About Enhancer
| Enhancer ID: | E_01_002 | |
| Enhancer symbol: | ERαenh588 | |
| Species: | Human | |
| Position : | chr11:69329684-69330954   |
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| Biosample name: | MCF-7,T-47D | |
| Experiment class : | Low+High throughput |
| Enhancer type: | Enhancer | |
| Disease: | Breast Cancer | |
| DO: | DOID:1612 | |
| Mesh: | D001943 | |
| Distance from TSS: | >2KB | |
| Pubmed ID: | 26751173 |
| Enhancer experiment: | ChIP-seq,CRISPR/Cas9,Luciferase Reporter Assay,Western blot,Competitive Proliferation Assay | |
| Enhancer experiment description: | To demonstrate the generalizability of our screening approach, we designed a dropout screen to identify novel ERα-bound enhancers. We started by cloning ERαenh588 WT region in a reporter vector and verified that it has strong ERα–dependent transcription-enhancing activity, since mutations in the ERα binding site completely abolish the enhancer activity and response to 17β-estradiol (Supplementary Fig. 4b). Next, we examined endogenous ERα binding at ERαenh588 region by ChIP-seq and observed a substantial decrease in both MCF-7 and T47D cells expressing sgRNA-ERαenh588 (Fig. 3e). Because CCND1 is a puttive target of ERαenh588, we verified the endogenous expression of ERαenh588 eRNAs and CCND1 mRNA and protein in MCF-7 and T47D cells transduced with sgRNA-ERαenh588.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–h). These results indicate that ERαenh588 is a bona fide enhancer element that regulates the expression of CCND1. |
About Target gene
| Target gene : | CCND1(BCL1,D11S287E,PRAD1,U21B31) | |
| Strong evidence: | -- | |
| Less strong evidence: | ChIP-seq,Western blot,qPCR | |
| Target gene experiment description: | Next, we examined endogenous ERα binding at ERαenh588 region by ChIP-seq and observed a substantial decrease in both MCF-7 and T47D cells expressing sgRNA-ERαenh588 (Fig. 3e).Because CCND1 is a putative target of ERαenh588, we verified the endogenous expression of ERαenh588 eRNAs and CCND1 mRNA and protein in MCF-7 and T47D cells transduced with sgRNA-ERαenh588. 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–h). These results indicate that ERαenh588 is a bona fide enhancer element that regulates the expression of CCND1.Next, we assessed the dependency of ERαenh588 and CCND1 endogenous expression on estrogen signaling by qPCR (Fig. 3i). As expected, we confirmed that both ERαenh588 eRNA and CCND1 mRNA are upregulated in MCF-7 cells upon treatment with 17β-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 ERαenh588 enhancer element. |
About TF
| TF name : | ESR1(ER,ESR,ESRA,ESTRR,Era,NR3A1) | |
| TF experiment: | Luciferase Reporter Assay,ChIP-seq | |
| TF experiment description: | We started by cloning ERαenh588 WT region in a reporter vector and verified that it has strong ERα–dependent transcription-enhancing activity, since mutations in the ERα binding site completely abolish the enhancer activity and response to 17β-estradiol (Supplementary Fig. 4b). Next, we examined endogenous ERα binding at ERαenh588 region by ChIP-seq and observed a substantial decrease in both MCF-7 and T47D cells expressing sgRNA-ERαenh588 (Fig. 3e). |
About Function
| Enhancer function : | The phenotypic outcome of disrupting ERαenh588 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. |
| Enhancer function experiment: | Flow Cytometry |
| Enhancer function experiment description: |
Finally,as CCND1 is a crucial component of the G1-S phase transition, we examined the phenotypic outcome of disrupting ERαenh588 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. |
About SNP
| SNP ID: | -- | |
| SNP position: | -- |
| SNP experiment: | -- |
Enhancer associated network
The number on yellow line represents the distance between enhancer and target gene
