Li Q, Hu N, Daggett M A, Chu W A, Bittel D, Johnson J A, Andrews G K
Department Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, 3901 Rainbow Boulevard, Kansas City, KS 66160-7421, USA.
Nucleic Acids Res. 1998 Nov 15;26(22):5182-9. doi: 10.1093/nar/26.22.5182.
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.
研究了碱性螺旋-环-螺旋拉链蛋白上游刺激因子（USF）在小鼠金属硫蛋白-I（MT-I）基因表达中的作用。该启动子包含一个假定的USF结合位点，该位点与位于相对于转录起始点-101 bp处的抗氧化反应元件（ARE）重叠。USF/ARE复合元件可增加小鼠MT-I基因的基础表达，并部分介导对氧化应激的反应。然而，该复合元件的其他功能以及USF在MT-I启动子功能中的体内作用尚未得到研究。我们报告的研究表明，USF通过USF/ARE元件参与小鼠MT-I启动子对镉的反应。在这些研究过程中，在-223 bp处鉴定出了第二个具有更高亲和力的USF结合位点。在小鼠肝癌细胞中进行的稳定和瞬时转染实验，使用最小启动子背景下的USF/ARE以及MT-I启动子的定点和截短突变体，结果显示USF和ARE位点有助于镉（2-30 microM）而非锌的反应，以及基础启动子活性。在共转染实验中过表达显性负性（dn）USF可显著减弱最小启动子背景下USF/ARE对镉的诱导作用，并减弱完整MT-I启动子对镉而非锌的诱导作用。MT-I启动子中-223 bp处的一个共有E盒（CACATG）在体外也被发现可结合USF，并且在体内持续被足迹分析检测到。USF与E盒1的相互作用明显比与USF/ARE的相互作用强10倍。然而，相比之下，E盒1不是一个强大的基础启动子元件，在小鼠Hepa细胞中也对金属离子无反应。总之，这些研究证明了USF在小鼠MT-I基因镉特异性诱导中的作用，但对USF在调节该基因基础活性中的必然作用提出了质疑。数据进一步表明，USF与ARE结合蛋白相互作用以影响MT-I基因表达。
