Rescuing DNMT1 Fails to Fully Reverse the Molecular and Functional Repercussions of Its Loss in Mouse Embryonic Stem Cells

SUMMARY Epigenetic mechanisms are crucial for developmental programming and can be disrupted by environmental stressors, increasing susceptibility to disease. This has sparked interest in therapies for restoring epigenetic balance, but it remains uncertain whether disordered epigenetic mechanisms can be fully corrected. Disruption of DNA methyltransferase 1 (DNMT1), responsible for DNA methylation maintenance, has particularly devastating biological consequences. Therefore, here we explored if rescuing DNMT1 activity is sufficient to reverse the effects of its loss utilizing mouse embryonic stem cells. However, only partial reversal could be achieved. Extensive changes in DNA methylation, histone modifications and gene expression were detected, along with transposable element de-repression and genomic instability. Reduction of cellular size, complexity and proliferation rate were observed, as well as lasting effects in germ layer lineages and embryoid bodies. Interestingly, by analyzing the impact on imprinted regions, we uncovered 20 regions exhibiting imprinted-like signatures. Notably, while many permanent effects persisted throughout Dnmt1 inactivation and rescue, others arose from the rescue intervention. Lastly, rescuing DNMT1 after differentiation initiation worsened outcomes, reinforcing the need for early intervention. Our findings highlight the far-reaching functions of DNMT1 and provide valuable perspectives on the repercussions of epigenetic perturbations during early development and the challenges of rescue interventions. HIGHLIGHTS - Extensive changes to epigenomic landscapes and gene expression following transient loss of DNMT1 activity - Dysregulation of known imprinted regions and identification of 20 regions with imprinted-like signatures - De-repression of MERVL and MT2 LTRs with evidence of chimeric gene transcript generation - Shorter telomeres, DNA damage accumulation and reduction of cell size, internal complexity and proliferation rate - Lasting effects upon differentiation toward germ layer lineages and embryoid bodies - Worsened molecular and cellular outcomes when delaying Dnmt1 rescue until after differentiation initiation GRAPHICAL ABSTRACT