Histone methylation is central to the regulation of eukaryotic transcription. In Saccharomyces cerevisiae, it is controlled by an evolutionarily conserved suite of histone methyltransferases (Set1p, Set2p, Dot1p, Set5p) and demethylases (Jhd1p, Jhd2p, Rph1p, Gis1p). While the histone targets for these enzymes are known, the post-translational regulation of the methylation enzymes is poorly understood in yeast and in all other eukaryotes (1,2).
Using enzyme purification and mass spectrometry, we identified a total of 75 phosphorylation sites on yeast histone methyltransferases and demethylases in vivo (3). To systematically investigate the functions of these sites, we constructed a phosphosite mutant library consisting of 82 genomically-edited yeast strains. Mutants were screened for changes in native H3K4, H3K36, and H3K79 methylation levels, and for sensitivity to environmental stress conditions. This revealed that multiple phosphosites on methyltransferase Set2p (Ser6, Ser8, Ser10, Thr127) modulate H3K36 methylation in vivo, and lead to downregulation of membrane-associated proteins and processes. We also found that specific phosphosites on Set2p (Ser6, Ser8, Ser10) and Jhd1p (Ser44) are required for a normal cellular response to diamide stress and cold stress, respectively. Finally, we showed that the yeast MAP kinase, Hog1p, phosphorylates H3K79 methyltransferase, Dot1p, at Ser565. This is the first kinase known to act on Dot1p and just the third kinase known to phosphorylate any histone methylation enzyme in S. cerevisiae.
Taken together, our findings represent the first systematic investigation into the phosphoregulation of the epigenetic network in any eukaryotic species, and establish phosphorylation as a key regulator of histone methylation in budding yeast.