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（申请人提供）：真核细胞解决了基因组包装问题，将染色质（由DNA和组蛋白组成的核蛋白纤维）与修饰和调节其动态的因子结合在一起。其中包括ATP依赖的染色质重塑复合体或“重塑者”，这是一种大型且保守的多亚基组合物，利用ATP水解非共价改变核小体结构。重塑剂可分为四科，它们的成分和在体内和体外产生的产物都不同。染色质重塑的机制是什么？不同的产品是如何产生的？这些问题仍然没有答案，在很大程度上，由于重塑者的复杂性和结构信息的缺乏，无法为积累的生化和遗传数据提供背景。目前的机械模型很难，如果不是不可能，单独用生化方法来测试。低温电子显微镜（Cryo-EM）非常适合分析重塑物和它们固有的构象灵活性。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，但更容易实现的低分辨率结构可以为我们的建模以及对重塑器专业化的见解提供重要的约束。我们将运用我们在大型，不对称和非均质大分子组装的电子显微镜方面的专业知识，开始对两种具有非常不同活性的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，都是~1MDa，是S. cerevisiae重塑者RSC，它滑动八聚体，SWR1，交换H2A。Z/H2B二聚体为原生H2A/H2B。在目的1中，我们将获得SWR1复合体本身和与核小体结合的复合体的重建。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的4亚基亚复合物（RSCsub）的结构，并将通过冷冻水合样品改进我们当前的RSC结构以获得更高的分辨率。在Aims 3中，我们将通过绘制SWR1和RSC/RSCsub中几个关键亚基的位置，从Aims 1和2中获得的结构中提取生物信息。我们已经选择了一些目标，这些目标将使我们能够获得的机械洞察力最大化，即使是在我们期望在这一资助期内获得的中等分辨率下。RSC的靶标也被设计为允许我们将RSC亚核小体结构对接到完整的RSC中。Aim 3的一个重要组成部分是开发在冷冻水合样品中标记亚基的新方法。