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Dynamic regulation of chromatin accessibility by pluripotency transcription factors across the cell cycle [OCT4OFF SOX2OFF ChIP-seq]

ABSTRACT: The pioneer activity of transcription factors allows for opening of inaccessible regulatory elements and has been extensively studied in the context of cellular differentiation and reprogramming. In contrast, the function of pioneer activity in self-renewing cell divisions and across the cell cycle is poorly understood. Here we assessed the interplay between OCT4 and SOX2 in controlling chromatin accessibility of mouse embryonic stem cells. We found that OCT4 and SOX2 operate in a largely independent manner even at co-occupied sites, and that their cooperative binding is mostly mediated indirectly through regulation of chromatin accessibility. Controlled protein degradation strategies revealed that the uninterrupted presence of OCT4 is required for post-mitotic re-establishment and interphase maintenance of chromatin accessibility, and that highly OCT4-bound enhancers are particularly vulnerable to transient loss of OCT4 expression. Our study sheds light on the constant pioneer activity required to maintain the dynamic pluripotency regulatory landscape in an accessible state.

ORGANISM(S): Mus musculus

PROVIDER: GSE134669 | GEO | 2019/11/26

REPOSITORIES: GEO

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Project description: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.

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Dynamic regulation of chromatin accessibility by pluripotency transcription factors across the cell cycle [OCT4OFF SOX2OFF ChIP-seq]

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