Structural and functional analysis of metal regulatory elements in the promoter region of genes encoding metallothionein isoforms in the Antarctic fish Chionodraco hamatus (icefish)

To investigate the regulation of Chionodraco hamatus metallothionein (MT) encoding genes about 1000-bp regions of both MT-I and MT-II gene promoters were cloned and sequenced. Both promoters were rich in A–T content, and lacked the canonical TATA box; several putative cis-regulatory sequences were also present. In the MT-I promoter, four MREs were identified within the first 300 bp from the ATG codon. In the MT-II promoter, seven MREs were organized into two clusters, one containing three MREs located close to the ATG codon, and the other consisting of four MREs lying 500–900 bp upstream of the transcription starting point. The alignment of the MT-I and MT-II promoter regions showed 57% identity, which increased to 87% in the 300-bp region upstream of the ATG. Only the three proximal putative MREs identified were conserved both in position and sequence. Functional analysis of MT-I and MT-II promoters was performed by introducing deletion mutants of the 5′-flanking regions into vector pGL-3, directly upstream of the firefly luciferase reporter gene. Each construct was tested in the HepG2 cell lines in the absence or presence of zinc or cadmium ions. Maximum inducibility of the MT-II gene promoter was achieved with a construct containing both the proximal and the distal MRE clusters. The lack of the most distally located MRE dramatically affected MT-II promoter sensitivity to metals; removal of the distal cluster of MREs also reduced metal inducibility. The MT-I promoter was more compact, since maximal activity and metal inducibility depended on the presence of the proximal cluster of four MREs. This study suggests that the different organization of the MT-I and MT-II gene promoter regions might account for the observed differences in the basal and metal-induced expression of MT-I and MT-II isoforms in the C. hamatus liver.