Proline-Adjacent Phosphosites on Saccharomyces cerevisiae Histone Demethylase Rph1p are Salt Stress Responsive and...

Phosphorylation of histone lysine demethylases is an important mechanism by which the cell modulates chromatin dynamics to regulate its response to stress. There is evidence that the Saccharomyces cerevisiae H3K36me2/3 demethylase, Rph1p, is an integrator of many signaling events. However, the regulatory function of most Rph1p phosphosites in stress response pathways remains unknown. Here, we investigated the role of Rph1p phosphorylation in the salt stress response. We showed that Rph1p is phosphorylated at seven sites in response to acute high salt stress, most of which are proline-adjacent. Genomic phosphonull mutations identified four salt-stress responsive phosphosites-S410, T411, S412, and S689-to be important for yeast cell growth in this condition. Phosphonull mutations at S412 or S689 were not associated with changes in the proteome in the chronic salt stress response. However, the Rph1p-S689A mutant downregulated a subset of 18 snoRNA genes in chronic salt stress compared to the wildtype, an effect absent in the Rph1p-S412A mutant. The downregulation of several snoRNA may cause changes to ribosomal RNA modifications and affect ribosome function. Consistent with these targeted transcriptional changes, neither mutant was associated with gross changes in H3K36 methylation in chronic salt stress. These findings suggest that S689 phosphorylation directs Rph1p to specific regions of the chromatin in the chronic salt stress response. Overall, our findings identify S689 as a key phosphorylation site linking Rph1p to salt stress-responsive gene regulation, offering new insights into stress-responsive mechanisms in eukaryotes.