# Identification and characterization of mCpH binding proteins in neurons

> **NIH NIH R01** · JOHNS HOPKINS UNIVERSITY · 2022 · $684,775

## Abstract

PROJECT SUMMARY
DNA methylation is a major epigenetic modification that plays an important role in key biological processes,
including genomic imprinting, X-chromosome inactivation, suppression of transposable elements, and
carcinogenesis. Although it has been traditionally considered to be restricted to CpG dinucleotides in metazoan
genomes, emerging evidence over the past decade has shown that CpH (H=A/C/T) methylation is present in
mammalian genomes, including cultured pluripotent stem cells, embryonic stem cells, induced pluripotent stem
cells, the mouse germ line, and especially at a relatively high level in human and mouse brains. Given that
CpGs only represent 4% of the metazoan genomes, CpH methylation greatly expands the proportion of the
genome that is subject to regulation by cytosine methylation and represents a new mechanism of
transcriptional regulation. In our previous studies, we generated neuronal DNA methylation profiles at a single
base-resolution of the adult mouse dentate gyrus in which 80-90% of the cells are NeuN positive granule
neurons, and our team was one of the first to show that ~25% of cytosine methylations are located in the CpH
context. Notably, we identified the first mCpH reader, MeCP2, both in vitro and in postmitotic neurons in vivo.
In addition, we found that CpH methylation was established postnatally and required DNMT3A for its active
maintenance in neurons in vivo. Mutations on both the reader and writer lead to neurodevelopmental disorders,
such as fragile X syndrome (FXS), amyotrophic lateral sclerosis (ALS), and Rett syndrome. Loss of either
Dnmt3A or MeCP2 in the mouse models causes overlapping and distinct phenotypes in behavioral and
molecular tests, suggesting the existence of additional mCpH-binding proteins. We believe that a critical step
towards understanding the biological functions of mCpH is to identify its binding proteins. In this proposal, our
goal is to identify additional mCpH binding proteins. We hypothesize that mCpH regulates transcription directly
or indirectly via recruiting sequence-independent and/or -dependent mCpH-binding proteins in neurons. We
will use protein array (Aim 1) and Digital Affinity Profiling via Proximity Ligation (DAPPL; Aim 2) to identify
sequence-independent and -dependent mCpH-binding proteins and validate candidates using gel-shift
(EMSA), OCTET and luciferase assays in vitro. We will employ a viral in vivo delivery system and high-
throughput sequencing technologies to characterize their roles in transcriptional and chromatin regulation in
the adult mouse brain (Aim 3). The effectiveness of our strategy will be rigorously evaluated via a series of in
vitro and in vivo assays. If funded, the success of this project is expected to provide a rich resource of
sequence-dependent and independent mCpH-binding proteins that will lay the foundation to elucidate the roles
of CpH methylation in neurons, stem cells and other tissues. The insights into the mechanism of Cp...

## Key facts

- **NIH application ID:** 10500331
- **Project number:** 1R01NS127913-01
- **Recipient organization:** JOHNS HOPKINS UNIVERSITY
- **Principal Investigator:** Yijing Su
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $684,775
- **Award type:** 1
- **Project period:** 2022-08-05 → 2027-07-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10500331

## Citation

> US National Institutes of Health, RePORTER application 10500331, Identification and characterization of mCpH binding proteins in neurons (1R01NS127913-01). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/10500331. Licensed CC0.

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