Systematic screening for new histone marks

系统筛选新的组蛋白标记

• 批准号: 7693723
• 负责人: YINGMING ZHAO
• 金额: $ 77.65万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-09-30 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7693723
• 关键词: ADP ribosylation; Acetylation; Algorithms; Analytical Chemistry; Animals; Atlases; Biochemistry; Bioinformatics; Biological; Biological Process; Calculi; Cell physiology; Cells; Chemical Structure; Chromatin Structure; Chromatography; Classification; Communities; Correlation Studies; DNA Microarray Chip; DNA Repair; DNA biosynthesis; Development; Disease; Disease Progression; Embryo; Epigenetic Process; Gene Expression; Genes; Genomics; High Pressure Liquid Chromatography; Histone H4; Histones; Immunochemistry; Investigation; Knowledge; Language; Liquid Chromatography; Lysine; Malignant Neoplasms; Maps; Methods; Methylation; Microarray Analysis; Mus; NMR Spectroscopy; Organic Synthesis; Output; Peptides; Phosphorylation; Post-Translational Protein Processing; Proline; Property; Proteomics; Regulation; Research; Resolution; Role; Screening procedure; Side; Signal Transduction; Source; Structure; Techniques; Undifferentiated; Ursidae Family; Western Blotting; base; chemical property; chromatin immunoprecipitation; embryonic stem cell; genome-wide; histone modification; improved; in vivo; interest; knowledge of results; mouse genome; novel; novel strategies; programs; public health relevance; research study; stem; stem cell differentiation

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.

描述(由申请人提供)： 越来越多的证据表明，表观遗传调控对不同的生物过程和疾病至关重要。组蛋白的翻译后修饰(PTM)通常被认为是一类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，这决定了不同的DNA模板生物输出。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，这些未被发现的信号在表观遗传现象中扮演什么角色。我们假设，在哺乳动物的组蛋白中存在调节表观遗传机制的未知的PTM。我们的假设是基于我们最近发现的两种新的组蛋白修饰，赖氨酸的丙酰化和丁酰化(KProp和KButy)以及组蛋白中其他未知的PTM，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，并随后通过一种新的方法来定义和验证它们的结构，涉及分析化学、有机合成、生物化学/免疫化学和新的生物信息学。这种综合的方法可以对组蛋白PTM进行全面、公正和灵敏的鉴定。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，这种系统的方法来发现新的组蛋白PTM以前从未被描述过。作为这项研究的结果，我们希望发现几个新的组蛋白PTM，扩大我们目前对组蛋白标记的了解。新的组蛋白PTM的鉴定将为研究界研究这些组蛋白PTM在表观遗传机制和疾病中的可能作用提供一个垫脚石。在这项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在小鼠胚胎干细胞中的基因组分布和动力学来研究它们潜在的表观遗传学作用。我们将使用蛋白质组学的方法来鉴定在未分化的MISH及其分化的细胞中的核心组蛋白中含有这两个PTM的所有残基。我们将通过研究PTM在MES细胞分化过程中的动态以及它们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。其次，我们将使用一种综合的方法来鉴定和提纯携带新的PTM的组蛋白多肽，该方法包括高效液相色谱、高效液相色谱/MS/MS分析和一种新的生物信息学算法。含有新的PTM的多肽将被分离出来用于结构测定。第三，我们将通过高分辨质谱分析和核磁共振光谱确定新的PTM部分的化学结构。新的PTM结构将通过各种分析技术和Western blotting分析进行验证。最后，我们将使用目标1中描述的实验，研究将在目标2和3中确定的新型组蛋白PTM在MES细胞中的动力学和潜在的表观遗传学作用。公共卫生相关性：组蛋白翻译后修饰(PTM)已知参与各种细胞过程和疾病的表观遗传调节。这项拟议的研究旨在鉴定和验证新的组蛋白PTM，并研究它们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PTMS的理解，并将刺激对新型组蛋白PTMS在疾病进展中的表观遗传学作用的研究。