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: 8008826
• 负责人: CHRISTOPHER JESS PAYNE
• 金额: $ 24.65万
• 依托单位: LURIE CHILDREN'S HOSPITAL OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-01-01 至 2012-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8008826
• 关键词: Address; Adult; Affect; Alleles; Award; Binding Sites; Candidate Disease Gene; Cell Culture Techniques; Cell Differentiation process; Cell Line; Cell Separation; Cells; Child; Chromatin Remodeling Factor; Collaborations; Complex; Coupled; Cultured Cells; Detergents; Development; EZH2 gene; Epigenetic Process; Equilibrium; Euchromatin; Fluorescence-Activated Cell Sorting; Gene Expression; Gene Expression Microarray Analysis; Gene Expression Profile; Gene Silencing; Generations; Genes; Germ Cells; Heterochromatin; Histone H3; Histones; Infertility; Knockout Mice; Link; Liquid Chromatography; 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; PRC1 Protein; Pattern; Phase; Play; Polycomb; Population; Process; Proteins; Proteome; Proteomics; Publishing; RNA Interference; Regenerative Medicine; Regulation; Regulator Genes; Reporting; Repression; Reproduction; Research; Role; Small Interfering RNA; Sorting - Cell Movement; Spermatogonia; Stem cells; Testing; Testis; Transcription Repressor/Corepressor; Transplantation; Undifferentiated; Universities; ZNF145 gene; chromatin immunoprecipitation; chromatin modification; design; embryonic stem cell; histone methyltransferase; histone modification; liquid chromatography mass spectrometry; loss of function; male; medical schools; member; mutant; protein complex; research study; self-renewal

Maintenance ofthe 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 separated by fluorescence-activated cell sorting (FACS) and analyzed by transcriptional profiling. Potential Polycomb group binding sites will then be identified by chromatin immunoprecipitation. 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 selfrenewing 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 (ROO), with a transition period built into the plan to successfully bridge the two phases. This project is now poised to enter the ROO phase, extending the progress made during the K99 phase and achieving the major aspects of both specific aims.

成年雄性种系的维持对于繁殖至关重要。仍然是一个知之甚少的过程， 生殖系干细胞自我更新似乎受到表观遗传染色质修饰的影响， 后来的核组织。这项建议旨在增加我们对这些问题的了解， 表观遗传调控过程我们假设组蛋白修饰和相关的核蛋白 包括转录抑制因子复合物和染色质重塑因子在内的蛋白质组成是重要的 决定干细胞是否自我更新或分化。我们以小鼠精原细胞为模型， 将在两个具体目标中解决干细胞自我更新的调节问题。第一个目标将审查 多梳族蛋白在自我更新精原细胞基因沉默中的作用。为了实现这一目标，两个不同的 精原细胞群将通过荧光激活细胞分选（FACS）分离，并通过 转录谱分析然后通过染色质鉴定潜在的Polycomb组结合位点 免疫沉淀。将通过以下两种方法之一来检查功能丧失效应： 条件性敲除小鼠或RNAi敲除并将培养的精原干细胞移植到 受体睾丸第二个目标是研究核基质蛋白在维持自我更新中的作用 精原细胞将核基质蛋白分级分离后进行蛋白质组学分析 并通过液相色谱-质谱法鉴定其组成。 然后将进行条件性敲除小鼠或RNAi的产生和移植实验， 评估基质相关蛋白的功能重要性。该奖项的建议研究是 旨在涵盖辅导阶段（K99）和独立阶段（ROO）， 在计划中加入了一个时间段，以成功地衔接这两个阶段。这个项目现在准备进入ROO 阶段，扩大K99阶段取得的进展，并实现两个具体的主要方面， 目标。