Promoter-dependent mechanism leading to selective hypomethylation within the 5' region of gene MAGE-A1 in tumor cells

Abstract

Several male germ line-specific genes, including MAGE-A1, rely on DNA methylation for their repression in normal somatic tissues. These genes become activated in many types of tumors in the course of the genome-wide demethylation process which often accompanies tumorigenesis. We show that in tumor cells expressing MAGE-A1, the 5' region is significantly less methylated than the other parts of the gene. The process leading to this site-specific hypomethylation does not appear to be permanent in these tumor cells, since in vitro-methylated MAGE-A1 sequences do not undergo demethylation after being stably transfected. However, in these cells there is a process that inhibits de novo methylation within the 5' region of MAGE-A1, since unmethylated MAGE-A1 transgenes undergo remethylation at all CpGs except those located within the 5' region. This local inhibition of methylation appears to depend on promoter activity. We conclude that the site-specific hypomethylation of MAGE-A1 in tumor cells relies on a transient process of demethylation followed by a persistent local inhibition of remethylation due to the presence of transcription factors.

FIG. 1.

Methylation patterns at the MAGE-A1 gene locus in normal tissues, melanoma cell lines, and melanoma tissues. The analyzed human genomic locus is represented at the top with vertical bars at the location of CpG dinucleotides, an arrowhead at the MAGE-A1 transcription start site, black boxes corresponding to the MAGE-A1 exons, and dark-grey boxes representing Alu repeats. The regions corresponding to the DNA segments that were amplified from bisulfite-treated DNA (SA to SK) are indicated by dashed lines. Amplified products were cloned, and several clones (7 to 12) were sequenced. The deduced methylation patterns are represented by the grids, where each line corresponds to one allele and each column represents a CpG site. Black squares correspond to methylated cytosines, and empty squares represent unmethylated cytosines. Hatched squares indicate incomplete sequence information for the corresponding CpG. The expression of MAGE-A1, obtained by RT-PCR, is indicated for each sample: no expression (−), weak expression (±), or strong expression (+++). Since MAGE-A1 is on the X chromosome, the content in sexual chromosomes of the samples is also given.

FIG. 2.

Lack of spontaneous demethylating activity targeted to the 5′ region of MAGE-A1 in cells that can express the gene. A cosmid carrying the MAGE-A1 gene within a 42-kb genomic insert was methylated in vitro prior to transfection into MZ2-MEL2.2.5 cells. (A) Expression of MAGE-A1 in these transfectants was analyzed by RT-PCR at two time points after transfection (days 29 and 82) and compared with that in MZ2-MEL2.2.5 cells transfected with the unmethylated MAGE-A1 cosmid (unmeth). (B) Bisulfite sequencing was used to test the methylation state of the in vitro-methylated MAGE-A1 transgene at different time points after transfection. The MAGE-A1 segments that were analyzed and the bisulfite sequencing results are represented as in Fig. 1.

FIG. 3.

The 5′ region of MAGE-A1 is protected against de novo methylation in cells capable of a high-level expression of the gene. The cosmid carrying MAGE-A1 was transfected unmethylated into MZ2-MEL2.2.5 cells. Bisulfite sequencing was used to test the methylation state of the MAGE-A1 transgene at two time points after transfection. The MAGE-A1 segments that were analyzed and the bisulfite sequencing results are represented as in Fig. 1. Histograms represent the overall percentages of methylated CpGs within each segment at the two points after transfection, deduced from the bisulfite sequencing results.

FIG. 4.

Treatment with 5-azadC leads to preferential hypomethylation of the 5′ region of MAGE-A1. (A) MAGE-A1 expression was tested in a clone derived from the MZ2-MEL2.2.5 population transfected with the in vitro-methylated MAGE-A1 cosmid (TM2.6), either before or after treatment with the methylation inhibitor 5-azadC. (B) Methylation analysis of the MAGEA1 transgene in the TM2.6 cell clone either untreated or after 4 days with 5-azadC. The MAGE-A1 segments that were analyzed and the bisulfite sequencing results are represented as in Fig. 1. Histograms compare the level of demethylation upon 5-azadC treatment in each segment with the overall level of demethylation in the entire transgene (dashed line). Only segment SF showed a significantly higher level of demethylation.

FIG. 5.

Protection of the MAGE-A1 5′ region from remethylation depends on promoter activity. (A) The MAGE-A1 gene fragment cloned in front of the EBFP transcription unit in the pEBFPM1-WT and pEBFPM1-mBB′ constructs is represented as in Fig. 1. Positions of the SF and SH segments are indicated by horizontal bars. Asterisks indicate the positions where short oligonucleotide tags were introduced. The wild-type and mutated MAGE-A1 promoter sequences (present in pEBFPM1-WT and pEBFPM1-mBB′, respectively) are given, with arrows indicating the positions and orientations of B and B′ regulatory elements. (B) MZ2-MEL3.1 and LB23-SAR cells were stably transfected with unmethylated pEBFPM1-WT or pEBFPM1-mBB′ plasmid DNA. Quantitative real-time RT-PCR was used to measure the relative MAGE-A1/EBFP expression levels in the transfectants at the earliest time point after transfection. MZ2-MEL3.1 transfected with pEBFPM1-WT was taken as the 100% reference. Values are the means for three separate RT-PCR experiments. (C) Methylation patterns in the SF and SH segments of the MAGE-A1 transgenes are represented by the grids, as in Fig. 1. Sequences deriving from the exogenous MAGE-A1 transgenes could be distinguished from those deriving from the endogenous MAGE-A1 gene by the presence of sequence tags. Asterisks above the grids indicate CpG sites deriving from these tag sequences. The mutations in the B and B′ promoter elements (m) resulted in the loss of two CpG sites, as indicated by the shading of the corresponding columns.