CpG Site-Specific Regulation of Metallothionein-1 Gene Expression

Metal-binding inducible proteins called metallothioneins (MTs) protect cells from heavy-metal toxicity. Their transcription is regulated by metal response element (MRE)-binding transcription factor-1 (MTF1), which is strongly recruited to MREs in the MT promoters, in response to Zn and Cd. Mouse gene promoter contains 5 MREs (a-e), and MTF1 has the highest affinity to MREd. Epigenetic changes like DNA methylation might affect transcription and, therefore, the cytoprotective function of MT genes. To reveal the CpG site(s) critical for transcription, we analyzed the methylation status of CpG dinucleotides in the gene promoter through bisulfite sequencing in P1798 mouse lymphosarcoma cells, with high or low MT expression. We found demethylated CpG sites near MREd and MREe, in cells with high expression. Next, we compared gene-promoter-driven Lucia luciferase gene expression in unmethylated and methylated reporter vectors. To clarify the effect of complete and partial CpG methylation, we used M.SssI (CG→ CG) and HhaI (GCGC→G CGC)-methylated reporter vectors. Point mutation analysis revealed that methylation of a CpG site near MREd and MREe strongly inhibited gene expression. Our results suggest that the methylation status of this site is important for the regulation of gene expression.