Single-molecule studies of ATP-dependent chromatin remodeling

ATP依赖性染色质重塑的单分子研究

• 批准号: 9053499
• 负责人: XIAOWEI ZHUANG
• 金额: $ 31.42万
• 依托单位: HARVARD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-08-01 至 2018-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9053499
• 关键词: ATP Hydrolysis; ATPase Domain; Address; Affect; Binding Sites; Biochemical; Biological Assay; Biological Process; Biophysics; Breast; Cell Fate Control; Cell Nucleus; Cell physiology; Cells; Chromatin; Chromatin Remodeling Factor; Chromatin Structure; Color; Complement; Complex; DNA; DNA Packaging; DNA biosynthesis; Derivation procedure; Development; Disease; Energy Transfer; Enzymes; Event; Family; Fluorescence; Fluorescence Resonance Energy Transfer; Free Energy; Functional disorder; Genetic Materials; Genetic Transcription; Goals; Health; Histone H2A; Histone H4; Histones; ISWI; Kinetics; Length; Light; Link; Linker DNA; Lung; Malignant Neoplasms; Methodology; Methods; Modeling; Molecular; Monitor; N-terminal; Nucleosomes; Pancreas; Pathway interactions; Physiology; Play; Positioning Attribute; Post-Translational Modification Site; Process; Prostate; Reaction; Regulation; Role; Second Messenger Systems; Side; Site; Structure; Tail; Techniques; Testing; Time; Variant; chromatin remodeling; combat; crosslink; developmental disease; histone modification; improved; insight; novel; novel therapeutics; recombinational repair; research study; second messenger; single molecule; single-molecule FRET

DESCRIPTION (provided by applicant): The packaging of DNA as chromatin regulates many important cellular processes that require access to the cell's genetic material. One major class of enzymes responsible for regulating the structure of chromatin is the ATP-dependent chromatin remodelers. These enzymes play essential roles in a variety of biological processes ranging from DNA replication, repair, recombination and transcription to the regulation of cell fate decisions. Dysfunction of chromatin remodeling enzymes can cause a variety of cancers, such as breast, lung, pancreatic, prostate, and rhabdoid cancers, as well as a number of multisystem developmental disorders. Dissecting the functional roles of chromatin remodelers and developing novel therapies to combat diseases related to remodeler dysfunction require a mechanistic understanding of the biochemical and biophysical principles underlying chromatin remodeling. Our long-term goal is to develop a detailed mechanistic understanding of how chromatin remodeling enzymes use the free energy of ATP hydrolysis to disrupt histone- DNA contacts and alter the position, structure, and composition of nucleosomes. In this project, we will focus on investigating the remodeling mechanisms of the ISWI and SWI/SNF family remodelers. Determining the mechanisms of chromatin remodeling requires quantitative characterizations of the dynamics of the remodeling reaction. Single-molecule techniques are well suited for this purpose as they allow us to monitor complex molecular processes in real time, directly observe intermediate states, and dissect reaction pathways. In this project, we will use single-molecule fluorescence resonance energy transfer, in conjunction with complementary biochemical assays, to study the mechanisms of chromatin remodeling and its regulation. We will address three specific aims. Aim 1: We will investigate the nucleosome remodeling dynamics catalyzed by ISWI family remodelers. In particular, we will determine the structural dynamics of the nucleosome during remodeling and probe how remodeling actions at different nucleosomal sites are coordinated. We aim to test different mechanistic models and advance our understanding of how ISWI family enzymes translocate nucleosomes along DNA. Aim 2: The activity of ATP-dependent chromatin remodelers is under intricate regulation by a variety of factors. In this aim, we plan to study how several biologically relevant nucleosomal features, including DNA linker length and histone modifications/variants, regulate ISWI remodeling activity. Aim 3: While remodeling enzymes from different families share a homologous ATPase domain, they display different remodeling activities and regulate different biological processes. In Aim 3, we will extend our studies to SWI/SNF family remodelers and compare them with the ISWI family, aiming to identify key commonalities and differences in the nucleosome remodeling mechanisms used by these two major families of chromatin remodelers.

描述（由申请人提供）：DNA作为染色质的包装调节许多重要的细胞过程，这些过程需要获得细胞的遗传物质。负责调节染色质结构的一个主要类别的酶是ATP依赖性染色质重塑物。这些酶在从DNA复制、修复、重组和转录到调节细胞命运决定的各种生物过程中发挥重要作用。染色质重塑酶的功能障碍可引起多种癌症，如乳腺癌、肺癌、胰腺癌、前列腺癌和横纹肌样癌，以及许多多系统发育障碍。剖析染色质重塑的功能作用和开发新的治疗方法来对抗与重塑功能障碍相关的疾病，需要对染色质重塑背后的生物化学和生物物理学原理有一个机械的理解。我们的长期目标是对染色质重塑酶如何使用ATP水解的自由能来破坏组蛋白- DNA接触并改变核小体的位置、结构和组成进行详细的机械理解。在本项目中，我们将重点研究ISWI和SWI/SNF家族重塑因子的重塑机制。确定染色质重塑的机制需要重塑反应动力学的定量表征。单分子技术非常适合这一目的，因为它们使我们能够真实的实时监测复杂的分子过程，直接观察中间状态，并剖析反应途径。在这个项目中，我们将 使用单分子荧光共振能量转移，结合互补的生化分析，研究染色质重塑及其调控的机制。我们将讨论三个具体目标。目的1：我们将研究ISWI家族重构因子催化的核小体重构动力学。特别是，我们将确定核小体在重塑过程中的结构动力学，并探讨如何在不同的核小体位点协调重塑行动。我们的目标是测试不同的机制模型，并推进我们的理解，如何ISWI家族酶易位核小体沿着DNA。目的2：ATP依赖性染色质重塑的活性受到多种因素的复杂调控。在这个目标中，我们计划研究几个生物学相关的核小体功能，包括DNA接头长度和组蛋白修饰/变体，调节ISWI重塑活动。目标3：虽然来自不同家族的重塑酶共享同源的ATP酶结构域，但它们显示不同的重塑活性并调节不同的生物过程。在目标3中，我们将把我们的研究扩展到SWI/SNF家族重塑，并将其与ISWI家族进行比较，旨在确定这两个主要染色质重塑家族所使用的核小体重塑机制的关键共性和差异。