Regulation of Male Germ Cell Development through DND1-Mediated Translation of Epigenetic Factors

项目摘要 雄性生殖细胞（MGCs）是精原干细胞（SSCs）的前体细胞，精原干细胞是一个干细胞群， 自我更新和分化，为男性的整个生殖寿命提供精子。 胎儿期MGCs发育中断导致人类和小鼠不育。就在出生前， MGCs经历相对较长的细胞静止期。这种G 0停滞期在小鼠之间是保守的 这被认为是MGC重新编程的阶段。在此期间，MGCs失去其早期GC命运 并通过大量的表观遗传修饰获得精原干细胞的分子特征。虽然增加了 各种表观遗传因子的mRNA水平发生在G 0期，目前尚不清楚表观遗传因子的转录物是如何在G 0期表达的。 在G 0期受到转录后调控。DND 1是许多RNA结合蛋白（RBP）之一， 对于MGC的发展，并且对于G 0阻滞和表观遗传调节因子的RNA表达都是必不可少的 在G 0期间。G 0期DND 1的转录靶点包括重要的表观遗传调节因子。在功能上，DND 1可以 促进转录物降解、保护转录物免于降解或促进其翻译。因为DND 1 DND 1具有多种调节作用，但目前尚不清楚DND 1在转录后如何调节其靶转录物。 G 0，以确保MGC-to-SSC的发展。在这项提议中，表观遗传因子的转录本是如何在 MGCs中转录调控的G 0将通过关注DND 1的翻译作用来确定， 有初步数据支持。核心假设是DND 1的一个重要作用是控制何时 在G 0期，其靶表观遗传调节因子被翻译，以指导MGCs的重编程 进入SSC。为了验证这一假设，在Aim 1中，DND 1结合的转录本编码表观遗传调节因子， 将定义在G 0期间转换的。蛋白质组将通过LC/MS-TOF在两个 G 0阶段，并将交叉参考先前获得的DND 1-靶转录物， 阶段，创建DND 1定向的翻译组。将评估这些翻译组中的表观遗传调节因子， 在整个G 0的新生翻译，以确定它们的时间翻译调节。在目标2中， 确定DND 1在何处以及如何促进其靶转录物的翻译。DND 1相互作用蛋白 将无偏见地确定，并使用各种高端显微镜工具，DND 1的定位，其目标 转录本和翻译机器将被确定。这些目标所提供的洞察力将提供一个 RBP如何调节MGCS的表观遗传重编程以确保 发展SSC。除了推进科学知识，这项奖学金提案还表现出很高的 训练潜力。培训计划描述了一个为期两年的蓝图，旨在加强技术，专业， 和概念技能，这将在杜克大学的布兰奇卡佩尔博士的实验室实施。 整体而言，封闭式的研究策略与训练计画，能将受训者培养成独立的研究者 同时大大提高了改善生殖健康的知识。

PROJECT SUMMARY Male germ cells (MGCs) are precursors to spermatogonial stem cells (SSCs), a stem cell population that both self-renews and differentiates, supplying spermatozoa for the entirety of a male’s reproductive lifespan. Disruptions in development of MGCs during fetal life leads to infertility in humans and mice. Just prior to birth, MGCs undergo a relatively long period of cellular quiescence. This G0 arrest phase is conserved between mice and humans and is considered an MGC reprogramming stage. During this time, MGCs lose their early GC fate and acquire the molecular profile of SSCs by undergoing vast epigenetic modifications. Although increases in mRNA levels of various epigenetic factors occur during G0, it is unclear how the transcripts of epigenetic factors are regulated post-transcriptionally throughout G0. DND1 is one of many RNA-binding proteins (RBPs) required for MGC development, and it is essential for both G0 arrest and for RNA expression of epigenetic regulators during G0. Transcript targets of DND1 during G0 include vital epigenetic regulators. Functionally, DND1 can promote transcript degradation, protect transcripts from degradation, or promote their translation. Because DND1 has multiple regulatory roles, it is unknown how DND1 post-transcriptionally regulates its target transcripts during G0 to ensure MGC-to-SSC development. In this proposal, how transcripts of epigenetic factors are post- transcriptionally regulated G0 in MGCs will be determined by focusing on the translational role of DND1, as supported by preliminary data. The central hypothesis is that an essential role of DND1 is to control when and where its target epigenetic regulators are translated during G0 to direct the reprogramming of MGCs into SSCs. To test this hypothesis, in Aim 1, DND1-bound transcripts that encode for epigenetic regulators and that are translated during G0 will be defined. The proteomes will be determined through LC/MS-TOF in two stages of G0 and will be cross-referenced to previously obtained DND1-target transcripts at their respective stages, creating DND1-directed translatomes. Epigenetic regulators in these translatomes will be assessed for nascent translation throughout G0 to determine their temporal translational regulation. In Aim 2, it will be determined where and how DND1 promotes the translation of its target transcripts. DND1-interacting proteins will be unbiasedly identified and using a variety of high-end microscopy tools, the localization of DND1, its target transcripts, and translational machinery will be determined. The insight provided by these aims will provide a novel paradigm for how an RBP regulates the epigenetic reprogramming of MGCS to ensure the development of SSCs. In addition to advancing scientific knowledge, this fellowship proposal also exhibits high training potential. The training plan describes a two-year blueprint designed to strengthen technical, professional, and conceptual skills, which will be implemented at Duke University in the laboratory of Dr. Blanche Capel. Overall, the enclosed research strategy and training plan can develop a trainee into an independent researcher while significantly advancing knowledge for improving reproductive health.