The Battle of Sexes: Studying the Regulation of Mammalian Sex Determination

Sex determination in mammals depends on a fine balance of gene expression in the early gonads being tipped one way by the action of several factors, such as Wnt signalling and FOXL2, that favors ovarian development or by the activity of the Y-linked gene SRY and its direct target SOX9 that promote testis development.

Understanding how these gene regulatory networks operate depends on identifying not just the critical downstream genes, but also the regulatory elements that control their activity. This understanding can help reveal novel causes of disorders of sex differentiation (DSDs).

In an attempt to explore the contribution of remote regulatory elements to the process of sex determination, we focused on the Sox9 gene. SOX9 is a transcription factor critical for several tissues including the testis with a complex cis-regulatory region controlling its expression. Using in vivo high-throughput chromatin accessibility techniques, transgenic reporter mice, and genome editing we revealed multiple gonadal regulatory elements in the 2 Mb gene desert upstream of Sox9. Enh13, located 565 kb 5’, was found to recapitulate early Sox9 testis expression. Unexpectedly, given the known role of a TES, a proximal enhancer, deletion of Enh13 results in complete XY male-to-female sex reversal. This conserved Enh13 is found embedded within XY SR, a 32.5 kb far upstream region whose deletion in patients lead to XY sex reversal, suggesting that Enh13 is also critical in humans. This shows that deleting a single enhancer can fully recapitulate the phenotype of mutating a gene in a tissue-specific manner.