Lin B, Morris D W, Chou J Y
Heritable Disorders Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892, USA.
Biochemistry. 1997 Nov 18;36(46):14096-106. doi: 10.1021/bi9703249.
The gene for glucose-6-phosphatase (G6Pase), the key enzyme in glucose homeostasis, is expressed in a tissue-specific manner in the liver and kidney. To understand the molecular mechanisms regulating liver-specific expression of the G6Pase gene, we characterized G6Pase promoter activity by transient expression assays. The G6Pase promoter is active in HepG2 hepatoma cells, but inactive in JEG3 choriocarcinoma or 3T3 cells. DNA elements essential for optimal and liver-specific expression of the G6Pase gene were contained within nucleotides -234 to +3. Deletion analysis revealed that the G6Pase promoter contained three activation elements (AEs) at nucleotides -234 to -212 (AE-I), -146 to -125 (AE-II), and -124 to -71 (AE-III). AE-I contains binding sites for hepatocyte nuclear factors (HNF) 1 and 4. Electromobility shift and cotransfection assays demonstrated that HNF1alpha, but not HNF4, bound to its cognate site and transactivated G6Pase gene expression. The G6Pase promoter contained five HNF3 motifs, 1 (-180/-174), 2 (-139/-133), 3 (-91/-85), 4 (-81/-75), and 5 (-72/-66), and all five sites bound HNF3gamma with high affinity. Transient expression and cotransfection assays showed that HNF3 site 1 is not required for basal promoter activity, but is essential for HNF3gamma-activated transcription from the G6Pase promoter. We further showed that HNF3 sites 3, 4, and 5 were essential for basal G6Pase promoter activity and transactivation by HNF3gamma. AE-II contains, in addition to a HNF3 motif, a cAMP-response element (CRE) and a C/EBP half-site. The G6Pase(-146/-116) DNA containing AE-II formed multiple protein-DNA complexes with HepG2 nuclear extracts, including HNF3gamma, CRE-binding protein (CREB), C/EBPalpha, and C/EBPbeta. We showed that AE-II mediated transcription activation of the G6Pase gene by cAMP.
葡萄糖-6-磷酸酶（G6Pase）是葡萄糖稳态中的关键酶，其基因在肝脏和肾脏中以组织特异性方式表达。为了解调节G6Pase基因肝脏特异性表达的分子机制，我们通过瞬时表达分析对G6Pase启动子活性进行了表征。G6Pase启动子在HepG2肝癌细胞中具有活性，但在JEG3绒毛膜癌细胞或3T3细胞中无活性。G6Pase基因最佳和肝脏特异性表达所必需的DNA元件包含在核苷酸-234至+3范围内。缺失分析表明，G6Pase启动子在核苷酸-234至-212（AE-I）、-146至-125（AE-II）和-124至-71（AE-III）处含有三个激活元件（AE）。AE-I包含肝细胞核因子（HNF）1和4的结合位点。电泳迁移率变动分析和共转染分析表明，HNF1α而非HNF4与其同源位点结合并反式激活G6Pase基因表达。G6Pase启动子包含五个HNF3基序，分别为1（-180/-174）、2（-139/-133）、3（-91/-85）、4（-81/-75）和5（-72/-66），所有这五个位点都与HNF3γ具有高亲和力结合。瞬时表达和共转染分析表明，HNF3位点1对于基础启动子活性不是必需的，但对于HNF3γ激活的G6Pase启动子转录是必需的。我们进一步表明，HNF3位点3、4和5对于基础G6Pase启动子活性和HNF3γ的反式激活是必需的。AE-II除了含有一个HNF3基序外，还含有一个cAMP反应元件（CRE）和一个C/EBP半位点。含有AE-II的G6Pase（-146/-116）DNA与HepG2核提取物形成多个蛋白质-DNA复合物，包括HNF3γ、CRE结合蛋白（CREB）、C/EBPα和C/EBPβ。我们表明，AE-II介导cAMP对G6Pase基因的转录激活。
