Epigenetic Regulation in Self-Renewing and Differentiating Male Germ Cells

描述（由申请人提供）：成年雄性生殖系的维持对生殖至关重要。生殖系干细胞自我更新似乎受到表观遗传染色质修饰和随后的核组织的影响，这仍然是一个知之甚少的过程。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，包括转录抑制复合物和染色质重塑因子，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，我们将从两个方面探讨干细胞自我更新的调控。第一个目的是研究Polycomb组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，将对两个不同的精原细胞群体进行FACS分选并通过转录谱分析进行分析。然后通过染色质免疫沉淀（ChIP）鉴定潜在的Polycomb组结合位点。将通过以下两种方法之一来检查功能丧失效应：产生条件性敲除小鼠或RNAi敲除以及将培养的精原干细胞移植到受体睾丸中。第二个目标是研究核基质蛋白在维持精原细胞自我更新中的作用。在从FACS分选的生殖细胞中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。然后将进行条件性敲除小鼠或RNAi的产生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（R 00），并在计划中设置过渡期，以成功衔接这两个阶段。相关性：更好地了解染色质是如何被修饰的，以及这些修饰如何影响干细胞自我更新过程中的基因表达，将大大有助于再生医学的新兴领域。最近的报告多能胚胎干细胞样集落来自培养的男性生殖系干细胞保证进一步调查睾丸干细胞的表观遗传控制。这一建议是高度相关和及时的，在这方面，通过解决有关的染色质结构和基因表达的自我更新精原细胞及其分化后代的基本生物学问题。此外，组蛋白修饰的缺陷与不育有关，而改变的核基质蛋白与癌症有关。因此，这项建议包括与NIH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.