ALTERNATIVE PROMOTERS OF GENE MAGE4A

Gene MAGE-4 (HGMW-approved symbol MAGE4) is expressed in several types of tumors, but not in normal tissues, except testis and placenta, The 5' end of this gene contains eight homologous exons spread over a 5.8 -kb region, These exons are alternatively spliced to a unique second e xon and a unique third exon, which encodes a protein of 317 amino acid s. The analysis of transcripts found in testis, placenta, and a sarcom a cell line showed that each of the alternative first exons is used in at least one of these tissues, Various regions of the promoter of the fifth alternative exon (1.5) were cloned in a luciferase reporter pla smid, and the constructs were transfected in a sarcoma cell. line that expresses MAGE-4, Two Ets motifs located between positions -70 and -2 9 relative to the transcription start site were found to drive 55% of the promoter activity, A region containing a Sp1 consensus binding sit e located upstream of the two Ets motifs was found to be responsible f or 44% of the transcriptional activity. MAGE-4a promoters 1.4 and 1.6, which also contain the Sp1 and the two Ets binding motifs, supported a level of transcription comparable to that of promoter 1.5, whereas p romoter 1.1, which contains only one Ets binding site, was sixfold les s active. In line with observations made with gene MAGE-1 (HGMW-approv ed symbol MAGE1), we found that promoter 1.5 stimulated a high level o f transcription in a melanoma cell line that does not express MAGE-4. This suggests that the tumor-specific expression of MAGE genes is not determined by the presence of specific transcription factors. (C) 1997 Academic Press.