Functional connections between histone variants and histone chaperones

描述（由申请人提供）：真核基因组被组织成染色质，染色质是一种复杂的核蛋白组装体，调节DNA的进入。染色质由核小体组成，核小体是由四种核心组蛋白中的每一种的两个拷贝组成的盘状八聚体，其外表面包裹着147 bp的DNA。核小体压缩成更高级的组装体，这些组装体由非组蛋白蛋白如连接体组蛋白H1稳定。我们的目标是发展一个定量的了解染色质如何组装和不可访问和可访问的状态之间的切换，从而调节必要的DNA代谢过程。不同组蛋白变体（具有不同序列和表达模式的核心组蛋白的非等位基因版本）在染色体中特定位置的掺入对许多细胞功能具有特定和深远的影响。组蛋白分子伴侣也影响基因组的可及性，并且是促进ATP非依赖性组蛋白交换、核小体组装和拆卸的结构多样的蛋白质组。组蛋白伴侣蛋白和组蛋白变体在功能上是相关的。在这里，我们测试的假设，纳入特定的组蛋白变体影响核小体的稳定性和动力学。这将导致变异核小体与接头组蛋白的相互作用改变，以及更高级结构的差异（目的1）。变体核小体的热力学和动力学性质的变化也可能影响它们被各种组蛋白伴侣作用的能力（目的2）。我们将量化几种组蛋白伴侣对主要类型和变体组蛋白的特异性，并测试关于其稳定组蛋白折叠状态的能力的新假设 并防止非经典组蛋白-DNA相互作用，无论是在体外使用我们的电池的分析方法和在体内的全基因组规模。单粒子荧光共振能量转移和小角X射线散射将用于表征变体核小体的溶液状态，而组蛋白伴侣对组蛋白变体复合物折叠状态的影响将通过氢氘交换耦合质谱法进行测定。我们的方法的优势在于结合了严格的分析方法和体内研究，以研究伴侣缺失对组蛋白分布的影响。这些研究具有重要的意义，因为它们系统地、定量地 测试关于组蛋白变体和组蛋白伴侣蛋白在核小体和更高级核小体组装的背景下的生物活性的相互关联的假设。我们挑战目前的核小体构象的观点，并描述具有巨大潜力的影响基因组的组织和可访问性的结构状态。

DESCRIPTION (provided by applicant): The eukaryotic genome is organized into chromatin, a complex nucleoprotein assembly that regulates access to the DNA. Chromatin consists of nucleosomes, disk-shaped octamers composed of two copies each of the four core histone proteins that wrap 147 bp of DNA around their outer surface. Nucleosomes compact into higher order assemblies that are stabilized by non-histone proteins such as linker histone H1. Our objective is to develop a quantitative understanding of how chromatin assembles and switches between inaccessible and accessible states, thereby regulating essential DNA metabolic processes. The incorporation of different histone variants (non-allelic versions of core histones with distinct sequence and expression pattern) at specific locations in the chromosome has specific and profound effects on many cellular functions. Histone chaperones also influence genome accessibility, and are a structurally diverse group of proteins that promote ATP-independent histone exchange, nucleosome assembly and disassembly. Histone chaperones and histone variants are functionally connected. Here we test the hypothesis that the incorporation of specific histone variants affects nucleosome stability and dynamics. This would result in altered interactions of variant nucleosomes with linker histones, and in differences in higher order structures (Aim 1). A change in thermodynamic and dynamic properties of variant nucleosomes also likely affects their ability to be acted upon by the various histone chaperones (Aim 2). We will quantify the specificity of several histone chaperones for major-type and variant histones, and test novel hypotheses regarding their ability to stabilize a folded state of histones and to prevent noncanonical histone-DNA interactions both in vitro using our battery of analytical approaches and in vivo on a genome-wide scale. Single-particle fluorescence resonance energy transfer and small angle x-ray scattering will be used to characterize in-solution states of variant nucleosomes, while the effect of histone chaperones on the folded state of histone variant complexes will be assayed by hydrogen-deuterium exchange coupled to mass spectrometry. The strength of our approach lies in the combination of rigorous analytical approaches with in vivo studies to investigate the effect of chaperone deletion on histone distribution. The proposed studies are highly significant as they systematically and quantitatively test interconnected hypotheses regarding the biological activities of histone variants and histone chaperones in the context of both nucleosomes and higher order nucleosome assemblies. We challenge current views of nucleosome conformation, and describe structural states that have enormous potential to impact genome organization and accessibility.