Functional connections between histone variants and histone chaperones

描述（由申请人提供）：真核生物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的获取。染色质由核小体组成，核小体是一种圆盘状的八聚体，由四个核心组蛋白的两个拷贝组成，它们在其外表面包裹着147bp的DNA。核小体紧密结合成高阶组合，由非组蛋白如连接组蛋白H1稳定。我们的目标是对染色质如何组装和在不可访问和可访问状态之间切换进行定量理解，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）在染色体特定位置的结合对许多细胞功能具有特异性和深远的影响。组蛋白伴侣也影响基因组的可及性，是一组结构多样的蛋白质，可促进不依赖atp的组蛋白交换、核小体组装和拆卸。组蛋白伴侣和组蛋白变体在功能上是相互联系的。在这里，我们测试了特定组蛋白变异的结合影响核小体稳定性和动力学的假设。这将导致变异核小体与连接体组蛋白相互作用的改变，以及高阶结构的差异（目的1）。变异核小体的热力学和动力学特性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蛋白对主要型组蛋白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

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.