Structural and Mechanistic Characterization of the Histone Chaperone FACT

组蛋白伴侣的结构和机制表征 FACT

• 批准号: 8059430
• 负责人: Duane David Winkler
• 金额: $ 4.84万
• 依托单位: COLORADO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-02-01 至 2013-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8059430
• 关键词: Affect; Affinity; Architecture; Base Pairing; Binding; Biological Assay; Cell Death; Cell Survival; Cell division; Cell physiology; Cells; Chromatin; Chromatin Modeling; Chromatin Structure; Complex; Crystallization; Crystallography; DNA; DNA Damage; DNA biosynthesis; Data; Development; Effectiveness; Eukaryotic Cell; Family; Fluorescence; Fluorescence Resonance Energy Transfer; Gene Expression; Genetic Materials; Genetic Transcription; Goals; Grant; Histones; Human; Image; Individual; Intention; Investigation; Label; Laboratories; Lead; Link; Malignant Neoplasms; Mediating; Metabolism; Molecular; Molecular Chaperones; Monitor; Morphologic artifacts; N-terminal; Nucleoproteins; Nucleosome Core Particle; Nucleosomes; Outcome; Pathway interactions; Process; Property; Recombinants; Research Project Grants; Resistance; Resolution; Roentgen Rays; Role; Shapes; Solutions; Structure; Titrations; Variant; Work; base; cancer therapy; carcinogenesis; chemotherapy; design; dimer; fluorophore; histone-binding proteins; improved; inhibitor/antagonist; insight; reconstitution; repaired; research study; stoichiometry; tumor growth; uncontrolled cell growth; wound

DESCRIPTION (provided by applicant): Chromatin is a densely packed and tightly regulated nucleoprotein complex that stores the genetic material of a cell in a stable yet readily accessible form. In eukaryotic cells, the repeating core subunit of chromatin is the nucleosome which is composed of 147 base pairs (bp) of DNA wound around a histone octamer in nearly two superhelical turns (1). Our laboratory focuses on nucleosome structure and dynamics and the accessory factors that promote these transitions. A group of these accessory factors termed histone chaperones are a diverse family of histone binding proteins that shield non-nucleosomal histone-DNA interactions. Histone chaperones sequester core histones from DNA until a more favorable nucleosomal arrangement becomes available (2). This work will focus on the histone chaperone FACT (FAcilitates Chromatin Transcription). FACT reorganizes components within the nucleosome and helps provide the cellular machinery access to DNA during replication, transcription, and repair (3-5). FACT also contributes to re-packaging chromatin after these critical processes are complete. Uncoordinated DNA accessibility can lead to aberrant gene expression and unrepaired DNA damage which are both prevalent markers in carcinogenesis. Changes in chromatin structure are essential for normal cellular processes such as gene expression and cell division. However, abnormal chromatin assembly can lead to cell death or uncontrolled cell growth leading to cancer. While it is generally accepted that nucleosome reorganization and changes in chromatin architecture can result from a direct interaction between FACT and nucleosomes, the mechanistic details of this process are poorly understood. Thus, the specific aims of this project are designed to better characterize FACT mediated nucleosome reorganization. First, quantitate the binding properties (affinities and stoichiometries) of FACT interactions with nucleosome sub-complexes via high-throughput fluorescence titration assays. Second, solution-based binding, competition, and fluorescence resonance energy transfer (FRET) assays will provide important mechanistic information on FACT mediated nucleosome assembly/disassembly. Third, the crystal structures of specific FACT-nucleosome related complexes will grant a first view of how FACT orchestrates nucleosome dynamics. The overriding goal of this research project is to understand the structure and function of the FACT complex and its role in cell viability, carcinogenesis, and resistance to cancer treatments. PUBLIC HEALTH RELEVANCE: The FACT complex was first discovered in 1998 as a factor essential for transcriptional elongation through chromatin, with similar roles in replication and repair; subsequent investigations have shown FACT activity levels affect tumor growth and even chemotherapy efficacy (6-8). Thus, structural and mechanistic details of FACT mediated nucleosome reorganization will not only give insight into general DNA replication, transcription, and repair processes in a chromatin context, they may also create a pathway for new or improved cancer treatments. Insights from these proposed aims could aid in the development of specific FACT inhibitors that have the potential to increase chemotherapy effectiveness while decreasing aberrant cancer-related processes.

描述（由申请人提供）：染色质是一种密集的填充且严格调节的核蛋白复合物，可将细胞的遗传物质存储在稳定但易于访问的形式中。在真核细胞中，染色质的重复核心亚基是核小体，该核小体由近两个超固定旋转的组蛋白八聚体周围围绕组蛋白的147个碱基对（BP）组成（1）。我们的实验室专注于核小体的结构和动力学以及促进这些过渡的辅助因子。这些称为组蛋白伴侣的附属因子组成的是一个多样化的组蛋白结合蛋白家族，可掩盖非核体组蛋白-DNA相互作用。组蛋白伴侣从DNA隔离到更有利的核小体排列（2）。这项工作将集中于组蛋白伴侣事实（促进染色质转录）。事实重组核小体内的组件，并有助于在复制，转录和修复过程中提供细胞机械访问DNA（3-5）。事实还有助于在完成这些关键过程后重新包装染色质。不协调的DNA可及性会导致异常的基因表达和未经修复的DNA损伤，这都是癌变中普遍的标记。染色质结构的变化对于正常细胞过程（例如基因表达和细胞分裂）至关重要。但是，异常的染色质组装会导致细胞死亡或不受控制的细胞生长导致癌症。虽然人们普遍认为，核小体的重组和染色质结构的变化可能是由于事实和核小体之间的直接相互作用而导致的，但该过程的机理细节知之甚少。因此，该项目的具体目的旨在更好地表征事实介导的核小体重组。首先，通过高通量荧光滴定测定法量化了与核小体亚复合物与核小体亚复合物相互作用的结合特性（亲和力和石化的）。其次，基于溶液的结合，竞争和荧光共振能量转移（FRET）测定将提供有关事实介导的核小体组装/拆卸的重要机械信息。第三，特定事实与核小体相关的复合物的晶体结构将对事实如何编排核小体动力学的第一视图。该研究项目的压倒性目标是了解事实复合物的结构和功能及其在细胞活力，致癌性和对癌症治疗的耐药性中的作用。 公共卫生相关性：事实综合体是在1998年首次发现的，是通过染色质转录伸长的重要因素，在复制和修复中具有相似的作用；随后的研究表明，事实活动水平会影响肿瘤的生长甚至化学疗法功效（6-8）。因此，事实介导的核小体重组的结构和机理细节不仅可以深入了解染色质的一般DNA复制，转录和修复过程，而且还可以为新的或改善的癌症治疗带来途径。这些拟议的目标的见解可以帮助开发特定的事实抑制剂，这些抑制剂有可能提高化学疗法有效性，同时减少与癌症相关的异常过程。