# DNA Methylation and Hydroxymethylation During Retinal Development and Stem Cell Maintenance

> **NIH NIH R01** · UNIVERSITY OF TEXAS AT AUSTIN · 2022 · $272,815

## Abstract

SUMMARY: The requirements and functions for DNA methylation (5mC) have been resolved in very few
organs and tissues, and even less is known about the roles of DNA hydroxymethylation (5hmC). Indeed, DNA
methylation and hydroxymethylation have not been well studied in the retina, and our preliminary studies
demonstrate that they play critical roles during retinal neurogenesis and in retinal stem cell maintenance.
Here, we focus on DNA methylation and hydroxymethylation during the transition from retinal progenitor cell
(RPC) to differentiated retinal neuron, and in regulating proliferation and differentiation of retinal stem cells. We
hypothesize that regulation of gene expression by DNA methylation and hydroxymethylation is critical for
retinal development and retinal stem cell maintenance, and that changes in methylation and
hydroxymethylation of specific loci in RPCs and retinal stem cells influence their abilities to differentiate as
retinal neurons. Aim1 utilizes state of the art next-generation sequencing and bioinformatics techniques to
determine genome-wide 5mC, 5hmC and transcriptome profiles from pure populations of early and late stage
RPCs, and early and late stage retinal ganglion cells (RGCs) from the zebrafish retina. These data are then
integrated to generate a genome-wide methylome, hydroxymethylome and transcriptome database for cells
during RPC maturation and the RPC to RGC transition. We then utilize an innovative human iPSC-derived
organoid model to determine genome-wide 5mC, 5hmC and transcriptome profiles from pure populations of
early and late stage human RPCs and leverage these data to identify genes with conserved methylation
changes during RPC maturation and thereby prioritize relevant loci for downstream functional analyses. Aim2
focuses on Ted-mediated DNA hydroxymethylation, an epigenetic process about which we have a limited
understanding during retinal development. Here, we will determine how tet2- and tet3-mediated DNA
hydroxymethylation facilitate early and late aspects of retinal development in zebrafish. Aim3 examines retinal
stem cell maintenance and determines how dnmt1-mediated methylation is required in retinal stem cells to
modulate proliferation and differentiation of cells within this niche. The results of this proposal will provide the
most detailed analyses of DNA methylation and hydroxymethylation during retinal development to date, and
functionally interrogate the requirements for these two key processes during retinal development and in retinal
stem cell maintenance. These data will be of broad interest to those working in the eye, CNS, and in other
organs and tissues, as well as more generally in epigenetic regulation of gene expression. Moreover, given
the pace at which regenerative therapies are being developed around stem cell and iPSC-based approaches,
our results will provide critical information about DNA methylation and hydroxymethylation changes occurring in
RPCs as they make decisions whethe...

## Key facts

- **NIH application ID:** 10747714
- **Project number:** 7R01EY029031-06
- **Recipient organization:** UNIVERSITY OF TEXAS AT AUSTIN
- **Principal Investigator:** Jeffrey Gross
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $272,815
- **Award type:** 7
- **Project period:** 2018-04-01 → 2025-03-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10747714

## Citation

> US National Institutes of Health, RePORTER application 10747714, DNA Methylation and Hydroxymethylation During Retinal Development and Stem Cell Maintenance (7R01EY029031-06). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10747714. Licensed CC0.

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