Participation of upstream stimulator factor (USF) in cadmium-induction of the mouse metallothionein-I gene.
The roles of the bHLH-Zip protein, upstream stimulatory factor (USF), in mouse metallothionein-I (MT-I) gene expression were examined. The promoter contains a putative USF binding site which overlaps an antioxidant response element (ARE) located at -101 bp relative to the transcription start point. The USF/ARE composite element increases basal expression of the mouse MT-I gene, and partly mediates response to oxidative stress. However, other functions of this composite element and the in vivo roles for USF in MT-I promoter functions have not been examined. We report studies which indicate that USF participates via the USF/ARE element in cadmium responsiveness of the mouse MT-I promoter. During the course of these studies a second, higher affinity USF binding site at -223 bp was identified. Stable and transient transfection assays in mouse hepatoma cells, using the USF/ARE in the context of a minimal promoter and site-directed and truncation mutants of the MT-I promoter, revealed that the USF and the ARE sites contribute to cadmium (2-30 microM) but not zinc responsiveness, and to basal promoter activity. Overexpression of dominant-negative (dn)USF in co-transfection assays significantly attenuated cadmium induction of the USF/ARE in the context of a minimal promoter, and attenuated cadmium, but not zinc, induction of the intact MT-I promoter. A consensus E-box (CACATG) at -223 bp in the MT-I promoter was also found to bind USF in vitro , and to be constitutively footprinted in vivo . The interaction of USF with E-box1 was apparently 10-fold stronger than that with the USF/ARE. However, in contrast, E-box1 was not a strong basal promoter element nor was it metal ions responsive in mouse Hepa cells. In conclusion, these studies demonstrate a role for USF in cadmium-specific induction of the mouse MT-I gene, but bring into question an obligate role for USF in regulating basal activity of this gene. The data further suggest that USF interacts with ARE-binding proteins to influence MT-I gene expression.
Li
Q
Q
Department Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, 3901 Rainbow Boulevard, Kansas City, KS 66160-7421, USA.
Hu
N
N
Daggett
M A
MA
Chu
W A
WA
Bittel
D
D
Johnson
J A
JA
Andrews
G K
GK
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002104:Cadmium / Q000633:toxicity*; D004247:DNA / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins; D015870:Gene Expression / Q000187:drug effects; D017930:Genes, Reporter; D008668:Metallothionein / Q000235:genetics*; D051379:Mice; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D051720:Upstream Stimulatory Factors
D004268:DNA-Binding Proteins; D014157:Transcription Factors; D051720:Upstream Stimulatory Factors; C497183:Usf1 protein, mouse; D002104:Cadmium; D004247:DNA; D008668:Metallothionein