Structural and Functional Analysis of the Chd1 Chromatin Remodeler

Chd1 染色质重塑剂的结构和功能分析

• 批准号: 8727583
• 负责人: GREGORY DEAN BOWMAN
• 金额: $ 29.4万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-04-01 至 2017-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8727583
• 关键词: ATP Hydrolysis; ATP phosphohydrolase; Affect; B-DNA; Base Pairing; Binding; Biochemical; C-terminal; Cell Survival; Cell physiology; Cells; Characteristics; Chromatin; Chromatin Structure; Chromosomes; Cleaved cell; Collection; Complex; DNA; DNA Binding; DNA Binding Domain; DNA Footprint; DNA Packaging; DNA Sequence; DNA biosynthesis; Defect; Deletion Mutation; Development; Disease; Eye; Face; Fluorescence; Fluorescence Resonance Energy Transfer; Funding; Gene Expression; Gene Expression Regulation; Gene Silencing; Genes; Genetic Transcription; Genitourinary system; Genome; Geometry; Health; Heart; Histone H4; Kinetics; Laboratories; Lead; Learning; Left; Length; Link; Major Groove; Malignant Neoplasms; Minor Groove; Molecular; Motor; N-terminal; Normal Cell; Nucleosomes; Pattern; Phase; Play; Positioning Attribute; Prevalence; Reaction; Regulation; Research; Research Proposals; Role; Site; Slide; Specificity; Speed; Staging; Structure; Substrate Specificity; Surface; Tail; Testing; Tissue Differentiation; Variant; Work; X-Ray Crystallography; biological systems; cancer type; cell growth; cell transformation; chromatin remodeling; developmental disease; insight; public health relevance; recombinational repair

DESCRIPTION (provided by applicant): Chromatin, the physical packaging of eukaryotic chromosomes, plays a major role in determining the patterns of gene silencing and expression across the genome. The active reorganization of chromatin structure, critical for gene regulation, is achieved by ATP-dependent machines called chromatin remodelers, which disassemble, slide, and reassemble nucleosomes on DNA. Disruptions in chromatin remodeler function perturb gene expression and have been directly linked with a number of cancers and developmental disorders. At present, it is not understood at a molecular level how remodelers reposition and reorganize nucleosomes, and what factors give rise to particular biochemical characteristics of remodelers. This proposal aims to uncover how different domains of the Chd1 remodeler participate in the nucleosome sliding reaction. X-ray crystallography will be used to visualize both the central ATPase motor as well as the C-terminal DNA-binding domain in complex with DNA substrates, which will reveal how remodelers recognize and distort duplex DNA. The contributions of the Chd1 DNA-binding domain to the speed, spacing, and direction of nucleosome sliding will be determined with Chd1 variants that have modified binding domains and/or variations in the linking segment to the ATPase motor. The ATPase motor is regulated by a pair of N-terminal chromodomains, which appear to enhance substrate specificity of the remodeler. Rapid kinetic analyses of nucleosome sliding reactions using stopped flow FRET will be used to identify stage(s) in the nucleosome sliding cycle affected by ATPase regulation. The results of this research will advance our understanding of how chromatin remodelers work and select their substrates, which are essential steps for interpreting changes in chromatin landscapes between healthy and diseased cells.

描述（由申请人提供）：染色质，真核染色体的物理包装在确定基因组中基因沉默和表达的模式中起着重要作用。染色质结构对基因调节至关重要的主动重组是通过称为染色质重塑剂的ATP依赖机器来实现的，该机器在DNA上拆卸，滑动和重新组装核小体。染色质重塑函数的破坏扰动基因表达，并已直接与许多癌症和发育障碍联系在一起。目前，在分子级别尚不清楚重塑器如何重新定位和重组核小体，以及哪些因素会导致重塑剂的特定生化特征。 该建议旨在发现CHD1重塑的不同结构域如何参与核小体滑动反应。 X射线晶体学将用于可视化中心ATPase运动以及与DNA底物复合物中的C末端DNA结合结构域，这将揭示重塑剂如何识别和扭曲双层DNA。 CHD1 DNA结合结构域对核体滑动速度，间距和方向的贡献将由CHD1变体确定，这些变体已修改了结合域和/或链接段中与ATP酶电动机的变化。 ATPase电动机受一对N末端染色体的调节，似乎增强了重塑剂的底物特异性。使用停止流体FRET对核小体滑动反应的快速动力学分析将用于识别受ATPase调节影响的核小体滑动周期中的阶段。这项研究的结果将促进我们对染色质重塑器如何工作并选择其底物的理解，这是解释健康和患病细胞之间染色质景观变化的重要步骤。