Characterization of Four Histone H3 Modifications in the DNA-Histone Interface

DNA-组蛋白界面中四种组蛋白 H3 修饰的表征

• 批准号: 7763218
• 负责人: Michael Guy Poirier
• 金额: $ 28.22万
• 依托单位: OHIO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-02-01 至 2013-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7763218
• 关键词: Affect; Biochemical; Biological Process; Cancer Research Project; Cell physiology; Chemicals; Chromatin; DNA; DNA Repair; DNA Structure; Digestion; Disease; Fluorescence; Fluorescence Resonance Energy Transfer; Fluorometry; Gene Expression; Gene Expression Regulation; Goals; Growth; Health; Histone H3; Histones; Knowledge; Lead; Ligation; Maps; Modification; Molecular; Molecular Conformation; Names; Nucleosomes; Organism; Positioning Attribute; Post-Translational Protein Processing; Principal Investigator; Proteins; Research; Research Project Grants; Role; Site; Spectrum Analysis; Structure; Syndrome; Transcriptional Regulation; histone modification; in vivo; insight; programs; reconstitution; research study; restriction enzyme

DESCRIPTION (provided by applicant): The long-term goal of our labs is to determine the molecular mechanisms by which post-translational modifications of histones regulate gene expression and DNA repair. These two cellular processes are vital for the growth and health of all organisms. Alterations in post-translational histone modifications affect the regulation of gene expression and DNA repair and can lead to diseases such as ICF, Rett and ATRX syndromes and, most predominantly, cancer. This research project will determine the molecular mechanisms by which four key post-translational modifications at histone H3 residues K56, K115, T118 and K122 function. These modifications were recently identified in structured regions of the nucleosome. They occur both individually and together in vivo, and are critical for transcriptional regulation and DNA repair. The mechanisms by which these modifications carry out vital biological functions remain poorly understood. However, each modification is buried beneath DNA in the histone-DNA interface at one of two critical regions of the nucleosome: the dyad symmetry axis and the DNA entry-exit region. This implies that the mechanisms by which these modifications function must require significant changes in nucleosome conformation and/or dynamics. Therefore, combined biochemical and biophysical studies are key to understanding the role of these modifications. The four modifications will be studied by reconstituting uniformly modified semi-synthetic nucleosomes using histone proteins that are constructed by expressed protein ligation and by sequential chemical ligation. Multiple approaches will be used to quantify modification-induced changes in chromatin conformation and dynamics: fluorescence resonance energy transfer, restriction enzyme digestions, nucleosome mapping, nucleosome competitive reconstitutions, stopped flow fluorometry and fluorescence correlation spectroscopy. These experiments will determine whether and how these four histone H3 modifications in the DNA-histone interface regulate, in Aim 1, Nucleosome Structure and DNA site Exposure, in Aim 2, Nucleosome Positioning and Stability and, in Aim 3, Nucleosome Dynamics. The successful completion of this research project will make a major impact on scientific knowledge and the molecular understanding of disease in two ways. First, it will determine how four modifications in histone H3 buried within the nucleosome DNA-histone interface facilitate both DNA repair and transcriptional regulation. Second, it will provide insight into the possible mechanisms of over 20 post-translational histone modifications that are known to be buried throughout the nucleosome DNA-histone interface.

描述（由申请人提供）：我们实验室的长期目标是确定组蛋白翻译后修饰调节基因表达和DNA修复的分子机制。这两个细胞过程对所有生物体的生长和健康至关重要。翻译后组蛋白修饰的改变影响基因表达和DNA修复的调节，并可导致诸如ICF、Rett和ATRX综合征等疾病，最主要的是癌症。