Hypoxia, an important stimulus in respiratory biology and medicine, uses reactive oxygen species (ROS) in signaling. ROS generated in hypoxia serve to stabilize the key transcription factor regulating hypoxia- induced gene expression, HIF-1. This application is founded on our discovery that, surprisingly, ROS generated in hypoxia cause transient oxidative DNA modifications that cluster in the hypoxic response elements (HREs) of multiple hypoxia-inducible genes, with specific bases near the HIF-1-DNA recognition sequence conspicuously targeted. Hypoxia-induced oxidative base modifications are removed by the Base Excision DNA Repair (BER) pathway in a step-wise process requiring formation and re-ligation of DNA strand breaks. Herein we propose to test the hypothesis that targeted DNA strand breaks in the HREs of the VEGF and ET-1 promoters, occurring as a consequence of BER-mediated repair of hypoxia-induced oxidative base damage, enable binding of transcription factors and chromatin remodeling enzymes. In the resting state, the close apposition between promoter DNA and nucleosome core particles physically obstructs transcription factor binding. It is evident that transcriptional activation requires the association between promoter DNA and nucleosomes to be altered such that the steric hindrance to transcription factor binding is removed, but it remains unclear just how this happens. One model holds that transcriptional regulators must first bind to DNA to initiate nucleosome repositioning. Alternatively, it is also possible that promoter DNA-nucleosome contacts must somehow be relaxed or loosened prior to assembly of transcriptional regulators on the responsive DNA sequence. Provocative observations made during the current award support the latter concept: We believe that strand breaks formed during BER-mediated repair of hypoxia-induced oxidative base modifications function to relax contacts between HREs and nucleosomes, thereby enhancing accessibility of the sequence to binding by transcriptional regulators. To test key elements of this hypothesis, we will use pulmonary artery endothelial cells as the model system and manipulate the strand break-forming and break re-ligating steps of the BER pathway. We will then monitor hypoxia-induced strand breaks and base oxidation products in the VEGF and ET-1 HREs, and define the outcome in terms of (1) transcriptional complex assembly and (2) binding of selected chromatin remodeling enzymes. Our concept that controlled DNA damage and repair govern gene expression in hypoxia has significant and potentially transformative implications: It reveals a fundamentally new mechanism of ROS-dependent gene expression linking physiologic signaling to the genomic instability characteristic of aging, cancer, and other diseases. The proposed research also points to previously unappreciated mechanisms of gene dysregulation;if DNA repair is required for normal transcription, then it follows that defective repair leads to transcriptional malfunction.

Public Health Relevance

Hypoxia is an important stimulus in respiratory biology and medicine, and much effort has been devoted to defining mechanisms of hypoxia-induced gene expression. This research, which tests the concept that controlled oxidant-mediated DNA damage and repair govern gene expression in hypoxia, has significant and potentially transformative implications: It reveals a fundamentally new mechanism of oxygen radical-dependent gene expression linking physiologic signaling to the genomic instability characteristic of aging, cancer, and other diseases. The proposed research also has practical ramifications;if DNA repair is required for normal gene expression, then it follows that defective DNA repair leads to dysregulated gene expression.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL058234-12
Application #
8307785
Study Section
Respiratory Integrative Biology and Translational Research Study Section (RIBT)
Program Officer
Moore, Timothy M
Project Start
1998-09-30
Project End
2014-07-31
Budget Start
2012-08-01
Budget End
2013-07-31
Support Year
12
Fiscal Year
2012
Total Cost
$367,538
Indirect Cost
$120,038
Name
University of South Alabama
Department
Pharmacology
Type
Schools of Medicine
DUNS #
172750234
City
Mobile
State
AL
Country
United States
Zip Code
36688
Pastukh, Viktor M; Gorodnya, Olena M; Gillespie, Mark N et al. (2016) Regulation of mitochondrial genome replication by hypoxia: The role of DNA oxidation in D-loop region. Free Radic Biol Med 96:78-88
Pastukh, Viktor; Roberts, Justin T; Clark, David W et al. (2015) An oxidative DNA ""damage"" and repair mechanism localized in the VEGF promoter is important for hypoxia-induced VEGF mRNA expression. Am J Physiol Lung Cell Mol Physiol 309:L1367-75
Yang, Xi-Ming; Cui, Lin; White, James et al. (2015) Mitochondrially targeted Endonuclease III has a powerful anti-infarct effect in an in vivo rat model of myocardial ischemia/reperfusion. Basic Res Cardiol 110:3
Kuck, Jamie L; Obiako, Boniface O; Gorodnya, Olena M et al. (2015) Mitochondrial DNA damage-associated molecular patterns mediate a feed-forward cycle of bacteria-induced vascular injury in perfused rat lungs. Am J Physiol Lung Cell Mol Physiol 308:L1078-85
Simmons, Jon D; Gillespie, Mark N (2015) Plasma nuclear and mitochondrial DNA levels in acute myocardial infarction patients. Coron Artery Dis 26:286-8
Lee, Yann-Leei; King, Madelyn B; Gonzalez, Richard P et al. (2014) Blood transfusion products contain mitochondrial DNA damage-associated molecular patterns: a potential effector of transfusion-related acute lung injury. J Surg Res 191:286-9
Schumacker, Paul T; Gillespie, Mark N; Nakahira, Kiichi et al. (2014) Mitochondria in lung biology and pathology: more than just a powerhouse. Am J Physiol Lung Cell Mol Physiol 306:L962-74
Hashizume, Masahiro; Mouner, Marc; Chouteau, Joshua M et al. (2013) Mitochondrial-targeted DNA repair enzyme 8-oxoguanine DNA glycosylase 1 protects against ventilator-induced lung injury in intact mice. Am J Physiol Lung Cell Mol Physiol 304:L287-97
Simmons, Jon D; Lee, Yann-Leei; Mulekar, Sujata et al. (2013) Elevated levels of plasma mitochondrial DNA DAMPs are linked to clinical outcome in severely injured human subjects. Ann Surg 258:591-6; discussion 596-8
Gillespie, Mark N; Al-Mehdi, Abu-Bakr; McMurtry, Ivan F (2013) Mitochondria in hypoxic pulmonary vasoconstriction: potential importance of compartmentalized reactive oxygen species signaling. Am J Respir Crit Care Med 187:338-40

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