ABSTRACT
Physiological stimuli using reactive oxygen species (ROS) as second messengers caused nucleotide-specific base modifications in the hypoxic response element of the VEGF gene in lung vascular cells, with the 3' guanine of the HIF-1 DNA recognition sequence uniformly targeted. Modeling this effect by replacing the targeted guanine with an abasic site increased incorporation of HIF-1 and the bi-functional DNA repair enzyme and transcriptional coactivator, Ref-1/Ape1, into the transcriptional complex and engendered more robust reporter gene expression. Oxidants generated in the context of physiological signaling thus affect nuclear DNA integrity and may facilitate gene expression by optimizing DNA-protein interactions.
Subject(s)
DNA Damage , Gene Expression Regulation , Oxidants/metabolism , Signal Transduction/physiology , Angiotensin II/pharmacology , Animals , Base Sequence , Binding Sites , Cell Hypoxia , Cells, Cultured , DNA/metabolism , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/genetics , Endothelial Cells/metabolism , Guanine/chemistry , Hypoxia-Inducible Factor 1 , Hypoxia-Inducible Factor 1, alpha Subunit , Molecular Sequence Data , Nuclear Proteins/chemistry , Nuclear Proteins/genetics , Platelet-Derived Growth Factor/pharmacology , Polymerase Chain Reaction , Promoter Regions, Genetic/genetics , Pulmonary Artery/cytology , Rats , Rats, Sprague-Dawley , Reactive Oxygen Species/metabolism , Response Elements , Thrombin/pharmacology , Transcription Factors/chemistry , Transcription Factors/genetics , Transfection , Vascular Endothelial Growth Factor A/geneticsABSTRACT
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.
