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: 8706191
• 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 2017-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8706191
• 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; 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; public health relevance; 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，这些过程需要访问细胞的遗传材料。负责调节染色质结构的一类主要酶是ATP依赖性染色质重塑剂。这些酶在从DNA复制，修复，重组和转录到细胞命运决策的各种生物学过程中起着至关重要的作用。染色质重塑酶的功能障碍会引起各种癌症，例如乳腺癌，肺，胰腺，前列腺和色齿癌，以及许多多系统发育障碍。剖析染色质重塑剂的功能作用并开发出新的疗法以对抗与重塑器功能障碍有关的疾病，需要对染色质重塑基础的生化和生物物理原理的机械理解。我们的长期目标是对染色质重塑酶如何利用ATP水解的自由能来破坏组蛋白接触并改变核小体的位置，结构和组成。在这个项目中，我们将专注于研究ISWI和SWI/SNF家庭改建器的重塑机制。确定染色质重塑的机制需要对重塑反应的动力学进行定量表征。单分子技术非常适合此目的，因为它们使我们能够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 将单分子荧光共振能量传递与互补的生化测定一起研究染色质重塑及其调节的机制。我们将解决三个具体目标。目标1：我们将研究ISWI家族重塑剂催化的核小体重塑动力学。特别是，我们将在重塑过程中确定核小体的结构动力学，并探测如何在不同核小体位点进行重塑作用。我们旨在测试不同的机械模型，并促进我们对ISWI家族酶如何沿DNA转移核小体的理解。 AIM 2：ATP依赖性染色质重塑剂的活性受到多种因素的复杂调节。在此目标中，我们计划研究几种与生物学相关的核小体特征（包括DNA接头长度和组蛋白修饰/变体）如何调节ISWI重塑活性。目标3：虽然来自不同家庭的重塑酶具有同源ATPase领域，但它们显示出不同的重塑活动并调节不同的生物过程。在AIM 3中，我们将把研究扩展到SWI/SNF家族重塑器，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剂家族使用的核小体重塑机制中的关键共同点和差异。