Using ChAP-MS to Study Macromolecular Chromatin Composition during Transcription

使用 ChAP-MS 研究转录过程中的大分子染色质组成

• 批准号: 8489555
• 负责人: Alan Tackett
• 金额: $ 30.92万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-07-01 至 2017-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8489555
• 关键词: Affinity; Affinity Chromatography; Animal Model; Antisense Oligonucleotides; Applications Grants; Award; Binding Sites; Biological Models; Biology; Cell Culture System; Cell Culture Techniques; Cell Line; Cells; Centers of Research Excellence; Chromatin; Chromatin Structure; Chromosomes; Collaborations; DNA Binding; DNA biosynthesis; Defect; Development; Disease; Engineering; Epigenetic Process; Eukaryotic Cell; Feasibility Studies; Funding; Future; Gene Targeting; Genes; Genetic; Genetic Engineering; Genetic Transcription; Genomics; Goals; Grant; Histones; Human; Human Cell Line; Laboratories; Link; Macromolecular Complexes; Malignant Neoplasms; Mammalian Cell; Mass Spectrum Analysis; Measures; Metabolism; Methodology; Modeling; Mus; Nature; Organism; Participant; Patients; Positioning Attribute; Post-Translational Protein Processing; Procedures; Protein Analysis; Proteins; Proteomics; Reagent; Regulation; Research Personnel; Saccharomyces cerevisiae; Saccharomycetales; Sampling; Sister Chromatid; Site; Structure; Techniques; Technology; Testing; Therapeutic; Tissues; Transcriptional Regulation; Translating; Triciribine Phosphate; United States National Institutes of Health; Work; Yeasts; combinatorial; epigenomics; human disease; human tissue; in vivo; insight; new technology; novel; programs; protein protein interaction; public health relevance; recombinational repair; research and development; segregation; skills; technology development; tissue/cell culture; tool

DESCRIPTION (provided by applicant): One of the most compositionally diverse structures in a eukaryotic cell is a chromosome. A multitude of macromolecular protein interactions must properly occur on chromatin to drive functional aspects of chromosome biology like gene transcription, DNA replication, recombination, repair, and sister chromatid segregation. Importantly, unwanted alterations in PTM states and defects in the enzymatic machineries that regulate chromatin metabolism are linked to a wide variety of illnesses and diseases that are epigenetic in scope, including cancer. However, analyzing how protein assemblies interact in vivo with chromatin to direct these activities remains a significant challenge due to the temporal and dynamic nature of their associations. The in vivo analysis of macromolecular protein interactions in the context of chromatin has been a primary collaborative focus of the Taverna and Tackett laboratories for the last 6 years. Together, using the complementary skill sets of our groups, we have developed novel tools to unambiguously identify protein- protein interactions of macromolecular complexes on chromatin. Most importantly, our combined efforts recently culminated in the development of a technique termed Chromatin Affinity Purification with Mass Spectrometry or ChAP-MS. ChAP-MS provides for the enrichment of a unique 1 kb section of a chromosome for site-specific identification of macromolecular protein interactions and associated histone posttranslational modifications. The establishment of ChAP-MS in human cells will permit unprecedented insight into mammalian transcription regulation as well as epigenetic disregulation in human disease, and potentially mechanisms of action for therapeutics. In this proposal, we hypothesize that ChAP-MS can be adapted to human cell culture and tissues to provide a novel tool for the analysis of in vivo macromolecular protein interactions. Our short-term goal is to apply ChAP-MS to human cell lines and tissues, while our long-term goal is to move development into animal models like mice and to use ChAP-MS to profile across an entire chromosome. To test our hypothesis and work towards our short term goal, we will pursue the following three Aims: (1) Develop and Apply ChAP-MS for the analysis of macromolecular chromatin composition at actively transcribing genes in mammalian cell lines, (2) Develop an antisense enrichment procedure for ChAP-MS and (3) Application of Multiplex ChAP-MS in mammalian cells and tissues.

描述（由申请人提供）：真核细胞中最多样化的结构之一是染色体。必须在染色质上正确发生多种大分子蛋白质相互作用，以驱动染色体生物学的功能方面，例如基因转录，DNA复制，重组，修复和姐妹染色质分离。重要的是，调节染色质代谢的PTM状态的不良改变和酶促机制的缺陷与包括癌症在内的范围表观遗传的多种疾病和疾病有关。但是，由于其关联的时间和动态性质，分析蛋白质组件如何与染色质在体内相互作用是指导这些活动的重大挑战。在过去的6年中，在染色质中对大分子蛋白相互作用的体内分析一直是Taverna和Tackett Laboratories的主要协作重点。共同使用我们组的互补技能集，我们开发了新颖的工具，可以明确鉴定染色质上大分子分子复合物的蛋白质蛋白质相互作用。最重要的是，我们最近的合并努力最终达到了一种用质谱或章节-MS的染色质亲和力纯化的技术的开发。 Chap-MS提供了染色体的独特的1 Kb截面的富集，用于特定于位点的大分子蛋白相互作用和相关组蛋白后翻译后修饰。在人类细胞中的建立将允许对哺乳动物转录调控以及人类疾病中的表观遗传学的前所未有的见解，以及对治疗疗法的可能作用机制。在此提案中，我们假设Chap-M可以适应人类细胞培养和组织，以提供一种新颖的工具，用于分析体内大分子蛋白质相互作用。我们的短期目标是在人类细胞系和组织中应用章节，而我们的长期目标是将发育转移到小鼠等动物模型中，并使用Chap-Ms在整个染色体上进行介绍。为了检验我们的假设并朝着短期目标努力，我们将追求以下三个目的：（1）开发和应用Chap-Ms，以分析在哺乳动物细胞系中主动转录基因的大分子染色质组成，（2）在Chap-Ms和（3）在Mammamal Chapue中应用抗浓度的富集程序，并在MAMMAMAL SILLIAIN中应用Mammamalian和组织中。