DNMT1 overexpression disrupts DNA methylation homeostasis in mouse embryonic stem cells

Abstract DNMT1 overexpression is frequently observed in cancer and other pathologies, yet its direct impact on DNA methylation remains poorly defined. Using whole-genome methyl sequencing, we show that overexpressing DNMT1 in mouse embryonic stem cells (mESCs) induces global hypomethylation, focal hypermethylation in promoters and CpG islands, and increased methylome variability—well-documented characteristics of cancer. We also find that differential promoter methylation is associated with developmental (axon guidance, Wnt) and disease (cancer, cardiomyopathy) pathways and correlates with altered gene expression. Additionally, DNMT3A/B levels are reduced, indicating that excess DNMT1 perturbs the broader DNA methylation machinery. Moreover, to model targeted therapies, we reveal that hypermethylation is mostly erased following DNMT1 depletion, but a substantial portion persists. Hypermethylation is then largely regained upon reinstating DNMT1 overexpression, only achieving permanent erasure at a minority of regions. Finally, promoter hypermethylation detected in mESCs is observed across diverse human cancers, supporting its biological significance and the translational relevance of this study. Overall, these findings illuminate how DNMT1 overexpression disrupts DNA methylation homeostasis, providing mechanistic insight into its pathogenic consequences and therapeutic targeting. Graphical abstract