A Novel Cysteine Cluster in Human Metal-responsive Transcription Factor 1 Is Required for Heavy Metal-induced Transcriptional Activation in Vivo

Xiaohua Chen,Bo Zhang,Philip M Harmon,Walter Schaffner,David O Peterson,David P Giedroc

doi:10.1074/jbc.m308924200

Xiaohua Chen, Bo Zhang + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m308924200

Copy DOI

Abstract

Metal-responsive transcription factor 1 (MTF-1) specifically binds to metal response elements (MREs) associated with a number of metal- and stress-responsive genes. Human MTF-1 contains a cysteine-rich cluster, -632Cys-Gln-Cys-Gln-Cys-Ala-Cys638-, conserved from pufferfish to humans far removed from the MRE-binding zinc finger domain and just C-terminal to a previously mapped serine/threonine-rich transcriptional activation domain. MTF-1 proteins containing two Cys-->Ala substitutions (C632A/C634A) or a deletion in this region altogether (Delta(632-644)) are significantly impaired in their ability to induce Zn(II)- and Cd(II)-responsive transcription of a MRE-linked reporter gene in transiently transfected mouse dko7 (MTF-1-/-) cells in culture under moderate metal stress but retain the ability to drive basal levels of transcription in a MRE-dependent manner in vivo and in vitro. In addition, the mutated proteins respond to induction by Zn(II) or Cd(II) with nuclear translocation and MRE binding activities comparable with wild-type MTF-1. Attempts to rescue the Delta(632-644) deletion mutant phenotype by inserting similar Cys-rich sequences from Drosophila MTF-1 were unsuccessful, suggesting that the structure of this motif within intact human MTF-1, rather than the simple presence of multiple closely spaced Cys residues, is required for function. This cysteine cluster therefore functions at a step subsequent to nuclear translocation and MRE-binding DNA to naked promoter-containing DNA and appears to be specifically required for MTF-1 to activate transcription in the presence of inducing heavy metal ions.

Highlights

Zinc and other transition metal ions play diverse roles in many biological processes, serving as structural components of proteins [1] or as essential cofactors in enzyme-catalyzed reactions [2, 3]
Treatment of cultured mammalian cells with zinc induces rapid nuclear localization of Metal-responsive transcription factor 1 (MTF-1) [23, 24], activates the specific metal response elements (MREs) binding activity of MTF-1 [45], and results in association of MTF-1 with MRE sequences linked to metal-responsive genes packaged in chromatin [32]
Studies with a purified recombinant zinc finger domain derived from the human protein and expressed in bacteria suggest that zinc fingers F1-F4 bind zinc tightly and form the core of the MRE-binding domain, while fingers F5 and F6 bind zinc with much lower affinity and increase the stability of the MRE complex formed by fingers F1-F4 (29 –31)

Summary

Introduction

Zinc and other transition metal ions play diverse roles in many biological processes, serving as structural components of proteins [1] or as essential cofactors in enzyme-catalyzed reactions [2, 3]. When transient transfection assays were performed with the wild-type MTF-1 expression plasmid (pChMTF-1), we observed metal-induced expression of the luciferase reporter gene (Fig. 2).

Results

Conclusion