6-Phosphofructo-2-kinase (pfkfb3) gene promoter contains hypoxia-inducible factor-1 binding sites necessary for transactivation in response to hypoxia.
The up-regulation of glycolysis to enhance the production of energy under reduced pO(2) is a hallmark of the hypoxic response. A key regulator of glycolytic flux is fructose-2,6-bisphosphate, and its steady state concentration is regulated by the action of different isozymes product of four genes (pfkfb1-4). pfkfb3 has been found in proliferating cells and tumors, being induced by hypoxia. To understand the organization of cis-acting sequences that are responsible for the oxygen-regulated pfkfb3 gene, we have studied its 5'-flanking region. Extensive analysis of the 5' pfkfb3 promoter sequence revealed the presence of putative consensus binding sites for various transcription factors that could play an important role in pfkfb3 gene regulation. These DNA consensus sequences included estrogen receptor, hypoxia response element (HRE), early growth response, and specific protein 1 putative binding sites. Promoter deletion analysis as well as putative HREs sequences (wild type and mutated) fused to a c-fos minimal promoter unit constructs demonstrate that the sequence located from -1269 to -1297 relative to the start site is required for hypoxia-inducible factor 1 (HIF-1) induction. The effective binding of HIF-1 transcription factor to the HREs at -1279 and -1288 was corroborated by electrophoretic mobility shift assay and biotinylated oligonucleotide pull-down. In addition, HIF-1alpha null mouse embryo fibroblasts transfected with a full-length pfkfb3 promoter-luciferase reporter construct further demonstrated that HIF-1 protein was critically involved for hypoxia transactivation of this gene. Altogether, these results demonstrate that pfkfb3 is a hypoxia-inducible gene that is stimulated through HIF interaction with the consensus HRE site in its promoter region.
Obach
Mercè
M
Unitat de Bioquímica i Biologia Molecular, Departament de Ciències Fisiològiques II, Campus de Bellvitge, Universitat de Barcelona, Feixa Llarga s/n, Pavelló de Govern, E-08907 L'Hospitalet, Spain.
Navarro-Sabaté
Aurea
A
Caro
Jaime
J
Kong
Xianguo
X
Duran
Joan
J
Gómez
Marta
M
Perales
Jose Carlos
JC
Ventura
Francesc
F
Rosa
Jose Luis
JL
Bartrons
Ramon
R
D000818:Animals; D001665:Binding Sites; D019921:Biotinylation; D015153:Blotting, Western; D015687:Cell Hypoxia / Q000502:physiology*; D002460:Cell Line; D049109:Cell Proliferation; D003035:Cobalt / Q000737:chemistry; D003676:Deferoxamine / Q000737:chemistry; D004742:Enhancer Elements, Genetic; D005635:Fructosediphosphates / Q000378:metabolism; D017353:Gene Deletion; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D005998:Glycine / Q000737:chemistry; D006801:Humans; D000860:Hypoxia; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D007502:Iron Chelating Agents / Q000494:pharmacology; D008156:Luciferases / Q000378:metabolism; D051379:Mice; D008957:Models, Genetic; D009841:Oligonucleotides / Q000737:chemistry; D010100:Oxygen / Q000378:metabolism; D025481:Phosphofructokinase-2; D010957:Plasmids / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000235:genetics* / Q000502:physiology*; D034622:RNA Interference; D011960:Receptors, Estrogen; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012984:Software; D013997:Time Factors; D014157:Transcription Factors / Q000737:chemistry*; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D014162:Transfection; D015854:Up-Regulation
D005635:Fructosediphosphates; C497442:HIF1A protein, human; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D007502:Iron Chelating Agents; D009841:Oligonucleotides; D020033:Protein Isoforms; D011506:Proteins; D011960:Receptors, Estrogen; D014157:Transcription Factors; D003035:Cobalt; C027652:fructose 2,6-diphosphate; D008156:Luciferases; C107345:PFKFB3 protein, human; D025481:Phosphofructokinase-2; C018021:cobaltous chloride; D003676:Deferoxamine; D010100:Oxygen; D005998:Glycine