Structural and Functional Analysis of the Chd1 Chromatin Remodeler

描述(申请人提供)：染色质，真核细胞染色体的物理包装，在决定整个基因组中基因沉默和表达的模式中发挥着重要作用。染色质结构的主动重组对基因调控至关重要，它是通过被称为染色质重构体的依赖于ATP的机器来实现的，它可以拆解、滑动和重组DNA上的核小体。染色质重塑功能的中断扰乱了基因的表达，并与许多癌症和发育障碍直接相关。目前，在分子水平上还不清楚重构体如何重新定位和重组核小体，以及是什么因素引起重构体的特殊生化特征。该建议旨在揭示Chd1重构体的不同结构域如何参与核小体滑动反应。X射线结晶学将被用来显示中央ATPase马达以及与DNA底物形成的复合体中的C-末端DNA结合域，这将揭示重构体如何识别和扭曲双链DNA。Chd1 DNA结合域对核小体滑动速度、间距和方向的贡献将由Chd1变异体决定，Chd1变异体具有修改的结合域和/或连接到ATPase马达的链段的变异。ATPase马达由一对N-末端的色素域调节，这似乎增强了底物重构体的特异性。使用停流FRET对核小体滑动反应的快速动力学分析将被用来识别受ATPase调节影响的核小体滑动周期中的阶段(S)。这项研究的结果将促进我们对染色质重构体如何工作的理解，并选择它们的底物，这是解释健康和疾病细胞之间染色质景观变化的关键步骤。

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.