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了许多需要访问细胞遗传物质的重要细胞过程。一类主要负责调节染色质结构的酶是依赖atp的染色质重塑酶。这些酶在多种生物过程中发挥重要作用，从DNA复制、修复、重组和转录到细胞命运决定的调节。染色质重塑酶的功能障碍可导致多种癌症，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。剖析染色质重塑者的功能作用和开发新的治疗方法来对抗与重塑者功能障碍相关的疾病，需要对染色质重塑背后的生化和生物物理原理有一个机制上的理解。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制理解。在本项目中，我们将重点研究ISWI和SWI/SNF家族重塑者的重塑机制。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这一目的，因为它们允许我们实时监测复杂的分子过程，直接观察中间状态，并解剖反应途径。在这个项目中，我们将