Epigenetic regulation is a complex process involving the interplay of multiple different cellular factors. Work described in this thesis concerned the characterisation of proteins involved in the binding to, and demethylation of, histone 3 (H3) tails modified by N-methylation. Initial work focussed on the biophysical characterisation of the tandem plant homeodomains (PHD) of the chromatin remodeller CHD4. NMR spectroscopy was used to investigate the solution structure of the tandem PHDs. Studies on a more native-like construct including the C terminal tandem chromodomains are also presented. Binding studies of the PHDs with H3 peptides reveal that the individual PHD fingers can independently bind a histone peptide. The remainder of the work involved characterisation of JmjC histone demethylases (KDMs), enzymes that catalyse removal of Nε-methyl groups from histone lysyl-residues. Initially, two members of the KDM7 subfamily, PHF8 and KIAA1718, were studied; a high throughput screening assay for them was developed, which enabled identification of a selective inhibitor of the KDM2/7 subfamilies of KDMs, the plant growth regulator Daminozide. A disease relevant mutation in PHF8 was studied and shown to cause mis-localisation of the enzyme to the cytoplasm, providing a potential explanation for the clinically observed phenotype. Subsequent chapters describe unprecedented activities for the JmjC KDMs. 2OG oxygenases catalyse a wide range of oxidative reactions, predominantly mediated by initial substrate hydroxylation. The activity of PHF8 with lysine analogous was tested; the results demonstrated that PHF8, and other KDMs, can oxidatively remove Nε-alkyl groups other than methyl groups, such as ethyl and isopropyl groups. The substrate scope of the JmjC KDMs thus has the potential to be wider than previously thought. Observation of β-hydroxylation of the Nε-isopropyl group of a histone peptide including Nε methylisopropyllysine by JMJD2A/E supports the presumed mechanism of histone lysine demethylation as proceeding via initial hydroxylation. This work led to the discovery that JmjC KDMs can catalyse arginine demethylation. This novel arginine demethylase activity by JmjC KDMs was characterised and the work extended to encompass potential arginine demethylase activity in cells. Biochemical characterisation of UTY, a homologue of the H3 K27 demethylases JMJD3 and UTX, which is reported to be inactive, was carried out; UTY was shown to catalyse demethylation at H3 trimethylated at K27 on peptidic substrates, albeit it at substantially lower rates than the other family members. To investigate the reason for this reduced activity, two variants were made, S1142G and P1214I; the latter variant was shown to be considerably more active than wildtype UTY, likely due to an increased peptide-binding interaction. Preliminary experiments in cells did not conclusively demonstrate histone demethylation, but a luciferase assay suggested that UTY may have catalytic activity in cells. Overall the findings in the thesis suggest that the process of cellular epigenetic regulation is likely even more complex than previously thought, with the potential that JmjC KDMs carry out multiple, context dependent functions.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:627813 |
| Date | January 2013 |
| Creators | Walport, Louise J. |
| Contributors | Schofield, Christopher J.; Redfield, Christina |
| Publisher | University of Oxford |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://ora.ox.ac.uk/objects/uuid:9738b29a-4a3d-420c-8eb7-76374ed4b692 |
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