Abstract
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 (kcat/KM) for monomethylation compared to di- and trimethylation on H3 peptide substrates. The favored monomethylation reaction was also reflected in kcat values, apart from SUV39H2 which displayed a similar kcat 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.