RNA Pol II Pausing is Critical for Spermatogenesis and Male Fertility

项目概要 精子发生的成功完成依赖于不同子集的精确时空表达 生精上皮内的分化标记。未能在正确的时间表达基因会导致 抑制精子发生和男性不育。然而，调节这一过程的转录机制 没有被很好地理解。我们的工作已证实 RNA Pol II 暂停对于维持精确度至关重要 精子发生过程中的时空基因表达。在启动子处暂停 RNA Pol II 确保精确 和基因转录的快速开始。该机制与精子发生特别相关，其中 基因组的同步转录对于形态发生和分化至关重要。中央 该提议的假设是，RNA Pol II 暂停发生在生殖细胞的全基因组范围内，并且对于 精子发生的成功。我们已经确定 43 kD 的 TAR DNA 结合蛋白 (TDP-43) 是一个关键参与者 维持雄性生殖细胞中靶基因启动子处暂停的 Pol II。推论是 TDP-43 调节 生殖细胞中一部分基因的暂停。 TDP-43 在进化上是保守的，并在小鼠中表达 人类睾丸。我们发现TDP-43对于精子发生至关重要；条件性敲除 TDP-43 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，通过确定全基因组占用率 Pol II 暂停生殖细胞中的机制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；检验 TDP-43 将暂停机制招募到 启动子子集，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 将测试以下假设：TDP-43 的丢失会破坏生殖细胞中 TDP-43 目标启动子处的 Pol II 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 暂停已被证明发挥作用 在基因转录调控中发挥关键作用。本研究意义重大，因为这是第一次 扩展有关调节雄性生殖细胞转录组的机制的知识。结果将产生重大影响 影响对特发性男性不育遗传基础的理解。因此，拟议的研究是 与 NICHD 生育与不孕 (FI) 部门确定的高度优先主题领域保持一致：遗传基础 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.