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Jin Song, Ray A. Enke, Shannath L. Merbs; Epigenetic Manipulation of the Expression of Retina-Specific Genes in Human Neuronal and Non-neuronal Cells. Invest. Ophthalmol. Vis. Sci. 2012;53(14):1577.
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DNA methylation is important for controlling gene expression. The purpose of this study is to determine whether the induction of DNA hypomethylation with DNA methyltransferase inhibitors could affect the expression of retina-specific genes in human neuronal and non-neuronal cells.
Two human retinoblastoma cell lines, WERI and Y79, and the non-neuronal human embryonic kidney cell line HEK 293 were grown in culture with 10% FBS. Cells were treated with 1, 2 µM 5-aza-2’-deoxycytidine or 40, 80 µM RG108 (both are DNA methyltransferase inhibitors) for 72 hours. The isolated genomic DNA was first subjected to a luminometric-based assay for global DNA methylation (LUMA) in drug-treated cells. Quantitation of DNA methylation of retina-specific genes was further performed by bisulfite conversion followed by pyrosequencing analysis. Real-time reverse transcription polymerase chain reaction (RT-PCR) was also used to examine the expression of retina-specific genes in drug-treated cells.
Treatment of WERI, Y79, and HEK 293 cells with 5-aza-2’-deoxycytidine showed a drug dependent global DNA hypomethylation. Furthermore, quantitation of DNA methylation of rhodopsin revealed a significant decrease in 5-aza-2’-deoxycytidine-treated WERI and HEK 293 cells compared to the control cells. Quantitative RT-PCR also demonstrated a significant increase of the expression of rhodopsin in 5-aza-2’-deoxycytidine-treated WERI and HEK 293 cells compared to the control cells.
Our results indicate that the induction of DNA hypomethylation with DNA methyltransferase inhibitors increases the expression of retina-specific genes in human neuronal and non-neuronal cells. The ability to manipulate the expression of retina-specific genes allows us to further explore the epigenetic regulatory mechanisms in human eye development and disease.
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