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

基本信息

批准号：
8197721
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
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-01-01 至 2013-12-31
项目状态：
已结题

项目摘要

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.

成年男性种系的维持对于繁殖至关重要。仍然是一个鲜为人知的过程，种系干细胞自我更新似乎受到表观遗传染色质修饰的影响和随后的核组织。该建议旨在增加我们对这些的理解表观遗传调节过程。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，很重要干细胞是自我更新还是分化的决定因素。使用鼠标精子作为模型，我们将解决两个具体目标中干细胞自我更新的调节。第一个目标将检查在自我更新精子中的基因沉默中，多肉瘤组蛋白。为了实现这一目标，两个明显的精子种群将通过荧光激活的细胞分选（FACS）分开，并通过转录分析。然后将通过染色质鉴定潜在的多孔组结合位点免疫沉淀。功能丧失效果将通过两种方法之一来检查：生成有条件的敲除小鼠或RNAi敲低以及将精子干细胞移植到接受者睾丸。第二个目的将研究核基质蛋白在维持自我修复中的作用精子。分馏核基质蛋白后将进行蛋白质组学分析从FACS分类的生殖细胞，并通过液相色谱 - 质谱法鉴定其组成。然后将进行有条件的敲除小鼠或RNAi和移植实验评估基质相关蛋白的功能重要性。该奖项的拟议研究是旨在涵盖指导阶段（K99）和独立阶段（ROO），并具有过渡计划中的时期成功地桥接了两个阶段。该项目现在准备进入Roo 阶段，扩展在K99阶段取得的进展，并实现两种特定的主要方面目标。