Conformational Control of Heterochromatin Formation by the HP-1 Protein from Fission Yeast

裂殖酵母 HP-1 蛋白对异染色质形成的构象控制

基本信息

批准号：
9568786
负责人：
John D Gross
金额：
$ 39.24万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-09-30 至 2021-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9568786
关键词：
Address Affinity Animal Model Architecture Binding Biochemical Biochemical Genetics Biological Biological Assay Cells Chromatin Chromatin Structure Chromosome Segregation Complex Computer-Assisted Image Analysis Cryoelectron Microscopy Crystallization DNA Data Dimerization Docking Essential Genes Euchromatin Fission Yeast Foundations Gene Silencing Genome Genome Stability Goals Hematopoietic Neoplasms Heritability Heterochromatin Histone H3 Histones Human In Vitro Knowledge Link Lysine Mass Spectrum Analysis Mediating Modeling Molecular Conformation NMR Spectroscopy Nature Normal Cell Nucleosome Core Particle Nucleosomes Pathology Play Polymers Protein Family Proteins Reporting Resolution Rest Roentgen Rays Role Schizosaccharomyces pombe Proteins Structural Models Structure Tail Telomere Maintenance Testing Work X-Ray Crystallography biophysical techniques cancer cell conformational conversion crosslink genetic analysis heterochromatin-specific nonhistone chromosomal protein HP-1 in vivo insight mutant polymerization recruit restraint

项目摘要

Project Summary Eukaryotic genomes are organized into active and inactive domains referred to euchromatin and heterochromatin. This functional organization plays an important role in chromosome segregation, telomere maintenance and genome stability. A key component of heterochromatin are the HP-1 family of proteins, which bind to a histone 3 lysine-9 methyl mark and act as a platform for diverse regulators. A biochemical activity thought to be important for the spread of heterochromatin is the ability of HP-1 proteins to polymerize. Recent work on the fission yeast HP-1 protein, Swi6, reveals that polymerization is regulated by autoinhibition. A critical and poorly understood question is the extent of the conformational transition between closed, inactive and open, active forms of Swi6 that drive heterochromatin spread. This gap in knowledge derives from the fact that HP-1 proteins and their complexes with nucleosomes are conformationally dynamic in solution and difficult to crystallize. Here we will define the structural dynamics of the Swi6-chromatin complex and link the structural states to function. In Aim 1 we will determine the structure of the autoinhibited form of Swi6 using an integrated modeling approach that employs restraints NMR spectroscopy and small-angle x-ray scattering in solution (SAXS). The biological significance of the structural models will be tested in gene silencing assays in the fission yeast S. pombe. In Aim 2, we will determine the degree of conformational rearrangements of chromatin when Swi6 engages the nucleosome to form a spreading competent state. The structure and dynamics of the nucleosome-Swi6 complex will be interrogated by a combination of biophysical methods, such as Methyl-TROSY NMR and HD-exchange mass-spectrometry (MS), that can provide residue specific structural information in solution. Cross-linking mass-spectrometry in conjunction with cryoEM will be employed to obtain models of the spreading competent form if Swi6 bound to nucleosomes. This approach will shed insights into conformational control of heterochromatin formation by Swi6 in fission yeast and provide a conceptual foundation for how heterochromatin is regulated in human cells.

项目概要真核基因组被组织成活性和非活性结构域，称为常染色质和异染色质。这种功能组织在染色体分离、端粒维护和基因组稳定性。异染色质的一个关键组成部分是 HP-1 蛋白家族，它与组蛋白 3 赖氨酸 9 甲基标记结合并充当多种调节因子的平台。生化活性 HP-1 蛋白的聚合能力被认为对于异染色质的传播很重要。最近的对裂殖酵母 HP-1 蛋白 Swi6 的研究揭示了聚合是通过自抑制来调节的。一个关键且知之甚少的问题是封闭的、不活跃的构象转变的程度以及驱动异染色质扩散的开放、活跃形式的 Swi6。这种知识差距源于以下事实 HP-1 蛋白及其与核小体的复合物在溶液中具有构象动态，并且难以结晶。在这里，我们将定义 Swi6-染色质复合物的结构动力学，并将结构链接起来状态才能发挥作用。在目标 1 中，我们将使用集成的方法确定 Swi6 自抑制形式的结构在溶液中采用约束 NMR 光谱和小角 X 射线散射的建模方法（萨克斯）。结构模型的生物学意义将在基因沉默测定中进行测试裂殖酵母粟酒裂殖酵母。在目标 2 中，我们将确定构象重排的程度当 Swi6 与核小体结合形成扩散活性状态时，染色质会发生变化。结构和核小体-Swi6 复合物的动力学将通过生物物理方法的组合进行研究，例如甲基-TROSY NMR 和 HD 交换质谱 (MS)，可以提供残留物特异性解决方案中的结构信息。将采用交联质谱法与冷冻电镜相结合获得 Swi6 与核小体结合时的传播能力形式的模型。这种方法会脱落深入了解裂殖酵母中 Swi6 对异染色质形成的构象控制，并提供人类细胞中异染色质如何调节的概念基础。