Regulation of human and rainbow trout metallothionein genes

Metallothionein (MT) genes encode small proteins that chelate metal ions through metal-thiolate bonds with cysteine residues. MTs may have a role in cellular zinc homeostasis and metal detoxification. Congruent with these putative functions, MT gene transcription is induced by metals via multiple Metal-Responsive Elements (MREs) present in the MT gene 5;The contributions of cis-acting elements to human (h)MT-IG promoter activity were examined using two functional assays: (i) transient transfection of human cells and (ii) in vivo transcription with rat liver nuclear extracts. In both systems, mutation of the TATA box or the proximal hMREa core sequence devastated promoter achieve suggesting that both elements function synergistically to achieve transcription initiation. The functional effects of the TATA box and hMREa mutations on hMT-IG promoter activity correlation with reduced physical interactions with the human TATA-Binding Protein and zinc-responsive hMREa binding factors from rat and human.;To study the functional interactions between MREs, the trout (t)MT-B promoter was mutated and analyzed using transient transfection of trout and mammalian cell lines. The tMREa, closest to the TATA box, directs basal promoter activity and also can mediate zinc induction independent of tMREb. In contrast, tMREb requires synergistic interactions with tMREa to contribute to zinc induction. Distal tMT-R regulatory elements depend on the proximal tMREs in order to contribute to basal promoter activity as well as to mediate metal and oxidant induction.;In vivo or in vitro zinc treatment activated mammalian MRE binding factors in nuclear extracts. However, cadmium did not increase MRE binding activity even though it induced MT gene expression. The molecular masses and MRE complex migration of the zinc-responsive factors are consistent with mouse and human MTF and expression of the MTF cDNAs increased transfected MT promoter activity in mammalian and trout cell lines. The results support a metal regulatory model whereby MTF is activated by direct association with zinc and, consequently, induces MRE-directed MT transcription.