Yamada K, Duong D T, Scott D K, Wang J C, Granner D K
Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0615, USA.
J Biol Chem. 1999 Feb 26;274(9):5880-7. doi: 10.1074/jbc.274.9.5880.
The cyclic AMP response element (CRE) of the rat phosphoenolpyruvate carboxykinase (PEPCK) gene promoter is required for a complete glucocorticoid response. Proteins known to bind the PEPCK CRE include the CRE-binding protein (CREB) and members of the CCAAT/enhancer-binding protein (C/EBP) family. We took two different approaches to determine which of these proteins provides the accessory factor activity for the glucocorticoid response from the PEPCK CRE. The first strategy involved replacing the CRE of the PEPCK promoter/chloramphenicol acetyltransferase reporter plasmid (pPL32) with a consensus C/EBP-binding sequence. This construct, termed pDeltaCREC/EBP, binds C/EBPalpha and beta but not CREB, yet it confers a nearly complete glucocorticoid response when transiently transfected into H4IIE rat hepatoma cells. These results suggest that one of the C/EBP family members may be the accessory factor. The second strategy involved co-transfecting H4IIE cells with a pPL32 mutant, in which the CRE was replaced with a GAL4-binding sequence (pDeltaCREGAL4), and various GAL4 DNA-binding domain (DBD) fusion protein expression vectors. Although chimeric proteins consisting of the GAL4 DBD fused to either CREB or C/EBPalpha are able to confer an increase in basal transcription, they do not facilitate the glucocorticoid response. In contrast, a fusion protein consisting of the GAL4 DBD and amino acids 1-118 of C/EBPbeta provides a significant glucocorticoid response. Additional GAL4 fusion studies were done to map the minimal domain of C/EBPbeta needed for accessory factor activity to the glucocorticoid response. Chimeric proteins containing amino acid regions 1-84, 52-118, or 85-118 of C/EBPbeta fused to the GAL4 DBD do not mediate a glucocorticoid response. We conclude that the amino terminus of C/EBPbeta contains a multicomponent domain necessary to confer accessory factor activity to the glucocorticoid response from the CRE of the PEPCK gene promoter.
大鼠磷酸烯醇式丙酮酸羧激酶（PEPCK）基因启动子的环磷酸腺苷反应元件（CRE）是糖皮质激素完整反应所必需的。已知与PEPCK CRE结合的蛋白质包括CRE结合蛋白（CREB）和CCAAT/增强子结合蛋白（C/EBP）家族成员。我们采用了两种不同的方法来确定这些蛋白质中的哪一种为来自PEPCK CRE的糖皮质激素反应提供辅助因子活性。第一种策略是用一个共有C/EBP结合序列替换PEPCK启动子/氯霉素乙酰转移酶报告质粒（pPL32）的CRE。这个构建体，称为pDeltaCREC/EBP，能结合C/EBPα和β，但不能结合CREB，然而当它瞬时转染到H4IIE大鼠肝癌细胞中时，能赋予几乎完整的糖皮质激素反应。这些结果表明C/EBP家族成员之一可能是辅助因子。第二种策略是将一个pPL32突变体与H4IIE细胞共转染，其中CRE被GAL4结合序列（pDeltaCREGAL4）取代，以及各种GAL4 DNA结合结构域（DBD）融合蛋白表达载体。尽管由GAL4 DBD与CREB或C/EBPα融合组成的嵌合蛋白能够增加基础转录，但它们并不能促进糖皮质激素反应。相反，由GAL4 DBD和C/EBPβ的1 - 118个氨基酸组成的融合蛋白能提供显著的糖皮质激素反应。进行了额外的GAL4融合研究，以将辅助因子活性所需的C/EBPβ的最小结构域定位到糖皮质激素反应上。含有C/EBPβ的1 - 84、52 - 118或85 - 118氨基酸区域与GAL4 DBD融合的嵌合蛋白不能介导糖皮质激素反应。我们得出结论，C/EBPβ的氨基末端包含一个多组分结构域，该结构域是赋予PEPCK基因启动子CRE的糖皮质激素反应辅助因子活性所必需的。
