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

 描述(由申请人提供)：染色质是DNA与蛋白质的真核复合体，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建块，每个核小体由~147bp的DNA组成，几乎两次包裹在一个组蛋白八聚体周围，组蛋白八聚体含有两个拷贝的组蛋白H_2A、H_2B、H_3和H_4。所有的组蛋白都含有无序的N末端尾部结构域，相当于其从核小体伸出的氨基酸序列的~15%-30%。组蛋白H3和H4的N末端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，介导染色质紧凑，招募各种染色质调节因子，并受大量翻译后修饰(PTM)调节其功能。虽然核小体的原子结构和以染色质纤维为代表的均匀排列的核小体的排列已经通过X射线结晶学和冷冻电子显微镜得到了解析，但组蛋白的N末端尾巴在密集排列的核小体阵列中逃脱了高分辨率的表征。后者是由于它们的内在无序性，加上它们是大型多兆吨蛋白质-DNA组装的组成部分。为了应对这些挑战并在生理浓度下直接研究染色质中的组蛋白尾部结构域，我们将魔角旋转(MAS)固体核磁共振(NMR)应用于由13C，15N富集组蛋白重组的核小体阵列。我们最近发表的高分辨率MAS核磁共振研究表明，即使在高度浓缩的染色质中，组蛋白H3和H4的N-末端结构域也是构象动态的。这些发现有力地表明，组蛋白尾巴并不是压紧染色质和招募PTM结合蛋白的静态纽带，这促使我们重新评估它们在染色质中的功能。这一提议的中心假设是，染色质中的组蛋白尾巴通过不同因素调节其构象动力学来发挥作用，这使得这些结构域能够调节染色质内部的相互作用，同时保持可被染色质调节复合体访问。为了研究这一假说，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子组蛋白一起调节高阶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。这项拟议的研究将首次提供高分辨率的见解，了解H3和H4尾巴如何控制调控转录的关键事件，包括染色质压缩和必要的PTM结合蛋白的招募，这对于理解组蛋白尾巴在染色质中的功能具有重要意义。最后，这些研究将提供一个重要的基础 用于在染色质环境中研究关键的组蛋白PTM结合复合体的未来工作。

 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.