Chen H, Walker G E, Taylor S I, McKeon C
National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892.
Diabetes. 1994 Jul;43(7):884-9. doi: 10.2337/diab.43.7.884.
The insulin receptor is a growth regulator present on the surface of most cells that transmits a mitogenic signal in response to insulin. Thus, the gene for the insulin receptor is constitutively expressed at low levels in all cells. We characterize a constitutive enhancer element that is present in the proximal promoter of the human insulin receptor gene. We have localized the enhancer to a 26-base-pair (26-bp) sequence from -528 to -503. When this sequence is inserted into the proximal promoter, a three- to fourfold increase in promoter activity is observed, and when two copies are inserted, a five- to sixfold increase is seen. Electrophoretic mobility shift analysis demonstrates that nuclear factors binding to this sequence are found in many different cell types. At least two proteins with different specificities bind within this 26-bp sequence. The identity of the predominant binding protein is Sp1, because an oligonucleotide composed of an Sp1 consensus binding sequence can compete for several of the DNA-protein complexes. In addition, we demonstrate that purified Sp1 can bind to the 26-bp oligonucleotide and that this complex comigrates with a DNA-protein complex formed with a HeLa nuclear extract. Finally, an antibody to human Sp1 protein is able to bind to the enhancer DNA/HeLa protein complex and supershift this complex. These findings suggest that this sequence corresponds to a general element that may contribute to the ubiquitous expression of the human insulin receptor gene.
胰岛素受体是一种存在于大多数细胞表面的生长调节因子，可响应胰岛素传递有丝分裂信号。因此，胰岛素受体基因在所有细胞中都以低水平组成性表达。我们鉴定了人类胰岛素受体基因近端启动子中存在的一个组成性增强子元件。我们已将该增强子定位到从-528至-503的一段26个碱基对（26-bp）的序列。当将此序列插入近端启动子时，可观察到启动子活性增加三到四倍，而插入两个拷贝时，则可看到增加五到六倍。电泳迁移率变动分析表明，在许多不同细胞类型中都发现了与该序列结合的核因子。至少有两种具有不同特异性的蛋白质在这26-bp序列内结合。主要结合蛋白的身份是Sp1，因为由Sp1共有结合序列组成的寡核苷酸可以竞争几种DNA-蛋白质复合物。此外，我们证明纯化的Sp1可以与26-bp寡核苷酸结合，并且该复合物与用HeLa核提取物形成的DNA-蛋白质复合物共迁移。最后，针对人类Sp1蛋白的抗体能够结合增强子DNA/HeLa蛋白复合物并使该复合物发生超迁移。这些发现表明，该序列对应于一个可能有助于人类胰岛素受体基因普遍表达的通用元件。
