Mechanisms of ubiquitin signaling in chromatin-mediated processes Diversity Supplement

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PROJECT ABSTRACT The mechanism by which eukaryotic genes are activated is inextricably linked with the packaging of DNA into chromatin. Coactivator complexes with histone-modifying activities play essential roles in this process by altering chromatin dynamics and interactions. Although there have been major advances in a molecular understanding many of the key macromolecular machines involved in eukaryotic transcription, progress in understanding coactivator function has lagged due to limited structural information on these large complexes and their interactions with chromatin. This project has focused on understanding the regulation of eukaryotic transcription and the connection between histone modifications and gene activation. A major focus has been on the role of histone ubiquitination in regulating transcription, and on the mechanism of cross-talk between histone ubiquitination, methylation and acetylation. Two major tools in our approach are cryo-electron microscopy (cryo-EM) and the development of a large array of designer chromatin templates, which have been instrumental in our ability to capture multisubunit complexes bound to modified chromatin templates. Since the start of this award we have made substantial progress in our structural and biochemical studies of enzyme complexes that mediate cross-talk between histone H2B ubiquitination and histone H3 methylation and have identified novel inhibitors of the SAGA complex’s histone H2B deubiquitinating module. Our ongoing studies are addressing the mechanism governing specificity of ubiquitin conjugating enzymes for histone H2B-K120, the role of this ubiquitin mark in regulating access to chromatin, and role of histone code cross-talk in regulating histone methyltransferases and demethylases. Many of the complexes we study, including MLL-1, Dot1L complexes, and the USP22/SAGA DUB module play a central role in a variety of cancers. Our mechanistic studies and drug discovery efforts are thus providing a foundation for developing novel therapeutics that target these chromatin-modifying enzymes.