Regulation of chromatin organization and dynamics by INO80

INO80 对染色质组织和动力学的调节

• 批准号: 9914760
• 负责人: Blaine Bartholomew
• 金额: $ 55.87万
• 依托单位: UNIVERSITY OF TX MD ANDERSON CAN CTR
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-05-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9914760
• 关键词: 3-Dimensional; ATP phosphohydrolase; ATPase Domain; Affect; Binding; Biochemical; Biological Assay; Catalytic Domain; Cells; Centromere; Chemicals; Chromatin; Chromosomes; Communication; Complex; Coronary; Coupling; Cryoelectron Microscopy; DNA; DNA Crosslinking; DNA Double Strand Break; DNA Repair; DNA Sequence; DNA biosynthesis; DNA-Protein Interaction; Data; Enhancers; Formaldehyde; Gatekeeping; Gene Expression; Genome; Genomics; Heterochromatin; Histone H4; Histones; Hot Spot; Human; Hydrolysis; In Vitro; Lead; Length; Linker DNA; Malignant Neoplasms; Mass Spectrum Analysis; Measures; Mediating; Methods; Molecular; Molecular Conformation; Motor; Movement; Mutate; Mutation; Nucleosome Core Particle; Nucleosomes; Outcome; Positioning Attribute; Promoter Regions; Protein-Protein Interaction Map; Publishing; Recombinants; Regulation; Resolution; Rest; Role; Series; Specificity; Structure; Surface; Testing; Time; To specify; Transcriptional Regulation; Vascular Diseases; Yeasts; analog; base; chromatin remodeling; crosslink; dimer; experimental study; human disease; improved; in vitro Assay; in vivo; innovation; insight; mutant; organizational structure; promoter; reconstitution; telomere; whole genome; yeast genome

Summary We know very little about the mechanism of INO80, how it disrupts nucleosomes and the factors governing its activity. We will take detailed “snapshots” of INO80 during nucleosome remodeling to find how INO80 and nucleosomes are moved during remodeling. A series of orthogonal approaches will be used to arrest INO80 remodeling at distinct stages and examine conformational changes in the core nucleosome and INO80. We will build on our recent observations of the motor domain being engaged at the H2A-H2B interface and persistently displacing DNA from this surface to find why displacement occurs, the factors that control displacement and whether this displacement weakens the interactions of H2A or H2A.Z dimers with the rest of the histone octamer or otherwise disrupts the nucleosome structure. Based on the proximity of Arp5 to nucleosomal DNA, we will test the premise of Arp5 as the “gatekeeper” regulating DNA traversing through the center of nucleosomes with wild type INO80 and mutant Arp5 in which either its histone or nucleosome binding regions have been deleted or mutated. We will also test whether the Arp8 module regulates Arp5 interactions with the acidic pocket of nucleosomes or nucleosomal DNA and if communication between these two domains is mediated by the Ino80 catalytic subunit. INO80 will be arrested at different stages in remodeling by limiting DNA translocations to specified distances, arresting with non-hydrolyzable ATP analogs, limiting linker DNA length and mutation of Arp8 and Arp5. We will probe the role of DNA sequence in INO80 remodeling because we observed coupling of ATPase activity to nucleosome movement being dramatically affected by the DNA sequence of the core nucleosome. We will find as suggested in these experiments if INO80 interactions and conformation varies depending on the DNA sequence bound by nucleosomes. In order to better examine the importance of DNA sequence in a “native” context, we will use yeast chromatin reconstituted with recombinant histones and simultaneously examine the differences of INO80 binding and remodeling with many thousands of nucleosomes, each with a different DNA sequence. We will use our expertise of mapping protein-DNA interactions in these genomic assays to sort with high precision the interactions of the INO80 subunits along with nucleosome movement, composition and structural features at ~bp resolution to provide a detailed analysis of each of these nucleosomes in a time resolved manner when remodeled. This approach will provide more insights into the DNA sequence specificity of INO80 and if there are “hot spots” for mobilizing/ destabilizing nucleosomes or exchanging H2A.Z in the yeast genome that doesn’t require additional factors. To confirm if INO80 behaves the same in vivo as in our in vitro assays, we will transfer several of these approaches to yeast cells so that we can measure chromatin dynamics in vivo with the same resolution as in vitro. We will compare how mutations in Arp5 and Arp8 change nucleosome dynamics in vivo, the importance of genomic position, and other factors for INO80 remodeling not present in our yeast reconstituted chromatin.

总结 我们对INO 80的机制、它如何破坏核小体以及控制其破坏的因素知之甚少。 活动我们将在核小体重塑过程中拍摄INO 80的详细“快照”，以了解INO 80和INO 80之间的关系。 核小体在重塑过程中移动。采用一系列正交实验方法， 重塑在不同的阶段，并检查在核心核小体和INO 80的构象变化。我们将 基于我们最近对H2 A-H2 B界面上的运动域的观察， 从这个表面置换DNA，以找出为什么发生置换，控制置换的因素， 这种置换是否减弱了H2 A或H2A.Z二聚体与组蛋白八聚体其余部分的相互作用 或者破坏核小体结构。根据Arp 5与核小体DNA的接近程度，我们将进行测试 Arp 5作为“看门人”调节DNA穿过核小体中心的前提是野生型的Arp 5是一种非编码的蛋白质。 INO 80型和突变型Arp 5，其中组蛋白或核小体结合区已缺失，或 变异了我们还将测试Arp 8模块是否调节Arp 5与 核小体或核小体DNA，并且如果这两个结构域之间的通讯由Ino 80介导， 催化亚单位INO 80将在重塑的不同阶段通过限制DNA易位而被阻止， 特定的距离，用不可水解的ATP类似物阻止，限制接头DNA长度和 Arp 8和Arp 5。我们将探讨DNA序列在INO 80重塑中的作用，因为我们观察到DNA序列与INO 80的偶联。 核心DNA序列对核小体运动的ATP酶活性有显著影响 核小体如这些实验所示，我们将发现INO 80相互作用和构象是否发生变化 这取决于核小体结合的DNA序列。为了更好地检验DNA的重要性 在“天然”环境中，我们将使用重组组蛋白重构的酵母染色质， 同时检查INO 80与数千个核小体的结合和重塑的差异， 每一个都有不同的DNA序列我们将利用我们在蛋白质-DNA相互作用方面的专业知识， 基因组测定以高精度分选INO 80亚基沿着与核小体的相互作用 在~bp分辨率下的运动、成分和结构特征，以提供这些中的每一个的详细分析 核小体以时间分辨的方式重构。这种方法将提供更多关于DNA的见解， INO 80的序列特异性，以及是否存在用于移动/去稳定核小体的“热点”， 在酵母基因组中交换H2A.Z，不需要额外的因子。为了确认INO 80是否表现出 与我们的体外试验相同，我们将把这些方法中的几种转移到酵母细胞中， 以与体外相同的分辨率测量体内染色质动力学。我们将比较Arp 5基因突变 和Arp 8改变核小体在体内的动力学，基因组位置的重要性，以及INO 80的其他因素 重塑不存在于我们的酵母重组染色质。