Regulation of chromatin organization and dynamics by INO80

摘要 我们对INO80的机制、它如何破坏核小体以及影响其的因素知之甚少。 活动。我们将在核小体重塑过程中拍摄详细的INO80快照，以找出INO80和INO80是如何 核小体在重塑过程中移动。将使用一系列正交方法来逮捕INO80 在不同的阶段重塑，并检查核心核小体和INO80的构象变化。我们会 基于我们最近观察到的运动域在H_2A-H_2B界面上的持续存在 从这个表面置换DNA，以找出置换发生的原因，控制置换的因素和 这种置换是否削弱了H_2A或H_2A·Z二聚体与组蛋白八聚体的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，我们将测试 Arp5作为“把关人”调控DNA穿越核小体中心的前提 INO80型和Arp5型突变体，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 催化亚基。INO80将通过将DNA易位限制在 特定距离，用非水解性ATP类似物阻止，限制接头DNA长度和突变 Arp8和Arp5。我们将探讨DNA序列在INO80重塑中的作用，因为我们观察到 核小体运动的ATPase活性受核心DNA序列的显著影响 核小体。我们将在这些实验中发现，如果INO80相互作用和构象发生变化 取决于核小体结合的DNA序列。为了更好地检验DNA的重要性 序列，我们将使用重组组蛋白和重组酵母染色质 同时检测INO80与数千个核小体结合和重塑的差异， 每一种都有不同的DNA序列。我们将利用我们绘制蛋白质-DNA相互作用图谱的专业知识 高精度分类INO80亚基与核小体相互作用的基因组分析 在~BP分辨率下的运动、组成和构造特征，以提供对每一个的详细分析 核小体在重塑时以时间分辨的方式。这种方法将提供对DNA的更多洞察力 INO80的序列特异性以及是否存在动员/破坏核小体稳定的“热点”或 在酵母基因组中交换不需要额外因子的H2A.Z。要确认INO80是否表现为 与我们的体外测试一样，我们将把这些方法中的几种转移到酵母细胞中，这样我们就可以 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Arp5基因的突变如何 和Arp8改变体内核小体动力学、基因组位置的重要性等因素对INO80的影响 我们的酵母重组染色质中不存在重塑。

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.