Structural and Mechanistic Characterization of the Histone Chaperone FACT

描述(申请人提供)：染色质是一种紧密堆积和严格调控的核蛋白复合体，它以稳定但容易获得的形式存储细胞的遗传物质。在真核细胞中，染色质的重复核心亚单位是核小体，它由147个碱基对(BP)的DNA缠绕在组蛋白八聚体周围，几乎有两个超螺旋旋转[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。一组被称为组蛋白伴侣的这些辅助因子是一个多样化的组蛋白结合蛋白家族，它可以屏蔽非核小体组蛋白与DNA的相互作用。组蛋白伴侣蛋白将核心组蛋白从DNA中隔离，直到更有利的核小体排列变得可用(2)。这项工作将集中在组蛋白伴侣事实(促进染色质转录)。FACT重组核小体内的成分，并在复制、转录和修复过程中帮助细胞机械访问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。DNA可及性不协调会导致基因表达异常和DNA损伤无法修复，这两个都是癌症发生的普遍标志。染色质结构的改变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，染色质组装异常会导致细胞死亡或细胞生长失控，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改变可能是事实和核小体之间直接相互作用的结果，但对这一过程的机制细节了解很少。因此，这个项目的具体目标是为了更好地描述事实介导的核小体重组。首先，通过高通量荧光滴定分析来定量FACT与核小体亚复合体相互作用的结合性质(亲和力和化学计量比)。其次，基于溶液的结合、竞争和荧光共振能量转移(FRET)分析将为事实介导的核小体组装/拆解提供重要的机制信息。第三，特定的FACT-核小体相关复合体的晶体结构将给予FACT如何协调核小体动力学的第一个视角。这项研究项目的首要目标是了解FACT复合体的结构和功能，以及它在细胞存活、癌变和抗癌治疗中的作用。 与公共卫生相关：该事实复合体于1998年首次被发现，是通过染色质进行转录延长的关键因素，在复制和修复中具有类似的作用；随后的研究表明，事实活动水平影响肿瘤生长，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将深入了解染色质背景下的一般DNA复制、转录和修复过程，还可能为新的或改进的癌症治疗创造一条途径。从这些拟议的目标中获得的见解可能有助于开发特定的事实抑制剂，这些药物有可能提高化疗效果，同时减少与癌症相关的异常过程。

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.