Immunogenetics (1995) 42:282-290 © Springer-Verlag 1995 Charles De Smet • St6phane J. Courtois Isabella Faraoni • Christophe Lurquin Jean-Pierre Szikora • Olivier De Backer • Thierry Boon Involvement of two Ets binding sites in the transcriptional activation of the MAGE1 gene Received: 30 May 1995 Abstract The MAGE1 gene codes for an antigen recog- nized on melanoma cell line MZ2-MEL by autologous cytolytic T lymphocytes. It is expressed in a number of tumors of different histological origins, but not in normal tissues except in testis. The MAGE1 promoter region was analyzed by performing transient transfections in MZ2- MEL cells with luciferase reporter plasmids. A fragment extending from nucleotide -792 to +118 exhibited high transcriptional activity. By deletional analysis of this frag- ment, we identified five activating regions designated C, A, B', B, and D. The activity of region A depends on the presence of region B' and vice versa. Two inverted Ets motifs contained in regions B' and B were found to drive 90% of the activity of the MAGE1 promoter in MZ2-MEL cells. Electrophoretic mobility shift assays performed with a nuclear extract from MZ2-MEL cells and with competitor oligonucleotides containing an Ets consensus site showed that nuclear proteins bind to the Ets motif of regions B' and B. Similar experiments suggested that region A binds transcription factors of the Spl family. The MAGE1 pro- moter was found to exert transcriptional activity in tumor cells where the MAGE1 gene is not expressed, suggesting that other mechanisms, such as demethylation, may con- tribute to the tumor-specific expression of the gene. C. De Smet (~) - S. J_ Courtois 1 • I. Faraoni2 • C. Lurquin • J.-P Szikora - O. De Backer - T. Boon Ludwig Institute for Cancer Research, 74 avenue Hippocrate - UCL 74.59, B-1200 Brussels, Belgium Cellular Genetics Unit, Universitd Catholique de Louvain, B-1200 Brussels, Belgium Present addresses: 1 Laboratorium voor Experimentele Geneeskunde en Endocrinoiogie, Katholieke Universiteit te Leuven, B-3000 Leuven, Belgium 2 Istituto di Medicina Spefimentale e Scienze Biochimiche, Universit~ di Roma "Tor Vergata", 00133 Rome, Italy Introduction It is now well established that human tumor cells express antigens recognized by cytolytic T lymphocytes derived from the blood of the tumor-bearing patient. Such antigens were identified on melanoma cell line MZ2-MEL (H&in et al. 1987; Van den Eynde et al. 1989). The MAGE1 gene which codes for two of these antigens was identified by an approach based on the transfection of cosmid libraries (van der Bruggen et al. 1991; Traversari et al. 1992). MAGE1 comprises three exons spread over 4.5 kilobases (kb) and shows no homology to any recorded gene. The entire open reading frame is contained in the last exon and codes for a protein of 309 amino acids. Nothing is known about the function of this protein. Expression of the MAGE1 gene was analyzed by reverse transcription and polymerase chain reaction (PCR) ampli- fication. The gene is silent in all the normal tissues that have been tested, with the exception of testis (De Smet et al. 1994). In contrast, MAGE1 is expressed in tumors of various histological types: 40% of the melanomas, 17% of the mammary carcinomas, 35% of the non-small cell lung carcinomas, and 21% of the transition-cell carcinomas of the bladder (van der Bruggen et al. 1991; Brasseur et al. 1992; Weynants et al. 1994; Patard et al. 1995). Other tumor types, such as colorectal carcinomas, kidney carci- nomas, or leukemias very rarely express MAGE1 (E Bras- seur, personal communication). The tumor-specific expression of MAGE1 is clearly of great importance for immunotherapy. As a first step in understanding the mechanisms governing this specificity, we analyzed the MAGE1 gene promoter region. Materials and methods Cells MZ2-MEL.2_2.5 is a subclone of human melanoma cell line MZ2- MEL (Van den Eynde et al. 1995). The loss of antigen MZ2-E on MZ2- MEL.2.2.5 was found to result from a deletion of the MAGE1 gene