TET1 controls maintenance DNA methylation via the regulation of Dppa3. TET1 controls maintenance DNA methylation

The transition from naive to primed pluripotency is accompanied by a dramatic resetting of global DNA modifications, closely mirroring the epigenomic reprogramming of peri-implantation development in vivo. The TET family of proteins catalyzes the oxidation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). Intriguingly, TET1 has been shown to control the expression of pluripotency as well as lineage markers. However, it remains unclear how catalytic and non-catalytic functions contribute to the regulation of gene expression and to which extent cytosine modifications represent intermediates of active DNA demethylation or can serve as stable epigenetic marks. Using catalytic mutant as well as knockout cell lines, we dissect the stage-specific contributions of TET1 catalytic activity to the regulation of the methylome and transcriptome of embryonic stem cells (ESCs) and epiblast-like cells (EpiLCs). Systematic transcriptome analysis of Tet1 knockouts and catalytic mutants reveals both catalytic-dependent and -independent transcriptional targets, which are largely distinct between ESCs and EpiLCs. While the catalytic activity of TET is necessary for the fine-tuning of naive pluripotency markers in ESCs, non-catalytic functions prevent the premature activation of developmental regulators in EpiLCs. Despite the global DNA hypermethylation present in Tet1 knockouts and catalytic mutants, both catalytically dependent and -independent gene regulation is uncoupled from DNA demethylation at promoters. Surprisingly, we can show that the observed global hypermethylation is not caused by a loss in active DNA demethylation due to loss of TET1 activity but is a result of premature activation of maintenance methylation. We demonstrate that the naïve pluripotency maker DPPA3 (STELLA) inhibits UHRF1 on a post-transcriptional level contributing to the establishment of the hypomethylated state of naïve ESCs. TET1 positively regulates the expression of Dppa3 in a catalytic dependent manner thereby establishing a feedback of TET1 catalytic activity into the DNA methylation maintenance machinery. In turn, loss of Dppa3 expression – not loss of Tet1 catalytic activity - in Tet1 knockout and catalytic mutants causes the global hypermethylation phenotype observed in these cells. Taken together, these findings question the role of oxidized cytosine derivatives as intermediates of DNA demethylation and underline their importance as stable epigenetic marks.