At embryonic day (E) 10.5, prior to gonadal sex determination, XX and XY gonads are bipotential and able to differentiate into either a testis or an ovary. At this point, they are transcriptionally and morphologically indistinguishable. Sex determination begins around E11.5 in the mouse when the supporting cell lineage commits to either Sertoli or granulosa cell fate. Testis-specific factors such as SRY and SOX9 drive differentiation of bipotential-supporting cells into the Sertoli cell pathway, whereas ovary-specific factors like WNT4 and FOXL2 guide differentiation into granulosa cells. It is known that these 2 pathways are mutually antagonistic, and repression of the alternative fate is critical for maintenance of the testis or ovary programs. While we understand much about the transcription factor networks guiding the process of sex determination, it is only more recently that we have begun to understand how this process is epigenetically controlled. Studies in the past decade have demonstrated the importance of the chromatin state for gene expression and cell fate commitment, with histone modifications and DNA accessibility having a direct role in gene regulation. It is now clear that the chromatin state during sex determination is dynamic and likely critical for the establishment and/or maintenance of the transcriptional programs. Prior to sex determination, supporting cells have similar chromatin structure and histone modification profiles, reflecting the bipotential nature of these cells. After differentiation to Sertoli or granulosa cells, the chromatin state acquires sex-specific profiles. The proteins that regulate the deposition of histone modifications or the opening of compact chromatin likely play an important role in Sertoli and granulosa cell fate commitment and gonad development. Here, we describe studies profiling the chromatin state during gonadal sex determination and one example in which depletion of <i>Cbx2</i>, a member of the Polycomb Repressive Complex 1 (PRC1), causes male-to-female sex reversal due to a failure to repress the ovarian pathway.

Mammalian sex is determined by an opposing networks of ovarian and testicular genes that are well characterized. However, its epigenetic regulation is still largely unknown, thus limiting our understanding of a fundamental process for species propagation. Here we explore the 3D chromatin landscape of sex determination in vivo, by profiling FACS-sorted embryonic mouse gonadal populations, prior and after sex determination, in both sexes. We integrate Hi-C with ChIP-seq experiments using METALoci, a novel genome spatial auto-correlation analysis that identifies 3D enhancer hubs across the genome. We uncover a prominent rewiring of chromatin interactions during sex determination, affecting the enhancer hubs of hundreds of genes that display temporal- and sex-specific expression. Moreover, the identification of the 3D enhancer hubs allows the reconstruction of regulatory networks, revealing key transcription factors involved in sex determination. By combining predictive approaches and validations in transgenic mice we identify a novel Fgf9 regulatory hub, deletion of which results in male-to-female sex reversal with the upregulation of ovarian-specific markers and the initiation of meiosis. Thus, spatial auto-correlation analysis is an effective strategy to identify regulatory networks associated to biological processes and to further characterize the functional role of the 3D genome.