Multiple elements within the 5' distal enhancer of the mouse heme oxygenase-1 gene mediate induction by heavy metals.
Multiple elements within the 5' distal enhancer of the mouse heme oxygenase-1 gene mediate induction by heavy metals.
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发表时间：
1994-10
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通讯作者：
J. Alam
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作者：
J. Alam
SX2是一个268个碱基对的5‘末端片段，它介导小鼠血红素加氧酶-1基因的基础水平和诱导剂依赖的激活，它包含两个激活蛋白-1(AP-1)结合位点(Alam，J.，和Zing，D.(1992)J.Biol)。化学。267、21894-21900)。在瞬时表达分析中，这两个AP-1结合元件的突变都会降低SX2的增强子活性(50-70%)，但不会消除SX2的增强子活性，这表明其他序列有助于增强子功能。AP-1结合位点的上游是一个序列基序的两个拷贝，TGAGGAAAT，它与已知与CCAAT/增强子结合蛋白(C/EBP)转录因子家族相互作用的细胞和病毒基因中的元件相似。这些SX2序列特异性地与肝脏富含的、热稳定的核蛋白结合，并赋予异源氯霉素乙酰转移酶(CAT)基因依赖于C/EBPα的反式激活。这些9碱基对元件的定点突变取消了蛋白质结合和反式激活，建立了这些序列作为功能C/EBP结合位点。稳定表达的SX2/CAT融合基因在小鼠肝癌、HEPA细胞中的诱导倍数为37-44倍，在小鼠成纤维细胞L929细胞中的诱导倍数为52-111倍。缺少AP-1结合元件的SX2亚片段不介导镉依赖的CAT基因激活，而含有AP-1结合元件但缺乏C/EBP结合位点的亚片段仅显示部分转录活性。一个或多个C/EBP和AP-1结合位点的定点突变表明，这些元件中的每一个都是SX2增强子片段最佳活性所必需的。然而，AP-1结合元件对于诱导似乎更重要，因为含有AP-1结合元件的多个副本的构建体，而不是C/EBP结合序列，很容易被CdCl2激活。用镉或血红素处理HEPA细胞不改变AP-1的核浓度或C/EBP结合活性。
A 268-base pair 5' distal fragment, SX2, which mediates basal level and inducer-dependent activation of the mouse heme oxygenase-1 gene, contains two activator protein-1 (AP-1) binding sites (Alam, J., and Zhining, D. (1992) J. Biol. Chem. 267, 21894-21900). Mutation of both AP-1 binding elements diminishes (by 50-70%), but does not abolish, the enhancer activity of SX2 in transient expression assays, suggesting that other sequences contribute to enhancer function. Directly upstream of the AP-1 binding sites are two copies of a sequence motif, TGAGGAAAT, which resemble elements found in cellular and viral genes that are known to interact with the CCAAT/enhancer-binding protein (C/EBP) family of transcription factors. These SX2 sequences bind specifically to liver-enriched, heat-stable nuclear proteins and confer C/EBP alpha-dependent transactivation of the heterologous chloramphenicol acetyltransferase (CAT) gene. Site-directed mutagenesis of these 9-base pair elements abolishes protein binding and transactivation, establishing these sequences as functional C/EBP binding sites. Stably transfected SX2/CAT fusion genes are induced between 37- and 44-fold in mouse hepatoma, Hepa, cells and between 52- and 111-fold in mouse fibroblast L929 cells in response to CdCl2 treatment. Subfragments of SX2 lacking the AP-1 binding elements do not mediate cadmium-dependent activation of the CAT gene, whereas subfragments containing the AP-1 binding elements, but lacking the C/EBP binding sites, exhibit only partial transcriptional activity. Site-directed mutagenesis of one or more of the C/EBP and AP-1 binding sites indicates that each of these elements is required for optimal activity of the SX2 enhancer fragment. The AP-1 binding elements, however, appear to be more important for induction as constructs containing multiple copies of either of the AP-1 binding elements, but not the C/EBP binding sequences, are readily activated by CdCl2. Treatment of Hepa cells with cadmium or heme does not alter the nuclear concentration of AP-1 or C/EBP binding activity.