A comparative structural study of ATP-dependent chromatin remodeling complexes

ATP依赖性染色质重塑复合物的比较结构研究

• 批准号: 8099202
• 负责人: Andres Leschziner
• 金额: $ 29.68万
• 依托单位: HARVARD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-04-01 至 2016-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8099202
• 关键词: ATP Hydrolysis; ATP phosphohydrolase; Address; Affinity; Binding; Binding Proteins; Binding Sites; Biochemical; Biochemical Genetics; Biological; Biological Models; Chromatin; Chromatin Remodeling Factor; Collaborations; Colorado; Complex; Cryoelectron Microscopy; DNA; Data; Development; Docking; Electron Microscopy; Epigenetic Process; Eukaryotic Cell; Family; Fiber; Freezing; Funding; General Hospitals; Genome; Goals; Gold; Histone H3; Histones; In Vitro; Label; Lead; Link; Location; Malignant Neoplasms; Maps; Massachusetts; Metals; Methods; Modeling; Molecular; N-terminal; Negative Staining; Nucleoproteins; Nucleosomes; Outcome; Peptides; Positioning Attribute; Proteins; Reaction; Reagent; Relative (related person); Research; Resolution; Saccharomyces cerevisiae; Sampling; Slide; Staining method; Stains; Structure; Tail; Testing; United States National Institutes of Health; Utah; Work; chromatin remodeling; comparative; density; design; dimer; flexibility; in vivo; insight; macromolecular assembly; novel; reconstruction

DESCRIPTION (provided by applicant): Eukaryotic cells have solved the genome-packaging problem combining chromatin, the nucleoprotein fiber consisting of DNA and histones, with factors that modify it and regulate its dynamics. Among them are the ATP- dependent chromatin remodeling complexes or "remodelers", large and conserved multi-subunit assemblies that use ATP hydrolysis to non-covalently alter nucleosome structure. Remodelers can be classified into four families and differ both in composition and the products they generate both in vivo and in vitro. What is the mechanism of chromatin remodeling? How are different products generated? These questions remain unanswered due, to a large extent, to a combination of the remodelers' complexity and a paucity of structural information to provide a context for the accumulated biochemical and genetic data. Current mechanistic models are difficult, if not impossible, to test with biochemical approaches alone. Cryo-electron microscopy (Cryo-EM) is ideally suited for the analysis of remodelers and of what appears to be their intrinsic conformational flexibility. While the high resolution required to answer many mechanistic questions is a challenging long-term goal, more easily achievable lower resolution structures can provide important constraints to our modeling as well as insights into remodeler specialization. We will apply our expertise in electron microscopy of large, asymmetric and heterogeneous macromolecular assemblies to begin a comparative structural study of two remodelers with very different activities: histone octamer sliding and histone dimer exchange. Our model systems, both ~1MDa, are the S. cerevisiae remodelers RSC, which slides octamers, and SWR1, which exchanges H2A.Z/H2B dimers for the native H2A/H2B. In Aim 1 we will obtain reconstructions of the SWR1 complex both by itself and bound to a nucleosome. In Aim 2 we will obtain the structure of a remodeling-competent 4-subunit subcomplex of RSC (RSCsub) bound to a nucleosome and will refine our current RSC structure to higher resolution with frozen-hydrated samples. In Aim 3 we will extract biological information from the structures obtained in Aims 1 and 2 by mapping the location of a few key subunits in SWR1 and RSC/RSCsub. We have selected targets that will maximize the mechanistic insight we can gain even at the medium-resolution we expect to obtain within this funding period. The targets for RSC are also designed to allow us to dock the RSCsub-nucleosome structure into full RSC. An important component of Aim 3 is the development of novel methods for labeling subunits in frozen-hydrated samples. PUBLIC HEALTH RELEVANCE: ATP-dependent chromatin remodeling complexes are large macromolecular assemblies capable of altering the structure of nucleosomes, the packaging units of DNA, to regulate DNA accessibility. We aim to understand, at the molecular level, how these complexes interact with and remodel nucleosomes. Our research could lead to new insights in the fields of epigenetics and cancer.

描述（由申请人提供）：真核细胞已经解决了将染色质（由 DNA 和组蛋白组成的核蛋白纤维）与修饰它并调节其动态的因子相结合的基因组包装问题。其中包括 ATP 依赖性染色质重塑复合物或“重塑者”，即利用 ATP 水解非共价改变核小体结构的大型且保守的多亚基组装体。重塑剂可分为四个家族，其成分和它们在体内和体外产生的产物均不同。染色质重塑的机制是什么？不同的产品是如何产生的？这些问题在很大程度上仍未得到解答，因为重塑者的复杂性和缺乏为积累的生化和遗传数据提供背景的结构信息。仅用生化方法来测试当前的机械模型即使不是不可能，也是很困难的。 冷冻电子显微镜 (Cryo-EM) 非常适合分析重塑蛋白及其内在构象灵活性。虽然回答许多机械问题所需的高分辨率是一个具有挑战性的长期目标，但更容易实现的较低分辨率结构可以为我们的建模提供重要的约束，并为重塑者专业化提供见解。我们将运用我们在大型、不对称和异质大分子组装体电子显微镜方面的专业知识，开始对具有截然不同活性的两种重塑剂进行比较结构研究：组蛋白八聚体滑动和组蛋白二聚体交换。我们的模型系统（均为 ~1MDa）是酿酒酵母重塑者 RSC（滑动八聚体）和 SWR1（将 H2A.Z/H2B 二聚体交换为天然 H2A/H2B）。 在目标 1 中，我们将获得 SWR1 复合物本身以及与核小体结合的重建。在目标 2 中，我们将获得与核小体结合的具有重塑能力的 RSC 4 亚基子复合物 (RSCsub) 的结构，并将利用冷冻水合样品将我们当前的 RSC 结构改进为更高分辨率。在目标 3 中，我们将通过绘制 SWR1 和 RSC/RSCsub 中几个关键亚基的位置，从目标 1 和 2 中获得的结构中提取生物信息。我们选择的目标将最大限度地提高我们可以获得的机械洞察力，即使在我们期望在本资助期内获得的中等分辨率下也是如此。 RSC 的目标还旨在允许我们将 RSC 亚核小体结构对接到完整的 RSC 中。目标 3 的一个重要组成部分是开发用于标记冷冻水合样品中亚基的新方法。 公共健康相关性：ATP 依赖性染色质重塑复合物是大型大分子组装体，能够改变核小体（DNA 的包装单位）的结构，以调节 DNA 的可及性。我们的目标是在分子水平上了解这些复合物如何与核小体相互作用并重塑核小体。我们的研究可能会给表观遗传学和癌症领域带来新的见解。