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Epigenetic regulation of the transition from naïve to formative pluripotency


Many epigenetic changes occur around implantation as pluripotent cells of the epiblast prepare for lineage specification. However, in vivo epigenetic studies during these early developmental stages is technically challenging. Here, we use an in vitro model of the transition from pre- to post-implantation epiblast where embryonic stem cells (ESCs) are differentiated into epiblast-like cells (EpiLCs) to profile the epigenetic changes that occur during this transition and elucidate specific mechanisms that underlie these changes. In Chapter 2, we use this in vitro system to perform an siRNA screen for chromatin modifiers that regulate this transition, identifying the cohesin complex as an important regulator. In Chapter 3, we profile changes in cohesin binding between ESCs and EpiLCs to identify regulators of enhancer activity and uncover the transcription factor GRHL2 as a driver of enhancer activation. We further show that GRHL2-mediated enhancer activation in EpiLCs is part of a larger process of enhancer switching where genes that are expressed at similar levels in ESCs and EpiLCs undergo a switch in enhancer usage from an ESC-specific enhancer to a GRHL2-bound enhancer in EpiLCs. GRHL2 targets are enriched for epithelial genes, indicating that this enhancer switch is necessary for maintaining an epithelial program characteristic of both cell states. In Chapter 4, we focus on the transcription factor, FOXD3, which primes enhancers during the ESC-to-EpiLC transition. We show that FOXD3 simultaneously recruits the nucleosome remodeler, BRG1, and the histone deacetylases, HDAC1/2, to target sites to establish nucleosome-depleted regions while preventing full enhancer activation by inhibiting histone acetylation. We further show that the cohesin complex is not yet present at FOXD3 binding sites, suggesting these enhancers are not yet engaged in stable interactions with their target promoters. Lastly, we provide evidence of an intermediate enhancer state between the traditional “primed” and “active” states that is cohesin-bound (K4me1+/cohesin+/K27Ac-), suggesting that enhancer-promoter looping may occur as a distinct step in enhancer priming. Together, these studies demonstrate the importance of epigenetic regulation during this developmental window and uncover novel mechanisms of enhancer regulation that we believe extend beyond the ESC-to-EpiLC transition.

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