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. 2015 Dec 1;309(11):L1367-75.
doi: 10.1152/ajplung.00236.2015. Epub 2015 Oct 2.

An Oxidative DNA "Damage" and Repair Mechanism Localized in the VEGF Promoter Is Important for Hypoxia-Induced VEGF mRNA Expression

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An Oxidative DNA "Damage" and Repair Mechanism Localized in the VEGF Promoter Is Important for Hypoxia-Induced VEGF mRNA Expression

Viktor Pastukh et al. Am J Physiol Lung Cell Mol Physiol. .
Free PMC article

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.

Keywords: DNA repair; hypoxia; reactive oxygen species; transcriptional regulation.

Figures

Fig. 1.
Fig. 1.
Temporal relationships between hypoxia (HYP)-induced VEGF mRNA expression (A) and formation of 8-oxoguanine (B), association of HYP inducible factor-1α (Hif-1α; C), the base excision DNA repair (BER) enzymes 8-oxoguanine glycosylase 1 (Ogg1; D), redox effector factor-1 (Ref-1)/apurinic/apyrimidinic endonuclease 1 (Ape1) (E), and formation of strand breaks (F) in the VEGF HYP response element (HRE). Values are means ± SE; N = 4–6 for each time point. *Different from normoxia (NORM) at P < 0.05.
Fig. 2.
Fig. 2.
Two-step chromatin immunoprecipitation (ChIP) analysis of Hif-1α with HRE sequences simultaneously associating with either Ogg1 or Ref-1/Ape1. The first immunoprecipitation step detected VEGF HRE sequences associating with either Hif-1α, Ogg1, or Ref-1/Ape1, whereas the second step determined whether Hif-1α colocalized on HRE sequences simultaneously associating with either Ogg1 or Ref-1/Ape1. Note the presence of Hif-1α on DNA sequences immunoprecipitating with an antibody to Ref-1/Ape1, but not with an antibody to Ogg1. Gel images are representative of three experiments. N, NORM; H, HYP.
Fig. 3.
Fig. 3.
A: abundance of 8-oxoguanine detected by ChIP analysis of the VEGF HRE from NORM and HYP pulmonary artery endothelial cells (PAECs) treated with transfection agent alone (TA), or small interfering RNA (siRNA) to affect Ogg1 depletion. B: HYP-induced, redox-sensitive green fluorescent protein (roGFP)-detected oxidant stress expressed as a function of the NORM baseline (designated as “time 0”) in TA-treated and Ogg1-depleted (siRNA) PAECs. C: abundance of Hif-1α detected by Western immunoblot analysis in NORM and HYP cells treated with TA alone or with siRNA to deplete Ogg1. Representative Western blots are shown at the top, and pooled data are depicted at the bottom. D: representative DNA affinity precipitation (DNAP) and Western immunoblot analysis for Hif-1α present in nuclear extract derived from TA-treated or Ogg1-depleted PAECs binding to an oligonucleotide model of the VEGF HRE. Note that Ogg1 depletion is associated with a rise in HYP-induced 8-oxoguanine formation in the VEGF HRE, but fails to impair HYP-induced oxidant stress, Hif-1α abundance, or Hif-1α binding to the VEGF HRE oligonucleotide. Values are means ± SE. With the exception of the DNA affinity/Western immunoblot analysis, which was representative of 3 experiments, N = 4–6 for each data set. *Different from NORM at P < 0.05. **Different from HYP and NORM at P < 0.05.
Fig. 4.
Fig. 4.
A: effect of treatment with TA or siRNA to deplete Ogg1 (siRNA) on VEGF mRNA expression in NORM and HYP PAECs, as assessed by quantitative RT-PCR. ChIP analyses are shown of Hif-1α (B) and Ref-1/Ape1 (C) association with the VEGF HRE in NORM and HYP cells treated with TA or depleted of Ogg1 using siRNA. Values are means ± SE; N = 4–6 for each experimental group. *Different from NORM at P < 0.05.
Fig. 5.
Fig. 5.
A: pie chart depicting ChIP-seq analysis of distribution of 8-oxoguanine (8-oxoG) between promoter and coding regions in genes from NORM and HYP PAECs. Nos. denote the no. of promoter or coding regions harboring 8-oxoG. Note particularly the preponderance of 8-oxoG in promoter regions and the fact that there were very few promoter or coding common to both NORM or HYP data sets. Data are representative of 3 separate ChIP-seq analyses. B: scatter diagram depicting relation between genes that acquired or lost 8-oxoG in HYP and gene expression in HYP. *The two data sets differed significantly at P < 0.05, as determined by the modified Kolmogorov-Smirnov test.
Fig. 6.
Fig. 6.
Proposed mechanism whereby HYP-induced formation and repair of oxidative base modifications in regulatory sequences govern the rate of transcriptional cycling and thereby mRNA abundance. When the density of regulatory sequence base damage is elevated by HYP-induced oxidative stress, BER enzymes are recruited to the sequence at a greater rate to restore and retain normal sequence integrity. In contrast, in the absence of ROS stress targeted at regulatory sequences, the rate of DNA repair enzyme recruitment to the sequence is lower. The rate of DNA damage and BER-mediated repair is envisioned to be a determinant of transcription complex assembly/stability and thereby directly impact the rate of transcriptional cycling and mRNA accumulation. See text for additional details.

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