Continuous pioneer activity is required for maintaining pluripotency specific gene expression [TTchem-seq]

ABSTRACT: Accessible chromatin enables the physical interaction of transcription factors with DNA. Mapping accessible chromatin regions with ATACseq can be used to identify the thousands of cis-regulatory elements in the mammalian genome. However, which of them contributes to gene expression remains an open question. In mouse embryonic stem cells (mESC), canonical transcription factor SOX2 is, among others, responsible for the establishment of chromatin accessibility. SOX2 acts as a pioneer factor that binds to inaccessible DNA and promotes the opening of the chromatin. However, not all regions bound by the pioneer factor undergo opening. To determine which accessible chromatin regions are directly controlled by SOX2, we depleted SOX2 from mESC using acute degradation by dTAG at high temporal resolution. The depletion leads to a rapid loss of chromatin accessibility (within 30 minutes) at SOX2 associated sites and a gradual gain of newly accessible sites. To understand the changes in transcription after SOX2 depletion we performed nascent transcript mapping using TTchemseq. This revealed that expression of hundreds of transcription units are also rapidly affected following SOX2 depletion.  The intersection of sites that lose accessibility and downregulated genes shows an enrichment within 40 kb around the TSS. Importantly, this is not the case for SOX2 binding sites as identified by ChIPseq. Therefore, accessible sites created by SOX2, rather than SOX2 occupancy, are relevant for gene expression. Furthermore, 60-70% of genes that lose expression can be explained by loss of accessibility. To validate this finding, we used CRISPR-Cas9 to delete an element that shows loss of accessibility in the vicinity of a downregulated gene. The deletion alone results in decreased expression of the gene, showing its importance in maintaining the transcription program. Our approach of acute depletion of a pioneer transcription factor combined with nascent transcript mapping allows us to predict functional regulatory elements. We show that constant maintenance of open chromatin regions relies directly on SOX2, and its disruption has drastic consequences on transcription.

ORGANISM(S): Mus musculus

PROVIDER: GSE209526 | GEO | 2023/07/24

REPOSITORIES: GEO