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Title: Biochemical and cellular studies of the KDM6 sub-family of JmjC histone lysine demethylases
Author: Dunne, Kate
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 2018
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The JmjC domain containing histone lysine demethylases (JmjC-KDMs) are a family of epigenetic enzymes which regulate gene expression by catalysing demethylation of N-terminal lysines on histone tails. KDM6A and KDM6B catalyse demethylation of histone H3 trimethyllysine (H3K27Me3) on the tail of histone H3, activating gene expression. The KDM6 family have various roles in development and disease, including in hypoxic disease states such as cancer and atherosclerosis. The aim of this project was to evaluate KDM6 activity in the context of cellular hypoxia, both in terms of reduced oxygen availability and alterations to the metabolic milieu. Efforts were made to establish a phenotypic cellular assay for atherosclerosis, a hypoxic disease state in which epigenetic regulation of macrophage functions, including by KDM6B, plays a central role. Expression and purification of recombinant KDM6A was undertaken, but low or no catalytic activity was observed, likely due to aggregation. Production of fusion-protein cleaved KDM6B resulted in inactive enzyme which was found to contain DNA contamination. Endonuclease treatment and heparin affinity purification restored KDM6B activity, indicating a catalytically inactivating interaction between KDM6B and contaminating DNA. The oxygen sensitivity of several JmjC-KDMs has been demonstrated. The oxygen dependence of KDM6B against global H3K27Me3 was assessed in HeLa and COS-7 cells. A graded response of KDM6B activity to oxygen concentrations (0.1-21%) was observed in both cell lines, indicating KDM6B activity can be modulated by oxygen availability in a physiologically relevant manner. Hypoxia impacts cellular metabolism, and leads to increased cellular concentration of tricarboxylic acid cycle intermediates (TCAI). Inhibition of various 2OG oxygenases by TCAI and related metabolites has been reported, and may contribute to reduction of activity seen in hypoxia. An AlphaScreen inhibition assay was used to evaluate KDM6B inhibition by 17 TCAI, with D-2HG, L-2HG and succinate identified as the most potent inhibitors (IC50 values of 9.7, 15, 20 and 20 μM). An orthogonal MALDI-MS assay validated potent inhibition by succinate, L-2HG and D-2HG. Cell permeable esters of TCAI were evaluated using the KDM6B immunofluorescence assay, but no strong cellular inhibition was observed, possibly due to saturating cellular 2OG conditions. Lipid-droplet filled foam cells make up the expanding core of an atherosclerotic plaque. Hypoxia was shown to stimulate foam cell formation using a cellular phenotypic assay. Inhibition of foam cell formation by small molecule inhibitors, including those of JmjC-KDMs, were evaluated but no modulation of foam cell formation was observed, in part due to the small dynamic range of the assay. The work described in this thesis demonstrates that KDM6B activity is reduced both by reduced oxygen availability in cells, and by TCAI inhibition in vitro. This may have important implications for gene regulation in the context of hypoxic disease states, where both reduced oxygen availability and dysregulation of metabolism occur.
Supervisor: Kawamura, Akane ; Flashman, Emily ; Schofield, Chris Sponsor: British Heart Foundation
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available