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于多种生物过程和疾病至关重要。组蛋白翻译后修饰（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，从而决定不同的 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，这些未发现的信号在表观遗传现象中的作用是什么。我们假设哺乳动物组蛋白中存在调节表观遗传机制的未识别 PTM。我们的假设基于我们最近鉴定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，其化学结构仍有待定义。因此，我们建议全面筛选新型组蛋白 PTM，并随后通过新方法定义和验证其结构，包括分析化学、有机合成、生物化学/免疫化学和新型生物信息学。这种集成方法可以全面、公正且灵敏地识别组蛋白 PTM。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，这种发现新组蛋白 PTM 的系统方法以前从未被描述过。我们希望通过这项研究鉴定出几种新的组蛋白 PTM，从而扩展我们目前对组蛋白标记的了解。新型组蛋白 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奠定基础。本申请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 (mES) 细胞中的基因组分布和动态来研究它们的潜在表观遗传作用。我们将使用蛋白质组学方法来鉴定来自未分化 mES 及其分化细胞的核心组蛋白中带有两个 PTM 的所有残基。我们将通过 ChIP-qPCR 和 ChIP-on-chip 研究 mES 细胞分化过程中 PTM 的动态及其对基因表达的贡献，从而研究这两个 PTM 在表观遗传调控中的可能作用。其次，我们将使用涉及液相色谱、HPLC/MS/MS 分析和新型生物信息学算法的综合方法来鉴定和纯化带有新型 PTM 的组蛋白肽。带有新型 PTM 的肽将被分离以进行结构测定。第三，我们将通过高分辨率质谱分析和核磁共振波谱确定新型 PTM 部分的化学结构。新的 PTM 结构将通过各种分析技术和蛋白质印迹分析进行验证。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 中鉴定的 mES 细胞中新型组蛋白 PTM 的动态和潜在表观遗传作用。 公共健康相关性：已知组蛋白翻译后修饰 (PTM) 参与多种细胞过程和疾病的表观遗传调控。拟议的研究旨在鉴定和验证新型组蛋白 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 PTM 的理解，并将刺激对新型组蛋白 PTM 在疾病进展中的表观遗传作用的研究。