone modification markers, including H3K4me1,H3K4me3, H3K27me3 and H3K27ac were presented at the Ang2 gene locus in liver tumor cells and were strongly colocal_x0002_ized with the two Arid1a-binding sites, suggesting an associa_x0002_tion between Arid1a binding and histone modification at the Ang2 gene locus (Fig. 4A). ChIP-qPCR analysis showed Arid1a knockdown increased the H3K27ac at the Ang2 proximal and distal regions." Enhancer Ang2 -- "ChIP-seq,ChIP-qPCR" "Notably, histone modification markers, including H3K4me1,H3K4me3, H3K27me3 and H3K27ac were presented at the Ang2 gene locus in liver tumor cells and were strongly colocal_x0002_ized with the two Arid1a-binding sites, suggesting an associa_x0002_tion between Arid1a binding and histone modification at the Ang2 gene locus (Fig. 4A). ChIP-qPCR analysis showed Arid1a knockdown increased the H3K27ac at the Ang2 proximal and distal regions." "Angrp,Raa3,Rnase5b" Hepatocellular Carcinoma DOID:684 D006528 -- -- -- Ep300 "A430090G16,A730011L11,KAT3B,p300,p300 HAT" "ChIP-seq,ChIP-PCR" "Interestingly,published ChIP-seq data also showed that p300 bound to the Ang2 gene locus, as we validated by ChIP-PCR." -- -- -- >2KB 33014656 E_02_205 29133016 -- mm10 chr2 122651534 122668846 Mouse Macrophage Low+High throughput "3C-seq,ChIP-seq" "Next, we identified an enhancer set associated with LPS-responsive pri-miRNAs based on publicly available H3K4 mono-methylation-specific ChIP-seq and GRO-seq datasets. This enhancer set was further characterized by the combination of publicly available ChIP and ATAC-seq datasets. Finally, direct interactions between the miR-155-coding genomic region and its distal regulatory elements were identified using a 3C-seq approach." Enhancer Tlr4 3C-seq "RT-qPCR,ChIP-seq,ATAC-seq" "This Enhancer set was further characterized by the combination of publicly available ChIP and ATAC-seq datasets.Finally,direct interactions between the miR-155-coding genomic region and its distal regulatory elements were identified using a 3C-seq approach." "Lps,Ly87,Ran/M1,Rasl2-8" -- -- -- -- -- -- "NFKB1,Junb" "CVID12,EBP-1,KBF1,NF-kB,NF-kB1,NF-kappa-B1,NF-kappaB,NFKB-p105,NFKB-p50,NFkappaB,p105,p50" ChIP-seq "As expected, de novo motif analysis under NFkB-p65 and JunB peaks revealed significant enrichment of NFkB-p65 and Jun-AP1 motifs (Supplementary Figure 3B and C). Targeted motif discovery at the LPS-responsive pri-miRNAs-associated enhancer subsets showed that both NFkB-p65 and Jun-AP1 motifs were found a higher number at the p65high enhancers compared to the p65low and LPS-repressed enhancers (Figure 3D)." -- -- -- >2KB 55832640 E_02_206 29141987 -- mm10 chr2 103887795 103888195 Mouse Embryo Low+High throughput ChIP-seq "We overlapped TBS with genome©\wide H3K4me1 peaks from T WT mesodermal cells and found the same trend of decreased H3K27ac within ¡À1 kb of TBS/H3K4me1 sites (Fig EV4E), suggesting that T functions to coordinate H3K27ac at putative enhancer regions throughout the genome, and may regulate the switch between poised H3K4me1(+) enhancers and active H3K27ac/H3K4me1(+) enhancer regions." Enhancer Lmo2 -- "RT-qPCR,ChIP-qPCR,ChIP-seq" "Both the ?70 enhancer and the TSS of Lmo2 display a decrease in H3K27ac in T Y88A mutant mesodermal cells, in the ChIP©\Seq data and confirmed by ChIP©\qPCR (Figs ?(Figs5A5A and B, and EV5A). We reasoned that T binding to the ?70 enhancer of Lmo2 may recruit permissive chromatin marks to the TSS to regulate expression, and that this regulation is disrupted in T Y88A mutant mesodermal cells." "Rbtn-2,Rbtn2,Rhom-2,Ttg2" -- -- -- -- -- -- T "Bra,D17Mit170,Low,Lr1,Tbxt,Tl2,Tl3,cou,me75,T" "ChIP-seq,ChIP-qPCR" "We performed a co-immunoprecipitation analysis from in vitrodifferentiated mesodermal cells and found an interaction between endogenous T and p300.Strikingly, loss of H3K27ac was found at TBS upstream of the T genomic locus itself by ChIP-Seq . We validated this decrease in H3K27ac at the T genomic locus by ChIP-qPCR in TY88A and TWT mesodermal cells differentiated in vitro." -- -- -- >2KB 69999 E_02_207 29141987 -- mm10 chr2 103886995 103888995 Mouse Embryo Low+High throughput ChIP-seq "We overlapped TBS with genome©\wide H3K4me1 peaks from T WT mesodermal cells and found the same trend of decreased H3K27ac within ¡À1 kb of TBS/H3K4me1 sites (Fig EV4E), suggesting that T functions to coordinate H3K27ac at putative enhancer regions throughout the genome, and may regulate the switch between poised H3K4me1(+) enhancers and active H3K27ac/H3K4me1(+) enhancer regions." Enhancer Lmo2 -- "RT-qPCR,ChIP-qPCR,ChIP-seq" "Both the ?70 enhancer and the TSS of Lmo2 display a decrease in H3K27ac in T Y88A mutant mesodermal cells, in the ChIP©\Seq data and confirmed by ChIP©\qPCR (Figs ?(Figs5A5A and B, and EV5A). We reasoned that T binding to the ?70 enhancer of Lmo2 may recruit permissive chromatin marks to the TSS to regulate expression, and that this regulation is disrupted in T Y88A mutant mesodermal cells." "Rbtn-2,Rbtn2,Rhom-2,Ttg2" -- -- -- -- -- -- T "Bra,D17Mit170,Low,Lr1,Tbxt,Tl2,Tl3,cou,me75,T" "ChIP-seq,ChIP-qPCR" "We performed a co-immunoprecipitation analysis from in vitrodifferentiated mesodermal cells and found an interaction between endogenous T and p300.Strikingly, loss of H3K27ac was found at TBS upstream of the T genomic locus itself by ChIP-Seq . We validated this decrease in H3K27ac at the T genomic locus by ChIP-qPCR in TY88A and TWT mesodermal cells differentiated in vitro." -- -- -- >2KB 69999 E_02_208 29254942 -- mm10 chr1 131878852 131942815 Mouse "Liver,Kidney" Low throughput "4C-seq,Transgenic mice,RNA-seq" Chromosome conformation capture (4C-seq) in liver and kidney identified liver-specific chromatin loops that recruited clock-bound enhancers to promoters to regulate liver-specific transcriptional rhythms.? Enhancer Slc45a3 4C-seq "Transgenic mice,RNA-seq" "Targeting the Slc45a3-short promoter with 4C-seq in liver and kidney showed liverspecific chromatin loops at three distal regions (two upstream, one downstream) (Figure 6A)." "2210413P12Rik,AU023994,AU043764,IPCA-6,PRST,Pcanap6" -- -- -- "Enhancers can contact a rhythmic promoter while looping out nearby nonrhythmic alternative promoters, confining rhythmic enhancer activity to specific promoters" 4C-seq Chromosome conformation capture (4C-seq) in liver and kidney identified liver-specific chromatin loops that recruited clock-bound Enhancers to promoters to regulate liver-specific transcriptional rhythms. "Rore,Onecut1,Foxa" "Rore,D9Ertd423e,HNF-6,Hfh12,Hnf6,Hnf6b,OC-1,Oc1,Foxa" ChIP-seq "These liver-specific DHSs were bound by liver-specific TFs, FOXA2 and ONECUT1, and clock TF, REVERBa, as shown in ChIP-seq." -- -- -- >2KB 52080 E_02_209 29254942 -- mm10 chr1 131920349 131962471 Mouse "Liver,Kidney" Low throughput "4C-seq,Transgenic mice,RNA-seq" Chromosome conformation capture (4C-seq) in liver and kidney identified liver-specific chromatin loops that recruited clock-bound enhancers to promoters to regulate liver-specific transcriptional rhythms.? Enhancer Slc45a3 4C-seq "Transgenic mice,RNA-seq" "Targeting the Slc45a3-short promoter with 4C-seq in liver and kidney showed liverspecific chromatin loops at three distal regions (two upstream, one downstream) (Figure 6A)." "2210413P12Rik,AU023994,AU043764,IPCA-6,PRST,Pcanap6" -- -- -- "Enhancers can contact a rhythmic promoter while looping out nearby nonrhythmic alternative promoters, confining rhythmic enhancer activity to specific promoters" 4C-seq Chromosome conformation capture (4C-seq) in liver and kidney identified liver-specific chromatin loops that recruited clock-bound Enhancers to promoters to regulate liver-specific transcriptional rhythms. "Rore,Onecut1,Foxa" "Rore,D9Ertd423e,HNF-6,Hfh12,Hnf6,Hnf6b,OC-1,Oc1,Foxa" ChIP-seq "These liver-specific DHSs were bound by liver-specific TFs, FOXA2 and ONECUT2, and clock TF, REVERBa, as shown in ChIP-seq." -- -- -- >2KB 21504 E_02_210 29256171 -- mm10 chr10 24357349 24375452 Mouse Embryonic Limb Low+High throughput "Transgenic mice,ChIP-seq,DNase I Hypersensitivity Assay" "Four functional enhancers were identified, with each driving distinct, tissue-specific patterns of transgene expression. An enhancer located -100 kb from the CCN2 transcription start site facilitated expression within vascular tissue. An enhancer -135 kb upstream of CCN2 drove expression within the articular chondrocytes of synovial joints.The other two enhancers, located at -198 kb and -229 kb, mediated transgene expression within dermal fibroblasts, however the most prevalent activity was found within hypertrophic chondrocytes and periosteal tissue, respectively. " Enhancer Ccn2 -- "PCR,Transgenic mice" "Four functional enhancers were identified, with each driving distinct, tissue-specific patterns of transgene expression. An enhancer located -100 kb from the CCN2 transcription start site facilitated expression within vascular tissue. An enhancer -135 kb upstream of CCN2 drove expression within the articular chondrocytes of synovial joints.The other two enhancers, located at -198 kb and -229 kb, mediated transgene expression within dermal fibroblasts, however the most prevalent activity was found within hypertrophic chondrocytes and periosteal tissue, respectively. " "Ctgf,Fisp12,Hcs24,fisp-12" -- -- -- -- -- -- -- -- -- -- -- -- -- >2KB 229041 E_02_211 29256171 -- mm10 chr10 24386336 24406594 Mouse Embryonic Limb Low+High throughput "Transgenic mice,ChIP-seq,DNase I Hypersensitivity Assay" "Four functional enhancers were identified, with each driving distinct, tissue-specific patterns of transgene expression. An enhancer located -100 kb from the CCN2 transcription start site facilitated expression within vascular tissue. An enhancer -135 kb upstream of CCN2 drove expression within the articular chondrocytes of synovial joints.The other two enhancers, located at -198 kb and -229 kb, mediated transgene expression within dermal fibroblasts, however the most prevalent activity was found within hypertrophic chondrocytes and periosteal tissue, respectively. " Enhancer Ccn2 -- "PCR,Transgenic mice" "Four functional enhancers were identified, with each driving distinct, tissue-specific patterns of transgene expression. An enhancer located -100 kb from the CCN2 transcription start site facilitated expression within vascular tissue. An enhancer -135 kb upstream of CCN2 drove expression within the articular chondrocytes of synovial joints.The other two enhancers, located at -198 kb and -229 kb, mediated transgene expression within dermal fibroblasts, however the most prevalent activity was found within hypertrophic chondrocytes and periosteal tissue, respectively. " "Ctgf,Fisp12,Hcs24,fisp-12" -- -- -- -- -- -- -- -- -- -- -- -- -- >2KB 198977 E_02_212 29256171 -- mm10 chr10 24449590 24469418 Mouse Embryonic Limb Low+High throughput "Transgenic mice,ChIP-seq,DNase I Hypersensitivity Assay" "Four functional enhancers were identified, with each driving distinct, tissue-specific patterns of transgene expression. An enhancer located -100 kb from the CCN2 transcription start site facilitated expression within vascular tissue. An enhancer -135 kb upstream of CCN2 drove expression within the articular chondrocytes of synovial joints.The other two enhancers, located at -198 kb and -229 kb, mediated transgene expression within dermal fibroblasts, however the most prevalent activity was found within hypertrophic chondrocytes and periosteal tissue, respectively. " Enhancer Ccn2 -- "PCR,Transgenic mice" "Four functional enhancers were identified, with each driving distinct, tissue-specific patterns of transgene expression. An enhancer located -100 kb from the CCN2 transcription start site facilitated expression within vascular tissue. An enhancer -135 kb upstream of CCN2 drove expression within the articular chondrocytes of synovial joints.The other two enhancers, located at -198 kb and -229 kb, mediated transgene expression within dermal fibroblasts, however the most prevalent activity was found within hypertrophic chondrocytes and periosteal tissue, respectively. " "Ctgf,Fisp12,Hcs24,fisp-12" -- -- -- -- -- -- -- -- -- -- -- -- -- >2KB 135938 E_02_213 29256171 -- mm10 chr10 24483642 24503469 Mouse Embryonic Limb Low+High throughput "Transgenic mice,ChIP-seq,DNase I Hypersensitivity Assay" "Four functional enhancers were identified, with each driving distinct, tissue-specific patterns of transgene expression. An enhancer located -100 kb from the CCN2 transcription start site facilitated expression within vascular tissue. An enhancer -135 kb upstream of CCN2 drove expression within the articular chondrocytes of synovial joints.The other two enhancers, located at -198 kb and -229 kb, mediated transgene expression within dermal fibroblasts, however the most prevalent activity was found within hypertrophic chondrocytes and periosteal tissue, respectively. " Enhancer Ccn2 -- "PCR,Transgenic mice" "Four functional enhancers were identified, with each driving distinct, tissue-specific patterns of transgene expression. An enhancer located -100 kb from the CCN2 transcription start site facilitated expression within vascular tissue. An enhancer -135 kb upstream of CCN2 drove expression within the articular chondrocytes of synovial joints.The other two enhancers, located at -198 kb and -229 kb, mediated transgene expression within dermal fibroblasts, however the most prevalent activity was found within hypertrophic chondrocytes and periosteal tissue, respectively. " "Ctgf,Fisp12,Hcs24,fisp-12" -- -- -- -- -- -- -- -- -- -- -- -- -- >2KB 101886 E_02_214 29272704 -- mm10 chr10 21410566 21412861 Mouse Primary Mouse Embryonic Fibroblasts Low+High throughput "ChIP-seq,ATAC-seq" "We mapped cell identity and LRG enhancers from Spret/EiJ MEFs (ATAC-seq, H3K4me1/2 ChIP-seq, ATAC-seq) to identify strain-specific cell identity and LRG enhancers (Figure 2a,b). We focused on cases in which there is no longer an ATAC-seq peak in the strain in which the enhancer sequence is no longer functional (see methods), reasoning that such examples would be most useful for determining which TF binding motifs are required for enhancer selection. In total, we identified 42 LRG enhancers and 363 cell identity enhancers that were selected in a strain-specific manner (Figure 2b)." Enhancer Lama2 -- "ChIP-seq,PCR" "Representative LRG Enhancers at the locus of the LRG Spred2. Scale bars indicate normalized read densities for each ChIP-seq and shaded boxes denote LRG Enhancers.To determine whether BAF is involved in inducible nucleosome remodeling at LRG enhancers, we performed ChIP-seq for Smarca4, a core component of BAF to determine if the complex is recruited to AP-1-bound LRG enhancers upon stimulation. This revealed that BAF levels are low at most LRG enhancers prior to stimulation but increase significantly upon stimulation (Figure 7a,b). " Lama2 -- -- -- -- -- -- Jun "AP-1c,c-jun,Jun" "ChIP-seq,ATAC-seq" "To identify fibroblast LDTFs that might be required for targeting AP-1 to fibroblast-specific Enhancers, we first performed ChIP-seq for the AP-1 TFs Fos/Jund in SPRET/EiJ MEFs to identify all strain-specific sites of AP-1 binding (n=1,224; Figure 5f). We next excluded all the strain-specific binding sites at which there was a SNP within an AP-1 motif that could directly explain the observed loss of AP-1 binding in one of the two strains (~47% of strain-specific AP-1 bound sites) (Figure 5g). " -- -- -- >2KB 5569570 E_02_215 29272704 -- mm10 chr11 19833945 19851027 Mouse Primary Mouse Embryonic Fibroblasts Low+High throughput "ChIP-seq,ATAC-seq" "We mapped cell identity and LRG enhancers from Spret/EiJ MEFs (ATAC-seq, H3K4me1/2 ChIP-seq, ATAC-seq) to identify strain-specific cell identity and LRG enhancers (Figure 2a,b). We focused on cases in which there is no longer an ATAC-seq peak in the strain in which the enhancer sequence is no longer functional (see methods), reasoning that such examples would be most useful for determining which TF binding motifs are required for enhancer selection. In total, we identified 42 LRG enhancers and 363 cell identity enhancers that were selected in a strain-specific manner (Figure 2b)." Enhancer Spred2 -- "ChIP-seq,PCR" "Representative LRG Enhancers at the locus of the LRG Spred2. Scale bars indicate normalized read densities for each ChIP-seq and shaded boxes denote LRG Enhancers.To determine whether BAF is involved in inducible nucleosome remodeling at LRG enhancers, we performed ChIP-seq for Smarca4, a core component of BAF to determine if the complex is recruited to AP-1-bound LRG enhancers upon stimulation. This revealed that BAF levels are low at most LRG enhancers prior to stimulation but increase significantly upon stimulation (Figure 7a,b). " C79158 -- -- -- -- -- -- Jun "AP-1c,c-jun,Jun" "ChIP-seq,ATAC-seq" "To identify fibroblast LDTFs that might be required for targeting AP-1 to fibroblast-specific Enhancers, we first performed ChIP-seq for the AP-1 TFs Fos/Jund in SPRET/EiJ MEFs to identify all strain-specific sites of AP-1 binding (n=1,224; Figure 5f). We next excluded all the strain-specific binding sites at which there was a SNP within an AP-1 motif that could directly explain the observed loss of AP-1 binding in one of the two strains (~47% of strain-specific AP-1 bound sites) (Figure 5g). " -- -- -- >2KB 81955 E_02_216 29272704 -- mm10 chr17 45790442 45800636 Mouse Primary Mouse Embryonic Fibroblasts Low+High throughput "ChIP-seq,ATAC-seq" "We mapped cell identity and LRG enhancers from Spret/EiJ MEFs (ATAC-seq, H3K4me1/2 ChIP-seq, ATAC-seq) to identify strain-specific cell identity and LRG enhancers (Figure 2a,b). We focused on cases in which there is no longer an ATAC-seq peak in the strain in which the enhancer sequence is no longer functional (see methods), reasoning that such examples would be most useful for determining which TF binding motifs are required for enhancer selection. In total, we identified 42 LRG enhancers and 363 cell identity enhancers that were selected in a strain-specific manner (Figure 2b)." Enhancer Vegfa -- "ChIP-seq,PCR" "Representative LRG Enhancers at the locus of the LRG Spred2. Scale bars indicate normalized read densities for each ChIP-seq and shaded boxes denote LRG Enhancers.To determine whether BAF is involved in inducible nucleosome remodeling at LRG enhancers, we performed ChIP-seq for Smarca4, a core component of BAF to determine if the complex is recruited to AP-1-bound LRG enhancers upon stimulation. This revealed that BAF levels are low at most LRG enhancers prior to stimulation but increase significantly upon stimulation (Figure 7a,b). " "Vegf,Vpf" -- -- -- -- -- -- Jun "AP-1c,c-jun,Jun" "ChIP-seq,ATAC-seq" "To identify fibroblast LDTFs that might be required for targeting AP-1 to fibroblast-specific Enhancers, we first performed ChIP-seq for the AP-1 TFs Fos/Jund in SPRET/EiJ MEFs to identify all strain-specific sites of AP-1 binding (n=1,224; Figure 5f). We next excluded all the strain-specific binding sites at which there was a SNP within an AP-1 motif that could directly explain the observed loss of AP-1 binding in one of the two strains (~47% of strain-specific AP-1 bound sites) (Figure 5g). " -- -- -- >2KB 221453 E_02_217 30545847 -- mm10 chr10 50623698 50625698 Mouse Hypothalamus Low+High throughput "ChIP-seq,ATAC-seq" "CRISPRa targeting of the Sim1 promoter or its hypothalamusspecific enhancer, which is 270 kilobases away from the gene, in Sim1 haploinsufficient mice increased the expression of the normal copy of Sim1. " Enhancer Sim1 -- "ChIP-seq,PCR,Transgenic mice" "We also carried out ChIP-seq using an antibody against S. pyogenes Cas9 in both CRISPRa-promoter and CRISPRa-Enhancer transfected cells and found on-target binding for the promoter and Enhancer,respectively." "bHLHe14, mSIM1" Haploinsufficiency -- D057895 "Transgenic-based CRISPRa targeting of the Sim1 promoter or its distant hypothalamic enhancer up-regulated its expression from the endogenous functional allele in a tissue-specific manner, rescuing the obesity phenotype in Sim1 heterozygous mice." "CRISPR/Cas9,ChIP-seq" "Transgenic-based CRISPRa targeting of the Sim1 promoter or its distant hypothalamic enhancer up-regulated its expression from the endogenous functional allele in a tissue-specific manner, rescuing the obesity phenotype in Sim1 heterozygous mice." Sim1 "bHLHe14,mSIM1" "CRISPR/Cas9,qPCR" "CRISPRa targeting of the Sim1 promoter or its hypothalamusspecific