HIF-2: Standing Guard at the Crossroads of Stress and Aging



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The capacity of mammalian organisms to cope with hypoxic or ischemic stress is in part mediated by stress-induced transcription factors. Hypoxia-induced mediators include transcription factors, such as the α (alpha) subunit of Hypoxia inducible factors (HIF-1alpha and HIF-2 alpha). HIF-1 alpha and HIF-2 alpha have similar structural organization, and after forming an obligate heterodimer with the common partner ARNT/HIF-1 alpha, bind to the same recognition element located in target gene promoter or enhancer regions. However, despite these similarities, HIF-1 alpha and HIF-2 alpha regulate distinct target genes. In previous studies from the Garcia laboratory using mouse knockout studies, we demonstrated the importance of HIF-2 alpha in the in vivo regulation of genes involved in the cellular response to hypoxic and oxidative stress. These genes include Erythropoietin (epo), vascular endothelial cell growth factor (Vegf), superoxide dismutase 2 (Sod2) and other genes encoding major antioxidant enzymes (AOE). Novel roles for HIF-2 alpha have been found not only in hematopoiesis, but also in the control of reactive oxygen species and mitochondrial homeostasis. The molecular mechanism by which HIF-2 alpha selectively regulates its target genes remains an exciting area of research. In the first part of my thesis, I identified a novel molecular mechanism regulating activity of the enhancer region in the Epo gene. First, by using bioinformatics to perform an unbiased sequence comparison of several mammalian 3 prime Epo enhancer region, we identified a previously unrecognized evolutionary conserved region. Second, we determined the functional significance of these conserved sequences using transient transfection and mutation analyses in cell culture studies and determined that these sequences contribute to HIF-2 alpha selectivity. Finally, using a candidate factor strategy, we determined that members of the early growth response (Egr) transcription factor family bind to these elements and act synergistically with HIF-2 alpha to augment Epo gene expression. In the second part of my thesis, we demonstrate that the redox-sensing, NAD+ dependent deacetylase enzyme Sirtuin 1, also known as Sirt1 or silent mating type information regulator 2 (Sir2) homolog 1, selectively stimulates HIF-2 alpha signaling during hypoxia. In lower organisms and cell culture models, the FoxO family of transcription factor regulates the transcription of SOD2 and other major AOE. During oxidative stress, Sirt1 modulates FoxO transcriptional activity, promoting the protective cellular response to oxidative stress. We hypothesized that Sirt1 would be activated by redox changes induced by hypoxia and that activated Sirt1 would in turn modulate HIF-2 signaling. We determined that HIF-2 alpha signaling is indeed increased by Sirt1 in transfection assays. Sirt1/HIF-2 alpha signaling does not involve previously described oxygen-dependent HIF-2 alpha modifications. Sirt1 augmentation of HIF-2 alpha transcriptional activity involves direct binding to and deacetylation of HIF-2 alpha. In cultured cells and in mice models, interventions that decrease or increase Sirt1 activity affect expression of the HIF-2 alpha target gene epo accordingly. Thus, Sirt1 is a molecular switch that promotes HIF-2 signaling during hypoxia and likely other environmental stresses.

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