Structural and Functional Analysis of the CHD1 Chromatin Remodeler

描述(申请人提供)：染色体的物理组织与基因调控密切相关。染色质重塑是指真核细胞染色体通过核小体的共价修饰和物理运动而发生的动态紧致和解聚。了解推动重塑的过程对于更有洞察力地理解人类疾病是至关重要的，这些疾病是由正常基因调控中断引起的，如癌症和遗传性发育障碍。关于ATP依赖的重塑因子改变核小体结构的机制，人们知之甚少，不了解不同的重塑结构域如何在功能上相互作用以促进重塑，关键的重塑结构域如何在空间中相互定位，也不了解重塑如何拥抱核小体底物。这项建议的长期目标是建立一个描述和理解染色质重塑过程所必需的生化和生物物理框架。这项建议集中在CHD1染色质重塑因子上，具体目的是(1)通过表征部分功能失调的变体来剖析CHD1重塑周期，(2)使用X射线结晶学确定CHD1重构体的结构，以及(3)使用小角X射线散射(SAXS)和DNA足迹确定CHD1：核小体复合体的组织。通过将CHD1的功能研究与通过X射线结晶学、SAXS和DNA足迹获得的结构信息相结合，我们有望阐明重塑反应的关键步骤。在未来，对染色质重塑的更深层次的了解将是开发操纵全基因组基因表达用于治疗人类疾病的治疗方法的关键。与公共健康相关人体内几乎所有细胞都拥有相同的一组基因，但在任何特定的组织中，只有这些基因的一个子集被“激活”。基因的“开”和“关”状态是以一种复杂和模糊的方式进行调节的，这种方式与染色体的物理包装直接相关，称为染色质结构。了解染色体被所谓的染色质重塑因子拆解和重新包装的过程对于理解并因此对抗许多疾病是必要的，例如癌症，这些疾病存在基因表达的不平衡。

DESCRIPTION (provided by applicant): The physical organization of chromosomes is intimately tied to gene regulation. Chromatin remodeling refers to the dynamic compaction and decondensation of eukaryotic chromosomes through the covalent modification and physical movement of nucleosomes. Knowledge of the processes that drive remodeling are essential for gaining a more insightful understanding of human diseases that result from disruptions of normal gene regulation, such as cancer and inherited developmental disorders. Relatively little is known regarding the mechanism by which ATP-dependent remodeling factors alter nucleosome structure, with no understanding of how distinct remodeler domains functionally interact to promote remodeling, how key remodeler domains are positioned in space with respect to one another, nor how remodelers embrace the nucleosome substrate. The long-term objective of this proposal is to establish a biochemical and biophysical framework necessary for describing and understanding the process of chromatin remodeling. This proposal focuses on the CHD1 chromatin remodeling factor, with specific aims to (1) dissect the CHD1 remodeling cycle by characterizing partially dysfunctional variants, (2) determine the architecture of the CHD1 remodeler using X-ray crystallography, and (3) determine the organization of a CHD1:nucleosome complex using small angle X-ray scattering (SAXS) and DNA footprinting. By coupling functional studies of CHD1 with structural information gained through X-ray crystallography, SAXS, and DNA footprinting, we expect to elucidate key steps of the remodeling reaction. In the future, a deeper understanding of chromatin remodeling will be essential for developing therapeutics that manipulate genome-wide gene expression for treatment of human disease. PUBLIC HEALTH RELEVANCE Nearly all cells in the human body possess the same set of genes, yet only a subset of these genes are turned "on" in any particular tissue. The "on" and "off" states of genes are regulated in a complex and ill-defined manner that directly correlates with the physical packaging of chromosomes, called the chromatin structure. Understanding the process by which chromosomes can be unpackaged and repackaged by so-called chromatin remodeling factors is necessary for understanding and therefore combatting many diseases such as cancer where there are imbalances in gene expression.