Transcriptional Regulation During Spermiogenesis

精子发生过程中的转录调控

• 批准号: 6773455
• 负责人: PRABHAKARA P REDDI
• 金额: $ 30.44万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-12-08 至 2009-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6773455
• 关键词: DNA binding protein; RNA interference; affinity chromatography; angiogenesis inhibitors; chromatin immunoprecipitation; gene expression; gene induction /repression; genetic promoter element; genetic regulatory element; genetically modified animals; immunoprecipitation; laboratory mouse; mass spectrometry; molecular genetics; nuclear proteins; spermatogenesis; spermatogenesis inhibitor; transcription factor; transfection

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.

描述（由申请人提供）：在精子发生过程中，精子获得顶体和鞭毛，染色质凝聚而成为精子。精细胞分化标记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的基因可作为实验模型。启动子分析表明，SP-10基因的近端启动子足以在精子中进行转录激活。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。核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志物的精确阶段和细胞特异性表达，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。本研究将验证以下假设：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/抑制子功能，同源转录激活子和抑制子交替占据启动子，分别诱导SP-10基因转录的ON和OFF状态。