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）：真核细胞已解决了结合染色质，核蛋白纤维由DNA和组蛋白组成的基因组包装问题，以及对其进行了修改并调节其动力学的因素。其中包括ATP依赖性染色质重塑络合物或“重塑剂”，即使用ATP水解以非共价改变核小体结构的大型和保守的多生物组件。可以将改建剂分为四个家庭，并且在组成和它们在体内和体外产生的产品都有不同。染色质重塑的机制是什么？如何生成不同的产品？这些问题在很大程度上尚未得到解答，这是由于改建商的复杂性和缺乏结构信息的结合，以提供累积的生化和遗传数据的背景。目前的机械模型很难（即使不是不可能）仅使用生化方法进行测试。 冷冻电子显微镜（Cryo-EM）非常适合分析重塑剂和似乎是其内在构象柔韧性。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限制，以及对改建器专业化的见解。我们将在大型，不对称和异质分子组件的电子显微镜中应用专业知识，以开始对具有非常不同活性的两个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（〜1MDA）都是S. cerevisiae Remodelers RSC，它滑动八聚体和SWR1，它换了H2A.Z/H2B二聚体为天然H2A/H2B。 在AIM 1中，我们将自身获得SWR1复合物的重建并与核小体结合。在AIM 2中，我们将获得与核小体结合的RSC（RSCSUB）重塑功能的4-亚基子复合物的结构，并将我们当前的RSC结构改进了使用冷冻水合样品的更高分辨率。在AIM 3中，我们将通过映射SWR1和RSC/RSCSUB中一些关键亚基的位置，从AIM 1和2中获得的结构中提取生物学信息。我们选择了目标，即使在我们期望在此资助期内获得的中期分辨率，我们也可以获得最大化的机械洞察力。 RSC的目标还旨在使我们可以将RSCSUB核体结构停靠为完整的RSC。 AIM 3的一个重要组成部分是开发了在冷冻水合样品中标记亚基的新方法。 公共卫生相关性：依赖ATP的染色质重塑络合物是能够改变核小体结构（DNA的包装单元）来调节DNA可及性的大分子分子组件。我们的目标是在分子水平上了解这些复合物与重塑核小体的相互作用和重塑。我们的研究可能导致对表观遗传学和癌症领域的新见解。