Epigenetic Inhibitor 5-Azacytidine Triggers DIM-2/DIM-5-Dependent Mutagenesis in H3K9me3-Enriched Regions of Neurospora crassa

The DNA methyltransferases inhibitor 5-azacytidine (5AzC), clinically used to treat hematopoietic malignancies, can elevate genomic mutational burden, raising safety concerns. To define the epigenetic specificity and mutagenic consequences of 5AzC, we performed multi-omics analyses in Neurospora crassa. Our data showed that 5AzC caused a non-selective, genome-wide reduction in both 5-methylcytosine (5mC; ~50% decrease) and the heterochromatin mark H3K9me3 (~65% decrease), indicating broad off-target demethylation that may transiently benefit therapy yet compromise genome stability. Whole-genome sequencing (WGS) revealed a ~290-fold increase in mutation rate under 5AzC, with a pronounced C->G transversion bias, a spectrum typically associated with higher functional burden. Strikingly, 5AzC-induced mutations were enriched in H3K9me3-marked domains, particularly pericentromeric regions characterized by low 5mC but high H3K9me3. Genetic analyses showed that the loss of DNA methyltransferase DIM-2 reduced 5AzC-induced mutations by ~64%, while individual or combined knockout of the histone methyltransferase DIM-5 with DIM-2 led to an 85% reduction. Thus, mutagenesis was markedly amplified by DIM-2 and DIM-5, with DIM-2 activity dependent on DIM-5. Collectively, DIM-2 and DIM-5 accounted for nearly all A/T-site and ~80% of G/C-site mutations. These results reveal that 5AzC drives genome-wide loss of 5mC and H3K9me3, with mutagenesis preferentially targeting H3K9me3-enriched regions via DIM-2 and DIM-5. This work clarifies a mechanistic basis for 5AzC-associated genomic risk and highlights strategies for next-generation epigenetic therapies that preserve heterochromatin integrity while minimizing mutational load.