About Enhancer
| Enhancer ID: | E_01_240 | |
| Enhancer symbol: | -- | |
| Species: | Human | |
| Position : | chr6:31139599-31139921   |
|
| Biosample name: | Embryonic Stem Cell | |
| Experiment class : | Low throughput |
| Enhancer type: | Enhancer | |
| Disease: | -- | |
| DO: | -- | |
| Mesh: | -- | |
| Distance from TSS: | >2KB | |
| Pubmed ID: | 26813977 |
| Enhancer experiment: | CRISPR/Cas9,Luciferase Reporter Assay | |
| Enhancer experiment description: | 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: 31,247,052–31,248,218) that controls POU5F1 expression in primed hESCs (Gafni et al. 2013). |
About Target gene
| Target gene : | POU5F1(OCT3,OCT4,OTF-3,OTF3,OTF4,Oct-3,Oct-4) | |
| Strong evidence: | 4C-seq | |
| Less strong evidence: | Luciferase Reporter Assay | |
| Target gene experiment description: | 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). |
About TF
| TF name : | -- | |
| TF experiment: | -- | |
| TF experiment description: | -- |
About Function
| Enhancer function : | -- |
| Enhancer function experiment: | -- |
| Enhancer function experiment description: |
-- |
About SNP
| SNP ID: | -- | |
| SNP position: | -- |
| SNP experiment: | -- |
Enhancer associated network
The number on yellow line represents the distance between enhancer and target gene
