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Ray A Enke, Ahmed Rashed G AlOtaibi, Sally J Coleman, Emily R Grunwald, Victoria D Shuklis; Epigenetic analysis of the developing chicken retina. Invest. Ophthalmol. Vis. Sci. 2014;55(13):4995.
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© ARVO (1962-2015); The Authors (2016-present)
Developmental patterning of the vertebrate retina requires complex temporal orchestration of DNA binding transcriptional activators and repressors. Recent studies have demonstrated that patterns of DNA methylation, an epigenetic modifier of genomic DNA, are inversely correlated with retina-specific gene expression in mammalian cells. These studies suggest DNA methylation may be a critical component of retinal development. The purpose of this study is to explore the use of the developing chicken embryo as a model system to investigate the role of DNA methylation in establishing retina-specific patterns of gene expression during normal retinal development.
Animal experiments were carried out in accordance with the ARVO statement on the use of animals in ophthalmic and visual research. Chicken retinas were harvested at embryonic days (E) 6-18. E18 brain tissue was also harvested and analyzed as a non-retinal control. Genomic DNA and total RNA were simultaneously harvested from tissues using a Qiagen AllPrep Mini kit. For DNA methylation analysis, genomic DNA was bisulfite converted and used as template for bisulfite PCR amplification of retina-specific genes. CG dinucleotide sites within amplicons were quantitatively assayed for DNA methylation using a Q24 Pyrosequencer. For gene expression analysis, total RNA was used as template for cDNA synthesis followed by real-time PCR amplification of retina-specific genes.
DNAs isolated from brain and early embryonic retina were densely methylated on sequences proximal to transcriptional start sites of the photoreceptor-specific genes RBP3, GNAT2, and PDE6c. The same sequences were hypomethylated during the course of normal retinal development. Preliminary RNA analysis demonstrates that hypomethylation of the RBP3 locus is associated with increased transcription. Ongoing studies will determine if a similar correlation is observed between gene expression of GNAT2 and PDE6c and their levels of DNA methylation during development.
Our data suggests that DNA methylation is a critical epigenetic regulator of the developing chicken retina. Further, this study demonstrates that the chicken embryo is a useful model for studying epigenetic regulation of retina-specific genes, particularly in cone photoreceptors.
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