Methionine deprivation reduces histone methylation and is rescuable with SAM. (A) Western blot for histone methylation levels in AML lines HL60, MOLM13, and K562 grown for 24 and 48 hours in the presence or absence of methionine. β-Actin and unmodified histone H3 used as loading controls (B) Western blot for H3K36me3 levels in a panel of 4 AML cell lines grown with and without the MAT2A inhibitor AG-270 (1 μM) for 48 hours. (C-D) Cell counts and viability of HL60 cells grown for 24 hours in the absence or presence of methionine and without methionine but supplemented with increasing concentrations of S-(5′-Adenosyl)-L-methionine p-toluenesulfonate salt (SAM-pTSA). Graphs directly correspond to the western blot experiment shown in (E), and are replicated in Figure 3B-C with appropriate statistics. (E) Western blot for histone methyl marks levels in the AML line HL60 grown for 24 hours in the absence or presence of methionine and without methionine but supplemented with increasing concentrations of SAM-pTSA. β-Actin and unmodified histone H3 were used as loading controls (F-G) Mass spectrometeric quantification of methylated (5meC/G) and hydroxymethylated (5hmC∖G) cystosines in the cell lines HL60, MOLM13, and K562 grown in the presence or absence of methionine for 24 and 48 hours. (H) Chromatin immunoprecipitation sequencing tracks of HL60 and CB-derived CD34⁺ cells grown in the presence or absence of methionine for 24 hours. Shown are tracks for H3K36me3, H3K4me3, and H3K4me1 marks on a number of gene loci such as BCL2L2, MYC, ENO, etc. (I) Gene ontology analysis of processes enriched for in HL60 and CB CD34+ cells, determined from gene loci with a greater than 4-fold decrease in H3K36me3 levels. All experiments: error bars represent mean ± SEM. Statistical analysis by Student t test, ∗P < .05, ∗∗P < .01, ∗∗∗∗P < .0001.