Who’s controlling the controllers? Constructing the regulatory network of histone methylation in yeast

Separovich RJ and Wilkins MR

Systems Biology Initiative, School of Biotechnology and Biomolecular Sciences, The University of New South Wales, Sydney, New South Wales 2052, Australia.

Methylation of histone proteins tightly regulates patterns of gene expression in the eukaryotic cell. The addition of methyl (CH3) groups to histone tails is catalysed by methyltransferase enzymes, while demethylase enzymes can reverse this modification. Together, the activity of these ’writer’ and ’eraser’ enzymes determines which histone methyl marks accumulate, and thus which genes are accessible for transcription. In the lower eukaryote, Saccharomyces cerevisiae, there are only four known histone lysine methyl writers (Set1, Set2, Set5, Dot1) and four erasers (Jhd1, Jhd2, Gis1, Rph1). While the catalytic activity and specificity of these enzymes have been established, knowledge of how they are post-translationally modified is surprisingly limited. Consequently, the regulatory network of histone methylation in yeast remains unknown. To this end, we aim to comprehensively characterise the eight writers and erasers in S. cerevisiae, with a focus on phosphorylation. This will be done through purification of these proteins, and their analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Through a series of gene knockout experiments, we will then seek to identify the upstream kinases responsible for the phosphorylation, and potential regulation, of the methyl writers and erasers. This will facilitate the integration of these enzymes into intracellular signalling pathways, and thereby enable the assembly of the first complete regulatory network of histone methylation in yeast. 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.