Loriot Axelle, De Plaen Etienne, Boon Thierry, De Smet Charles
Ludwig Institute for Cancer Research, Brussels and Cellular Genetics Unit, Université Catholique de Louvain, 74 Avenue Hippocrate, B1200 Brussels, Belgium.
J Biol Chem. 2006 Apr 14;281(15):10118-26. doi: 10.1074/jbc.M510469200. Epub 2006 Feb 22.
MAGE-A1 belongs to a group of germ line-specific genes that rely primarily on DNA methylation for repression in somatic tissues. In many types of tumors, the promoter of these genes becomes demethylated and transcription becomes activated. We showed previously that, although MZ2-MEL melanoma cells contain an active unmethylated MAGE-A1 gene, they lack the ability to induce demethylation of newly integrated MAGE-A1 transgenes that were methylated in vitro before transfection. In the same cells, unmethylated MAGE-A1 transgenes were protected against remethylation, and this appeared to depend on the level of transcriptional activity. We therefore proposed that hypomethylation of MAGE-A1 in tumors relies on a past demethylation event and on the presence of appropriate transcription factors that maintain the promoter unmethylated. Here, we tested this hypothesis further by examining whether induction of a transient demethylation phase in MZ2-MEL would suffice to convert a previously methylated MAGE-A1 transgene into a permanently hypomethylated and active one. For induction of the demethylation phase, we used antisense oligonucleotides targeting the three known human DNA methyltransferases. We found that down-regulation of DNMT1, but not of DNMT3A and DNMT3B, induces activation of the MAGE-A1 transgene, suggesting that DNMT1 has a predominant role for methylation maintenance in MZ2-MEL cells. By using a selectable MAGE-A1 transgene construct, we were able to isolate a cell population in which DNMT1 depletion had resulted in transgene activation. The promoter region of the transgene was almost completely unmethylated in these cells, and this active and unmethylated state was maintained for over 60 days after restoration of normal DNMT1 expression.
MAGE - A1属于一组生殖系特异性基因，在体细胞组织中，这些基因主要依靠DNA甲基化来实现抑制作用。在许多类型的肿瘤中，这些基因的启动子会发生去甲基化，转录得以激活。我们之前表明，尽管MZ2 - MEL黑色素瘤细胞含有一个活跃的未甲基化MAGE - A1基因，但它们缺乏诱导新整合的MAGE - A1转基因去甲基化的能力，这些转基因在转染前体外是甲基化的。在相同细胞中，未甲基化的MAGE - A1转基因可免受重新甲基化影响，这似乎取决于转录活性水平。因此，我们提出肿瘤中MAGE - A1的低甲基化依赖于过去的去甲基化事件以及维持启动子未甲基化的适当转录因子的存在。在此，我们通过检查在MZ2 - MEL中诱导短暂的去甲基化阶段是否足以将先前甲基化的MAGE - A1转基因转化为永久低甲基化且活跃的转基因，进一步验证了这一假设。为诱导去甲基化阶段，我们使用了针对三种已知人类DNA甲基转移酶的反义寡核苷酸。我们发现，DNMT1的下调而非DNMT3A和DNMT3B的下调会诱导MAGE - A1转基因的激活，这表明DNMT1在MZ2 - MEL细胞的甲基化维持中起主要作用。通过使用一个可选择的MAGE - A1转基因构建体，我们能够分离出一个细胞群体，其中DNMT1的缺失导致了转基因的激活。在这些细胞中，转基因的启动子区域几乎完全未甲基化，并且在恢复正常DNMT1表达后，这种活跃且未甲基化的状态维持了60多天。
