High throughput screening identifies SOX2 as a Super Pioneer Factor that inhibits DNA methylation maintenance at its binding sites
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PROVIDER: PRJNA603914 | ENA |
REPOSITORIES: ENA
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Project description:Access of mammalian transcription factors (TFs) to regulatory regions, an essential event for transcription regulation, is hindered by chromatin compaction involving nucleosome wrapping, repressive histone modifications and DNA methylation. Moreover, methylation of TF binding sites (TBSs) affects TF binding affinity to these sites. Remarkably, a special class of TFs called pioneer transcription factors (PFs) can access nucleosomal DNA, leading to nucleosome remodelling and chromatin opening. However, whether PFs can bind to methylated sites and induce DNA demethylation is largely unknown. Here, we set up a highly parallelized approach to investigate PF ability to bind methylated DNA and induce demethylation. Our results indicate that the interdependence between DNA methylation and TF binding is more complex than previously thought, even within a select group of TFs that have a strong pioneering activity; while most PFs do not induce changes in DNA methylation at their binding sites, we identified PFs that can protect DNA from methylation and PFs that can induce DNA demethylation at methylated binding sites. We called the latter “super pioneer transcription factors” (SPFs), as they are seemingly able to overcome several types of repressive epigenetic marks. Importantly, while most SPFs induce TET-dependent active DNA demethylation, SOX2 binding leads to passive demethylation by inhibition of the maintenance methyltransferase DNMT1 during replication. This important finding suggests a novel mechanism allowing TFs to interfere with the epigenetic memory during DNA replication.
2021-03-18 | GSE144524 | GEO
Project description: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.
2023-10-24 | PXD043672 | Pride
Project description:Binding of mammalian transcription factors (TFs) to regulatory regions is hindered by chromatin compaction and DNA methylation of their binding sites. Nevertheless, pioneer transcription factors (PFs), a distinct class of TFs, have the ability to access nucleosomal DNA, leading to nucleosome remodelling and enhanced chromatin accessibility. Whether PFs can bind to methylated sites and induce DNA demethylation is largely unknown. Using a highly parallelized approach to investigate PF ability to bind methylated DNA and induce DNA demethylation, we show that the interdependence between DNA methylation and TF binding is more complex than previously thought, even within a select group of TFs displaying pioneering activity; while some PFs do not affect the methylation status of their binding sites, we identified PFs that can protect DNA from methylation and others that can induce DNA demethylation at methylated binding sites. We call the latter super pioneer transcription factors (SPFs), as they are seemingly able to overcome several types of repressive epigenetic marks. Finally, while most SPFs induce TET-dependent active DNA demethylation, SOX2 binding leads to passive demethylation, an activity enhanced by the co-binding of OCT4. This finding suggests that SPFs could interfere with epigenetic memory during DNA replication.
| S-EPMC8184831 | biostudies-literature
Project description: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.
2023-07-24 | GSE234703 | GEO
Project description:Pioneer transcription factors, by interacting with nucleosomes, scan silent, compact chromatin to target regulatory sequences, enabling cooperative binding events that modulate local chromatin structure and gene activity. However, not all cognate motifs are targeted by pioneer factors and dynamic scanning parameters required to scan compact chromatin are unknown. A combined genomics and single-molecule tracking approach shows that to target DNase-resistant, low-histone turnover sites, pioneer factors FOXA1 and SOX2 display opposite dynamics of chromatin scanning: slow, with low nucleoplasmic diffusion and stable interactions, versus fast, with high nucleoplasmic diffusion and transient interactions, respectively. Despite such differences, the ability of FOXA1 and SOX2 to scan low-mobility chromatin, mediated by protein domains outside of the respective DNA binding domains, leads to targeting silent chromatin. By contrast, non-pioneer HNF4A predominantly targets DNase-sensitive, nucleosome-depleted regions. We conclude that the targeting of compact chromatin sites by pioneer factors can be performed through diverse dynamic processes. Cleavage under Targets and Release Using Nuclease (CUT&RUN) of SOX2 and SOX2-DBD after ectopic expression in BJ fibroblasts.
2023-06-07 | GSE220568 | GEO
Project description:Pioneer transcription factors, by interacting with nucleosomes, scan silent, compact chromatin to target regulatory sequences, enabling cooperative binding events that modulate local chromatin structure and gene activity. However, not all cognate motifs are targeted by pioneer factors and dynamic scanning parameters required to scan compact chromatin are unknown. A combined genomics and single-molecule tracking approach shows that to target DNase-resistant, low-histone turnover sites, pioneer factors FOXA1 and SOX2 display opposite dynamics of chromatin scanning: slow, with low nucleoplasmic diffusion and stable interactions, versus fast, with high nucleoplasmic diffusion and transient interactions, respectively. Despite such differences, the ability of FOXA1 and SOX2 to scan low-mobility chromatin, mediated by protein domains outside of the respective DNA binding domains, leads to targeting silent chromatin. By contrast, non-pioneer HNF4A predominantly targets DNase-sensitive, nucleosome-depleted regions. We conclude that the targeting of compact chromatin sites by pioneer factors can be performed through diverse dynamic processes. Micrococcol nuclease digestion and sequencing (MNase-seq) of human BJ fibroblasts
2023-06-07 | GSE220569 | GEO
Project description:Pioneer transcription factors, by interacting with nucleosomes, scan silent, compact chromatin to target regulatory sequences, enabling cooperative binding events that modulate local chromatin structure and gene activity. However, not all cognate motifs are targeted by pioneer factors and dynamic scanning parameters required to scan compact chromatin are unknown. A combined genomics and single-molecule tracking approach shows that to target DNase-resistant, low-histone turnover sites, pioneer factors FOXA1 and SOX2 display opposite dynamics of chromatin scanning: slow, with low nucleoplasmic diffusion and stable interactions, versus fast, with high nucleoplasmic diffusion and transient interactions, respectively. Despite such differences, the ability of FOXA1 and SOX2 to scan low-mobility chromatin, mediated by protein domains outside of the respective DNA binding domains, leads to targeting silent chromatin. By contrast, non-pioneer HNF4A predominantly targets DNase-sensitive, nucleosome-depleted regions. We conclude that the targeting of compact chromatin sites by pioneer factors can be performed through diverse dynamic processes. Chromatin immunoprecipitation DNA-sequencing (ChIP-seq) for HNF4A, FOXA1, FOXA1-DBD, FOXA1-NHAA, FOXA1-RRAA, as well as H2B-HALO after ectopic expression in human BJ fibroblast cells.
