Cloning of zebrafish metallothionein gene and characterization of its gene promoter region in HepG2 cell line.

The coding region of cDNA and genomic DNA, with its promoter region, of zebrafish metallothionein (zMT) gene homologous to the piscine MT-II was obtained. The A/T-rich promoter region contains four metal regulatory elements (MREs), three activator protein 1 (AP1) and one specific protein 1 (Sp1) binding sites. The four MREs are organized into two clusters, a distal cluster with one MRE lying around 740 bp upstream of the transcription start point and a proximal cluster with three MREs located close to the TATA box. The metal induction ability of the promoter was assessed by transient luciferase gene expression assays in HepG2 cells. The zMT promoter was inducible by Zn2+, Cd2+, Cu2+ and Hg2+ ions in decreasing inducibility, while inert to Ni2+, Pb2+ and Co2+ ions, and H2O2 treatment in vitro. Deletion of the putative cis-acting elements in the promoter region revealed that the distal MRE (MREd) was important in mediating metal inducibility. Despite the binding of HepG2 cell nuclear protein factors to all MREs as confirmed by electrophoretic mobility shift assay (EMSA), the proximal MREs did not provide significant contribution to metal induction of zMT gene in HepG2 cells. The metal inducibility of zMT promoter required the cooperative effect of at least three MRE sites.