Methyl-CpG binding domain proteins and their involvement in the regulation of the MAGE-A1, MAGE-A2, MAGE-A3, and MAGE-A12 gene promoters.
Promoter hypermethylation is responsible for the restricted expression of the tumor-associated MAGE antigens. In order to elucidate the mechanism underlying methylation-dependent repression, we examined the involvement of methyl-CpG binding proteins, MBD1, MBD2a, and MeCP2, in silencing of MAGE-A1, MAGE-A2, MAGE-A3, and MAGE-A12 genes. Electrophoretic mobility shift assays displayed binding of MBD1 to the methylated and unmethylated MAGE-A promoters. Using chromatin immunoprecipitation assays, in vivo binding of MBD1 and MeCP2 to the promoters could be observed in MCF-7 and T47D cells. Transient transfection assays of MCF-7 cells were done with the transcriptional repression domains (TRD) of MBD1, MBD2a, and MeCP2, and MAGE-A1, MAGE-A2, MAGE-A3, and MAGE-A12 promoters. Whereas the TRD of MBD1 and MeCP2 repressed the MAGE-A promoters, the TRD of MBD2 had no inhibiting effect on the promoter activity. Furthermore, cotransfections of Mbd1-deficient mouse fibroblasts and MCF-7 cells with MBD2a, MeCP2, and the MBD1 splice variants, 1v1 and 1v3, showed that strong methylation-dependent repression of the MAGE-A promoters could not be further down-regulated by these proteins. However, the two MBD1 splice variants, 1v1 and 1v3, were able to repress the basal activity of unmethylated MAGE-A promoters. Additional cotransfection experiments with both isoforms of MBD1 and the transcription factor Ets-1 showed that Ets-1 could not abrogate the MBD1-mediated suppression. In contrast with the repressive effect mediated by MBD1, MBD2a was found to up-regulate the basal activity of the promoters. In conclusion, these data show, for the first time, the involvement of methyl-CpG binding domain proteins in the regulation of the MAGE-A genes.
Wischnewski
Frank
F
Institute of Tumor Biology, University Medical Center Hamburg-Eppendorf, Martinstrasse 52, 20246 Hamburg, Germany.
Friese
Olaf
O
Pantel
Klaus
K
Schwarzenbach
Heidi
H
D000818:Animals; D000951:Antigens, Neoplasm / Q000235:genetics; D002457:Cell Extracts; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism; D018899:CpG Islands / Q000235:genetics*; D019175:DNA Methylation; D004268:DNA-Binding Proteins / Q000378:metabolism*; D015972:Gene Expression Regulation, Neoplastic; D058950:Melanoma-Specific Antigens; D051783:Methyl-CpG-Binding Protein 2 / Q000378:metabolism; D051379:Mice; D009363:Neoplasm Proteins / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D050784:Proto-Oncogene Protein c-ets-1 / Q000378:metabolism; D012326:RNA Splicing; D012097:Repressor Proteins / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic
D000951:Antigens, Neoplasm; D002457:Cell Extracts; D004268:DNA-Binding Proteins; C072011:MAGEA1 protein, human; C409589:MAGEA12 protein, human; C072013:MAGEA3 protein, human; C066141:MBD1 protein, human; C115368:MBD2 protein; C420909:Mage-a2 antigen; D058950:Melanoma-Specific Antigens; D051783:Methyl-CpG-Binding Protein 2; D009363:Neoplasm Proteins; D050784:Proto-Oncogene Protein c-ets-1; D012097:Repressor Proteins; D014157:Transcription Factors