# Proline-Adjacent Phosphosites on Saccharomyces cerevisiae Histone Demethylase Rph1p are Salt Stress Responsive and Important for Cell Growth Under Salt Stress

## TL;DR

This study shows that phosphorylation of the yeast histone demethylase Rph1p, especially at site S689, is crucial for cell growth and gene regulation under salt stress.

## Contribution

The study identifies S689 as a key phosphorylation site in Rph1p that regulates snoRNA gene expression during chronic salt stress.

## Key findings

- Rph1p is phosphorylated at seven sites, including S410, T411, S412, and S689, in response to acute salt stress.
- The S689A phosphonull mutant downregulates a subset of 18 snoRNA genes in chronic salt stress.
- Phosphonull mutations at S412 and S689 do not affect H3K36 methylation or the proteome in chronic salt stress.

## Abstract

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.

•Histone demethylase Rph1p shows extensive salt stress-responsive phosphorylation.•Phosphonull mutations of Rph1p impaired growth in salt stress.•Rph1p phosphonull mutations did not affect the proteome in chronic salt stress.•Phosphonull mutations of Rph1p did not affect the gross methylation of H3K36.•Rph1p phosphonull S689A affected snoRNA gene transcription in chronic salt stress.

Histone demethylase Rph1p shows extensive salt stress-responsive phosphorylation.

Phosphonull mutations of Rph1p impaired growth in salt stress.

Rph1p phosphonull mutations did not affect the proteome in chronic salt stress.

Phosphonull mutations of Rph1p did not affect the gross methylation of H3K36.

Rph1p phosphonull S689A affected snoRNA gene transcription in chronic salt stress.

Phosphorylation of histone demethylases is an important mechanism for modulating chromatin dynamics in response to stress. Yeast H3K36me2/3 demethylase, Rph1p, is extensively phosphorylated in stress however the function of most phosphosites remains unknown. Mutation of phosphosites showed their importance for cell growth in salt stress. Investigation of the proteome and transcriptome of mutants revealed the S689 phosphosite to be critical for snoRNA gene regulation. Our findings highlight that phosphorylation sites in histone demethylating enzymes can regulate expression of specific genes.