# The mechanism and modulation of 5-methylcytosine oxidation by ATET family enzymes

> **NIH NIH R01** · UNIVERSITY OF PENNSYLVANIA · 2020 · $309,925

## Abstract

PROJECT SUMMARY
This proposal aims to elucidate the mechanisms and implications of step-wise oxidation of 5-methylcytosine by
TET family enzymes. Modifications to cytosine bases play an important role in diverse processes, including
embryonic development, pluripotency and oncogenesis. The best studied cytosine modification is methylation
at the 5-position (5mC), which typically occurs at cytosine-guanine dinucleotides (CpGs) in the genome. While
stable CpG methylation can mediate gene silencing in processes such as imprinting, dynamic changes in CpG
methylation are also important in cellular differentiation and induced pluripotency. The recent discovery that
5mC can be oxidized by TET family enzymes has shed new light on the mechanisms involved in DNA
methylation dynamics and has greatly increased the potential coding capacity of CpGs. There are three
different TET family members that are differentially expressed and likely have both complementary and
redundant roles. These enzymes are Fe(II)/α-ketoglutarate-dependent dioxygenases that can oxidize 5mC to
generate 5-hydroxymethylcytosine (5hmC). Importantly, 5hmC is itself a substrate for further oxidation,
generating 5-formylcytosine (5fC), and 5fC can be oxidized yet further to 5-carboxylcytosine (5caC). These
three different oxidized 5mC bases (ox-mCs) are postulated to play two important biological roles: as
intermediates in the process of DNA demethylation and as independent epigenetic marks. A critical goal in the
field is understanding what distinct roles are played by each of the ox-mC bases. However, we currently lack
an understanding of the mechanisms that regulate which modification is introduced at a given CpG by the
different isozymes, nor can we dissociate these three intricately linked ox-mCs from one another. Our proposal
aims to fill this gap by using a combination of innovative enzymatic assays and targeted active site
manipulation. In order to track oxidation at a single site, we have developed isotopologue-based assays using
specific radiolabeled or heavy-atom labeled 5mC. To inform how the extended epigenome is established and
maintained, we will exploit our assays to determine the preferences for 5mC, 5hmC or 5fC as substrates and
how opposite strand CpG modification influences each TET isozyme. To connect the biochemical preferences
to patterns of activity in cells, we will pursue a cellular model that allows for expression of each TET isozyme in
isolation and characterize the pattern of ox-mCs and their coupling on opposite strands. By manipulating the
TET active site, we have also discovered remarkable mutants that are specifically deficient in later oxidation
steps and now aim to decipher the mechanism at play in these “5hmC-dominant” variants. We propose to
exploit our discovery to determine whether 5hmC alone, or 5fC/5caC also, are required for reprogramming of
fibroblasts to induced pluripotent stem cells. Thus, together our aims encompass mechanism and function: to
el...

## Key facts

- **NIH application ID:** 9848584
- **Project number:** 5R01GM118501-04
- **Recipient organization:** UNIVERSITY OF PENNSYLVANIA
- **Principal Investigator:** Rahul Manu Kohli
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $309,925
- **Award type:** 5
- **Project period:** 2017-01-01 → 2021-05-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9848584

## Citation

> US National Institutes of Health, RePORTER application 9848584, The mechanism and modulation of 5-methylcytosine oxidation by ATET family enzymes (5R01GM118501-04). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/9848584. Licensed CC0.

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