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T. D. Petkova, G. M. Seigel, D. C. Otteson; Transcriptional Regulation of Eph Receptor Expression by DNA Methylation in Embryonic Retinal Cells and Müller-Derived Retinal Stem Cells. Invest. Ophthalmol. Vis. Sci. 2010;51(13):4305.
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© ARVO (1962-2015); The Authors (2016-present)
Understanding gene regulation in the retina during development and in retinal stem cells will be key for successful optic nerve regeneration. EPH receptor signaling functions in axon guidance, formation of retinotopic maps and maintaining stem cell characteristics. We hypothesize that differential DNA methylation of CpG islands regulates Eph promoter activity in retinal development and retinal stem cells.
Genomic DNA and RNA were isolated from mouse Müller glia-derived neurospheres (ImM10 cell line) and embryonic retina (E17). DNA was bisulfite converted (ZymoGold), CpG islands were PCR amplified, subcloned and sequenced. Gene expression was analyzed by quantitative RT-PCR. Statistical analysis used SPSS. EphA5 promoter activity, with and without in vitro promoter methylation, was assayed by dual luciferase assays in R28 cells.
In Müller-derived neurospheres, EphA5 and EphB1 promoters were heavily methylated (48%-67%) and no mRNA expression was detected. The EphB2 promoter was minimally methylated (0.5%) and mRNA expression was detected (mean Ct=29.5). In E17 retina, CpG methylation of EphA5 and EphB1 was 1.2% and both genes were expressed (mean Ct=21.7, EphA5; 20.1, EphB1). Pearson correlation of CpG methylation vs. mean Ct across all samples was 0.876 (p=0.026), R square=0.767. Total CpG methylation by SssI decreased EphA5 and EphB1-luc promoter activity by 97% and 87% respectively (p<0.0001, T-test). Partial methylation using HhaI decreased EphA5 promoter activity by 20%, while partial methylation using HpaII increased activity by 35%.
The inverse relationship between promoter methylation and mRNA expression in Müller glia-derived retinal stem cells and embryonic retina supports a role for CpG methylation in regulating Eph promoter activity. Differential effects of partial methylation on EphA5 promoter activity suggests site specificity of CpG methylation.
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