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.

项目摘要/摘要 生殖细胞具有产生体内每种细胞类型的非凡潜力。但是，分子程序 这可以保护和自杀这种希望在生殖细胞中能付的诺言，这可以持续40多年 人类，知之甚少。为了面对这一挑战，种系调节器在维持胚芽中扮演双重角色 细胞身份：激活种系转录程序，同时保护生殖细胞免受 重新编程到体细胞命运。实际上，涉及种系规范的基因，例如配置 RNA结合蛋白Nanos和可转座元件（TE）调节剂PIWI被错误地更新 某些人类肿瘤。这些肿瘤被认为会经历soma to-germline转化以获取 更不朽的类系状状态。但是，生殖细胞转录激活的特定程序尚未 待描述，主要是因为缺少生殖细胞命运的“主调节转录因子”。这 提案使用多收益的方法系统地表征原始中的基因表达程序 生殖细胞（PGC）并鉴定生殖细胞命运的主要调节剂。目标是区分“默认” 模型，通过该模型，躯体程序的表达“允许” PGC程序和“启发性”模型。 主动控制哪种PGC规范。在AIM1中，我们探究了指导性模型以发现种系 转录程序。通过单细胞RNA测序，我们检测到了特定的，暂时调节的 种系基因。为了确定交易调节因素，我们将使用ATAC-severing，以表征已知 具有匹配结合活性的因素，并确定种系基因的潜在新调节候选。 AIM2，我们解决了生殖细胞命运（配置的RNA调节剂纳米）的关键调节剂的功能。使用 RNA靶标识别方法高部位和核糖体分析，我们将确定纳米靶标和 在促进种系程序和/或反映躯体的那些目标之间有功能区分 程序。 AIM3专注于PIRNA途径，该途径主要提供针对TE活动的免疫力 通过控制TE元素的转录，特别是在生殖细胞中。使用新颖的退化策略 特别降低了PGC中piRNA途径的成分而不会影响卵子发生，我们将确定 该途径在超出控制之外的胚胎PGC基因表达的作用。通过整合多重发现， 该提案将揭示早期生殖细胞的调节景观，从而提供必要的 了解性腺发生过程，最终是该物种的生存。