Characterization of the human liver fructose-1,6-bisphosphatase gene promoter.
Fructose-1,6-bisphosphatase (FBPase; EC 3.1.3.11), an important gluconeogenic enzyme, catalyses the hydrolysis of fructose 1, 6-bisphosphate to fructose 6-phosphate and P(i). Enzyme activity is mainly regulated by the allosteric inhibitors fructose 2, 6-bisphosphate and AMP. Although some observations about hormonal regulation of the enzyme have been published, the FBPase promoter has not been studied in detail. Here we report an in vitro characterization of the FBPase promoter with respect to the elements that are required for basal promoter activity. Transient transfection of H4IIE rat hepatoma cells, combined with site-directed mutagenesis, demonstrated that an enhancer box, three GC-boxes and a nuclear factor kappaB (NF-kappaB)-binding element are important for hepatic FBPase promoter activity. These elements are found in the region located between -405 to +25 bp relative to the transcription start site. Electrophoretic-mobility-shift assays and supershift analysis confirmed that upstream stimulatory factor 1 (USF1)/USF2, specificity protein 1 (Sp1)/Sp3 and NF-kappaB respectively bind to these sites. The present study provides the basis for a more comprehensive screening for mutations in FBPase-deficient patients and for further studies of the transcriptional regulation of this gene.
Herzog
B
B
Institute of Biochemistry, School of Medicine, University of Leipzig, Liebigstrasse 16, D-04103 Leipzig, Germany.
Waltner-Law
M
M
Scott
D K
DK
Eschrich
K
K
Granner
D K
DK
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D006528:Carcinoma, Hepatocellular / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D004586:Electrophoresis; D004742:Enhancer Elements, Genetic; D006597:Fructose-Bisphosphatase / Q000235:genetics*; D017353:Gene Deletion; D017930:Genes, Reporter; D006801:Humans; D006868:Hydrolysis; D008099:Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic; D020033:Protein Isoforms; D051381:Rats; D016329:Sp1 Transcription Factor / Q000378:metabolism; D051705:Sp3 Transcription Factor; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic; D014162:Transfection; D014407:Tumor Cells, Cultured; D051720:Upstream Stimulatory Factors
D004268:DNA-Binding Proteins; D016328:NF-kappa B; D020033:Protein Isoforms; C497121:SP3 protein, human; D016329:Sp1 Transcription Factor; C497123:Sp3 protein, rat; D014157:Transcription Factors; C497181:USF1 protein, human; C497184:USF2 protein, human; D051720:Upstream Stimulatory Factors; C497182:Usf1 protein, rat; C497187:Usf2 protein, rat; D051705:Sp3 Transcription Factor; D006597:Fructose-Bisphosphatase