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首页> 外文期刊>Biochemistry >The role of HNF1alpha, HNF3gamma, and cyclic AMP in glucose-6-phosphatase gene activation
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The role of HNF1alpha, HNF3gamma, and cyclic AMP in glucose-6-phosphatase gene activation

机译：HNF1alpha，HNF3gamma和环状AMP在葡萄糖6磷酸酶基因激活中的作用

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摘要

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.
机译：葡萄糖稳态中的关键酶葡萄糖6磷酸酶（G6Pase）的基因在肝脏和肾脏中以组织特异性的方式表达。为了了解调节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表征了G6Pase启动子活性。 G6Pase启动子在HepG2肝癌细胞中有活性，但在JEG3绒癌或3T3细胞中无活性。 G6Pase基因的最佳表达和肝脏特异性表达所必需的DNA元素包含在-234至+3核苷酸内。缺失分析显示，G6Pase启动子在核苷酸-234至-212（AE-1），-146至-125（AE-II）和-124至-71（AE-III）的核苷酸处包含三个激活元件（AE）。 AE-1包含肝细胞核因子（HNF）1和4的结合位点。电迁移和共转染试验表明，HNF1alpha而非其HNF4与其同源位点结合并反式激活了G6Pase基因表达。 G6Pase启动子包含五个HNF3基序，1（-180 / -174），2（-139 / -133），3（-91 / -85），4（-81 / -75）和5（-72 / -66），并且所有五个位点均以高亲和力结合HNF3gamma。瞬时表达和共转染测定法表明，基础启动子活性不需要HNF3位点1，但对于HNF3γ激活的G6Pase启动子转录来说是必不可少的。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的反式激活是必不可少的。 AE-II除包含HNF3基序外，还包含cAMP反应元件（CRE）和C / EBP半位。含有AE-II的G6Pase（-146 / -116）DNA与HepG2核提取物形成了多种蛋白质-DNA复合物，包括HNF3γ，CRE结合蛋白（CREB），C / EBPalpha和C / EBPbeta。我们表明，AE-II通过cAMP介导G6Pase基因的转录激活。

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