ABSTRACT The Ten-Eleven Translocation (Tet1/2/3) family of enzymes promote DNA demethylation and are highly expressed in neural stem cells (NSCs). They catalyze the stepwise oxidization of 5-methylcytosine (5mC) to 5- hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC). These modified cytosines are removed by thymine DNA glycosylases (TDG) to promote active demethylation. They also interfere with recruitment of Dnmt1 during replication thereby promoting passive DNA demethylation. 5hmC is stable and abundant in various cell types, particularly in the nervous system, which suggests that not all DNA hydroxylation leads to demethylation. By uncoupling Tet-mediated DNA hydroxylation from formylation and carboxylation, we may elucidate novel functions of these enzymatic processes in the epigenetic regulation of gene expression in NSC biology. We hypothesize that Tet-mediated DNA hydroxylation vs. formylation/ carboxylation contribute uniquely to gene regulation and biology in NSCs. To dissect these dual catalytic functions, we have introduced a mutation in all three Tet genes in ESCs that abrogates Tet’s ability to formylate and carboxylate, which we call Tet1/2/3 Formylation/Carboxylation (FoCa) mutant (Tet-FoCam/m) ESCs. This line can generate 5hmC but not 5fC and 5caC in contrast to our Tet1/2/3 full catalytic mutant ESCs (Tet-Catm/m) ESCs that lack all oxidative activities. We will differentiate these lines to NSCs to: (1) establish the biological requirements of Tet-mediated DNA hydroxylation vs formylation/carboxylation in the self-renewal and multipotency of NSCs, and (2) define how Tet-mediated DNA hydroxylation vs formylation/carboxylation regulate neural genes critical for NSC biology. Findings from these studies will establish the individual contributions of Tet-mediated DNA hydroxylation vs formylation/carboxylation and uncover the significance of 5hmC, 5fC, and 5caC in NSC gene regulation and biology. Under the joint mentorship of Drs. Meelad Dawlaty and Bernice Morrow, I will successfully complete the proposed research and training plan. This research program will enhance my knowledge of stem cell biology and facilitate my scientific and professional development by equipping me with the necessary skills to become a physician-scientist.