Covalent histone modifications such as methylation, acetylation as well as differential incorporation of histone variants are shown to coincide with different chromatin compartments and mark active or repressed genes. Msc1 is one of the seven JmjC Domain Proteins (JDPs) in Fission Yeast. JDPs are known to function in chromatin and some act as histone demethylases. We found that Msc1 is a member of Swr1 Complex which is known to exchange histone H2A variant H2A.z in nucleosomes. We purified H2A.z as a member of Swr1 Complex and its interaction with Swr1 Complex depends on Swr1. We’ve shown that histone H4 Lysine 20 trimethylation (H4 K20 Me3) is lost in h2A.z and msc1 deletion strains and these strains are sensitive to UV. Deletion strain of h2A.z is sensitive to Camptothecin. Histones H3 and H4 are obtained in Msc1 and H2A.z purifications and we’ve shown that histone H4 from these purifications has low level of Lysine 16 acetylation (H4 K16 Ac). Deletion strains of h2A.z, swr1 and msc1 are shown to be sensitive to TSA, a histone deacetylase (HDAC) inhibitor suggesting that H2A.z cooperates with HDACs. TSA treatment of wild type cells cause an increase in H4 K16 Ac and a decrease in H4 K20 Me3. Gene expression profiles of h2A.z, swr1 and msc1 are significantly similar and upregulated genes in deletion strains localize at chromosome ends (a region of 160 kb for each end). The number of stress or meiotic inducible genes is increased in deletion strains suggesting that H2A.z has a role in regulation of inducible genes. We suggest that H2A.z, in cooperation with HDACs, functions in regulation of chromatin accessibility of inducible promoters.

Covalent histone modifications such as methylation, acetylation as well as differential incorporation of histone variants are shown to coincide with different chromatin compartments and mark active or repressed genes. Msc1 is one of the seven JmjC Domain Proteins (JDPs) in Fission Yeast. JDPs are known to function in chromatin and some act as histone demethylases. We found that Msc1 is a member of Swr1 Complex which is known to exchange histone H2A variant H2A.z in nucleosomes. We purified H2A.z as a member of Swr1 Complex and its interaction with Swr1 Complex depends on Swr1. We’ve shown that histone H4 Lysine 20 trimethylation (H4 K20 Me3) is lost in h2A.z and msc1 deletion strains and these strains are sensitive to UV. Deletion strain of h2A.z is sensitive to Camptothecin. Histones H3 and H4 are obtained in Msc1 and H2A.z purifications and we’ve shown that histone H4 from these purifications has low level of Lysine 16 acetylation (H4 K16 Ac). Deletion strains of h2A.z, swr1 and msc1 are shown to be sensitive to TSA, a histone deacetylase (HDAC) inhibitor suggesting that H2A.z cooperates with HDACs. TSA treatment of wild type cells cause an increase in H4 K16 Ac and a decrease in H4 K20 Me3. Gene expression profiles of h2A.z, swr1 and msc1 are significantly similar and upregulated genes in deletion strains localize at chromosome ends (a region of 160 kb for each end). The number of stress or meiotic inducible genes is increased in deletion strains suggesting that H2A.z has a role in regulation of inducible genes. We suggest that H2A.z, in cooperation with HDACs, functions in regulation of chromatin accessibility of inducible promoters.

