Active turnover of genomic methylcytosine in pluripotent cells
Epigenetic plasticity underpins cell potency, but the extent to which active turnover of DNA methylation contributes to such plasticity is not known and the underlying pathways are poorly understood. Here we use metabolic labelling with stable isotopes and mass spectrometry to quantitatively address the global turnover of genomic methylcytidine (mdC), hydroxymethylcytidine (hmdC) and formylcytidine (fdC) across mouse pluripotent cell states. High rates of mdC/hmdC oxidation and fdC turnover characterize a formative-like pluripotent state. In primed pluripotent cells the global mdC turnover rate is about 3-6% faster than can be explained by passive dilution through DNA synthesis. While this active component is largely dependent on Tet-mediated mdC oxidation, we unveil additional oxidation-independent mdC turnover, possibly through DNA repair. This process accelerates upon acquisition of primed pluripotency and returns to low levels in lineage committed cells. Thus, in pluripotent cells active mdC turnover involves both mdC oxidation-dependent and  independent processes. Overall design: High throughput sequencing of a bisulfite amplicon library was used to measure CpG methylation levels at selected secondary imprints in mouse pluripotent stem cells, before and at three and five days during the transition from alternative naïve/formative-like state to primed state; mouse primary embryonic fibroblasts were used as somatic cell reference; no independent replicates were analyzed