Sequence-specific oxidative base modifications in hypoxia-inducible genes.
Reactive oxygen species associated with hypoxic signaling in pulmonary arterial endothelial cells (PAECs) oxidatively modify specific nucleotides in the hypoxic response element (HRE) of the VEGF gene (FASEB J.19:387-394; 2005). In this study, we determined in PAECs if hypoxia caused genome-wide oxidative modifications or if they were restricted to the promoters of genes differentially regulated by hypoxia. Comet assays indicated that there were no differences between normoxic and hypoxic PAECs in terms of global DNA damage. However, a simple PCR-based method involving DNA amplification before and after treatment with formamidopyrimidine DNA glycosylase (Fpg), a bacterial DNA repair enzyme that cleaves at sites of purine base oxidation, revealed that hypoxia caused modifications in the HREs of the hypoxia-inducible VEGF, HO-1, and ET-1 genes which coincided with accumulation of their respective mRNA transcripts. Promoter sequences not involved with hypoxic induction and coding regions of these genes failed to display Fpg-sensitive sites. Oxidative modifications also were not detected in sequences of the hypoxia down-regulated ornithine decarboxylase and TFAM genes or the constitutively expressed beta-actin gene. These findings show that hypoxia-mediated oxidative DNA modifications cluster in functionally relevant promoter sequences in hypoxia-inducible genes and suggest that such oxidative modifications may be biologically significant.
Pastukh
Viktor
V
Department of Pharmacology and Center for Lung Biology, University of South Alabama College of Medicine, Mobile, AL 36688, USA.
Ruchko
Mykhaylo
M
Gorodnya
Olena
O
Wilson
Glenn L
GL
Gillespie
Mark N
MN
D000818:Animals; D001483:Base Sequence; D015687:Cell Hypoxia / Q000235:genetics* / Q000502:physiology*; D002478:Cells, Cultured; D004247:DNA / Q000737:chemistry / Q000235:genetics; D004249:DNA Damage; D017931:DNA Primers / Q000235:genetics; D042783:Endothelial Cells / Q000378:metabolism; D019332:Endothelin-1 / Q000235:genetics; D005786:Gene Expression Regulation; D006419:Heme Oxygenase (Decyclizing) / Q000235:genetics; D010084:Oxidation-Reduction; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D017382:Reactive Oxygen Species / Q000378:metabolism; D015398:Signal Transduction; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics
D017931:DNA Primers; D019332:Endothelin-1; D012333:RNA, Messenger; D017382:Reactive Oxygen Species; D042461:Vascular Endothelial Growth Factor A; C467498:vascular endothelial growth factor A, rat; D004247:DNA; D006419:Heme Oxygenase (Decyclizing); C491523:Hmox1 protein, rat