Gene expression profiling of TET1 KO induced pluripotent stem cells (iPSCs) reprogrammed from WT mouse embryonic fibroblasts (MEFs)

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 are insufficient using existing stage markers. 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. A transient phase of global gene repression accompanies full activation of Tet1, precedes activation of meiotic and gametogenesis genes, and distinguishes phases of global DNA demethylation reminiscent of germ-line reprogramming. Loss of Tet1 is compatible with reprogramming towards full Oct4 gene activation, but generates iPSCs with epigenetic defects. Therefore, the transcriptional logic of Tet1 expression signals an epigenetic roadmap towards efficient reprogramming. Transcriptome analysis of Cas9 TET1 KO and non-targeted iPSCs all in biological triplicates by RNA-seq.