Deciphering the functional significance and physico-chemical mechanisms of histone post-translational modification cross-talk

在真核细胞中，DNA被包装在染色质中。核小体是染色质的亚单位，是表观遗传信号传递的枢纽。它们由四种类型的核心组蛋白的组蛋白八聚体组成，每个组蛋白两个拷贝，周围包裹着约147个碱基对的DNA。所有组蛋白的侧翼都是固有无序的尾部，这些尾部携带多种翻译后修饰（PTM-甲基化、乙酰化等）。通过它们的PTM，组蛋白可以破坏或介导与其他染色质因子的相互作用，从而在时间和空间上协调途径。它需要一个开关，可以通过多个组蛋白修饰之间的串扰来实现。组蛋白尾部可以广泛地与核小体和接头DNA相互作用，并且这些相互作用可以极大地改变DNA和组蛋白位点对阅读器、写入器和擦除器的可接近性。然而，在核小体的背景下，对组蛋白尾部及其与DNA结合的研究很少，这是由于其实验和计算机表征的困难。我们建议发起一项新的合作，以确定通过组蛋白尾部修饰及其串扰进行调控的分子基础。我们测试的主要假设，根据组蛋白尾部的动力学和它们与核小体中的DNA的相互作用是一个重要因素，解释组蛋白尾部修饰的串扰。 我们将进行计算机模拟、体外和体内实验。将使用增强的采样方法和全原子分子动力学模拟对核小体和有和没有PTM的游离肽中的尾部构象进行采样。PTM对尾部柔性和与DNA相互作用的影响将通过NMR光谱和无偏质谱分析进行验证。该项目的成果将提供深入了解的物理化学机制的参与染色质因子的转录和复制。这项研究将把组蛋白修饰的某些组合与不同的染色质状态联系起来，并将为实验和计算机模拟方法、基础科学和应用科学提供联系。

In eukaryotic cells DNA is packed in chromatin. Nucleosomes represent subunits of chromatin and hubs in epigenetic signalling. They are comprised of a histone octamer of four types of core histones, two copies each, with ~147 base pairs of DNA wrapped around them. All histones are flanked by the intrinsically disordered tails that carry multiple post-translational modifications (PTMs - methylation, acetylation and others). Through their PTMs, histones can disrupt or mediate the interactions with other chromatin factors, thereby coordinating pathways in time and space. It requires a switch that can be achieved by a cross-talk between multiple histone modifications. Histone tails can extensively interact with the nucleosomal and linker DNA and these interactions can drastically change the DNA and histone site accessibility to readers, writers and erasers. However, there are very few studies on histone tails and their binding to DNA in the context of nucleosomes, which is explained by the difficulty in their experimental and in silico characterization. We propose to initiate a new collaboration to identify the molecular basis for regulation via histone tail modifications and their cross-talk. We test the main hypothesis according to which the dynamics of histone tails and their interactions with DNA in nucleosomes is an important factor explaining the cross-talk of histone tail modifications. We will perform in silico, in vitro and in vivo experiments. The sampling of tail conformations in nucleosomes and in a free peptide with and without PTMs will be done using enhanced sampling methods and all-atom molecular dynamics simulations. The effects of PTMs on tail flexibility and interactions with DNA will be verified by NMR spectroscopy and unbiased mass spectrometry analyses. Outcomes of this project will provide insights into physico-chemical mechanisms of engagement of chromatin factors in transcription and replication. The study will link certain combinations of histone modifications with distinct chromatin states and will provide liaisons to bridge experimental and in silico approaches, basic and applied science.