Global DNA Methylation Level Monitoring by methyl-CpG Binding Domain-Fused Luciferase |
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Authors: | Yuji Baba Isao Karube |
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Institution: | School of Bioscience and Biotechnology, Graduate School of Bionics, Tokyo University of Technology, Tokyo, Japan |
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Abstract: | DNA methylation is an epigenetic modification that represses gene expression. In cancer cells, alterations of the DNA methylation state in promoter regions and repetitive DNA sequences are observed; therefore, DNA methyltransferase inhibitors have been the focus of interest as potential anticancer drugs. We previously reported a simple global DNA methylation level-sensing assay using methyl-CpG binding domain (MBD) fused to luciferase (MBD-luciferase). In the assay, the MBD-luciferase binds to methyl-CpG sites on genomic DNA. Subsequently, bioluminescence resonance energy transfer (BRET) between the luciferase and a fluorescent DNA intercalating dye generates a signal that is dependent on DNA methylation level. In this study, we investigated whether global DNA hypomethylation induced by a DNA methyltransferase inhibitor or nutrient can be monitored by the BRET assay. 5-Aza-2′-deoxycytidine and folic acid were utilized as the DNA-methyltransferase inhibitor and nutrient that affect DNA methylation in cells. The HeLa cells were cultured with the inhibitor or in folic acid-deficient medium and their global DNA methylation levels measured. Both time- and concentration-dependent hypomethylation were detected by the BRET assay. These results demonstrate that global DNA hypomethylation can be monitored by the BRET assay, indicating that the assay is applicable to cell-based screening of DNA-methyltransferase inhibitors. |
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Keywords: | 5-Aza-2′-deoxycytidine bioluminescence resonance energy transfer DNA methylation folic acid luciferase methyl-CpG binding domain |
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