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

Raphael Pantier, Tulin Tatar, Douglas Colby, Ian Chambers

Research output: Contribution to journalArticlepeer-review

Abstract

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 Tetfamily alleles using CRISPR/Cas9, and characterised TET protein expression in distinct pluripotent cell culture conditions.Whereas TET1 is abundantly expressed in both na¨ıve and primed pluripotent cells, TET2 expression is restricted to the na¨ıvestate. Moreover, TET2 is expressed heterogeneously in embryonic stem cells (ESCs) cultured in serum/leukemia inhibitory factor, with expression correlating with na¨ıve pluripotency markers. FACS-sorting of ESCs carrying a Tet2Flag-IRES-EGFP reporter demonstrated that TET2-negative cells have lost theability 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 naıve pluripotency genes.
Original languageEnglish
Article numbere201900516
JournalLife Science Alliance
Volume2
Issue number5
DOIs
Publication statusPublished - 3 Oct 2019

Keywords

  • embryonic stem cells
  • naive pluriptency
  • NANOG
  • primed pluriptency
  • TET proteins

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