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

Biochem J. 2007 Jul 15;405(2):359-67. doi: 10.1042/BJ20070276.

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 HNF3beta (hepatocyte nuclear factor 3beta)/Foxa2 (forkhead/winged helix transcription factor box2) and USFs (upstream stimulatory factors), USF1 and USF2, respectively. Chromatin immunoprecipitation assays using antisera against HNF3beta/Foxa2, USF1 and USF2 demonstrated that endogenous HNF3beta/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 beta-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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Base Sequence
  • Cell Line, Tumor
  • Electrophoretic Mobility Shift Assay
  • Gene Expression Regulation / drug effects*
  • Hepatocyte Nuclear Factor 3-beta / physiology*
  • Insulinoma
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Pancreatic Neoplasms
  • Promoter Regions, Genetic / drug effects*
  • Promoter Regions, Genetic / genetics
  • Pyruvate Carboxylase / genetics*
  • Rats
  • Upstream Stimulatory Factors / physiology*

Substances

  • Foxa2 protein, rat
  • Upstream Stimulatory Factors
  • Usf1 protein, rat
  • Usf2 protein, rat
  • Hepatocyte Nuclear Factor 3-beta
  • Pyruvate Carboxylase