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.
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.
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