Savoldi G, Fenaroli A, Ferrari F, Rigaud G, Albertini A, Di Lorenzo D
Laboratory of Hormonology and Toxicology, Civic Hospital of Brescia, Italy.
DNA Cell Biol. 1997 Dec;16(12):1467-76. doi: 10.1089/dna.1997.16.1467.
A complex interaction between the Glucocorticoid Receptor (GR), C/EBPbeta, and other transcription factors activate the Alpha-1 Acid Glycoprotein (AGP) promoter in HTC(JZ-1) rat hepatoma culture cells. This effect is mediated by the so-called Steroid Responsive Unit (SRU) of the AGP promoter that contains several binding sites for C/EBP transcription factors, some of which overlap with the Glucocorticoid Responsive Element (GRE). Our in vivo footprinting experiments revealed that the GRE- and the C/EBP-binding sites were already occupied glucocorticoid dependently in HTC(JZ-1) cells 10 min after dexamethasone administration (10(-6) M). Furthermore, local changes in the chromatine structure shown by the appearance of DNAse I hypersensitive sites (HS sites) also took place. These changes were probably dependent on a tissue-specific organization of the chromatine at the SRU because they were not detectable in a different glucocorticoid-responsive cell line (PC12) that did not express AGP. Here, we have also shown that withdrawal of dexamethasone or addition of the anti-glucocorticoid RU486 were able to revert the pattern induced by dexamethasone in vivo. The disappearance of the protected region and the hypersensitive sites, typical of the hormone activated promoter, confirmed the necessity of the GR to be bound by the agonist and the inability of the GR-antagonist complex to bind the DNA. By functional assays, we showed that the occupancy of the SRU by these transcriptional proteins in vivo correlated with the activation of the AGP gene transcription. With these results, we have shown that one of the functions of the GR to activate transcription of the AGP gene is to recruit C/EBPbeta and to maintain it bound at its target DNA sequences (SRU). This process was not accomplished by RU486.
在HTC（JZ - 1）大鼠肝癌培养细胞中，糖皮质激素受体（GR）、C/EBPβ和其他转录因子之间的复杂相互作用激活了α1酸性糖蛋白（AGP）启动子。这种效应由AGP启动子的所谓类固醇反应元件（SRU）介导，该元件包含多个C/EBP转录因子的结合位点，其中一些与糖皮质激素反应元件（GRE）重叠。我们的体内足迹实验表明，地塞米松给药（10^(-6) M）10分钟后，HTC（JZ - 1）细胞中GRE和C/EBP结合位点已依赖糖皮质激素被占据。此外，还出现了DNA酶I超敏位点（HS位点）所显示的染色质结构局部变化。这些变化可能依赖于SRU处染色质的组织特异性，因为在不表达AGP的不同糖皮质激素反应细胞系（PC12）中未检测到这些变化。在这里，我们还表明，撤去地塞米松或添加抗糖皮质激素RU486能够逆转地塞米松在体内诱导的模式。激素激活启动子典型的受保护区域和超敏位点的消失，证实了GR需要被激动剂结合以及GR - 拮抗剂复合物无法结合DNA。通过功能测定，我们表明这些转录蛋白在体内对SRU的占据与AGP基因转录的激活相关。通过这些结果，我们表明GR激活AGP基因转录的功能之一是招募C/EBPβ并使其维持在其靶DNA序列（SRU）上。而RU486无法完成这一过程。
