Structural and Mechanistic Characterization of the Histone Chaperone FACT

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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.