Global DNA Methylation Level Monitoring by methyl-CpG Binding Domain-Fused Luciferase
Global DNA Methylation Level Monitoring by methyl-CpG Binding Domain-Fused Luciferase
复制标题
DOI：
10.1080/00032719.2018.1494739
复制
发表时间：
2019-03-24
影响因子：
2
通讯作者：
Yoshida, Wataru
中科院分区：
文献类型：
--
作者：
Baba, Yuji;Karube, Isao;Yoshida, Wataru
DNA甲基化是一种抑制基因表达的表观遗传修饰。在癌细胞中，观察到启动子区域DNA甲基化状态的改变和重复的DNA序列，因此，DNA甲基转移酶抑制剂作为潜在的抗癌药物一直是人们感兴趣的焦点。我们之前报道了一种简单的利用甲基-CpG结合域(MBD)融合到荧光素酶(MBD-荧光素酶)的全球DNA甲基化水平检测方法。在检测中，MBD-荧光素酶结合到基因组DNA上的甲基-CpG位点。随后，荧光素酶和荧光DNA嵌入染料之间的生物发光共振能量转移(BRET)产生依赖于DNA甲基化水平的信号。在这项研究中，我们研究了由DNA甲基转移酶抑制剂或营养物质引起的整体DNA低甲基化是否可以通过Bret试验进行监测。利用5-氮杂-2‘-脱氧胞苷和叶酸作为DNA甲基转移酶抑制剂和营养物质，影响细胞内DNA甲基化。用该抑制剂或在叶酸缺乏的培养液中培养HeLa细胞，检测其整体DNA甲基化水平。Bret试验检测到时间和浓度依赖的低甲基化。这些结果表明，Bret法可以监测到DNA的整体甲基化，这表明该方法适用于基于细胞的DNA甲基转移酶抑制剂的筛选。
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.