Being able to respond to extracellular cues such as nutrients and growth factors is of vital importance to all living cells. Pathways have therefore evolved which can sense the extracellular status, transmit a signal through the cell and affect gene expression, which ultimately enables adaptation. Intriguingly, research has revealed that such signaling pathways responding to nutrient status are intrinsically linked to the lifespan of organisms, a phenomenon known as caloric restriction. This thesis utilizes budding yeast, Saccharomyces cerevisiae, as a model system to investigate how transcription factors affect gene expression in response to nutrient signaling pathways. Paper I investigates the role of the three homologous transcription factors Mig1, Mig2 and Mig3 in regulating gene expression in response to glucose. This is done by transcriptional profiling with microarrays of wild type yeast, as well as mutant strains where the MIG1, MIG2 and MIG3 genes have been deleted in all possible combinations. The results reveal that Mig1 and Mig2 act together, with Mig1 having a larger effect in general while Mig2 has a role specialized for high-glucose conditions. Using a strategy similar to that in paper I, paper II examines the roles of the two homologous transcription factors Gis1 and Rph1 in gene regulation. This study shows that Gis1 and Rph1 are both involved in nutrient signaling, acting in parallel with a large degree of redundancy. Furthermore, we find that these two transcription factors change both target genes as well as the effects on transcription when the yeast cell transitions through different growth phases. Rph1 is a functional JmjC histone demethylase, and paper III investigates the connection between this activity and the transcriptional regulation studied in paper II. We find that rendering Rph1 catalytically inactive has little effect on its role in nutrient signaling and gene regulation, but subtly affects certain groups of genes. Paper IV reveals that Rph1 does not affect the chronological lifespan of yeast as does its homolog Gis1. However, deleting or overexpressing RPH1 has effects on the response to rapamycin and caffeine, inhibitors of the evolutionary conserved TORC1 complex affecting lifespan in both yeast and mammals.

Epigenetic control of gene expression by histone post-translational modifications (PTMs) is a complex process regulated by proteins that can 'read', 'write' or 'erase' these PTMs. The histone lysine demethylase (KDM) family of epigenetic enzymes remove methyl modifications from lysines on histone tails. The Jumonji C domain (JmjC) family is the largest family of KDMs. Investigating the scope and mechanisms of the JmjC KDMs is of interest for understanding the diverse functions of the JmjC KDMs in vivo, as well as for the application of the basic science to medicinal chemistry design. The work described in this thesis aimed to biochemically investigate the inhibitor and substrate diversity of the JmjC KDMs, it led to the identification of new inhibitors and substrates and revealed a potential combinatorial dependence between adjacent histone PTMs. Structure-activity relationship studies gave rise to an n-octyl ester form of IOX1 with improved cellular potency and selectivity towards the KDM4 subfamily. This compound should find utility as a basis for the development of JmjC inhibitors and as a tool compound for biological studies. The rest of this thesis focused on the biochemical investigations of potential substrates and inhibitors for KDM3A, a JmjC demethylase with varied physiological functions. Kinetic characterisation of reported KDM3A substrates was used as the basis for evaluations of novel substrates and inhibitors. Further studies found TCA cycle intermediates to be moderate co-substrate competitive inhibitors of KDM3A. Biochemical investigations were carried out to study potential protein-protein interactions of KDM3A with intraflagellar transport proteins (IFTs), non-histone proteins involved in the formation of sperm flagellum. Work then addressed the exploration of novel in vitro substrates for KDM3 (KDM3A and JMJD1C) mediated catalysis, including: methylated arginines, lysine analogues, acetylated and formylated lysines. KDM3A, and other JmjC KDMs, were found to catalyse novel arginine demethylation reaction in vitro. Knowledge gained from studies with unnatural lysine analogues was utilised to search for additional novel PTM substrates for KDM3A. These results constitute the first evidence of JmjC KDM catalysed hydroxylation of an Nε-acetyllysine residue. The H3 K4me3 position seems to be required for acetyllysine substrate recognition, implying a combinatorial effect between PTMs. Preliminary results provide evidence that JMJD1C, a KDM3 protein previously reported to be inactive, may catalyse deacetylation in vitro. An additional novel reaction, observed with both KDM3A and JMJD1C, is deformylation of N^ε-formyllysine residues on histone H3 fragment peptides. Interestingly, H3 K4 methylation was also observed to enhance the apparent deformylation of both KDM3A and JMJD1C catalysed reactions. Overall, findings in this thesis suggest that the catalytic activity of JmjC KDMs extends beyond lysine demethylation. In a cellular context, members of the KDM3 subfamily might provide a regulatory link between methylation and acylation marks. Such a link will further highlight the complex relationships between histone PTMs and the epigenetic enzymes that regulate them. The observed dependency of H3 K9 catalysis on H3 K4 methylation adds another layer of complexity to the epigenetic regulation by histone PTMs.