The Activity of JmjC Histone Lysine Demethylase KDM4A is Highly Sensitive to Oxygen Concentrations.
The Activity of JmjC Histone Lysine Demethylase KDM4A is Highly Sensitive to Oxygen Concentrations.
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DOI：
10.1021/acschembio.6b00958
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发表时间：
2017-04-21
影响因子：
4
通讯作者：
Kawamura A
中科院分区：
文献类型：
--
作者：
Hancock RL;Masson N;Dunne K;Flashman E;Kawamura A
JmjC组蛋白赖氨酸脱甲基酶（KDM）是表观遗传调节剂，其参与从组蛋白尾部内的后修饰的赖氨酰残基去除甲基，从而调节基因转录。这些酶需要分子氧来进行催化活性，并且作为2-酮戊二酸（2 OG）依赖性加氧酶，与细胞氧传感HIF羟化酶PHD 2和FIH相关。最近的研究表明，包括假基因编码的KDM 4 E在内的一些KDM的活性可能对氧浓度的变化敏感。在这里，我们报告了氧可用性对KDM 4亚家族成员KDM 4A活性的影响的详细分析，重要的是证明了分离的蛋白质和细胞中的高水平的O2敏感性。重组酶的动力学分析显示高KMapp（O2）为173 ± 23 μM，表明该酶的活性能够对氧浓度的降低敏感地响应。此外，在U2 OS细胞中条件性过表达KDM 4A的免疫荧光实验表明，KDM 4A对其主要底物H3 K9 me 3的细胞活性显示出对耗尽氧浓度的分级反应，与使用分离的蛋白质获得的数据一致。这些结果表明，KDM 4A具有作为染色质修饰背景下的氧传感器的潜力，可能影响缺氧疾病状态下的表观遗传调控。重要的是，在生物化学和细胞测定中KDM 4A的催化活性的氧敏感性之间的这种相关性证明了生物化学研究在理解促成2 OG加氧酶的不同生物学功能和不同活性的因素中的效用。
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.