An oxidative DNA “damage” and repair mechanism localized in the VEGF promoter is important for hypoxia-induced VEGF mRNA expression

Oct 4, 2015American journal of physiology. Lung cellular and molecular physiology

A DNA damage and repair process in the VEGF gene helps increase VEGF mRNA during low oxygen

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Abstract

Hypoxia leads to the formation of 8-oxoguanine in hypoxia-inducible gene promoters.

Reactive oxygen species generated by mitochondria during hypoxia stimulate the accumulation of the transcription regulator Hif-1.
The presence of oxidative base modifications in hypoxia response elements affects the binding of Hif-1 to gene promoters.
Inhibition of base excision DNA repair enzymes results in increased oxidative base modifications and decreased VEGF mRNA expression.
8-oxoguanine accumulation in VEGF promoter regions is associated with the binding of Hif-1α and specific DNA repair enzymes.
The distribution of 8-oxoG is primarily localized to hypoxic promoters, indicating a significant difference from normoxic conditions.
Transcription of genes with decreased 8-oxoG during hypoxia is reduced, while those with increased 8-oxoG are elevated.

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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.

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An oxidative DNA “damage” and repair mechanism localized in the VEGF promoter is important for hypoxia-induced VEGF mRNA expression

Abstract

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