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

在真核细胞中，DNA被染色质包裹。在表观遗传信号中，核小体代表染色质的亚单位和中枢。它们由四种核心组蛋白的组蛋白八聚体组成，每个拷贝两个拷贝，周围包裹着大约147个碱基对的DNA。所有组蛋白的两侧都有固有的无序的尾巴，这些尾巴携带着多种翻译后修饰(PTMS-甲基化、乙酰化和其他)。通过它们的PTM，组蛋白可以破坏或调节与其他染色质因子的相互作用，从而协调时间和空间的途径。它需要一种可以通过多个组蛋白修饰之间的串扰实现的切换。组蛋白尾巴可以广泛地与核小体和连接物DNA相互作用，这些相互作用可以极大地改变DNA和组蛋白位点对读者、作家和擦除者的可及性。然而，在核小体的背景下，关于组蛋白尾巴及其与DNA结合的研究很少，这是由于它们的实验和电子表征的困难。我们建议启动一项新的合作，通过组蛋白尾部修饰及其相互作用来确定调控的分子基础。我们检验了主要假说，根据该假说，组蛋白尾巴的动力学及其与核小体中DNA的相互作用是解释组蛋白尾巴修饰的串扰的重要因素。我们将在硅胶、体外和体内实验中进行。使用改进的采样方法和全原子分子动力学模拟，将对核小体和具有和不具有PTMS的游离肽中的尾部构象进行采样。PTMS对尾巴柔韧性的影响以及与DNA的相互作用将通过核磁共振光谱分析和无偏质谱分析得到验证。该项目的成果将为染色质因子参与转录和复制的物理化学机制提供深入的见解。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，并将为连接实验和电子方法、基础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.