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

通过 DND1 介导的表观遗传因子翻译调节雄性生殖细胞发育

• 批准号: 10748520
• 负责人: Talia Lee Hatkevich
• 金额: $ 7.18万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-01 至 2025-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10748520
• 关键词: Binding; Birth; Cell Reprogramming; Chromatin; Complex; Confocal Microscopy; Cytoplasm; Cytoplasmic Granules; DNMT3a; Data; Data Set; Development; Diffuse; Embryo; Ensure; Epigenetic Process; Exhibits; Fellowship; Fertility; Genetic Transcription; Genome; Germ; Goals; HDAC6 gene; Human; Infertility; KDM5B gene; Knowledge; Laboratories; Lead; Life; Locales; Longevity; Mediating; Mediator; Messenger RNA; Metabolism; Methylation; Methyltransferase; Microscopy; Modification; Molecular Profiling; Mus; Phase; Play; Post-Transcriptional Regulation; Proteins; Proteome; RNA immunoprecipitation sequencing; RNA-Binding Proteins; Regulation; Repression; Reproductive Health; Research; Research Personnel; Resolution; Role; SETDB1 gene; SMARCA4 gene; SMARCA5 gene; Scientific Advances and Accomplishments; Site; Spermatocytes; Stem Cell Development; Testing; Time; Training; Transcript; Translating; Translation Initiation; Translational Derepression; Translational Regulation; Translational Repression; Translations; Universities; Work; design; experimental study; fetal; histone modification; improved; insight; liquid chromatography mass spectrometry; male; novel; postnatal; posttranscriptional; precursor cell; programs; reproductive; self-renewal; skills; sperm cell; stem cell population; stem cells; tool; transcriptomics; translatome

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.

项目概要 雄性生殖细胞 (MGC) 是精原干细胞 (SSC) 的前体，精原干细胞是一种干细胞群， 自我更新和分化，为男性的整个生殖寿命提供精子。 胎儿期 MGC 发育中断会导致人类和小鼠不育。就在出生之前， MGC 经历相对较长的细胞静止期。 G0 停滞期在小鼠之间是保守的 和人类，被认为是 MGC 重编程阶段。在此期间，MGC 失去了早期 GC 的命运 并通过进行大量的表观遗传修饰来获取 SSC 的分子特征。虽然增加 各种表观遗传因子的mRNA水平发生在G0期间，目前尚不清楚表观遗传因子的转录如何 在整个 G0 期间进行转录后调节。 DND1 是所需的众多 RNA 结合蛋白 (RBP) 之一 对于 MGC 发育，它对于 G0 停滞和表观遗传调节因子的 RNA 表达至关重要 G0期间。 G0 期间 DND1 的转录目标包括重要的表观遗传调节因子。从功能上讲，DND1 可以 促进转录本降解、保护转录本免遭降解或促进其翻译。因为免打扰1 具有多种调节作用，目前尚不清楚 DND1 在转录后如何调节其目标转录本 G0 确保 MGC 到 SSC 的发展。在这个提案中，表观遗传因子的转录本是如何被后处理的？ MGC 中转录调节的 G0 将通过关注 DND1 的翻译作用来确定，如 有初步数据支持。中心假设是 DND1 的一个重要作用是控制何时 其目标表观遗传调节因子在 G0 期间被翻译以指导 MGC 的重编程 进入 SSC。为了检验这一假设，在目标 1 中，编码表观遗传调节因子的 DND1 结合转录本和 在 G0 期间翻译的内容将被定义。蛋白质组将通过 LC/MS-TOF 分两步测定 G0 阶段，并将与之前获得的 DND1 目标转录本在各自的位置进行交叉引用 阶段，创建 DND1 定向翻译组。这些翻译组中的表观遗传调节因子将被评估 整个 G0 的新生翻译以确定其时间翻译调节。在目标 2 中，它将是 确定了 DND1 在何处以及如何促进其目标转录本的翻译。 DND1 相互作用蛋白 将被公正地识别并使用各种高端显微镜工具，定位 DND1，其目标 转录本和翻译机制将被确定。这些目标提供的见解将提供 RBP 如何调节 MGCS 的表观遗传重编程以确保 发展南南合作中心。除了推进科学知识之外，该奖学金提案还表现出很高的水平 培训潜力。该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，这些将在杜克大学布兰奇·卡佩尔博士的实验室中实施。 总体而言，随附的研究策略和培训计划可以将实习生培养成独立的研究员 同时显着提高改善生殖健康的知识。