Temporal Dynamics of Tet1 and Oct4 Gene Activation Resolve Distinct Stages of Global DNA Demethylation and Transcriptomic Changes in the Final Phases of Induced Pluripotency

The generation of induced pluripotent stem cells (iPSCs) involves activation of the endogenous pluripotency circuitry and global DNA demethylation late in reprogramming, but temporal resolution of these events using existing markers is insufficient. Here, we generated murine transgenic lines harboring dual fluorescent reporters reflecting cell-state specific expression of the master pluripotency factor Oct4 and the 5-methylcytosine dioxygenase Tet1. By assessing reprogramming intermediates based on dual reporter patterns, we identified a sequential order of Tet1 and Oct4 gene activation at proximal and distal regulatory elements following pluripotency entry. Full induction of Tet1 marks a pivotal late stage occurring after a phase of global gene repression, and preceding full activation of Oct4 along with late naive pluripotency and germ cell-specific genes. Sequential activation of Tet1 further distinguishes two waves of global DNA demethylation that target distinct genomic features and are largely uncoupled from transcriptional changes, showing both molecular parallels with and distinction from physiological reprogramming. Loss of Tet1 affects both waves of demethylation and generates iPSCs with chromosomal instability during lineage priming and defective differentiation potential. Therefore, the transcriptional logic of Tet1 expression signals a deterministic epigenetic roadmap towards generation of high quality iPSCs.