Wischnewski Frank, Friese Olaf, Pantel Klaus, Schwarzenbach Heidi
Institute of Tumor Biology, University Medical Center Hamburg-Eppendorf, Martinstrasse 52, 20246 Hamburg, Germany.
Mol Cancer Res. 2007 Jul;5(7):749-59. doi: 10.1158/1541-7786.MCR-06-0364.
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.
启动子高甲基化导致肿瘤相关MAGE抗原的表达受限。为了阐明甲基化依赖性抑制的潜在机制，我们研究了甲基化CpG结合蛋白MBD1、MBD2a和MeCP2在MAGE-A1、MAGE-A2、MAGE-A3和MAGE-A12基因沉默中的作用。电泳迁移率变动分析显示MBD1与甲基化和未甲基化的MAGE-A启动子结合。使用染色质免疫沉淀分析，在MCF-7和T47D细胞中可观察到MBD1和MeCP2在体内与启动子的结合。用MBD1、MBD2a和MeCP2的转录抑制结构域（TRD）以及MAGE-A1、MAGE-A2、MAGE-A3和MAGE-A12启动子对MCF-7细胞进行瞬时转染分析。虽然MBD1和MeCP2的TRD抑制了MAGE-A启动子，但MBD2的TRD对启动子活性没有抑制作用。此外，将Mbd1缺陷型小鼠成纤维细胞和MCF-7细胞与MBD2a、MeCP2以及MBD1剪接变体1v1和1v3共转染，结果表明这些蛋白不能进一步下调MAGE-A启动子强烈的甲基化依赖性抑制。然而，两个MBD1剪接变体1v1和1v3能够抑制未甲基化MAGE-A启动子的基础活性。用MBD1的两种同工型和转录因子Ets-1进行的额外共转染实验表明，Ets-1不能消除MBD1介导的抑制作用。与MBD1介导的抑制作用相反，发现MBD2a上调启动子的基础活性。总之，这些数据首次表明甲基化CpG结合结构域蛋白参与了MAGE-A基因的调控。
