# Labeling and sequencing of 5hmC and 5mC in DNA-Renewal

> **NIH NIH R01** · UNIVERSITY OF CHICAGO · 2023 · $567,580

## Abstract

Project Summary/Abstract
DNA cytosine methylation (5-methylcytosine or 5mC) is a key epigenetic modification in the
regulation of human gene expression. It plays critical roles in suppressing transcription of a
large portion of human genome, including repetitive elements. 5mC can be reversed through
oxidation by the human TET family enzymes, which utilize dioxygen to sequentially oxidize 5mC
to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and finally 5-carboxycytosine
(5caC). Both 5fC and 5caC can be recognized and excised by human thymine DNA glycosylase
(TDG), followed by base excision repair (BER) to replace the modified cytosine with a normal
cytosine, in an active demethylation process. Cell-type specific DNA methylation has been
studied and applied as a biomarker for disease diagnosis and prognosis. Antibody-based
MeDIP immunoprecipitation followed by high-throughput sequencing is a common method for
genome-wide mapping of DNA 5mC distribution, but it is not quantitative and requires a
significant amount of input material. Bisulfite sequencing is the gold standard approach, widely
applied in quantitative 5mC sequencing; however, whole-genome bisulfite sequencing is
expensive and causes severe DNA degradation. To overcome these critical barriers to
progress, we propose to develop new methods that combine 5mC enrichment with quantitative
5mC sequencing, using limited starting material. The potential application of these new methods
in circulating cell-free DNA (cfDNA) analyses stands to potentially revolutionize human disease
diagnosis and prognosis. Whereas the 5mC modification suppress activation of a majority of
human genome, studies from us and others revealed that 5hmC marks active loci. We
previously developed robust procedures to map 5hmC genome-wide using 1,000 cells.
However, quantitative methods that determine the presence and stoichiometry of 5hmC at the
single-cell level are still lacking. Our ongoing efforts to overcome this challenge recently led to a
chemical solution that yields base-resolution 5hmC information with modification stoichiometry,
using limited input material. In this renewal, we propose base resolution sequencing of 5hmC at
the single-cell level, enabling the detection of intercellular differences that are otherwise missed,
including the epigenome remodeling that underlies the initiation of cell fates during early
embryogenesis. The success of this program will provide new methods for 5mC and 5hmC
mapping to enable breakthrough discoveries in both basic research and clinical applications.

## Key facts

- **NIH application ID:** 10692575
- **Project number:** 5R01HG006827-12
- **Recipient organization:** UNIVERSITY OF CHICAGO
- **Principal Investigator:** CHUAN HE
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2023
- **Award amount:** $567,580
- **Award type:** 5
- **Project period:** 2012-08-07 → 2026-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10692575

## Citation

> US National Institutes of Health, RePORTER application 10692575, Labeling and sequencing of 5hmC and 5mC in DNA-Renewal (5R01HG006827-12). Retrieved via AI Analytics 2026-05-21 from https://api.ai-analytics.org/grant/nih/10692575. Licensed CC0.

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