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Uncovering Chromatin-Mediated Regulatory Mechanisms of Mammalian Sex Determination

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A fundamental goal in biology is to understand how distinct cell types containing the same genetic information arise from a single stem cell throughout development. Sex determination is a key developmental process that requires a unidirectional commitment of an initially bipotential gonad towards either the male or female fate. This makes sex determination a unique model to study cell fate commitment and differentiation in vivo. Based on the accumulating evidence that dynamic chromatin structure regulates cell fate decisions, our overarching goal was to identify epigenetic mechanisms and cis-regulatory elements that contribute to the bipotential state of the fetal gonad and to the establishment and maintenance of the male or female fate during sex determination in mice. In this study, we developed a quantitative genome-wide profile of the active H3K4me3 and the repressive H3K27me3 histone modifications in isolated XY and XX gonadal supporting cells before and after sex determination. We show that male- and female-promoting genes are bivalent before sex determination, providing insight into how the bipotential state of the gonad is established at the epigenetic level. Surprisingly, after sex determination, many genes that promote the alternate pathway remain bivalent, possibly contributing to the ability of these cells to transdifferentiate even in adults. The finding that bivalency is retained at female-promoting genes whose expression declines after commitment to the male fate led us to question what could be silencing these bivalent genes throughout sex determination. It is known that the Polycomb group of proteins (PcG) mediate repression of bivalent genes in ESCs, and that loss of CBX2, the PcG subunit that binds H3K27me3 and mediates silencing, leads to upregulation of the female pathway and ovary development in XY individuals. We found that many genes in the Wnt signaling pathway were targeted for H3K27me3-mediated repression in Sertoli (XY) cells, leading us to test whether deletion of the ovarian-promoting gene Wnt4 could rescue male development in Cbx2 mutants. In accordance with our hypothesis, we show that expression of the male-determining gene Sry and testis development were rescued in XY Cbx2-/-;Wnt4-/- mice. We also show that CBX2 binds the downstream Wnt signaler Lef1. Our findings suggest that Cbx2 induces Sry indirectly by repressing the key ovarian-promoting/anti-testis Wnt pathway. In addition to histone modifications that regulate chromatin accessibility at promoter regions, the precise spatiotemporal regulation of genes during development is orchestrated by cis-regulatory genomic elements (such as enhancers, silencers and insulators) that function at a distance from the transcription start site. However, identifying functional regulatory sites that drive cell differentiation in vivo has been complicated by the high numbers of cells required for whole-genome epigenetic assays. Our inability to pinpoint the location of these sites has limited our capacity to study the mechanisms that regulate the expression of sex-determining genes. To address this limitation, we performed an Assay for Transposase-Accessibility followed by next-generation sequencing (ATAC-seq), a low-input assay that identifies regulatory elements, in purified XX and XY gonadal supporting cells before and after sex determination in mice. To distinguish enhancers from silencers and insulators, we also performed Chromatin Immunoprecipitation followed by next-generation sequencing (ChIP-seq) for H3K27ac, a histone modification that marks active enhancers. We show that XX and XY supporting cells initiate sex determination with similar chromatin landscapes in accordance to their bipotential nature, and acquire sex-specific regulatory elements as they commit to the male or female fate. To validate our approach, we identified two functional gonad-specific enhancers: one downstream of Bmp2, an ovary-promoting gene, and one upstream of Sox9, a testis-promoting gene. Deletion of the distal enhancer upstream of Sox9, named Enh13, led to development of phenotypically-normal XY females. This remarkable finding suggests that Enh13 alone is sufficient to upregulate Sox9 and is necessary for testis development. The work presented in this dissertation greatly increases our understanding of the complex regulatory network underlying mammalian sex determination. In addition to providing insight into the role that chromatin dynamics plays in regulating cell fate decisions during a key developmental event in vivo, our datasets provide a powerful resource for identifying non-coding regulatory elements that could lead to Disorders of Sexual Development when disrupted.

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