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S. L. Merbs, M. A. Khan, D. J. Zack; DNA Methylation Patterns in the Retina of the Nrl-/- Mouse. Invest. Ophthalmol. Vis. Sci. 2009;50(13):471.
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© ARVO (1962-2015); The Authors (2016-present)
DNA methylation is an epigenetic phenomenon that controls various genomic functions without altering the DNA sequence and plays a role in transcriptional regulation. The promoter sequences of housekeeping genes are typically unmethylated, and aberrant methylation is known to shut down expression of tumor suppressor genes in cancer. To broaden our investigation of the role DNA methylation plays in cell-specific expression of photoreceptor genes, we investigated the methylation status of photoreceptor-specific genes in the Nrl-/- retina. Deletion of Nrl in the mouse results in a retina that lacks rods and has a predominance of S-cones.
Photoreceptors from the outer nuclear layer (ONL) and non-photoreceptors cells from the inner nuclear layer (INL) were isolated by LCM from wildtype (WT) and Nrl-/- retinas. Genomic DNA, isolated from each cell type, was modified by bisulfite treatment. Primers were designed to amplify only the bisulfite modified DNA in 300-500bp segments of the 1000bp around the transcription start sites (TSS) of Rho, Opn1sw, and Rbp3. After confirmation of a single product by gel electrophoresis, amplified sequences were subcloned and a minimum of 10 clones for each gene were sequenced.
Corresponding to their expression patterns, Rho was unmethylated in the ONL of WT mice but not in Nrl-/- mice, Opn1sw was unmethylated in the ONL only in Nrl-/- mice, and Rbp3 was unmethylated in the ONL of both WT and Nrl-/- mice but methylated in their INL.
We have previously demonstrated that the relative lack of DNA methylation in some photoreceptor-specific genes correlates with the expression of those genes at the rod-specific (Rho) or photoreceptor-specific (Rbp3) level. In the Nrl-/- photoreceptor layer, which is comprised primarily of S-cones, we have now shown cone-specific lack of DNA methylation of Opn1sw. Whether the observed DNA methylation differences of these genes accounts, in part, for their photoreceptor cell type-specific expression patterns is under investigation.
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