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Akira Matsuda, Nobuyuki Ebihara, Akira Murakami; Genomewide DNA methylation analysis of primary human trabecular meshwork cells with dexamethasone stimulation. Invest. Ophthalmol. Vis. Sci. 2014;55(13):5658.
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© ARVO (1962-2015); The Authors (2016-present)
We reported the change of DNA methylation status during glucocorticoid exposure using immortalized human trabecular meshwork (TM) cells. To exclude the effect of immortalization, we investigated genomewide DNA methylation analysis using primary TM cells.
Three batches human primary trabecular meshwork (PTM) cells were cultured with and without 100nM dexamethasone (DEX) for 14 days. Genome wide methylation analysis was carried out using Illumina 450K methylation analysis. Gene expression analysis was also carried out with Agilent 80K whole human genome array using cultured TM cells. The effect of DNA methylation in response to DEX treatments for gene expression was verified by DNA methyltransferase inhibitor (5-aza-2’-deoxycytidine: 5-aza-dC) treatment and subsequent realtime PCR analysis.
In response to 2 week-DEX treatment, we found cytosine-phosphate-guanine (CpG) sites on COPS8, PDE3A, TEAD1, TSEN2 genes showing significant demethylation in all three batches of PTM cells. In addition, CpG sites on 10 genes (including HDAC4, IGF2 genes) showing significant methylation in all three batches of PTM. We found 3 sets of demethylated-CpG sites after DEX exposure with significant changes of corresponding gene expression (COPS8, FKBP5 and SAA1). DNA demethylation of CpG sites of these three genes were also observed in immortalized TM cells during DEX exposure. Among them, we further evaluated the effect of epigenetic modification for the expression of FKBP5 and SAA1. 5-aza-dC treatment of PTM cells increased both FKBP5 and SAA1 mRNA expression.
DEX treatments induce the alteration of DNA methylation status in human PTM cells. Epigenetic modification could affect TM gene expression profile not only in immortalized TM cells but also primary cultured TM cells.
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