Mouse embryonic stem cells exhibit cancer-like DNA methylation landscapes upon DNMT1 overexpression

DNA methyltransferase 1 (DNMT1) overexpression is a recurrent feature and an emerging therapeutic target in cancer, yet its direct impact on DNA methylation regulation remains poorly defined. Using enzymatic methyl sequencing, we show that DNMT1 overexpression in mouse embryonic stem cells leads to DNA methylation landscapes strikingly reminiscent of cancer methylomes, characterized by global hypomethylation, focal hypermethylation in promoters and CpG islands, and increased CpG-to-CpG variability. Differentially methylated regions in promoters correlate with altered gene expression and intersect with binding sites of key transcriptional regulators, pointing to potential underlying mechanisms of DNMT1 overexpression. We also show that DNMT3A and DNMT3B levels become lowered, suggesting that the broader DNA methylation machinery is perturbed when DNMT1 is in excess. Finally, we reveal that hypermethylation is partially retained following acute DNMT1 depletion and conserved across diverse human cancer methylomes, highlighting the translational relevance of this study for understanding the pathological mechanisms of DNMT1 overexpression in cancer and the limitations of DNMT1-targeted therapeutic strategies.