Native chromatome profiling reveals hundreds of metabolic enzymes in the nucleus across tissues.

Metabolic and epigenetic rewiring are hallmarks of cancer, with increasing evidence suggesting crosstalk between these processes. While previous studies have hinted at the role of metabolic enzymes in the nucleus, the extent and functional relevance of their nuclear localization remain unclear. In this study, we present a comprehensive chromatome proteomic analysis across cancer lineages and healthy samples, revealing that metabolic enzyme moonlighting on chromatin is widespread across various tissues and metabolic pathways. We show that the abundance of metabolic enzymes on chromatin is tissue-specific, with oxidative phosphorylation proteins notably depleted in lung cancer, suggesting a link between nuclear metabolism and cell identity. Further, we explore the dynamic chromatin association of one-carbon folate enzymes, demonstrating their involvement in DNA damage and repair processes. Finally, we asked whether restricting metabolic enzymes to specific subcellular compartments rewires the transcriptome, thereby decoupling the observed transcriptional changes from mere metabolite diffusion. Our findings propose and validate novel non-canonical nuclear roles for several metabolic enzymes, providing new insights into the functional relationship between metabolism and chromatin regulation. This study underscores the hypothesis that the nucleus is populated by metabolic enzymes, offering new avenues for understanding how nuclear metabolism impacts chromatin function and cancer progression.