Differential regulation of zebrafish metallothionein-II (zMT-II) gene transcription in ZFL and SJD cell lines by metal ions

Two alleles of a zebrafish metallothionein II gene (zMT-II) promoter (zMT-IIA and zMT-IIB) containing 10 MREs in the 5′-flanking region (1514 bp) were identified in zebrafish. These putative MREs were confirmed via electrophoretic mobility shift assay (EMSA) to have binding activities from the cellular and nuclear extracts of a zebrafish cell line, ZFL. Transient gene expression studies using zebrafish liver (ZFL) and caudal fin (SJD) cell lines also confirmed that the most distal cluster of MREs contributed to the maximal induction of zMT-IIA activity by Zn2+ and that this Zn2+ induction was dose-dependent. Further transient gene expression assay of the zMT-IIA gene promoter was carried out to study the effects of various metal ions (Zn2+, Cd2+, Cu2+, Hg+, As3+, As5+, Cr3+ and Cr6+), and Zn2+ and Cd2+ were found to be the most efficient MT gene inducers of zMT-II. As3+ was a weak inducer of zMT-II in the two cell lines, and Hg+ caused significant induction only in the SJD cells. No significant induction was found in the other metal ion exposures. EMSA also identified transcription factor(s) of two different sizes from the cytoplasmic and nuclear extracts of the ZFL cells that were able to bind with the MREs, but no increase in MRE binding was detected in the extracts of these cells after Zn2+ or Cd2+ treatment, compared with untreated control cells. The mechanisms of MT gene transcription induction via metal ions are discussed herein.