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Core sex determining gene, SOX9, regulates novel target genes during testis development
thesisposted on 23.02.2017, 23:57 by Hewa Alankarage, Dimuthu
SOX9 is a transcriptional activator, expressed in the XY gonads, that plays an essential role during male sex development. In humans, dysregulation of SOX9 is accompanied by sex reversal (46, XY female, 46, XX male). In mice, early or late loss of SOX9 leads to sex reversal or male infertility, respectively. SOX9 functions as the key regulator of testis development and fertility, driving differentiation of Sertoli cells directly, and the development and maintenance of other testicular cell types by juxtacrine and paracrine signalling. However, the mechanisms by which SOX9 regulate downstream testicular functions remain poorly understood. The use of transcriptomic analysis has become a useful tool in the exploration of transcription factor function. Here, we have utilised human Sertoli-like NT2/D1 cells to transiently over-express SOX9 and performed microarray analysis of the RNA. In order to uncover genes that are positively regulated by SOX9, genes that increased in expression were pursued further. One such gene, Ets variant factor 5 (ETV5), is explored in chapter 3. We show that SOX9 binds to and activates the proximal ETV5 promoter via a conserved SOX site in vitro. Examination of Etv5 expression during mouse embryonic development indicates that Etv5 is strongly expressed by the XY gonads. ETV5 protein localises to Sertoli cell nuclei, as well as germ cells and interstitial cells of the embryonic testis. In two Sox9 KO XY mouse models, Etv5 expression is decreased before and after sex determination. Results of this chapter reveal SOX9 as a key regulator of ETV5 expression and therefore of ETV5 mediated functions in the testis. Chapter 4 describes the investigation of desert hedgehog (DHH) as a target gene of SOX9. DHH is an important signalling factor for testis organogenesis and maintenance of fertility. We show that DHH is a target of SOX9 transcriptional activity in human and mouse Sertoli cell lines. In the human Sertoli-like cell line, NT2/D1, transfection of a mutant SOX9 associated with a 46,XY DSD patient (SOX9-A158T) could not increase DHH expression. Chromatin immunoprecipitation revealed that SOX9 was bound to the endogenous DHH promoter at a conserved SOX consensus DNA binding site. Dhh expression was down-regulated in foetal XY gonads of two Sox9 knock-out mouse models before and after sex determination. Conversely, ectopic expression of Sox9 in XX mouse gonads resulted in Dhh up-regulation. Findings of this chapter suggest that SOX9 is a transcriptional regulator of Dhh expression in Sertoli cells of the developing testis and thereby controls DHH-mediated paracrine signalling required for the differentiation of multiple cell types in the testis. That SOX9 mediates its action via DHH is consistent with the overlapping reproductive phenotypes observed in 46,XY DSD patients with mutations in either gene. In conclusion, the work within this thesis details the regulation of novel target genes of SOX9 and adds to the current knowledge regarding molecular mechanisms of testis development. Ultimately, it is hoped that the identification of these target genes would contribute to the knowledge of human sex development and human disorders, facilitating easier diagnosis of DSD.