A novel cysteine cluster in human metal-responsive transcription factor 1 is required for heavy metal-induced transcriptional activation in vivo.
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.
Chen
Xiaohua
X
Department of Biochemistry and Biophysics, Texas A&M University, College Station, Texas 77843-2128, USA.
Zhang
Bo
B
Harmon
Philip M
PM
Schaffner
Walter
W
Peterson
David O
DO
Giedroc
David P
DP
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002478:Cells, Cultured; D017124:Conserved Sequence; D003545:Cysteine / Q000737:chemistry; D004247:DNA / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins; D006801:Humans; D051379:Mice; D018345:Mice, Knockout; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014157:Transcription Factors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D015533:Transcriptional Activation; D014162:Transfection; D000097809:Transcription Factor MTF-1
D003545:Cysteine; D004247:DNA; D004268:DNA-Binding Proteins; D011994:Recombinant Proteins; D014157:Transcription Factors; D000097809:Transcription Factor MTF-1