Mechanistic studies of chromatin modification in transcription regulation

PROJECT SUMMARY Post-translational modifications of the histone proteins that package DNA play a central role in regulating transcription and repair of DNA damage. The goal of this research program is to determine the molecular mechanism by which attachment of the small protein, ubiquitin, to histone proteins functions in concert with modifications including methylation and acetylation to regulate gene expression. Since misregulation of histone ubiquitination and methylation characterizes many cancers, the results of these studies can be used to design new drugs that target the molecular machines involved in these pathways. A major focus will be on the mechanisms by which histone H2B-K120 monoubiquitination orchestrates multiple events during transcription that result in gene activation. A multidisciplinary approach combining cryo-electron microscopy, x-ray crystallography, solution biochemistry and cell-based approaches will be used to study the mechanism by which ubiquitin is specifically attached to histone H2B during transcription and the distinct mechanisms by which different deubiquitinating enzymes distinguish between H2B in intact versus partially disassembled nucleosomes. We will also determine the nature of intra- and inter- nucleosomal cross-talk between histone ubiquitination, methylation and acetylation mediated by transcriptional activators that act downstream of H2B ubiquitination. Building upon the recent discovery of first-in-class macrocyclic inhibitors of the human SAGA H2B deubiquitinating enzyme, we will determine their mechanism of action and derive principles that will drive further drug discovery of multi- subunit deubiquitinating enzymes. To elucidate the molecular basis by which H2B ubiquitination drives cell proliferation in MLL-rearranged leukeumia, we will uncover the role of histone ubiquitination in stimulating the activity of MLL1 fusion complexes that result from chromosomal rearrangements. The molecular insights that will result from the proposed studies will provide a foundation for developing new chemotherapies that target cancers for which there currently are no effective treatments.