Chemical Reporters for the Analysis of Lysine Methylation in Epigenetics

描述（由申请人提供）：用于表观遗传学中赖氨酸甲基化分析的化学报告基因 生物信息代代相传的调节超出了DNA序列的水平。这一基本原理已在许多生物学背景中观察到，并被称为“表观遗传学”，它开始描述在给定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基因表达的调节，这体现在对核小体结构和功能（染色体的核心单元）的精细控制上。调节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中发挥着重要作用 [3, 4]。调节组蛋白赖氨酸甲基化的酶家族——赖氨酸甲基转移酶 (KMT) 和赖氨酸去甲基化酶 (KDM) 现已被鉴定，并且与转录调节、x 染色体失活和异染色质形成相关 [3, 4]。有趣的是，最近也描述了几种非组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，缺乏表征蛋白质及其各自酶上赖氨酸甲基化的通用方法，阻碍了对这种 PTM 如何调节信号转导和表观遗传机制的更全面的了解。为了充分了解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化学报告基因，以便能够使用生物正交标记方法快速检测和识别复杂混合物中的甲基化蛋白质（目标 1）。此外，我们将开发正交酶-底物对来识别单个赖氨酸甲基转移酶的选择性蛋白质底物（目标 2）。这些研究应该揭示新的赖氨酸甲基化蛋白质，并确定调节其在基本细胞途径和表型遗传的表观遗传机制中的功能的特定酶。最终，这些工具应该提供对正常生理和疾病中蛋白质甲基化的更普遍的了解。 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于人类健康至关重要。蛋白质上的可逆赖氨酸甲基化已成为调节表型遗传的重要 PTM，然而，赖氨酸甲基化的分析需要更通用的方法来表征 KMT 和 KDM 的特定底物。为了解决这个问题，该提案描述了化学报告基因的开发，以识别赖氨酸甲基化蛋白质和 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.