2023-06-07 | GSE220567 | GEO
Project description:The pluripotency transcription factor SOX2 is essential for the maintenance of glioblastoma stem cells (GSC), which drive tumor growth and treatment resistance.To understand how SOX2 is regulated in GSCs, we utilized a proteomic approach and identified the E3 ubiquitin ligase TRIM26 as a direct SOX2-interacting protein. Unexpectedly, we found TRIM26 depletion decreased SOX2 protein levels and increased SOX2 polyubiquitination in patient-derived GSCs, suggesting TRIM26 promotes SOX2 protein stability. Accordingly, TRIM26 knockdown reduced SOX2 transcriptional activity, self-renewal capacity, and in vivo tumorigenicity in multiple GSC lines. Mechanistically, we found TRIM26, via its C-terminal PRYSPRY domain, but independent of its RING domain, stabilizes SOX2 protein by directly inhibiting the interaction of SOX2 with WWP2, which we identify as a bona fide SOX2 E3 ligase in GSCs. Our work identifies E3 ligase competition as a critical mechanism of SOX2 regulation, with functional consequences for GSC identity and maintenance.
2021-11-04 | PXD028933 | Pride
Project description:Squamous cell carcinomas (SCCs) are aggressive malignancies. Previous report demonstrated that the transcription factors TP63 and SOX2 exhibited overlapping genomic occupancy in SCCs. Our recent study have identified TP63 and SOX2 as super-enhancer-associated genes. However, functional consequences of their frequent co-localization at super-enhancers region remains unexplored. Here, ChIP-seq result indicated TP63 and SOX2 co-occupied peaks are significantly located the super enhancer region compare with unique of TP63 and SOX2 signaling, and combined RNA-seq analyses of different types of SCCs reveal that TP63 and SOX2 cooperatively regulate expression of the super-enhancer-associated the long non-coding RNA (lncRNA) gene, CCAT1. CCAT1 is highly expressed in SCCs compared to either other tumor types or normal tissues. CCAT1 expression strongly correlates with expression levels of TP63 and SOX2. Silencing of CCAT1 significantly reduces cellular growth both in vitro and in vivo, phenotyping the effect of silencing either TP63 or SOX2. Furthermore, ChIP-Seq and luciferase reporter assays demonstrate TP63 and SOX2 co-occupy the promoter and super-enhancer regions of CCAT1. Interaction of CCAT1 with TP63 and SOX2 are validated by RNA immunoprecipitation. In addition, ChIRP analysis suggests that CCAT1 regulates EGFR expression by binding to the super-enhancer of EGFR. Further investigations shows that CCAT1 activates both the MEK/ERK1/2 and PI3K/AKT signaling pathways through up-regulation of EGFR. In conclusion, CCAT1 is driven by master transcription factors TP63 and SOX2 and is recruited to the super-enhancer of EGFR, promoting SCC tumorigenesis through activation of the MEK/ERK1/2 and PI3K/AKT signaling pathways. These studies help to explain the pathogenesis of SCC and aids in providing novel therapeutic strategy.
2018-09-17 | GSE106564 | GEO
Project description:Squamous cell carcinomas (SCCs) are aggressive malignancies. Previous report demonstrated that the transcription factors TP63 and SOX2 exhibited overlapping genomic occupancy in SCCs. Our recent study have identified TP63 and SOX2 as super-enhancer-associated genes. However, functional consequences of their frequent co-localization at super-enhancers region remains unexplored. Here, ChIP-seq result indicated TP63 and SOX2 co-occupied peaks are significantly located the super enhancer region compare with unique of TP63 and SOX2 signaling, and combined RNA-seq analyses of different types of SCCs reveal that TP63 and SOX2 cooperatively regulate expression of the super-enhancer-associated the long non-coding RNA (lncRNA) gene, CCAT1. CCAT1 is highly expressed in SCCs compared to either other tumor types or normal tissues. CCAT1 expression strongly correlates with expression levels of TP63 and SOX2. Silencing of CCAT1 significantly reduces cellular growth both in vitro and in vivo, phenotyping the effect of silencing either TP63 or SOX2. Furthermore, ChIP-Seq and luciferase reporter assays demonstrate TP63 and SOX2 co-occupy the promoter and super-enhancer regions of CCAT1. Interaction of CCAT1 with TP63 and SOX2 are validated by RNA immunoprecipitation. In addition, ChIRP analysis suggests that CCAT1 regulates EGFR expression by binding to the super-enhancer of EGFR. Further investigations shows that CCAT1 activates both the MEK/ERK1/2 and PI3K/AKT signaling pathways through up-regulation of EGFR. In conclusion, CCAT1 is driven by master transcription factors TP63 and SOX2 and is recruited to the super-enhancer of EGFR, promoting SCC tumorigenesis through activation of the MEK/ERK1/2 and PI3K/AKT signaling pathways. These studies help to explain the pathogenesis of SCC and aids in providing novel therapeutic strategy.
2018-09-17 | GSE106563 | GEO