Counter to traditional concepts, recent advances in DNA sequencing show that the majority of tissue-specific somatic mutations underlying cancer and other disorders reside not within coding regions, but in regulatory sequences. This conspicuous preponderance of somatic mutation in regulatory sequences may point to their probable cause; based on studies begun over a decade ago on Reactive Oxygen Species (ROS) as a signaling event in hypoxic pulmonary artery endothelial and smooth muscle cells (PAECs and PASMCs), we propose to test the hypothesis that a pathway of oxidative DNA damage and repair pathway targeted to specific promoter sequences contributes to transcriptional regulation in response to pathophysiologically-relevant stimuli. The overall goals of the proposed research are to determine in pulmonary arterial cells from rats and human subjects whether the DNA damage and repair pathway is of general significance to transcriptional regulation and to establish the mechanism linking BER to transcription complex assembly and gene expression.
Aim 1 will compare the pathway initiated by hypoxia to another stimulus incriminated in pulmonary vascular disease, estrogen (E2), testing the idea that hypoxia and E2 employ distinct mechanisms to activate the DNA damage and repair pathway of transcription.
The second Aim i s based on the recognition that accurate and expeditious completion of DNA repair is essential for maintenance of DNA integrity. But, is this true for the postulated DNA damage and repair mechanism of transcription localized to non-coding regulatory regions? While some observations emphasize the importance fully prosecuting of BER in transcriptional activation mediated by the pathway, other studies suggest that only the first enzyme in BER, Ogg1, is involved. Because understanding how BER contributes to oxidant DNA damage-related gene expression is important for defining its role in transcriptional dysregulation and somatic mutation, Aim 2 will determine if complete prosecution of BER is necessary for hypoxia- and E2- mediated transcription. The last Aim is predicated on the the need to establish whether the targeted DNA damage and repair pathway of transcription is operative in human cells, and assess whether its malfunction could contribute to transriptional dysregulation and/or acquisition of somatic mutations in human disease.
Aim 3 will therefore test the hypothesis that the genome-wide landscape of oxidative promoter modifications in normal human PAECs aligns predictably with transcriptional responses evoked by hypoxia and E2. These studies will also provide hypothesis-generating insight into the link between the damage and repair pathway to disease by comparing DNA damage landscapes in normal human PAECs to those in PAECs from patients with pulmonary arterial hypertension. Completion of this work will not only define a new epigenetic mechanism governing transcription, but will points to the prospect that malfunction of the pathway could be linked to disease-related transcriptional dysregulation and somatic mutation.
This study tests the new idea that oxygen radical-mediated damage and repair in regions of genes controlling gene expression is a normal part of physiological signaling. Validation of this hypothesis could have transformative implications; if DNA damage and repair in gene regulatory regions is shown to dictate gene expression, this will uncover an entirely new level of genetic control with the prospect of leading to new drugs to correct inadequate gene expression.
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