Structural and Functional Analysis of the Rainbow Trout (Oncorhyncus mykiss) Metallothionein-A Gene

Abstract

In the present study, the distal part of the 5′‐flanking region of the rainbow trout metallothionein‐A promoter was sequenced in order to identify cis‐acting regulatory elements. Analysis of this sequence combined with that previously reported for the 5′‐flanking region directly proximal to the start of transcription revealed several putative regulatory sequences. In total, six metal‐responsive elements (MREs) were identified; these sequences were organised into two clusters, one containing two copies of MRE and located close to the predicted TATA box sequence, and a second consisting of four MREs and lying 500–700 bp upstream from the start of transcription. In addition, the 5′‐flanking region contained sequences sharing high similarity with the activator protein 1 consensus sequence as well as one nuclear‐factor‐interleukin‐6‐responsive element. Functional analysis of the promoter was performed by introducing deletion mutants of the 5′‐flanking region into the vector pGL‐2, directly upstream from the luciferase reporter gene. Both MRE clusters were needed for maximal metal inducibility in both rainbow trout hepatoma (RTH‐149) and human hepatoblastoma (Hep G2) cell lines. Furthermore, the distal region was found to be functional in promoting gene transcription following exposure of RTH‐149 cells to hydrogen peroxide.

Cortisol stimulates the zinc signaling pathway and expression of metallothioneins and ZnT1 in rainbow trout gill epithelial cells

“…The 5Ј-flanking regions of ZnT1 and MTB do not possess any obvious GREs; however, similar to MTA, they are characterized by the presence of multiple MREs. The smaller effect of cortisol and zinc treatment on MTB expression compared with MTA expression may be explained by the presence of three tandem MREs in the promoter of MTA compared with one tandem MRE in the MTB promoter (36,37,45). However, there are two caveats to our conclusions.…”

“…To provide more mechanistic evidence as to whether cortisol can directly induce MTA promoter activity, a reporter plasmid (pGLMT-LUC) was constructed containing the Ϫ1042-bp 5Ј-flanking region of the MTA gene (37) linked to the firefly luciferase gene. Zinc treatment induced transactivation in COS-7 cells transiently transfected with the plasmid, demonstrating that the promoter was functional.…”

“…Given the equivocal data on cortisol-mediated MT expression in teleost fish, we hypothesized that cortisol-induced MT expression may not be directly regulated via glucocorticoid receptor (GR) binding to the GRE in the promoter region of the MT gene but via interaction between the glucocorticoid and zinc signaling pathways. In this scenario, cortisol would cause an increase in the intracellular concentration of labile zinc, triggering MT expression via the classical MTF1/MRE pathway.In rainbow trout, there are two MT isoforms, MTA and MTB (7), which both possess tandem MREs located within 1-100 bp of the transcription initiation site and which are especially important for metal inducibility of the genes (33,37,45,46). Rainbow trout MTA and MTB also have distal MRE clusters (less than Ϫ500 bp), and these are also characteristic of teleost fish MT promoters (21,31,41,50).…”

“…In rainbow trout, there are two MT isoforms, MTA and MTB (7), which both possess tandem MREs located within 1-100 bp of the transcription initiation site and which are especially important for metal inducibility of the genes (33,37,45,46). Rainbow trout MTA and MTB also have distal MRE clusters (less than Ϫ500 bp), and these are also characteristic of teleost fish MT promoters (21,31,41,50).…”

“…Zinc causes MTF1 to translocate to the nucleus (47, 48) and enhances MTF1 binding to metal response elements (MRE) of target genes, including MT (2). Similar to the situation in mammals, MT genes in fish and many other species characteristically possess multiple MRE that drive MT transcription in response to MTF1 binding (7,31,37,41). MTF1 also enhances expression of a growing list of genes involved in zinc homeostasis and responses to metal toxicity and oxidative stress (1, 47).…”