Epigenetic hierarchy within the MAGEA1 cancer-germline gene: promoter DNA methylation dictates local histone modifications

Abstract

Gene MAGEA1 belongs to a group of human germline-specific genes that rely on DNA methylation for repression in somatic tissues. Many of these genes, termed cancer-germline (CG) genes, become demethylated and activated in a wide variety of tumors, where they encode tumor-specific antigens. The process leading to DNA demethylation of CG genes in tumors remains unclear. Previous data suggested that histone acetylation might be involved. Here, we investigated the relative contribution of DNA methylation and histone acetylation in the epigenetic regulation of gene MAGEA1. We show that MAGEA1 DNA hypomethylation in expressing melanoma cells is indeed correlated with local increases in histone H3 acetylation (H3ac). However, when MAGEA1-negative cells were exposed to a histone deacetylase inhibitor (TSA), we observed only short-term activation of the gene and detected no demethylation of its promoter. As a more sensitive assay, we used a cell clone harboring a methylated MAGEA1/hph construct, which confers resistance to hygromycin upon stable re-activation. TSA induced only transient de-repression of the transgene, and did not lead to the emergence of hygromycin-resistant cells. In striking contrast, transient depletion of DNA-methyltransferase-1 in the reporter cell clone gave rise to a hygromycin-resistant population, in which the re-activated MAGEA1/hph transgene displayed not only marked DNA hypomethylation, but also significant reversal of histone marks, including gains in H3ac and H3K4me2, and losses of H3K9me2. Collectively, our results indicate that DNA methylation has a dominant role in the epigenetic hierarchy governing MAGEA1 expression.

Figure 1. Histone changes associated with the activation of gene MAGEA1 in melanoma cell lines.

A, MAGEA1 mRNA expression levels (upper panel) and 5′-region DNA methylation levels (lower panel) were evaluated in three non-expressing cell lines (HFF2-hTERT, SK-MEL-23 and EB16-MEL) as well as in three expressing cell lines (MZ2-MEL3.1, BB74-MEL and Mi13443-MEL). Values represent the mean (± sem) of two independent qRT-PCR or qMS-PCR experiments, each in duplicate. B, ChIP-quantitative PCR was applied to the same cell lines to evaluate enrichment of the indicated histone modifications within either the MAGEA1 5′-region or the GAPDH promoter (representing a ubiquitously active promoter). Data derive from at least two independent ChIP experiments, with two duplicate qPCR measures in each case.

Figure 2. TSA induces transient activation of MAGEA1 in melanoma cells.

SK-MEL-23 and EB16-MEL cell lines were exposed to either 300 nM TSA during 24h (A), or 1 µM 5-azadC during 96h (B), and RNA was extracted from the cells at days 1, 4 and 7 after TSA treatment or at days 4, 7, 10 after 5-azadC treatment. MAGEA1 expression levels were determined by quantitative RT-PCR. Values, which derive from three independent experiments, were normalized by the ACTINB expression level, and are expressed relative to the levels found in non-treated cells (ctrl). * P<0.05, ** P<0.01, *** P<0.001. C, The effect of TSA and 5-azadC on MAGEA1 5′-region DNA demethylation was assessed by applying MS-PCR to DNA samples extracted 10 days after the beginning of the treatments. Data (fold demethylation) correspond to the relative amount of unmethylated sequences in treated cells reported to that in untreated cells (ctrl). Values represent the mean (± sem) of three independent qMS-PCR experiments. * P<0.05, ** P<0.01.

Figure 3. Histone modifications associated with an in vitro methylated MAGEA1 transgene.

