# Tet-mediated DNA hydroxylation vs formylation and carboxylation in NSC biology

> **NIH NIH F30** · ALBERT EINSTEIN COLLEGE OF MEDICINE · 2024 · $52,694

## Abstract

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.

## Key facts

- **NIH application ID:** 10892389
- **Project number:** 1F30HD113183-01A1
- **Recipient organization:** ALBERT EINSTEIN COLLEGE OF MEDICINE
- **Principal Investigator:** Blake C Ebert
- **Activity code:** F30 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $52,694
- **Award type:** 1
- **Project period:** 2024-04-16 → 2027-04-15

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10892389

## Citation

> US National Institutes of Health, RePORTER application 10892389, Tet-mediated DNA hydroxylation vs formylation and carboxylation in NSC biology (1F30HD113183-01A1). Retrieved via AI Analytics 2026-05-29 from https://api.ai-analytics.org/grant/nih/10892389. Licensed CC0.

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