To investigate the regulation of metallothionein genes (HsMTs) of Hyriopsis schlegelii, 1,121-bp and 1,270-bp regions of the HsMT1 and HsMT2 promoters were cloned and analyzed, respectively. The two promoters shared partially conserved features and possessed distinct characteristics such as the number or position of metal response elements (MREs). Further analysis of the HsMT1 and HsMT2 promoters was performed by the reporter assay using the luciferase gene. Both promoters were activated by various metals, and presented different levels of metal ions inducibility in human hepatoblastoma cells. Deletion mutant assays demonstrated that both the longest promoter regions achieved the maximum inducibility, and the metal inducibility was dependent on the presence of the MRE in HsMT1 and the distal MRE in HsMT2. In addition, we cloned a putative metal responsive transcription factor (hereby designated as HsMTF-like) and studied its effect on HsMTs expression in human hepatoblastoma cells. An in vivo assay demonstrated that HsMTF-like activates basal HsMTs transcription level, and the MRE in the HsMTs promoter mediates this activation process. Moreover, this basal transcription level can be further boosted by zinc treatment. In conclusion, the regulation mechanism for MT activation in H. schlegelii should be evolutionarily conserved.
Keywords: HsMTF-like; Luciferase reporter gene; MREs; Metallothionein promoter; Transfection.