Poster Presentation 28th Annual Lorne Proteomics Symposium 2023

The phospho-regulation of histone demethylases in Saccharomyces cerevisiae (#132)

Nicola M Karakatsanis 1 , Joshua J Hamey 1 , Marc R Wilkins 1
  1. Systems Biology Initiative, School of Biotechnology and Biomolecular Sciences, UNSW, Sydney, NSW, Australia

Histone methylation is a dynamic regulator of transcription that has been correlated with both gene activation and gene silencing (1,2). Dysregulation of the histone methylation system has been linked to the aetiology and progression of several diseases, including cancer and neurodegenerative disorders (1,2). In the model organism, Saccharomyces cerevisiae, histone methylation is mediated by a total of eight evolutionarily conserved methyltransferases and demethylases (3-6). There is emerging evidence that histone methylation itself is regulated by the kinase signalling network via phosphorylation (7,8). Despite this, the function of many phospho-sites on histone methylation enzymes – particularly demethylases – in S. cerevisiae is still unknown. Consequently, we seek to investigate the phospho-regulatory system of histone demethylases in S. cerevisiae and its role in modulating cellular response phenotypes. To start, we conducted a comprehensive analysis of the literature to identify candidate conditions in which demethylase knock outs have shown abnormal growth phenotypes. Using spot plate growth assays, we then showed that almost all known S. cerevisiae demethylase kockouts (Rph1p, Jhd1p and Jhd2p) had clear phenotypes in at least one condition tested. In particular, the H3K36 demethylase, Rph1p, was found to be essential to the galactose genetic switch. However, subsequent analysis of H3K36 methylation using liquid chromatography tandem mass spectrometry (LS-MS/MS) showed no significant changes in global methylation when grown in media supplemented with galactose instead of glucose. This suggested that changes in H3K36 methylation were probably localised to specific regions of the chromatin (ie. the regulatory GAL genes). To determine whether this is the case, we intend to measure changes in methylation of K36 at histone H3 bound at specific genes/coding sequences under these conditions. Our future work will then look to link observed changes in histone methylation to changes in Rph1p phosphorylation at specific sites using a combination of phospho-site mutants and LC-MS/MS analysis of purified Rph1p expressed under the same conditions. Ultimately, our aim is to identify the kinases that act on candidate phospho-sites to induce changes in gene expression and cellular phenotypes. This research will allow us to identify which histone methylation regulatory pathways may be conserved across eukaryotes, as well as identify those that are unique to yeast.

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