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J. Biol. Chem., Vol. 281, Issue 15, 10118-10126, April 14, 2006

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Transient Down-regulation of DNMT1 Methyltransferase Leads to Activation and Stable Hypomethylation of MAGE-A1 in Melanoma Cells^*

From the Ludwig Institute for Cancer Research, Brussels and Cellular Genetics Unit, Université Catholique de Louvain, B1200 Brussels, Belgium

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.

Received for publication, September 23, 2005 , and in revised form, January 20, 2006.

^* This work was supported in part by grants from the Fédération Belge contre le Cancer and the Fonds National de la Recherche Scientifique (FNRS, Belgium). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Supported by a Télévie grant from the FNRS.

² To whom correspondence should be addressed: Ludwig Inst. for Cancer Research, Brussels branch, and Cellular Genetics Unit, Université Catholique de Louvain, 74 Ave. Hippocrate, B1200 Brussels, Belgium. Tel.: 32-2-7647443; Fax: 32-2-7629405; E-mail: charles.desmet{at}bru.licr.org.

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