Structural and functional analyses of the trout metallothionein A gene: Promoter interactions with the mammalian transcription factors

Metallothioneins (MTs) are small proteins that can bind heavy metal ions such as zinc, cadmium, and copper through interactions with their multiple cysteine residues. MTs probably play a role in metal ion homeostasis and detoxification of poisonous metal ions as well as providing protection from the effects of oxidative stress. MT gene expression can be induced by the same metals that the protein binds as well as some oxidants. The activation of transcription from MT genes is mediated by multiple cis-acting elements called Metal Responsive Elements (MREs) found in the 5;The tMT-A gene was structurally analyzed and found to be highly homologous to the tMT-B gene. The 5;The contributions of various tMT-A promoter elements were examined by a series of deletion mutants ligated to reporter genes and transiently transfected into trout cells. The results indicate that at least two MREs are required for high levels of metal inducible expression but a single MRE can support low basal levels of transcription. When all MREs are removed from the tMT-A promoter all transcriptional activity is abolished. For oxidants to induce transcription from the tMT-A promoter both an ARE half-site and a single MRE are minimally required.;The function of mMTF and hMTF in trout cells was assayed by co-transfecting their respective cDNAs with a tMT-A promoter deletion. Both mammalian factors were active in trout cells and increased the transcription from the tMT-A promoter without the addition of exogenous zinc. Recombinant, in vitro translated MTFs would bind trout MREs in mobility shift assays in a zinc dependent manner. Treatment of the in vitro translated MTFs with cadmium, however, abolished the DNA binding activity. It was also found that cadmium can displace zinc and zinc can displace cadmium from the zinc finger moieties of MTF. This suggests that MTF transiently and reversibly binds metal ions but DNA binding activity is only seen when the fingers are occupied by zinc. These results support a model of metal directed transcription whereby MTF is activated directly by association with zinc and as such becomes competent to bind MREs and activate transcription from MT promoters.