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 O2 sensitivity 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.

Lysine‐241 Has a Role in Coupling 2OG Turnover with Substrate Oxidation During KDM4‐Catalysed Histone Demethylation

“…Studies were then carried out to evaluate the ability of the two KDM4A proteins to catalyse the demethylation of a histone peptide substrate. The two proteins (1 μ m ) were individually incubated with 2OG (100 μ m , a concentration well above its WT K M value of 26±7 μ m ), ascorbate (100 μ m ), ferrous iron (10 μ m ) and a 15‐residue H3K9me3 peptide (100 μ m , sequence: ARTKQTARKme3STGGKA) in HEPES buffer (50 m m , pH 7.5) at 37 °C; the extent of the reaction was determined by using MALDI‐TOF mass spectrometry after 40 min. Substantial demethylation of the H3K9me3 peptide was observed in the sample with WT KDM4A, resulting in formation of both di‐ and monomethylated products (H3K9me2 and H3K9me1 respectively, Figure A).…”

“…MS studies were then carried out to investigate whether demethylation by K241A KDM4A is stimulated by increasing the O 2 concentration. KDM assays (using the same final concentrations of components as above) were carried out with the use of a Mass Flow Controller (Brooks Instruments) to equilibrate the reactions at either 20 or 80 % O 2 , as described; reactions were left for 10 min before quenching (with methanol) and MALDI‐TOF MS analysis. As reported, the KDM activity of WT KDM4A increased with increasing O 2 concentration (from 31 to 46 % demethylation, Figure E) .…”

“…KDM assays (using the same final concentrations of components as above) were carried out with the use of a Mass Flow Controller (Brooks Instruments) to equilibrate the reactions at either 20 or 80 % O 2 , as described; reactions were left for 10 min before quenching (with methanol) and MALDI‐TOF MS analysis. As reported, the KDM activity of WT KDM4A increased with increasing O 2 concentration (from 31 to 46 % demethylation, Figure E) . However, only trace demethylation (<5 %) was observed in the samples with K241A KDM4A at either 20 or 80 % oxygen (Figure E); this indicated that oxygen binding/recruitment does not limiting the KDM activity of the K241A variant.…”

Adventures in Defining Roles of Oxygenases in the Regulation of Protein Biosynthesis

“…As we, and others, have found, epigenetic factors, including histone tail modifications and epigenetic enzymes likely also play important roles in regulating HIF target gene expression . However, as yet no other hypoxia/dioxygen sensors apart from the PHDs/FIH have been identified for the HIF system, though other 2OG oxygenases have been shown to have the potential to act in this capacity (e. g. histone demethylases) …”

Hypoxia and Inflammation in Cancer, Focus on HIF and NF-кB

“…Additionally, NF-κB controls gene expression of G1 cyclins, such as cyclin demethylase activity of these enzymes (JmjC domain) has a fold that is remarkably similar to the catalytic core of FIH [56,57]. In addition, investigation of the oxygen dependency of two of these enzymes revealed a graded drop in activity over physiologically relevant ranges of oxygen [58,59]. These studies indicate the potential of JmjC enzymes to link chromatin structure to oxygen sensing and participate in the hypoxia mediated transcriptional response.…”