title: The Activity of JmjC Histone Lysine Demethylase KDM4A is Highly Sensitive to Oxygen Concentrations.
abstract: The JmjC histone lysine demethylases (KDMs) are epigenetic regulators involved in the removal of methyl groups from post-translationally modified lysyl residues within histone tails, modulating gene transcription. These enzymes require molecular oxygen for catalytic activity and, as 2-oxoglutarate (2OG)-dependent oxygenases, are related to the cellular oxygen sensing HIF hydroxylases PHD2 and FIH. Recent studies have indicated that the activity of some KDMs, including the pseudogene-encoded KDM4E, may be sensitive to changing oxygen concentrations. Here, we report detailed analysis of the effect of oxygen availability on the activity of the KDM4 subfamily member KDM4A, importantly demonstrating a high level of O2sensitivity both with isolated protein and in cells. Kinetic analysis of the recombinant enzyme revealed a high KMapp(O2) of 173 ± 23 μM, indicating that the activity of the enzyme is able to respond sensitively to a reduction in oxygen concentration. Furthermore, immunofluorescence experiments in U2OS cells conditionally overexpressing KDM4A showed that the cellular activity of KDM4A against its primary substrate, H3K9me3, displayed a graded response to depleting oxygen concentrations in line with the data obtained using isolated protein. These results suggest that KDM4A possesses the potential to act as an oxygen sensor in the context of chromatin modifications, with possible implications for epigenetic regulation in hypoxic disease states. Importantly, this correlation between the oxygen sensitivity of the catalytic activity of KDM4A in biochemical and cellular assays demonstrates the utility of biochemical studies in understanding the factors contributing to the diverse biological functions and varied activity of the 2OG oxygenases.
authors.affiliation: Chemistry Research Laboratory , 12 Mansfield Road, Oxford OX1 3TA, United Kingdom.|Radcliffe Department of Medicine, Division of Cardiovascular Medicine, BHF Centre of Research Excellence, Wellcome Trust Centre for Human Genetics , Roosevelt Drive, Oxford OX3 7BN, United Kingdom.
authors.forename: Rebecca L
authors.initials: RL
authors.lastname: Hancock
authors.affiliation: Target Discovery Institute, NDM Research Building, University of Oxford , Roosevelt Drive, Oxford OX3 7BN, United Kingdom.
authors.forename: Norma
authors.initials: N
authors.lastname: Masson
authors.affiliation: Chemistry Research Laboratory , 12 Mansfield Road, Oxford OX1 3TA, United Kingdom.|Radcliffe Department of Medicine, Division of Cardiovascular Medicine, BHF Centre of Research Excellence, Wellcome Trust Centre for Human Genetics , Roosevelt Drive, Oxford OX3 7BN, United Kingdom.
authors.forename: Kate
authors.initials: K
authors.lastname: Dunne
authors.affiliation: Chemistry Research Laboratory , 12 Mansfield Road, Oxford OX1 3TA, United Kingdom.
authors.forename: Emily
authors.initials: E
authors.lastname: Flashman
authors.affiliation: Chemistry Research Laboratory , 12 Mansfield Road, Oxford OX1 3TA, United Kingdom.|Radcliffe Department of Medicine, Division of Cardiovascular Medicine, BHF Centre of Research Excellence, Wellcome Trust Centre for Human Genetics , Roosevelt Drive, Oxford OX3 7BN, United Kingdom.
authors.forename: Akane
authors.identifier: 0000-0003-1169-5082
authors.initials: A
authors.lastname: Kawamura
mesh_terms: D045744:Cell Line, Tumor; D002843:Chromatin / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D006801:Humans; D056484:Jumonji Domain-Containing Histone Demethylases / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D010100:Oxygen / Q000378:metabolism*; D011544:Pseudogenes
chemical_list: D002843:Chromatin; D056484:Jumonji Domain-Containing Histone Demethylases; C494424:KDM4A protein, human; D010100:Oxygen