Systematic screening for new histone marks

描述（由申请人提供）： 越来越多的证据表明，表观遗传调控对各种生物过程和疾病至关重要。组蛋白的翻译后修饰（PTM）通常被认为是一组主要的表观遗传标记。许多组蛋白PTM已被证明有助于“组蛋白语言”和表观遗传程序，决定了不同的DNA模板的生物输出。然而，目前尚不清楚细胞中是否存在额外的组蛋白标记，如果存在，这些未发现的信号在表观遗传现象中的作用是什么。我们假设，未鉴定的PTM调节表观遗传机制存在于哺乳动物组蛋白。我们的假设是基于我们最近发现的两种新的组蛋白修饰，丙酰化和丁酰化的赖氨酸（KProp和KButy）和其他未知的PTM组蛋白，其化学结构仍有待确定。因此，我们建议全面筛选新的组蛋白PTM，并随后定义和验证其结构的新方法，涉及分析化学，有机合成，生物化学/免疫化学，和一个新的生物信息学。这种综合的方法允许全面，公正和敏感的组蛋白PTM的鉴定。我们已经证明了这种新方法的主要组成部分的可行性。据我们所知，这样一个系统的方法来发现新的组蛋白PTM还没有描述过。我们希望通过这项研究确定几种新的组蛋白PTM，扩展我们目前对组蛋白标记的了解。新的组蛋白PTM的鉴定将为研究界提供一个垫脚石，以调查这些组蛋白PTM在表观遗传机制和疾病中的可能作用。在本申请中提出了四个具体目标。首先，我们将通过研究组蛋白KProp和KButy在小鼠胚胎干细胞（mES）中的基因组分布和动态来研究其潜在的表观遗传作用。我们将使用蛋白质组学的方法来确定所有的残基，携带两个PTM的核心组蛋白从未分化的胚胎干细胞和他们的分化细胞。我们将通过ChIP-qPCR和ChIP-on-chip研究mES细胞分化过程中PTM的动力学及其对基因表达的贡献来研究这两种PTM在表观遗传调控中的可能作用。第二，我们将使用液相色谱、HPLC/MS/MS分析和新型生物信息学算法等综合方法鉴定和纯化具有新型PTM的组蛋白肽。将分离携带新型PTM的肽用于结构测定。第三，我们将通过高分辨率质谱分析和NMR光谱确定新型PTM部分的化学结构。新的PTM结构将通过各种分析技术和Western印迹分析进行验证。最后，我们将使用目标1中所述的实验，研究新组蛋白PTM在目标2和3中鉴定的mES细胞中的动力学和潜在表观遗传作用。公共卫生相关性：组蛋白翻译后修饰（PTM）参与多种细胞过程和疾病的表观遗传调控。该研究旨在鉴定和验证新的组蛋白PTM，并研究其在小鼠干细胞分化中的可能作用。由此产生的知识将扩大我们对组蛋白PTM的理解，并将刺激对新的组蛋白PTM在疾病进展中的表观遗传作用的研究。

DESCRIPTION (provided by applicant): Mounting evidence suggests that epigenetic regulation is critical to diverse biological processes and diseases. Post-translational modifications (PTM) in histones are generally considered to be a major group of epigenetic marks. Many histone PTMs have been shown to contribute to the "histone language" and epigenetic program that dictates diverse DNA-templated biological outputs. Nevertheless, it remains unclear whether additional histone marks exist in cells and, if so, what are the roles of these undiscovered signals in epigenetic phenomena. We hypothesize that unidentified PTMs that regulate epigenetic mechanisms are present in mammalian histones. Our hypothesis is based on our recent identification of two novel histone modifications, propionylation and butyrylation of lysine (KProp and KButy) and other unknown PTMs in histones, the chemical structures of which remain to be defined. We therefore propose to comprehensively screen for novel histone PTMs, and subsequently define and verify their structures by a novel approach, involving analytical chemistry, organic synthesis, biochemistry/immunochemistry, and a novel bioinformatics. This integrated approach allows for comprehensive, unbiased, and sensitive identification of histone PTMs. We have demonstrated the feasibility of major components of this novel method. To our knowledge, such a systematic approach to the discovery of new histone PTMs has not been described before. We expect to identify several novel histone PTMs as a result of this study, expanding our current knowledge of histone marks. Identification of novel histone PTMs will provide a stepping stone to the research community toward investigation of the possible roles of these histone PTMs in epigenetic mechanisms and diseases. Four specific aims are proposed in this application. First, we will investigate potential epigenetic roles of histone KProp and KButy by studying their genomic distribution and dynamics in mouse embryonic stem (mES) cells. We will use a proteomics approach to identify all the residues that bear the both PTMs in the core histones from undifferentiated mESs and their differentiated cells. We will examine the possible roles of the two PTMs in epigenetic regulation by studying the dynamics of the PTMs during mES cell differentiation and their contributions to the gene expression by ChIP- qPCR and ChIP-on-chip. Second, we will identify and purify histone peptides bearing novel PTMs using an integrated approach involving liquid chromatography, HPLC/MS/MS analysis, and a novel bioinformatics algorithm. The peptide bearing a novel PTM will be isolated for structure determination. Third, we will determine the chemical structures of the novel PTM moieties by high-resolution mass spectrometric analysis and NMR spectroscopy. The novel PTM structures will be verified by various analytical techniques and Western blotting analysis. Finally, we will study dynamics and potential epigenetic roles of the novel histone PTMs in mES cells that will be identified in Aim 2 and 3, using the experiments as described in Aim 1. PUBLIC HEALTH RELEVANCE: Histone post-translational modifications (PTMs) are known to be involved in the epigenetic regulation of diverse cellular processes and diseases. The proposed study aims to identify and validate novel histone PTMs, and to study their possible roles in mouse stem cell differentiation. The resulting knowledge will expand our understanding on histone PTMs and will stimulate the studies on the epigenetic roles of the novel histone PTMs in disease progression.