Abstract
Purpose::
One element of transcriptional regulation is the local epigenetic configuration of a gene. DNA methylation is an epigenetic phenomenon that controls various genomic functions without altering the DNA sequence. 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 investigate whether DNA methylation might play a role in tissue-specific expression of photoreceptor genes, we investigated the methylation status of photoreceptor-specific genes and correlated the degree of methylation with the expression of those genes.
Methods::
Genomic DNA from WERI and Y79 human retinoblastoma cell lines and 293 cells were treated with bisulfite. Primers were designed to amplify only the bisulfite modified DNA in 300-500bp segments of the 1000bp upstream of the TSS through the first exon of selected photoreceptor-specific genes (RPB3, RHO, OPN1SW, OPN1MW, and OPN1LW). Direct sequencing of the PCR products was performed. The ratio of unmethylated/methylated (T/C) DNA was calculated for each CpG site. Expression of the same genes was measured by real time PCR. The cell line experiments were then reproduced in the mouse, comparing the methylation status of RBP3, RHO, OPN1SW and OPN1MW in retina, brain, kidney and testes with the expression level of those genes.
Results::
None of the photoreceptor-specific genes were detected in 293 cells, Y79 cells expressed RPB3 but only very low levels of the opsins, and WERI cells expressed RBP3, OPN1MW and OPN1LW. The CpG sites 1000bp upstream of the TSS through the end of the first exon in all photoreceptor-specific genes were primarily methylated in 293 cells, consistent with the non-expression. In Y79 cells, the photoreceptor-specific genes were primarily methylated, except for RPB3, which was primarily unmethylated. The RPB3, OPN1MW, and OPN1LW genes were primarily unmethylated in WERI cells, and the RHO and OPN1SW genes were methylated. The same correlation between expression and the lack of methylation was found in corresponding experiments in mice. Photoreceptor-specific genes were overall less methylated in the retina, than in the other non-expressing tissues.
Conclusions::
We have demonstrated that the relative lack of DNA methylation in some photoreceptor-specific genes correlates with the expression of those genes, both in human tissue culture cells and in several mouse tissues. Whether the methylation in part directs the tissue-specific non-expression, or is simply a consequence of the non-expression is yet to be determined.
Keywords: gene/expression • photoreceptors