Regulation of chromatin organization and dynamics by INO80

INO80 对染色质组织和动力学的调节

基本信息

批准号：
10321641
负责人：
Blaine Bartholomew
金额：
$ 45.52万
依托单位：
UNIVERSITY OF TX MD ANDERSON CAN CTR
依托单位国家：
美国
项目类别：
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/10321641
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 promoter reconstitution telomere whole genome yeast genome

项目摘要

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.

概括我们对 INO80 的机制、它如何破坏核小体以及控制其影响的因素知之甚少。我们将在核小体重塑过程中拍摄 INO80 的详细“快照”，以了解 INO80 和核小体在重塑过程中发生移动，将使用一系列正交方法来抑制 INO80。我们将在不同阶段进行重构并检查核心核小体和 INO80 的构象变化。基于我们最近对 H2A-H2B 界面上运动域的观察，并持续从该表面置换 DNA，找出发生置换的原因、控制置换的因素以及这种位移是否削弱了 H2A 或 H2A.Z 二聚体与组蛋白八聚体其余部分的相互作用或以其他方式破坏核小体结构基于 Arp5 与核小体 DNA 的接近程度，我们将进行测试。 Arp5作为调节DNA穿过核小体中心的“看门人”的前提 INO80 型和突变体 Arp5，其中组蛋白或核小体结合区已被删除，或我们还将测试 Arp8 模块是否调节 Arp5 与酸性口袋的相互作用。核小体或核小体 DNA 以及这两个结构域之间的通讯是否由 Ino80 介导 INO80 将通过限制 DNA 易位而在重塑的不同阶段被阻止。指定的距离，用不可水解的 ATP 类似物进行抑制，限制连接子 DNA 长度和突变由于我们观察到了 Arp8 和 Arp5 的耦合，因此我们将探讨 DNA 序列在 INO80 重塑中的作用。核小体运动的 ATP 酶活性受到核心 DNA 序列的显着影响如果 INO80 相互作用和构象发生变化，我们将发现如这些实验中所建议的那样。取决于核小体结合的DNA序列，以便更好地检验DNA的重要性。在“天然”环境中的序列，我们将使用用重组组蛋白重构的酵母染色质和同时检查 INO80 与数千个核小体结合和重塑的差异，每个都有不同的 DNA 序列，我们将利用我们在绘制蛋白质-DNA 相互作用方面的专业知识。基因组分析可高精度分类 INO80 亚基与核小体的相互作用〜bp 分辨率的运动、组成和结构特征，以提供对其中每一项的详细分析核小体在重构时以时间分辨的方式进行，这种方法将为 DNA 提供更多的见解。 INO80 的序列特异性以及是否存在动员/不稳定核小体的“热点”或在酵母基因组中交换 H2A.Z 不需要额外的因素来确认 INO80 是否表现出与我们的体外测定相同，我们将把其中几种方法转移到酵母细胞中，以便我们可以以与体外相同的分辨率测量体内染色质动态我们将比较 Arp5 的突变情况。 Arp8 改变体内核小体动力学、基因组位置的重要性以及 INO80 的其他因素我们的酵母重构染色质中不存在重塑。