enhancer, which is 270 kilobases away from the gene, in Sim1 haploinsufficient mice increased the expression of the normal copy of Sim1. This up-regulation was sufficient to rescue the obesity phenotype of Sim1 heterozygous mice and led to significantly reduced food intake and body fat content in thesemice." -- -- -- >2KB 269976 E_02_218 29273679 -- mm10 chr2 75120051 75165771 Mouse Murine Limb Bud Cells Low+High throughput "Hi-C,4C,ChIP-seq" "In order to gain insights into TAD organization around the HoxD locus during limb bud development, we performed Hi-C (chromosome capture followed by high-throughput sequencing) on microdissected distal and proximal limb bud cells isolated from embryonic day 12.5 (E12.5) embryos. At this stage, T-DOM enhancers regulate Hoxd gene expression in proximal cells and are silent in distal cells, whereas C-DOM enhancers control Hoxd gene targets in future digit cells and are silent in proximal cells. Therefore, the two TADs are either transcriptionally active or inactive in an exclusive manner in the two tissue samples (Fig. 1A,B, top schemes)." Enhancer Hoxd 4C "Hi-C,qPCR" "Consequently,under wild-type conditions, Hoxd4 is expressed only in proximal limb cells under the control of the T-DOM,while C-DOM Enhancers control Evx2 transcripts in dis_x0002_tal cells exclusively." "Hox-4,Hox-5,Ul,ulnaless" -- -- -- -- -- -- -- -- -- -- -- -- -- >2KB 489923 E_02_219 29273679 -- mm10 chr2 75413472 75451553 Mouse Murine Limb Bud Cells Low+High throughput "Hi-C,4C,ChIP-seq" "In order to gain insights into TAD organization around the HoxD locus during limb bud development, we performed Hi-C (chromosome capture followed by high-throughput sequencing) on microdissected distal and proximal limb bud cells isolated from embryonic day 12.5 (E12.5) embryos. At this stage, T-DOM enhancers regulate Hoxd gene expression in proximal cells and are silent in distal cells, whereas C-DOM enhancers control Hoxd gene targets in future digit cells and are silent in proximal cells. Therefore, the two TADs are either transcriptionally active or inactive in an exclusive manner in the two tissue samples (Fig. 1A,B, top schemes)." Enhancer Hoxd 4C "Hi-C,qPCR" "Consequently,under wild-type conditions, Hoxd4 is expressed only in proximal limb cells under the control of the T-DOM,while C-DOM Enhancers control Evx2 transcripts in dis_x0002_tal cells exclusively." "Hox-4,Hox-5,Ul,ulnaless" -- -- -- -- -- -- -- -- -- -- -- -- -- >2KB 779525 E_02_220 29277702 -- mm10 chr2 126601468 126603468 Mouse Connective Tissue Mast Cell Low+High throughput "ChIP-seq,Luciferase Reporter Assay" "Chromatin immunoprecipitation sequencing analysis identified putative Hdc enhancers.Luciferase reporter transcription assay confirmed enhancer activities of putative enhancers in the Hdc gene.MITF bound to an enhancer located 8.8 kb upstream of the transcription start site of the Hdc gene and directed enhancer activity.To determine whether the putative Hdc Enhancers function in Hdc gene transcription, we constructed a luciferase reporter gene in which luciferase expression is under the control of the Hdc minimal promoter (-44 to +1 relative to the TSS) and the -8.8 or +0.3 Hdc Enhancer. We found that the -8.8 Hdc Enhancer dramatically increased the activity of the Hdc minimal promoter (13.4-fold increase) , whereas the +0.3 Hdc Enhancer failed to show significant Enhancer activity." Enhancer Hdc -- "ChIP-qPCR,Luciferase Reporter Assay" Chromatin immunoprecipitation sequencing analysis identified putative Hdc enhancers.Luciferase reporter transcription assay confirmed enhancer activities of putative enhancers in the Hdc gene.MITF bound to an enhancer located 8.8 kb upstream of the transcription start site of the Hdc gene and directed enhancer activity. "AW108189-a,?Hdc-c,Hdc-e,?Hdc-s,?Hdc" IgE/mast cell-mediated anaphylaxis -- -- -- -- -- "Gata2,Mitf" "Gata-2,BCC2,Bhlhe32,Gsfbcc2,Vitiligo,Wh,bw,mi,vit" "ChIP-qPCR,shRNA,Luciferase Reporter Assay" "Our ChIP-qPCR analysis of MITF binding in BMMCs revealed that among five predicted MITF-binding sites located within the -8.8 and +0.3 Hdc Enhancers.We found that Hdc mRNA expression was significantly diminished (70% reduction) in TBMMCs with knocked down Mitf mRNA expression.Conversely, overexpression of MITF in a non-mast cell line greatly 383 increased the activity of the -8.8 Hdc Enhancer." -- -- -- >2KB 8810 E_02_221 29277702 -- mm10 chr2 126593905 126595430 Mouse Connective Tissue Mast Cell Low+High throughput "ChIP-seq,Luciferase Reporter Assay" "Chromatin immunoprecipitation sequencing analysis identified putative Hdc enhancers.Luciferase reporter transcription assay confirmed enhancer activities of putative enhancers in the Hdc gene.MITF bound to an enhancer located 8.8 kb upstream of the transcription start site of the Hdc gene and directed enhancer activity.To determine whether the putative Hdc Enhancers function in Hdc gene transcription, we constructed a luciferase reporter gene in which luciferase expression is under the control of the Hdc minimal promoter (-44 to +1 relative to the TSS) and the -8.8 or +0.3 Hdc Enhancer. We found that the -8.8 Hdc Enhancer dramatically increased the activity of the Hdc minimal promoter (13.4-fold increase) , whereas the +0.3 Hdc Enhancer failed to show significant Enhancer activity." Enhancer Hdc -- "ChIP-qPCR,Luciferase Reporter Assay" Chromatin immunoprecipitation sequencing analysis identified putative Hdc enhancers.Luciferase reporter transcription assay confirmed enhancer activities of putative enhancers in the Hdc gene.MITF bound to an enhancer located 8.8 kb upstream of the transcription start site of the Hdc gene and directed enhancer activity. "AW108189-a,?Hdc-c,Hdc-e,?Hdc-s,?Hdc" IgE/mast cell-mediated anaphylaxis -- -- -- -- -- "Gata2,Mitf" "Gata-2,BCC2,Bhlhe32,Gsfbcc2,Vitiligo,Wh,bw,mi,vit" "ChIP-qPCR,shRNA,Luciferase Reporter Assay" "Our ChIP-qPCR analysis of MITF binding in BMMCs revealed that among five predicted MITF-binding sites located within the -8.8 and +0.3 Hdc Enhancers.We found that Hdc mRNA expression was significantly diminished (70% reduction) in TBMMCs with knocked down Mitf mRNA expression.Conversely, overexpression of MITF in a non-mast cell line greatly 383 increased the activity of the -8.8 Hdc Enhancer." -- -- -- <2KB 1010 E_02_222 29284139 -- mm10 chr7 143459172 143459787 Mouse Mouse Embryonic Fibroblasts Low throughput Luciferase Reporter Assay "As shown in Fig 5A, enh1 is in the second intron of Cdkn1c while enh2 is located in the 3¡¯ UTR. Luciferase reporter constructs were transfected into iMEFs stably overexpressing FOXD1 or a negative control vector." Enhancer Cdkn1c CRISPR/Cas9 "Luciferase Reporter Assay,RT-PCR" "We tested whether FOXD1 directly binds these enhancer sequences using established luciferase reporter constructs. As shown in Fig. 5A, enh1 is in the second intron of Cdkn1c while enh2 is located in the 3¡ä UTR. Luciferase reporter constructs were transfected into iMEFs stably overexpressing FOXD1 or a negative control vector. FOXD1-expressing cells exhibited higher activity of enh1-driven luciferase than negative controls while no difference was observed in luciferase activity driven by enh2(Fig. 5B)." "AL024410,CDKI,Kip2,p57(kip2),p57Kip2" Malignancies -- -- -- -- -- Foxd1 "AI385632,BF-2,FREAC4,Hfh10,Hfhbf2" Luciferase Reporter Assay "Firefly luciferase reporter assay driven by Cdkn1c enhancer elements, enh1 and enh2, in iMEFs overexpressing empty pLenti construct or FOXD1." -- -- -- <2KB 1142 E_02_223 29284139 -- mm10 chr7 143462624 143463585 Mouse Mouse Embryonic Fibroblasts Low throughput Luciferase Reporter Assay "As shown in Fig 5A, enh1 is in the second intron of Cdkn1c while enh2 is located in the 3¡¯ UTR. Luciferase reporter constructs were transfected into iMEFs stably overexpressing FOXD1 or a negative control vector." Enhancer Cdkn1c CRISPR/Cas9 "Luciferase Reporter Assay,RT-PCR" "We tested whether FOXD1 directly binds these enhancer sequences using established luciferase reporter constructs. As shown in Fig. 5A, enh1 is in the second intron of Cdkn1c while enh2 is located in the 3¡ä UTR. Luciferase reporter constructs were transfected into iMEFs stably overexpressing FOXD1 or a negative control vector. FOXD1-expressing cells exhibited higher activity of enh1-driven luciferase than negative controls while no difference was observed in luciferase activity driven by enh2(Fig. 5B)." "AL024410,CDKI,Kip2,p57(kip2),p57Kip2" Malignancies -- -- -- -- -- Gli1 "AV235269,Zfp-5,Zfp5" "RT-PCR,CRISPR/Cas9" "SHH-regulation of Ccnd1 and Cdkn1c also appeared dependent upon the GLI transcription factors, as SHH ligand addition in Gli2 iMEFs did not change their expression." -- -- -- >2KB 4767 E_02_224 29342133 -- mm10 chr15 63714919 63952099 Mouse Bone Marrow Low+High throughput ChIP-seq Here we show that an evolutionarily conserved region located 1.7 megabases downstream of the Myc gene that has previously been labelled as a ¡®super_x0002_enhancer¡¯2 is essential for the regulation of Myc expression levels in both normal haematopoietic and leukaemic stem cell hierarchies in mice and humans. Deletion of this region in mice leads to a complete loss of Myc expression in haematopoietic stem cells and progenitors. Super-Enhancer Myc -- "ChIP-seq,ATAC-seq,RT-qPCR" "In line with this hypothesis, chromatin conformation capture performedon mouse leukaemic cells2 as well as DNA proximity analysis using fluorescence in situ hybridization (DNA FISH) in LSK cells showed that the Myc promoter and BENC are in close physical proximity to each other, suggesting that cis-regulation is mediated by chromosomal looping in haematopoietic stem and progenitor cells." "AU0167572,Niard,Nird,bHLHe39,?Myc" Leukaemia -- D007938 "Clusters of enhancers, such as BENC, form highly combinatorial systems that allow precise control of gene expression across normal cellular hierarchies and which also can be hijacked in malignancies." "ATAC-seq,RT-qPCR" "Analysis of mice carrying deletions of individual Enhancer modules suggests that specific Myc expression levels throughout most of the haematopoietic hierarchy are controlled by the combinatorial and additive activity of individual Enhancer modules, which collectively function as a ¡®blood Enhancer cluster¡¯(BENC)." -- -- -- -- -- -- -- >2KB 1848169 E_02_225 29358217 MdE Enhancer mm10 chr1 133347574 133364574 Mouse "As4.1,Tsukuba hypertensive mice" Low throughput "Luciferase Reporter Assay,ChIP" "Because the 17-kb sequence exhibited Enhancer activity in vivo and in vitro,we narrowed down the Enhancer to a 2.3-kb core using Luciferase assays in As4.1 cells. When this 2.3-kb sequence was removed from the endogenous mRen gene in the mouse,its basal expression was dramatically reduced,and the hypertension esponsiveness was significantly attenuated." Enhancer Ren1 CRISPR/Cas9 "qRT-PCR,ChIP" "While the mRen gene bearing the 3' deletion was appropriately downregulated, the one bearing the 5'deletion lost this hypertension responsiveness. Because the 17-kb sequence exhib_x0002_ited enhancer activity in vivo and in vitro, we narrowed down the enhancer to a 2.3-kb core using luciferase assays in As4.1 cells. When this 2.3-kb sequence was re_x0002_moved from the endogenous mRen gene in the mouse, its basal expression was dra_x0002_matically reduced, and the hypertension responsiveness was significantly attenuated." "D19352,Ren,Ren-1,Ren-Ac,Ren1d,Rn-1,Rnr,?Ren1" Hypertension DOID:10763 D006973 This finding sheds light on the unexplored distal regulatory region of the mRen gene and provides in vivo evidence that a novel 5' enhancer plays an essential role in the physiological response to an AII-induced hypertensive environment. qRT-PCR "We propose that in a hypertensive environment, the activity of this novel enhancer is attenuated, and, as a consequence, mRen gene transcription is suppressed to maintain blood pressure." -- -- -- -- -- -- -- >2KB 5401 E_02_226 29514092 -- mm10 chr19 61151074 61161722 Mouse "Monocytes,Dendritic Cells" Low+High throughput ChIP-seq "Active enhancers shared by monocytes and DCs (but not neutrophils) and those specific for monocytes were primed and activated with similar kinetics(Figure 2A, left and center). H3K4me1 and H3K27ac ChIP-seq tag densities in these enhancer regions initially increased at the MDP stage.Notably, the expression of genes with primed but not active (often called poised) enhancers was barely induced in monocytes and DCs (Figure S2). We found that the kinetics of DC-specific active enhancers were slower(Figure 2A, right);" Super-Enhancer Csf2ra -- "ChIP-seq,Transgenic mice,RT-qPCR" "Representative genome browser images of monocyte- or DC-specific en_x0002_hancers, associated with the Bfsp1 and Ehf genes, respec_x0002_tively, are depicted in Figures 2B and 2C.Thus, monocyte_x0002_and/or DC-specific active enhancers are gradually established at progenitor stages prior to the expression of associated genes." "CD116,Csfgmra,GM-CSFR,GM-CSF-Ra" -- -- -- -- -- -- Irf8 "AI893568,ICSBP,IRF-8,Icsbp1,Myls" "ChIP-seq,Transgenic mice" "Analysis of Enhancer landscapes in Irf8¨C/¨C mononuclear phagocyte progenitors and ChIP-seq of IRF8 binding sites in wild-type (WT) cells uncovered a critical role for IRF8.Using an in vitro monocyte differentiation system, we additionally showed that IRF8 binds to promoter-distal enhancers and promotes H3K4me1 enrichment at its binding sites.Consistent with these data, the correlation coefficient of H3K4me1 ChIP-seq tag densities between WT MDPs and Irf8¨C/¨C MDPs was lower than that of the microarray data." -- -- -- >2KB 60792 E_02_227 29514092 -- mm10 chr19 61158643 61164569 Mouse "Monocytes,Dendritic Cells" Low+High throughput ChIP-seq "Active enhancers shared by monocytes and DCs (but not neutrophils) and those specific for monocytes were primed and activated with similar kinetics(Figure 2A, left and center). H3K4me1 and H3K27ac ChIP-seq tag densities in these enhancer regions initially increased at the MDP stage.Notably, the expression of genes with primed but not active (often called poised) enhancers was barely induced in monocytes and DCs (Figure S2). We found that the kinetics of DC-specific active enhancers were slower(Figure 2A, right);" Super-Enhancer Csf2ra -- "ChIP-seq,Transgenic mice,RT-qPCR" "Representative genome browser images of monocyte- or DC-specific en_x0002_hancers, associated with the Bfsp1 and Ehf genes, respec_x0002_tively, are depicted in Figures 2B and 2C.Thus, monocyte_x0002_and/or DC-specific active enhancers are gradually established at progenitor stages prior to the expression of associated genes." "CD116,Csfgmra,GM-CSFR,GM-CSF-Ra" -- -- -- -- -- -- Irf8 "AI893568,ICSBP,IRF-8,Icsbp1,Myls" "ChIP-seq,Transgenic mice" "Analysis of Enhancer landscapes in Irf8¨C/¨C mononuclear phagocyte progenitors and ChIP-seq of IRF8 binding sites in wild-type (WT) cells uncovered a critical role for IRF8.Using an in vitro monocyte differentiation system, we additionally showed that IRF8 binds to promoter-distal enhancers and promotes H3K4me1 enrichment at its binding sites.Consistent with these data, the correlation coefficient of H3K4me1 ChIP-seq tag densities between WT MDPs and Irf8¨C/¨C MDPs was lower than that of the microarray data." -- -- -- >2KB 55584 E_02_228 29514092 -- mm10 chr19 61165425 61172555 Mouse "Monocytes,Dendritic Cells" Low+High throughput ChIP-seq "Active enhancers shared by monocytes and DCs (but not neutrophils) and those specific for monocytes were primed and activated with similar kinetics(Figure 2A, left and center). H3K4me1 and H3K27ac ChIP-seq tag densities in these enhancer regions initially increased at the MDP stage.Notably, the expression of genes with primed but not active (often called poised) enhancers was barely induced in monocytes and DCs (Figure S2). We found that the kinetics of DC-specific active enhancers were slower(Figure 2A, right);" Super-Enhancer Csf2ra -- "ChIP-seq,Transgenic mice,RT-qPCR" "Representative genome browser images of monocyte- or DC-specific en_x0002_hancers, associated with the Bfsp1 and Ehf genes, respec_x0002_tively, are depicted in Figures 2B and 2C.Thus, monocyte_x0002_and/or DC-specific active enhancers are gradually established at progenitor stages prior to the expression of associated genes." "CD116,Csfgmra,GM-CSFR,GM-CSF-Ra" -- -- -- -- -- -- Irf8 "AI893568,ICSBP,IRF-8,Icsbp1,Myls" "ChIP-seq,Transgenic mice" "Analysis of Enhancer landscapes in Irf8¨C/¨C mononuclear phagocyte progenitors and ChIP-seq of IRF8 binding sites in wild-type (WT) cells uncovered a critical role for IRF8.Using an in vitro monocyte differentiation system, we additionally showed that IRF8 binds to promoter-distal enhancers and promotes H3K4me1 enrichment at its binding sites.Consistent with these data, the correlation coefficient of H3K4me1 ChIP-seq tag densities between WT MDPs and Irf8¨C/¨C MDPs was lower than that of the microarray data." -- -- -- >2KB 48200 E_02_229 29514092 -- mm10 chr2 144000110 144014615 Mouse "Monocytes,Dendritic Cells" Low+High throughput ChIP-seq "Active enhancers shared by monocytes and DCs (but not neutrophils) and those specific for monocytes were primed and activated with similar kinetics(Figure 2A, left and center). H3K4me1 and H3K27ac ChIP-seq tag densities in these enhancer regions initially increased at the MDP stage.Notably, the expression of genes with primed but not active (often called poised) enhancers was barely induced in monocytes and DCs (Figure S2). We found that the kinetics of DC-specific active enhancers were slower(Figure 2A, right);" Super-Enhancer Bfsp1 -- "ChIP-seq,Transgenic mice,RT-qPCR" "Representative genome browser images of monocyte- or DC-specific en_x0002_hancers, associated with the Bfsp1 and Ehf genes, respec_x0002_tively, are depicted in Figures 2B and 2C.Thus, monocyte_x0002_and/or DC-specific active enhancers are gradually established at progenitor stages prior to the expression of associated genes." CP95 -- -- -- -- -- -- Irf8 "AI893568,ICSBP,IRF-8,Icsbp1,Myls" "ChIP-seq,Transgenic mice" "Analysis of Enhancer landscapes in Irf8¨C/¨C mononuclear phagocyte progenitors and ChIP-seq of IRF8 binding sites in wild-type (WT) cells uncovered a critical role for IRF8.Using an in vitro monocyte differentiation system, we additionally showed that IRF8 binds to promoter-distal enhancers and promotes H3K4me1 enrichment at its binding sites.Consistent with these data, the correlation coefficient of H3K4me1 ChIP-seq tag densities between WT MDPs and Irf8¨C/¨C MDPs was lower than that of the microarray data." -- -- -- >2KB 180836 E_02_230 29514092 -- mm10 chr2 144014506 144023297 Mouse "Monocytes,Dendritic Cells" Low+High throughput ChIP-seq "Active enhancers shared by monocytes and DCs (but not neutrophils) and those specific for monocytes were primed and activated with similar kinetics(Figure 2A, left and center). H3K4me1 and H3K27ac ChIP-seq tag densities in these enhancer regions initially increased at the MDP stage.Notably, the expression of genes with primed but not active (often called poised) enhancers was barely induced in monocytes and DCs (Figure S2). We found that the kinetics of DC-specific active enhancers were slower(Figure 2A, right);" Super-Enhancer Bfsp1 -- "ChIP-seq,Transgenic mice,RT-qPCR" "Representative genome browser images of monocyte- or DC-specific en_x0002_hancers, associated with the Bfsp1 and Ehf genes, respec_x0002_tively, are depicted in Figures 2B and 2C.Thus, monocyte_x0002_and/or DC-specific active enhancers are gradually established at progenitor stages prior to the expression of associated genes." CP95 -- -- -- -- -- -- Irf8 "AI893568,ICSBP,IRF-8,Icsbp1,Myls" "ChIP-seq,Transgenic mice" "Analysis of Enhancer landscapes in Irf8¨C/¨C mononuclear phagocyte progenitors and ChIP-seq of IRF8 binding sites in wild-type (WT) cells uncovered a critical role for IRF8.Using an in vitro monocyte differentiation system, we additionally showed that IRF8 binds to promoter-distal enhancers and promotes H3K4me1 enrichment at its binding sites.Consistent with these data, the correlation coefficient of H3K4me1 ChIP-seq tag densities between WT MDPs and Irf8¨C/¨C MDPs was lower than that of the microarray data." -- -- -- >2KB 192375 E_02_231 29514092 -- mm10 chr2 103464661 103471206 Mouse "Monocytes,Dendritic Cells" Low+High throughput ChIP-seq "Active enhancers shared by monocytes and DCs (but not neutrophils) and those specific