Structural and dynamic studies of histone tails in chromatin by magnetic resonance spectroscopy

磁共振波谱法对染色质组蛋白尾部的结构和动态研究

基本信息

批准号：
9082087
负责人：
Christopher P Jaroniec
金额：
$ 32.43万
依托单位：
OHIO STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-09-16 至 2018-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9082087
关键词：
Acetylation Address Amides Amino Acid Sequence Amino Acids Binding Binding Proteins Biological Assay Chromatin Chromatin Fiber Chromatin Structure Complex Computational Technique Coupled Cryoelectron Microscopy DNA Disease Drug resistance Electron Spin Resonance Spectroscopy Environment Epigenetic Process Event Foundations Future Gene Expression Regulation Genetic Transcription Genome Stability Higher Order Chromatin Structure Histone H1 Histone H1(s)Histone H2A Histone H3 Histone H4 Histones Label Lysine Magic Magnetic Resonance Spectroscopy Measurement Mediating Methodology Methods Methylation Modeling Molecular Monitor N-terminal NMR Spectroscopy Nature Nuclear Magnetic Resonance Nucleosomes Peptides Physiological Positioning Attribute Post-Translational Protein Processing Proteins Publishing RNA Reader Recombinants Recruitment Activity Regulation Relaxation Reporting Resolution Site Spin Labels Structure Tail Time Transcriptional Activation Transcriptional Regulation Vertebral column Work X-Ray Crystallography anticancer research cancer therapy chromatin protein computer studies design flexibility insight molecular dynamics mutant novel anticancer drug protein complex public health relevance reconstitution repaired solid state nuclear magnetic resonance stem tumorigenesis

项目摘要

DESCRIPTION (provided by applicant): PROJECT SUMMARY Chromatin is the eukaryotic complex of DNA with proteins that regulates transcription, replication and repair through dynamic changes in its structure. The DNA in chromatin is packaged into repeat nucleosome building blocks, with each nucleosome consisting of ~147 bp of DNA wrapped nearly twice around a histone protein octamer containing two copies each of histones H2A, H2B, H3 and H4. All histones contain disordered N- terminal tail domains, corresponding to ~15-30% of their amino acid sequences that protrude out from the nucleosome. The N-terminal tails of histones H3 and H4 are essential regulators of chromatin function. These domains interact with DNA and other histones to mediate chromatin compaction, recruit a variety of chromatin regulatory factors, and have their functions regulated by numerous post-translational modifications (PTMs). While the atomic structure of the nucleosome and arrangements of nucleosomes within evenly spaced arrays representative of chromatin fibers have been resolved by X-ray crystallography and cryo-electron microscopy, the histone N-terminal tails have escaped high-resolution characterization in densely packed nucleosome arrays. The latter is due to their intrinsic disorder coupled with the fact that they are an integral part of large multi-megadalton protein-DNA assemblies. To address these challenges and directly investigate histone tail domains in chromatin at physiological concentrations, we have applied magic-angle spinning (MAS) solid-state nuclear magnetic resonance (NMR) to recombinant nucleosome arrays reconstituted with 13C,15N-enriched histones. Our recently published initial high-resolution MAS NMR studies revealed that N-terminal domains of histones H3 and H4 are conformationally dynamic even in highly condensed chromatin. These findings strongly suggest that histone tails do not act as static tethers to compact chromatin and recruit PTM-binding proteins and have caused us to reevaluate their function in chromatin. The central hypothesis of this proposal is that histone tails in chromatin function through the modulation of their conformational dynamics by different factors, which allows these domains to mediate interactions within chromatin while remaining accessible to chromatin regulatory complexes. To investigate this hypothesis we will pursue the following three aims: (1) determine how the conformational flexibility of histone tails functions with nucleosome positioning and linker histones to regulate higher order chromatin structure and dynamics, (2) determine how acetylation of histone H4 lysine 16 regulates chromatin compaction, and (3) determine the regulation of H3 tail dynamics by trimethylated lysine 36 and PHF1. The proposed studies will provide the first high-resolution insights into how H3 and H4 tails control critical events that regulate transcription including chromatin compaction and recruitment of an essential PTM-binding protein, and are highly significant for understanding the function of histone tails in chromatin. Finally, these studies will provide an important foundation for future work on key histone PTM-binding complexes in the chromatin environment.

描述（适用提供）：项目摘要染色质是DNA的真核复合物，其蛋白质通过动态变化的结构变化来调节转录，复制和修复。将染色质中的DNA包装到重复的核小体构建块中，每个核小体由〜147 bp的DNA组成，几乎包裹在组蛋白蛋白八聚体周围，其中包含两个组蛋白H2A，H2B，H3和H4的两份副本。所有组蛋白都包含无序的N末端尾域，对应于从核小体中突出的氨基酸序列的约15-30％。组蛋白H3和H4的N末端是染色质功能的必不可少的调节剂。这些结构域与DNA和其他组蛋白相互作用，以介导染色质压实，募集各种染色质调节因子，并受到许多翻译后修饰（PTM）的调节功能。虽然X射线晶体学和冷冻电子显微镜在代表染色质纤维的均匀间隔阵列中的核小体的原子结构和核小体的排列已经解决，但组蛋白N末端尾巴却逐渐脱离了无填充的核小组阵列中的高分辨率表征。后者是由于它们的固有疾病以及它们是大型多麦达尔顿蛋白-DNA组件的组成部分的事实。为了应对这些挑战，并直接研究了物理浓度的染色质组蛋白尾域，我们已经应用了魔法旋转（MAS）固态核磁共振（NMR），以重组核共振阵阵列，以13C，15N增强的组蛋白重新构成。我们最近发表的初始高分辨率MAS NMR研究表明，即使在高度凝结的染色质中，组蛋白H3和H4的N末端结构域也是构象动态的。这些发现强烈表明，组蛋白的尾巴不充当静态泪液，可促进染色质并募集PTM结合蛋白，并导致我们在染色质中重新评估其功能。该提议的中心假设是，通过不同因素调节其构象动力学的组蛋白尾巴在染色质功能中尾巴，这使这些结构域可以介导染色质中的相互作用，同时仍可以通过染色质调节络合物访问。 To investigate this hypothesis we will pursue the following three aims: (1) determine how the conformational flexibility of histone tails functions with nuclearsome positioning and linker histones to regulate higher order chromatin structure and dynamics, (2) determine how acetylation of histone H4 lysine 16 regulates chromatin compaction, and (3) determine the regulation of H3 tail dynamics by trimethylated lysine 36 and PHF1.拟议的研究将提供第一个高分辨率见解，以了解H3和H4尾部如何控制调节转录的关键事件，包括染色质压实和必需的PTM结合蛋白的募集，并且对于理解组蛋白尾巴在染色质中的功能非常重要。最后，这些研究将为重要的基础提供重要的基础对于染色质环境中关键组蛋白PTM结合复合物的未来工作。