title: Ref-1/Ape is critical for formation of the hypoxia-inducible transcriptional complex on the hypoxic response element of the rat pulmonary artery endothelial cell VEGF gene.
abstract: The co-transcription factor and DNA repair enzyme, Redox effector factor-1/apurinic/apyrimidinic endonuclease (Ref-1/Ape), facilitates DNA binding and transcriptional activity of a number of transactivating factors, including those governing hypoxia-induced gene expression HIF-1. It is not known, however, whether Ref-1/Ape is a component of the hypoxic transcriptional complex. Electrophoretic mobility shift assays failed to detect direct DNA binding of Ref-1/Ape to either the HIF-1 or AP1 DNA recognition sequences present in the hypoxic response element of the VEGF gene. However, immunodepletion of Ref-1/Ape from nuclear extract prevented DNA binding of ATF/CREB and HIF-1 to the HIF-1 DNA recognition sequence. DNA affinity-precipitation analyses showed that Ref-1/Ape was part of the multiprotein transcriptional complex forming on a 64-mer sequence encompassing a minimal hypoxic response element. Immunodepletion of Ref-1/Ape prevented probe association with HIF-1, p300, ATF, and CREB. Co-immunoprecipitation experiments indicated that Ref-1/Ape present in nuclear extract interacted with HIF-1 and p300 but not ATF/CREB. However, when Ref-1/Ape was immunoprecipitated from the oligonucleotide probe, both HIF-1 and p300 remained probe-associated while ATF/CREB co-immunoprecipitated. These findings suggest that Ref-1/Ape is a critical component of the hypoxia-inducible transcriptional complex forming on the VEGF gene's hypoxic response element and that the presence of Ref-1/Ape in the complex is required for the apparent high affinity association between HIF-1 and its DNA recognition sequence.
authors.affiliation: Department of Pharmacology, University of South Alabama College of Medicine, Mobile, Alabama 36688, USA.
authors.forename: Kathryn A
authors.initials: KA
authors.lastname: Ziel
authors.forename: Clayton C
authors.initials: CC
authors.lastname: Campbell
authors.forename: Glenn L
authors.initials: GL
authors.lastname: Wilson
authors.forename: Mark N
authors.initials: MN
authors.lastname: Gillespie
mesh_terms: D051697:Activating Transcription Factor 1; D000818:Animals; D001483:Base Sequence; D015687:Cell Hypoxia / Q000502:physiology*; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D042783:Endothelial Cells / Q000378:metabolism*; D051793:Hypoxia-Inducible Factor 1; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D046912:Multiprotein Complexes; D009687:Nuclear Proteins / Q000378:metabolism; D010100:Oxygen / Q000494:pharmacology; D011485:Protein Binding; D011651:Pulmonary Artery; D051381:Rats; D017207:Rats, Sprague-Dawley; D020218:Response Elements / Q000235:genetics*; D014157:Transcription Factors / Q000378:metabolism*; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics*
chemical_list: D051697:Activating Transcription Factor 1; D017362:Cyclic AMP Response Element-Binding Protein; D004268:DNA-Binding Proteins; C497444:Hif1a protein, rat; D051793:Hypoxia-Inducible Factor 1; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D046911:Macromolecular Substances; D046912:Multiprotein Complexes; D009687:Nuclear Proteins; D014157:Transcription Factors; D042461:Vascular Endothelial Growth Factor A; C470319:Apex1 protein, rat; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D010100:Oxygen