Structural and Functional Analysis of the Chd1 Chromatin Remodeler

描述（由申请人提供）：染色质是真核生物染色体的物理包装，在决定基因组中基因沉默和表达模式方面起主要作用。染色质结构的主动重组，对基因调控至关重要，是由ATP依赖性的机器实现的，称为染色质重塑剂，它在DNA上分解，滑动和重新组装核小体。染色质重塑功能的破坏扰乱基因表达，并与许多癌症和发育障碍直接相关。目前，在分子水平上尚不清楚重塑者如何重新定位和重组核小体，以及哪些因素引起重塑者的特定生化特征。 该提案旨在揭示Chd 1重塑剂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央ATP酶马达以及与DNA底物复合的C-末端DNA结合结构域，这将揭示重塑者如何识别和扭曲双链DNA。Chd 1 DNA结合结构域对核小体滑动的速度、间距和方向的贡献将用具有修饰的结合结构域和/或ATP酶马达连接片段的变化的Chd 1变体来确定。ATP酶马达由一对N-末端的染色体结构域调节，这似乎增强了重塑剂的底物特异性。使用停流FRET的核小体滑动反应的快速动力学分析将用于鉴定受ATP酶调节影响的核小体滑动循环中的阶段。这项研究的结果将促进我们对染色质重塑如何工作和选择其底物的理解，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。

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.