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A biofilm is a complex community of cells enveloped in a self-produced polymeric matrix. Entry into a biofilm is exquisitely controlled at the level of transcription and in Bacillus subtilis it requires the concerted efforts of several major transcription factors including the repressor SinR and activator DegU. I initially identified that these transcriptional regulators control biofilm formation via parallel pathways. Through investigating the regulation of biofilm formation by SinR and DegU, I discovered that biofilm formation is also regulated at the post-translational level. This was achieved by identifying three key proteins which are needed for biofilm formation. These proteins are PtkA, a bacterial tyrosine kinase; TkmA, the cognate modulator of PtkA; and PtpZ, a bacterial tyrosine phosphatase. By introducing amino acid point mutations within the catalytic domains of PtkA and PtpZ it was identified that the kinase phosphatase activities, respectively, are essential function.In addition, PtkA contains a conserved C-tyrosine cluster that is the site autophosphorylation. Investigation of the role of the C-terminal tyrosine cluster tentatively suggests that this domain acts to block access to the active site of PtkA, thus affecting the ability of PtkA to phosphorylate its targets. Deletion of the gene coding for TkmA demonstrated that this modulator was also required for biofilm formation. It was also demonstrated that TkmA may interact with other protein partners, at least in the absence of PtkA, raising the question of how signal specificity is maintained. Finally, a systematic mutagenesis approach was used with the aim of identifying the target(s) of PtkA and PtpZ during biofilm formation but,despite extensive efforts, it remained elusive. The findings presented in this thesis highlight the complexity of biofilm formation by B. subtilis by revealing an additional level of regulation in the form of protein tyrosine phosphorylation.

Histone methylation is a key epigenetic modification that regulates eukaryotic transcription. It is involved in many cellular processes, including growth, development, and differentiation, and its dysregulation is linked to the aetiology and progression of disease. In the budding yeast and model eukaryote, Saccharomyces cerevisiae, there are six histone lysine methylation sites (H3K4, H3K36, H3K79, H4K5, H4K8, and H4K12) that are controlled by an evolutionarily conserved suite of methyltransferase (Set1p, Set2p, Dot1p, and Set5p) and demethylase (Jhd1p, Jhd2p, Rph1p, and Gis1p) enzymes. While the histone targets of these enzymes are known, their regulation is poorly understood. To this end, we performed a systematic investigation into the post-translational regulation of the histone lysine methylation network in S. cerevisiae, with a particular focus on the role of phosphorylation and its connection with upstream signalling pathways. First, we used targeted mass spectrometry to identify a total of 75 phosphorylation sites occurring on yeast histone methyltransferase and demethylase enzymes in vivo. To investigate the functional effects of these sites, we constructed and screened a phosphosite mutant library, consisting of 82 genomically-edited yeast strains, using mass spectrometry, immunoblotting, and spot plate growth assays. This revealed the importance of phosphosites on methyltransferase Set2p, at residues S6, S8, S10, and T127, and demethylase Jhd1p, at residue S44, in the regulation of H3K36 methylation and in the coordination of specific stress response pathways. Finally, we employed in vitro and in vivo kinase discovery approaches to show that the mitogen-activated protein kinase, Hog1p, phosphorylates methyltransferase Dot1p at multiple proline-adjacent sites. Taken together, our findings establish phosphorylation as a key regulator of histone methylation enzymes in budding yeast, and contextualise the histone-based gene regulatory system within the signalling system of the cell.

Cholesterol is an essential biological molecule, important in maintaining cell membrane integrity and fluidity, cell signalling, and being a precursor for bile acids and steroid hormones. However, high cholesterol levels correlate with several disease states, including heart disease, cancer and neurodegeneration. The cell therefore tightly regulates cholesterol levels at both the transcriptional and post-translational levels. Cholesterol synthesis involves over 20 enzymatic reactions. Previously, research focused on 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), the first control point in cholesterol synthesis. However, our lab recently identified a second rate-limiting step, catalysed by squalene monooxygenase (SM). Statins, the cholesterol-lowering drugs of choice, target HMGCR. However, SM lies further downstream, beyond a critical branch point. SM therefore presents as a possible alternative drug target to HMGCR, with the potential to have fewer side-effects. In this thesis, we looked closer at the cholesterol regulation of SM. First, we investigated the transcriptional regulation of both HMGCR and SQLE (the gene encoding SM) by sterol response element binding protein-2 (SREBP-2). We found that both human HMGCR and SQLE promoters contain two adjacent SREBP-2 binding sites (sterol response elements, SREs). We also identified a nuclear factor Y (NF-Y) binding site in the human HMGCR promoter, and two NF-Ys in human SQLE. NF-Y is known to enhance SREBP-2 binding. In addition, we identified a transcriptional mechanism whereby cells preferentially take up pre-made cholesterol via low density lipoprotein receptor (LDLR), ensuring energetically-expensive cholesterol synthesis is only activated when necessary. Next, we explored the membrane topology of the cholesterol regulatory domain of SM, SM N100 (the first 100 amino acids of SM). We showed that SM N100 is anchored to the endoplasmic reticulum (ER) membrane by a re-entrant loop. We subsequently found that cholesterol induces a conformational change in SM N100, causing it to be degraded by the proteasome, shutting down the cholesterol synthesis pathway when it is not needed. Overall, we increase the understanding of the transcriptional regulation of the two rate-limiting steps of cholesterol synthesis, and demonstrate how excess cholesterol levels negatively regulate SM protein abundance. Hence, we have contributed to the fundamental understanding of the regulation of cholesterol synthesis.