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: 8209458
• 负责人: Duane David Winkler
• 金额: $ 5.22万
• 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/8209458
• 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; public health relevance; 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）：染色质是一种密集包装和严格调控的核蛋白复合物，以稳定但易于获取的形式储存细胞的遗传物质。在真核细胞中，染色质的重复核心亚基是核小体，其由147个碱基对（bp）的DNA以近两个超螺旋圈缠绕在组蛋白八聚体周围组成（1）。我们的实验室专注于核小体结构和动力学以及促进这些转变的辅助因素。一组这些辅助因子称为组蛋白伴侣是一个不同的组蛋白结合蛋白家族，屏蔽非核小体组蛋白-DNA相互作用。组蛋白伴侣将核心组蛋白与DNA隔离，直到获得更有利的核小体排列（2）。这项工作将集中在组蛋白伴侣FACT（FACILITates染色质转录）。FACT重组核小体内的组分，并在复制、转录和修复过程中帮助细胞机器接近DNA（3-5）。FACT还有助于在这些关键过程完成后重新包装染色质。不协调的DNA可及性可导致基因表达异常和DNA损伤未修复，这两者都是肿瘤发生的普遍标志。染色质结构的变化对于正常的细胞过程如基因表达和细胞分裂是必不可少的。然而，异常的染色质组装可导致细胞死亡或不受控制的细胞生长，从而导致癌症。虽然人们普遍认为核小体重组和染色质结构的变化可能是由FACT和核小体之间的直接相互作用引起的，但对该过程的机制细节知之甚少。因此，该项目的具体目标是更好地表征FACT介导的核小体重组。首先，通过高通量荧光滴定测定定量FACT与核小体子复合物相互作用的结合特性（亲和力和化学计量）。其次，基于溶液的结合、竞争和荧光共振能量转移（FRET）测定将提供关于FACT介导的核小体组装/拆卸的重要机制信息。第三，特定的FACT-nucleosome相关的复合物的晶体结构将授予FACT如何编排核小体动力学的第一个视图。该研究项目的首要目标是了解FACT复合物的结构和功能及其在细胞活力，致癌作用和对癌症治疗的抗性中的作用。 公共卫生关系：FACT复合物于1998年首次被发现是通过染色质进行转录延伸所必需的因子，在复制和修复中具有相似的作用;随后的研究表明FACT活性水平影响肿瘤生长，甚至影响化疗疗效（6-8）。因此，FACT介导的核小体重组的结构和机制细节不仅可以深入了解染色质背景下的一般DNA复制，转录和修复过程，还可以为新的或改进的癌症治疗创造途径。从这些拟议目标中获得的见解可以帮助开发特异性FACT抑制剂，这些抑制剂有可能增加化疗的有效性，同时减少异常的癌症相关过程。