Transcriptional Regulation During Spermiogenesis

描述(申请人提供)：在精子发生过程中，精子细胞获得顶体和鞭毛，浓缩其染色质，成为精子。精子细胞分化标志物的精确表达对精子的正常形成至关重要。我们研究的总体目标是了解精子发生的转录调控机制。编码顶体蛋白SP-10的基因作为实验模型。启动子分析表明，SP-10基因的近端启动子足以在精子细胞中转录激活。令人惊讶的是，同样的启动子通过充当绝缘体在所有其他细胞中引起转录抑制。中心假设是，睾丸特异性转录激活因子和普遍表达的转录抑制因子的协同作用决定了精子细胞分化标记的精确阶段和细胞特异性表达，而生殖细胞特异性基因在近端启动子中保留了必要的顺式元件。这一建议将检验这样的假设，即在体内，SP-10近端启动子中的特定顺式元件介导精子细胞的增强子功能和所有其他细胞的绝缘/抑制功能，以及同源转录激活因子和抑制因子交替启动分别诱导SP-10基因转录的开启和关闭状态。 这项研究将集中在-186/28 SP-10启动子，它同时显示了增强子和绝缘体的活性，并鉴定了介导这些功能的转录因子。TNP47是一个47kD的睾丸核蛋白，在体外与-186/-148区特异性结合，将用DNA亲和法克隆并确定其在SP-10转录中的作用(目标1)。利用-186/-148 DNA克隆的转录抑制因子TDP43和PURpha是否介导SP-10的绝缘体功能将通过共转染和RNAi来确定，它们在体内的启动子占有率将由染色质IP(Aim 2)来确定。假设TNP47、TDP43和PURpha的协调相互作用足以确保精子细胞特异性转录将在转基因小鼠中使用其天然(SP-10)和异源核心启动子(AIM 3)的-186/-148调节启动子进行测试。这些结果将列举精子发生的分子机制。这项研究与男性生殖健康有关，因为铅可以应用于开发新的男性避孕药。SP-10启动子将用于治疗不育症或生殖细胞缺陷的基因治疗。

DESCRIPTION (provided by applicant): During spermiogenesis, spermatids acquire the acrosome and flagellum, condense their chromatin and become spermatozoa. Precise expression of spermatid differentiation markers is critical for proper formation of sperm. The overall goal of our research is to understand the mechanisms of transcriptional regulation of spermiogenesis. The gene encoding the acrosomal protein SP-10 serves as an experimental model. Promoter analysis showed that the proximal promoter of the SP-10 gene was sufficient for transcriptional activation in spermatids. Surprisingly, the same promoter caused transcriptional repression in all other cells by acting as an insulator. The central hypothesis is that coordinated interplay of testis-specific transcriptional activators and ubiquitously expressed transcriptional repressors dictates the precise stage- and cell-specific expression of spermatid differentiation markers and that the germ cell-specific genes retain the necessary cis-elements in the proximal promoters. This proposal will test the hypothesis that in vivo, specific cis elements in the SP-10 proximal promoter mediate the enhancer function in spermatids and insulator / repressor function in all other cells, and that the cognate transcriptional activators and repressors alternate promoter occupancy to induce ON and OFF states of SP-10 gene transcription, respectively. The study will focus on the -186/+28 SP-10 promoter, which showed both enhancer and insulator activities, and characterize transcription factors mediating these functions. TNP47, a 47kD testis nuclear protein, which specifically binds the -186/-148 region in vitro, will be cloned by DNA affinity method and its role in SP-10 transcription determined (Aim 1). Whether TDP43 and PURalpha, transcriptional repressors cloned using the -186/-148 DNA, mediate the SP-10 insulator function will be determined by cotransfections and RNAi, their promoter occupancy in vivo will be determined by chromatin IP (Aim 2). The hypothesis that the coordinated interplay of TNP47, TDP43 and PURalpha is sufficient to ensure spermatid-specific transcription will be tested in transgenic mice using the -186/-148 regulatory promoter in the context of its native (SP-10) as well as heterologous core promoters (Aim 3). The results will enumerate molecular mechanisms underlying spermiogenesis. The study is relevant to male reproductive health as leads can be applied to develop novel male contraceptives. The SP-10 promoter will be useful for gene therapy to treat infertility or germ cell defects.