Target gene search for the metal-responsive transcription factor MTF-1.
Activation of genes by heavy metals, notably zinc, cadmium and copper, depends on MTF-1, a unique zinc finger transcription factor conserved from insects to human. Knockout of MTF-1 in the mouse results in embryonic lethality due to liver decay, while knockout of its best characterized target genes, the stress-inducible metallothionein genes I and II, is viable, suggesting additional target genes of MTF-1. Here we report on a multi-pronged search for potential target genes of MTF-1, including microarray screening, SABRE selective amplification, a computer search for MREs (DNA-binding sites of MTF-1) and transfection of reporter genes driven by candidate gene promoters. Some new candidate target genes emerged, including those encoding alpha-fetoprotein, the liver-enriched transcription factor C/EBPalpha and tear lipocalin/von Ebner's gland protein, all of which have a role in toxicity/the cell stress response. In contrast, expression of other cell stress-associated genes, such as those for superoxide dismutases, thioredoxin and heat shock proteins, do not appear to be affected by loss of MTF-1. Our experiments have also exposed some problems with target gene searches. First, finding the optimal time window for detecting MTF-1 target genes in a lethal phenotype of rapid liver decay proved problematical: 12.5-day-old mouse embryos (stage E12.5) yielded hardly any differentially expressed genes, whereas at stage 13.0 reduced expression of secretory liver proteins probably reflected the onset of liver decay, i.e. a secondary effect. Likewise, up-regulation of some proliferation-associated genes may also just reflect responses to the concomitant loss of hepatocytes. Another sobering finding concerns gamma-glutamylcysteine synthetase(hc) (gamma-GCS(hc)), which controls synthesis of the antioxidant glutathione and which was previously suggested to be a target gene contributing to the lethal phenotype in MTF-1 knockout mice. gamma-GCS(hc) mRNA is reduced at the onset of liver decay but MTF-1 null mutant embryos manage to maintain a very high glutathione level until shortly before that stage, perhaps in an attempt to compensate for low expression of metallothioneins, which also have a role as antioxidants.
Lichtlen
P
P
Institute of Molecular Biology, University of Zürich, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland,
Wang
Y
Y
Belser
T
T
Georgiev
O
O
Certa
U
U
Sack
R
R
Schaffner
W
W
D000818:Animals; D001667:Binding, Competitive; D022763:CCAAT-Enhancer-Binding Protein-alpha / Q000235:genetics / Q000378:metabolism; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D018076:DNA, Complementary / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins; D004622:Embryo, Mammalian / Q000378:metabolism; D005260:Female; D020869:Gene Expression Profiling; D018507:Gene Expression Regulation, Developmental; D005978:Glutathione / Q000378:metabolism; D006801:Humans; D054835:Lipocalin 1; D008099:Liver / Q000196:embryology / Q000378:metabolism; D008297:Male; D051379:Mice; D018345:Mice, Knockout; D020411:Oligonucleotide Array Sequence Analysis; D009841:Oligonucleotides / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism; D000509:alpha-Fetoproteins / Q000235:genetics / Q000378:metabolism; D000097809:Transcription Factor MTF-1
D022763:CCAAT-Enhancer-Binding Protein-alpha; D002352:Carrier Proteins; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D005978:Glutathione; D054835:Lipocalin 1; D009841:Oligonucleotides; D012333:RNA, Messenger; D014157:Transcription Factors; D000509:alpha-Fetoproteins; D000097809:Transcription Factor MTF-1; C518443:LCN1 protein, human; C518449:Lcn1 protein, rat