Using ChAP-MS to Study Macromolecular Chromatin Composition during Transcription

描述(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。染色质上必须适当地发生大量的大分子蛋白质相互作用，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状态的不必要变化和调节染色质新陈代谢的酶机制的缺陷与各种疾病和范围内的表观遗传疾病有关，包括癌症。然而，分析蛋白质组件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的挑战，因为它们之间的关联具有时间性和动态性。在过去6年中，在染色质背景下对大分子蛋白质相互作用的活体分析一直是Taverna和Tackett实验室的主要合作重点。共同利用我们团队的互补技能，我们开发了新的工具，以明确地识别染色质上的大分子复合体的蛋白质-蛋白质相互作用。最重要的是，我们的共同努力最近达到了顶峰，开发了一种名为染色质亲和纯化与质谱学或CHAP-MS的技术。CHAP-MS提供了一段独特的1kb染色体的富集物，用于大分子蛋白质相互作用和相关的组蛋白翻译后修饰的位点特异性鉴定。CHAP-MS在人类细胞中的建立将使人们能够前所未有地深入了解哺乳动物的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。在这个提议中，我们假设CHAP-MS可以适用于人类细胞培养和组织，为分析体内大分子蛋白质相互作用提供一种新的工具。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发育转移到小鼠等动物模型中，并使用CHAP-MS来分析整个染色体。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的反义富集法；(3)多重CHAP-MS在哺乳动物细胞和组织中的应用。

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.