Dataset Information

Pioneer activity distinguishes activating from non-activating SOX2 binding sites

ABSTRACT: Genome-wide transcriptional activity involves the binding of many transcription factors to thousands of sites in the genome. Determining which sites are directly driving transcription remains a challenge. Here we use acute protein depletion of the pioneer transcription factor SOX2 to establish its functionality in maintaining chromatin accessibility. We show that thousands of accessible sites are lost within an hour of protein depletion, indicating rapid turnover of these sites in the absence of pioneer factors. To understand the relationship with transcription we performed nascent transcription analysis and found that open chromatin sites that are maintained by SOX2 are highly predictive of gene expression, in contrast to all other SOX2 binding sites. We use CRISPR-Cas9 genome editing in the Klf2 locus to functionally validate a predicted regulatory element. We conclude that the regulatory activity of SOX2 is exerted largely at sites where it maintains accessibility and that other binding sites are largely dispensable for gene regulation.

INSTRUMENT(S): Orbitrap Exploris 480

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Embryonic Stem Cell

SUBMITTER: Liesbeth Hoekman

LAB HEAD: Onno Bleijerveld

PROVIDER: PXD043672 | Pride | 2023-10-24

REPOSITORIES: pride

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			Action	DRS
	20210927_Exploris_Project_528_Michela_sample_S_D1.raw	Raw
	20210927_Exploris_Project_528_Michela_sample_S_D2.raw	Raw
	20210927_Exploris_Project_528_Michela_sample_S_D3.raw	Raw
	20210927_Exploris_Project_528_Michela_sample_S_D4.raw	Raw
	20210927_Exploris_Project_528_Michela_sample_S_G1.raw	Raw

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Publications

Pioneer activity distinguishes activating from non-activating SOX2 binding sites.

Maresca Michela M van den Brand Teun T Li Hangpeng H Teunissen Hans H Davies James J de Wit Elzo E

The EMBO journal 20230911 20

Genome-wide transcriptional activity involves the binding of many transcription factors (TFs) to thousands of sites in the genome. Pioneer TFs are a class of TFs that maintain open chromatin and allow non-pioneer TFs access to their target sites. Determining which TF binding sites directly drive transcription remains a challenge. Here, we use acute protein depletion of the pioneer TF SOX2 to establish its functionality in maintaining chromatin accessibility. We show that thousands of accessible ...[more]

PMID: 37691488

Similar Datasets

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.