Dissecting the regulatory mechanisms governing the histone 3 lysine 9 methyltransferase Clr4

The histone methyltransferase Clr4 is essential for heterochromatin formation in Schizosaccharomyces pombe, catalysing the methylation of histone H3 at lysine 9 (H3K9). Recent studies have shown that the ubiquitination of histone H3 at lysine 14 (H3K14Ub) stimulates Clr4’s catalytic activity, while phosphorylation of its autoregulatory loop (ARL) modulates activity during meiosis. This thesis focuses on exploring the structural dynamics and regulatory mechanisms of Clr4 using NMR spectroscopy, with a focus on how post-translational modifications influence its function. I optimised recombinant expression of the C-terminal catalytic domain of Clr4 (Clr4-SET) using a Golden Gate cloning system to screen purification and solubility tags. While this did not markedly improve solubility, it provided a useful tool for future applications. A key achievement was the synthesis at milligram-scale, of site-specific H3K14Ub conjugates using halogen ketone-mediated cross-linking. Additionally, I successfully expressed and purified isotopically labelled Clr4-SET and obtained 2D and 3D NMR spectra—making this work the first detailed NMR study of Clr4. Titration experiments revealed significant chemical shift perturbations upon H3K14Ub binding and CDK1-mediated phosphorylation, indicating conformational changes linked to activation. These findings provide a foundation for future structural studies aimed at understanding Clr4’s regulatory mechanisms and their broader implications for chromatin regulation in S. pombe.