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

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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.