Protecting and Sustaining Germ Cell Identity

保护和维持生殖细胞身份

• 批准号: 10736134
• 负责人: RUTH LEHMANN
• 金额: $ 41.93万
• 依托单位: WHITEHEAD INSTITUTE FOR BIOMEDICAL RES
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-11 至 2028-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10736134
• 关键词: ATAC-seq; Address; Binding; Binding Sites; Biological Assay; Cell Reprogramming; Cells; Chromatin; Chromatin Remodeling Factor; DNA Transposable Elements; DNA damage checkpoint; Deposition; Development; Elements; Embryo; Embryonic Development; Enzymes; Face; Gene Activation; Gene Expression; Gene Expression Regulation; Genes; Genetic; Genetic Transcription; Genome; Germ; Germ Cells; Goals; Human; Immunity; Inherited; Longevity; Mediating; Methods; Modeling; Molecular; Mus; Mutation; Oogenesis; Pathway interactions; Phenotype; Process; Proteins; RNA; RNA Editing; RNA-Binding Proteins; Regulation; Regulatory Pathway; Reporter; Repression; Role; Site; Small RNA; Somatic Cell; Specific qualifier value; Structure of primordial sex cell; System; Testing; Totipotency; Trans-Activators; Transact; Transcriptional Activation; Transcriptional Regulation; Translational Repression; Translations; Validation; candidate identification; cell type; egg; expectation; experimental study; fly; member; mutant; nano; nanobodies; neuronal cell body; next generation; novel; permissiveness; piRNA; preempt; premature; prevent; programs; reproductive; ribosome profiling; single-cell RNA sequencing; sperm cell; transcription factor; transmission process; tumor

Project Summary/Abstract Germ cells have the extraordinary potential to generate every cell type in the body. Yet, the molecular program that protects and sustains this promise for totipotency in germ cells, which can last more than forty years in humans, is poorly understood. To face this challenge, germline regulators assume dual roles in maintaining germ cell identity: activating a germline transcriptional program while simultaneously protecting germ cells from reprogramming to a somatic cell fate. Indeed, genes involved in germline specification, such as the conserved RNA-binding protein Nanos and the transposable element (TE) regulator Piwi, are erroneously upregulated in certain human tumors. These tumors are thought to undergo soma-to-germline transformations to acquire a more immortal, germline-like state. However, a specific program for germ cell transcriptional activation has yet to be described, mainly because a ‘master-regulator transcription factor’ for germ cell fate is missing. This proposal uses a multipronged approach to systematically characterize the gene expression program in primordial germ cells (PGCs) and identify prime regulators of germ cell fate. The goal is to distinguish between a ‘default’ model, by which repression of the somatic program ‘allows’ the PGC program, and an ‘instructive’ model, by which PGC specification is actively controlled. In Aim1, we probe the instructive model to uncover the germline transcriptional program. By single-cell RNA sequencing, we have detected specific, temporally-regulated germline genes. To identify the transacting regulatory factors, we will use ATAC-sequencing, characterize known factors with matching binding activities, and identify potential new regulatory candidates for germline genes. In Aim2, we address the function of a critical regulator of germ cell fate, the conserved RNA regulator Nanos. Using the RNA target identification method hyper-TRIBE and ribosome profiling, we will identify Nanos targets and functionally distinguish between those targets that promote the germline program and/or repress the somatic program. Aim3 focuses on the piRNA pathway, which provides maternally inherited immunity against TE activity by controlling the transcription of TE elements, specifically in germ cells. Using a novel degradation strategy to specifically degrade components of the piRNA pathway in PGCs without impacting oogenesis, we will determine the role of this pathway for embryonic PGC gene expression beyond TE control. By integrating multiplex findings, this proposal will uncover the regulatory landscape of early germ cells, thereby providing a necessary understanding of the process of gonadogenesis and, ultimately, the survival of the species.

项目总结/摘要 生殖细胞具有产生体内每种细胞类型的非凡潜力。然而，分子程序 保护和维持生殖细胞全能性的承诺，这种承诺可以持续四十多年， 人，知之甚少。为了应对这一挑战，生殖系调节因子在维持生殖系统中扮演着双重角色。 细胞身份：激活生殖系转录程序，同时保护生殖细胞免受 重编程为体细胞的命运事实上，涉及生殖细胞特化的基因，如保守的 RNA结合蛋白Nanos和转座因子（TE）调节因子Piwi在哺乳动物中被错误地上调， 某些人类肿瘤这些肿瘤被认为经历了体细胞到生殖细胞的转化，以获得一种新的细胞因子。 更加不朽的、类似生殖细胞的状态。然而，生殖细胞转录激活的特定程序还没有 这主要是因为缺少了一个控制生殖细胞命运的“主调节转录因子”。这 该提案使用多管齐下的方法系统地表征原始细胞中的基因表达程序， 生殖细胞（PGCs），并确定生殖细胞命运的主要调节因子。我们的目标是区分“违约” 模型，通过抑制体细胞程序“允许”PGC程序，和一个“指导性”模型，通过 主动控制PGC规范。在目标1中，我们探索了揭示生殖系的指导性模型 转录程序通过单细胞RNA测序，我们已经检测到特异性的，时间调节的 生殖系基因为了鉴定反式调节因子，我们将使用ATAC测序，表征已知的 因子与匹配的结合活性，并确定潜在的新的调控候选人的生殖系基因。在 目的2，我们解决生殖细胞命运的关键调节器，保守的RNA调节Nanos的功能。使用 RNA靶向鉴定方法hyper-TRIBE和核糖体分析，我们将鉴定Nanos靶点， 在功能上区分促进种系程序和/或抑制体细胞程序的那些靶标， 程序. Aim 3专注于皮尔纳途径，其提供针对TE活性的母系遗传免疫 通过控制TE元件的转录，特别是在生殖细胞中。使用新的降解策略， 特异性降解PGC中皮尔纳途径的组分而不影响卵子发生，我们将确定 这一途径在胚胎PGC基因表达中的作用超出TE控制。通过整合多重发现， 这项提议将揭示早期生殖细胞的调控前景，从而提供必要的 了解性腺发生的过程，最终，物种的生存。