GSE209527
Continuous pioneer activity is required for maintaining pluripotency specific gene expression [ATAC-Seq]
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. Overall design: Analysis of accessible chromatin at a timeseries of depletion of the SOX2, OCT4 and NANOG transcription factors using the dTAG depletion system in mESC. Depletion happens on addition of dTAG-13, hence untreated samples serve as controls.  In addition, an analysis of accessible chromatin with ectopic expression vectors with (parts of) SOX2 after depletion of SOX2, wherein DMSO treated samples are controls.  Lastly, a profile of accessible chromatin after knockout of a differentially accessible region nearby the Klf2 gene.