Disorders of Sex Development (DSDs) encompass a wide spectrum of conditions and often manifest with atypical gonads or genitalia. The majority of DSD patients cannot be given an accurate diagnosis, which severely comprises their clinical management. While mutations in coding regions of gonad genes have been important in understanding the aetiology of DSD little attention has focussed on the regulatory regions of gonad genes.
It has been 27 years since the Y-linked SRY gene and its role as the master of mammalian sex determination were discovered. Studies in mice implicate Sox9 as a direct target of Sry, and while SOX9 upregulation in the early gonad is crucial for testis development, the SOX9 sequences bound by SRY during human testis determination have not been identified. We analysed copy number variations (CNVs) in the upstream regulatory region of SOX9 in DNA from patients with DSD, allowing us to define several minimal critical regions for sex-reversal.
Using a combination of luciferase tiling and bioinformatics approaches we identified three novel human testis enhancers 5’ of SOX9. In cell-based reporter assays these enhancers responded to different combinations of testis specific regulators including SRY, NR5A1 (SF1) and SOX9 itself. Enhancers that showed the strongest activity in vitro were used to generate transgenic mice. In vivo, two enhancers drove reporter gene expression in mouse embryonic gonads, while mice null for the third enhancer showed reduced Sox9 transcription. Given that duplications and deletions of these enhancers in DSD patients result in sex reversal (SRY negative, 46,XX males or 46,XY females respectively) our results suggest a mechanism by which these upstream enhancers play crucial roles in human sex development. Together these three enhancers may provide the missing link between SRY and SOX9 initiation, upregulation and maintenance – all fundamental to testis development.