for monocytes were primed and activated with similar kinetics(Figure 2A, left and center). H3K4me1 and H3K27ac ChIP-seq tag densities in these enhancer regions initially increased at the MDP stage.Notably, the expression of genes with primed but not active (often called poised) enhancers was barely induced in monocytes and DCs (Figure S2). We found that the kinetics of DC-specific active enhancers were slower(Figure 2A, right);" Super-Enhancer Ehf -- "ChIP-seq,Transgenic mice,RT-qPCR" "Representative genome browser images of monocyte- or DC-specific en_x0002_hancers, associated with the Bfsp1 and Ehf genes, respec_x0002_tively, are depicted in Figures 2B and 2C.Thus, monocyte_x0002_and/or DC-specific active enhancers are gradually established at progenitor stages prior to the expression of associated genes." "9030625L19Rik,AU019492" -- -- -- -- -- -- Irf8 "AI893568,ICSBP,IRF-8,Icsbp1,Myls" "ChIP-seq,Transgenic mice" "Analysis of Enhancer landscapes in Irf8¨C/¨C mononuclear phagocyte progenitors and ChIP-seq of IRF8 binding sites in wild-type (WT) cells uncovered a critical role for IRF8.Using an in vitro monocyte differentiation system, we additionally showed that IRF8 binds to promoter-distal enhancers and promotes H3K4me1 enrichment at its binding sites.Consistent with these data, the correlation coefficient of H3K4me1 ChIP-seq tag densities between WT MDPs and Irf8¨C/¨C MDPs was lower than that of the microarray data." -- -- -- >2KB 204502 E_02_232 29527594 -- mm10 chr6 127033997 127036097 Mouse Osteocyte Cell Lines Low+High throughput "ChIP-seq,CRISPR/Cas9" "We therefore utilized chromatin immunoprecipitation followed by DNA sequencing (ChIP-seq) data from an osteocyte cell line to identify potential regulatory regions of the Fgf23 gene. Based on ChIP-seq analysis of enhancer_x0002_associated histone modifications, including H3K4 methylation and H3K9 acetylation, we discovered several potential enhancers for Fgf23, one of which was located 16kb upstream of the gene¡¯s transcriptional start site." Enhancer Fgf23 CRISPR/Cas9 "ChIP-seq,RT-PCR" "Nevertheless, lack of the _x0003_16kb enhancer blunted FGF23 upregulation in a tissue-specific manner by the acute inflammatory inducers lipopolysaccharide (LPS), interleukin-1-beta (IL-1b), and tumor necrosis factor-alpha (TNFa) in bone, non-osseous tissues,and in circulation. Lack of the _x0003_16kb enhancer also inhibited PTH-induced bone Fgf23 mRNA. Moreover, the absence of this Fgf23 enhancer in an oxalate diet-induced murine CKD model prevented the early onset induction of osseous, renal, and thymic Fgf23 mRNA levels and led to a significant blunting of elevated circulating intact FGF23 levels. These results suggest that _x0003_16kb enhancer mediates the induction of Fgf23 by inflammation and PTH and facilitates the increase in FGF23 expression in a murine model of CKD." Fgf23 Chronic Kidney Disease DOID:784 D051436 16kb Enhancer mediates the induction of Fgf23 by inflammation and PTH and facilitates the increase in FGF23 expression in a murine model of CKD ChIP-seq These results suggest that 16kb Enhancer mediates the induction of Fgf23 by inflammation and PTH and facilitates the increase in FGF23 expression in a murine model of CKD. "Nfkb1,Hif1a" "NF-KB1,NF-kappaB,NF-kappaB1,p105,p50,p50/p105;AA959795,HIF-1-alpha,HIF1-alpha,HIF1alpha,MOP1,bHLHe78" ChIP-seq "Advances in genome©\wide techniques such as chromatin immunoprecipitation followed by DNA sequencing (ChIP©\seq) have revealed that the transcriptional regulation of many genes, including receptor activator of NF©\¦ÊB ligand (Tnfsf11), vitamin D receptor (Vdr), or matrix metallopeptidase 13 (Mmp13), are mediated through a complex network of Enhancers each providing distinct factor©\ and cell©\type specificity." -- -- -- >2KB 37854 E_02_233 29903884 -- mm10 chr11 112217210 112217767 Mouse Testis Low+High throughput "DNaseI-seq,ChIP-seq" "In vivo high-throughput chromatin accessibility techniques, transgenic assays, and genome editing revealed several novel gonadal regulatory elements in the 2 Mb gene desert upstream of Sox9.Although others are redundant, Enh13, a 557 bp element located 565 kb 5¡¯, is essential to initiate mouse testis development." Enhancer Sox9 CRISPR/Cas9 "ChIP-seq,RT-PCR" "There was a 30 to 50% decrease in Sox9 mRNA levels in E11.5 XX Enh13-/- gonads compared to the wild type, as reflected by reduced immunofluorescence for SOX9 protein (fig. S11). These data indicate that Enh13 also plays a role in the very early expression of Sox9 in the XX gonad." "2010306G03Rik,AV220920,mKIAA4243" -- -- -- "Enh13 is conserved and embedded within a 32.5-kilobase region whose deletion in humans is associated with XY sex reversal, suggesting that it is also critical in humans." "CRISPR/Cas9,RT-qPCR" This again supports the conclusion that Enh13 plays a more substantial role than TES during early gonadal development. Sry "Tdf,Tdy" ChIP-qPCR "We performed ChIP-qPCR on E11.5 gonads dissected from Sry-Myc transgenic embryos by using a specific antibody against the MYC tag.SRY-MYC¨Cpositive gonads had an 11-fold enrichment versus SRY-MYC¨Cnegative gonads with primers spanning the SOX9 consensus site and a sixfold enrichment with primers spanning the SRY site, whereas primers against the strongest SRY binding site in TESCO showed fivefold enrichment.This reveals the strong binding of SRY to Enh13 at E11.5, with a preference for the SOX9 consensus site, possibly due to the adjacent SF1 binding site." -- -- -- >2KB 564720 E_02_234 29967281 -- mm10 chr17 85527960 85530660 Mouse The Subventricular Zone Of Lateral Ganglionic Eminence??LGE SVZ?? Low+High throughput "Luciferase Reporter Assay,ChIP-seq" "We found an SP9 ChIP-Seq peak at the Six3 promoter region (?5 to +411 of the TSS) (Fig. 9C), and several additional peaks upstream of the Six3 gene (regions 1, 2 and 3) (Fig. 9C);Thus, these noncoding domains are strong candidates for the Six3 promoter and three Six3 enhancers." Enhancer Six3 -- "ChIP-seq,Luciferase Reporter Assay" "Together,the results of ChIP-seq and Dual-Luciferase assay provide evidence that SP9 promotes Six3 expression in the LGE SVZ through its direct binding to the promoter and possibly through binding to regulatory elements within the Six3 locus." "E130112M24Rika,Six3alpha,Six3b,Six3beta,?Six3" -- -- -- -- -- -- Six3 E130112M24Rika£¬Six3alpha£¬Six3b£¬Six3beta "RNA-seq,ChIP-seq" "SP8 and SP9 together drive expression of the transcription factor Six3 in a spatially restricted domain of the LGE subventricular zone. Conditional deletion of Six3 also prevents the formation of most D2 MSNs, phenocopying the Sp8/9 mutants.This implies that there are additional mechanisms that regulate the generation of D2 MSNs in Sp9-KO mice, one of which was suggested by the LGE RNA-Seq analysis. We identified that expression of the TF Six3 was decreased ?5-fold in E16.5 Sp8/9-DCKO mice compared with control mice." -- -- -- >2KB 84297 E_02_235 29967281 -- mm10 chr17 85463916 85466386 Mouse The Subventricular Zone Of Lateral Ganglionic Eminence??LGE SVZ?? Low+High throughput "Luciferase Reporter Assay,ChIP-seq" "We found an SP9 ChIP-Seq peak at the Six3 promoter region (?5 to +411 of the TSS) (Fig. 9C), and several additional peaks upstream of the Six3 gene (regions 1, 2 and 3) (Fig. 9C);Thus, these noncoding domains are strong candidates for the Six3 promoter and three Six3 enhancers." Enhancer Six3 -- "ChIP-seq,Luciferase Reporter Assay" "Together,the results of ChIP-seq and Dual-Luciferase assay provide evidence that SP9 promotes Six3 expression in the LGE SVZ through its direct binding to the promoter and possibly through binding to regulatory elements within the Six3 locus." "E130112M24Rika,Six3alpha,Six3b,Six3beta,?Six3" -- -- -- -- -- -- Six3 E130112M24Rika£¬Six3alpha£¬Six3b£¬Six3beta "RNA-seq,ChIP-seq" "SP8 and SP9 together drive expression of the transcription factor Six3 in a spatially restricted domain of the LGE subventricular zone. Conditional deletion of Six3 also prevents the formation of most D2 MSNs, phenocopying the Sp8/9 mutants.This implies that there are additional mechanisms that regulate the generation of D2 MSNs in Sp9-KO mice, one of which was suggested by the LGE RNA-Seq analysis. We identified that expression of the TF Six3 was decreased ?5-fold in E16.5 Sp8/9-DCKO mice compared with control mice." -- -- -- >2KB 148456 E_02_236 29967281 -- mm10 chr17 85392908 85395234 Mouse The Subventricular Zone Of Lateral Ganglionic Eminence??LGE SVZ?? Low+High throughput "Luciferase Reporter Assay,ChIP-seq" "We found an SP9 ChIP-Seq peak at the Six3 promoter region (?5 to +411 of the TSS) (Fig. 9C), and several additional peaks upstream of the Six3 gene (regions 1, 2 and 3) (Fig. 9C);Thus, these noncoding domains are strong candidates for the Six3 promoter and three Six3 enhancers." Enhancer Six3 -- "ChIP-seq,Luciferase Reporter Assay" "Together,the results of ChIP-seq and Dual-Luciferase assay provide evidence that SP9 promotes Six3 expression in the LGE SVZ through its direct binding to the promoter and possibly through binding to regulatory elements within the Six3 locus." "E130112M24Rika,Six3alpha,Six3b,Six3beta,?Six3" -- -- -- -- -- -- Six3 E130112M24Rika£¬Six3alpha£¬Six3b£¬Six3beta "RNA-seq,ChIP-seq" "SP8 and SP9 together drive expression of the transcription factor Six3 in a spatially restricted domain of the LGE subventricular zone. Conditional deletion of Six3 also prevents the formation of most D2 MSNs, phenocopying the Sp8/9 mutants.This implies that there are additional mechanisms that regulate the generation of D2 MSNs in Sp9-KO mice, one of which was suggested by the LGE RNA-Seq analysis. We identified that expression of the TF Six3 was decreased ?5-fold in E16.5 Sp8/9-DCKO mice compared with control mice." -- -- -- >2KB 219536 E_02_237 29979962 DRR Enhancer mm10 chr7 46371356 46373481 Mouse C2C12 Low throughput ChIRP "To directly evaluate whether the DRReRNA 2Kb transcript could undergo splicing, we cloned it into the splicing reporter plasmid RHCglo.The resulting plasmid (RHCglo-DRR) or the parental vector were transiently transfected in human embryonic kidney 293 cells, in C2C12 MB or C2C12 cells induced to differentiate. RNA recovered from RHCglo-DRR-transfected cells and amplified with vector-specific primers gave rise to a single band of ~1.5Kb (Figure S1B,C), the same length observed for endogenous DRReRNA." Enhancer Myod1 -- "Western blot,RT-qPCR,ChIP" "DRReRNA influences transcription of Myogenin in trans without significantly affecting that of MyoD in cis;This analysis revealed that the top two DRReRNA-enriched ChIRP regions were observed at Myogenin and DRR, whereas the MyoD gene located 5 kb downstream of DRR was devoid of DRReRNA binding." "AI503393,MYF3,MyoD,?Myod-1,bHLHc1" -- -- -- DRR eRNA Promotes Cohesin Recruitment and Maintenance at the Myogenin Locus qRT-PCR A non-coding RNA transcribed from an Enhancer region on mouse chromosome 7 (eRNA) recruits cohesin to regulate transcription of the Myogenin gene located on chromosome 1. -- -- -- -- -- -- -- >2KB 4054 E_02_238 30008200 -- mm10 chr13 56243248 56243833 Mouse P19 Low throughput ChIP "In control cells, the enrichment of H3K27Ac, an active Enhancer marker, was significantly increased at the Neurog1 promoter, as well as other Enhancers, upon RA treatment." Enhancer Neurog1 -- ChIP-qPCR "Genome-wide CTCF occupancy patterns, which have been mapped across more than 100 mammalian cell types, re_x0002_vealed prominent CTCF binding to the -14.4 kb and -7.6 kb transcription start sites of Neurog1.We performed ChIP-qPCR analysis to validate the in vivo binding of CTCF in P19 cells, and observed strong occupancy of CTCF to these binding sites." "AKA,Math4C,Neurod3,bHLHa6,ngn1" -- -- -- -- -- -- Ctcf AW108038 ChIP-qPCR "Genome-wide CTCF occupancy patterns, which have been mapped across more than 100 mammalian cell types, re_x0002_vealed prominent CTCF binding to the -14.4 kb and -7.6 kb transcription start sites of Neurog1.We performed ChIP-qPCR analysis to validate the in vivo binding of CTCF in P19 cells, and observed strong occupancy of CTCF to these binding sites." -- -- -- >2KB 6950 E_02_239 30008200 -- mm10 chr13 56244114 56244357 Mouse P19 Low throughput ChIP "In control cells, the enrichment of H3K27Ac, an active Enhancer marker, was significantly increased at the Neurog1 promoter, as well as other Enhancers, upon RA treatment." Enhancer Neurog1 -- ChIP-qPCR "Genome-wide CTCF occupancy patterns, which have been mapped across more than 100 mammalian cell types, re_x0002_vealed prominent CTCF binding to the -14.4 kb and -7.6 kb transcription start sites of Neurog1.We performed ChIP-qPCR analysis to validate the in vivo binding of CTCF in P19 cells, and observed strong occupancy of CTCF to these binding sites." "AKA,Math4C,Neurod3,bHLHa6,ngn1" -- -- -- -- -- -- Ctcf AW108038 ChIP-qPCR "Genome-wide CTCF occupancy patterns, which have been mapped across more than 100 mammalian cell types, re_x0002_vealed prominent CTCF binding to the -14.4 kb and -7.6 kb transcription start sites of Neurog1.We performed ChIP-qPCR analysis to validate the in vivo binding of CTCF in P19 cells, and observed strong occupancy of CTCF to these binding sites." -- -- -- >2KB 6255 E_02_240 30008200 -- mm10 chr13 56245174 56245857 Mouse P19 Low throughput ChIP "In control cells, the enrichment of H3K27Ac, an active Enhancer marker, was significantly increased at the Neurog1 promoter, as well as other Enhancers, upon RA treatment." Enhancer Neurog1 -- ChIP-qPCR "Genome-wide CTCF occupancy patterns, which have been mapped across more than 100 mammalian cell types, re_x0002_vealed prominent CTCF binding to the -14.4 kb and -7.6 kb transcription start sites of Neurog1.We performed ChIP-qPCR analysis to validate the in vivo binding of CTCF in P19 cells, and observed strong occupancy of CTCF to these binding sites." "AKA,Math4C,Neurod3,bHLHa6,ngn1" -- -- -- -- -- -- Ctcf AW108038 ChIP-qPCR "Genome-wide CTCF occupancy patterns, which have been mapped across more than 100 mammalian cell types, re_x0002_vealed prominent CTCF binding to the -14.4 kb and -7.6 kb transcription start sites of Neurog1.We performed ChIP-qPCR analysis to validate the in vivo binding of CTCF in P19 cells, and observed strong occupancy of CTCF to these binding sites." -- -- -- >2KB 4975 E_02_241 30017589 -- mm10 chr13 30108722 30109111 Mouse "Embryonic Stem Cell,Epiblast-like Cells (EpiLCs)" Low+High throughput ChIP-seq "Our findings were corroborated by H3K4me1 and H3K27ac ChIP-seq data from an alternative na?ve-to-formative differentiation system. As a control, we found little change in these marks at SMC1 sites common to both states. These results indicate that GRHL2 binding correlates with full enhancer activation, suggesting a role for GRHL2 not only in regulation of cohesin binding, but also in regulation of other crucial steps in enhancer activation.Together, these results suggest that our SMC1 ChIP-seq data reflect changing enhancer activity and that cohesin binding at enhancers is highly dynamic during the ESC to EpiLC transition." Enhancer Dsp CRISPR/Cas9 "ChIP-seq,qRT-PCR" "We then assessed the effects of each Enhancer deletion on Dsp expression in ES cells and EpiLCs by qRT-PCR.Indeed, we found that deletion of one or both KLF4-bound enhancers led to a significant reduction in Dsp expression in ESCs but had no effect in EpiLCs(Figure 6B). Conversely, deletion of the GRHL2-bound enhancer resulted in significantly reduced Dsp expression in the EpiLC state but had no effect in ESCs (Figure 6B). We observed similar state-specific reductions in target gene expression with deletions of the KLF4-bound and GRHL2-bound enhancers at the Cdh1 and Jam1 loci (Figure 6C and D, S6A and B)." "2300002E22Rik,5730453H04Rik,AA407887,AA407888,AW109828,DP,rul" -- -- -- GRHL2 therefore control over a subset of the naive network via Enhancer switching to maintain expression of epithelial genes upon exit from naive pluripotency ChIP-seq "Together,these results suggest that our SMC1 ChIP-seq data reflect changing Enhancer activity and that cohesin binding at Enhancers is highly dynamic during the ESC to EpiLC transition." Grhl2 "0610015A08Rik,BOM,Tcfcp2l3,clft3" "ChIP-seq,ATAC-seq" "Given the strong association between GRHL2 binding and various events in enhancer activation, we asked whether GRHL2 binding is necessary for each of these events. To address this question, we assessed levels of each active enhancer mark in wildtype and GHRL2 KO EpiLCs by performing ATAC-seq and ChIP-seq for H3K4me1, H3K27ac, and SMC1. There was a strong reduction in all active enhancer marks (chromatin accessibility, H3K4me1, H3K27ac, and SMC1 levels) at GRHL2 sites in GRHL2 KO EpiLCs, suggesting a near complete block in enhancer activation in the absence of GRHL2. Together, these results indicate an absolute requirement for GRHL2 for enhancer activation specifically at its target sites." -- -- -- >2KB 8042376 E_02_242 30017589 -- mm10 chr13 30123333 30124861 Mouse "Embryonic Stem Cell,Epiblast-like Cells (EpiLCs)" Low+High throughput ChIP-seq "Our findings were corroborated by H3K4me1 and H3K27ac ChIP-seq data from an alternative na?ve-to-formative differentiation system. As a control, we found little change in these marks at SMC1 sites common to both states. These results indicate that GRHL2 binding correlates with full enhancer activation, suggesting a role for GRHL2 not only in regulation of cohesin binding, but also in regulation of other crucial steps in enhancer activation.Together, these results suggest that our SMC1 ChIP-seq data reflect changing enhancer activity and that cohesin binding at enhancers is highly dynamic during the ESC to EpiLC transition." Enhancer Dsp CRISPR/Cas9 "ChIP-seq,qRT-PCR" "We then assessed the effects of each Enhancer deletion on Dsp expression in ES cells and EpiLCs by qRT-PCR.Indeed, we found that deletion of one or both KLF4-bound enhancers led to a significant reduction in Dsp expression in ESCs but had no effect in EpiLCs(Figure 6B). Conversely, deletion of the GRHL2-bound enhancer resulted in significantly reduced Dsp expression in the EpiLC state but had no effect in ESCs (Figure 6B). We observed similar state-specific reductions in target gene expression with deletions of the KLF4-bound and GRHL2-bound enhancers at the Cdh1 and Jam1 loci (Figure 6C and D, S6A and B)." "2300002E22Rik,5730453H04Rik,AA407887,AA407888,AW109828,DP,rul" -- -- -- GRHL2 therefore control over a subset of the naive network via Enhancer switching to maintain expression of epithelial genes upon exit from naive pluripotency ChIP-seq "Together,these results suggest that our SMC0 ChIP-seq data reflect changing Enhancer activity and that cohesin binding at Enhancers is highly dynamic during the ESC to EpiLC transition." Grhl2 "0610015A08Rik,BOM,Tcfcp2l3,clft3" "ChIP-seq,ATAC-seq" "Given the strong association between GRHL2 binding and various events in enhancer activation, we asked whether GRHL2 binding is necessary for each of these events. To address this question, we assessed levels of each active enhancer mark in wildtype and GHRL2 KO EpiLCs by performing ATAC-seq and ChIP-seq for H3K4me1, H3K27ac, and SMC1. There was a strong reduction in all active enhancer marks (chromatin accessibility, H3K4me1, H3K27ac, and SMC1 levels) at GRHL2 sites in GRHL2 KO EpiLCs, suggesting a near complete block in enhancer activation in the absence of GRHL2. Together, these results indicate an absolute requirement for GRHL2 for enhancer activation specifically at its target sites." -- -- -- >2KB 8027196 E_02_243 30017589 -- mm10 chr13 30144166 30145833 Mouse Embryonic Stem Cell Low+High throughput ChIP-seq "Our findings were corroborated by H3K4me1 and H3K27ac ChIP-seq data from an alternative na?ve-to-formative differentiation system. As a control, we found little change in these marks at SMC1 sites common to both states. These results indicate that GRHL2 binding correlates with full enhancer activation, suggesting a role for GRHL2 not only in regulation of cohesin binding, but also in regulation of other crucial steps in enhancer activation.Together, these results suggest that our SMC1 ChIP-seq data reflect changing enhancer activity and that cohesin binding at enhancers is highly dynamic during the ESC to EpiLC transition." Enhancer Dsp CRISPR/Cas9 "ChIP-seq,qRT-PCR" "We then assessed the effects of each Enhancer deletion on Dsp