Lightning Oral Talk 25th Annual Lorne Proteomics Symposium 2020

The role of upstream phosphorylation in the regulation of histone methylation (#60)

Ryan 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) using a multi-protease and multi-fragmentation type approach. With respect to phosphorylation, to date, we have identified modification sites on the histone methyltransferases Set2 (12 sites), Set5 (14 sites) and Dot1 (11 sites), and the demethylases Jhd1 (one site), Jhd2 (two sites) and Rph1 (19 sites). Of these 62 phosphorylation sites, 35 validate those observed previously in high throughput phosphoproteomic screens, and 27 sites are novel. To determine the upstream kinases responsible for the phosphorylation, and potential regulation of these enzymes, mass spectrometric analysis was employed to monitor levels of histone methylation in kinase knockout yeast strains. As a proof of concept, quantification of H3K79 methylation in the knockout cells established 43 kinases that are not responsible for the regulation of Dot1 methyltransferase activity. The screening of all other non-essential kinases is in progress. We plan to extend this methodology to the other yeast histone methyltransferases and demethylases in order to comprehensively integrate these enzymes into intracellular signalling pathways, and ultimately facilitate the assembly of the first regulatory network of histone methylation in eukaryotes.