Methyl-CpG Binding Domain Proteins and Their Involvement in the Regulation of the MAGE-A1, MAGE-A2, MAGE-A3, and MAGE-A12 Gene Promoters

Abstract

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.

Demethylation of (Cytosine-5-C-methyl) DNA and regulation of transcription in the epigenetic pathways of cancer development

“…Because enucleated sheep oocytes are also able to demethylate serum-starved fibroblast nuclei by~50%, either sheep ooplasm has considerable residual demethylating activity or the sheep fibroblast DNA is particularly prone to demethylation ( [106] and references therein). Wischnewski et al [108] have elucidated the mechanistic roles of MBD proteins on methylation-dependent restricted expression of the tumor-associated MAGE antigens, including the possibility that MBD2 protein may play a role on silencing of MAGE-genes. Their results displayed that the transcriptional repression domains of MBD2 had no inhibiting effect on the promoter activity.…”

Coordinated expression of clustered cancer/testis genes encoded in a large inverted repeat DNA structure

“…MAGEA1 or A4) react similar to MAGEAs encoded in the IR structure (e.g. MAGEA2 or A3) indicating that the individual promoter elements are comparable (Sigalotti et al, 2002;Vatolin et al, 2005;Wischnewski et al, 2007). This, however, is in contrast to the observed extremely heterogeneous expression pattern of the 12 MAGEA genes.…”

Androgen receptor molecular biology and potential targets in prostate cancer

“…This is supported by evidence that the demethylating agent 5-aza-2-deoxycytidine increases cancer testis antigen expression [Shichijo et al 1996]. MAGE genes are normally repressed by DNA methylation and histone deacetylation [Wischnewski et al 2006;De Smet et al 1999] that may involve methyl-CpG binding proteins that recruit histone deacetylases [Wischnewski et al 2007]. In agreement with these findings, the MAGE-11 promoter has a high CpG content that is subject to DNA methylation.…”