expression in ES cells and EpiLCs by qRT-PCR.Indeed, we found that deletion of one or both KLF4-bound enhancers led to a significant reduction in Dsp expression in ESCs but had no effect in EpiLCs(Figure 6B). Conversely, deletion of the GRHL2-bound enhancer resulted in significantly reduced Dsp expression in the EpiLC state but had no effect in ESCs (Figure 6B). We observed similar state-specific reductions in target gene expression with deletions of the KLF4-bound and GRHL2-bound enhancers at the Cdh1 and Jam1 loci (Figure 6C and D, S6A and B)." "2300002E22Rik,5730453H04Rik,AA407887,AA407888,AW109828,DP,rul" -- -- -- GRHL2 therefore control over a subset of the naive network via Enhancer switching to maintain expression of epithelial genes upon exit from naive pluripotency ChIP-seq Deletion of the GRHL2-bound Enhancer resulted in significantly reduced Dsp expression in the EpiLC state but had no effect in ESCs. Grhl2 "0610015A08Rik,BOM,Tcfcp2l3,clft3" "ChIP-seq,ATAC-seq" "Given the strong association between GRHL2 binding and various events in enhancer activation, we asked whether GRHL2 binding is necessary for each of these events. To address this question, we assessed levels of each active enhancer mark in wildtype and GHRL2 KO EpiLCs by performing ATAC-seq and ChIP-seq for H3K4me1, H3K27ac, and SMC1. There was a strong reduction in all active enhancer marks (chromatin accessibility, H3K4me1, H3K27ac, and SMC1 levels) at GRHL2 sites in GRHL2 KO EpiLCs, suggesting a near complete block in enhancer activation in the absence of GRHL2. Together, these results indicate an absolute requirement for GRHL2 for enhancer activation specifically at its target sites." -- -- -- >2KB 8006293 E_02_244 30017589 -- mm10 chr8 106608533 106609800 Mouse Embryonic Stem Cell Low+High throughput ChIP-seq "Our findings were corroborated by H3K4me1 and H3K27ac ChIP-seq data from an alternative na?ve-to-formative differentiation system. As a control, we found little change in these marks at SMC1 sites common to both states. These results indicate that GRHL2 binding correlates with full enhancer activation, suggesting a role for GRHL2 not only in regulation of cohesin binding, but also in regulation of other crucial steps in enhancer activation.Together, these results suggest that our SMC1 ChIP-seq data reflect changing enhancer activity and that cohesin binding at enhancers is highly dynamic during the ESC to EpiLC transition." Enhancer Cdh1 CRISPR/Cas9 "ChIP-seq,qRT-PCR" "We then assessed the effects of each Enhancer deletion on Dsp expression in ES cells and EpiLCs by qRT-PCR.Indeed, we found that deletion of one or both KLF4-bound enhancers led to a significant reduction in Dsp expression in ESCs but had no effect in EpiLCs(Figure 6B). Conversely, deletion of the GRHL2-bound enhancer resulted in significantly reduced Dsp expression in the EpiLC state but had no effect in ESCs (Figure 6B). We observed similar state-specific reductions in target gene expression with deletions of the KLF4-bound and GRHL2-bound enhancers at the Cdh1 and Jam1 loci (Figure 6C and D, S6A and B)." "AA960649,ARC-1,E-cad,Ecad,L-CAM,UVO,Um" -- -- -- GRHL2 therefore control over a subset of the naive network via Enhancer switching to maintain expression of epithelial genes upon exit from naive pluripotency ChIP-seq "Together,these results suggest that our SMC1 ChIP-seq data reflect changing Enhancer activity and that cohesin binding at Enhancers is highly dynamic during the ESC to EpiLC transition." Grhl2 "0610015A08Rik,BOM,Tcfcp2l3,clft3" "ChIP-seq,ATAC-seq" "Given the strong association between GRHL2 binding and various events in enhancer activation, we asked whether GRHL2 binding is necessary for each of these events. To address this question, we assessed levels of each active enhancer mark in wildtype and GHRL2 KO EpiLCs by performing ATAC-seq and ChIP-seq for H3K4me1, H3K27ac, and SMC1. There was a strong reduction in all active enhancer marks (chromatin accessibility, H3K4me1, H3K27ac, and SMC1 levels) at GRHL2 sites in GRHL2 KO EpiLCs, suggesting a near complete block in enhancer activation in the absence of GRHL2. Together, these results indicate an absolute requirement for GRHL2 for enhancer activation specifically at its target sites." -- -- -- >2KB 5818 E_02_245 30017589 -- mm10 chr8 106610533 106611666 Mouse Embryonic Stem Cell Low+High throughput ChIP-seq "Our findings were corroborated by H3K4me1 and H3K27ac ChIP-seq data from an alternative na?ve-to-formative differentiation system. As a control, we found little change in these marks at SMC1 sites common to both states. These results indicate that GRHL2 binding correlates with full enhancer activation, suggesting a role for GRHL2 not only in regulation of cohesin binding, but also in regulation of other crucial steps in enhancer activation.Together, these results suggest that our SMC1 ChIP-seq data reflect changing enhancer activity and that cohesin binding at enhancers is highly dynamic during the ESC to EpiLC transition." Enhancer Cdh1 CRISPR/Cas9 "ChIP-seq,qRT-PCR" "We then assessed the effects of each Enhancer deletion on Dsp expression in ES cells and EpiLCs by qRT-PCR.Indeed, we found that deletion of one or both KLF4-bound enhancers led to a significant reduction in Dsp expression in ESCs but had no effect in EpiLCs(Figure 6B). Conversely, deletion of the GRHL2-bound enhancer resulted in significantly reduced Dsp expression in the EpiLC state but had no effect in ESCs (Figure 6B). We observed similar state-specific reductions in target gene expression with deletions of the KLF4-bound and GRHL2-bound enhancers at the Cdh1 and Jam1 loci (Figure 6C and D, S6A and B)." "AA960649,ARC-1,E-cad,Ecad,L-CAM,UVO,Um" -- -- -- GRHL2 therefore control over a subset of the naive network via Enhancer switching to maintain expression of epithelial genes upon exit from naive pluripotency ChIP-seq Deletion of the GRHL2-bound Enhancer resulted in significantly reduced Dsp expression in the EpiLC state but had no effect in ESCs. Grhl2 "0610015A08Rik,BOM,Tcfcp2l3,clft3" "ChIP-seq,ATAC-seq" "Given the strong association between GRHL2 binding and various events in enhancer activation, we asked whether GRHL2 binding is necessary for each of these events. To address this question, we assessed levels of each active enhancer mark in wildtype and GHRL2 KO EpiLCs by performing ATAC-seq and ChIP-seq for H3K4me1, H3K27ac, and SMC1. There was a strong reduction in all active enhancer marks (chromatin accessibility, H3K4me1, H3K27ac, and SMC1 levels) at GRHL2 sites in GRHL2 KO EpiLCs, suggesting a near complete block in enhancer activation in the absence of GRHL2. Together, these results indicate an absolute requirement for GRHL2 for enhancer activation specifically at its target sites." -- -- -- >2KB 7751 E_02_246 30285185 -- mm10 chr11 6639091 6639657 Mouse Mammary Low+High throughput ChIP-seq "Employ_x0002_ing ChIP-seq, we uncover a changing transcription factor occupancy at mammary enhancers, suggest_x0002_ing that their activities evolve with advancing dif_x0002_ferentiation." Enhancer Wap CRISPR/Cas9 "qRT-PCR,ChIP-seq" "Most profoundly, a seed enhancer, which is manda_x0002_tory for the activation of the Wap super-enhancer during pregnancy, is not required during lactation,suggesting compensatory flexibility. Combinatorial deletions of structurally equivalent constituent en_x0002_hancers demonstrated differentiation-specific com_x0002_pensatory activities during lactation." Wap -- -- -- "Wap super-enhancer evolve as lactation progresses. " CRISPR/Cas9 "Using mouse genetics, we demonstrate that the functions of individual enhancers within the Wap super-enhancer evolve as lactation progresses." "Stat5a,Nr3c1" "AA959963,STAT5,GR,Grl-1,Grl1 " ChIP-seq "Employ_x0002_ing ChIP-seq, we uncover a changing transcription factor occupancy at mammary enhancers, suggest_x0002_ing that their activities evolve with advancing dif_x0002_ferentiation." -- -- -- >2KB 3892 E_02_247 30285185 -- mm10 chr11 6639824 6640424 Mouse Mammary Low+High throughput ChIP-seq "Employ_x0002_ing ChIP-seq, we uncover a changing transcription factor occupancy at mammary enhancers, suggest_x0002_ing that their activities evolve with advancing dif_x0002_ferentiation." Enhancer Wap CRISPR/Cas9 "qRT-PCR,ChIP-seq" "Most profoundly, a seed enhancer, which is manda_x0002_tory for the activation of the Wap super-enhancer during pregnancy, is not required during lactation,suggesting compensatory flexibility. Combinatorial deletions of structurally equivalent constituent en_x0002_hancers demonstrated differentiation-specific com_x0002_pensatory activities during lactation." Wap -- -- -- "Wap super-enhancer evolve as lactation progresses. " CRISPR/Cas9 "Using mouse genetics, we demonstrate that the functions of individual enhancers within the Wap super-enhancer evolve as lactation progresses." "Stat5a,Nr3c1" "AA959963,STAT5,GR,Grl-1,Grl1 " ChIP-seq "Employ_x0002_ing ChIP-seq, we uncover a changing transcription factor occupancy at mammary enhancers, suggest_x0002_ing that their activities evolve with advancing dif_x0002_ferentiation." -- -- -- >2KB 4642 E_02_248 30285185 -- mm10 chr11 6644257 6644824 Mouse Mammary Low+High throughput ChIP-seq "Employ_x0002_ing ChIP-seq, we uncover a changing transcription factor occupancy at mammary enhancers, suggest_x0002_ing that their activities evolve with advancing dif_x0002_ferentiation." Super-Enhancer Wap CRISPR/Cas9 "qRT-PCR,ChIP-seq" "Most profoundly, a seed enhancer, which is manda_x0002_tory for the activation of the Wap super-enhancer during pregnancy, is not required during lactation,suggesting compensatory flexibility. Combinatorial deletions of structurally equivalent constituent en_x0002_hancers demonstrated differentiation-specific com_x0002_pensatory activities during lactation." Wap -- -- -- "Wap super-enhancer evolve as lactation progresses. " CRISPR/Cas9 "Using mouse genetics, we demonstrate that the functions of individual enhancers within the Wap super-enhancer evolve as lactation progresses." "Stat5a,Nr3c1" "AA959963,STAT5,GR,Grl-1,Grl1 " ChIP-seq "Employ_x0002_ing ChIP-seq, we uncover a changing transcription factor occupancy at mammary enhancers, suggest_x0002_ing that their activities evolve with advancing dif_x0002_ferentiation." -- -- -- >2KB 9059 E_02_249 30285185 -- mm10 chr11 6639029 6644397 Mouse Mammary Low+High throughput ChIP-seq "Employ_x0002_ing ChIP-seq, we uncover a changing transcription factor occupancy at mammary enhancers, suggest_x0002_ing that their activities evolve with advancing dif_x0002_ferentiation." Super-Enhancer Wap CRISPR/Cas9 "qRT-PCR,ChIP-seq" "Most profoundly, a seed enhancer, which is manda_x0002_tory for the activation of the Wap super-enhancer during pregnancy, is not required during lactation,suggesting compensatory flexibility. Combinatorial deletions of structurally equivalent constituent en_x0002_hancers demonstrated differentiation-specific com_x0002_pensatory activities during lactation." Wap -- -- -- "Wap super-enhancer evolve as lactation progresses. " CRISPR/Cas9 "Using mouse genetics, we demonstrate that the functions of individual enhancers within the Wap super-enhancer evolve as lactation progresses." "Stat5a,Nr3c1" "AA959963,STAT5,GR,Grl-1,Grl1 " ChIP-seq "Employ_x0002_ing ChIP-seq, we uncover a changing transcription factor occupancy at mammary enhancers, suggest_x0002_ing that their activities evolve with advancing dif_x0002_ferentiation." -- -- -- >2KB 6231 E_02_250 30295986 Gata3 distal Enhancers mm10 chr2 9838078 9840078 Mouse Mouse Embryos Low throughput "Luciferase Reporter Assay,qPCR" We next employed a transient transfection assay to test putative Gata3 Enhancers in Gata3-expressing 293T cells using a heterologous TATA promoter that drives Luciferasegene expression. Here we describe the identification of a novel bipartite Gata3 lens-specific Enhancer located ~18 kb upstream from its transcriptional start site. We also found that a 5 kb Gata3 promoter possesses low activity in the lens. Enhancer Gata3 -- "Luciferase Reporter Assay,qPCR" "Here we describe the identification of a novel bipartite Gata3 lens-specific enhancer located 18 kb upstream from its transcriptional start site. We also found that a 5-kb Gata3 promoter possesses low activity in the lens. The bipartite enhancer contains arrays of AP-1, Ets-, and Smad1/5-binding sites as well as binding sites for lensassociated DNA-binding factors." "Gata-3,jal" -- -- -- -- -- -- -- -- -- -- -- -- -- >2KB 17999 E_02_251 30327417 Ciita Enhancer mm10 chr16 10440279 10442279 Mouse Macrophage Low throughput qChIP "We then analyzed peak A and Ciita promoter I by qChIP in NFAT5-deficient or control macrophages for different histone modifications that are enriched in gene enhancers (H3K4me1) or promoters (H3K4me3) and a modification that marks transcribed regions and active enhancers.We observed that peak A region had a slightly higher content in H3K4me1 compared with Ciita promoter I, and this was NFAT5 independent." Enhancer Ciita -- ChIP-seq ChIP-seq analysis of wild-type and NFAT5-deficient BMDMs showing the position of NFAT5-binding site peak A 47 kb upstream of the Ciita locus. "C2ta,EG669998,Gm9475,Mhc2ta" -- -- -- -- -- -- Nfat5 "AI225870,B130038B15Rik,CAG-8,CAG80,NFATL1,OREBP,TonEBP,mKIAA0827,nfatz" qChIP "We therefore asked whether NFAT5 bound these regulatory regions. Quantitative chromatin immunoprecipitation (qChIP) with a combination of two polyclonal antibodies to NFAT5 detected its specific binding to promoter I of Ciita and the promoter of H2-Ab both in untreated macrophages as well as in macrophages treated with IFN¦Ã.This result indicated that NFAT5 is a relevant factor to control Ciita expression in macrophages through its myeloid promoter I. Altogether,our analysis of primary myeloid and lymphoid APCs uncovered a selective requirement of NFAT5 in macrophages for expressing CIITA and MHCII." -- -- -- >2KB 38735 E_02_252 30328555 STAT5 Enhancers mm10 chr11 100854851 100856851 Mouse Mammary Low+High throughput "ChIP-seq,CRISPR/Cas9" "Autoregulation of the Stat5 locus in mammary tissue. a ChIP-seq profiles of an auto-regulatory Enhancer at the Stat5 locus. Mammary-specific STAT5 binding coincides with two adjacent GAS motifs,3.5 kb upstream of Stat5a." Enhancer Stat5a -- ChIP-seq "We have further provided biological evidence supporting the in vivo function of a STAT5-driven super_x0002_enhancer with the aid of CRISPR/Cas9 genome editing.Finally, we discuss how the functions of mammary-specific super_x0002_enhancers are confined by the zinc finger protein, CTCF, to allow exclusive activation of mammary-specific genes without affecting common neighboring genes. " "AA959963,STAT5" -- -- -- -- -- -- Stat5a "AA959963,STAT5" "ChIP-seq,CRISPR/Cas9" We have further provided biological evidence supporting the in vivo function of a STAT5-driven super-Enhancer with the aid of CRISPR/Cas9 genome editing. -- -- -- >2KB 3499 E_02_253 30407507 E¦Ì Enhancer mm10 chr12 114640000 114670000 Mouse B Cell Low throughput ChIP "Chromatin immunoprecipitation analyses of activated B cells identified significant ER¦Á binding to estrogen response elements (ERE) centered within enhancer elements of the immunoglobulin heavy chain locus, including the E¦Ì enhancer and hypersensitive site 1,2 (HS1,2) in the 3¡¯ regulatory region." Enhancer Aicda -- "ChIP-qPCR,Transgenic mice,ChIP-seq" "Chromatin immunoprecipitation analyses of activated B cells identified significant ER¦Á binding to estrogen response elements (ERE) centered within enhancer elements of the immunoglobulin heavy chain locus, including the E¦Ì enhancer and hypersensitive site 1,2 (HS1,2) in the 3¡¯ regulatory region. The ERE in HS1,2 was conserved across animal species, and in humans marked a site of polymorphism associated with the estrogen-augmented autoimmune disease, lupus." "Aid,Arp2" Autoimmune Diseases -- D001327 -- -- -- Esr1 "ER,ER-alpha,ERa,ERalpha,ESR,Estr,Estra,Nr3a1" "ChIP,ChIP-qPCR" "Our ChIP studies identified ER¦Á binding to Enhancer elements in the immunoglobulin heavy chain locus.Indeed, we observed that when estrogen was added to B cell cultures, there was repositioning of ER¦Á and RNA Pol II within the locus, an upregulation of sterile transcription, and a biased representation of the female-preferred IgG2b in tissue culture supernatants.Direct influences of estrogen and ER¦Á on antibody activities can help explain, at least in part, why males and females are differentially vulnerable to infectious pathogens and autoimmune disease." -- -- -- >2KB 7898808 E_02_254 30407507 -- mm10 chr12 114460000 114490000 Mouse B Cell Low throughput ChIP "Chromatin immunoprecipitation analyses of activated B cells identified significant ER¦Á binding to estrogen response elements (ERE) centered within enhancer elements of the immunoglobulin heavy chain locus, including the E¦Ì enhancer and hypersensitive site 1,2 (HS1,2) in the 3¡¯ regulatory region." Enhancer Aicda -- "ChIP-qPCR,Transgenic mice,ChIP-seq" "Chromatin immunoprecipitation analyses of activated B cells identified significant ER¦Á binding to estrogen response elements (ERE) centered within enhancer elements of the immunoglobulin heavy chain locus, including the E¦Ì enhancer and hypersensitive site 1,2 (HS1,2) in the 3¡¯ regulatory region. The ERE in HS1,2 was conserved across animal species, and in humans marked a site of polymorphism associated with the estrogen-augmented autoimmune disease, lupus." "Aid,Arp2" Autoimmune Diseases -- D001327 -- -- -- Esr1 "ER,ER-alpha,ERa,ERalpha,ESR,Estr,Estra,Nr3a1" "ChIP,ChIP-qPCR" "Our ChIP studies identified ER¦Á binding to Enhancer elements in the immunoglobulin heavy chain locus.Indeed, we observed that when estrogen was added to B cell cultures, there was repositioning of ER¦Á and RNA Pol II within the locus, an upregulation of sterile transcription, and a biased representation of the female-preferred IgG2b in tissue culture supernatants.Direct influences of estrogen and ER¦Á on antibody activities can help explain, at least in part, why males and females are differentially vulnerable to infectious pathogens and autoimmune disease." -- -- -- >2KB 8078808 E_02_255 30416088 -- mm10 chr17 85795937 85796217 Mouse Metanephric Mesenchymal (MM) cells Low+High throughput ChIP-seq "We conducted comprehensive analyses of the chromatin signature and transcriptome by RNA_x0002_seq and ChIP-seq.We found seventeen lncRNAs that were expressed specifically in MM cells with an active chromatin signature, while remaining silenced in a bivalent chromatin state in non-MM cells.Out of these MM specific lncRNAs, we identified a lncRNA, Gm29418, in a distal enhancer region of Six2,a key regulatory gene of MM cells." Enhancer Six2 -- "ChIP-seq,RNA-seq,qRT-PCR" "We conducted comprehensive analyses of the chromatin signature and transcriptome by RNA_x0002_seq and ChIP-seq.We found seventeen lncRNAs that were expressed specifically in MM cells with an active chromatin signature, while remaining silenced in a bivalent chromatin state in non-MM cells.Out of these MM specific lncRNAs, we identified a lncRNA, Gm29418, in a distal enhancer region of Six2,a key regulatory gene of MM cells." Six2 -- -- -- -- -- -- -- -- -- -- -- -- -- >2KB 133336 E_02_256 30416088 -- mm10 chr17 85797325 85797927 Mouse Metanephric Mesenchymal (MM) cells Low+High throughput ChIP-seq "We conducted comprehensive analyses of the chromatin signature and transcriptome by RNA_x0002_seq and ChIP-seq.We found seventeen lncRNAs that were expressed specifically in MM cells with an active chromatin signature, while remaining silenced in a bivalent chromatin state in non-MM cells.Out of these MM specific lncRNAs, we identified a lncRNA, Gm29418, in a distal enhancer region of Six2,a key regulatory gene of MM cells." Enhancer Six2 -- "ChIP-seq,RNA-seq,qRT-PCR" "We conducted comprehensive analyses of the chromatin signature and transcriptome by RNA_x0002_seq and ChIP-seq.We found seventeen lncRNAs that were expressed specifically in MM cells with an active chromatin signature, while remaining silenced in a bivalent chromatin state in non-MM cells.Out of these MM specific lncRNAs, we identified a lncRNA, Gm29418, in a distal enhancer region of Six2,a key regulatory gene of MM cells." Six2 -- -- -- -- -- -- -- -- -- -- -- -- -- >2KB 134885 E_02_257 30527662 BdR Enhancer mm10 chr17 30249928 30278499 Mouse Mouse Embryonic Stem Cell