Histone methylation is a dynamic regulator of transcription in eukaryotes and has been correlated with both gene activation and gene silencing. Dysregulation of the histone methylation system has been linked to the aetiology and progression of several diseases, including cancer and neurodegenerative disorders. There is evidence that histone methylation itself is regulated by the kinase signalling network via phosphorylation. Rph1p is a yeast histone lysine demethylase (human KDM4 homolog) that removes di- and tri-methyl groups from lysine 36 of the histone H3 (H3K36). It is of particular interest due to its high number of phosphosites and thus likelihood that it is an integrator of signalling information. However, few Rph1p phosphorylation events have been functionally characterised, or their respective kinases identified. The aim of this project is to find regulatory Rph1p phosphosites responsive to chromatin-modulating stresses. To determine which phosphosites are regulatory we analysed the effect of 1M sodium chloride and 15mM caffeine on Rph1p phosphorylation. This was done by purifying Rph1p expressed in cells subject to these different growth stressors for 15 minutes and analysing resulting phosphorylation levels using liquid chromatography tandem mass spectrometry (LC-MS/MS). Candidate stress-responsive phosphosites were then mutated to create a library of 10 Rph1p phospho-null mutants covering 25 phosphosites, which were tested for growth phenotypes under the same conditions. Ongoing work aims to link observed changes in Rph1p phosphorylation under specific conditions to changes in H3K36 methylation and the proteome profile. This will include using LC-MS/MS to measure global changes in H3K36 methylation states of wildtype Rph1p and Rph1p phospho-null mutants, as well as using chromatin immunoprecipitation coupled to DNA sequencing to measure changes in Rph1p association to specific regions of the genome. LC-MS/MS-based techniques will also be used to conduct differential expression analysis of Rph1p phospho-null strains. Ultimately, our aim is to identify the kinases and upstream signalling pathways that mediate Rph1p phosphorylation and thus influence H3K36 methylation and down gene expression profiles.