Interaction of upstream stimulatory factor proteins with an E-box located within the human CYP1A2 5'-flanking gene contributes to basal transcriptional gene activation.
Interaction of upstream stimulatory factor proteins with an E-box located within the human CYP1A2 5'-flanking gene contributes to basal transcriptional gene activation.
复制标题
上游刺激因子蛋白与位于人 CYP1A2 5 侧翼基因内的 E-box 的相互作用有助于基础转录基因激活。
DOI：
10.1016/s0006-2952(03)00037-6
复制
发表时间：
2003
影响因子：
5.8
通讯作者：
Quattrochi,LindaC
中科院分区：
文献类型：
--
作者：
Pickwell,GeorgeV;Shih,Hsueh;Quattrochi,LindaC
细胞色素P450（CYP）1A 2在所有脊椎动物的肝脏中大量表达。在大多数情况下，其表达仅限于肝脏。对人类CYP 1A 2 5′侧翼区+3至-3201的序列分析确定了3-甲基胆蒽（MC）增强子元件内的6个E盒基序（-1987至-3201）。E-box基序被转录因子的碱性螺旋-环-螺旋（bHLH）家族的成员识别。凝胶迁移率变化和抗体超移测定被用来检查每个的六个上游E盒基序的能力，结合核蛋白和竞争的无处不在的表达bHLH蛋白，上游刺激因子（USF），结合。我们发现USF-1和USF-2蛋白与上游E-box基序EB 2、EB 3和EB 4结合。在HepG 2细胞中进行瞬时转染试验，将人CYP 1A 2 5′-侧翼区的不同片段与荧光素酶报告基因连接。定点诱变的E盒基序之一，EB 2，导致在基础报告基因活性的60%的减少。EB 3和EB 4的突变没有影响。我们发现转染含有USF-1或USF-2 cDNA的表达载体可激活CYP 1A 2报告基因活性，而显性负性USF-2表达载体可阻断这种活性。染色质免疫沉淀试验证实USF蛋白与CYP 1A 2 EB 2位点的相互作用发生在体内。这些数据支持USF作为人CYP 1A 2的组成型转录激活因子的作用。
Cytochrome P450 (CYP)1A2 is abundantly expressed in the liver of all vertebrate species. In most, its expression is restricted to the liver. Sequence analysis of the human CYP1A2 5′-flanking region from +3 to −3201 identified six E-box motifs within the 3-methylcholanthrene (MC) enhancer element (−1987 to −3201). The E-box motif is recognized by members of the basic helix-loop-helix (bHLH) family of transcription factors. Gel mobility shift and antibody supershift assays were used to examine each of the six upstream E-box motifs for their ability to bind nuclear proteins and to compete with the ubiquitously expressed bHLH protein, upstream stimulatory factor (USF), for binding. We found that USF-1 and USF-2 proteins bind to the upstream E-box motifs EB2, EB3, and EB4. Transient transfection assays in HepG2 cells were performed with different segments of the human CYP1A2 5′-flanking region linked to a luciferase reporter gene. Site-directed mutagenesis of one of the E-box motifs, EB2, resulted in a 60% reduction in basal reporter gene activity. Mutations in EB3 and EB4 had no effect. We found that transfection of expression vectors containing USF-1 or USF-2 cDNAs activated CYP1A2 reporter gene activity, while a dominant-negative USF-2 expression vector blocked such activity. Chromatin immunoprecipitation assays confirmed that the interaction of USF proteins with the CYP1A2 EB2 site occurs in vivo. These data support the role of USF as a constitutive transcriptional activator of human CYP1A2.