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.
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.
Boonsaen
Thirajit
T
Department of Biochemistry, Faculty of Science, Mahidol University, Rama 6 Road, Bangkok 10400, Thailand.
Rojvirat
Pinnara
P
Surinya
Kathy H
KH
Wallace
John C
JC
Jitrapakdee
Sarawut
S
D000818:Animals; D001483:Base Sequence; D045744:Cell Line, Tumor; D024202:Electrophoretic Mobility Shift Assay; D005786:Gene Expression Regulation / Q000187:drug effects*; D051542:Hepatocyte Nuclear Factor 3-beta / Q000502:physiology*; D007340:Insulinoma; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010190:Pancreatic Neoplasms; D011401:Promoter Regions, Genetic / Q000187:drug effects* / Q000235:genetics; D011766:Pyruvate Carboxylase / Q000235:genetics*; D051381:Rats; D051720:Upstream Stimulatory Factors / Q000502:physiology*
C496504:Foxa2 protein, rat; D051720:Upstream Stimulatory Factors; C497182:Usf1 protein, rat; C497187:Usf2 protein, rat; D051542:Hepatocyte Nuclear Factor 3-beta; D011766:Pyruvate Carboxylase