RNA Pol II Pausing is Critical for Spermatogenesis and Male Fertility

项目摘要 精子发生的成功完成依赖于不同亚群的精确时空表达， 生精上皮内的分化标志物。如果不能在正确的时间表达基因， 精子生成受阻和男性不育然而，调控这一过程的转录机制， 并没有得到很好的理解。我们的工作已经确定，RNA Pol II暂停对于维持精确的 精子发生过程中的时空基因表达。在启动子处暂停的RNA Pol II确保了精确的 和基因转录的快速启动。该机制特别与精子发生相关，其中 基因群的同步转录对于形态发生和分化是至关重要的。中央 该建议的假设是RNA Pol II暂停发生在生殖细胞的全基因组范围内，并且对于生殖细胞的发育至关重要。 精子发生的成功。我们已经确定了43 kD的TAR DNA结合蛋白（TDP-43）作为关键参与者 在雄性生殖细胞中维持暂停的Pol II在靶基因启动子处。一个推论是TDP-43调节 在生殖细胞中的一个基因子集的暂停。TDP-43在进化上是保守的，并在小鼠中表达， 人类睾丸我们已经发现TDP-43对精子发生是必需的; 生殖细胞导致成熟停滞和男性不育。具体目标1将检验总体假设， Pol II暂停是时空基因转录的关键调节因子，通过决定全基因组的占据率 的Pol II暂停机制在生殖细胞，并寻求暂停的生化验证。目标2将研究 Pol II在生殖细胞中暂停的机制;检验TDP-43招募暂停机制以使其在生殖细胞中暂停的假设。 启动子的子集，绘制其与暂停因子的相互作用并确定功能意义。具体目标 3将检验TDP-43的缺失破坏Pol II在生殖细胞中TDP-43靶启动子处的暂停的假设 导致男性不育。在包括胚胎干细胞在内的人类细胞中， 在基因转录调控中起着关键作用。这项研究意义重大，因为它将首次 扩大对男性生殖细胞转录组调节机制的了解。结果将有一个重大的 对理解特发性男性不育症的遗传基础的影响。因此，拟议的研究是 与NICHD生育和不育（FI）分支确定的高优先主题领域一致：遗传基础 和不孕症模型。

PROJECT SUMMARY Successful completion of spermatogenesis relies upon precise spatiotemporal expression of distinct subsets of differentiation markers within the seminiferous epithelium. Failure to express genes at the correct time leads to arrested spermatogenesis and male infertility. The transcriptional mechanisms regulating this process, however, are not well understood. Our work has established that RNA Pol II pausing is critical for maintaining precise spatiotemporal gene expression during spermatogenesis. Paused RNA Pol II at the promoter ensures precise and rapid onset of gene transcription. This mechanism is particularly relevant to spermatogenesis wherein synchronous transcription of cohorts of genes is critical for morphogenesis and differentiation. The central hypothesis of this proposal is that RNA Pol II pausing occurs genome-wide in germ cells and is critical for the success of spermatogenesis. We have identified the TAR DNA binding protein of 43 kD (TDP-43) as a key player in maintaining paused Pol II at a target gene promoter in male germ cells. A corollary is that TDP-43 regulates pausing of a subset of genes in germ cells. TDP-43 is evolutionarily conserved and expressed in the mouse and human testis. We have found that TDP-43 is essential for spermatogenesis; conditional knockout of TDP-43 in germ cells led to maturation arrest and male infertility. Specific Aim 1 will test the overarching hypothesis that Pol II pausing is a key regulator of spatiotemporal gene transcription by determining the genome-wide occupancy of Pol II pause machinery in germ cells and seek biochemical validation for pausing. Aim 2 will study the mechanism of Pol II pausing in germ cells; test the hypothesis that TDP-43 recruits the pause machinery to a subset of promoters, map its interactions with pause factors and determine functional significance. Specific Aim 3 will test the hypothesis that loss of TDP-43 disrupts Pol II pausing at TDP-43 target promoters in germ cells leading to male infertility. In human cells including embryonic stem cells, Pol II pausing has been shown to play a pivotal role in regulation of gene transcription. The present study is significant because for the first time it will expand knowledge on a mechanism regulating the male germ cell transcriptome. The outcome will have a major impact on the understanding of the genetic basis of idiopathic male infertility. Thus, the proposed studies are aligned with high priority topic areas identified by the Fertility and Infertility (FI) Branch of NICHD: Genetic basis of idiopathic infertility and Models for infertility.