Targeted Suppression of the Transcription Factor Sox9

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DESCRIPTION (provided by applicant): The transcription factor Sox9 is critical for the male gonadal development during embryogenesis. Sox9 expression also persists in some adult tissues, particularly in the testes, but its role after birth is not known, as conventional Sox9 knockout (KO) mice die during embryogenesis. In this application, I propose to determine Sox9's role in the postnatal and adult testes. I propose to accomplish this task by using a newly developed tissue specific RNA interference (RNAi) approach with a tool that I recently identified - a highly active promoter selectively expressed in Sertoli cells, the site of Sox9 expression in the testes. We have shown in our recent studies that only 0.6-kb 5'-flanking sequences from the proximal promoter of the RhoxS homeobox gene(RhoxS Pp) is sufficient to drive strong expression specifically in postnatal and adult Sertoli cells in a developmentally regulated manner in vivo. I propose to use RhoxS Pp to generate transgenic mice that express short hairpin (sh) RNAs specific for Sox9 in vivo. In addition to this study providing the first information on the role of Sox9 in the testes after birth, it will be a proof of principle for my newly developed in vivo RNAi approach that I used to knock down Wilms' tumor 1 gene (WT1) in a tissue-specific manner. This RNAi approach will be a technical advance, as to date no laboratory has reported the development of a generally applicable method for stable tissue-specific RNAi in vivo. My approach stems from the means by which naturally occurring short RNAs - micro RNAs (miRNAs) - are generated from Pol II transcripts. In my approach, a shRNA molecule targeting Sox9 will be processed from a RhoxS Pp-driven precursor transcript by Drosha, a ubiquitously expressed RNase-lll enzyme that processes out naturally occurring miRNAs. Because the Sox9 shRNA transcript will be specifically expressed from the RhoxS Pp, I predict it will suppress Sox9 expression specifically in Sertoli cells within the testes. I hypothesize that Sox9 functions in Sertoli cells to direct specific events during spermatogenesis. There are at least two lines of evidence that support my hypothesis : first, Sox9 is highly expressed in the adult Sertoli cells in a stage-specific manner suggesting that it might have a pivotal role in germ cell differentiation. Second, I recently showed that WT1, which is known to be highly expressed in adult Sertoli cells and plays a crucial role in gonadal development during embryogenesis like Sox9, is also important for maintenance of spermatogenesis. Therefore, I propose that Sox9 plays an important role in spermatogenesis. The specific aim of this application is: (1) to elucidate the function of transcription factor Sox9 in adult Sertoli cells by using tissue-specific RNAi. The elucidation of Sox9 function in postnatal testes may lead to better understanding of the pathophysiology of gonadal dysgenesis and sex-reversal commonly found in human patients with mutated Sox9. In addition, revelation of Sox9 function in reproduction may lead to treatments for infertility.