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：
10546440
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 2024-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10546440
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 Length Linker DNA Malignant Neoplasms Maps 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 Sorting Specific qualifier value Specificity Structure Surface Testing Time Transcriptional Regulation Vascular Diseases Visualization Yeasts analog 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的机制知之甚少，它如何破坏核小体和控制其的因素活动。我们将在核小体重塑过程中详细介绍Ino80的“快照”，以查找Ino80和核小体在重塑过程中移动。一系列正交方法将用于逮捕INO80 在不同的阶段进行重塑，并检查核心核小体和INO80的构象变化。我们将建立在我们最近对H2A-H2B接口的电机域的观察结果，并持续从该表面置换DNA，以了解为什么发生位移，控制位移和这种位移是否会削弱H2A或H2A.Z二聚体与其余组蛋白八聚体的相互作用或否则会破坏核小体结构。基于ARP5与核小体DNA的接近性，我们将测试 ARP5的前提为调节DNA穿过核心体中心的“守门人” INO80类型和突变体ARP5，其中已删除其Hisstone或核小体结合区域突变。我们还将测试ARP8模块是否调节ARP5与ARP5的相互作用核小体或核小体DNA，如果这两个结构域之间的通信是由INO80介导的催化亚基。 INO80将通过将DNA易位限制在重塑时在不同阶段被捕指定的距离，用不可氢化的ATP类似物阻止，限制了接头DNA的长度和突变 ARP8和ARP5。我们将探测DNA序列在INO80重塑中的作用，因为我们观察到偶联的耦合 ATPase活性对核小体运动受到核心的DNA序列的极大影响核小体。如果Ino80相互作用和构象各种取决于由核小体结合的DNA序列。为了更好地检查DNA的重要性在“天然”上下文中的序列，我们将使用重组组蛋白重组的酵母染色质和类似地检查INO80结合和重塑与数千个核组的差异，每个都有不同的DNA序列。我们将在这些中使用我们的绘制蛋白-DNA相互作用的专家基因组刺激以高精度排序INO80亚基与核小体的相互作用以〜bp分辨率的运动，组成和结构特征，以提供对每种的详细分析重塑时，核小组以时间分辨的方式解决。这种方法将为DNA提供更多的见解 INO80的序列特异性，如果有“热点”用于动员/破坏核小体的稳定或在不需要其他因素的酵母基因组中交换H2A.Z。确认Ino80是否行为与体外测定中的体内相同，我们将其中几种方法转移到酵母细胞上，以便我们可以在体内测量染色质动力学，其分辨率与体外相同。我们将比较ARP5中的突变 ARP8在体内改变核小体动力学，基因组位置的重要性以及其他因素对INO80 我们的酵母重构染色质中不存在重塑。

项目成果

期刊论文数量（4）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

INO80 Chromatin Remodeler Facilitates Release of RNA Polymerase II from Chromatin for Ubiquitin-Mediated Proteasomal Degradation.

INO80 染色质重塑器促进 RNA 聚合酶 II 从染色质中释放，以实现泛素介导的蛋白酶体降解。

DOI：
10.1016/j.molcel.2015.10.028
发表时间：
2015-12-03
期刊：
Molecular cell
影响因子：
16
作者：
Lafon A;Taranum S;Pietrocola F;Dingli F;Loew D;Brahma S;Bartholomew B;Papamichos-Chronakis M
通讯作者：
Papamichos-Chronakis M

INO80 exchanges H2A.Z for H2A by translocating on DNA proximal to histone dimers.

DOI：
10.1038/ncomms15616
发表时间：
2017-06-12
期刊：
Nature communications
影响因子：
16.6
作者：
Brahma S;Udugama MI;Kim J;Hada A;Bhardwaj SK;Hailu SG;Lee TH;Bartholomew B
通讯作者：
Bartholomew B

The Arp8 and Arp4 module acts as a DNA sensor controlling INO80 chromatin remodeling.

DOI：
10.1038/s41467-018-05710-7
发表时间：
2018-08-17
期刊：
Nature communications
影响因子：
16.6
作者：
Brahma S;Ngubo M;Paul S;Udugama M;Bartholomew B
通讯作者：
Bartholomew B

New insights into the mechanism and DNA-sequence specificity of INO80 chromatin remodeling.

对 INO80 染色质重塑机制和 DNA 序列特异性的新见解。

DOI：
10.21203/rs.3.rs-3443329/v1
发表时间：
2023
期刊：
Research square
影响因子：
0
作者：
Bartholomew,Blaine;Shukla,Shagun;Ngubo,Mzwanele;Paul,Somnath;Persinger,Jim;Brahma,Sandipan
通讯作者：
Brahma,Sandipan