Low+High throughput "ChIP-seq,ChIA-PET" "The data show that the promoters are located within Smc1 ChIA-PET loops,the anchors of which align with strong cohesin and CTCF ChIP-seq peaks lacking H3K27ac ( These insulated neighborhoods encompass the TSS(arrow),promoters (vertical gray bars),and potential Enhancers of Mitf and Sik1 (vertical green bar). View of a genomic region around Mitf integrating Smc1 ChIA-PET,and Smc1,Smc3,Rad21,CTCF,Med1,and H3K27ac ChIP-seq with 4C data and promoter capture Hi-C data." Enhancer Sik1 4C "Hi-C,ChIP-seq,ChIA-PET" "High-resolution 4C was employed using promoter viewpoints to determine whether individual genes fit into the framework described in Figure 1.Figures 2A and 2B compare the Mitf and Sik1 genes expressed at low(RPKM = 2.2) and modest (RPKM = 21) levels, respectively, as measured by mRNA-seq and nascent RNA-seq (RPKM 0.53 and 4.2 for Mitf and Sik1). The data show that the promoters are located within Smc1 ChIA-PET loops, the anchors of which align with strong cohesin and CTCF ChIP-seq peaks (vertical red bars) lacking H3K27ac." "Hrt-20,Msk,Sik,Sik-1,Snf1lk" -- -- -- -- -- -- "Esrrb,Med1" "Err2,Errb,Estrrb,Nr3b2,AI480703,CRSP210,DRIP205,PBP,Pparbp,TRAP220,TRIP-2,l11Jus15" "ChIP,Immobilized Template Assay,siRNA,Western blot" "Consistent with the biochemical data, knockdown of Esrrb by small interfering RNA (siRNA) (Esrrb KD) in cells causes diminished recruitment of Mediator (Med1 and Med12) to Esrrb binding sites genome-wide.For example, upon Esrrb KD, Med1 and Med12 binding decreases at Esrrb sites within the enhancer (Enh) of the divergently transcribed Slc13a5 and Xaf1 genes.In sum, on select genes, Esrrb KD can be used to deplete the Mediator and inactivate Enhancers, either partially or near fully, to downregulate transcription of their target genes." -- -- -- >2KB 1580035 E_02_258 30527662 BdR Enhancer mm10 chr6 98336842 98365566 Mouse Mouse Embryonic Stem Cell Low+High throughput "ChIP-seq,ChIA-PET" "The data show that the promoters are located within Smc1 ChIA-PET loops,the anchors of which align with strong cohesin and CTCF ChIP-seq peaks lacking H3K27ac ( These insulated neighborhoods encompass the TSS(arrow),promoters (vertical gray bars),and potential Enhancers of Mitf and Sik1 (vertical green bar). View of a genomic region around Mitf integrating Smc1 ChIA-PET,and Smc1,Smc3,Rad21,CTCF,Med1,and H3K27ac ChIP-seq with 4C data and promoter capture Hi-C data." Enhancer Mitf 4C "Hi-C,ChIP-seq,ChIA-PET" "High-resolution 4C was employed using promoter viewpoints to determine whether individual genes fit into the framework described in Figure 1.Figures 2A and 2B compare the Mitf and Sik1 genes expressed at low(RPKM = 2.2) and modest (RPKM = 21) levels, respectively, as measured by mRNA-seq and nascent RNA-seq (RPKM 0.53 and 4.2 for Mitf and Sik1). The data show that the promoters are located within Smc1 ChIA-PET loops, the anchors of which align with strong cohesin and CTCF ChIP-seq peaks (vertical red bars) lacking H3K27ac." "BCC2,Bhlhe32,Gsfbcc2,Vitiligo,Wh,bw,mi,vit" -- -- -- -- -- -- "Esrrb,Med1" "Err2,Errb,Estrrb,Nr3b2,AI480703,CRSP210,DRIP205,PBP,Pparbp,TRAP220,TRIP-2,l11Jus15" "ChIP,Immobilized Template Assay,siRNA,Western blot" "Consistent with the biochemical data, knockdown of Esrrb by small interfering RNA (siRNA) (Esrrb KD) in cells causes diminished recruitment of Mediator (Med1 and Med12) to Esrrb binding sites genome-wide.For example, upon Esrrb KD, Med1 and Med12 binding decreases at Esrrb sites within the enhancer (Enh) of the divergently transcribed Slc13a5 and Xaf1 genes.In sum, on select genes, Esrrb KD can be used to deplete the Mediator and inactivate Enhancers, either partially or near fully, to downregulate transcription of their target genes." -- -- -- >2KB 544202 E_02_259 30544251 -- mm10 chr12 35939690 35990427 Mouse ST2 Low+High throughput ChIP-seq "The four adjacent merged SEs (SE283,SE284,SE285,and SE286) at the Ahr locus together cover a continuous region over 300 kb of active enhancer signal downstream of the Ahr gene in the ST2 cells.Moreover, all four SEs showed a very high correlation (r ¡Ý 0.95) with Ahr mRNA levels as measured by RNA-seq (Figure 5C and D, upper panels) and validated by RT-qPCR." Super-Enhancer Ahr -- "RT-qPCR,ChIP,RNA-seq" Ahr is regulated by multiple SEs with lineage-specific dynamics The Ahr mRNA level was measured across the differentiation by RNA-seq and RT-qPCR in both adipocyte and osteoblast differentiation and is indicated astheintactline. "Ah,Ahhe,In,bHLHe76,Ahr" -- -- -- -- -- -- "Ahr,Glis1" "Ah, Ahhe, In, bHLHe76, Ahr,Gli5, Gli6, GliH1" RT-qPCR "To confirm the observed differentiation defects in the presence of high AHR and GLIS1 levels, RT-qPCR analysis of the known adipocyte marker gene Lpl was performed. In ST2-TetOn-GFP cells Lpl was upregulated by D5 of differentiation and remained elevated in D9 cells both in presence and absence of doxycycline." -- -- -- >2KB 467081 E_02_260 30544251 -- mm10 chr12 35841201 35921783 Mouse ST2 Low+High throughput ChIP-seq "The four adjacent merged SEs (SE283,SE284,SE285,and SE286) at the Ahr locus together cover a continuous region over 300 kb of active enhancer signal downstream of the Ahr gene in the ST2 cells.Moreover, all four SEs showed a very high correlation (r ¡Ý 0.95) with Ahr mRNA levels as measured by RNA-seq (Figure 5C and D, upper panels) and validated by RT-qPCR." Super-Enhancer Ahr -- "RT-qPCR,ChIP,RNA-seq" Ahr is regulated by multiple SEs with lineage-specific dynamics The Ahr mRNA level was measured across the differentiation by RNA-seq and RT-qPCR in both adipocyte and osteoblast differentiation and is indicated astheintactline. "Ah,Ahhe,In,bHLHe76,Ahr" -- -- -- -- -- -- "Ahr,Glis1" "Ah, Ahhe, In, bHLHe76, Ahr,Gli5, Gli6, GliH1" RT-qPCR "To confirm the observed differentiation defects in the presence of high AHR and GLIS1 levels, RT-qPCR analysis of the known adipocyte marker gene Lpl was performed. In ST2-TetOn-GFP cells Lpl was upregulated by D5 of differentiation and remained elevated in D9 cells both in presence and absence of doxycycline." -- -- -- >2KB 383514 E_02_261 30544251 -- mm10 chr12 35769572 35823293 Mouse ST2 Low+High throughput ChIP-seq "The four adjacent merged SEs (SE283,SE284,SE285,and SE286) at the Ahr locus together cover a continuous region over 300 kb of active enhancer signal downstream of the Ahr gene in the ST2 cells.Moreover, all four SEs showed a very high correlation (r ¡Ý 0.95) with Ahr mRNA levels as measured by RNA-seq (Figure 5C and D, upper panels) and validated by RT-qPCR." Super-Enhancer Ahr -- "RT-qPCR,ChIP,RNA-seq" Ahr is regulated by multiple SEs with lineage-specific dynamics The Ahr mRNA level was measured across the differentiation by RNA-seqand RT-qPCR in both adipocyte and osteoblastdifferentiation and is indicated astheintactline. "Ah,Ahhe,In,bHLHe76,Ahr" -- -- -- -- -- -- "Ahr,Glis1" "Ah, Ahhe, In, bHLHe76, Ahr,Gli5, Gli6, GliH1" RT-qPCR "To confirm the observed differentiation defects in the presence of high AHR and GLIS1 levels, RT-qPCR analysis of the known adipocyte marker gene Lpl was performed. In ST2-TetOn-GFP cells Lpl was upregulated by D5 of differentiation and remained elevated in D9 cells both in presence and absence of doxycycline." -- -- -- >2KB 298455 E_02_262 30544251 -- mm10 chr12 35690979 35768472 Mouse ST2 Low+High throughput ChIP-seq "The four adjacent merged SEs (SE283,SE284,SE285,and SE286) at the Ahr locus together cover a continuous region over 300 kb of active enhancer signal downstream of the Ahr gene in the ST2 cells.Moreover, all four SEs showed a very high correlation (r ¡Ý 0.95) with Ahr mRNA levels as measured by RNA-seq (Figure 5C and D, upper panels) and validated by RT-qPCR." Super-Enhancer Ahr -- "RT-qPCR,ChIP,RNA-seq" Ahr is regulated by multiple SEs with lineage-specific dynamics The Ahr mRNA level was measuredacross the differentiation by RNA-seq and RT-qPCR in both adipocyte and osteoblast differentiation and is indicatedastheintactline. "Ah,Ahhe,In,bHLHe76,Ahr" -- -- -- -- -- -- "Ahr,Glis1" "Ah, Ahhe, In, bHLHe76, Ahr,Gli5, Gli6, GliH1" RT-qPCR "To confirm the observed differentiation defects in the presence of high AHR and GLIS1 levels, RT-qPCR analysis of the known adipocyte marker gene Lpl was performed. In ST2-TetOn-GFP cells Lpl was upregulated by D5 of differentiation and remained elevated in D9 cells both in presence and absence of doxycycline." -- -- -- >2KB 231748 E_02_263 19141476 -- mm10 chr5 147205131 147212149 Mouse Pancreas Low+High throughput "ChIP,ChIP-seq" "Using chromatin immunoprecipitations (ChIP) and ChIP sequencing (ChIP-Seq) of fetal pancreas and islet chromatin, we demonstrate that Foxa1 and Foxa2 predominantly occupy a distal Enhancer at -6.4 kb relative to the transcriptional start site in the Pdx1 gene." Enhancer Pdx1 -- ChIP We performed chromatin immunoprecipitation (ChIP) as_x0002_says with antibodies specific to either Foxa1 or Foxa2 on chromatin isolated from primary mouse islets. Both Foxa1 and Foxa2 bound to the Area I¨CII¨CIII enhancer of Pdx1 in vivo. "IDX-1,IPF-1,Ipf1,Mody4,STF-1,pdx-1" Pancreatic Hypoplasia -- -- -- -- -- "Foxa2,Foxa1" "HNF3-beta,HNF3beta,Hnf-3b,Hnf3b,Tcf-3b,Tcf3b,Hnf-3a,Hnf3a,Tcf-3a,Tcf3a" ChIP "We performed chromatin immunoprecipitation (ChIP) assays with antibodies specific to either Foxa1 or Foxa2 on chromatin isolated from primary mouse islets. Both Foxa1 and Foxa2 bound to the Area I¨CII¨CIII Enhancer of Pdx1 in vivo .Notably, the binding of Foxa2 to Area IV or Area I¨CII¨CIII is signifi-cantly enhanced in pancreatic islets compared with fetal pancreas." -- -- -- >2KB 61490 E_02_264 19141476 -- mm10 chr5 147263601 147264086 Mouse Pancreas Low+High throughput "ChIP,ChIP-seq" "Using chromatin immunoprecipitations (ChIP) and ChIP sequencing (ChIP-Seq) of fetal pancreas and islet chromatin, we demonstrate that Foxa1 and Foxa2 predominantly occupy a distal Enhancer at -6.4 kb relative to the transcriptional start site in the Pdx1 gene." Enhancer Pdx1 -- ChIP We performed chromatin immunoprecipitation (ChIP) as_x0002_says with antibodies specific to either Foxa1 or Foxa2 on chromatin isolated from primary mouse islets. Both Foxa1 and Foxa2 bound to the Area I¨CII¨CIII enhancer of Pdx1 in vivo. "IDX-1,IPF-1,Ipf1,Mody4,STF-1,pdx-1" Pancreatic Hypoplasia -- -- -- -- -- "Foxa2,Foxa1" "HNF3-beta,HNF3beta,Hnf-3b,Hnf3b,Tcf-3b,Tcf3b,Hnf-3a,Hnf3a,Tcf-3a,Tcf3a" ChIP "We performed chromatin immunoprecipitation (ChIP) assays with antibodies specific to either Foxa1 or Foxa2 on chromatin isolated from primary mouse islets. Both Foxa1 and Foxa2 bound to the Area I¨CII¨CIII Enhancer of Pdx1 in vivo .Notably, the binding of Foxa2 to Area IV or Area I¨CII¨CIII is signifi-cantly enhanced in pancreatic islets compared with fetal pancreas." -- -- -- >2KB 6286 E_02_265 21185279 Enhancer N2 mm10 chr3 34645597 34646134 Mouse Mouse Embryos Low throughput ChIP "ChIP assay demonstrating Pou5f1 binding to the Enhancer N2 region.In transgenic mouse embryos, mouse enhancer N2 showed activity in the entire epiblast at E6.5, and ANP area at E7.75, as indicated by the expression of the N2-hspLacZ transgene, coincident with Sox2 expression in the epiblast and forming ANP." Enhancer Sox2 -- "ChIP,RT-PCR" "ChIP assay demonstrating Pou5f1 binding to the Enhancer N2 region. Embryos were injected with pou5f1-FLAG mRNA or left non-injected, and ChIP was performed using anti-FLAG antibody. qPCR analysis shows enrichment of the Enhancer N2 region sequence (N2) in injected embryos without enrichment of the control region (Rpl5b)." "Sox-2,lcc,ysb" -- -- -- -- -- -- Pou5f1 "NF-A3,Oct-3,Oct-3/4,Oct-4,Oct3,Oct3/4,Oct4,Otf-3,Otf-4,Otf3,Otf3-rs7,Otf3g,Otf4" EMSA "These observations indicated that simultaneous binding of POU factors to the core sequence is essential for eliciting enhancer N2 activity in all examined developmental stages of the mouse embryos.these EMSA data in conjunction with the enhancer activity analyses shown in Fig. 3 indicate that the simultaneous binding of POU factors to POU-1 and POU-2/3 sites is required for the activation of the 176-bp enhancer in ES, epiblast/EpiSC and ANP cells." -- -- -- >2KB 4128 E_02_266 21527504 -- mm10 chr14 67745323 67746327 Mouse GT1-1 Low throughput "Luciferase Reporter Assay,ChIP" "To examine the enhancer activity of intron A, we constructed reporter vectors containing the full-length mouse intron A sequence (1003 bp) in either the forward or reversed direction followed by the simian vacuolating virus 40 (SV40) minimal promoter (SV40p) driving luciferase expression.These findings strongly suggest that GnRH intron A contains a putative transcriptional enhancer that is likely involved in GnRH gene regulation. In an ensuing experiment, serially deleted and truncated reporter constructs revealed that 315 nucleotides (nt) of GnRH intron A, located proximal to Ex1, were responsible for its transcription enhancing activity; the enhancing activity of this region was comparable with that of the full-length intron A." Enhancer Gnrh1 -- "ChIP,RT-qPCR" "GnRH is a pivotal hypothalamic neurohormone governing reproduction and sexual development. Because transcriptional regulation is crucial for the spatial and temporal expression of the GnRH gene, a region approximately 3.0 kb upstream of the mammalian GnRH promoter has been extensive studied. In the present study, we demonstrate a transcription-enhancer located in the first intron (intron A) region of the GnRH gene. " "Gnrh,Gnrh2,LHRH,Lhrh1,Lnrh,hpg" -- -- -- -- -- -- Qsox1 "1300003H02Rik,QSOX,Qscn6,SOx,b2b2673Clo" "ChIP,EMSA" "Chromatin immunoprecipitation (ChIP) assay revealed that SOX11 binds to the Ex1-proximal Enhancer region of intron A, and overexpression of SOX11 in creases this binding in GT1-1 cells. " -- -- -- <2KB 645 E_02_267 25801169 -- mm10 chr17 35501113 35507015 Mouse Embryonic Stem Cell Low+High throughput "Luciferase Reporter Assay,ChIP-seq" "We cloned individual constituent Enhancers of the five super-Enhancers into Enhancer-reporter vectors,and found that the majority (21/24) of super-Enhancer constituents were active in luciferase reporter assays (>1.5 fold over control, P<0.01 Student¡¯s t-test) in ESCs ." Super-Enhancer Pou5f1 -- "Luciferase Reporter Assay,ChIP-seq" "We used the reporter system to investigate how various combinations of the constituents of the Pou5f1 super-enhancer behaved in this assay. The Pou5f1 super-enhancer was selected for this experiment because its SE was small enough to be fully accommodated by the reporter vector. The results revealed that the three constituent enhancers produced slightly less activity than E2 alone, which produced the largest signal." "NF-A3,Oct-3,Oct-3/4,Oct-4,Oct3,Oct3/4,Oct4,Otf-3,Otf-4,Otf3,Otf3-rs7,Otf3g,Otf4" -- -- -- -- -- -- -- -- -- -- -- -- -- <2KB 1967 E_02_268 24385922 -- mm10 chr6 52407532 52459468 Mouse "Limb,Head" Low+High throughput "ChIP-seq,3C,5C" "Active enhancers are characterized by the binding of several proteins including RNA polymerase II (RNAP2), and subunits of Mediator like Med12.We therefore mapped candidate enhancer sequences by identifying genomic sites enriched in these proteins using chromatin immunoprecipitation combined with deep sequencing(ChIP-seq) in cells isolated from E12.5 distal limb buds.Candidate HoxA enhancers therefore reside amidst other genes including Hibadh, Tax1bp1, and Jazf1, for which expression has been reported in the limb. Consistent with our 3C data, this 5C analysis revealed a similar contact pattern between the 59 HoxA genes and upstream regulatory region in the Shh2/2 mutant and wt limbs." Enhancer Hoxa "5C,3C" -- "We profiled the interaction pattern of the HoxA cluster with the upstream 850 Kb region in distal limb buds and head tissues using 5C technology combined with deep sequencing.We found that the 59 part of HoxA, containing Hoxa9 to Hoxa13, frequently interacts with several regions upstream of the cluster,and that most of these regions contain the candidate limb enhancers." Hox-1 Limb malformation -- D017880 -- -- -- Ctcf AW108038 5C "Interestingly,loci bound either by CTCF or cohesin in limb buds have been recently identified and comparison with our data shows that almost all loci interacting with 59 HoxA genes overlap with either CTCF cohesin binding." -- -- -- -- -- E_02_269 24385922 -- mm10 chr6 52527210 52556565 Mouse "Limb,Head" Low+High throughput "ChIP-seq,3C,5C" "Active enhancers are characterized by the binding of several proteins including RNA polymerase II (RNAP2), and subunits of Mediator like Med12.We therefore mapped candidate enhancer sequences by identifying genomic sites enriched in these proteins using chromatin immunoprecipitation combined with deep sequencing(ChIP-seq) in cells isolated from E12.5 distal limb buds.Candidate HoxA enhancers therefore reside amidst other genes including Hibadh, Tax1bp1, and Jazf1, for which expression has been reported in the limb.Consistent with our 3C data, this 5C analysis revealed a similar contact pattern between the 59 HoxA genes and upstream regulatory region in the Shh2/2 mutant and wt limbs." Enhancer Hoxa "5C,3C" -- "We profiled the interaction pattern of the HoxA cluster with the upstream 850 Kb region in distal limb buds and head tissues using 5C technology combined with deep sequencing.We found that the 59 part of HoxA, containing Hoxa9 to Hoxa13, frequently interacts with several regions upstream of the cluster,and that most of these regions contain the candidate limb enhancers." Hox-1 Limb malformation -- D017880 -- -- -- Ctcf AW108038 5C "Interestingly,loci bound either by CTCF or cohesin in limb buds have been recently identified and comparison with our data shows that almost all loci interacting with 59 HoxA genes overlap with either CTCF cohesin binding." -- -- -- <2KB 963 E_02_270 26855180 miR-290-295 SE mm10 chr7 3193003 3218182 Mouse Embryonic Stem Cell Low+High throughput "CRISPR/Cas9,RT-qPCR" "We performed a functional dissection of miRNA SEs in vivo by generating cell lines depleted of individual miRNA SE constituents (about 400¨C700 bp) in multiple cell types through a CRISPR/Cas9-based approach (Table S5). We analyzed three miRNA SEs: miR-290-295 SE in mESCs, miR-1 SE in myotubes, and miR-148a SE in Pro-B cells. Among seven constituents of miR-290-295 SE (E1-E7, Figure 4A), deletion of individual elements except for E6 led to substantial decrease (ranging from 50% to 80%) in de novo production of mature miRNAs in TT-FHAgo2 mESC background without concomitant changes in other miRNA production (Figures 4B and S5A). A 5-fold reduction in expression upon deletion of one constituent of a group of six indicates cooperative interactions.As expected from a decrease in these miRNAs, these deletions also suppressed expression of Ago2-inducible gene Lin28a, a regulator of ES cells (Figure 4B). By comparing pri-miRNAs, pre-miRNAs, andmaturemiRNAs levels, we found that deletion of several miR-290-295 SE constituents modestly (2 to 3 fold) suppressed pri-miRNA production and further attenuated the ratio of pre-miRNA and mature miRNA to pri-miRNA (2 to 3 fold) (Figure 4G). This trend was also observed formiR-1 SE inmyotubes (Figure 4H)." Super-Enhancer "MiR-290,291a,292,291b,293,294,295-295" CRISPR/Cas9 RT-qPCR "We performed a functional dissection of miRNA SEs in vivo by generating cell lines depleted of individual miRNA SE constituents (about 400¨C700 bp) in multiple cell types through a CRISPR/Cas9-based approach (Table S5). We analyzed three miRNA SEs: miR-290-295 SE in mESCs, miR-1 SE in myotubes, and miR-148a SE in Pro-B cells. Among seven constituents of miR-290-295 SE (E1-E7, Figure 4A), deletion of individual elements except for E6 led to substantial decrease (ranging from 50% to 80%) in de novo production of mature miRNAs in TT-FHAgo2 mESC background without concomitant changes in other miRNA production (Figures 4B and S5A). A 5-fold reduction in expression upon deletion of one constituent of a group of six indicates cooperative interactions.As expected from a decrease in these miRNAs, these deletions also suppressed expression of Ago2-inducible gene Lin28a, a regulator of ES cells (Figure 4B). By comparing pri-miRNAs, pre-miRNAs, andmaturemiRNAs levels, we found that deletion of several miR-290-295 SE constituents modestly (2 to 3 fold) suppressed pri-miRNA production and further attenuated the ratio of pre-miRNA and mature miRNA to pri-miRNA (2 to 3 fold) (Figure 4G). This