Publication: 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
Issued Date
2007-07-15
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ISSN
02646021
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2-s2.0-34447116301
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Mahidol University
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SCOPUS
Bibliographic Citation
Biochemical Journal. Vol.405, No.2 (2007), 359-367
Suggested Citation
Thirajit Boonsaen, Pinnara Rojvirat, Kathy H. Surinya, John C. Wallace, Sarawut Jitrapakdee 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. Biochemical Journal. Vol.405, No.2 (2007), 359-367. doi:10.1042/BJ20070276 Retrieved from: https://repository.li.mahidol.ac.th/handle/20.500.14594/24162
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Title
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
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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. © 2007 Biochemical Society.
