TET family regulates the embryonic pluripotency of porcine preimplantation embryos by maintaining the DNA methylation level of <i>NANOG</i>

The ten-eleven translocation (TET) family (TET1/2/3) initiates conversion of 5-methylcytosine to 5-hydroxymethylcytosine, thereby orchestrating the DNA demethylation process and changes in epigenetic marks during early embryogenesis. In this study, CRISPR/Cas9 technology and a TET-specific inhibitor were applied to elucidate the role of TET family in regulating pluripotency in preimplantation embryos using porcine embryos as a model. Disruption of <i>TET1</i> unexpectedly resulted in the upregulation of <i>NANOG</i> and <i>ESRRB</i> transcripts, although there was no change to the level of DNA methylation in the promoter of <i>NANOG</i>. Surprisingly, a threefold increase in the transcript level of <i>TET3</i> was observed in blastocysts carrying modified TET1, which may explain the upregulation of <i>NANOG</i> and <i>ESRRB</i>. When the activity of TET enzymes was inhibited by dimethyloxalylglycine (DMOG) treatment, a dioxygenase inhibitor, to investigate the role of TET1 while eliminating the potential compensatory activation of TET3, reduced level of pluripotency genes including <i>NANOG</i> and <i>ESRRB</i>, and increased level of DNA methylation in the <i>NANOG</i> promoter was detected. Blastocysts treated with DMOG also presented a lower inner cell mass/TE ratio, implying the involvement of TET family in lineage specification in blastocysts. Our results indicate that the TET family modulates proper expression of <i>NANOG</i>, a key pluripotency marker, by controlling its DNA methylation profile in the promoter during embryogenesis. This study suggests that TET family is a critical component in pluripotency network of porcine embryos by regulating gene expression involved in pluripotency and early lineage specification.