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）：真核细胞中组成最多样化的结构之一是染色体。许多大分子蛋白质相互作用必须正确地发生在染色质上，以驱动染色体生物学的功能方面，如基因转录，DNA复制，重组，修复和姐妹染色单体分离。重要的是，PTM状态的不必要改变和调节染色质代谢的酶机制的缺陷与表观遗传范围内的各种疾病有关，包括癌症。然而，分析蛋白质组装体如何在体内与染色质相互作用以指导这些活动仍然是一个重大挑战，因为它们的关联具有时间和动态性质。染色质背景下大分子蛋白质相互作用的体内分析一直是Taverna和Tackett实验室在过去6年中的主要合作重点。利用我们团队的互补技能，我们开发了新的工具来明确识别染色质上大分子复合物的蛋白质-蛋白质相互作用。最重要的是，我们的共同努力，最近在开发一种技术，称为染色质亲和纯化与质谱或ChAP-MS的高潮。ChAP-MS提供了一个独特的富集1 kb的部分染色体的位点特异性鉴定的大分子蛋白质相互作用和相关的组蛋白翻译后修饰。在人类细胞中建立ChAP-MS将允许前所未有地深入了解哺乳动物转录调控以及人类疾病中的表观遗传失调，以及潜在的治疗作用机制。在这个建议中，我们假设，ChAP-MS可以适应人类细胞培养和组织，提供一种新的工具，在体内大分子蛋白质相互作用的分析。我们的短期目标是将ChAP-MS应用于人类细胞系和组织，而我们的长期目标是将开发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分析。为了验证我们的假设并朝着我们的短期目标努力，我们将追求以下三个目标：（1）开发和应用ChAP-MS分析哺乳动物细胞系中活跃转录基因的大分子染色质组成，（2）开发ChAP-MS的反义富集程序和（3）多重ChAP-MS在哺乳动物细胞和组织中的应用。