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，表观遗传调节对于多种生物学过程和疾病至关重要。组蛋白中的翻译后修饰（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，该程序决定了不同的DNA模拟生物产量。然而，尚不清楚细胞中是否存在其他组蛋白标记，如果是，这些未被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鉴定的PTM。我们的假设是基于我们最近对两种新型组蛋白修饰的鉴定，赖氨酸（Kprop和kbuty）的丙酰化和丁酰化和组蛋白中其他未知的PTM的化合物，其化学结构仍有待定的定义。因此，我们建议全面筛选新的组蛋白PTM，然后通过新颖的方法来定义和验证其结构，涉及分析化学，有机合成，生物化学/免疫化学和新型生物信息学。这种综合方法允许对组蛋白PTM的全面，公正和敏感的识别。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，以前没有描述过这种系统的发现新组蛋白PTM的方法。我们希望由于这项研究而识别几个新型的组蛋白PTM，从而扩展了我们当前对组蛋白标记的知识。新型组蛋白PTM的识别将为研究界提供一块垫脚石，以研究这些组蛋白PTM在表观遗传机制和疾病中的可能作用。在本应用程序中提出了四个具体目标。首先，我们将通过研究其基因组分布和小鼠胚胎干（MES）细胞中的基因组分布和动力学来研究组蛋白KPROP和KBUTY的潜在表观遗传作用。我们将使用蛋白质组学方法来识别从未分化的混乱及其分化细胞中核心组蛋白中两个PTM的所有残基。我们将通过研究MES细胞分化过程中PTM的动力学及其对ChIP-QPCR和Chip-n-Chip中PTM对基因表达的贡献，从而检查两个PTM在表观遗传调节中的可能作用。其次，我们将使用涉及液相色谱，HPLC/MS/MS分析和新型的生物信息学算法的集成方法来识别和纯化具有新型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NMR光谱来确定新型PTM部分的化学结构。新型的PTM结构将通过各种分析技术和Western印迹分析来验证。最后，我们将使用AIM1。公共卫生相关性的实验研究，将在AIM 2和3中鉴定出在AIM 2和3中鉴定的新型组蛋白PTM的动力学和潜在的表观遗传作用：已知组蛋白的翻译后修饰（PTM）与多种细胞过程和疾病的表观遗传调节有关。拟议的研究旨在鉴定和验证新型组蛋白PTM，并研究其在小鼠干细胞分化中的可能作用。所得的知识将扩大我们对组蛋白PTM的理解，并刺激有关新型组蛋白PTM在疾病进展中的表观遗传作用的研究。