This proposal seeks to elucidate the dynamics and function of histone H3 lysine 36 methylation (H3K36me) and demethylation. Proper methylation and demethylation of H3K36 are critical for regulation of gene expression and organismal development. Defects in genes responsible for adding and removing this post- translational modification lead to developmental disorders and cancer. Understanding the dynamic nature of H3K36me is the focus of this study. Gaps in the field include understanding how demethylation relates to transcription, when and where demethylation functions across the genome and how this impacts organogenesis. While addition and removal of H3K36me are known to occur, the kinetics of this process remains largely unexplored. This project focuses on understanding these rapid dynamics using recently developed, innovative optogenetic tools that offer unprecedented control of enzymatic activity.
The first aim seeks to understand the role of this dynamic equilibrium in transcription and gene expression regulation in the genetically tractable model organism Saccharomyces cerevisiae. The hypothesis is that histone demethylases function collaboratively to erase H3K36me co-transcriptionally, which then permits proper histone acetylation required for chromatin remodeling during transcription elongation. To test this idea, a photoactivatable variant of Set2, the enzyme that catalyzes H3K36me, will permit characterization of the kinetics of loss of the modification, quantification of the impact of known and uncharacterized demethylases, clarification of the co-transcriptional role of these demethylases and identification of locations across the genome where they function. Preliminary results demonstrate rapid removal of H3K36me upon removal of Set2 and also suggest a role for the uncharacterized demethylase Ecm5 in removing H3K36me2.
The second aim seeks to fill the knowledge gap linking dynamics of methylation and demethylation to the developing vulva in Caenorhabditis elegans, a paradigm for understanding signal transduction and cell specification in a multicellular organism. The hypothesis is that light-control of MET-1, an enzyme that catalyzes H3K36me3 in worms, will enable a new capability to study kinetics of this epigenetic mark and its role in cell fate determination in vulval development. Toward development of a photoactivatable variant of MET-1, pilot studies in tissue culture will guide engineering of C. elegans using the CRISPR/Cas9 system to effect whole- gene replacement of met-1, rendering MET-1 under optogenetic control. Using light-activatable MET-1, reciprocal shifts at defined stages of vulval development will test the kinetics of chromatin alterations and their impact on organogenesis. Finally, a reporter gene expression system and RNAi-mediated knockdown of known and uncharacterized demethylases will elucidate the impact of methylation dynamics on gene expression. Ultimately, this study will advance our understanding of epigenetic regulation of gene expression and development and establish new optogenetic methods to study dynamics of epigenetic modifications. !
Dysfunction in the machinery that drives the establishment and removal of histone post-translational modifications underlies many human diseases including cancer. One histone-modifying enzyme in particular, SETD2, is responsible for histone H3 lysine 36 methylation during gene transcription and is mutated in a variety of cancers. The goal of this project is to elucidate the dynamic process of H3 lysine 36 methylation establishment and removal in yeast and worms in order to better understand the role of this modification in gene expression and organismal development. The results from this work may not only uncover novel targets of therapeutic intervention, but may lead to the treatment of diseases where SETD2, or the enzymes that are responsible for histone H3 lysine 36 methylation removal, are mutated.
