# Single-cell approaches to probe the function of the unique neuronal epigenome

> **NIH NIH R21** · WASHINGTON UNIVERSITY · 2022 · $196,875

## Abstract

Abstract
The recent explosion of single cell techniques has revealed striking cellular diversity and complexity in the
mammalian nervous system. Neurons are driven to develop their unique features by intrinsic factors, but also
must adapt to local environmental features to produce functional circuits. This raises the question: how do
neurons maintain or return to stable cellular states in the context of variable inputs? Evidence suggests that this
process requires transcriptional regulation by DNA methylation and the methyl-binding protein MeCP2. The
levels of 5-methylcytosine (mC) and its oxidized form, 5-hydroxymethylcytosine (hmC), dramatically change in
the developing mammalian brain in response to intrinsic cues and environmental input, and the genomic profiles
of these marks appear to show unique patterns in different cell-types. MeCP2 modulates the effects of mC and
hmC in neurons and is commonly mutated in Rett syndrome. Further, mutations in the TET enzymes responsible
for converting mC to hmC have been recently associated with neurological disorders, indicating that hmC may
play a key epigenetic role in gene regulation in the brain. Despite this, we have little understanding of why mC
and hmC patterns are unique to individual cell-types and how they function in the brain. Recent results from us
and others indicate that hmC plays a dual role as a context-specific activator of mC repressed genes and as a
stable repressor during neuronal development. Emerging evidence further indicates MeCP2 as an important
reader of mC and hmC signals. Meanwhile, developmental studies have shown that a neuron-specific buildup of
a unique form of non-CG methylation (mCH), as well as hmC and MeCP2 occurs during the postnatal period,
when neurons integrate into circuits and complete their final maturation into specific functional subtypes. This
leads to the intriguing hypothesis that mCH, hmC, and MeCP2 are critical for the establishment and maintenance
of diverse, specialized neuronal subtypes within microcircuits. However, a critical barrier in understanding mC,
hmC, and MeCP2’s function in the brain, and to testing this hypothesis, is the lack of available methods to
simultaneously analyze mC, hmC, and gene expression genome-wide in individual cells. Here we propose to
develop a novel experimental and computational approach to perform integrative mC, hmC, and gene expression
analysis at the single-cell level. We will apply this method to parvalbumin positive interneurons in the visual
cortex to determine the patterns of mC and hmC during postnatal neuronal subtype specification and probe how
disruption or rescue of MeCP2 in these cells impacts gene regulation at the highest levels of cellular resolution.
Together these studies will provide key insights into the function of MeCP2 in the brain, while developing a new
technology that can be used to comprehensively assess the unique neuronal methylome and its impact on
transcription in normal and disease stat...

## Key facts

- **NIH application ID:** 10440762
- **Project number:** 1R21NS127191-01
- **Recipient organization:** WASHINGTON UNIVERSITY
- **Principal Investigator:** John R. Edwards
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $196,875
- **Award type:** 1
- **Project period:** 2022-03-01 → 2024-02-29

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10440762

## Citation

> US National Institutes of Health, RePORTER application 10440762, Single-cell approaches to probe the function of the unique neuronal epigenome (1R21NS127191-01). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10440762. Licensed CC0.

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