GENOMICS 40, 305–313 (1997) ARTICLE NO. GE964566 Alternative Promoters of Gene MAGE4a E TIENNE DE P LAEN,BE ´ NE ´ DICTE NAERHUYZEN,C HARLES DE S M ET,J EAN-P IERRE S ZIKORA, AND T HIERRY B OON 1 Ludwig Institute for Cancer Research, Brussels Branch, 74 avenue Hippocrate, UCL 74.59, B1200 Brussels, Belgium; and Cellular Genetics Unit, Universite ´ Catholique de Louvain, B1200 Brussels, Belgium Received July 22, 1996; accepted December 5, 1996 nificant proportion of tumors of different histological Gene MAGE-4 (HGMW-approved symbol MAGE4) is types, such as melanomas, breast carcinomas, and non- expressed in several types of tumors, but not in normal small cell lung cancers. No expression was detected in tissues, except testis and placenta. The 5 end of this normal cells, except in testis. gene contains eight homologous exons spread over a Gene MAGE-1 belongs to a family of 12 closely re- 5.8-kb region. These exons are alternatively spliced to lated genes that are located in Xq28 (De Plaen et al., a unique second exon and a unique third exon, which 1994; Rogner et al., 1995). Genes MAGE-1, -2, -3, -4, encodes a protein of 317 amino acids. The analysis of -6, and -12 are transcribed at a significant level in a transcripts found in testis, placenta, and a sarcoma number of tumors of various histological origins. None cell line showed that each of the alternative first exons of these genes are expressed in normal tissues, except is used in at least one of these tissues. Various regions in testis, and, for MAGE-3 and -4, in placenta. Two of the promoter of the fifth alternative exon (1.5) were additional MAGE-related genes have been identified in cloned in a luciferase reporter plasmid, and the con- the Xp21.3 region (Dabovic et al., 1995; Muscatelli et structs were transfected in a sarcoma cell line that al., 1995). They were named MAGE-Xp/DAM10 and expresses MAGE-4. Two Ets motifs located between po- DAM6. These genes are also not expressed in any nor- sitions 070 and 029 relative to the transcription start mal tissue, except in testis. They are rarely expressed site were found to drive 55% of the promoter activity. A in tumors, except in lung carcinoma samples (Dabovic region containing a Sp1 consensus binding site located et al., 1995). upstream of the two Ets motifs was found to be respon- All MAGE genes contain small untranslated exons sible for 44% of the transcriptional activity. MAGE-4a followed by a large last exon, which contains the entire promoters 1.4 and 1.6, which also contain the Sp1 and coding sequence (Dabovic et al., 1995; De Plaen et al., the two Ets binding motifs, supported a level of tran- scription comparable to that of promoter 1.5, whereas 1994; Muscatelli et al., 1995). The coding sequences are promoter 1.1, which contains only one Ets binding highly conserved, suggesting that the proteins encoded site, was sixfold less active. In line with observa- by these genes exert a similar function. This function tions made with gene MAGE-1 (HGMW-approved sym- is currently unknown. The 5 ends of MAGE genes lo- bol MAGE1), we found that promoter 1.5 stimulated a cated in Xq28 were found to be highly variable with high level of transcription in a melanoma cell line that large deletions, insertions, and in MAGE-2 an addi- does not express MAGE-4. This suggests that the tu- tional exon, which is homologous to intronic regions of mor-specific expression of MAGE genes is not deter- the other genes. mined by the presence of specific transcription factors. We reported before that MAGE-4a has several alter-  1997 Academic Press native first exons (De Plaen et al., 1994). We report here the detailed structure of the 5 end of gene MAGE- 4a, and we present an analysis of promoter regions of INTRODUCTION this gene. The characterization of tumor antigens of human MATERIALS AND METHODS melanoma cell line MZ2-MEL led to the discovery of a Cell lines and cell transfection. MZ2-MEL.2.2.5 is a subclone of gene named MAGE-1 2 (Traversari et al., 1992; van der human melanoma cell line MZ2-MEL that was derived from an ab- Bruggen et al., 1991). This gene is expressed in a sig- dominal metastasis of patient MZ2. Sarcoma cell line LB23-SAR was derived from a rhabdomyosarcoma of patient LB23. Culture conditions of the MZ2-MEL.2.2.5 and LB23-SAR cells have been de- Sequence data from this article have been deposited with the EMBL/GenBank Data Libraries under Accession No. U10687. scribed (De Smet et al., 1995; He ´rin et al., 1987; Van den Eynde et al., 1989). 1 To whom correspondence should be addressed. Telephone: (32 2) 764 75 80. Fax: (32 2) 764 75 90. MZ2-MEL and LB23-SAR cells were transfected as described (De Smet et al., 1995). pTKluc is a gift from S. J. Courtois and F. P. 2 The HGMW-approved symbols for the genes described in this paper are MAGE1 and MAGE4. Lemaigre (UCL and ICP, Brussels). 305 0888-7543/97 $25.00 Copyright  1997 by Academic Press All rights of reproduction in any form reserved.