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

> **NIH NIH R01** · UNIVERSITY OF CHICAGO · 2021 · $625,702

## Abstract

Project Summary/Abstract
DNA cytosine methylation (5-methylcytosine or 5mC) is the main epigenetic mechanism in
human gene expression regulation. This methylation is oxidized by the human TET family
enzymes to 5-hydroxymethylcytosine (5hmC) in an active demethylation process. While 5mC is
a mark for gene repression in general and is also used to suppress repetitive elements in the
human genome, recent studies showed that 5hmC tends to mark active loci, as gene activation
requires removal of 5mC in gene bodies, promoters, and enhancers. The presence of 5hmC
indicates open chromatin loci, whereas heavy 5mC methylation often marks heterochromatin
regions. Therefore, genomic locations of 5hmC and 5mC provide genome-wide, comprehensive
information on chromatin activation and repression, which are suitable marks for classifying
individual human cells. The locations of 5hmC and 5mC can also store information of gene
activation in frozen or fragmented genomic samples, such as DNA from biopsy samples or cell-
free DNA (cfDNA), providing additional advantages in clinical diagnosis and prognosis of
various human diseases.
Building on our previous successes in inventing enabling technologies that label and sequence
5hmC and 5mC, we have recently developed robust procedures to map 5hmC and related
cytosine modifications genome-wide using 1,000 cells. In this application we propose new
approaches that can map both 5hmC and 5hmC at base-resolution down to the single-cell level.
We plan to test and validate these new methods by investigating cell-cell heterogeneity using
Tet2-mutant acute myeloid leukemia stem cells versus controls. We will also validate the
new methods to map 5hmC and 5mC in other systems with limited DNA. In addition, we
propose a new and highly useful method for single-cell bisulfite sequencing of 5mC in DNA by
pre-amplify the limited DNA samples to much higher abundances compatible with most
downstream analysis methods, with 5mC sites replicated faithfully during the amplification. The
proposed research will provide urgently needed tools for the PI's group and the broader
scientific community to study a range of questions in biomedical research and clinical biomarker
discoveries using limited input DNA and at the single-cell level.

## Key facts

- **NIH application ID:** 10225996
- **Project number:** 5R01HG006827-10
- **Recipient organization:** UNIVERSITY OF CHICAGO
- **Principal Investigator:** CHUAN HE
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $625,702
- **Award type:** 5
- **Project period:** 2012-08-07 → 2022-05-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10225996

## Citation

> US National Institutes of Health, RePORTER application 10225996, Labeling and sequencing of 5hmC and 5mC in DNA (5R01HG006827-10). Retrieved via AI Analytics 2026-05-25 from https://api.ai-analytics.org/grant/nih/10225996. Licensed CC0.

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