Transcriptional regulation of the distal promoter of the rat pyruvate carboxylase gene by hepatocyte nuclear factor 3β/Foxa2 and upstream stimulatory factors in insulinoma cells

Abstract

PC (pyruvate carboxylase) plays a crucial role in intermediary metabolism including glucose-induced insulin secretion in pancreatic islets. In the present study, we identified two regions of the 1.2 kb distal promoter, the −803/−795 site and the −408/−403 E-box upstream of the transcription start site, as the important cis-acting elements for transcriptional activation of the luciferase reporter gene. Site-directed mutagenesis of either one of these sites in the context of this 1.2 kb promoter fragment, followed by transient transfections in the insulinoma cell line, INS-1, abolished reporter activity by approx. 50%. However, disruption of either the −803/−795 or the −408/−403 site did not affect reporter gene activity in NIH 3T3 cells, suggesting that this promoter fragment is subjected to cell-specific regulation. The nuclear proteins that bound to these −803/−795 and −408/−403 sites were identified by gel retardation assays as HNF3β (hepatocyte nuclear factor 3β)/Foxa2 (forkhead/winged helix transcription factor box2) and USFs (upstream stimulatory factors), USF1 and USF2, respectively. Chromatin immunoprecipitation assays using antisera against HNF3β/Foxa2, USF1 and USF2 demonstrated that endogenous HNF3β/Foxa2 binds to the −803/−795 Foxa2 site, and USF1 and USF2 bind to the −408/−403 E-box respectively in vivo, consistent with the gel retardation assay results. Although there are weak binding sites located at regions −904 and −572 for PDX1 (pancreatic duodenal homeobox-1), a transcription factor that controls expression of β-cell-specific genes, it did not appear to regulate PC expression in INS-1 cells in the context of the 1.2 kb promoter fragment. The results presented here show that Foxa2 and USFs regulate the distal promoter of the rat PC gene in a cell-specific manner.