Chemical Reporters for the Analysis of Lysine Methylation in Epigenetics

描述(申请人提供)：用于表观遗传学中赖氨酸甲基化分析的化学记者生物信息的世代传递受到超出DNA序列水平的调控。这一基本原理已经在许多生物学背景下被观察到，并被称为“表观遗传学”，它开始描述在给定的遗传背景下环境对表型的基本贡献[1]。表观遗传机制的核心是调控基因表达，这表现在对染色体的核心单位--核小体结构和功能的精细控制。调控转录的关键因素之一是染色质相关蛋白(组蛋白)的翻译后修饰(PTM)[2]。特别是，组蛋白尾部的动态赖氨酸甲基化似乎在调节基因表达和表观遗传现象方面发挥了重要作用[3，4]。赖氨酸甲基转移酶(KMTs)和赖氨酸去甲基酶(KDms)是调节组蛋白上赖氨酸甲基化的酶家族，现已被鉴定，并与转录调节、x染色体失活和异染色质形成有关[3，4]。有趣的是，最近还描述了几种非组蛋白蛋白上的赖氨酸甲基化，这引发了许多关于KMT和KDM的特异性以及它们在表观遗传学中的离散作用的问题[2-5]。不幸的是，缺乏通用的方法来表征蛋白质及其相应酶上的赖氨酸甲基化，阻碍了对这种PTM如何调节信号转导和表观遗传机制的更广泛的理解。为了充分认识赖氨酸甲基化在生物途径中的作用，需要具有更高灵敏度和通用性的新方法。因此，我们建议开发赖氨酸甲基化的化学报告程序，这将使使用生物正交标记方法快速检测和鉴定复杂混合物中的甲基化蛋白成为可能(目标1)。此外，我们将开发正交酶-底物对来确定单个赖氨酸甲基转移酶的选择性蛋白质底物(目标2)。这些研究应该发现新的赖氨酸甲基化蛋白，并确定在表型遗传的基本细胞途径和表观遗传机制中调节它们功能的特定酶。最终，这些工具将提供对正常生理和疾病中蛋白质甲基化的更全面的了解。 公共卫生相关性：表观遗传机制对表型的调节是许多生物过程和疾病的核心。因此，详细了解控制表观遗传学的潜在机制对人类健康至关重要。蛋白质的可逆赖氨酸甲基化已成为调节表型遗传的一种重要的PTM，然而，对赖氨酸甲基化的分析需要更通用的方法来表征KMT和KDMS的特定底物。为了解决这个问题，这项建议描述了化学报告的发展，以确定赖氨酸甲基化的蛋白质和KMT的特定底物。如果成功，这些化学方法将为科学界提供一套新的工具来分析蛋白质甲基化在基本细胞途径和表观遗传过程中的作用。

DESCRIPTION (provided by applicant): Chemical Reporters for the Analysis of Lysine Methylation in Epigenetics The transmission of biological information over generations is regulated beyond the level of the DNA sequence. This fundamental principle has been observed in many biological contexts and has been termed "epigenetics", which begins to describe the basic contribution of environment to phenotype under a given genetic background [1]. At the heart of epigenetic mechanisms is regulated gene expression, which manifests itself in the fine control of nucleosome structure and function, the core unit of chromosomes. One of the key factors that modulate transcription is the posttranslational modification (PTM) of chromatin-associated proteins (histones) [2]. In particular, dynamic lysine methylation of histone tails appears to play essential roles in regulating gene expression and epigenetic phenomena [3, 4]. The families of enzymes that regulate lysine methylation on histones, lysine methyltransferases (KMTs) and lysine demethylases (KDMs), have now been identified and are associated with transcriptional regulation, x-chromosomal inactivation and heterochromatin formation [3, 4]. Interestingly, lysine methylation on several non-histone proteins has also been recently described, which has raised many questions regarding the specificity of KMTs and KDMs as well as their discrete roles in epigenetics [2-5]. Unfortunately, the lack of general methods to characterize lysine methylation on proteins and their respective enzymes has hindered a more general understanding of how this PTM regulates signal transduction and epigenetic mechanisms. To fully appreciate the roles of lysine methylation in biological pathways, new methods with higher sensitivity and generality are required. We therefore propose to develop chemical reporters for lysine methylation that will enable rapid detection and identification of methylated proteins in complex mixtures using bioorthogonal labeling methods (Aim 1). Furthermore, we will develop orthogonal enzyme-substrate pairs to identify selective protein substrates of individual lysine methyltransferases (Aim 2). These studies should uncover novel lysine-methylated proteins and identify specific enzymes that regulate their function in fundamental cellular pathways and epigenetic mechanisms of phenotypic inheritance. Ultimately, these tools should provide a more general understanding of protein methylation in normal physiology and disease. PUBLIC HEALTH RELEVANCE: The modulation of phenotypes by epigenetic mechanisms is central to many biological processes and diseases. A detail understanding of the underlying mechanisms that control epigenetics is therefore essential to human health. Reversible lysine methylation on proteins has emerged as an important PTM that regulates the inheritance of phenotypes, however, the analysis of lysine methylation requires more general methods to characterize specific substrates for KMTs and KDMs. To address this problem, this proposal describes the development of chemical reporters to identify lysine-methylated proteins and specific substrates of KMTs. If successful, these chemical approaches would provide the scientific community with a new set of tools to analyze the role of protein methylation in fundamental cellular pathways and in epigenetic processes.