DNA Methylation Is the Primary Silencing Mechanism for a Set of Germ Line- and Tumor-Specific Genes with a CpG-Rich Promoter

ABSTRACT

A subset of male germ line-specific genes, theMAGE-type genes, are activated in many human tumors, where they produce tumor-specific antigens recognized by cytolytic T lymphocytes. Previous studies on gene MAGE-A1 indicated that transcription factors regulating its expression are present in all tumor cell lines whether or not they express the gene. The analysis of two CpG sites located in the promoter showed a strong correlation between expression and demethylation. It was also shown thatMAGE-A1 transcription was induced in cell cultures treated with demethylating agent 5′-aza-2′-deoxycytidine. We have now analyzed all of the CpG sites within the 5′ region of MAGE-A1 and show that for all of them, demethylation correlates with the transcription of the gene. We also show that the induction ofMAGE-A1 with 5′-aza-2′-deoxycytidine is stable and that in all the cell clones it correlates with demethylation, indicating that demethylation is necessary and sufficient to produce expression. Conversely, transfection experiments with in vitro-methylatedMAGE-A1 sequences indicated that heavy methylation suffices to stably repress the gene in cells containing the transcription factors required for expression. Most MAGE-type genes were found to have promoters with a high CpG content. Remarkably, although CpG-rich promoters are classically unmethylated in all normal tissues, those of MAGE-A1 and LAGE-1 were highly methylated in somatic tissues. In contrast, they were largely unmethylated in male germ cells. We conclude that MAGE-type genes belong to a unique subset of germ line-specific genes that use DNA methylation as a primary silencing mechanism.