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Verity F. Oliver, Kieron Torres, Mariam S. Assadian, Ray A. Enke, Shannath L. Merbs; DNA Methylation Patterns During Retinal Development In Mice. Invest. Ophthalmol. Vis. Sci. 2011;52(14):5979.
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© ARVO (1962-2015); The Authors (2016-present)
The retina is a highly organized tissue with specialized cells types that develop from a common retinal progenitor cell type. Our previous studies have shown that in the adult mouse, genes that are preferentially expressed in photoreceptors (PRs) from the outer nuclear layer (ONL) are unmethylated in PRs but are methylated in non-expressing, non-PR cells from the inner nuclear layer (INL). To understand when this differential DNA methylation pattern is established and to begin to understand its significance to retinal development, we investigated the methylation status of PR-specific genes in the developing mouse retina.
PRs (~97% rods) from the ONL and non-PR cells from the INL were isolated by laser capture microdissection from wild-type retina of mice aged E11.5, E17.5, P0 and P6 and were compared to adult whole retina, brain, kidney and testes. S-cones (which comprise only 1% of the PRs in the wild type ONL), were isolated from adult Nrl-/- mice ONL (~98% S-cones). Genomic DNA was isolated from each cell type and modified by bisulfite treatment. 200-500 bp regions within 1000 bp of the TSS of Rho (rod-specific), Rbp3 (rod/ cone-specific) and Opn1sw (cone-specific) were sequenced.
At least 3 different DNA methylation patterns of PR-specific genes were observed: one specific for non-retinal tissues and non-PR retinal cells (methylation), another specific for expressing PR cells (hypomethylation), and a third pattern that did not simply correlate with expression (regional hypomethylation). This regional hypomethylation was seen close to the TSS of Opn1sw (cone-specific) in wild-type ONL (~97% rods) and in the promoter of Rho (rod-specific) in Nrl-/- ONL (~98% S-cones). Hypomethylation of the promoter region of Rho and Rbp3 was also seen in developing cells not yet expressing these genes.
We hypothesize that the regional hypomethylation of Rho and Opn1sw is characteristic of PR precursor cells and once cell fate is determined, these early methylation patterns become completely unmethylated in expressing PRs but persist in the non-expressing PRs. Additionally, the regional hypomethylation near the TSS of Rbp3 and Rho that did not correlate with expression at time points as early as E11.5 likely represent differential methylation that leads to lineage determination by regulating gene expression in the developing retina.
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