Poster Presentation 24th Annual Lorne Proteomics Symposium 2019

The role of upstream phosphorylation in the regulation of histone methylation (#106)

Ryan J Separovich 1 , Joshua J Hamey 1 , Marc R Wilkins 1
  1. School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, NSW, Australia

Histone methylation is a central means by which gene expression is controlled. In the lower eukaryote, Saccharomyces cerevisiae, histone methylation is regulated by a reduced, but evolutionarily-conserved set of methyltransferases (Set1, Set2, Set5, Dot1) and demethylases (Jhd1, Jhd2, Rph1, Gis1). While the catalytic activity and specificity of these enzymes have been established, knowledge of how they themselves are regulated by post-translational modification is surprisingly limited. Consequently, the regulatory network of histone methylation in yeast remains unknown and is also unknown in all other eukaryotes. To this end, we aimed to comprehensively characterise the modifications occurring on the eight yeast histone methyltransferases and demethylases in vivo. This was achieved by purification of these proteins, and their analysis by targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS). With respect to phosphorylation, to date, we have identified modification sites on the histone methyltransferases Set5 (nine sites) and Dot1 (four sites), and the demethylases Gis1 (seven sites) and Jhd2 (two sites). Fourteen of these sites validate those observed previously in high-throughput phosphorylation screens, while eight sites are novel. By purifying these proteins from single gene knockout yeast strains, we now seek to determine the upstream kinases responsible for the phosphorylation, and potential regulation of Set5, Dot1, Gis1 and Jhd2. This will facilitate the integration of these enzymes into intracellular signalling pathways, and thereby aid the assembly of the first regulatory network of histone methylation. Given the evolutionary conservation of this histone modification, the foundational insights gained through this work will be relevant to other eukaryotes and may prompt similar in-depth analyses in these organisms.