April 2011
Volume 52, Issue 14
Free
ARVO Annual Meeting Abstract  |   April 2011
DNA Demethylation Increases Expression of Pluripotent and Retinal Ganglion Cell-Specific Genes in ImM10 Müller Glia-Derived Sphere Cultures
Author Affiliations & Notes
  • Tihomira D. Petkova
    College of Optometry, University of Houston, Houston, Texas
  • Deborah C. Otteson
    College of Optometry, University of Houston, Houston, Texas
Investigative Ophthalmology & Visual Science April 2011, Vol.52, 5986. doi:
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      Tihomira D. Petkova, Deborah C. Otteson; DNA Demethylation Increases Expression of Pluripotent and Retinal Ganglion Cell-Specific Genes in ImM10 Müller Glia-Derived Sphere Cultures. Invest. Ophthalmol. Vis. Sci. 2011;52(14):5986.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: : In the mammalian retina, Müller glia have limited stem cell properties and the mechanisms that restrict their potential to proliferate and differentiate into retinal ganglion cells (RGCs) are poorly understood. The purpose of this study was to determine if key pluripotency and developmental genes are transcriptionally silenced by DNA methylation in Müller-derived retinal stem cells.

Methods: : Genomic DNA and RNA were isolated from untreated or 5-Aza-deoxycytidine (AzadC) (1 and 10µM) treated, conditionally immortalized mouse Müller glia (ImM10) cultured as spheres in serum-free medium with 20 ng/ml EGF and FGF2. DNA was bisulfite converted (ZymoGold), CpG islands were PCR amplified, subcloned and sequenced. Gene expression was analyzed by quantitative RT-PCR. Statistical analyses used the Mann-Whitney U-test for methylation analysis and Student's T-tests for expression analysis.

Results: : In Müller-derived spheres Atoh7 was hypermethylated (66%) and Atoh7 mRNA expression was not detected. AzadC treatment decreased methylation of Atoh7 to 26% (p<0.0001) and increased mRNA expression by at least 97-fold (p<0.0001). AzadC treatment also increased expression of several pluripotency genes: Pou5f1 (from no expression to 35.3Ct, p<0.0001), Nanog2 (from 34.7 to 32.6 Ct, 2.7-fold, p=0.0003) and Sox2 (from 23.5 to 22.5 Ct, 1.7-fold, p=0.0007) and retinal ganglion cell specific genes: Isl1 (from 35.6 to 29.6 Ct, 26.8-fold, p<0.0001), Pou4f3 (from 37.5 to 29.8 Ct, 110-fold; p<0.0001). Pou4f2 mRNA was not detected in control or AzadC-treated ImM10 spheres.

Conclusions: : The inverse relationship between DNA methylation and mRNA expression of Atoh7 in Müller glia-derived sphere cultures supports a role for DNA methylation in regulating Atoh7 gene expression in vitro. Up-regulation of pluripotent and RGC developmental genes following DNA demethylation suggests that epigenetic mechanisms could limit the proliferative and neurogenic potential of Müller glia. Reversing epigenetic silencing of neuronal and pluripotency genes offers a potential strategy for increasing neurogenesis from Müller-derived stem cells and enhancing retinal regeneration in mammals.

Keywords: gene/expression • Muller cells • regeneration 
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