Epigenetic Regulation in Self-Renewing and Differentiating Male Germ Cells

描述(由申请人提供)：保持成年男性生殖系对于繁殖是必不可少的。生殖系干细胞的自我更新仍然是一个知之甚少的过程，似乎受到表观遗传的染色质修饰和随后的核组织的影响。这项建议旨在增加我们对这些表观遗传调控过程的理解。我们假设，组蛋白修饰和相关的核蛋白组成，包括转录抑制物复合体和染色质重塑因子，是干细胞自我更新或分化的重要决定因素。以小鼠精原细胞为模型，我们将从两个特定的目标来研究干细胞自我更新的调控。第一个目标是研究多梳蛋白在精原细胞自我更新过程中基因沉默中的作用。为了实现这一目标，将对两个不同的精原细胞群体进行FAC分类，并通过转录图谱进行分析。然后，将通过染色质免疫沉淀(CHIP)确定潜在的多梳基结合位点。功能丧失的影响将通过两种方法之一进行检测：产生条件基因敲除小鼠或RNAi敲除，以及将培养的精原干细胞移植到受体睾丸中。第二个目的将研究核基质蛋白在维持自我更新的精原细胞中的作用。蛋白质组学分析将在从FACS分选的生殖细胞中分离出核基质蛋白并用液相色谱-质谱仪鉴定其组成后进行。然后将进行条件基因敲除小鼠或RNAi的生成和移植实验，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)，并在计划中加入过渡期，以成功地弥合这两个阶段。相关性：更好地了解染色质是如何修改的，以及这些修改如何在干细胞自我更新过程中影响基因表达，将极大地促进再生医学的新兴领域。最近关于多能胚胎干细胞样集落起源于培养的男性生殖系干细胞的报道值得进一步研究睾丸干细胞的表观遗传控制。这项建议在这方面是高度相关和及时的，因为它解决了关于自我更新的精原细胞及其分化的后代中染色质结构和基因表达的基本生物学问题。此外，组蛋白修饰的缺陷与不孕症有关，而核基质蛋白的改变与癌症有关。因此，该提案包含了与美国国立卫生研究院的目标高度相关的研究。

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.