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

总结：我们身体中的每个细胞都接收信号，这些信号告诉它是否生长和分裂，分化成特定的细胞类型（例如肌肉细胞或神经元），甚至自杀。这些信号由蛋白质传递，蛋白质是负责大多数细胞活动的演员。在细胞中，蛋白质是通过首先将DNA转录成中间分子（RNA），然后将其转录成蛋白质而产生的。因此，转录过程是生命的核心。我的研究计划的长期目标是更好地理解转录。为此，该应用程序旨在创建新的工具，使我的团队能够研究一种参与转录的蛋白质，即含有溴结构域的蛋白质。Bromodomain是一种在40多种人类蛋白质中发现的蛋白质结构域，在蛋白质中形成一个深腔（如锁），这对于与乙酰赖氨酸结合其他蛋白质（在这种类比中充当钥匙）至关重要。我的研究计划的长期目标是获得个体基因转录的详细机制理解以及它如何受到细胞环境的影响。在短期内，本申请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溴结构域蛋白质的蛋白质-蛋白质相互作用。在目标2中，我们将使用裂解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邻近生物素化实验。最后，在目标3中，我们将设计一种方法，将Cas9的基因组靶向与邻近生物素化相结合，以识别与特定基因组位点相关的蛋白质。重要性：这里概述的工作旨在开发三个互补的技术，这将提高我的小组的能力，以确定，定量和表征的相互作用网络，涉及表观遗传调节。它将利用我在生化纯化和质谱分析方面的经验来产生蛋白质组学界目前尚不具备的新方法。它们的使用将使我的研究小组能够对含有布罗莫结构域的蛋白质进行详细研究，以定义各个结构域对其相互作用组的贡献;允许特定蛋白质-蛋白质相互作用的环境;以及特定基因组位点上围绕它们的蛋白质组。**培训内容：本研究适合于培养蛋白质组学、分子生物学、生物化学和遗传学方法领域的4名研究生。此外，它将使我的团队在制备和分析用于质谱分析的蛋白质样品方面获得实践经验，这些技能将有助于下一代加拿大基础和临床研究人员的发展。

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.**