A, Schematic representation of the structure and DNA methylation status of the active unmethylated (empty circles) MAGEA1 gene and the inactive methylated (filled circles) MAGEA1/hph transgene in MZ2-MEL.TrHM cells. Black boxes correspond to the MAGEA1 exons, the dark gray box within the MAGEA1/hph transgene represents the hph transcription unit, and the asterisk (*) is the site where a 12-bp tag sequence (carrying a XbaI restriction site) was inserted. The lower panel is an enlargement of the amplicon that was amplified in ChIP experiments, and indicates the expected fragment sizes following XbaI digestion. B, ChIP experiments were applied to MZ2-MEL.TrHM. The resulting MAGEA1 amplicons were digested with XbaI and separated on agarose gels, thereby revealing relative enrichment of the indicated histone modifications within either the MAGEA1 gene (upper band) or the MAGEA1/hph transgene (lower band). C, Relative enrichment of histone marks on the transgene was deduced by quantifying band intensities in gel electrophoresis pictures (ImageJ software), and calculating the lower/upper ratio. Data, which were normalized by the lower/upper ratio in input samples, derive from at least two independent ChIP experiments, with two PCR/XbaI/electrophoresis analyses in each case. * P<0.05, *** P<0.001.

Figure 4. Lack of long-term activation of MAGEA1/hph following TSA treatment.

A, Schematic outline of the experiment. MZ2-MEL.TrHM cells were treated or not (control) with 300 nM of TSA for 24h, 80 nM of TSA for 72h, or with 20 nM of 5-azadC for 72h. After three days, 10⁶ cells from each group were transferred into two flasks (75 cm²) and were selected in a medium containing hygromycin (180 µg/mL) during 13 days. B, The level of expression of the MAGEA1/hph transgene was quantified by qRT-PCR at the indicated time point (d1 or d3) in each group of cells. Data represent the mean (± sem) of al least three independent experiments. *** P<0.001. C, The level of DNA demethylation of the MAGEA1/hph 5′-region in the different groups of cells was evaluated by quantitative MS-PCR, using primers that specifically amplify the tagged transgene sequence. The data (fold DNA demethylation) were calculated as in Fig 2C, and correspond to the mean (± sem) of at least three independent experiments. * P<0.05. D, The number of clones that survived hygromycin selection were counted at day 16 in the three groups of cells. Data derive from at least two independent experiments, each in duplicate.

Figure 5. DNA demethylation induces reversal of histone marks in MAGEA1/hph.

A, Schematic outline of the derivation of the H^r population and H^r clone 1 from MZ2-MEL.TrHM cells (see reference for details). MZ2-MEL.TrHM cells were repeatedly tranfected with antisense oligonucleotides directed against DNMT1 (DNMT1-AS) during 7 days, and were thereafter transferred into medium containing hygromycin. After 9 days of hygromycin selection, a resistant population emerged (H^r population), and a clone (H^r clone 1) was isolated from this population by limiting dilution. The H^r population and H^r clone 1 were subsequently cultured without hygromycin selection. B, The mRNA expression level (ratio to 10⁴ ACTINB) and 5′-region DNA methylation status (% methylated CpGs) of the MAGEA1/hph transgene were determined in MZ2-MEL.TrHM cells, the H^r population and H^r clone 1 by qRT-PCR and bisulfite sequencing, respectively. C, ChIP-qPCR was used to evaluate enrichment of H3K9me2 within the MAGEA1/hph transgene in the three groups of cells (see text for details). Fold enrichment levels were obtained by reporting the MAGEA1 5′-region enrichment values to that of the GAPDH 5′-region in the same sample. Data represent the mean (± sem) of two to three ChIP experiments, with two duplicate qPCR measurements in each case. *** P<0.001. D, The ChIP/PCR/XbaI procedure (see Fig. 3A, B) was applied to evaluate enrichment of H3ac and H3K4me2 within the 5′-region of the MAGEA1/hph transgene in the three group of cells. E, ImageJ analyses of gel electrophoresis pictures were used to quantify the MAGEA1 5′-region transgene/gene ratio. Data represent the mean (± sem) of two independent ChIP experiments, with two PCR/XbaI/electrophoresis analyses in each case. ** P<0.01, *** P<0.001.