HYPOMETHYLATION OF THE XMRK ONCOGENE PROMOTER IN MELANOMA-CELLS OF XIPHOPHORUS

Melanoma formation in Xiphophorus is caused by the tissue-specific ove rexpression of the Xmrk oncogene. This gene arose by a non-homologous recombination event between the Xmrk proto-oncogene and an unrelated l ocus, D, during which the Xmrk transcribed regions were fused to new u pstream sequences. Functional analyses of this newly acquired promoter revealed the presence of positive regulatory elements. Since these el ements did not activate transcription in a melanoma-specific manner, t he methylation state of the Xmrk oncogene promoter in different tissue s was analyzed. Digestion with methylation sensitive restriction enzym es followed by PCR amplification demonstrated differential methylation in a melanoma cell line and non-melanoma tissue. The methylation stat e of single CpG-dinucleotides in genomic DNA from different cell types was investigated by bisulfite genomic sequencing. This analysis revea led a high degree of methylation of the Xmrk oncogene promoter in nont ransformed tissues. In contrast, the DNA-region analyzed was completel y unmethylated in the melanoma cell line PSM. This effect was oncogene -specific, since the Xmrk proto-oncogene showed CpG-methylation in the se cells. Studies with melanoma tissue demonstrated that hypomethylati on of the Xmrk oncogene promoter can also be found in DNA from these t umors. This correlation suggests that the methylation status of the pr omoter might play a role in the overexpression of the Xmrk oncogene in vivo.