# DNA hydroxymethylation and post stroke brain damage

> **NIH NIH R01** · UNIVERSITY OF WISCONSIN-MADISON · 2020 · $415,351

## Abstract

Epigenetic changes in DNA and histones are known to significantly influence the gene expression and outcome
after many diseases. The role of epigenetics in ischemic brain damage is not yet fully understood. Of particular
interest, the cytosine in DNA undergoes methylation to form 5-methylcytosine (5mC) which is known to be a
transcriptional silencer. Recent studies showed that 5mC will be oxidized by ten-eleven translocation (TET)
hydroxylases to form 5-hydroxymethylcytosine (5hmC). This epigenetic change is considered as a transcriptional
derepression mark that increases cell survival under adverse conditions. In particular, brain contains ~10 fold
higher 5hmC levels than other organs of the body. Preliminary studies showed that transient focal ischemia in
adult rodents significantly increase the genomic 5hmC levels in the peri-infarct cortex. TET3 knockdown
decreased 5hmC levels, and exacerbated post-ischemic mortality and infarction in both male and female mice.
On the other hand, increasing 5hmC levels by treatment with ascorbate (a TET inducer) significantly protected
the brain after focal ischemia in a TET3-dependent manner. Hence, we hypothesize that “Tet3 mediated
induction of 5hmC is a neuroprotective adaptation that can be potentiated to protect brain after stroke.”
The major neuronal isoform of TET3 lacks DNA binding domains. Our preliminary data show that TET3 binds to
lncRNAs with high affinity. The lncRNAs are known to act as scaffolds to bring DNA/RNA/protein together
enabling their action. LncRNAs are also known to modulate post-stroke outcome. Hence, we further hypothesize
that “lncRNAs play a vital role in scaffolding and guiding TET3 to specific genomic sites, and thus
modulate 5hmC levels and functional outcome after stroke.”
Aim 1: To evaluate if DNA hydroxymethylation is neuroprotective after stroke. We will test the functional
significance of 5hmC in post-stroke pathophysiology by loss of function and gain of function of TET3. Genomic
sites where 5hmC is increased after stroke will be mapped by chromatin immunoprecipitation combined with
massively parallel DNA sequencing (ChIP-seq) following TET3 knockdown and induction.
Aim 2: To study if lncRNAs regulate TET3-mediated DNA hydroxymethylation and the ensuing neuroprotection
after stroke. We will determine the genomic locations modulated by the TET3-interacting lncRNAs by high
throughput sequencing method chromatin isolation by RNA purification (ChiRP-seq). We will further study if
lncRNA function is essential for TET3/5hmC mediated neuroprotection after stroke.
The long-term goal is to define the role of 5hmC in post-ischemic pathology and to test if increasing 5hmC levels
is beneficial after stroke.

## Key facts

- **NIH application ID:** 10001037
- **Project number:** 5R01NS109459-03
- **Recipient organization:** UNIVERSITY OF WISCONSIN-MADISON
- **Principal Investigator:** Raghu VEMUGANTI
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $415,351
- **Award type:** 5
- **Project period:** 2018-09-01 → 2023-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10001037

## Citation

> US National Institutes of Health, RePORTER application 10001037, DNA hydroxymethylation and post stroke brain damage (5R01NS109459-03). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/10001037. Licensed CC0.

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