Rescuing DNMT1 Fails to Fully Reverse the Molecular and Functional Repercussions of Its Loss in Mouse Embryonic Stem Cells [mRNA-seq]

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. Overall design: Dnmt1 was inactivated and rescued in mouse embryonic stem cells (mESCs) using the Tet-Off system, i.e. Dnmt1tet/tet mESCs developed and published by Borowczyk et al. (Borowczyk et al. Proc Natl Acad Sci U S A. 2009. doi:10.1073/pnas.0905668106). Samples were collected prior to Dnmt1 inactivation (i.e. Dnmt1 control, CTL), after Dnmt1 inactivation (i.e. Dnmt1 inactive, INV) and after rescuing Dnmt1 (i.e. Dnmt1 rescue, RES). Various sequencing experiments were then conducted. DNA methylation profiles were assessed by EM-seq, histone modification (H3K4me3, H3K27ac, H3K4me1, H3K27me3, H3K9me3) landscapes by ChIP-seq, gene expression levels by mRNA-seq and transposable element transcription levels by RNA-seq. The impact of inactivation and rescue of Dnmt1 in mESCs on gene expression profiles in germ layers was also assessed by mRNA-seq; CTL and RES mESCs were differentiated towards endoderm, mesoderm, and ectoderm lineages. Finally, we investigated whether rescuing Dnmt1 after differentiation initiation during embryoid body formation would worsen outcomes; embryoid bodies were derived from CTL, INV and RES mESCs and grown for 10 days, reactivating Dnmt1 on day 5 in a subset of INV embryoid bodies for post-differention rescue (PostDiff). Embryoid bodies were harvested and pooled on day 5 and day 10 for mRNA sequencing.