title: Participation of upstream stimulator factor (USF) in cadmium-induction of the mouse metallothionein-I gene.
abstract: 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.
authors.affiliation: Department Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, 3901 Rainbow Boulevard, Kansas City, KS 66160-7421, USA.
authors.forename: Q
authors.initials: Q
authors.lastname: Li
authors.forename: N
authors.initials: N
authors.lastname: Hu
authors.forename: M A
authors.initials: MA
authors.lastname: Daggett
authors.forename: W A
authors.initials: WA
authors.lastname: Chu
authors.forename: D
authors.initials: D
authors.lastname: Bittel
authors.forename: J A
authors.initials: JA
authors.lastname: Johnson
authors.forename: G K
authors.initials: GK
authors.lastname: Andrews
mesh_terms: 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
chemical_list: D004268:DNA-Binding Proteins; D014157:Transcription Factors; D051720:Upstream Stimulatory Factors; C497183:Usf1 protein, mouse; D002104:Cadmium; D004247:DNA; D008668:Metallothionein