# Defining the regulation of UHRF1 and DNMT1 for maintenance of the epigenome

> **NIH NIH F32** · VAN ANDEL RESEARCH INSTITUTE · 2021 · $66,390

## Abstract

PROJECT SUMMARY
In multicellular organisms, methylation of DNA on the 5’ carbon of cytosine helps ensure proper gene expression.
Methylation occurs symmetrically on both DNA strands, and the maintenance methyltransferase DNMT1 is
responsible for copying the methylation pattern from the parent strand to the daughter strand during DNA
replication. Maintenance methylation by DNMT1 requires the chromatin-associated ubiquitin ligase UHRF1,
which catalyzes ubiquitination of histone H3 (H3ub). Many questions remain about how the various domain
functions of DNMT1 and UHRF1 are regulated and which are necessary to maintain DNA methylation. The
objective of this proposal is to define the mechanistic relationship between UHRF1, DNMT1, and H3
ubiquitination in the maintenance of DNA methylation. The first aim investigates the hypothesis that ubiquitination
of histone H3 by UHRF1 regulates the catalytic activity of DNMT1 and that maintenance methylation by DNMT1
depends on both UHRF1 ubiquitin ligase-dependent and independent mechanisms. Aim 1 will use in vitro
biochemical assays to characterize point mutations in DNMT1 and UHRF1 that compromise individual domains
of these proteins. Transgenes encoding these mutant enzymes will then be tested in cancer cells using western
blot analysis of H3ub and EPIC array analysis of DNA methylation. These experiments will define the contribution
of individual functions of DNMT1 and UHRF1 to H3 ubiquitination and DNA methylation in vitro and during DNA
maintenance methylation in cancer cells. The second aim tests the hypothesis that allosteric activation of
UHRF1’s ubiquitin ligase activity by hemimethylated DNA (heDNA) operates in cells and regulates genome
architecture in cooperation with CTCF/cohesin. Aim 2 will use UHRF1 mutant transgenes to test whether heDNA
stimulates UHRF1’s enzymatic activity in cancer cells using western blot analysis of H3ub and hairpin bisulfite
sequencing to determine levels of heDNA. The contribution of UHRF1 function to genome architecture through
recently identified stable heDNA at CTCF/cohesin sites will also be defined using CTCF ChIP-seq and Hi-C.
These experiments will determine if heDNA regulates the activity of UHRF1 in cancer cells and test the
contribution of stable heDNA at CTCF/cohesin sites to genome architecture through UHRF1. Misregulation of
DNA methylation is a driving force in many cancers. Thus, insight into how DNA methylation is synthesized and
propagated is central to understanding how cancer begins and progresses. The studies proposed here will
advance our knowledge of the mechanisms involved in methylation maintenance to allow the rational design of
therapies that halt or reverse the formation of oncogenic DNA methylation patterns.

## Key facts

- **NIH application ID:** 10234536
- **Project number:** 1F32CA260116-01A1
- **Recipient organization:** VAN ANDEL RESEARCH INSTITUTE
- **Principal Investigator:** Joel Hrit
- **Activity code:** F32 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $66,390
- **Award type:** 1
- **Project period:** 2021-04-01 → 2024-03-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10234536

## Citation

> US National Institutes of Health, RePORTER application 10234536, Defining the regulation of UHRF1 and DNMT1 for maintenance of the epigenome (1F32CA260116-01A1). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10234536. Licensed CC0.

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