Targeted Suppression of the Transcription Factor Sox9

描述(申请人提供)：转录因子Sox9在胚胎发育过程中对男性性腺发育至关重要。Sox9也在一些成年组织中持续表达，特别是在睾丸中，但其在出生后的作用尚不清楚，因为传统的Sox9基因敲除(KO)小鼠在胚胎发育过程中死亡。在这项申请中，我建议确定Sox9的S在出生后和成人睾丸中的作用。我建议通过使用一种新开发的组织特异性RNA干扰(RNAi)方法来完成这一任务，该方法使用我最近发现的一种工具-一种在支持细胞中选择性表达的高活性启动子，支持细胞是Sox9在睾丸中表达的位置。我们最近的研究表明，RhoxS Homeobox基因(RhoxS PP)近端启动子(RhoxS PP)仅0.6kb的5‘侧翼序列就足以在体内以发育调节的方式在出生后和成年Sertoli细胞中特异性地驱动强表达。我建议使用RhoxS PP在体内产生表达Sox9特异的短发夹(Sh)RNA的转基因小鼠。除了这项研究提供了关于Sox9在出生后睾丸中的作用的第一个信息外，它还将是我新开发的体内RNAi方法的原理证明，我使用该方法以组织特异性的方式敲除Wilms的肿瘤1基因(WT1)。这种RNAi方法将是一项技术进步，因为到目前为止，还没有实验室报道开发出一种普遍适用的方法来在体内进行稳定的组织特异性RNAi。我的方法源于自然产生的短RNA-微RNA(MiRNAs)-从Pol II转录本产生的方法。在我的方法中，靶向Sox9的shRNA分子将由RhoxS PP驱动的前体转录本由DROSHA处理，DROSHA是一种普遍表达的RNase-111酶，可以处理出自然产生的miRNAs。由于Sox9 shRNA转录本将从RhoxS PP特异表达，我预测它将特异性地抑制Sox9在睾丸内的Sertoli细胞中的表达。我推测，Sox9在Sertoli细胞中发挥功能，指导精子发生过程中的特定事件。至少有两条证据支持我的假设：首先，Sox9以一种特定阶段的方式在成年Sertoli细胞中高度表达，这表明它可能在生殖细胞分化中发挥关键作用。其次，我最近发现，WT1在成年支持细胞中高表达，在Sox9等胚胎发生期间对性腺发育起关键作用，对维持精子发生也很重要。因此，我认为Sox9在精子发生中起重要作用。本研究的具体目的是：(1)利用组织特异性RNAi技术研究转录因子Sox9在成人支持细胞中的功能。阐明Sox9在出生后睾丸中的功能可能有助于更好地理解SOX9突变患者常见的性腺发育不全和性反转的病理生理学机制。此外，揭示Sox9在生殖中的作用可能会导致不孕症的治疗。

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.