TET proteins regulate T cell and iNKT cell lineage specification in a TET2 catalytic dependent manner [CATCH-SEQ]

TET proteins mediate DNA demethylation by oxidizing 5-methylcytosine to 5-hydroxymethylcytosine (5hmC) and other oxidative derivatives. We have previously demonstrated a dynamic enrichment of 5hmC during T and invariant natural killer T cell lineage specification. Here, we investigate shared signatures in gene expression of Tet2/3 DKO CD4 single positive (SP) and iNKT cells in the thymus. We discover that TET proteins exert a fundamental role in regulating the expression of the lineage specifying factor Th-POK, which is encoded by Zbtb7b. We demonstrate that TET proteins mediate DNA demethylation - surrounding a proximal enhancer, critical for the intensity of Th-POK expression. In addition, TET proteins drive the DNA demethylation of site A at the Zbtb7b locus to facilitate GATA3 binding. GATA3 induces Th-POK expression in CD4 SP cells. Finally, by introducing a novel mouse model that lacks TET3 and expresses full length, catalytically inactive TET2, we establish a causal link between TET2 catalytic activity and lineage specification of both conventional and unconventional T cells. Overall design: We compared the methylation status across the Zbtb7b locus, that encodes for Th-POK, in CD4 SP cells isolated from 4 control (wt), 3 Tet2-/-Tet3flx/flxCD4 cre (DKO) mice and 1 Tet2CDTet3flx/flxCD4 cre (TET2CDTET3KO) mouse by using CATCHseq