Transcriptional Regulation During Spermiogenesis

描述（由申请人提供）：在精子发生过程中，精子细胞获得顶体和鞭毛，浓缩其染色质并成为精子。精子细胞分化标记物的精确表达对于精子的正确形成至关重要。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进行转录激活。令人惊讶的是，相同的启动子通过充当绝缘体而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调相互作用决定了精细胞分化标记物的精确阶段和细胞特异性表达，并且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。该提案将测试以下假设：在体内，SP-10近端启动子中的特定顺式元件介导精子细胞中的增强子功能和所有其他细胞中的绝缘子/阻遏子功能，并且同源转录激活子和阻遏子交替占据启动子以分别诱导SP-10基因转录的ON和OFF状态。 该研究将重点关注 -186/ 28 SP-10 启动子，该启动子显示出增强子和绝缘子活性，并表征介导这些功能的转录因子。 TNP47 是一种 47kD 睾丸核蛋白，在体外特异性结合 -186/-148 区域，将通过 DNA 亲和方法克隆并确定其在 SP-10 转录中的作用（目标 1）。使用 -186/-148 DNA 克隆的转录抑制因子 TDP43 和 PURα 是否介导 SP-10 绝缘子功能将通过共转染和 RNAi 确定，它们在体内的启动子占据情况将通过染色质 IP 确定（目标 2）。 TNP47、TDP43 和 PURα 的协调相互作用足以确保精子细胞特异性转录的假设将在转基因小鼠中使用 -186/-148 调节启动子在其天然 (SP-10) 以及异源核心启动子的背景下进行测试 (目标 3)。结果将列举精子发生的分子机制。该研究与男性生殖健康相关，因为线索可用于开发新型男性避孕药。 SP-10启动子将可用于治疗不孕症或生殖细胞缺陷的基因疗法。

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.