Abstract
Purpose: :
The promoter regions of tissue-specific genes are thought to be methylated during early development, becoming hypomethylated in the expressed tissue shortly before the onset of expression and remaining methylated in non-expressing adult somatic tissues. Little is known about the temporal relationship between DNA hypomethylation and establishment and maintenance of cell type-restricted gene expression in the retina. Our previous work in mice (ARVO 2007) showed that Rbp3 (IRBP) and Rho (rhodopsin), two important photoreceptor (PR) genes with well-characterized spatial and temporal expression patterns, are overall less methylated in the retina than in other non-expressing tissues. Additionally, Liou et al. showed that during embryonic mouse development, Rbp3 demonstrates retina-specific DNA hypomethylation of several promoter CpG sites, the timing of which correlates with the beginning of Rbp3 expression. To expand upon these prior studies, we are investigating whether DNA methylation of the transcriptional start site (TSS) region of PR-specific genes contributes to the establishment of their tissue-specific expression.
Methods: :
PRs from the outer nuclear layer and non-PR cells from the inner nuclear layer were isolated by LCM of developing and adult murine retina. 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 TSS of Rbp3 and Rho. Direct sequencing of the PCR products was performed. The unmethylated to methylated ratio of the T to C peak heights from each chromatogram was calculated for each CpG site. For several embryonic and postnatal stages, DNA methylation patterns are being compared to stage-related changes in gene expression determined by real-time quantitative PCR.
Results: :
In adult retina, Rpb3 and Rho are both largely unmethylated and expressed in PR cells, while they are methylated and unexpressed in non-PR cells from the inner nuclear layer. The onset of these differential DNA methylation patterns and their temporal relationship to Rbp3 and Rho expression in the developing mammalian retina are being determined.
Conclusions: :
We demonstrated that the TSS region of the PR-specific genes Rbp3 and Rho are unmethylated in PRs and methylated in non-PR retinal cells. When these disparate DNA methylation patterns arise in cell types originating from a common precursor is unknown and under investigation. Whether differential DNA methylation in part directs their cell type-specific expression pattern, or is simply a consequence of non-expression, is also yet to be determined.Liou et al. Dev Biol. 1994 161:345.
Keywords: gene/expression • photoreceptors