High throughput screening identifies SOX2 as a Super Pioneer Factor that inhibits DNA methylation maintenance at its binding sites

Abstract

19Access of mammalian transcription factors (TFs) to regulatory regions, an essential 20 event for transcription regulation, is hindered by chromatin compaction involving 21 nucleosome wrapping, repressive histone modifications and DNA methylation. 22TET-dependent active DNA demethylation, SOX2 binding leads to passive 37 demethylation by inhibition of the maintenance methyltransferase DNMT1 during 38 replication. This important finding suggests a novel mechanism allowing TFs to 39 interfere with the epigenetic memory during DNA replication. 40 41 42 deemed necessary prior to TF binding to compact chromatin 2 . However, several 54 studies identified a new class of TFs, called pioneer transcription factors (PFs) that 55 access their target sites in condensed chromatin. This results in chromatin "de-56 condensation" by nucleosome remodelling, recruitment of "settler" TFs that are 57 unable to access condensed chromatin, and transcription activation 3-6 . These 58 findings hint for a more complex relationship between epigenetic-and genetic-based 59 mechanisms in transcription regulation than previously thought. It is therefore 60 important to establish whether and when epigenetic mechanisms constitute a 61 primary event in the regulation of transcription and when do they simply result from 62 previous events governed by the genetic composition of the regulatory regions (i.e. 63 TF binding). 64DNA methylation is an essential epigenetic modification that was hypothesized not 65 only to inhibit the accessibility of DNA but also its affinity to TFs 7,8 . Remarkably, 66 recent studies have shown that not all TFs show sensitivity to DNA methylation. 67 4 Moreover, some TFs were shown to preferentially bind to methylated sites 9-11 . 68However, it is currently less clear whether TFs, notably those that can bind to 69 methylated DNA, lead to changes in the methylation status of their binding sites (i.e. 70DNA demethylation), and if so, how. More precisely, can PFs, in addition to their 71 ability to remodel the nucleosomes, induce DNA demethylation? 72Using a high throughput approach, our study methodically determined the ability of 73reported PFs to induce DNA demethylation at their binding sites in mouse embryonic 74 stem cells (ESCs) and in vitro differentiated neuronal progenitors (NPs). Results 75show that, while many PFs do not affect the methylation status of their binding sites, 76 a group of PFs that we call "protective pioneer transcription factors (PPFs)" protect 77 DNA from de novo acquisition of methylation, while another group called "super 78 pioneer transcription factors (SPFs)" induce DNA demethylation at their methylated 79 binding sites. Importantly, we show that most SPF-driven demethylation is TET-80 dependent, except for SOX2 that inhibits DNMT1 at the replication fork, thus leading 81 to replication-dependent passive DNA demethylation. 82 5 Results: 83Hi-TransMet: a high throughput assay for the analysis of transcription factor 84 binding effect on DNA methylation 85We developed a method called Hi-Tra...

Pioneer transcription factors are associated with the modulation of DNA methylation patterns across cancers

“…The expression of the majority of the emTFs exhibited anti-correlation with CpG methylation close to their TFBSs, indicating that these emTFs are likely inducing local DNA demethylation. This is in agreement with previous studies that reported RUNX1 [7,61], RUNX3 [6], SPI1 [6,20,61], BHLHE40 [61], and FOXA1 [22] to induce binding-site directed DNA demethylation.…”

“…The strategy relies on the integration of a sequence backbone with known methylation status but containing diverse TF binding motifs followed by bisulfite sequencing of PCR amplicons. The study revealed pioneer TFs that can induce local DNA demethylation and pioneer TFs whose binding have a protective effect against de novo DNA methylation [22]. Using a computational approach, the ELMER (Enhancer Linking by Methylation/Expression Relationships) tool allowed for the large-scale identification of transcriptional enhancers and their target genes based on DNA methylation data (at enhancers) and gene expression [23].…”

“…Motif enrichment analysis at the enhancers predicted pan-cancer by ELMER inferred TFs that could act as upstream regulators of DNA methylation patterns at these enhancers [23]. Despite continuous efforts to unravel the molecular mechanism by which DNA methylation is regulated, the current understanding of how DNA methylation is regulated and its interplay with TF-binding in cancer patients is limited [6,7,18,22]. We hypothesized that a pan-cancer and genome-wide investigation of the interplay between TF binding and resulting local DNA methylation patterns in cancer genomes could reveal key regulatory processes that are critical for an improved molecular understanding of cancers.…”

“…This strategy identified a set of developmental (cell fate determining) TFs that were associated with binding site-directed DNA demethylation [6]. Another high-throughput screening strategy investigated the interplay between TF binding and DNA methylation for hundreds of TFs [22]. The strategy relies on the integration of a sequence backbone with known methylation status but containing diverse TF binding motifs followed by bisulfite sequencing of PCR amplicons.…”

SOX transcription factors direct TCF-independent WNT/beta-catenin transcription

“…There is evidence that SOX TFs can act as pioneering factors by directly engaging nucleosomes to regulate chromatin accessibility [45][46][47][48] , which might explain in part the loss of ßcatenin binding in SOX17KO cells. To address this possibility, we performed ATAC-Seq 49 to predict nucleosome occupancy at both classes of enhancers.…”