Epigenetic Regulation in Self-Renewing and Differentiating Male Germ Cells

说明（由申请人提供）：维持成年雄性生殖系对繁殖至关重要。生殖系干细胞的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。这一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，包括转录抑制因子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，我们将在两个特定的目标中解决干细胞自我更新的调节。第一个目标是研究Polycomb蛋白在自我更新精原细胞中基因沉默的作用。为了实现这一目标，两个不同的精原种群将被facs分类并通过转录谱分析。然后通过染色质免疫沉淀（ChIP）鉴定潜在的Polycomb基团结合位点。功能丧失效应将通过以下两种方法之一进行检测：产生条件敲除小鼠或敲除RNAi和将培养的精原干细胞移植到受体睾丸中。第二个目标将检查核基质蛋白在维持自我更新精原细胞中的作用。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，进行蛋白质组学分析。随后将进行条件敲除小鼠或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包括指导阶段（K99）和独立阶段（R00），并在计划中建立一个过渡期，以成功地将这两个阶段连接起来。相关性：更好地了解染色质如何被修饰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，将极大地促进再生医学的新兴领域。最近报道了从培养的男性生殖系干细胞中产生的多能胚胎干细胞样菌落，这为进一步研究睾丸干细胞的表观遗传控制提供了依据。本研究解决了有关自我更新精原细胞及其分化后代染色质结构和基因表达的基本生物学问题，具有很强的相关性和时代性。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.