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

项目总结 男性生殖细胞(MGCs)是精原干细胞(SSCs)的前体，SSCs是一种干细胞群体，既 自我更新和分化，为男性整个生殖寿命提供精子。 胎儿期mGCS发育受阻会导致人类和小鼠不孕。就在出生之前， MGC经历了相对较长的细胞静止期。这种G0停滞期在小鼠之间是保守的 和人类，被认为是MGC重新编程的阶段。在此期间，MGC失去了早期的GC命运 并通过进行大量的表观遗传修饰来获得SSCs的分子图谱。尽管增加了 各种表观遗传因子的mRNA水平在G0期间发生，尚不清楚表观遗传因子的转录本是如何 在整个G0中都受到转录后调控。DND1是许多必需的RNA结合蛋白(RBPs)之一 对于MGC的发育，它对G0停滞和表观遗传调节因子的RNA表达都是必不可少的 在G0期间。DND1在G0期间的转录靶标包括重要的表观遗传调控因子。在功能上，DND1可以 促进文字记录的降级，保护记录不被降级，或促进其翻译。因为DND1 具有多种调节作用，目前尚不清楚DND1在转录后如何调节其靶向转录本 G0，以确保MGC到SSC的发展。在这项提案中，表观遗传因子的转录是如何后- MGCS中转录调控的G0将通过关注DND1的翻译作用来确定，AS 有初步数据支持。核心假设是DND1的一个基本作用是控制何时 并在G0期间翻译其目标表观遗传调控因子以指导mGCS的重新编程 变成了SSCs。为了验证这一假设，在目标1中，DND1结合的转录本编码表观遗传调节因子和 在G0期间翻译的将被定义。蛋白质组将通过LC/MS-TOF分两部分进行测定 G0的阶段，并将与之前获得的DND1目标转录本在各自的 阶段，产生DND1导向的翻译体。将对这些翻译体中的表观遗传调控因子进行评估 整个G0的新生翻译，以确定他们的时间翻译调节。在《目标2》中，它将是 确定了DND1在哪里以及如何促进其目标转录的翻译。DND1相互作用蛋白 将不偏不倚地鉴定并使用各种高端显微镜工具，定位DND1，其靶标 成绩单和翻译机器将被确定。这些目标提供的洞察力将提供 RBP如何调节mGC的表观遗传重编程以确保 SSCS的发展。除了促进科学知识，这项奖学金提案还显示出很高的 培训潜力。培训计划描述了一项为期两年的蓝图，旨在加强技术、专业、 和概念技能，将在杜克大学布兰奇·卡佩尔博士的实验室实施。 总体而言，所附研究战略和培训计划可以将受训人员培养为独立研究人员 同时极大地促进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.