Functional Analysis of Two Zinc (Zn) Transporters (ZIP3andZIP8) Promoters and Their Distinct Response toMTF1andRREB1in the Regulation of Zn Metabolism

ZIP(zinc-regulated transporters, iron-regulated transporter-like protein) family plays an important role in organism Zn balance. This research identified the promoter regions ofZIP3andZIP8, two members ofZIPfamily, from a freshwater teleost yellow catfishPelteobagrus fulvidraco, characterized the binding sequences of the metal-responsive transcription factor-1 (MTF-1) and Ras responsive element binding protein 1 (RREB1) on their promoter regions. The present study cloned and obtained the 2027 bp ofZIP3promoter and 1664 bp of ZIP8 promoter, and predicted several key elements on their promoters, such as the binding sites ofCREB(cAMP-response element binding protein),KLF4(Kruppel like factor 4),MTF-1andRREB1. The sequence deletion from -361 bp to -895 bp down-regulated the luciferase activity ofZIP3promoter, and the deletion from -897 bp to -1664 bp down-regulated the luciferase activity ofZIP8promoter. Within different deletion plasmids, the relative luciferase activities ofZIP3andZIP8promoters changes to Zn incubation in a Zn concentration-dependent manner. The site mutagenesis and EMSA (electrophoretic mobility shift assay) found that the -1327 bp/-1343 bpMTF-1binding site and the -248 bp/-267 bpRREB1binding site on the ZIP3 promoter, and the -1543 bp/-1557 bpMTF-1binding site on theZIP8promoter are functional sites. Low Zn increased the binding capability betweenMTF-1and its responsive site on theZIP3promoter, and high Zn increased the transcriptional activationZIP3byRREB1; Zn also promoted the binding ability betweenMTF-1and its responsive element on theZIP8promoter. This study provides the first direct evidence for the response elements ofMTF-1andRREB1onZIP3andMTF-1onZIP8to Zn, which are very important for the evaluation of Zn nutrition and toxicity in vertebrates.