Epigenetic regulation of metallothionein‐i gene expression: Differential regulation of methylated and unmethylated promoters by DNA methyltransferases and methyl CpG binding proteins

Abstract

Metallothioneins (MTs) are a group of cysteine-rich stress response proteins that scavenge reactive oxygen species and heavy metals. Recently, we have shown that MT-I promoter is methylated and suppressed in some solid and liquid tumors and can be robustly activated following treatment with inhibitors of DNA methyltransferase (DNMT) and histone deacetylase (HDAC). Here, we have analyzed MT-I chromatin structure in active, unmethylated (Hepa cells) and in repressed, methylated state (lymphosarcoma cells). Restriction enzyme accessibility assay showed that the MT-I promoter has an open conformation in unmethylated state as opposed to refractory chromatin structure in methylated state. Positioning of nucleosomal arrays on the methylated promoter further confirmed the closed chromatin structure of the methylated promoter. Chromatin immunoprecipitation (ChIP) assay demonstrated that the unmethylated promoter is associated with K9-acetyl, K4-methyl, and S10-phospho histone H3 whereas the methylated promoter is predominantly associated with K9-methyl H3. HP1alpha that recognizes K9-methyl H3 inhibited methylated MT-1 promoter activity whereas closely related HP1gamma repressed the promoter irrespective of its methylation status. Ubiquitously expressed DNA methyltransferase 1 (DNMT1) suppressed MT-I promoter activity irrespective of its methylation status that does not require its catalytic activity. The DNMT1-mediated repression of MT-I promoter was relieved by trichostatin A, an HDAC inhibitor. Among the methyl CpG binding proteins, MBD2 and MBD4 specifically associated with the methylated promoter and inhibited its activity. In contrast, MBD1 and MeCP2 interacted with both promoters and suppressed the promoter activity irrespective of its methylation status. These results demonstrate that the methylated and unmethylated MT-I promoter are differentially regulated by DNA methyltransferase and methyl-CpG binding proteins, and DNMT1 could suppress MT promoter by a transcriptional mechanism independent of its enzymatic function. These studies suggest that the components of epigenetic machinery differentially regulate methylated and unmethylated MT-I gene expression.

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

“…Moreover, our binding analyses of EMSA show that MBD1 bound to methylated as well as to unmethylated MAGE-A promoters, and sustains the transfection data that binding of MBD1 to unmethylated DNA may lead to repression of the promoters. The ability of MBD1 to repress unmethylated promoters in vitro is supported by a recently published study showing methylation-independent repression (35). Repression of unmethylated genes depends on the third CXXC domain, and repression of methylated genes requires the methyl-CpG binding domain (26,36).…”

“…Thus far, these epigenetic factors have been found to be mainly associated with methylated promoters of tumor suppressor genes (32)(33)(34)(35)(36).…”

“…The discrepancy of our results could allude to the availability of endogenous MeCP2 in MCF-7 cells and Mbd1-deficient mouse fibroblasts. Besides its affinity to hypermethylated DNA (36, 39, 40), the binding of MeCP2 to unmethylated promoters was recently described for the metallothionein I gene, and was explained by its TRD which harbors a DNA binding motif in addition to the NH 2 -terminal methylCpG binding domain (35).…”

DNA methylation profile of 28 potential marker loci in malignant mesothelioma

“…It indicates that CpG islands within the MT cluster are not necessarily coordinately regulated, an observation supported by our analysis of MT1A and MT2A methylation. Epigenetic regulation has been implicated in the control of various MT genes [49]. Hypermethylation of the MT1G promoter has been reported to be associated with higher tumor stage in prostate cancer [50], and MT3 and MT1G expression are downregulated by methylation in oesophageal cancer and thyroid carcinoma respectively [51,52].…”

Silent beginning: Early silencing of the MED1/MBD4 gene in colorectal tumorigenesis

“…It has been shown that MED1 is also capable of interacting with corepressor SIN3A and HDAC1 and to repress transcription at the hyper-methylated promoter of such genes as CDKN2A, MLH1 and MT-1 genes. 8,9 The C-terminal catalytic domain, homologous to base excision repair proteins, shows DNA N-glycosylase activity. MED1 acts especially as a thymine and uracil DNA N-glycosylase on T:G and U:G mismatches that occur at CpG methylation sites due to spontaneous deamination of 5-methylcytosine and cytosine, respectively.…”