Functional connections between histone variants and histone chaperones

描述(申请人提供)：真核基因组被组织成染色质，这是一种复杂的核蛋白组合，调节对DNA的访问。染色质由核小体组成，盘状八聚体由四个核心组蛋白各两个副本组成，外表面包裹着147bp的DNA。核小体紧凑成由非组蛋白蛋白(如连接物组蛋白H1)稳定的高阶组合体。我们的目标是对染色质如何组装并在不可及和可及状态之间切换，从而调节必要的DNA代谢过程有一个定量的了解。不同的组蛋白变体(核心组蛋白的非等位基因版本，具有不同的序列和表达模式)在染色体上的特定位置融合在一起，对许多细胞功能具有特定而深远的影响。组蛋白伴侣蛋白也影响基因组的可及性，是一组结构不同的蛋白质，促进不依赖于ATP的组蛋白交换、核小体组装和分解。组蛋白伴侣蛋白和组蛋白变体在功能上是相连的。在这里，我们测试了一个假设，即特定的组蛋白变体的加入影响核小体的稳定性和动力学。这将导致不同的核小体与连接子组蛋白的相互作用改变，并导致更高级别结构的差异(目标1)。变异核小体的热力学和动力学性质的改变也可能影响它们被不同的组蛋白伴侣蛋白作用的能力(目标2)。我们将量化几个组蛋白伴侣蛋白对主要类型和变异组蛋白的特异性，并测试关于它们稳定组蛋白折叠状态的能力的新假设。 用我们的一系列分析方法在体外和体内在全基因组范围内防止非规范的组蛋白-DNA相互作用。单粒子荧光共振能量转移和小角x射线散射将被用来表征不同核小体的溶液状态，而组蛋白伴侣对组蛋白不同复合体折叠状态的影响将通过氢-氢交换-质谱联用来分析。我们方法的优势在于将严格的分析方法与体内研究相结合，以研究伴侣蛋白缺失对组蛋白分布的影响。拟议的研究具有非常重要的意义，因为它们是系统的和定量的 在核小体和高阶核小体组装的背景下，测试关于组蛋白变体和组蛋白伴侣蛋白的生物学活性的相互关联的假设。我们挑战了当前对核小体构象的看法，并描述了具有巨大潜力影响基因组组织和可获得性的结构状态。

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.