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 Mt1 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 Mt1 transcription, we analyzed the methylation status of CpG dinucleotides in the Mt1 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 Mt1 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→5mCG) and HhaI (GCGC→G5mCGC)-methylated reporter vectors. Point mutation analysis revealed that methylation of a CpG site near MREd and MREe strongly inhibited Mt1 gene expression. Our results suggest that the methylation status of this site is important for the regulation of Mt1 gene expression.

Heat shock selectively inhibits ribosomal RNA gene transcription and down-regulates E1BF/Ku in mouse lymphosarcoma cells

The effect of heat shock on RNA polymerase I (pol I)-directed transcription of the rRNA gene was studied in S-100 extract derived from mouse lymphosarcoma cells, and by in vivo labelling of rRNA. Exposure of cells to 42 °C for 2 h resulted in complete inhibition of rRNA synthesis in vivo. Pol I transcription was inhibited by 50% within 2 h of heat shock and was abolished after 3 h exposure at 42 °C. Under this condition, the core-promoter-binding activity of the factor (CPBF) that modulates pol I transcription was unaffected. In contrast, the promoter-binding activity of enhancer-1-binding factor, a protein related to the Ku autoantigen, which is involved in pol I transcription initiation, was reduced by 50 and 90% after 2 and 3 h of heat shock respectively. Western-blot analysis with antibodies specific for the two subunits of Ku protein showed the absence of p72 subunit after 3 h of heat shock. Under this condition, pol II transcription from the adenovirus major late promoter and pol III transcription of 5 S RNA gene remained unaffected. Mixing experiments ruled out the possibility that the inhibition of transcription was due to activation of nucleases or other inhibitors. This is the first report to show selective down-regulation of pol I transcription in vitro by heat shock and of the potential involvement of a pol I transcription factor in this process.