RNA Pol II Pausing is Critical for Spermatogenesis and Male Fertility

RNA Pol II 暂停对于精子发生和男性生育能力至关重要

基本信息

批准号：
9767846
负责人：
PRABHAKARA P REDDI
金额：
$ 33.4万
依托单位：
UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-08-21 至 2023-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9767846
关键词：
Address Affect Area Binding Sites Biochemical Candidate Disease Gene Cell Differentiation process Cells ChIP-seq Complex Couples DNA Polymerase II DNA-Directed RNA Polymerase Data Differentiation Antigens Disease Ensure Failure Fertility Gene Expression Gene Expression Regulation Genes Genetic Genetic Transcription Germ Cells Human Infertility Knock-out Knockout Mice Knowledge Link Male Infertility Maps Mass Spectrum Analysis Mediating Messenger RNA Modeling Molecular Profiling Morphogenesis Mus Mutation National Institute of Child Health and Human Development Nature Nuclear Proteins Outcome Phenotype Play Positive Transcriptional Elongation Factor B Post-Translational Protein Processing Process Production Proteins Publishing RNA Regulation Regulator Genes Reproductive Health Research Priority Role Signal Transduction Spermatids Spermatocytes Spermatogenesis Spermatogonia System Testing Testis Therapeutic Time Transcription Initiation Site Transcriptional Regulation Validation Work age group clinically significant cohort embryonic stem cell genome-wide human embryonic stem cell idiopathic infertility improved in vivo male male fertility men negative elongation factor novel nucleic acid binding protein premature promoter protein TDP-43 public health relevance recruit reproductive sertoli cell spatiotemporal sperm cell spermatogenic epithelium structure success transcriptome transcriptome sequencing

项目摘要

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.

项目摘要成功完成精子发生依赖于不同的时空表达分化标志物中的上皮。未能在正确时间表达基因导致被捕的精子发生和男性不育症。但是，转录机制调节了这一过程不太了解。我们的工作已经确定RNA pol II暂停对于保持精确精子发生过程中时空基因的表达。暂停的RNA Pol II在发起人确保精确和基因转录的快速发作。该机制与精子发生特别相关，其中基因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暂停在生殖细胞中发生全基因组，对精子发生的成功。我们已经将43 kD（TDP-43）的焦油DNA结合蛋白确定为关键参与者在雄性生殖细胞中的靶基因启动子维持暂停的POL II时。推论是TDP-43调节暂停生殖细胞中基因的子集。 TDP-43在进化上保守并在小鼠中表达人睾丸。我们发现TDP-43对于精子发生至关重要。 TDP-43的有条件淘汰生殖细胞导致了成熟的停滞和男性不育症。具体目标1将检验总体假设 Pol II暂停是通过确定全基因组占用率的时空基因转录的关键调节剂生殖细胞中的Pol II暂停机械，并寻求生化验证以暂停。 AIM 2将研究 Pol II在生殖细胞中暂停的机制；测试TDP-43将暂停机械招募到一个的假设启动子的子集，绘制其与暂停因素的相互作用，并确定功能意义。具体目标 3将检验以下假设：TDP-43的损失破坏了在TDP-43生殖细胞中暂停的POL II 导致男性不育症。在包括胚胎干细胞在内的人类细胞中，Pol II暂停已显示在基因转录调节中的关键作用。本研究很重要，因为这是第一次扩大有关调节男性生殖细胞转录组的机制的知识。结果将有一个专业影响对特发性男性不育症的遗传基础的理解。因此，拟议的研究是与NICHD的生育和不育（FI）分支确定的高优先级主题领域：遗传基础特发性不育症和不育模型。