Regulation of mouse Ah receptor (Ahr) gene basal expression by members of the Sp family of transcription factors
Regulation of mouse Ah receptor (Ahr) gene basal expression by members of the Sp family of transcription factors
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DOI：
10.1089/dna.1998.17.811
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发表时间：
1998-09-01
影响因子：
3.1
通讯作者：
Puga, A
中科院分区：
文献类型：
--
作者：
Fitzgerald, CT;Nebert, DW;Puga, A
芳香烃受体（AHR）是一种配体激活的转录因子，它调节多种药物代谢酶的表达，并与免疫抑制、致畸、细胞特异性增生以及某些类型的恶性肿瘤和毒性有关。小鼠Ahr基因5'端启动子区含有4个潜在的Spl基序，是有效基础表达所必需的。利用跨越Ahr启动子的nt-174至+70区域的片段，我们发现，使用来自MLE-12（肺）、F9（胚胎癌）、Hepa-1（肝细胞瘤）和41-5a（表皮）细胞的核提取物，对应于四个Spl位点的四个区域被保护免于DNA酶I消化。Hepa-1和F9细胞系通过逆转录-聚合酶链反应和Western印迹显示含有Sp1和Sp3的mRNA和蛋白，但不含Sp2和Sp 4。在电泳迁移率变动分析中，使用对应于四个Ahr Sp1位点的寡核苷酸探针，从Hepa-1和F9细胞的核提取物形成复合物，免疫学上确定含有Sp1和Sp3蛋白。两个Ahr近端Sp1位点（A和B）显示结合Sp1和Sp3蛋白，而更远端的位点（C和D）仅结合Sp1。竞争凝胶迁移实验表明，位点A和B对Sp因子的亲和力比位点C和D高10倍。为了确定每个的四个Ahr Sp1位点的反式激活潜力，我们融合的Ahr启动子的荧光素酶（LUC）报告基因和转染到果蝇细胞系Schneider-2，其中不含Sp1或Sp1样因子的构建。共转染该构建体与每个Sp因子的表达质粒显示，Sp3在Ahr反式激活中比Sp1效率高约1.6倍。单独和组合的四个Sp1位点的突变表明，每个位点有助于报告基因的表达的总体水平，这些位点之间的相互作用在调节Ahr-LUC构建体中起次要作用。这些结果表明，基础Ahr的表达可能是由Sp1样因子的表达和分布调节。
The aromatic hydrocarbon receptor (AHR) is a ligand-activated transcription factor that regulates the expression of several drug-metabolizing enzymes and has been implicated in immunosuppression, teratogenesis, cell-specific hyperplasia, and certain types of malignancies and toxicities, The mouse Ahr gene 5' proximal promoter region, which contains four potential Spl motifs, is required for efficient basal expression. Using a fragment spanning the region from nt -174 to +70 of the Ahr promoter, we found that four regions corresponding to four Spl sites were protected from DNase I digestion using nuclear extracts from MLE-12 (lung), F9 (embryonal carcinoma), Hepa-1 (hepatoma), and 41-5a (epidermal) cells. The Hepa-1 and F9 cell lines were shown by reverse transcriptase-polymerase chain reaction and Western blot to contain mRNA and protein for Sp1 and Sp3, but not Sp2 and Sp4. In electrophoretic mobility shift assays using oligonucleotide probes corresponding to the four Ahr Sp1 sites, nuclear extracts from Hepa-1 and F9 cells formed complexes that were determined immunologically to contain both Sp1 and Sp3 protein. The two Ahr proximal Sp1 sites (A and B) were shown to bind both Sp1 and Sp3 proteins, whereas the more distal sites (C and D) bound only Sp1. Competition gel shift experiments showed that sites A and B had 10-fold higher affinity for Sp factors than did sites C and D. To determine the transactivation potential of each of the four Ahr Sp1 sites, we fused the Ahr promoter to a luciferase (LUC) reporter gene and transfected the construct into the Drosophila cell line Schneider-2, which contains no Sp1 or Sp1-like factors. Cotransfection of this construct with expression plasmids for each of the Sp factors revealed that Sp3 was approximately 1.6-fold more efficient than Sp1 in Ahr transactivation. Mutation of the four Sp1 sites individually and in combination demonstrated that each site contributes to the overall level of expression of the reporter gene and that interactions between these sites play a minor role in regulation of the Ahr-LUC construct. These results suggest that basal Ahr expression may be regulated by the expression and distribution of Sp1-like factors.