Cheng Lin-ling, Li Bing, Tu Hong-bin, Liu Qi-cai, Ran Pi-xin
Guangzhou Institute of Respiratory Disease, Guangzhou Medical College, Guangzhou 510182, China.
Zhonghua Yi Xue Za Zhi. 2005 Mar 2;85(8):560-3.
To validate that the E-box element in the -745-705 region of rat gamma-GCS catalytic subunit gene (GCLC gene), already found to be a negative regulatory region, is an important transcriptional suppressor element.
Electrophoretic mobility shift assays (EMSA) and antibody supershift assay were used to confirm the specific binding of USF, a transcription factor that binds the regulatory sequence -745 - 705, and E-box element. Rat alveolar epidermal cells of the line CCL-149 were cultured and then divided into 4 groups: group 1 to be transfected with GCLC-Luc, group 2 to be co-transfected with pGCLC-Luc and pCMV-USF1, group 3 to be co-transfected with pGCLC-Luc and pCMV-USF2, and group 4 to be co-transfected with pGCLC-Luc and blank vector pCMV. Recombinant retrovirus vector PLXSN-USF1 and recombinant retrovirus vector PLXSN-USF2 were constructed. Another CCL-149 cells were divided into 4 groups: group A to be infected with recombinant USF1 virus, group B to be infected with recombinant USF2 virus, group C to be infected with blank vector, and group D not to be infected with any plasmid. Western blotting was used to detect the concentrations of GCLC protein.
EMSA showed that only the probes with complete E-box element could be bound by the USFs. Supershift assay showed that the transcription factor binding the probe was USF. The luciferase activity of the CCL-149 cells co-transfected with pCMV-USF1/USF2 and GCLC-Luc were decreased by 66.4% and 63.2% respectively in comparison with those transfected with GCLC-Luc only (both P < 0.05) and decreased by 54.5% and 61.1% respectively in comparison with those of the cells of the blank vector group (both P < 0.05). Western blotting showed that the expression of GCLC protein of the CCL-149 cells transfected with PLXSN-USF1 and PLXSN-USF2 were decreased in comparison with that of the control cells.
The interaction between E-box and USF suppresses the expression of GCLC gene. E-box is an important transcriptional suppressor element of gamma-GCS gene.
验证大鼠γ-谷氨酰半胱氨酸合成酶催化亚基基因（GCLC基因）-745至-705区域中的E盒元件是一个重要的转录抑制元件，该区域已被发现是一个负调控区域。
采用电泳迁移率变动分析（EMSA）和抗体超迁移分析来确认与调控序列-745至-705结合的转录因子USF与E盒元件的特异性结合。培养大鼠肺泡上皮细胞系CCL-149，然后分为4组：第1组转染GCLC-Luc，第2组共转染pGCLC-Luc和pCMV-USF1，第3组共转染pGCLC-Luc和pCMV-USF2，第4组共转染pGCLC-Luc和空载体pCMV。构建重组逆转录病毒载体PLXSN-USF1和重组逆转录病毒载体PLXSN-USF2。另取CCL-149细胞分为4组：A组感染重组USF1病毒，B组感染重组USF2病毒，C组感染空载体，D组不感染任何质粒。采用蛋白质免疫印迹法检测GCLC蛋白浓度。
EMSA显示只有带有完整E盒元件的探针能与USFs结合。超迁移分析显示与探针结合的转录因子是USF。与仅转染GCLC-Luc的细胞相比，共转染pCMV-USF1/USF2和GCLC-Luc的CCL-149细胞的荧光素酶活性分别降低了66.4%和63.2%（均P<0.05），与空载体组细胞相比分别降低了54.5%和61.1%（均P<0.05）。蛋白质免疫印迹法显示，与对照细胞相比，转染PLXSN-USF1和PLXSN-USF2的CCL-149细胞中GCLC蛋白的表达降低。
E盒与USF之间的相互作用抑制了GCLC基因的表达。E盒是γ-谷氨酰半胱氨酸合成酶基因重要的转录抑制元件。
