Structural and Mechanistic Characterization of the Histone Chaperone FACT

描述（由申请人提供）：染色质是一种密集堆积且受到严格调控的核蛋白复合物，以稳定且易于获取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基是核小体，它由147个碱基对（bp）的DNA以近两个超螺旋的方式缠绕在组蛋白八聚体上(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转变的辅助因素。一组被称为组蛋白伴侣的辅助因子是一个不同的组蛋白结合蛋白家族，它保护非核小体组蛋白- dna相互作用。组蛋白伴侣将核心组蛋白从DNA中隔离出来，直到获得更有利的核小体排列(2)。这项工作将重点关注组蛋白伴侣FACT（促进染色质转录）。FACT重组核小体内的成分，并帮助在复制、转录和修复过程中提供细胞机制进入DNA的途径（3-5）。在这些关键过程完成后，FACT也有助于重新包装染色质。不协调的DNA可接近性可导致基因表达异常和DNA损伤未修复，这两者都是致癌的普遍标志。染色质结构的改变是正常细胞过程如基因表达和细胞分裂所必需的。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FACT和核小体之间直接相互作用的结果，但这一过程的机制细节尚不清楚。因此，该项目的具体目的是为了更好地表征FACT介导的核小体重组。首先，通过高通量荧光滴定测定FACT与核小体亚复合物相互作用的结合特性（亲和力和化学计量学）。其次，基于溶液的结合、竞争和荧光共振能量转移（FRET）分析将提供FACT介导的核小体组装/拆卸的重要机制信息。第三，特定的FACT-核小体相关复合物的晶体结构将提供FACT如何协调核小体动力学的第一个视图。本研究项目的首要目标是了解FACT复合体的结构和功能及其在细胞活力、癌变和对癌症治疗的耐药性中的作用。

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.