title: An oxidative DNA "damage" and repair mechanism localized in the VEGF promoter is important for hypoxia-induced VEGF mRNA expression.
abstract: In hypoxia, mitochondria-generated reactive oxygen species not only stimulate accumulation of the transcriptional regulator of hypoxic gene expression, hypoxia inducible factor-1 (Hif-1), but also cause oxidative base modifications in hypoxic response elements (HREs) of hypoxia-inducible genes. When the hypoxia-induced base modifications are suppressed, Hif-1 fails to associate with the HRE of the VEGF promoter, and VEGF mRNA accumulation is blunted. The mechanism linking base modifications to transcription is unknown. Here we determined whether recruitment of base excision DNA repair (BER) enzymes in response to hypoxia-induced promoter modifications was required for transcription complex assembly and VEGF mRNA expression. Using chromatin immunoprecipitation analyses in pulmonary artery endothelial cells, we found that hypoxia-mediated formation of the base oxidation product 8-oxoguanine (8-oxoG) in VEGF HREs was temporally associated with binding of Hif-1α and the BER enzymes 8-oxoguanine glycosylase 1 (Ogg1) and redox effector factor-1 (Ref-1)/apurinic/apyrimidinic endonuclease 1 (Ape1) and introduction of DNA strand breaks. Hif-1α colocalized with HRE sequences harboring Ref-1/Ape1, but not Ogg1. Inhibition of BER by small interfering RNA-mediated reduction in Ogg1 augmented hypoxia-induced 8-oxoG accumulation and attenuated Hif-1α and Ref-1/Ape1 binding to VEGF HRE sequences and blunted VEGF mRNA expression. Chromatin immunoprecipitation-sequence analysis of 8-oxoG distribution in hypoxic pulmonary artery endothelial cells showed that most of the oxidized base was localized to promoters with virtually no overlap between normoxic and hypoxic data sets. Transcription of genes whose promoters lost 8-oxoG during hypoxia was reduced, while those gaining 8-oxoG was elevated. Collectively, these findings suggest that the BER pathway links hypoxia-induced introduction of oxidative DNA modifications in promoters of hypoxia-inducible genes to transcriptional activation.
authors.affiliation: Department of Pharmacology and Center for Lung Biology, University of South Alabama College of Medicine, Mobile, Alabama; and.
authors.forename: Viktor
authors.initials: V
authors.lastname: Pastukh
authors.affiliation: Department of Biology, College of Arts and Sciences, University of South Alabama, Mobile, Alabama.
authors.forename: Justin T
authors.initials: JT
authors.lastname: Roberts
authors.affiliation: Department of Pharmacology and Center for Lung Biology, University of South Alabama College of Medicine, Mobile, Alabama; and.
authors.forename: David W
authors.initials: DW
authors.lastname: Clark
authors.affiliation: Department of Pharmacology and Center for Lung Biology, University of South Alabama College of Medicine, Mobile, Alabama; and.
authors.forename: Gina C
authors.initials: GC
authors.lastname: Bardwell
authors.affiliation: Department of Pharmacology and Center for Lung Biology, University of South Alabama College of Medicine, Mobile, Alabama; and.
authors.forename: Mita
authors.initials: M
authors.lastname: Patel
authors.affiliation: Department of Pharmacology and Center for Lung Biology, University of South Alabama College of Medicine, Mobile, Alabama; and.
authors.forename: Abu-Bakr
authors.initials: AB
authors.lastname: Al-Mehdi
authors.affiliation: Department of Pharmacology and Center for Lung Biology, University of South Alabama College of Medicine, Mobile, Alabama; and Department of Biology, College of Arts and Sciences, University of South Alabama, Mobile, Alabama.
authors.forename: Glen M
authors.initials: GM
authors.lastname: Borchert
authors.affiliation: Department of Pharmacology and Center for Lung Biology, University of South Alabama College of Medicine, Mobile, Alabama; and mgillesp@southalabama.edu.
authors.forename: Mark N
authors.initials: MN
authors.lastname: Gillespie
mesh_terms: D000818:Animals; D001665:Binding Sites; D015687:Cell Hypoxia / Q000235:genetics; D047369:Chromatin Immunoprecipitation; D004249:DNA Damage / Q000235:genetics*; D004260:DNA Repair / Q000235:genetics*; D042783:Endothelial Cells / Q000378:metabolism; D005786:Gene Expression Regulation; D006147:Guanine / Q000031:analogs & derivatives / Q000378:metabolism; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000378:metabolism; D059372:Nucleotide Motifs; D010084:Oxidation-Reduction; D011401:Promoter Regions, Genetic; D011651:Pulmonary Artery / Q000473:pathology; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism; D051381:Rats; D020218:Response Elements / Q000235:genetics; D013997:Time Factors; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics* / Q000378:metabolism
chemical_list: D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D042461:Vascular Endothelial Growth Factor A; C024829:8-hydroxyguanine; D006147:Guanine
keywords: DNA repair; hypoxia; reactive oxygen species; transcriptional regulation