Pronounced specificity of the TET enzyme catalytic domain guides cellular function

Epigenetic memory in the form of cytosine methylation is essential for vertebrate development and the formation of cellular identity. Active removal of DNA methylation by the action of the TET hydroxylase family helps enable developmental potency, both in vivo and during creation of induced pluripotent stem cells. Despite this, little is known about how TET proteins are targeted to DNA. We report that mammalian TET enzymes show strong preference (>200 fold) for oxidising certain CG-containing hexamers in vitro, and during global methylation reprogramming in cultured cells and during embryogenesis. These preferred sequences, and also the most poorly targeted motifs, constitute recognition sites for developmental transcription factors whose binding activity is sensitive to DNA methylation. X-ray structural analysis and molecular dynamics simulations suggest that TET proteins use indirect readout to sense the sequence context flanking CG sites. These results are significant for understanding epigenetic reprogramming during development and the biological role TET enzymes play in modulating epigenetic memory. 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. Wild-type V6.5 hybrid embryonic stem cells (i.e. C57BL/6 X 129/sv cross) were treated with decitabine (5-aza-2’-deoxycytidine). Genome-wide bisulfite sequencing was used to uncover the rate at which DNA demethylation occurred in these samples.