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

(zinc-regulated transporters, iron-regulated transporter-like protein) family plays an important role in organism Zn balance. This research identified the promoter regions of and , two members of family, from a freshwater teleost yellow catfish , characterized the binding sequences of the metal-responsive transcription factor-1 () and Ras responsive element binding protein 1 () on their promoter regions. The present study cloned and obtained the 2027 bp of promoter and 1664 bp of ZIP8 promoter, and predicted several key elements on their promoters, such as the binding sites of (cAMP-response element binding protein), (Kruppel like factor 4), and . The sequence deletion from -361 bp to -895 bp down-regulated the luciferase activity of promoter, and the deletion from -897 bp to -1664 bp down-regulated the luciferase activity of promoter. Within different deletion plasmids, the relative luciferase activities of and promoters 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 bp binding site and the -248 bp/-267 bp binding site on the ZIP3 promoter, and the -1543 bp/-1557 bp binding site on the promoter are functional sites. Low Zn increased the binding capability between and its responsive site on the promoter, and high Zn increased the transcriptional activation by ; Zn also promoted the binding ability between and its responsive element on the promoter. This study provides the first direct evidence for the response elements of and on and on to Zn, which are very important for the evaluation of Zn nutrition and toxicity in vertebrates.