Characterization of the scaffolding roles of bromodomain containing proteins at the level of chromatin

摘要：我们身体中的每个细胞都会收到信号，告诉它是否生长和分裂，是否分化为特定的细胞类型(例如肌肉细胞或神经元)，甚至是自杀。这些信号是由蛋白质传递的，蛋白质是负责大多数细胞活动的因素。在细胞中，蛋白质是通过首先将DNA转录成中间分子(RNA)，然后再转录成蛋白质而产生的。因此，转录过程是生命的核心。我的研究项目的长期目标是更好地理解抄写。为此，这一应用程序旨在创造新的工具，使我的团队能够研究一种参与转录的蛋白质，即含有溴域的蛋白质。溴域是在40多种人类蛋白质中发现的蛋白质结构域，它在蛋白质中形成一个深腔(根据锁)，这是与乙酰赖氨酸结合其他蛋白质所必需的(在这个类比中，这将是一个关键)。我的研究计划的长期目标是获得对单个基因转录的详细机制理解，以及它是如何受到细胞环境的影响的。短期内，这项应用的目标是通过开发和实施创新的蛋白质组学方法，在染色质水平上表征含溴结构域蛋白质的支架作用。在目标1中，我们将系统地绘制含有溴域的蛋白质的蛋白质-蛋白质相互作用图。在目标2中，我们将使用分裂的辣根过氧化物酶试剂，以允许在一对诱饵蛋白的背景下进行接近生物素化实验。最后，在目标3中，我们将设计一种将Cas9的基因组靶向与邻近生物素化相结合的方法，以允许识别与特定基因组位点相关的蛋白质。*意义：这里概述的工作旨在开发三项互补技术，将提高我的团队识别、量化和表征涉及表观遗传学调节的相互作用网络的能力。它将利用我在生化提纯和质谱学方面的经验，产生蛋白质组学社区目前无法获得的新方法。它们的使用将使我的团队能够对含有溴域的蛋白质进行详细的研究，以确定单个结构域对其相互作用组的贡献；允许特定蛋白质-蛋白质相互作用的背景；以及它们周围特定基因组位置的蛋白质组。*培训：拟议的研究适合于培训蛋白质组学、分子生物学、生物化学和遗传方法领域的4名研究生。此外，它还将使我的团队在为质谱分析准备和分析蛋白质样本方面获得实践经验，这些技能将有助于培养下一代加拿大基础和临床研究人员。**

Summary: Every cell in our bodies receives signals which tell it whether to grow and divide, to differentiate into a specific cell type (e.g. a muscle cell or a neuron) or even to commit suicide. These signals are transmitted by proteins, the actors responsible for most cellular activity. In cells, proteins are generated by first transcribing DNA to an intermediate molecule (RNA) which is subsequently transcribed into proteins. The process of transcription is thus central to life. The long-term goal of my research program is to better understand transcription. To do so, this application aims at creating new tools that will allow my team to study one type of proteins involved in transcription, proteins containing bromodomain. Bromodomain, a protein domain found in over 40 human proteins, form a deep cavity in proteins (as per a lock) which is essential for binding to other protein with acetyl lysines (which would act as a key in this analogy). The long-term goal of my research program is to obtain detailed mechanistic understanding of individual gene transcription and how it is impacted by cellular context. In the short-term, the goal of this application is to characterize the scaffolding role for bromodomain containing proteins at the level of chromatin through the development and implementation of innovative proteomics approaches. In Aim 1, we will systematic map protein-protein interactions for bromodomain-containing proteins. In Aim 2, we will implement the use of split horseradish peroxidase reagents to allow proximity biotinylation experiments to be performed in the context of a pair of bait proteins. Finally in Aim 3, we will engineer an approach combining genome targeting by Cas9 with proximity biotinylation to allow the identification of proteins associated with a particular genomic locus.******Significance: The work outlined here aims to develop three complimentary techniques that will improve my group capacity to identify, quantitate and characterize the interaction networks involving epigenetics regulators. It will leverage my experience in biochemical purifications and mass spectrometry to generate novel approaches not currently available to the proteomics community. Their usage will enable my group to performed detailed study of bromodomain containing proteins to define the contributions of individual domains to their interactome; the context allowing for specific protein-protein interactions; and the proteome surrounding them at specific genomic loci.******Training: The proposed research is appropriate for the training of 4 graduate students in the field of proteomics, molecular biology, biochemistry and genetic methods. In addition, it will allow my group to gain hands-on experience in the preparation and analysis of protein samples for mass spectrometry analysis, skills which will aid in the development of the next generation of Canadian basic and clinical researchers.**