trend was also observed formiR-1 SE inmyotubes (Figure 4H)." "Mir290, Mirn290, mir-290a, mmu-miR-290, mmu-mir-290a" -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- E_02_271 26855180 miR-290-295 SE (E1) mm10 chr7 3199567 3201439 Mouse Embryonic Stem Cell Low+High throughput "CRISPR/Cas9,RT-qPCR" "Among seven constituents of miR-290-295 SE (E1-E7, Figure 4A), deletion of individual elements except for E6 led to substantial decrease (ranging from 50% to 80%) in de novo production of mature miRNAs in TT-FHAgo2 mESC background without concomitant changes in other miRNA production (Figures 4Band S5A). A 5-fold reduction in expression upon deletion of one constituent of a group of six indicates cooperative interactions.As expected from a decrease in these miRNAs, these deletions also suppressed expression of Ago2-inducible gene Lin28a, a regulator of ES cells (Figure 4B)." Enhancer "MiR-290,291a,292,291b,293,294,295-295" CRISPR/Cas9 RT-qPCR "Among seven constituents of miR-290-295 SE (E1-E7, Figure 4A), deletion of individual elements except for E6 led to substantial decrease (ranging from 50% to 80%) in de novo production of mature miRNAs in TT-FHAgo2 mESC background without concomitant changes in other miRNA production (Figures 4Band S5A). A 5-fold reduction in expression upon deletion of one constituent of a group of six indicates cooperative interactions.As expected from a decrease in these miRNAs, these deletions also suppressed expression of Ago2-inducible gene Lin28a, a regulator of ES cells (Figure 4B)." "Mir290, Mirn290, mir-290a, mmu-miR-290, mmu-mir-290a" -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- E_02_272 26855180 miR-290-295 SE (E2) mm10 chr7 3202646 3204360 Mouse Embryonic Stem Cell Low+High throughput "CRISPR/Cas9,RT-qPCR" "Among seven constituents of miR-290-295 SE (E1-E7, Figure 4A), deletion of individual elements except for E6 led to substantial decrease (ranging from 50% to 80%) in de novo production of mature miRNAs in TT-FHAgo2 mESC background without concomitant changes in other miRNA production (Figures 4Band S5A). A 5-fold reduction in expression upon deletion of one constituent of a group of six indicates cooperative interactions.As expected from a decrease in these miRNAs, these deletions also suppressed expression of Ago2-inducible gene Lin28a, a regulator of ES cells (Figure 4B)." Enhancer "MiR-290,291a,292,291b,293,294,295-295" CRISPR/Cas9 RT-qPCR "Among seven constituents of miR-290-295 SE (E1-E7, Figure 4A), deletion of individual elements except for E6 led to substantial decrease (ranging from 50% to 80%) in de novo production of mature miRNAs in TT-FHAgo2 mESC background without concomitant changes in other miRNA production (Figures 4Band S5A). A 5-fold reduction in expression upon deletion of one constituent of a group of six indicates cooperative interactions.As expected from a decrease in these miRNAs, these deletions also suppressed expression of Ago2-inducible gene Lin28a, a regulator of ES cells (Figure 4B)." "Mir290, Mirn290, mir-290a, mmu-miR-290, mmu-mir-290a" -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- E_02_273 26855180 miR-290-295 SE (E3) mm10 chr7 3205726 3207280 Mouse Embryonic Stem Cell Low+High throughput "CRISPR/Cas9,RT-qPCR" "Among seven constituents of miR-290-295 SE (E1-E7, Figure 4A), deletion of individual elements except for E6 led to substantial decrease (ranging from 50% to 80%) in de novo production of mature miRNAs in TT-FHAgo2 mESC background without concomitant changes in other miRNA production (Figures 4Band S5A). A 5-fold reduction in expression upon deletion of one constituent of a group of six indicates cooperative interactions.As expected from a decrease in these miRNAs, these deletions also suppressed expression of Ago2-inducible gene Lin28a, a regulator of ES cells (Figure 4B)." Enhancer "MiR-290,291a,292,291b,293,294,295-295" CRISPR/Cas9 RT-qPCR "Among seven constituents of miR-290-295 SE (E1-E7, Figure 4A), deletion of individual elements except for E6 led to substantial decrease (ranging from 50% to 80%) in de novo production of mature miRNAs in TT-FHAgo2 mESC background without concomitant changes in other miRNA production (Figures 4Band S5A). A 5-fold reduction in expression upon deletion of one constituent of a group of six indicates cooperative interactions.As expected from a decrease in these miRNAs, these deletions also suppressed expression of Ago2-inducible gene Lin28a, a regulator of ES cells (Figure 4B)." "Mir290, Mirn290, mir-290a, mmu-miR-290, mmu-mir-290a" -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- E_02_274 26855180 miR-290-295 SE (E4) mm10 chr7 3209567 3211439 Mouse Embryonic Stem Cell Low+High throughput "CRISPR/Cas9,RT-qPCR" "Among seven constituents of miR-290-295 SE (E1-E7, Figure 4A), deletion of individual elements except for E6 led to substantial decrease (ranging from 50% to 80%) in de novo production of mature miRNAs in TT-FHAgo2 mESC background without concomitant changes in other miRNA production (Figures 4Band S5A). A 5-fold reduction in expression upon deletion of one constituent of a group of six indicates cooperative interactions.As expected from a decrease in these miRNAs, these deletions also suppressed expression of Ago2-inducible gene Lin28a, a regulator of ES cells (Figure 4B)." Enhancer "MiR-290,291a,292,291b,293,294,295-295" CRISPR/Cas9 RT-qPCR "Among seven constituents of miR-290-295 SE (E1-E7, Figure 4A), deletion of individual elements except for E6 led to substantial decrease (ranging from 50% to 80%) in de novo production of mature miRNAs in TT-FHAgo2 mESC background without concomitant changes in other miRNA production (Figures 4Band S5A). A 5-fold reduction in expression upon deletion of one constituent of a group of six indicates cooperative interactions.As expected from a decrease in these miRNAs, these deletions also suppressed expression of Ago2-inducible gene Lin28a, a regulator of ES cells (Figure 4B)." "Mir290, Mirn290, mir-290a, mmu-miR-290, mmu-mir-290a" -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- E_02_275 26855180 miR-290-295 SE (E6) mm10 chr7 3212448 3214558 Mouse Embryonic Stem Cell Low+High throughput "CRISPR/Cas9,RT-qPCR" "Among seven constituents of miR-290-295 SE (E1-E7, Figure 4A), deletion of individual elements except for E6 led to substantial decrease (ranging from 50% to 80%) in de novo production of mature miRNAs in TT-FHAgo2 mESC background without concomitant changes in other miRNA production (Figures 4Band S5A). A 5-fold reduction in expression upon deletion of one constituent of a group of six indicates cooperative interactions.As expected from a decrease in these miRNAs, these deletions also suppressed expression of Ago2-inducible gene Lin28a, a regulator of ES cells (Figure 4B)." Enhancer "MiR-290,291a,292,291b,293,294,295-295" CRISPR/Cas9 RT-qPCR "Among seven constituents of miR-290-295 SE (E1-E7, Figure 4A), deletion of individual elements except for E6 led to substantial decrease (ranging from 50% to 80%) in de novo production of mature miRNAs in TT-FHAgo2 mESC background without concomitant changes in other miRNA production (Figures 4Band S5A). A 5-fold reduction in expression upon deletion of one constituent of a group of six indicates cooperative interactions.As expected from a decrease in these miRNAs, these deletions also suppressed expression of Ago2-inducible gene Lin28a, a regulator of ES cells (Figure 4B)." "Mir290, Mirn290, mir-290a, mmu-miR-290, mmu-mir-290a" -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- E_02_276 26855180 miR-290-295 SE (E7) mm10 chr7 3216607 3218399 Mouse Embryonic Stem Cell Low+High throughput "CRISPR/Cas9,RT-qPCR" "Among seven constituents of miR-290-295 SE (E1-E7, Figure 4A), deletion of individual elements except for E6 led to substantial decrease (ranging from 50% to 80%) in de novo production of mature miRNAs in TT-FHAgo2 mESC background without concomitant changes in other miRNA production (Figures 4Band S5A). A 5-fold reduction in expression upon deletion of one constituent of a group of six indicates cooperative interactions.As expected from a decrease in these miRNAs, these deletions also suppressed expression of Ago2-inducible gene Lin28a, a regulator of ES cells (Figure 4B)." Enhancer "MiR-290,291a,292,291b,293,294,295-295" CRISPR/Cas9 RT-qPCR "Among seven constituents of miR-290-295 SE (E1-E7, Figure 4A), deletion of individual elements except for E6 led to substantial decrease (ranging from 50% to 80%) in de novo production of mature miRNAs in TT-FHAgo2 mESC background without concomitant changes in other miRNA production (Figures 4Band S5A). A 5-fold reduction in expression upon deletion of one constituent of a group of six indicates cooperative interactions.As expected from a decrease in these miRNAs, these deletions also suppressed expression of Ago2-inducible gene Lin28a, a regulator of ES cells (Figure 4B)." "Mir290, Mirn290, mir-290a, mmu-miR-290, mmu-mir-290a" -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- E_02_277 26855180 miR-1 SE mm10 chr2 180362582 180377003 Mouse myotubes Low+High throughput "CRISPR/Cas9,RT-qPCR" "We performed a functional dissection ofmiRNA SEs in vivo by generating cell lines depleted of individual miRNA SE constituents (about 400¨C700 bp) in multiple cell types through a CRISPR/Cas9-based approach (Table S5). We analyzed three miRNA SEs: miR-290-295 SE in mESCs, miR-1 SE in myotubes, and miR-148a SE in Pro-B cells. In the C2C12 cell line, a model for myotube differentiation, SE was only observed for one of the two miR-1 genes (miR-1a-1/133a-2) and myotube differentiation strongly induced primary miRNAs of only this miRNA gene (Fig-ure S5B).Deletion of all four constituents of miR-1 SE dramatically suppressed induction of miR-1 and miR-133 and myogenic differentiationmarkers such asMyogenin andmuscle creatine kinase (MCK), phenocopying miR-1 knockdown phenotype (Chen et al., 2006)(Figures 4C and4D and S5C). " Super-Enhancer "MiR-1a-1,MiR-133a-2" CRISPR/Cas9 RT-qPCR "We performed a functional dissection ofmiRNA SEs in vivo by generating cell lines depleted of individual miRNA SE constituents (about 400¨C700 bp) in multiple cell types through a CRISPR/Cas9-based approach (Table S5). We analyzed three miRNA SEs: miR-290-295 SE in mESCs, miR-1 SE in myotubes, and miR-148a SE in Pro-B cells. In the C2C12 cell line, a model for myotube differentiation, SE was only observed for one of the two miR-1 genes (miR-1a-1/133a-2) and myotube differentiation strongly induced primary miRNAs of only this miRNA gene (Fig-ure S5B).Deletion of all four constituents of miR-1 SE dramatically suppressed induction of miR-1 and miR-133 and myogenic differentiationmarkers such asMyogenin andmuscle creatine kinase (MCK), phenocopying miR-1 knockdown phenotype (Chen et al., 2006)(Figures 4C and4D and S5C). " "Mir1-1, Mirn1-1, Mirn1b, Mirn1c, Mirn1d,?mir-1a-1, mmu-mir-1-1, mmu-mir-1a-1" -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- E_02_278 26855180 miR-1 SE(M1) mm10 chr2 180361998 180364335 Mouse myotubes Low+High throughput "CRISPR/Cas9,RT-qPCR" "In the C2C12 cell line, a model for myotube differentiation, SE was only observed for one of the two miR-1 genes (miR-1a-1/133a-2) and myotube differentiation strongly induced primary miRNAs of only this miRNA gene (Fig-ure S5B).Deletion of all four constituents of miR-1 SE dramatically suppressed induction of miR-1 and miR-133 and myogenic differentiationmarkers such asMyogenin andmuscle creatine kinase (MCK), phenocopying miR-1 knockdown phenotype (Chen et al., 2006)(Figures 4C and4D and S5C). " Enhancer "MiR-1a-1,MiR-133a-2" CRISPR/Cas9 RT-qPCR "In the C2C12 cell line, a model for myotube differentiation, SE was only observed for one of the two miR-1 genes (miR-1a-1/133a-2) and myotube differentiation strongly induced primary miRNAs of only this miRNA gene (Fig-ure S5B).Deletion of all four constituents of miR-1 SE dramatically suppressed induction of miR-1 and miR-133 and myogenic differentiationmarkers such asMyogenin andmuscle creatine kinase (MCK), phenocopying miR-1 knockdown phenotype (Chen et al., 2006)(Figures 4C and4D and S5C). " "Mir1-1, Mirn1-1, Mirn1b, Mirn1c, Mirn1d,?mir-1a-1, mmu-mir-1-1, mmu-mir-1a-2" -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- E_02_279 26855180 miR-1 SE(M2) mm10 chr2 180366734 180368371 Mouse myotubes Low+High throughput "CRISPR/Cas9,RT-qPCR" "In the C2C12 cell line, a model for myotube differentiation, SE was only observed for one of the two miR-1 genes (miR-1a-1/133a-2) and myotube differentiation strongly induced primary miRNAs of only this miRNA gene (Fig-ure S5B).Deletion of all four constituents of miR-1 SE dramatically suppressed induction of miR-1 and miR-133 and myogenic differentiationmarkers such asMyogenin andmuscle creatine kinase (MCK), phenocopying miR-1 knockdown phenotype (Chen et al., 2006)(Figures 4C and4D and S5C). " Enhancer "MiR-1a-1,MiR-133a-2" CRISPR/Cas9 RT-qPCR "In the C2C12 cell line, a model for myotube differentiation, SE was only observed for one of the two miR-1 genes (miR-1a-1/133a-2) and myotube differentiation strongly induced primary miRNAs of only this miRNA gene (Fig-ure S5B).Deletion of all four constituents of miR-1 SE dramatically suppressed induction of miR-1 and miR-133 and myogenic differentiationmarkers such asMyogenin andmuscle creatine kinase (MCK), phenocopying miR-1 knockdown phenotype (Chen et al., 2006)(Figures 4C and4D and S5C). " "Mir1-1, Mirn1-1, Mirn1b, Mirn1c, Mirn1d,?mir-1a-1, mmu-mir-1-1, mmu-mir-1a-3" -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- E_02_280 26855180 miR-1 SE(M3) mm10 chr2 180367903 180369540 Mouse myotubes Low+High throughput "CRISPR/Cas9,RT-qPCR" "In the C2C12 cell line, a model for myotube differentiation, SE was only observed for one of the two miR-1 genes (miR-1a-1/133a-2) and myotube differentiation strongly induced primary miRNAs of only this miRNA gene (Fig-ure S5B).Deletion of all four constituents of miR-1 SE dramatically suppressed induction of miR-1 and miR-133 and myogenic differentiationmarkers such asMyogenin andmuscle creatine kinase (MCK), phenocopying miR-1 knockdown phenotype (Chen et al., 2006)(Figures 4C and4D and S5C). " Enhancer "MiR-1a-1,MiR-133a-2" CRISPR/Cas9 RT-qPCR "In the C2C12 cell line, a model for myotube differentiation, SE was only observed for one of the two miR-1 genes (miR-1a-1/133a-2) and myotube differentiation strongly induced primary miRNAs of only this miRNA gene (Fig-ure S5B).Deletion of all four constituents of miR-1 SE dramatically suppressed induction of miR-1 and miR-133 and myogenic differentiationmarkers such asMyogenin andmuscle creatine kinase (MCK), phenocopying miR-1 knockdown phenotype (Chen et al., 2006)(Figures 4C and4D and S5C). " "Mir1-1, Mirn1-1, Mirn1b, Mirn1c, Mirn1d,?mir-1a-1, mmu-mir-1-1, mmu-mir-1a-4" -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- E_02_281 26855180 miR-1 SE(M4) mm10 chr2 180375974 180377844 Mouse myotubes Low+High throughput "CRISPR/Cas9,RT-qPCR" "In the C2C12 cell line, a model for myotube differentiation, SE was only observed for one of the two miR-1 genes (miR-1a-1/133a-2) and myotube differentiation strongly induced primary miRNAs of only this miRNA gene (Fig-ure S5B).Deletion of all four constituents of miR-1 SE dramatically suppressed induction of miR-1 and miR-133 and myogenic differentiationmarkers such asMyogenin andmuscle creatine kinase (MCK), phenocopying miR-1 knockdown phenotype (Chen et al., 2006)(Figures 4C and4D and S5C). " Enhancer "MiR-1a-1,MiR-133a-2" CRISPR/Cas9 RT-qPCR "In the C2C12 cell line, a model for myotube differentiation, SE was only observed for one of the two miR-1 genes (miR-1a-1/133a-2) and myotube differentiation strongly induced primary miRNAs of only this miRNA gene (Fig-ure S5B).Deletion of all four constituents of miR-1 SE dramatically suppressed induction of miR-1 and miR-133 and myogenic differentiationmarkers such asMyogenin andmuscle creatine kinase (MCK), phenocopying miR-1 knockdown phenotype (Chen et al., 2006)(Figures 4C and4D and S5C). " "Mir1-1, Mirn1-1, Mirn1b, Mirn1c, Mirn1d,?mir-1a-1, mmu-mir-1-1, mmu-mir-1a-5" -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- E_02_282 26855180 miR-148a SE1 mm10 chr6 51108708 51130031 Mouse Pro-B cell Low+High throughput "CRISPR/Cas9,RT-qPCR" "We performed a functional dissection ofmiRNA SEs in vivo by generating cell lines depleted of individual miRNA SE constituents (about 400¨C700 bp) in multiple cell types through a CRISPR/Cas9-based approach (Table S5). We analyzed three miRNA SEs: miR-290-295 SE in mESCs, miR-1 SE in myotubes, and miR-148a SE in Pro-B cells.Furthermore, in Pro-B cells, deletion of four out of ?ve miR-148a SE constituents dynamically suppressed induction of miR-148a during differenti- ation and affected its downstream genes Bach2 and Blimp1(Porstner et al., 2015)(Figures 4E and 4F). Interestingly, in Pro-Bcells,the effects of deletions of SE constituents on pri-miRNAs were minor, but the reductions in pri-miRNA processing were more pronounced (Figure 4I). " Super-Enhancer MiR-148a CRISPR/Cas9 RT-qPCR "We performed a functional dissection ofmiRNA SEs in vivo by generating cell lines depleted of individual miRNA SE constituents (about 400¨C700 bp) in multiple cell types through a CRISPR/Cas9-based approach (Table S5). We analyzed three miRNA SEs: miR-290-295 SE in mESCs, miR-1 SE in myotubes, and miR-148a SE in Pro-B cells.Furthermore, in Pro-B cells, deletion of four out of ?ve miR-148a SE constituents dynamically suppressed induction of miR-148a during differenti- ation and affected its downstream genes Bach2 and Blimp1(Porstner et al., 2015)(Figures 4E and 4F). Interestingly, in Pro-Bcells,the effects of deletions of SE constituents on pri-miRNAs were minor, but the reductions in pri-miRNA processing were more pronounced (Figure 4I). " "Mirn148, Mirn148a,?mir-148a" -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- E_02_283 26855180 miR-148a SE2 mm10 chr6 51168034 51174811 Mouse Pro-B cell Low+High throughput "CRISPR/Cas9,RT-qPCR" "We performed a functional dissection ofmiRNA SEs in vivo by generating cell lines depleted of individual miRNA SE constituents (about 400¨C700 bp) in multiple cell types through a CRISPR/Cas9-based approach (Table S5). We analyzed three miRNA SEs: miR-290-295 SE in mESCs, miR-1 SE in myotubes, and miR-148a SE in Pro-B cells.Furthermore, in Pro-B cells, deletion of four out of ?ve miR-148a SE constituents dynamically suppressed induction of miR-148a during differenti- ation and affected its downstream genes Bach2 and Blimp1(Porstner et al., 2015)(Figures 4E and 4F). Interestingly, in Pro-Bcells,the effects of deletions of SE constituents on pri-miRNAs were minor, but the reductions in pri-miRNA processing were more pronounced (Figure 4I). " Super-Enhancer MiR-148a CRISPR/Cas9 RT-qPCR "We performed a functional dissection ofmiRNA SEs in vivo by generating cell lines depleted of individual miRNA SE constituents (about 400¨C700 bp) in multiple cell types through a CRISPR/Cas9-based approach (Table S5). We analyzed three miRNA SEs: miR-290-295 SE in mESCs, miR-1 SE in myotubes, and miR-148a SE in Pro-B cells.Furthermore, in Pro-B cells, deletion of four out of ?ve miR-148a SE constituents dynamically suppressed induction of miR-148a during differenti- ation and affected its downstream genes Bach2 and Blimp1(Porstner et al., 2015)(Figures 4E and 4F). Interestingly, in Pro-Bcells,the effects of deletions of SE constituents on pri-miRNAs were minor, but the reductions in pri-miRNA processing were more pronounced (Figure 4I). " "Mirn148, Mirn148a,?mir-148a" -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- E_02_284 26855180 miR-148a SE(B1) mm10 chr6 51117747 51120656 Mouse Pro-B cell Low+High throughput "CRISPR/Cas9,RT-qPCR" "Furthermore, in Pro-B cells, deletion of four out of ?ve miR-148a SE constituents dynamically suppressed induction of miR-148a during differenti- ation and affected its downstream genes Bach2 and Blimp1(Porstner et al., 2015)(Figures 4E and 4F)." Enhancer MiR-148a CRISPR/Cas9 RT-qPCR "Furthermore, in Pro-B cells, deletion of four out of ?ve miR-148a SE constituents dynamically suppressed induction of miR-148a during differenti- ation and affected its downstream genes Bach2 and Blimp1(Porstner et al., 2015)(Figures 4E and 4F)." "Mirn148, Mirn148a,?mir-148a" -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- E_02_285 26855180 