April 2009
Volume 50, Issue 13
ARVO Annual Meeting Abstract  |   April 2009
Regulation of Retinal EphA5 Receptor Expression by CpG Methylation
Author Affiliations & Notes
  • T. D. Petkova
    College of Optometry, University of Houston, Houston, Texas
  • L. Pillai
    College of Optometry, University of Houston, Houston, Texas
  • G. M. Seigel
    Ross Eye Institute, University at Buffalo, SUNY, Buffalo, New York
  • D. C. Otteson
    College of Optometry, University of Houston, Houston, Texas
  • Footnotes
    Commercial Relationships  T.D. Petkova, None; L. Pillai, None; G.M. Seigel, None; D.C. Otteson, None.
  • Footnotes
    Support  Thomas R. Lee Award for National Glaucoma Research, AHAF (DCO); NIH NEI P30 EY07551 (core UHCO); NIH CA127061, EY016662, Research to Prevent Blindness (GMS).
Investigative Ophthalmology & Visual Science April 2009, Vol.50, 473. doi:
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    • Get Citation

      T. D. Petkova, L. Pillai, G. M. Seigel, D. C. Otteson; Regulation of Retinal EphA5 Receptor Expression by CpG Methylation. Invest. Ophthalmol. Vis. Sci. 2009;50(13):473.

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

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Purpose: : Gradients of Eph receptor expression regulate the topographic mapping of connections in the developing retina. We have cloned the EphA5 promoter and identified a 511bp CpG island in the EphA5 proximal promoter region. We hypothesize that differential CpG methylation is an epigenetic mechanism contributing to the nasal-low/temporal-high gradients of Eph5 receptor expression in the retina.

Methods: : Activity of mock methylated or mSssI methylated EphA5 promoter-luciferase plasmids was measured by dual luciferase assays of transiently transfected R28 retinal cells. Mouse genomic DNA purified from whole retinas (E17, P0 and 8 months), nasal, temporal, dorsal and ventral thirds of retinas (P0) was bisulfite converted (ZymoGold), EphA5 CpG islands were PCR amplified, subcloned into TOPO PCR2.1 (Invitrogen). CpG methylation frequencies were analyzed in 10 subclones for each sample by ANOVA (SPSS). Eph gene expression was determined by quantitative real-time PCR.

Results: : Relative luciferase activity of mock-methylated EphA5-luc reporters was 8.5 (0.5 µg/well) to 12.5-fold (0.1 and 0.25 µg/well) higher than promoterless pGL2 controls at the same concentrations. SssI methlyation reduced EphA5-luc activity to levels comparable to pGL2 alone at all concentrations. Retinal CpG methylation in the EphA5 CpG island increased from 0.4% (E17), 0.8% (P0) to 1.8% (8 months) (p=0.011). EphA5 CpG methylation at P0 varied topographically: nasal 2.2%, temporal 1.0%, dorsal 0.5% and ventral 0.2% (p=.005). EphA5 mRNA expression was increased by 90% in temporal vs nasal retina.

Conclusions: : Complete CpG methylation abolishes EphA5 promoter activity in vitro. The overall density of CpG methylation of the EphA5 promoter in vivo was low. However, the small but statistically significant increase in CpG methylation in the nasal retina at P0 and in whole retinas at 8 months suggests a role for low density CpG methylation in modulating spatial and developmental expression of EphA5 in the retina.

Keywords: transcription • gene/expression • retinal development 

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