# New approaches to determine the function of neuronal epigenetic marks

> **NIH NIH R21** · WASHINGTON UNIVERSITY · 2024 · $194,375

## Abstract

Abstract
Single cell profiling has revealed a striking cellular diversity and complexity in the mammalian nervous system.
Neurons 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 these marks show unique genomic 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. Meanwhile, developmental studies indicate that neuron-specific non-CG methylation (mCH), as well
as hmC and MeCP2 build up 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 mC, hmC, and
MeCP2 are critical for the establishment and maintenance of diverse, specialized neuronal subtypes within
microcircuits. Recent results from us and others indicate that mC and hmC play distinct roles in neuron-specific
gene regulation and function depending on sequence context (CG vs CH) and the genomic feature (promoter,
enhancer, gene body). However, a critical barrier to testing our hypothesis is the lack of available methods to
simultaneously analyze mC and hmC across different genomic contexts on an individual allele. The primary
limitation is that the bisulfite chemistry that underlies all high-resolution mC profiling methods shears DNA to
under 300 bp. Here we propose to take advantage of the long-read capabilities of nanopore sequencers to
develop a method to accurately simultaneously profile mC and hmC in both CG and non-CG contexts across
gene promoters and bodies (Aim 1). This will allow us to address how mC and hmC coordinate in different
contexts to affect MeCP2 binding and transcriptional programs in Granule and Pukinje neuronal subtypes in the
cerebellum (Aim 2). More specifically we will address how differences in mC and hmC profiles across cell types
are read out by MeCP2 to maintain differential subtype-specific transcriptomes. We will further dissect how allele
specific patterns of this methylation, which can only be decoded using long read sequencing, contribute to
neuronal gene regulation. In the future, our approach, which can be easily implemented into standard nanopore
workflows, will enable complete sequencing and methylation analysis of clinical or research samples in a single
assay. This could lead to a profound i...

## Key facts

- **NIH application ID:** 10879358
- **Project number:** 1R21NS137254-01
- **Recipient organization:** WASHINGTON UNIVERSITY
- **Principal Investigator:** John R. Edwards
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $194,375
- **Award type:** 1
- **Project period:** 2024-05-01 → 2026-04-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10879358

## Citation

> US National Institutes of Health, RePORTER application 10879358, New approaches to determine the function of neuronal epigenetic marks (1R21NS137254-01). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10879358. Licensed CC0.

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