Abstract
Purpose:
We have previously shown that Rhodopsin (RHO) has a cell-specific DNA methylation pattern within the retina and is methylated in non-expressing cells. We aimed to determine if DNA methylation contributes directly to the suppression of RHO expression.
Methods:
Cells expressing low levels of RHO (HEK 293, WERI and Y79) were treated with the DNA methyltransferase inhibitor 5-aza-2’-deoxycytidine (5-aza-dc; 72 h treatment) to induce hypomethylation. Global DNA methylation was measured by LUMA (luminometric methylation assay). RHO methylation was measured by bisulfite pyrosequencing, and the expression level determined by quantitative real-time PCR (qPCR). To explore whether the addition of transcription factors could further increase endogenous RHO expression in 5-aza-dc-treated HEK 293 cells, a bovine Rho promoter reporter (Gluc luciferase) was co-transfected with CRX and NRL expression vectors. Endogenous RHO methylation and expression was examined.
Results:
Treatment of HEK 293, WERI, and Y79 cells with 5-aza-dc induced a drug-dependent global DNA hypomethylation in all three cell lines. At 17 CpG sites of RHO, DNA methylation was reduced in 5-aza-dc -treated cells compared to controls and qPCR demonstrated a concomitant increase in RHO expression. In 5-aza-dc-treated HEK 293 cells, addition of CRX and NRL greatly increased Gluc activity from the reporter plasmid and increased expression of endogenous RHO. Additional chromatin remodeling drugs, including HDAC inhibitors, are currently being examined for possible synergistic effects in the regulation of RHO gene expression.
Conclusions:
Our results show that DNA hypomethylation by 5-aza-dc can induce RHO expression from low-expressing cells. Endogenous RHO expression can be further increased by the addition of transcription factors required during normal gene expression. Our results suggest that DNA methylation plays an important role in the suppression of RHO gene expression and is not a passive marker of RHO silencing. The ability to manipulate the expression of retina-specific genes using epigenetic modifiers currently in therapeutic use enables further study into the role that these mechanisms may play in both normal eye development and disease.
Keywords: 688 retina •
539 genetics •
533 gene/expression