Role of the ubiquitin system in epigenetic regulation

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Ubiquitin, a 76 amino acid small protein, is well conserved from yeast to humans. It can be covalently added onto thousands of different proteins through a process known as ubiquitination to provide diversity and dynamics to cell regulation. Histones are a group of basic nuclear proteins that bind and organize the linear DNA into the structural units called nucleosomes, which are further condensed into chromatin. Ubiquitin modification of histones is directly involved in many vital cellular functions including transcription, DNA replication, repair and gene silencing and therefore plays a critical role in the regulation of cell division, growth and differentiation. The mechanism of this histone modification and the players involved in this process are poorly characterized. The goal of our research program is to understand the mechanism and the importance of ubiquitin modification of histones in cellular regulation.In this application, we will focus on elucidation of the ubiquitin system involved in ubiquitination of histone H2A. H2A ubiquitination is involved in the maintenance of genome integrity, developmental patterning, X-chromosome inactivation and maintenance of pluripotency of stem cells. The proteins responsible for H2A ubiquitination include a cascade of enzymes, E1 (activating enzyme), E2 (conjugating enzymes) and E3 (ligases), which covalently attaching ubiquitin to specific lysine residues of H2A. Through proteomic, biochemical and cellular approaches, we propose to characterize the mechanism of H2A ubiquitination, to reveal the roles of each protein player in this process and to understand the function of H2A ubiquitination in cellular regulation. Specific aims include: (1) to reveal specific enzymes required for H2A ubiquitination in vitro and in vivo; (2) to characterize the molecular basis of H2A ubiquitination and the roles of these enzymes in gene regulation; and (3) to investigate specific enzymes that are responsible for ubiquitination of the H2A variants. By studying how these enzymes work, we will gain a better understanding of the importance of histone ubiquitination and how it is involved in the regulation of gene expression in different biological processes.