Endogenous epitope-tagging of Tet1, Tet2 and Tet3 identifies TET2 as a naive pluripotency marker

Tet1 Tet2, and Tet3 encode DNA demethylases that play critical roles during stem cell differentiation and reprogramming to pluripotency. Although all three genes are transcribed in pluripotent cells, little is known about the expression of the corresponding proteins. Here, we tagged all the endogenous Tet family alleles using CRISPR/Cas9, and characterised TET protein expression in distinct pluripotent cell culture conditions. Whereas TET1 is abundantly expressed in both naive and primed pluripotent cells, TET2 expression is restricted to the naive state. Moreover, TET2 is expressed heterogeneously in embryonic stem cells (ESCs) cultured in serum/leukemia inhibitory factor, with expression correlating with naive pluripotency markers. FACS-sorting of ESCs carrying a Tet2(Flag-IRES-EGFP) reporter demonstrated that TET2-negative cells have lost the ability to form undifferentiated ESC colonies. We further show that TET2 binds to the transcription factor NANOG. We hypothesize that TET2 and NANOG co-localise on chromatin to regulate enhancers associated with naive pluripotency genes.