Characterization of Drosophila Germline Stem Cell Chromatin Using ChIP-Seq

描述（由申请人提供）：使用ChIP-Seq对果蝇生殖系干细胞染色质进行表征总结。我们对调节人类干细胞活性的遗传和分子机制的理解在很大程度上依赖于使用模式生物的工作。果蝇是研究干细胞生物学各个方面的强大系统。在果蝇卵巢的生殖系干细胞中，已经发现了几种复杂的调控干细胞行为的网络。一个跨物种的新兴主题是染色质介导的基因调控在干细胞维持中的作用。虽然一些参与建立和调节染色质结构的个别基因已知对干细胞维持是重要的，但缺乏对成体干细胞中染色质结构的全面看法。在这里，我们建议纯化卵巢生殖干细胞（GSC）和调查他们的染色质景观全基因组染色质免疫沉淀和大规模平行测序。我们将研究纯化的GSC中聚合酶II的分布，以及与沉默染色质（例如组蛋白H3在赖氨酸27上的三甲基化（H3 K27 me 3））和活性染色质（例如组蛋白H3在赖氨酸4上的三甲基化（H3 K4 me 3））相关的组蛋白修饰。这些研究将确定果蝇干细胞是否含有二价染色质结构域，被H3 K27 me 3和H3 K4 me 3占据，以及被平衡的聚合酶占据的基因，这两个特征被认为有助于哺乳动物培养细胞中的干细胞身份。鉴定跨系统保守的染色质特征可能揭示干细胞特有的基因调控的重要机制。我们还将研究GSC的分化，以描述染色质编程如何随发育时间而变化。我们的建议，以分析从成人组织中分离的高度纯化的干细胞的染色质将是第一次检查的染色质环境中的干细胞在其天然环境。这些研究应该揭示干细胞是否具有独特的染色质特征，从而有助于我们理解支配干细胞自我更新的基本原理。 公共卫生相关性：再生医学依赖于对管理干细胞自我更新分裂的基本原理的理解。该项目旨在从成人组织中分离纯干细胞，并描述自我更新和分化干细胞之间DNA环境中发生的变化。

DESCRIPTION (provided by applicant): Characterization of Drosophila Germline Stem Cell Chromatin Using ChIP-Seq Summary. Our understanding of the genetic and molecular mechanisms that regulate the activity of human stem cells relies heavily on work using model organisms. Drosophila represents a powerful system for the study of diverse aspects of stem cell biology. Several complex regulatory networks governing stem cell behavior have been identified and characterized in the germline stem cells of the Drosophila ovary. One emerging theme across species is a role for chromatin-mediated gene regulation in stem cell maintenance. While a few individual genes that participate in establishing and regulating chromatin structure are known to be important for stem cell maintenance, a comprehensive view of the chromatin architecture in adult stem cells is lacking. Here we propose to purify ovarian germline stem cells (GSCs) and survey their chromatin landscape by genome-wide chromatin immunoprecipitation and massive parallel sequencing. We will examine the distribution of Polymerase II, as well as histone modifications associated with silence chromatin (e.g. trimethylation of histone H3 on lysine 27 (H3K27me3)) and active chromatin (e.g. trimethylation of histone H3 on lysine 4 (H3K4me3)) in purified GSCs. These studies will establish whether Drosophila stem cells contain bivalent chromatin domains, occupied by both H3K27me3 and H3K4me3, as well as genes occupied by poised polymerases, two features proposed to contribute to stem cell identity in mammalian cultured cells. The identification of chromatin features conserved across systems may reveal important mechanisms of gene regulation that are unique to stem cells. We will also examine GSCs as they differentiate, to describe how chromatin programming changes over developmental time. Our proposal to assay chromatin from highly purified stem cells isolated from adult tissues would be the first examination of the chromatin environment from stem cells in their native environment. These studies should reveal whether stem cells share unique chromatin features, and thus contribute to our understanding of the basic principles that govern stem cell self-renewal. PUBLIC HEALTH RELEVANCE: Regenerative medicine relies on an understanding of the basic principles that govern the self-renewing divisions of stem cells. This project aims to isolate pure stem cells from adult tissues and characterize the changes that occur in the DNA environment between self-renewing and differentiating stem cells.