Cloning and characterization of the human beta2-glycoprotein I (beta2-GPI) gene promoter: roles of the atypical TATA box and hepatic nuclear factor-1alpha in regulating beta2-GPI promoter activity

摘要

pβsub2/sub-Glycoprotein I (βsub2/sub-GPI) is a plasma glycoprotein primarily synthesized in the liver. The interindividual variability of βsub2/sub-GPI expression in subjects with various metabolic syndromes and disease states suggests that it may have clinical importance. However, the regulation of βsubi2/i/sub-iGPI/i gene expression has not been clarified. To gain more insight into the control of βsubi2/i/sub-iGPI/i gene expression, we cloned the 4.1-kb 5′-flanking region and characterized the proximal promoter of the βsubi2/i/sub-iGPI/i gene in this study. iCis/i-acting elements required for βsubi2/i/sub-iGPI/i promoter activity were identified with transient transfection assays in the hepatoma cell lines HepG2 and Huh7 and in non-hepatic HeLa cells. Serial deletion analyses of the βsubi2/i/sub-iGPI/i 5′-flanking sequence revealed that the region from ?197 to +7 had strong promoter activity in hepatoma cells but not in HeLa cells. Truncation and site-directed mutagenesis of putative icis/i-elements within this region showing an atypical TATA box and a HNF-1 (hepatic nuclear factor-1) element were both essential for the βsubi2/i/sub-iGPI/i promoter activity. Subsequent gel mobility shift assays confirmed the interaction of HNF-1α with the HNF-1 site residing downstream of the TATA box. Co-transfection of βsubi2/i/sub-iGPI/i promoter-luciferase vector with HNF-1α expression vector in Huh7 and HNF-1-deficient HeLa cells demonstrated the transactivation effect of HNF-1α on βsubi2/i/sub-iGPI/i promoter activity. In addition, overexpression of HNF-1α enhanced the endogenous βsub2/sub-GPI expression. These results suggest that the atypical TATA box and HNF-1 icis/i-element are critical for βsubi2/i/sub-iGPI/i transcription and HNF-1α may play an important role in cell-specific regulation of βsubi2/i/sub-iGPI/i gene expression./p