Several post-translational modifications exist on histones. While some of these modifications have been associated with various cellular processes, the function of many of them remains poorly understood. Additionally, novel mass spectrometry techniques continue to find previously undiscovered modifications. This work describes the function of two modifications on histone H3 in the budding yeast Saccharomyces cerevisie. The first modification is the conserved methylation of lysine 36 of histone H3 (H3-K36). The second is a newly discovered modification, formylation of H3-K64. We describe novel functions for both of these modifications.

In yeast, methylation of H3-K36 is catalyzed by the NSD1 leukemia oncoprotein homolog Set2. The histone deacetylase complex Rpd3S is recruited to chromatin via binding of the chromodomain protein Eaf3 to methylated H3-K36 to prevent ectopic initiation of transcription within coding sequences. Here we identify a distinct function for H3-K36 methylation. We used random mutagenesis of histones H3 and H4 followed by a reporter-based screen to identify residues necessary to prevent the ectopic spread of silencing from the silent-mating type locus HMRa into flanking euchromatin. Mutations in H3-K36 or deletion of SET2 caused ectopic silencing of a heterochromatin-adjacent reporter. Transcriptional profiling revealed that telomere-proximal genes are enriched for those that display decreased expression in a set2-delta strain. Deletion of SIR4 rescued the expression defect of 26 out of 37 of telomere-proximal genes with reduced expression in set2-delta cells, implying that H3-K36 methylation prevents the spread of telomeric silencing. Indeed, Sir3 spreads from heterochromatin into neighboring euchromatin in set2-delta cells. Furthermore, genetic experiments demonstrated that cells lacking the Rpd3S-specific subunits Eaf3 or Rco1 did not display the anti-silencing phenotype of mutations in SET2 or H3-K36. Thus, H3-K36 methylation antagonizes silencing and prevents erroneous transcriptional initiation by two distinct mechanisms, suggesting a broader range of functions for this conserved modification than previously appreciated.

Formylation on H3-K64 is a recently discovered modification and is conserved between yeast and humans. Mutations in this residue impart telomeric silencing defects on the cell, implying that this modification may play a role in heterochromatic silencing in budding yeast.