Wisconsin Institute for Discovery, University of Wisconsin-Madison, Madison, Wisconsin, USA; Department of Biomolecular Chemistry, SMPH, University of Wisconsin-Madison, Madison, Wisconsin, USA.
Department of Mathematics, University of Wisconsin-Madison, Madison, Wisconsin, USA.
J Biol Chem. 2023 Jul;299(7):104938. doi: 10.1016/j.jbc.2023.104938. Epub 2023 Jun 17.
S-adenosylmethionine (SAM) is the methyl donor for site-specific methylation reactions on histone proteins, imparting key epigenetic information. During SAM-depleted conditions that can arise from dietary methionine restriction, lysine di- and tri-methylation are reduced while sites such as Histone-3 lysine-9 (H3K9) are actively maintained, allowing cells to restore higher-state methylation upon metabolic recovery. Here, we investigated if the intrinsic catalytic properties of H3K9 histone methyltransferases (HMTs) contribute to this epigenetic persistence. We employed systematic kinetic analyses and substrate binding assays using four recombinant H3K9 HMTs (i.e., EHMT1, EHMT2, SUV39H1, and SUV39H2). At both high and low (i.e., sub-saturating) SAM, all HMTs displayed the highest catalytic efficiency (k/K) for monomethylation compared to di- and trimethylation on H3 peptide substrates. The favored monomethylation reaction was also reflected in k values, apart from SUV39H2 which displayed a similar k regardless of substrate methylation state. Using differentially methylated nucleosomes as substrates, kinetic analyses of EHMT1 and EHMT2 revealed similar catalytic preferences. Orthogonal binding assays revealed only small differences in substrate affinity across methylation states, suggesting that catalytic steps dictate the monomethylation preferences of EHMT1, EHMT2, and SUV39H1. To link in vitro catalytic rates with nuclear methylation dynamics, we built a mathematical model incorporating measured kinetic parameters and a time course of mass spectrometry-based H3K9 methylation measurements following cellular SAM depletion. The model revealed that the intrinsic kinetic constants of the catalytic domains could recapitulate in vivo observations. Together, these results suggest catalytic discrimination by H3K9 HMTs maintains nuclear H3K9me1, ensuring epigenetic persistence after metabolic stress.
S-腺苷甲硫氨酸（SAM）是组蛋白蛋白上特定位置甲基化反应的甲基供体，赋予关键的表观遗传信息。在饮食蛋氨酸限制可能导致的 SAM 耗尽条件下，赖氨酸二甲基化和三甲基化减少，而组蛋白-3 赖氨酸-9（H3K9）等位点则被积极维持，使细胞能够在代谢恢复后恢复更高状态的甲基化。在这里，我们研究了 H3K9 组蛋白甲基转移酶（HMTs）的固有催化特性是否有助于这种表观遗传持久性。我们使用四种重组 H3K9 HMT（即 EHMT1、EHMT2、SUV39H1 和 SUV39H2）进行了系统的动力学分析和底物结合测定。在高和低（即亚饱和）SAM 下，所有 HMT 在 H3 肽底物上的单甲基化比二甲基化和三甲基化具有最高的催化效率（k/K）。除了 SUV39H2 之外，所有 HMT 的 favored 单甲基化反应也反映在 k 值上，SUV39H2 无论底物甲基化状态如何，k 值都相似。使用差异甲基化核小体作为底物，EHMT1 和 EHMT2 的动力学分析显示出相似的催化偏好。正交结合测定显示，不同甲基化状态下的底物亲和力只有微小差异，表明催化步骤决定了 EHMT1、EHMT2 和 SUV39H1 的单甲基化偏好。为了将体外催化速率与核甲基化动力学联系起来，我们构建了一个数学模型，该模型包含了测量的动力学参数和基于质谱的 H3K9 甲基化测量的时间过程，这些测量是在细胞 SAM 耗尽后进行的。该模型表明，催化结构域的固有动力学常数可以再现体内观察结果。总之，这些结果表明 H3K9 HMT 的催化区分维持了核 H3K9me1，确保了代谢应激后表观遗传的持久性。