miR-148a SE(B3) mm10 chr6 51168147 51170512 Mouse Pro-B cell Low+High throughput "CRISPR/Cas9,RT-qPCR" "Furthermore, in Pro-B cells, deletion of four out of ?ve miR-148a SE constituents dynamically suppressed induction of miR-148a during differenti- ation and affected its downstream genes Bach2 and Blimp1(Porstner et al., 2015)(Figures 4E and 4F)." Enhancer MiR-148a CRISPR/Cas9 RT-qPCR "Furthermore, in Pro-B cells, deletion of four out of ?ve miR-148a SE constituents dynamically suppressed induction of miR-148a during differenti- ation and affected its downstream genes Bach2 and Blimp1(Porstner et al., 2015)(Figures 4E and 4F)." "Mirn148, Mirn148a,?mir-148a" -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- E_02_286 26855180 miR-148a SE(B4) mm10 chr6 51171474 51173111 Mouse Pro-B cell Low+High throughput "CRISPR/Cas9,RT-qPCR" "Furthermore, in Pro-B cells, deletion of four out of ?ve miR-148a SE constituents dynamically suppressed induction of miR-148a during differenti- ation and affected its downstream genes Bach2 and Blimp1(Porstner et al., 2015)(Figures 4E and 4F)." Enhancer MiR-148a CRISPR/Cas9 RT-qPCR "Furthermore, in Pro-B cells, deletion of four out of ?ve miR-148a SE constituents dynamically suppressed induction of miR-148a during differenti- ation and affected its downstream genes Bach2 and Blimp1(Porstner et al., 2015)(Figures 4E and 4F)." "Mirn148, Mirn148a,?mir-148a" -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- E_02_287 26855180 miR-148a SE(B5) mm10 chr6 51173363 51175544 Mouse Pro-B cell Low+High throughput "CRISPR/Cas9,RT-qPCR" "Furthermore, in Pro-B cells, deletion of four out of ?ve miR-148a SE constituents dynamically suppressed induction of miR-148a during differenti- ation and affected its downstream genes Bach2 and Blimp1(Porstner et al., 2015)(Figures 4E and 4F)." Enhancer MiR-148a CRISPR/Cas9 RT-qPCR "Furthermore, in Pro-B cells, deletion of four out of ?ve miR-148a SE constituents dynamically suppressed induction of miR-148a during differenti- ation and affected its downstream genes Bach2 and Blimp1(Porstner et al., 2015)(Figures 4E and 4F)." "Mirn148, Mirn148a,?mir-148a" -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- E_02_288 26912657 mm10 chr2 129350852 129356319 Mouse Macrophage Low+High throughput "ChIP-qPCR,ChIP-seq" "Based on the ENCODE/UCSDbonemarrow-derivedmacrophageH3K27Ac chromatin immunoprecipitation sequencing (ChIP-seq) database, two peaks of DNA sequencing frequency were found in area, -10 kb (peak 1) and -2 kb (peak 2) upstream of the pro-IL-1beta transcription start site (TSS) (Fig. 4A). These peaks also coincided with the genomic regions highly associated with H3K4me1 but not with H3Kme3, indicating active enhancers." enhancer Il1b -- qPCR "Therefore, we designed 11 qPCR primer sets encom-passing the genomic area to analyze the production of pro-IL-1 beta -associated eRNAs.These results suggest that the production of peak 2 eRNA was required for the optimal production of pro-IL-1beta mRNA." "IL-1beta, Il-1b" -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- E_02_289 26728555 mm10 chr12 109528035 109532509 Mouse Embryonic Stem Cell Low+High throughput "ChIP-seq,ChIP" "By performing ChIP-seq in different mESC lines and using two different antibodies, we were able to identify 973 high-confidence AFF3-binding sites.AFF3 is also de_x0002_tected in the intergenic regions within the Dlk1-Dio3 locus, which harbors a paternally methylated gDMR/ ICR (Fig. 1A). Two discrete AFF3-binding sites are located 12 kb (distal AFF3 peak) and 10 kb (proximal AFF3 peak)upstream of the promoter region of the maternally ex_x0002_pressed gene Meg3 (Fig. 1B).This AFF3 peak resides within the intergenic DMR (IG-DMR) of the Dlk1-Dio3 cluster and corresponds to the Meg3-proximal enhancer peak located just 2.4 kb from the Meg3-distal gDMR peak (Figs. 1B, 3A). While bisulfite sequencing of ChIP DNA demonstrated that AFF3 is on the inactive allele at the Meg3-distal gDMR,AFF3 is restricted to the unmethylated allele at the Meg3-proximal enhancer (Fig. 3B,C; Supplemental Fig. S4)." Enhancer Meg3 -- "ChIP-seq,ChIP" "This AFF3 peak resides within the intergenic DMR (IG-DMR) of the Dlk1-Dio3 cluster and corresponds to the Meg3-proximal enhancer peak located just 2.4 kb from the Meg3-distal gDMR peak (Figs. 1B, 3A). While bisulfite sequencing of ChIP DNA demonstrated that AFF3 is on the inactive allele at the Meg3-distal gDMR,AFF3 is restricted to the unmethylated allele at the Meg3-proximal enhancer (Fig. 3B,C; Supplemental Fig. S4)." "2900016C05Rik,3110050O07Rik,6330408G06Rik,AI425946,AW108224,D12Bwg1266e,Gtl2,R74756,R75394" -- -- -- -- -- -- Aff3 "LAF4,MLLT2-like" "ChIP,ChIP-seq" "This AFF3 peak resides within the intergenic DMR (IG-DMR) of the Dlk1-Dio3 cluster and corresponds to the Meg3-proximal enhancer peak located just 2.4 kb from the Meg3-distal gDMR peak (Figs. 1B, 3A). While bisulfite sequencing of ChIP DNA demonstrated that AFF3 is on the inactive allele at the Meg3-distal gDMR,AFF3 is restricted to the unmethylated allele at the Meg3-proximal enhancer (Fig. 3B,C; Supplemental Fig. S4).Knockdown of AFF3 leads to reduced H3K27ac at the Meg3-proximal enhancer, indicating that AFF3 is required to maintain its active chromatin state (Fig. 3D; Supple_x0002_mental Table 2). Furthermore, the expression levels of Meg3 and the downstream maternally expressed noncod_x0002_ing genes within the Dlk1-Dio3 locus are also reduced in AFF3 knockdown ESCs (Fig. 3E; Supplemental Fig. S5A),suggesting that an activating function of AFF3 might beexerted from the nearby Meg3-proximal enhancer." -- -- -- >2KB 10724 E_02_290 2835657 -- mm10 chr12 103852772 103852898 Mouse Hepatoma Cells low throughput "Transient Assay,EMSA" "To test whether the more distant upstream sequences could function as an enhancer, the -523- to -168-bp restriction fragment was attached in either orientation (at -341 bp)to a mouse ,beta-globin promoter.The results of transfection experiments showed a 10- to 15-fold enhancement of beta-globin transcription when this region was present in either orientation ." enhancer Serpina1a -- "Transient Assay,EMSA" Proteins present in nuclear extracts bind to the functionally defined elements present within the al-AT enhancer. Nuclear extracts from Hep-G2 cells were incubated with end-labeled DNA probes from the enhancer region to detect nuclear protein-binding sites by the gel retardation or gel shift assay. Each protein-binding site correlated with a functionally importantregion necessary for full enhancer activity. These experiments demonstrated a complex arrangement of regulatory elements comprising the al-antitrypsin enhancer. "Aat-2,Aat2,Dom1,PI1,Spi1-1,Spi1-3" -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- E_02_291 2835657 mm10 chr12 103852898 103853032 Mouse Hepatoma Cells low throughput "Transient Assay,EMSA" "To test whether the more distant upstream sequences could function as an enhancer, the -523- to -168-bp restriction fragment was attached in either orientation (at -341 bp)to a mouse ,beta-globin promoter.The results of transfection experiments showed a 10- to 15-fold enhancement of beta-globin transcription when this region was present in either orientation ." enhancer Serpina1a -- "Transient Assay,EMSA" Proteins present in nuclear extracts bind to the functionally defined elements present within the al-AT enhancer. Nuclear extracts from Hep-G2 cells were incubated with end-labeled DNA probes from the enhancer region to detect nuclear protein-binding sites by the gel retardation or gel shift assay. Each protein-binding site correlated with a functionally importantregion necessary for full enhancer activity. These experiments demonstrated a complex arrangement of regulatory elements comprising the al-antitrypsin enhancer. "Aat-2,Aat2,Dom1,PI1,Spi1-1,Spi1-3" -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- E_02_292 2835657 mm10 chr12 103853032 103853127 Mouse Hepatoma Cells low throughput "Transient Assay,EMSA" "To test whether the more distant upstream sequences could function as an enhancer, the -523- to -168-bp restriction fragment was attached in either orientation (at -341 bp)to a mouse ,beta-globin promoter.The results of transfection experiments showed a 10- to 15-fold enhancement of beta-globin transcription when this region was present in either orientation ." enhancer Serpina1a -- "Transient Assay,EMSA" Proteins present in nuclear extracts bind to the functionally defined elements present within the al-AT enhancer. Nuclear extracts from Hep-G2 cells were incubated with end-labeled DNA probes from the enhancer region to detect nuclear protein-binding sites by the gel retardation or gel shift assay. Each protein-binding site correlated with a functionally importantregion necessary for full enhancer activity. These experiments demonstrated a complex arrangement of regulatory elements comprising the al-antitrypsin enhancer. "Aat-2,Aat2,Dom1,PI1,Spi1-1,Spi1-3" -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- E_02_293 12861010 mm10 chr7 103771431 103772043 Mouse "Pleen Cells,MEL cells" Low throughput "PCR,DNaseI-seq" "We have also found DNase I HSs at kb -85.5 (HS-85.5) and -84.5 in mouse, but there is no corresponding se_x0002_quence or structure in human for these HSs.By comparison,neither the active _x0002_maj-globin gene promoter nor a region located ~1 kb from HS5 shows significant enrichment. HS-85.5 exhibits a modest (twofold) enrichment." Enhancer Hbb-ar -- "DNaseI-seq,Southern blot,PCR" "In ery_x0002_throid cells, the active _x0002_-globin locus contains several DNase I HSs, which have been shown to map to sequences with regu_x0002_latory function.In addition, histone hyperacetylation and dimethylation of histone H3 K4 are not uniform features of the nuclease-sensitive mouse _x0001_-globin domain but rather define distinct subdomains within it. Our results reveal a complex chromatin landscape for the active _x0001_-globin locus and illustrate the complexity of broad structural changes that accompany gene activation." Hbb-lcr -- -- -- -- -- -- Ctcf AW108038 ChIP "To determine whether CTCF binds to these sequences in vivo, we performed ChIP analysis using antibodies to CTCF." -- -- -- >2kb 81029 E_02_294 15798207 Ei mm10 chr6 67556736 67558736 Mouse "B Cell,T Cell,MPC-11" Low throughput "3C,PCR" "The mouse immunoglobulin kappa (Igk) gene contains an intronic enhancer and two enhancers downstream of its transcription unit. Using chromosome conformation capture technology, we demonstrate that rearranged and actively transcribed Igk alleles in MPC-11 plasmacytoma cells exhibit mutual interactions over 22 kb between these three enhancers and Vk gene promoters. We also observe interactions between Ei and E3'with 3'boundary sequences 24 kb downstream of Ed, adjacent to a neighboring house_x0002_keeping gene." enhancer Igk 3C "ChIP,Southern blot" Chromatin segments within the transcription unit exhibit interactions throughout regions downstream of the tran_x0002_scription termination region specifically in active Igk loci. kappa -- -- -- "We observe interactions between Ei and E3' with 3' boundary sequences 24 kb downstream of Ed, adjacent to a neighboring house-keeping gene." 3C These results fit a looping mechanism for enhancer function like in the beta-globin locus and suggest a dynamic modulation of the spatial organization of the active Igkappa locus. "Rela,Tcf3,Tfap4 " "p65,p65 NF-kappa B,p65 NFkB,A1,AA408400,ALF2,AW209082,E12,E12/E47,E2A,E47,KA1,ME2,Pan1,Pan2, TCF-3,Tcfe2a,VDIR,bHLHb21,AI642933,AP-4,D930048N17Rik,Tcfap4,bHLHc41" "ChIP,PCR" "Taken together with the hypothesis that proteins that may be recruited by NF-kB and/or E2A might exhibit mutual bridging interactions between these transcription factors on different cis elements, we decided to first assay for the presence of NF-kB and E2A on the Igk gene enhancers and the Vk19-17 gene by performing ChIP experiments." -- -- -- Intron 2100 E_02_295 15798207 E3' mm10 chr6 67561736 67563736 Mouse "B Cell,T Cell,MPC-11" Low throughput "3C,PCR" "The mouse immunoglobulin kappa (Igk) gene contains an intronic enhancer and two enhancers downstream of its transcription unit. Using chromosome conformation capture technology, we demonstrate that rearranged and actively transcribed Igk alleles in MPC-11 plasmacytoma cells exhibit mutual interactions over 22 kb between these three enhancers and Vk gene promoters. We also observe interactions between Ei and E3'with 3'boundary sequences 24 kb downstream of Ed, adjacent to a neighboring house_x0002_keeping gene." enhancer Igk 3C "ChIP,Southern blot" Chromatin segments within the transcription unit exhibit interactions throughout regions downstream of the tran_x0002_scription termination region specifically in active Igk loci. kappa -- -- -- "We observe interactions between Ei and E3' with 3' boundary sequences 24 kb downstream of Ed, adjacent to a neighboring house-keeping gene." 3C These results fit a looping mechanism for enhancer function like in the beta-globin locus and suggest a dynamic modulation of the spatial organization of the active Igkappa locus. "Rela,Tcf3,Tfap4 " "p65,p65 NF-kappa B,p65 NFkB,A1,AA408400,ALF2,AW209082,E12,E12/E47,E2A,E47,KA1,ME2,Pan1,Pan2, TCF-3,Tcfe2a,VDIR,bHLHb21,AI642933,AP-4,D930048N17Rik,Tcfap4,bHLHc41" "ChIP,PCR" "Taken together with the hypothesis that proteins that may be recruited by NF-kB and/or E2A might exhibit mutual bridging interactions between these transcription factors on different cis elements, we decided to first assay for the presence of NF-kB and E2A on the Igk gene enhancers and the Vk19-17 gene by performing ChIP experiments." -- -- -- >2KB 7100 E_02_296 15798207 Ed mm10 chr6 67560136 67562136 Mouse "B Cell,T Cell,MPC-11" Low throughput "3C,PCR" "The mouse immunoglobulin kappa (Igk) gene contains an intronic enhancer and two enhancers downstream of its transcription unit. Using chromosome conformation capture technology, we demonstrate that rearranged and actively transcribed Igk alleles in MPC-11 plasmacytoma cells exhibit mutual interactions over 22 kb between these three enhancers and Vk gene promoters. We also observe interactions between Ei and E3'with 3'boundary sequences 24 kb downstream of Ed, adjacent to a neighboring house_x0002_keeping gene." enhancer Igk 3C "ChIP,Southern blot" Chromatin segments within the transcription unit exhibit interactions throughout regions downstream of the tran_x0002_scription termination region specifically in active Igk loci. kappa -- -- -- "We observe interactions between Ei and E3' with 3' boundary sequences 24 kb downstream of Ed, adjacent to a neighboring house-keeping gene." 3C These results fit a looping mechanism for enhancer function like in the beta-globin locus and suggest a dynamic modulation of the spatial organization of the active Igkappa locus. "Rela,Tcf3,Tfap4 " "p65,p65 NF-kappa B,p65 NFkB,A1,AA408400,ALF2,AW209082,E12,E12/E47,E2A,E47,KA1,ME2,Pan1,Pan2, TCF-3,Tcfe2a,VDIR,bHLHb21,AI642933,AP-4,D930048N17Rik,Tcfap4,bHLHc41" "ChIP,PCR" "Taken together with the hypothesis that proteins that may be recruited by NF-kB and/or E2A might exhibit mutual bridging interactions between these transcription factors on different cis elements, we decided to first assay for the presence of NF-kB and E2A on the Igk gene enhancers and the Vk19-17 gene by performing ChIP experiments." -- -- -- >2KB 5500 E_02_297 27166834 -- mm10 chr8 120876144 120876856 Mouse Mouse Embryos Low+High throughput "3C,ChIP-seq" "To identify the presence of physical interaction between the 11 enhancer candidates and the Foxf1 promoter, 3C assays were performed. Among 11 selected regions, three enhancer candidates demonstrated interactions with the Foxf1 promoter." Enhancer Foxf1 3C "RT-qPCR,DNaseI-seq,Transgenic mice" "To investigate the in vivo activity of the discovered enhanc_x0002_ers and compare the location of activity with Foxf1-express_x0002_ing regions, we generated enhancer-driven lacZ-reporter mice 5¡äe207mFoxf1-lacZ and 5¡äe201mFoxf1-lacZ.Foxf1 mRNA was expressed in the maxilla, mandible, limbs, sclerotome and gut of wild-type mouse embryos at E12 (Fig. 2a), whereas lacZ-positive cells were observed in part of the forebrain and neural tube in 5¡äe207mFoxf1-lacZ transgenic mice at E11 and E12 as well as 5¡äe201mFoxf1-lacZ transgenic mouse embryos at E11 after x-gal staining.Foxf1 was also expressed in the mesenchyme surrounding epithelial buds in lungs at E12." "AI450827,FREAC1a,Freac-1,HFH-8,Hfh8,Foxf1" -- -- -- -- -- -- "Gli1,Gli2,Gli3 " "AV235269,Zfp-5,Zfp5;Gli2,AI854843,AU023367,Bph,GLI3-190,GLI3FL,Pdn,Xt,add" "RT-qPCR,Luciferase Reporter Assay" Schematic drawing of luciferase reporter constructs containing 112 bp 5¡äe207mFoxf1 and 173 bp 5¡äe201mFoxf1 fragments with Gli binding sites is shown. Luciferase reporter constructs were assayed for luciferase activity without or with Gli1 over-expression vector at 50 or 100 ng. -- -- -- >2kb 207886 E_02_298 27166834 -- mm10 chr8 120882734 120883410 Mouse Mouse Embryos Low+High throughput "3C,ChIP-seq" "To identify the presence of physical interaction between the 11 enhancer candidates and the Foxf1 promoter, 3C assays were performed. Among 11 selected regions, three enhancer candidates demonstrated interactions with the Foxf1 promoter." Enhancer Foxf1 3C "RT-qPCR,DNaseI-seq,Transgenic mice" "To investigate the in vivo activity of the discovered enhanc_x0002_ers and compare the location of activity with Foxf1-express_x0002_ing regions, we generated enhancer-driven lacZ-reporter mice 5¡äe207mFoxf1-lacZ and 5¡äe201mFoxf1-lacZ.Foxf1 mRNA was expressed in the maxilla, mandible, limbs, sclerotome and gut of wild-type mouse embryos at E12 (Fig. 2a), whereas lacZ-positive cells were observed in part of the forebrain and neural tube in 5¡äe207mFoxf1-lacZ transgenic mice at E11 and E12 as well as 5¡äe201mFoxf1-lacZ transgenic mouse embryos at E11 after x-gal staining.Foxf1 was also expressed in the mesenchyme surrounding epithelial buds in lungs at E12." "AI450827,FREAC1a,Freac-1,HFH-8,Hfh8,Foxf1" -- -- -- -- -- -- "Gli1,Gli2,Gli3 " "AV235269,Zfp-5,Zfp5;Gli2,AI854843,AU023367,Bph,GLI3-190,GLI3FL,Pdn,Xt,add" "RT-qPCR,Luciferase Reporter Assay" Schematic drawing of luciferase reporter constructs containing 112 bp 5¡äe207mFoxf1 and 173 bp 5¡äe201mFoxf1 fragments with Gli binding sites is shown. Luciferase reporter constructs were assayed for luciferase activity without or with Gli1 over-expression vector at 50 or 100 ng. -- -- -- >2kb 201314 E_02_299 27166834 -- mm10 chr8 121088830 121089174 Mouse Mouse Embryos Low+High throughput "3C,ChIP-seq" "To identify the presence of physical interaction between the 11 enhancer candidates and the Foxf1 promoter, 3C assays were performed. Among 11 selected regions, three enhancer candidates demonstrated interactions with the Foxf1 promoter." Enhancer Foxf1 3C "RT-qPCR,DNaseI-seq,Transgenic mice" "To investigate the in vivo activity of the discovered enhanc_x0002_ers and compare the location of activity with Foxf1-express_x0002_ing regions, we generated enhancer-driven lacZ-reporter mice 5¡äe207mFoxf1-lacZ and 5¡äe201mFoxf1-lacZ.Foxf1 mRNA was expressed in the maxilla, mandible, limbs, sclerotome and gut of wild-type mouse embryos at E12 (Fig. 2a), whereas lacZ-positive cells were observed in part of the forebrain and neural tube in 5¡äe207mFoxf1-lacZ transgenic mice at E11 and E12 as well as 5¡äe201mFoxf1-lacZ transgenic mouse embryos at E11 after x-gal staining.Foxf1 was also expressed in the mesenchyme surrounding epithelial buds in lungs at E12. " "AI450827,FREAC1a,Freac-1,HFH-8,Hfh8,Foxf1" -- -- -- -- -- -- "Gli1,Gli2,Gli3 " "AV235269,Zfp-5,Zfp5;Gli2,AI854843,AU023367,Bph,GLI3-190,GLI3FL,Pdn,Xt,add" "RT-qPCR,Luciferase Reporter Assay" Schematic drawing of luciferase reporter constructs containing 112 bp 5¡äe207mFoxf1 and 173 bp 5¡äe201mFoxf1 fragments with Gli binding sites is shown. Luciferase reporter constructs were assayed for luciferase activity without or with Gli1 over-expression vector at 50 or 100 ng. -- -- -- >2kb 4616 E_02_300 26957309 -- mm10 chr17 63168191 63175262 Mouse Embryonic Stem Cell Low+High throughput "Luciferase Reporter Assay,PCR,ChIP-seq" "To independently test the predictive power of H2BK20ac, we again used luciferase enhancer assaysas above to test 18 genomic regions randomly chosen from among the top 20,000 H2BK20ac ChIP--seq peaks in human embryonic stem cells (H1--ESCs)." Enhancer Fbxl17 -- ChIP-seq "As above, we noticed that some of the validated forebrain enhancers showed H2BK20ac even in the absence of H3K27ac (Fig. 2C).Notably, the top H3K4me1 peaks were less predictive of forebrain enhancer function, perhaps due to the presence of this mark at in_x0002_active but poised enhancers.Again, H2BK20ac was observed to be the most predictive mark of forebrain enhancers (Fig. 2B). As above, we noticed that some of the validated forebrain enhancers showed H2BK20ac even in the absence of H3K27ac." "6330576B01Rik, AI452053, BB073797, C130023C01Rik, Fbx13, Fbxo13" -- -- -- -- -- -- -- -- -- -- -- -- -- >2kb 125778 E_02_301 26957309 -- mm10 chr10 87283358 87312833 Mouse Embryonic Stem Cell Low+High throughput "Luciferase Reporter Assay,PCR,ChIP-seq" "To independently test the predictive power of H2BK20ac, we again used luciferase enhancer assaysas above to test 18 genomic regions randomly chosen from among the top 20,000 H2BK20ac ChIP--seq peaks in human embryonic stem cells (H1--ESCs)." Enhancer Ak157291 -- ChIP-seq "As above, we noticed that some of the validated forebrain enhancers showed H2BK20ac even in the absence of H3K27ac (Fig. 2C).Notably, the top H3K4me1 peaks were less predictive of forebrain enhancer function, perhaps due to the presence of this mark at in_x0002_active but poised enhancers.Again, H2BK20ac was observed to be the most predictive mark of forebrain enhancers (Fig. 2B). As above, we noticed that some of the validated forebrain enhancers showed H2BK20ac even in the absence of H3K27ac." Ak157291 -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- E_02_302 26787680 MCE mm10 chr17 26829232 26829742 Mouse Cardiac Progenitor Cell Low throughput "Luciferase Reporter Assay,Transient Transfection Assay" "The influence of desmin on nkx2.5 gene expression was monitored first with the luciferase (LUC) reporter plasmid pNKE24 (Searcy et al., 1998), containing the proximal enhancerand promoter region (PEPR) of the nkx2.5 gene and then with the LUC reporter plasmid pMCE, containing the minimal cardiac specific enhancer region(MCE) (Lien et al., 1999) in addition to the PEPR (Fig. 1A; for precise localization along the nkx2.5 gene see Fig. 4A).Co-transfection of 10T1/2 fibroblasts with pNKE24 and a desmin-expressing plasmid (desmin-ect.) resulted in a significant decrease of the LUC activity (Fig. 1B)." enhancer Nkx2-5 -- "Luciferase Reporter Assay,PCR,ChIP-seq" "The influence of desmin on nkx2.5 gene expression was monitored first with the luciferase (LUC) reporter plasmid pNKE24 (Searcy et al., 1998), containing the proximal enhancerand promoter region (PEPR) of the nkx2.5 gene and then with the LUC reporter plasmid pMCE, containing the minimal cardiac specific enhancer region(MCE) (Lien et al., 1999) in addition to the PEPR (Fig. 1A; for precise localization along the nkx2.5 gene see Fig. 4A).Co-transfection of 10T1/2 fibroblasts with pNKE24 and a desmin-expressing plasmid (desmin-ect.) resulted in a significant decrease of the LUC activity (Fig. 1B)." "Csx,Nkx-2.5,Nkx2.5,tinman" -- -- -- -- -- -- Desmin Desmin "ChIP,PCR" Desmin is a component of transcription factor complexes interacting with regulatory regions of the nkx2.5 gene at the beginning of cardiomyogenesis. -- -- -- >2kb 9177 E_02_303 26771354 -- mm10 chr2 152735054 152735521 Mouse Embryonic Stem Cell Low throughput "qRT-PCR,ChIP" "To address the roles of the BMP-SMAD pathway, we performed ChIP-seq analyses (Figures 1C and S1D).SMAD1/5 were enriched in the promoter regions of Pou5f1 (which encodes OCT4) and Nanog in naive mESCs,as well as a positive control region in the Id1 promoter.Consistent with previous findings (Chen et al., 2008), the regions bound by SMAD1/5 were enriched with active enhancer marks (H3K4me1, H3K27ac, and co-activator p300)." Enhancer Id1 -- ChIP-seq "Impor_x0002_tantly, expression levels of BMP target genes, Id1 and Dusp9, were comparable or even higher in mESD-EpiSCs treated with BMP-4 (24 hr) than in mESCs, suggesting that induction of these genes is not associated with the maintenance or reversion to the naive state.Consistent with previous findings,the regions bound by SMAD1/5 were enriched with active enhancer marks (H3K4me1, H3K27ac, and co-activator p300)." "AI323524,D2Wsu140e,Idb1,bHLHb24" -- -- -- -- -- -- Smad1/5 "AI528653, Mad1, Madh1, Madr1, Mlp1, MusMLP, dwf-A, mMad1,1110051M15Rik, AI451355, Dwf-C, Madh5, MusMLP" ChIP-seq "In the present study, we have performed both RNA_x0002_sequencing and SMAD1/5 genome-wide chromatin immu_x0002_noprecipitation and sequencing (ChIP-seq) analyses of mESCs in the naive or primed states, and have employed a genome editing method. We show that the BMP_x0002_SMAD pathway is dispensable for maintaining naive pluripotency." -- -- -- -- -- E_02_304 26774474 -- mm10 chr13 43362622 43364444 Mouse "C2C12,MEFs" Low+High throughput "ChIP-qPCR,ChIP-seq,ChIP,EMSA,Luciferase Reporter Assay,qPCR" "Interactions of methylated PGC-1a[K779me] with the Spt-Ada_x0002_Gcn5-acetyltransferase (SAGA) complex, the Medi_x0002_ator members MED1 and MED17, and the NOP2/ Sun RNA methytransferase 7 (NSUN7) reinforce tran_x0002_scription, and are concomitant with the m5C mark on enhancer RNAs (eRNAs)." Enhancer Sirt5 -- qPCR "To further evaluate the physiological function of Sirt5 eRNA, we tested the lysine de_x0002_glutarylase activity of SIRT5 upon knockdown of its associated eRNA. We found that carbamoyl phosphate synthase1 (CPS1), a specific substrate of SIRT5, was glutary_x0002_lated upon depletion of Sirt5 eRNA, leading to an overall diminishment in CPS1 activity. Overall, these results suggest that eRNAs are important sensors of the meta_x0002_bolic state." "0610012J09Rik, 1500032M05Rik, AV001953" -- -- -- -- -- -- "Tfiid,Lxr,nrf2,Ppar" "GTF2D1, Gtf2d, SCA17, TFIID,AU018371, LXR, RLD1, Unr1,Nrf2,4933429D07Rik, AW742785, Nr1c1, PPAR-alpha, PPARalpha, Ppar" ChIP-seq "urthermore, DNA motifs overrepresented in PGC-1¦Á[K779me] fell into four dominant motif consensus classes, including the TFIID, LXR, NRF2, and the PPAR family of transcription factors (Figure 3F, left and middle), which were localized in four metabolic genes identified as specific targets of Pgc-1¦Á by knockdown experiments (Figure 3F, right; Figure S3A): 6-phosphofructokinase (Pfkl), Sirtuin 5 (Sirt5), isocitrate dehydrogenase 3 (Idh3b), and Heme oxygenase (decycling) 2 (Hmox2).Thus, depletion of Pgc-1¦Á led to a decrease of Pfkl, Sirt5, Idh3b, and Hmox2 expression, which was rescued with wild-type PGC-1¦Á, but not mutated PGC-1¦Á[K779R], overexpression." -- -- -- <2KB 1856 E_02_305 26774474 -- mm10 chr2 130280565 130281887 Mouse "C2C12,MEFs" Low+High throughput "ChIP-qPCR,ChIP-seq,ChIP,EMSA,Luciferase Reporter Assay,qPCR" "Interactions of methylated PGC-1a[K779me] with the Spt-Ada_x0002_Gcn5-acetyltransferase (SAGA) complex, the Medi_x0002_ator members MED1 and MED17, and the NOP2/ Sun RNA methytransferase 7 (NSUN7) reinforce tran_x0002_scription, and are concomitant with the m5C mark on enhancer RNAs (eRNAs)." Enhancer Idh3b -- qPCR "Direct knockdown studies combined with ChIP analysis revealed some bona fide targets as Pfkl,Sirt5,Idh3b,and Hmox2 genes.Although a significant pool of PGC-1a[K779me] resides within the TSS, we were able to localize a significant binding at enhancer regions that allowed the characterization of eRNAs overlapping those PGC-1a[K779me] peaks. Thus, complex ncRNA-protein interactions appear consequential to the fidelity of transcription and to the cellular metabolic fate. " C78231 -- -- -- -- -- -- "Tfiid,Lxr,nrf2,Ppar" "GTF2D1, Gtf2d, SCA17, TFIID,AU018371, LXR, RLD1, Unr1,Nrf2,4933429D07Rik, AW742785, Nr1c1, PPAR-alpha, PPARalpha, Ppar" ChIP-seq "urthermore, DNA motifs overrepresented in PGC-1¦Á[K779me] fell into four dominant motif consensus classes, including the TFIID, LXR, NRF2, and the PPAR family of transcription factors (Figure 3F, left and middle), which were localized in four metabolic genes identified as specific targets of Pgc-1¦Á by knockdown experiments (Figure 3F, right; Figure S3A): 6-phosphofructokinase (Pfkl), Sirtuin 5 (Sirt5), isocitrate dehydrogenase 3 (Idh3b), and Heme oxygenase (decycling) 2 (Hmox2).Thus, depletion of Pgc-1¦Á led to a decrease of Pfkl, Sirt5, Idh3b, and Hmox2 expression, which was rescued with wild-type PGC-1¦Á, but not mutated PGC-1¦Á[K779R], overexpression." -- -- -- <2KB 1917 E_02_306 26774474 -- mm10 chr16 4721118 4724819 Mouse "C2C12,MEFs" Low+High throughput "ChIP-qPCR,ChIP-seq,ChIP,EMSA,Luciferase Reporter Assay,qPCR" "Interactions of methylated PGC-1a[K779me] with the Spt-Ada_x0002_Gcn5-acetyltransferase (SAGA) complex, the Medi_x0002_ator members MED1 and MED17, and the NOP2/ Sun RNA methytransferase 7 (NSUN7) reinforce tran_x0002_scription, and are concomitant with the m5C mark on enhancer RNAs (eRNAs)." Enhancer Hmox2 -- qPCR "Direct knockdown studies combined with ChIP analysis revealed some bona fide targets as Pfkl,Sirt5,Idh3b,and Hmox2 genes.Although a significant pool of PGC-1a[K779me] resides within the TSS, we were able to localize a significant binding at enhancer regions that allowed the characterization of eRNAs overlapping those PGC-1a[K780me] peaks. Thus, complex ncRNA-protein interactions appear consequential to the fidelity of transcription and to the cellular metabolic fate. " "HO-2, HO2" -- -- -- -- -- -- "Tfiid,Lxr,nrf2,Ppar" "GTF2D1, Gtf2d, SCA17, TFIID,AU018371, LXR, RLD1, Unr1,Nrf2,4933429D07Rik, AW742785, Nr1c1, PPAR-alpha, PPARalpha, Ppar" ChIP-seq "urthermore, DNA motifs overrepresented in PGC-1¦Á[K779me] fell into four dominant motif consensus classes, including the TFIID, LXR, NRF2, and the PPAR family of transcription factors (Figure 3F, left and middle), which were localized in four metabolic genes identified as specific targets of Pgc-1¦Á by knockdown experiments (Figure 3F, right; Figure S3A): 6-phosphofructokinase (Pfkl), Sirtuin 5 (Sirt5), isocitrate dehydrogenase 3 (Idh3b), and Heme oxygenase (decycling) 2 (Hmox2).Thus, depletion of Pgc-1¦Á led to a decrease of Pfkl, Sirt5, Idh3b, and Hmox2 expression, which was rescued with wild-type PGC-1¦Á, but not mutated PGC-1¦Á[K779R], overexpression." -- -- -- >2KB 3392 E_02_307 26774474 -- mm10 chr10 77989870 77993100 Mouse "C2C12,MEFs" Low+High throughput "ChIP-qPCR,ChIP-seq,ChIP,EMSA,Luciferase Reporter Assay,qPCR" "Interactions of methylated PGC-1a[K779me] with the Spt-Ada_x0002_Gcn5-acetyltransferase (SAGA) complex, the Medi_x0002_ator members MED1 and MED17, and the NOP2/ Sun RNA methytransferase 7 (NSUN7) reinforce tran_x0002_scription, and are concomitant with the m5C mark on enhancer RNAs (eRNAs)." Enhancer Pfkl -- qPCR "qPCR of the Pfkl-associated eRNA and the Dlx2 enhancer-associated Evf-2 (Dlx6os1) transcript used as a negative control, after RIP with PGC-1a[K779me] antibody upon Set7/9 depletion in both Hepa 1-6 and C2C12 cells.Direct knockdown studies combined with ChIP analysis revealed some bona fide targets as Pfkl,Sirt5,Idh3b,and Hmox2 genes.Although a significant pool of PGC-1a[K779me] resides within the TSS, we were able to localize a significant binding at enhancer regions that allowed the characterization of eRNAs overlapping those PGC-1a[K780me] peaks. Thus, complex ncRNA-protein interactions appear consequential to the fidelity of transcription and to the cellular metabolic fate. " "AA407869, ATP-PFK, PFK-B, PFK-L" -- -- -- -- -- -- "Tfiid,Lxr,nrf2,Ppar" "GTF2D1, Gtf2d, SCA17, TFIID,AU018371, LXR, RLD1, Unr1,Nrf2,4933429D07Rik, AW742785, Nr1c1, PPAR-alpha, PPARalpha, Ppar" ChIP-seq "urthermore, DNA motifs overrepresented in PGC-1¦Á[K779me] fell into four dominant motif consensus classes, including the TFIID, LXR, NRF2, and the PPAR family of transcription factors (Figure 3F, left and middle), which were localized in four metabolic genes identified as specific targets of Pgc-1¦Á by knockdown experiments (Figure 3F, right; Figure S3A): 6-phosphofructokinase (Pfkl), Sirtuin 5 (Sirt5), isocitrate dehydrogenase 3 (Idh3b), and Heme oxygenase (decycling) 2 (Hmox2).Thus, depletion of Pgc-1¦Á led to a decrease of Pfkl, Sirt5, Idh3b, and Hmox2 expression, which was rescued with wild-type PGC-1¦Á, but not mutated PGC-1¦Á[K779R], overexpression." -- -- -- -- -- E_02_308 26809507 -- mm10 chr4 115040426 115042426 Mouse HPC-7 Low+High throughput "DNaseI-seq,ChIP,ChIP-seq,Hi-C,Transgenic mice" "Identification of putative enhancers based on ChIP-Seq data alone cannot assign distal regions to specific genes with confidence because enhancers are known.to have the ability to act over large distances, and may loop over intervening genes.39 To overcome this limitation, we made use of our CHi-C interaction list, and filtered our list of putative enhancers to only retain those that looped to the promoter regions of known regulators of HSPC function." Enhancer Scl -- "ChIP-seq,Hi-C" "Visualization of the interaction files together with our previously published 10 TF ChIP-Seq demonstrated specific interactions of the Scl (also known as Tal1) and Lmo2 promoters with the previously characterized enhancer regions at Scl -15 kb,+19 kb, and +40 kb, as well as Lmo2 -75 kb,-70 kb,-64 kb, and the proximal promoter (pPex)." "Hpt,SCL/tal-1,Scl,bHLHa17,tal-1" Malignant Hematopoiesis -- -- "Scl Enhancer elements also interact with the promoter of the neighboring Pdzk1ip1 gene, consistent with previous reports suggesting that Scl and Pdzk1ip4 form a single transcriptional domain." "Hi-C,ChIP-seq" Analysis of well_x0002_characterized gene loci encoding key HSPC regulators therefore suggests that the newly generated CHi-C data set represents a valuable resource to advance our understanding of transcriptional control mechanisms in HSPCs. "Cebpa,Cebpb,Fos,Myc,E2f4,Egr1,Elf1,Cbfa2t3,Jun,Ldb1,Max,Myb,Nfe2,Trp53,Rad21,Stat3" "C/ebpalpha, CBF-A, Cebp£¬C/EBPbeta, CRP2, IL-6DBP, LAP, LIP, NF-IL6, NF-M, Nfil6£¬D12Rfj1,c-fos,cFos,AU0167572, Niard, Nird, bHLHe39, Myc" "DNaseI-seq,ChIP-seq,Hi-C" "Given that HPC-7 represents one of the best in vitro models for HSPCs, we wanted to bring genomic information for these cells up to a similar level of completeness, and therefore performed ChIP-Seq experiments for a further 17 TFs (CEBPa,CEBPb, cFOS, cMYC, E2F4, EGR1, ELF1, ETO2,c-JUN,LDB1,MAX, MYB, NFE2, p53, RAD21, pSTAT1, and STAT3) as well as genome-wide DNase I hypersensitive mapping and 3 additional his_x0002_tone marks (H2AK5ac, H3K4me3, and H3K36me3)." -- -- -- -- -- E_02_309 26883548 -- mm10 chr12 113302447 113305102 Mouse Blood Low throughput "PCR,ChIP" "Extracted DNA was amplified by PCR and submitted to high_x0002_throughput sequencing to evaluate SHM. As SHM in light chains is not under the 3¡¯RR control9, IgH SHM values along rearranged JH4 sequences were normalised to Jk5 SHM values. Mutation frequencies of 1.45% and 0.07% were found in wt and AID _x0002_ -/-_x0002_mice, respectively. SHM frequency was markedly reduced (by more than fourfold, at 0.33%) in DleftPAL mice (Fig. 2a,b).The presence of hs3a and hs1,2 enhancers in DIRIS mice maintained SHM frequency at 0.75%, that is, at an intermediate level higher than DleftPAL mice (Fig. 2a,b) but markedly lower(by about twofold) than in wt mice (Fig. 2a,b)." Enhancer Ighg3 -- "RNA-seq,ChIP" "RNA-seq experiments showed that Cg3 and Cg2b transcription(Fig. 5a and d, respectively) was markedly reduced in DleftPAL mice compared with wt mice. The presence of hs3a and hs1,2 enhancers in DIRIS mice preserved Cg3 and Cg2b transcription.Levels of H3K4me3 (Fig. 5b,e) and H3K27ac (Fig. 5c,f) in the Ig3- S g3-C g3 and Ig2b-S g2b-C g2b regions (during IgG3 and IgG2b CSR,respectively) were reduced only on deletion of 3¡¯RR enhancers but preserved in DIRIS mice." IgH -- -- -- "This shows a synergistic role of enhancers and of the palindromic architecture This shows a synergistic role of enhancers and of the palindromic architecture for induction of epigenetic modifications and chromatin accessibility." ChIP "In conclusion, this work indicates that the IgH 3'RR does not simply pile up enhancer units but also optimally exposes them into a functional architecture of crucial importance." -- -- -- -- -- -- -- >2KB 53667 E_02_310 26883548 -- mm10 chr12 113305522 113308796 Mouse Blood Low throughput "PCR,ChIP" "Extracted DNA was amplified by PCR and submitted to high_x0002_throughput sequencing to evaluate SHM. As SHM in light chains is not under the 3¡¯RR control9, IgH SHM values along rearranged JH4 sequences were normalised to Jk5 SHM values. Mutation frequencies of 1.45% and 0.07% were found in wt and AID _x0002_ -/-_x0002_mice, respectively. SHM frequency was markedly reduced (by more than fourfold, at 0.33%) in DleftPAL mice (Fig. 2a,b).The presence of hs3a and hs1,2 enhancers in DIRIS mice maintained SHM frequency at 0.75%, that is, at an intermediate level higher than DleftPAL mice (Fig. 2a,b) but markedly lower(by about twofold) than in wt mice (Fig. 2a,b)." Enhancer Ighg2b -- "RNA-seq,ChIP" "RNA-seq experiments showed that Cg3 and Cg2b transcription(Fig. 5a and d, respectively) was markedly reduced in DleftPAL mice compared with wt mice. The presence of hs3a and hs1,2 enhancers in DIRIS mice preserved Cg3 and Cg2b transcription.Levels of H3K4me3 (Fig. 5b,e) and H3K27ac (Fig. 5c,f) in the Ig3- S g3-C g3 and Ig2b-S g2b-C g2b regions (during IgG3 and IgG2b CSR,respectively) were reduced only on deletion of 3¡¯RR enhancers but preserved in DIRIS mice." "IgG2b, Igh-3, gamma2b" -- -- -- "This shows a synergistic role of enhancers and of the palindromic architecture This shows a synergistic role of enhancers and of the palindromic architecture for induction of epigenetic modifications and chromatin accessibility." ChIP "In conclusion, this work indicates that the IgH 3'RR does not simply pile up enhancer units but also optimally exposes them into a functional architecture of crucial importance." -- -- -- -- -- -- -- >2KB 2845 E_02_311 26883548 -- mm10 chr12 113314217 113314925 Mouse Blood Low throughput "PCR,ChIP" "Extracted DNA was amplified by PCR and submitted to high_x0002_throughput sequencing to evaluate SHM. As SHM in light chains is not under the 3¡¯RR control9, IgH SHM values along rearranged JH4 sequences were normalised to Jk5 SHM values. Mutation frequencies of 1.45% and 0.07% were found in wt and AID _x0002_ -/-_x0002_mice, respectively. SHM frequency was markedly reduced (by more than fourfold, at 0.33%) in DleftPAL mice (Fig. 2a,b).The presence of hs3a and hs1,2 enhancers in DIRIS mice maintained SHM frequency at 0.75%, that is, at an intermediate level higher than DleftPAL mice (Fig. 2a,b) but markedly lower(by about twofold) than in wt mice (Fig. 2a,b)." Enhancer Ighg2b -- "RNA-seq,ChIP" "RNA-seq experiments showed that Cg3 and Cg2b transcription(Fig. 5a and d, respectively) was markedly reduced in DleftPAL mice compared with wt mice. The presence of hs3a and hs1,2 enhancers in DIRIS mice preserved Cg3 and Cg2b transcription.Levels of H3K4me3 (Fig. 5b,e) and H3K27ac (Fig. 5c,f) in the Ig3- S g3-C g3 and Ig2b-S g2b-C g2b regions (during IgG3 and IgG2b CSR,respectively) were reduced only on deletion of 3¡¯RR enhancers but preserved in DIRIS mice." "IgG2b, Igh-3, gamma2b" -- -- -- "This shows a synergistic role of enhancers and of the palindromic architecture This shows a synergistic role of enhancers and of the palindromic architecture for induction of epigenetic modifications and chromatin accessibility." ChIP "In conclusion, this work indicates that the IgH 3'RR does not simply pile up enhancer units but also optimally exposes them into a functional architecture of crucial importance." -- -- -- -- -- -- -- >2KB 10257 E_02_312 26446995 -- mm10 chr11 100859351 100885169 Mouse Mammary Epithelium Low+High throughput "PCR,ChIP-seq,RT-PCR,DNase-seq" MED1 binding coincided with STAT5 occupancy. RNA Pol II binding also coincided with these sequences. Enhancer Stat5 -- "PCR,ChIP-seq,RT-PCR,DNase-seq" MED1 binding coincided with STAT5 occupancy. RNA Pol II binding also coincided with these sequences. "AA959963,STAT5" -- -- -- "Transcriptome analysis identified a class of mammary-restricted genes that was particularly dependent on high STAT5 levels as a result of the intergenic enhancer." CRISPR/Cas9 "Taken together, the mammary_x0002_specific enhancer enables a positive feedback cir_x0002_cuit that contributes to the remarkable abundance of STAT5 and, in turn, to the efficacy of STAT5-dependent mammary physiology." -- -- -- -- -- -- -- -- --