Epigenetic Regulation in Self-Renewing and Differentiating Male Germ Cells

雄性生殖细胞自我更新和分化的表观遗传调控

• 批准号: 7385698
• 负责人: CHRISTOPHER JESS PAYNE
• 金额: $ 8.63万
• 依托单位: JACKSON LABORATORY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-04-01 至 2010-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7385698
• 关键词: Address; Adult; Affect; Alleles; Award; Binding Sites; Biological; Candidate Disease Gene; Cell Differentiation process; Cell Line; Cells; Chromatin; Chromatin Remodeling Factor; Chromatin Structure; Collaborations; Complex; Condition; Coupled; Detergents; Development; Environment; Epigenetic Process; Equilibrium; Faculty; Gene Expression; Gene Expression Microarray Analysis; Gene Expression Profile; Gene Silencing; Generations; Genes; Genetic; Germ Cells; Goals; Heterochromatin; Histone H3; Histones; Infertility; Investigation; Knockout Mice; Laboratories; Letters; Link; Liquid Chromatography; Localized; Lysine; Maintenance; Malignant Neoplasms; Mass Spectrum Analysis; Mentors; Methods; Methylation; Modeling; Modification; Molecular; Mus; Mutant Strains Mice; Nuclear; Nuclear Matrix; Nuclear Matrix-Associated Proteins; Nuclear Protein; Nuclear Proteins; Nuclear Structure; Operative Surgical Procedures; Pattern; Phase; Play; Polycomb; Population; Positioning Attribute; Process; Protein Sortings; Proteins; Proteome; Proteomics; Publishing; RNA Interference; Regenerative Medicine; Regulation; Regulator Genes; Reporter; Reporting; Repression; Reproduction; Research; Research Personnel; Research Project Grants; Research Training; Resources; Role; Small Interfering RNA; Sorting - Cell Movement; Spermatogonia; Stem cells; Testing; Testis; Training; Transcription Repressor/Corepressor; Transgenic Mice; Transplantation; Undifferentiated; United States National Institutes of Health; Universities; Washington; cell type; chromatin immunoprecipitation; design; embryonic stem cell; histone methyltransferase; interest; liquid chromatography mass spectrometry; loss of function; male; member; mutant; reproductive; research study; self-renewal; skills

DESCRIPTION (provided by applicant): Maintenance of the adult male germline is essential for reproduction. Still a poorly understood process, germline stem cell self-renewal appears to be influenced by epigenetic chromatin modifications and subsequent nuclear organization. This proposal is designed to increase our understanding of these epigenetic regulatory processes. We hypothesize that histone modification and associated nuclear protein composition, including transcriptional repressor complexes and chromatin remodeling factors, are important determinants in whether stem cells self-renew or differentiate. Using mouse spermatogonia as a model, we will address the regulation of stem cell self-renewal in two specific aims. The first aim will examine the role of Polycomb group proteins on gene silencing in self-renewing spermatogonia. To achieve this aim, two distinct spermatogonial populations will be FACS-sorted and analyzed by transcriptional profiling. Potential Polycomb group binding sites will then be identified by chromatin immunoprecipitation (ChIP). Loss-of-function effects will be examined by one of two methods: generation of conditional knockout mice or RNAi knockdown and transplantation of cultured spermatogonial stem cells into recipient testes. The second aim will examine the role of nuclear matrix proteins in the maintenance of self-renewing spermatogonia. Proteomic profiling will be performed after fractionating nuclear matrix proteins from FACS-sorted germ cells and identifying their composition by liquid chromatography-mass spectrometry. Generation of conditional knockout mice or RNAi and transplantation experiments will then be performed to assess the functional importance of matrix-associated proteins. The proposed research for this award is designed to encompass both the mentored phase (K99) and the independent phase (R00), with a transition period built into the plan to successfully bridge the two phases. Relevance: A better understanding of how chromatin is modified and how these modifications influence gene expression during the self-renewal of stem cells will greatly contribute to the emerging field of regenerative medicine. Recent reports of pluripotent embryonic stem cell-like colonies emanating from cultured male germline stem cells warrant further investigation into the epigenetic control of stem cells in the testis. This proposal is both highly relevant and timely in this regard by addressing fundamental biological questions concerning the chromatin structure and gene expression in self-renewing spermatogonia and their differentiated progeny. Furthermore, deficiencies in histone modifications have been linked to infertility, while altered nuclear matrix proteins have been implicated in cancer. This proposal thus encompasses research highly relevant to the goals of the NIH.

描述（由申请人提供）：成年男性种系的维持对于繁殖至关重要。生殖线干细胞自我更新似乎仍然受到表观遗传染色质修饰和随后的核组织的影响。该建议旨在增加我们对这些表观遗传调节过程的理解。我们假设组蛋白修饰和相关的核蛋白组成，包括转录阻遏物复合物和染色质重塑因子，是干细胞是自我更新还是分化的重要决定因素。使用小鼠精子症作为模型，我们将在两个特定目标中解决干细胞自我更新的调节。第一个目的将研究多肉体蛋白在基因沉默中的作用在自我更新精子中。为了实现这一目标，将通过转录分析对两个截然不同的精子种群进行FACS分类和分析。然后，将通过染色质免疫沉淀（CHIP）鉴定潜在的多孔组结合位点。功能丧失效应将通过两种方法之一来检查：有条件的基因敲除小鼠或RNAi敲低以及将精子干细胞移植到受体睾丸中。第二个目标将研究核基质蛋白在维持自我更新精子中的作用。从FACS分级生殖细胞分离核基质蛋白并通过液相色谱 - 质谱法鉴定其组成后，将进行蛋白质组学分析。然后将进行有条件的基因敲除小鼠或RNAi和移植实验，以评估基质相关蛋白的功能重要性。该奖项的拟议研究旨在涵盖指导阶段（K99）和独立阶段（R00），并在计划中内置了一个过渡期，以成功地桥接了两个阶段。相关性：更好地理解如何修饰染色质以及这些修饰如何影响干细胞自我更新期间基因表达，将极大地有助于再生医学的新兴领域。最近关于培养的雄性种系干细胞产生的多能胚胎干细胞样菌落的报道值得进一步研究睾丸中干细胞的表观遗传控制。通过解决有关自我更新精子中的染色质结构和基因表达的基本生物学问题，这一建议在这方面既高度相关又及时。此外，组蛋白修饰的缺乏与不育症有关，而改变的核基质蛋白已与癌症有关。因此，该提案涵盖了与NIH目标高度相关的研究。