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

 描述（由申请人提供）：项目概述染色质是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，每个核小体由约147 bp的DNA组成，DNA围绕组蛋白八聚体包裹近两次，组蛋白八聚体含有两个拷贝的组蛋白H2 A，H2 B，H3和H4。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其氨基酸序列的约15-30%。组蛋白H3和H4的N-末端尾部是染色质功能的重要调节剂。这些结构域与DNA和其他组蛋白相互作用以介导染色质致密化，募集各种染色质调节因子，并且具有由许多翻译后修饰（PTM）调节的功能。虽然核小体的原子结构和核小体在代表染色质纤维的均匀间隔阵列内的排列已经通过X射线晶体学和冷冻电子显微镜解析，但组蛋白N-末端尾部在密集排列的核小体阵列中已经逃脱了高分辨率表征。后者是由于它们的内在无序加上它们是大型多兆道尔顿蛋白质-DNA组装体的组成部分的事实。为了解决这些挑战，并直接调查组蛋白尾域在染色质中的生理浓度，我们已经应用魔角旋转（MAS）固态核磁共振（NMR）重组核小体阵列重建与13 C，15 N-丰富的组蛋白。我们最近发表的初步高分辨率MAS NMR研究表明，组蛋白H3和H4的N-末端结构域是构象动态的，即使在高度浓缩的染色质。这些发现强烈表明，组蛋白尾部不作为静态系链压缩染色质和招募PTM结合蛋白，并使我们重新评估其在染色质中的功能。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对其构象动力学的调节来发挥功能，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接头组蛋白一起发挥作用，以调节更高级的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致密化，以及（3）确定三甲基化赖氨酸36和PHF 1对H3尾部动力学的调节。拟议的研究将提供第一个高分辨率的见解，了解H3和H4尾巴如何控制调节转录的关键事件，包括染色质压缩和募集一个必要的PTM结合蛋白，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，这些研究将提供重要的基础 为将来在染色质环境中的关键组蛋白PTM结合复合物的工作。

 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.