Transcriptional activity and regulatory protein binding of metal‐responsive elements of the human metallothionein‐IIA gene

Multiple copies of a cis-acting DNA element, metal-responsive element (MRE) are required for heavy metal-induced transcriptional activation of mammalian metallothionein genes. To approach the regulatory mechanism mediated by these multiple elements, we studied the properties of seven MREs located upstream of the human metallothionein-IIA (hMT-IIA) gene in detail. Transfection assays of reporter gene constructs each containing one of these MREs as the promoter element revealed that only four MREs can mediate zinc response. With respect to the distribution of active MREs over the promoter region, the hMT-IIA gene is largely different from the mouse metallothionein-I gene, suggesting that MRE arrangement is not an important factor for metal regulation. Experiments using various model promoters showed that multiple MRE copies act highly synergistically, supporting the biological significance of the multiplicity. Only the four active MREs efficiently bound the purified transcription factor human MTF-1, and MRE mutants defective in binding this protein lost the ability to support zinc-induced reporter gene expression, strongly suggesting that the direct interaction between human MTF-1 and a set of the selected MREs plays the major role in heavy metal regulation. In protein/DNA binding reactions in vitro, the purified human MTF-1 was activated by zinc but not by other metallothionein-inducing heavy metals, supporting the idea that zinc is the direct modulator of human MTF-1.