| 研究実績の概要 |
We have identified H4K5acK8ac positive regions in mESCs and GSCs and performed bioinformatics analyses to prove our hypothesis that H4K5acK8ac positive regions associate with Pol II, TIP60. To demonstrate the enhancer function of H4K5acK8ac+ regulatory regions, we perform loss-of-function experiments via CRISPR-Cas9 mediated genome deletion of selected genome loci.
We identified super-enhancers with the ranking of H4K5acK8ac and compared with known H3K27ac marks. We found that, when SEs were defined as having the top ranks for H4K5acK8ac or H3K27ac signal, 43% of H4K5acK8ac-ranked SEs were distinct from H3K27ac-ranked SEs in a GSC (0316-GSC) line. To understand whether the H4K5acK8ac-preferred SEs regulate the expression of the associated genes, we performed CRISPR-Cas9 mediated SE deletion in GSCs. To this end, we selected paired guide RNAs (gRNAs) flanking the H4K5acK8ac-preferred SEs associated with putative target genes in 0316-GSC cells. The CRISPR-Cas9 mediated deletion of a single targeted H4K5acK8ac-preferred SE resulted in significant downregulation of putative target genes, compared with the unedited cells. However, these SE deletions did not change the expression of the putative non-target genes in 0316-GSC cells. Supporting our purposes, we have revealed the existence of H4K5acK8ac-preferred enhancers and SEs, and their involvement in associated gene expression.
Currently, we focus to unravel the long-range interaction of H4K5acK8ac-enriched enhancer and their involvement in associated gene transcription, especially in mESCs.
|
| 今後の研究の推進方策 |
As the contribution of H4K5acK8ac at enhancer regulatory elements is not well known. As lineage specific transcription factors (LTF) and Pol II are recruited to enhancers, but how does such LTF and Pol II interaction with H4K5acK8ac-associated enhancers favor transcription is not known. Therefore, the present proposal focuses to redefine the active enhancer enriched with H4K5acK8ac. In future, we focus to unravel the long-range interaction of H4K5acK8ac-enriched enhancer with its promoter and crosstalk those functions with lineage specific TFs, that regulate the associated transcription.
|
