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

• Published in: The FASEB journal Vol. 18; no. 9; p. 986
• Main Authors: Ziel, Kathryn A, Campbell, Clayton C, Wilson, Glenn L, Gillespie, Mark N
• Format: Journal Article
• Language: English
• Published: United States 01.06.2004
• Subjects: Activating Transcription Factor 1; Animals; Base Sequence; Cell Hypoxia - physiology; Cyclic AMP Response Element-Binding Protein - metabolism; DNA-(Apurinic or Apyrimidinic Site) Lyase - metabolism; DNA-Binding Proteins - metabolism; Electrophoretic Mobility Shift Assay; Endothelial Cells - metabolism; Hypoxia-Inducible Factor 1; Hypoxia-Inducible Factor 1, alpha Subunit; Macromolecular Substances; Molecular Sequence Data; Multiprotein Complexes; Nuclear Proteins - metabolism; Oxygen - pharmacology; Protein Binding; Pulmonary Artery; Rats; Rats, Sprague-Dawley; Response Elements - genetics; Transcription Factors - metabolism; Vascular Endothelial Growth Factor A - genetics
• ISSN: 1530-6860
• DOI: 10.1096/fj.03-1160fje

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.