S-SOX5对运动纤毛基因的转录调节及对精子鞭毛结构和功能的影响

The axoneme of motile cilia is composed of a "9+2" array, which includes nine peripheral doublet microtubules and two central microtubules. Defect in motile cilia gives rise to primary ciliary dyskinesia (PCD),such as male infertility, and infection in upper respiratory tract.The mechanism of PCD is a research focus of foundation study on motile cilia. SOX5 gene transcribes two major isoforms, the full length L-SOX5 and shorter length S-SOX5, in which S-SOX5 has one unique non-coding exon, and it is highly expressed in the tissues with motile cilia.It has been shown that several sperm associated antigens,including Spag6, Spag16L,and Spag17, regulate motile ciliary function by forming a network, and these genes have multiple SOX5 binding sites in their promoter regions.It has been confirmed that S-SOX5 could regulate Spag6 transcription by binding directly to its promoter in our previous study, indicating that S-SOX5 is a potentially transcriptional factor to regulate motile ciliary function. The purpose of this project is to study the role of S-SOX5 on transcriptional regulation of SPAG16L and SPAG17 ,and its effect on sperm flagellar structure and function in vitro and in vivo by comprehensive techniques, such as RNAi,and gene targeting. This study will dissect a common regulatory mechanism of motile cilia genes, and provide foundation to treat ciliopathy and male infertility.

运动纤毛轴突为"9+2"结构(9个双微管+2个中央微管)，其功能障碍可引起纤毛不动综合症(PCD)，表现为男性不育、呼吸系统炎症等，引起PCD的机制研究是目前运动纤毛基础研究的热点。SOX5主要转录全长L-SOX5和较短S-SOX5，其中S-SOX5有一个独特的非编码蛋白外显子，主要在含运动纤毛的组织表达。研究表明精子相关抗原6(SPAG6)、SPAG16L、SPAG17等可形成网络共同调节运动纤毛功能，且这些基因启动子均含有转录因子SOX5的多个结合位点。我们前期研究证明S-SOX5可转录调节SPAG6，提示S-SOX5可能是调节运动纤毛基因的转录因子。本研究将在细胞和小鼠整体水平，结合RNA干扰、基因敲除等手段全面而系统研究S-SOX5对SPAG16L、SPAG17的转录调节及对精子鞭毛结构及功能的影响，从而揭示运动纤毛基因的一种共同转录机制，为PCD特别是男性不育防治提供科学依据。

精子相关抗原（Sperm associated antigens,SPAG）是一组主要在睾丸/精子高度表达的蛋白。其中，SPAG6、SPAG16、SPAG17 同时位于纤毛/精子鞭毛的一对中心微管上，形成一个网络共同动态调节纤毛和鞭毛运动。SOX蛋白是一组拥有一个或多个DNA结合结构域即HMG DNA结合区域的蛋白家族，它们能通过HMG结构域与DNA结合。SOX 家族分为10 组，其中，SOX5 是SOXD 亚家族的一员。小鼠Sox5 有两个转录子：一个短的转录子 (2 kb)命名为S-SOX5，仅在睾丸、脑、支气管/肺等具有运动纤毛的组织表达；另一个长的转录子 (6 kb)在其它组织表达。S-SOX5与运动纤毛有着非常密切的关系，已被证实可与SPAG6启动子直接结合进行转录调节，鉴于SPAG6、SPAG16L、SPAG17的启动子有相似的SOX5的转录结合位点，本研究主要探讨S-SOX5转录通过调节SPAG16L、SPAG17表达，并在体内影响精子鞭毛结构形成，进而影响精子发生过程。本研究计划包括：①转录因子S-SOX5通过直接与运动纤毛基因SPAG16L/SPAG17的启动子区域结合，调控其表达；②S-SOX5改变精子鞭毛结构，降低小鼠精子运动功能。结果发现，当S-SOX5与人类SPAG16L质粒共转染到BEAS-2B细胞后，荧光素酶标记的SPAG16L启动子活性增加了将近100倍，说明S-SOX5可以激活SPAG16L的启动子。腺病毒感染实验证实，Ad/S-SOX5可使细胞的SPAG16L的mRNA水平显著增加，表明外源性S-SOX5上调SPAG16L的mRNA水平。RNAi干扰实验证实S-SOX5被抑制后，SPAG16L的mRNA水平显著降低，特别是RNAi（1109-1130）质粒组更为明显。染色体免疫共沉淀实验表明S-SOX5直接结合在SPAG16L的启动子区域。当启动子区域SOX5结合位点突变后，S-SOX5不再能激活SPAG16L启动子活性。体外细胞实验已证实S-SOX5可通过直接与SPAG16L结合发挥转录调节作用。此外，人类SPAG17启动区多种质粒构建已完成，正在进行以上实验证实S-Sox5对SPAG17的转录调节作用；用CRISPER-Cas9系统建立生精细胞特异性S-Sox5基因敲除小鼠正在进行中，约2个月后可获得小鼠，进行后续功能性实验。