ABSTRACT Sex determination in mammals is a complex biological process leading to the differentiation of the bipotential gonad into a testis or ovary. It is initiated by a cell fate decision in a subset of somatic cells called the supporting cell lineage. These cells carry bivalent histone marks at many genes associated with sex determination pathways and are poised for direction into either the Sertoli cell lineage (leading to testis development) or the granulosa cell lineage (leading to ovary development). Commitment to Sertoli or granulosa fate is characterized by the loss of repressive histone marks at genes associated with the active pathway, and expansion of repressive marks at genes associated with the alternative pathway. Repression, which we hypothesize is mediated in part by chromatin conformational changes, is important to stabilize fate commitment. However, how epigenetic modifications and transcription factor cascades are integrated to drive sex determination is not understood. It is well established that the key step in activating Sertoli cell differentiation is the transient activation of the Y-encoded transcription factor (TF), SRY, and its direct downstream target, SOX9. However, since our discovery that supporting cells are derived from the coelomic epithelium (CE), it has been unclear why Sry and female pathway genes are only activated in cells once they leave the CE and enter the gonad. We recently showed that NUMB, an inhibitor of the Notch signaling pathway, is asymmetrically distributed to cells that enter the gonad from the CE and is required for gonadal cell differentiation. These findings imply that a factor(s) activated in Numb+/Notchlo cells confers competence for differentiation. Exploration of our transcriptome, ATAC-seq, and histone modification datasets, revealed that 2 E-box family HLH transcription factors, Tcf4 and Tcf12, are expressed at high levels in the bipotential gonad, remain highly expressed in the granulosa lineage, but are downregulated in the Sertoli lineage where they map as binding targets of SOX9 and could be targets of feedback repression to block the granulosa pathway in XY gonads. E-box binding motifs are enriched in the nucleosome-depleted regions near granulosa genes but are absent from Sertoli genes, with the exception of Sry itself. Our findings suggest that TCF4 and TCF12, activated in Numb+/Notchlo cells, license differentiation of the supporting cell lineage, serving as activators of the granulosa pathway and of Sry in the XY gonad; whereas, SOX9, activated by SRY, acts in a feedback loop to down-regulate Tcf4 and Tcf12 transcription. In Aim 1, we will test the hypothesis that bHLH TFs are downstream of Numb, comprise an HLH “code” for differentiation of the supporting cell lineage, and are repressed by SOX9. In Aim 2, we will test the hypothesis that epigenetic and chromatin conformation changes occur during Sertoli fate commitment, mediated in part by competition between SOX9 and HLH proteins, and function to stabilize the male pathway by modulating accessibility of promoters and enhancers and the expansion of silencing marks at genes associated with the alternative granulosa pathway.

NARRATIVE Our previous work suggests that there is a pathway downstream of NUMB that confers “gonadal” competence on cells that have entered the gonad from the coelomic epithelium. Many lines of evidence point to the hypothesis that bHLH proteins are this missing link and are opposed by SOX factors in the male pathway. We will test this hypothesis and also discover how these transcription factors mediate chromatin conformational changes that alter accessibility of genes associated with the male or female pathways.