Ion chromatography-ultra-high-resolution mass spectrometry reveals EZH2-driven reprogramming of nucleic acid and...

ENHANCER OF ZESTE: homolog 2 (EZH2), a histone H3K27 trimethyltransferase, is a key epigenetic regulator frequently dysregulated in cancer. To determine its impact on nucleotide biosynthesis and nucleic acid methylation in intact cells requires highly sensitive, isomer-resolving analytical workflows. We developed a targeted ion chromatography-ultra-high-resolution Fourier transform mass spectrometry (IC-UHR-FTMS) workflow with lower limits of quantification down to 9 fmol on-column to determine changes in methylation of DNA, total RNA, and mRNA in A549 cells following EZH2 knockdown (KD). Using dual stable isotope tracers, l-methionine-(methyl-¹³C) and l-glutamine-(¹⁵N₂), in a multiplexed stable isotope-resolved metabolomics (SIRM) design, we quantified positionally-resolved ¹³C/¹⁵N labeling of methylated nucleotides and their precursors. EZH2 KD reduced ¹⁵N incorporation into deoxynucleotides, indicating impaired de novo synthesis from glutamine. It also attenuated ¹⁵N and/or ¹³C labeling of nucleotides and methylated nucleotides in total RNA and mRNA at various atomic positions, reflecting global losses in biosynthesis and S-adenosylmethionine (SAM)-dependent methylation. Notably, AMP methylation at N6 and 2'-O positions was most responsive to EZH2 KD, implicating reduced capped-RNA translation. Some of the EZH2 KD-induced changes in RNA methylation corresponded with the altered expression of their writer or eraser enzymes. This study demonstrates multiplex stable isotope tracers-coupled IC-UHR-MS as a powerful tool for comprehensive tracing of methylation dynamics in mammalian cells and reveals EZH2's role in metabolic-epitranscriptomic regulation by modulating SAM availability via glutamine-fueled de novo purine biosynthesis and RNA methylation.