Identification of the Interactome of Methylated Histones from Human Proteome

描述(申请人提供)：由组蛋白赖氨酸甲基化介导的分子网络错综复杂。目前，组蛋白赖氨酸甲基化介导的信号通路的作用机制仍很不清楚。假设组蛋白在不同赖氨酸残基和不同状态(如二甲基化和三甲基化)的甲基化被相应的结合效应器识别，从而开启和关闭相关的下游信号通路。一个有待解决的主要问题是如何定义每个甲基化的组蛋白尾巴的甲基化依赖的相互作用组。该项目旨在开发转化技术，以鉴定与赖氨酸残基上每个甲基化组蛋白状态结合的效应蛋白的自然谱系。在化学和生物学的界面上集成了强大的技术来解决这个问题。具体地说，我们将使用一种名为mRNA展示的新蛋白质组学技术，在这种技术中，每个蛋白质都与自己的mRNA共价连接。结合不同甲基化状态的蛋白质序列将从一个显示mRNA的人类蛋白质组文库中分离出来。这些选择将在非常特殊的条件下进行。在捕获甲基化的组蛋白尾部/结合伙伴复合体后，将使用未经修饰的组蛋白多肽去除甲基化无关的结合子，而依赖于所需甲基化状态的结合伙伴将使用相应的二甲基化或三甲基化的组蛋白多肽被特异性洗脱。由于基因和表型之间的结合，所选择的序列可以很容易地被扩增，用于反复几轮选择，使我们能够解决在蛋白质组大范围内识别相对较弱的甲基化依赖的蛋白质-蛋白质相互作用的困难。作为备用计划，我们将使用含有可交联组蛋白亮氨酸部分的甲基化的组蛋白尾肽来促进捕获与甲基化的组蛋白尾部相互作用相对较弱的低丰度蛋白质。虽然我们在这个项目中使用了人类蛋白质组来解决这个问题，但该方法可以很容易地应用于模式生物，如线虫、斑马鱼和果蝇。 公共卫生相关性：甲基化依赖的相互作用组的获得将使我们能够破译每个组蛋白甲基化状态的结合效应，从而极大地促进我们对组蛋白赖氨酸甲基化所介导的信号通路的理解。它还应该帮助开发治疗和成像试剂，使其能够操纵与包括癌症在内的许多人类疾病有关的表观遗传过程。

DESCRIPTION (provided by applicant): The molecular networks mediated by histone lysine methylation are intricate and extremely complex. Currently, the action mechanisms of the signaling pathways mediated by histone lysine methylation are still very elusive. It is hypothesized that the histone methylation at different lysine residues and at different states (e.g., di- and trimethylation) are recognized by the corresponding binding effectors that subsequently turn on and off the related downstream signaling pathways. One major problem that remains to be addressed is how to define the methylation-dependent interactome for each methylated histone tail. This project is aimed at developing transforming technologies that allow the identification of the natural repertoire of the effector proteins that bind to each of the methylated histone states at lysine residues. Powerful techniques at the interface of chemistry and biology are integrated to address the problem. Specifically, we will use a novel proteomic technique called mRNA-display in which each protein is covalently linked to its own mRNA. The protein sequences that bind to distinct methylation states will be isolated from an mRNA-displayed human proteome library. The selections will be performed under very specific conditions. After capturing the methylated histone tail/binding partner complexes, the methylation-independent binders will be removed by using the unmodified histone peptide, while those binding partners that are dependent on the desired methylation state will be specifically eluted using the corresponding di- or trimethylated histone peptide. Due to the conjugation between the genotype and the phenotype, the selected sequences can be readily amplified for iterative rounds of selection, allowing us to address the difficulty in identifying the relatively weak methylation-dependent protein-protein interactions on a proteome wide scale. As a backup plan, we will use the methylated histone tail peptides that contain a crosslinkable photo-leucine moiety to facilitate the capture of low abundant proteins whose interactions with methylated histone tails are relatively weak. Although we address the problem using the human proteome in this project, the method can be readily applied to model organisms such as C. elegans, zebrafish and Drosophila. PUBLIC HEALTH RELEVANCE: The availability of the methylation-dependent interactome will allow us to decipher the binding effectors of each histone methylation state and therefore greatly facilitate our understanding of the signaling pathways mediated by histone lysine methylation. It should also aid in the development of therapeutic and imaging agents that allow the manipulation of epigenetic processes involved in a number of human diseases including cancer.