Regulation of Histone Chaperone Function by Glutamylation

谷氨酰化调节组蛋白伴侣功能

• 批准号: 10321249
• 负责人: DAVID SHECHTER
• 金额: $ 36.96万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10321249
• 关键词: Affinity; Animals; Binding; Biological; Biological Process; Cell Cycle; Cell physiology; Cells; Chromatin; Chromatin Modeling; Cytoskeleton; DNA; Data; Deposition; Development; Diagnosis; Disease; Epigenetic Process; Event; Gene Expression; Genes; Genetic Transcription; Genome; Histone H2A; Histones; Human; In Vitro; Knowledge; Liquid substance; Measures; Mediating; Modeling; Molecular; Molecular Chaperones; Molecular Conformation; Nature; Nuclear; Nucleosomes; Pathology; Pathway interactions; Phase; Physiological; Post-Translational Protein Processing; Process; Protein Region; Proteins; Rana; Recycling; Regulation; Role; Site; Surface; Testing; Vision; Work; Xenopus; Xenopus laevis; egg; epigenetic regulation; experimental study; genome integrity; in vivo; insight; novel; nucleoplasmin; protein function; repaired; tool; vertebrate genome

ABSTRACT Histone chaperones escort histones into and out of chromatin, the physiological form of the eukaryotic genome. Many chaperones use acidic intrinsically disordered regions (IDRs) to interact with histones. These IDRs contain the post-translational modification glutamylation. Glutamylation’s role in modulating chaperone function is poorly understood. The perplexing observations that chaperone mono-glutamylation–catalyzed by TTLL4 and removed by CCP5–enhances both histone affinity and deposition highlight a central enigma of histone chaperone mechanisms: the tighter that a chaperone binds to histones, the better a chaperone is at releasing them into chromatin. To explain this, our studies suggest that histone chaperones use their glutamylated acidic IDRs as a DNA-like surface to: 1) capture histones; 2) shield non-specific histone interactions; and 3) structurally stabilize and orient bound histones. Therefore, we hypothesize that glutamylated chaperone IDRs mimic DNA to facilitate binding and pre-organization of histones into a nucleosome-compatible conformation. We will test this hypothesis in the following aims. In Aim 1, we will determine how glutamylated acidic IDRs from the chaperones Nap1 and Nucleoplasmin (Npm2) modulate histone H2A/H2B and linker histone interactions. Using our comprehensive in vitro platform for studying the disorder and function of histone chaperones, we will determine how chaperone IDR glutamylation modulates its histone interaction affinity; we will use NMR to determine where glutamylated IDRs interact with histones and how IDRs structurally stabilize histones; and we will measure the impact of chaperone glutamylation on chromatin assembly in vitro. In Aim 2, we will probe the biological function of chaperone acidic IDR glutamylation. Using our Xenopus laevis cell-free extract model, we will determine when and how chaperone glutamylation modulates chromatin assembly in vivo. We will: identify sites of endogenous chaperone glutamylation; probe the regulation and biological role of chaperone glutamylation during the cell cycle; and measure the impact of chaperone glutamylation on chromatin assembly in egg extract. Our study will move the field forward by answering the central enigma of chaperone function: how is it that the best chaperones for depositing histones also bind histones the tightest? We will similarly advance our knowledge by understanding a novel means of regulating histone chaperone function. Because histone chaperones are critical for genome integrity and gene expression, our work has wide significance. Understanding how chaperones are regulated during the cell cycle will provide key insight for studies of animal development, chromosomal replication, gene expression, and damage repair. IDRs and their PTMs–found in over 30% of eukaryotic proteins–are key components of many regulatory processes, such as liquid-liquid phase-separations. Chaperones are now an important example of this class of proteins. As our work will contribute to the emerging and generalizable concept that the dynamic nature of disordered protein regions is at the core of their function, this proposal is broadly significant.

抽象的 组蛋白伴侣护送组蛋白进入染色质，这是真核的物理形式 基因组。许多伴侣使用酸性固有无序区域（IDR）与组蛋白相互作用。这些 IDR包含翻译后修饰谷氨酸。谷氨酸在调节链酮中的作用 功能知之甚少。令人困惑的观察结果表明，由 TTLL4并被CCP5增强 - Hisstone亲和力和沉积物均突出了一个中心的谜 Hisstone伴侣机制：伴侣与组蛋白结合的较紧，伴侣越好 将它们释放到染色质中。为了解释这一点，我们的研究表明，Hisstone伴侣使用他们 谷氨酸化的酸性IDR作为DNA样表面至：1）捕获组蛋白； 2）盾牌非特异性组蛋白 互动； 3）在结构上稳定和向前结合的组蛋白。因此，我们假设 谷氨酸的伴侣IDRS模拟DNA，以促进组蛋白的结合和预组织 核小体兼容构象。我们将在以下目的中检验这一假设。在AIM 1中，我们将 确定来自伴侣NAP1和核纤维蛋白（NPM2）的谷氨酸化酸性IDR如何调节 Hisstone H2A/H2B和Linker Hisstone互动。利用我们全面的体外平台来研究 组蛋白伴侣的障碍和功能，我们将确定伴侣IDR谷氨酸如何调节 它的Hisstone互动亲和力；我们将使用NMR确定谷氨酸IDR与组蛋白相互作用 以及IDR如何在结构上稳定组蛋白；我们将测量伴侣谷氨酸对 体外染色质组装。在AIM 2中，我们将探测伴侣酸性IDR的生物学功能 谷氨酸。使用我们的Xenopus laevis无细胞提取物模型，我们将确定何时以及如何方式 谷氨酸在体内调节染色质组装。我们将：识别内源性链酮的位置 谷氨酸；探测细胞周期期间伴侣谷氨酸的调节和生物学作用；和 测量伴侣谷氨酸对鸡蛋提取物中染色质组件的影响。我们的研究将移动 通过回答伴侣功能的中心谜来向前介绍：如何最好的伴侣 沉积组蛋白也将组蛋白结合最紧吗？我们将通过理解来提高我们的知识 一种新型的调节组蛋白链函数的方法。因为组蛋白链对于基因组至关重要 完整性和基因表达，我们的工作具有广泛的意义。了解如何调节伴侣 在细胞周期期间，将为动物发育，染色体复制，基因的研究提供关键见解 表达和损坏修复。 IDR及其PTMS - 在30％以上的真核蛋白中发现 - 关键 许多调节过程的组成部分，例如液 - 液相分离。伴侣现在是 这类蛋白质的重要​​例子。因为我们的工作将有助于新兴和可推广 无序蛋白质区域的动态性质是其功能的核心，该建议是 广泛意义。