Functional Analysis of Two Zinc (Zn) Transporters (<i>ZIP3</i> and <i>ZIP8</i>) Promoters and Their Distinct Response to <i>MTF1</i> and <i>RREB1</i> in the Regulation of Zn Metabolism

Abstract

ZIP (zinc-regulated transporters, iron-regulated transporter-like protein) family plays an important role in organism Zn balance. This research identified the promoter regions of ZIP3 and ZIP8, two members of ZIP family, from a freshwater teleost yellow catfish Pelteobagrus 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 of ZIP3 promoter and 1664 bp of ZIP8 promoter, and predicted several key elements on their promoters, such as the binding sites of CREB (cAMP-response element binding protein), KLF4 (Kruppel like factor 4), MTF-1 and RREB1. The sequence deletion from −361 bp to −895 bp down-regulated the luciferase activity of ZIP3 promoter, and the deletion from −897 bp to −1664 bp down-regulated the luciferase activity of ZIP8 promoter. Within different deletion plasmids, the relative luciferase activities of ZIP3 and ZIP8 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 MTF-1 binding site and the −248 bp/−267 bp RREB1 binding site on the ZIP3 promoter, and the −1543 bp/−1557 bp MTF-1 binding site on the ZIP8 promoter are functional sites. Low Zn increased the binding capability between MTF-1 and its responsive site on the ZIP3 promoter, and high Zn increased the transcriptional activation ZIP3 by RREB1; Zn also promoted the binding ability between MTF-1 and its responsive element on the ZIP8 promoter. This study provides the first direct evidence for the response elements of MTF-1 and RREB1 on ZIP3 and MTF-1 on ZIP8 to Zn, which are very important for the evaluation of Zn nutrition and toxicity in vertebrates.

Keywords

• fish
• Zn homeostasis
• <i>ZIP</i> transporter
• transcriptional regulation
• Zn toxicity