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

• 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
• 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

 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（由申请人提供）： 项目摘要 染色质是 DNA 与蛋白质的真核复合物，通过其结构的动态变化来调节转录、复制和修复。染色质中的 DNA 被包装成重复的核小体构件，每个核小体由约 147 bp 的 DNA 组成，在组蛋白八聚体周围包裹近两圈，其中组蛋白 H2A、H2B、H3 和 H4 各有两个拷贝。所有组蛋白都含有无序的 N 末端尾部结构域，对应于从核小体突出的约 15-30% 的氨基酸序列。组蛋白 H3 和 H4 的 N 末端尾部是染色质功能的重要调节因子。这些结构域与 DNA 和其他组蛋白相互作用，介导染色质压缩，招募多种染色质调节因子，并通过大量翻译后修饰 (PTM) 调节其功能。虽然核小体的原子结构和代表染色质纤维的均匀间隔阵列内核小体的排列已通过 X 射线晶体学和冷冻电子显微镜解析，但组蛋白 N 末端尾部在密集堆积的核小体阵列中未能进行高分辨率表征。后者是由于它们的内在无序以及它们是大型多兆道尔顿蛋白质-DNA 组装体的组成部分这一事实造成的。为了解决这些挑战并直接研究生理浓度下染色质中的组蛋白尾部结构域，我们将魔角旋转 (MAS) 固态核磁共振 (NMR) 应用于用 13C,15N 富集组蛋白重建的重组核小体阵列。我们最近发表的初始高分辨率 MAS NMR 研究表明，即使在高度浓缩的染色质中，组蛋白 H3 和 H4 的 N 末端结构域也具有构象动态。这些发现强烈表明，组蛋白尾部并不充当压缩染色质和招募 PTM 结合蛋白的静态系绳，并促使我们重新评估它们在染色质中的功能。该提议的中心假设是，染色质中的组蛋白尾部通过不同因素调节其构象动力学来发挥功能，这使得这些结构域能够介导染色质内的相互作用，同时保持对染色质调节复合物的可及性。为了研究这一假设，我们将追求以下三个目标：(1) 确定组蛋白尾部的构象灵活性如何与核小体定位和连接组蛋白一起发挥作用，以调节高级染色质结构和动力学，(2) 确定组蛋白 H4 赖氨酸 16 的乙酰化如何调节染色质压缩，以及 (3) 确定三甲基化赖氨酸 36 和 PHF1 对 H3 尾部动力学的调节。拟议的研究将首次提供关于 H3 和 H4 尾如何控制调节转录的关键事件的高分辨率见解，包括染色质压缩和必需 PTM 结合蛋白的招募，并且对于理解组蛋白尾在染色质中的功能具有非常重要的意义。最后，这些研究将为 用于染色质环境中关键组蛋白 PTM 结合复合物的未来工作。