Yamaguchi H, Ikeda Y, Moreno J I, Katsumura M, Miyazawa T, Takahashi E, Imakawa K, Sakai S, Christenson R K
Laboratory of Animal Breeding, Faculty of Agriculture, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
Biochem J. 1999 Jun 15;340 ( Pt 3)(Pt 3):767-73.
To examine regulatory mechanisms of sheep interferon tau (oIFNtau) gene expression, potential enhancer/silencer elements of the oIFNtau gene were examined using a transient transfection system with oIFNtau gene-chloramphenicol acetyltransferase (oIFNtau-CAT) reporter constructs in human choriocarcinoma cells, JEG3. Experiments with 5'-deletion constructs revealed that the upstream regions from bases -654 to -607 and from bases -606 to -555 were essential for oIFNtau gene expression. In a heterologous transcriptional system in which the upstream regions of oIFNtau were inserted in front of simian virus 40 (SV40) promoter, the regions between bases -654 and -555 were determined as being the enhancer region required for oIFNtau-SV40-CAT transactivation. A subsequent study with the oIFNtau-CAT constructs lacking the upstream region between bases -542 and -124 revealed that, in addition to the further upstream region between bases -1000 and -654, the sequences from bases -543 to -452 seemed to act as silencer regions. The oIFNtau-CAT constructs with site-specific mutagenesis revealed that multiple enhancer elements existed between bases -654 and -555 of the oIFNtau gene. On the basis of nucleotide sequence analysis, there are numerous sites between bases -654 and -555 to which potential transcriptional factors, AP-1, GATA and GATA-related proteins, could bind. Furthermore, gel mobility-shift assays revealed that AP-1 or other nuclear factors could bind to these elements. In co-transfection studies, the expression of c-Jun plus c-Fos enhanced the transactivation of oIFNtau-CAT but the expression of GATA-1, GATA-2 or GATA-3 did not. Taken together, these results suggest that the upstream region between bases -654 and -555 could be considered as the enhancer region for oIFNtau gene transactivation.
为研究绵羊干扰素τ（oIFNtau）基因表达的调控机制，在人绒毛膜癌细胞JEG3中使用oIFNtau基因-氯霉素乙酰转移酶（oIFNtau-CAT）报告基因构建体的瞬时转染系统，对oIFNtau基因潜在的增强子/沉默子元件进行了研究。对5'-缺失构建体的实验表明，从碱基-654至-607以及从碱基-606至-555的上游区域对于oIFNtau基因表达至关重要。在将oIFNtau上游区域插入猿猴病毒40（SV40）启动子之前的异源转录系统中，碱基-654和-555之间的区域被确定为oIFNtau-SV40-CAT反式激活所需的增强子区域。随后对缺失碱基-542和-124之间上游区域的oIFNtau-CAT构建体的研究表明，除了碱基-1000和-654之间更远的上游区域外，碱基-543至-452的序列似乎充当沉默子区域。具有位点特异性诱变的oIFNtau-CAT构建体表明，oIFNtau基因的碱基-654和-555之间存在多个增强子元件。基于核苷酸序列分析，在碱基-654和-555之间有许多位点，潜在的转录因子AP-1、GATA和GATA相关蛋白可以结合到这些位点上。此外，凝胶迁移率变动分析表明AP-1或其他核因子可以结合到这些元件上。在共转染研究中，c-Jun加c-Fos的表达增强了oIFNtau-CAT的反式激活，但GATA-1、GATA-2或GATA-3的表达没有增强。综上所述，这些结果表明碱基-654和-555之间的上游区域可被视为oIFNtau基因反式激活的增强子区域。
