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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.

项目总结