RNA Pol II Pausing is Critical for Spermatogenesis and Male Fertility

项目总结 精子发生的成功完成依赖于不同亚型的精确时空表达 生精上皮内的分化标志物。未能在正确的时间表达基因会导致 精子发生受阻和男性不育。然而，调控这一过程的转录机制， 都没有被很好地理解。我们的工作已经确定，RNA Pol II的停顿对于保持准确 精子发生过程中的时空基因表达。在启动子上暂停的RNA POL II确保准确 以及基因转录的快速启动。这种机制与精子发生特别相关，在这种情况下 一组基因的同步转录对形态发生和分化至关重要。中环 这一提议的假设是，RNA Pol II暂停发生在整个基因组的生殖细胞中，这对 精子发生的成功。我们已经确定43kD的TAR DNA结合蛋白(TDP-43)是一个关键角色 在雄性生殖细胞的靶基因启动子上维持暂停的POL II。推论是TDP-43调节 生殖细胞中基因子集的暂停。TDP-43在进化上是保守的，并在小鼠和 人类的睾丸。我们已经发现TDP-43对精子发生是必不可少的；条件基因敲除TDP-43在 生殖细胞导致成熟停滞和男性不育。具体目标1将检验压倒一切的假设 POL II停顿是决定全基因组占有率的时空基因转录的关键调节因子 Pol II暂停生殖细胞中的机械，并寻求暂停的生化验证。目标2将研究 生殖细胞中Pol II暂停的机制；验证TDP-43将暂停机制招募到 启动子的子集，映射其与停顿因子的相互作用，并确定功能意义。特定目标 3将验证假设，即TDP-43的丢失扰乱了生殖细胞中Pol II在TDP-43靶向启动子处的暂停 导致男性不育。在包括胚胎干细胞在内的人类细胞中，Pol II停顿已被证明发挥作用 在基因转录调控中起着关键作用。这项研究意义重大，因为它将第一次 扩大关于男性生殖细胞转录组调控机制的知识。结果将会有一个重大的 对特发性男性不育遗传学基础认识的影响。因此，拟议的研究是 与NICHD生育和不孕处确定的高度优先主题领域保持一致：遗传学基础 特发性不孕症和不孕症模型。

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.