A comparative structural study of ATP-dependent chromatin remodeling complexes

描述(申请人提供)：真核细胞已经解决了基因组包装问题，结合了染色质，由DNA和组蛋白组成的核蛋白纤维，以及修饰它和调节其动态的因素。其中包括依赖于三磷酸腺苷的染色质重塑复合体或“重构体”，这是一种大的、保守的多亚基组合体，它利用三磷酸腺苷的水解来非共价改变核小体的结构。重构体可以分为四个家族，它们的组成和它们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？这些问题在很大程度上仍然没有得到回答，这在很大程度上是由于改构体的复杂性，以及缺乏为积累的生化和遗传数据提供背景的结构信息。目前的机械模型很难，如果不是不可能的话，仅用生化方法进行测试。低温电子显微镜(Cryo-EM)非常适合分析改构剂及其固有的构象柔性。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建器专业化的洞察提供重要的约束。我们将应用我们在大型、不对称和非均相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构体进行比较结构研究：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，是酿酒酵母重构体RSC，它滑动八聚体，和SWR1，它交换H_2A.Z/H_2B二聚体为天然的H_2A/H_2B。在目标1中，我们将获得SWR1复合体的重建，既有自身的，也有与核小体结合的。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亚单位亚基复合体(RSCSub)的结构，并将用冷冻水合样品将我们目前的RSC结构提炼到更高的分辨率。在目标3中，我们将通过定位SWR1和RSC/RSCsubs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扩大我们能够获得的机械性洞察力，即使是在我们预期在这一供资期间内获得的中等决议的情况下也是如此。RSC的靶点也被设计成允许我们将RSC亚核小体结构对接成完整的RSC。目标3的一个重要组成部分是开发新的方法来标记冷冻水合样品中的亚基。 与公共健康相关：依赖于ATP的染色质重塑复合体是一种大的大分子组件，能够改变核小体的结构，核小体是DNA的包装单位，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体如何与核小体相互作用并重塑核小体。我们的研究可能会在表观遗传学和癌症领域带来新的见解。

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.