Pronounced specificity of the TET enzyme catalytic domain guides cellular function

TET (ten-eleven translocation) enzymes catalyze the oxidation of 5-methylcytosine bases in DNA, thus driving active and passive DNA demethylation. Here, we report that the catalytic domain of mammalian TET enzymes favor CGs embedded within bHLH and bZIP transcription factor binding sites, with up to 250-fold preference in vitro. Crystal structures and molecular dynamics calculations show that sequence preference is caused by intrasubstrate interactions and CG flanking sequence indirectly affecting enzyme conformation. TET sequence preferences are physiologically relevant as they explain the rates of DNA demethylation in TET-rescue experiments in culture and in vivo within the zygote and germline. Most and least favorable TET motifs represent DNA sites that are bound by methylation-sensitive immediate-early transcription factors and OCT4, respectively, illuminating TET function in transcriptional responses and pluripotency support. Overall design: An ESC line with TET1-3 genetically inactivated (TET-TKO) was rescued using an inducible PiggyBAC transposon based vector containing the catalytic domain from mouse TET3 induced by doxicycline and transcriptionally linked to an mCherry reporter gene. Cells without doxycicline treatment were used as control. APOBEC-coupled epigenetic sequencing (ACE-seq) was used to quantitate 5-Hydroxymethylcytosine (5hmC) levels.