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

Shu-Wei Chen,Fang Chen,Zhi Luo,Chang-Chun Song,Wu-Hong Lv,Kun Wu

doi:10.3390/ijms21176135

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.

Highlights

Zn is a trace element essential for all the organisms because of its important roles in many important physiological processes [1,2]
This study provides an innovative insight into the mechanism by which Zn regulates the transcriptional activities of ZIP3 and ZIP8, which are very important for the evaluation of Zn nutrition and toxicity in vertebrates
The 2027 bp of ZIP3 promoter and 1664 bp of ZIP8 promoter were successfully cloned (Figure 1A), and the first nucleotide of 5 cDNA of ZIP3 and ZIP8 were designated as +1

Summary

Introduction

Zn is a trace element essential for all the organisms because of its important roles in many important physiological processes [1,2]. Zn uptake is mediated by Zn transporters of the ZIP (zinc-regulated transporters, iron-regulated transporter-like protein) family, which increase the cytoplasmic Zn concentration by transporting Zn from the extracellular space or organelles into cytoplasma [3,4]. Among these members, ZIP3 and ZIP8 were widely expressed in many tissues and involved in Zn acquisition by many cells of the body [4,5]. ZIP3 and ZIP8 possess many differences in the tissue distribution, expression, subcellular localization and regulation These differences have important implications for their functions in maintaining Zn homeostasis. This study provides an innovative insight into the mechanism by which Zn regulates the transcriptional activities of ZIP3 and ZIP8, which are very important for the evaluation of Zn nutrition and toxicity in vertebrates

Results

Animals and Reagents

Promoter Cloning and Plasmid Construction

Sequence Analysis

Plasmid Transfections and Assays of Luciferase Activities

Statistical Analysis