Establishment of neuronal and glial competence of neural stem cells requires distinct enzymatic activities of TET enzymes.

Ten-eleven translocation (TET1/2/3) enzymes are expressed in neural stem cells (NSCs). They iteratively oxidize 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC). The significance of hydroxymethylation (i.e., 5hmC) versus formylation and carboxylation (i.e., active demethylation) is undefined. We generated NSCs lacking only TET formylation and carboxylation activities (Tet-TFoCa) and compared them to NSCs lacking all three TET activities (Tet-TMut). Tet-TFoCa NSCs could differentiate into neurons but not into glial cells, while Tet-TMut NSCs could not form either cell type. Mechanistically, neuronal genes retained 5hmC at their enhancers in Tet-TFoCa NSCs and were expressed normally, consistent with the ability of these cells to form neurons. In contrast, enhancers of glial genes were hypermethylated in both Tet-TFoCa and Tet-TMut NSCs underpinning downregulation of these genes and the glial block in these cells. Our findings implicate TET-driven hydroxymethylation in establishing NSC neuronal competence and formylation and carboxylation in defining NSC glial competence.