The Nrf2 transcription factor promotes survival following cellular insults that trigger oxidative damage. Nrf2 activity is opposed by the BTB/POZ domain protein Keap1. Keap1 is proposed to regulate Nrf2 activity strictly through its capacity to inhibit Nrf2 nuclear import. Recent work suggests that inhibition of Nrf2 may also depend upon ubiquitin-mediated proteolysis. To address the contribution of Keap1-dependent sequestration versus Nrf2 proteolysis, we identified the E3 ligase that regulates Nrf2 ubiquitination. We demonstrate that Keap1 is not solely a cytosolic anchor; rather, Keap1 is an adaptor that bridges Nrf2 to Cul3. We demonstrate that Cul3-Keap1 complexes regulate Nrf2 polyubiquitination both in vitro and in vivo. Inhibition of either Keap1 or Cul3 increases Nrf2 nuclear accumulation, leading to promiscuous activation of Nrf2-dependent gene expression. Our data demonstrate that Keap1 restrains Nrf2 activity via its capacity to target Nrf2 to a cytoplasmic Cul3-based E3 ligase and suggest a model in which Keap1 coordinately regulates both Nrf2 accumulation and access to target genes.The Nrf2 transcription factor regulates the expression of antioxidant genes following cellular insults that induce oxidative stress (2, 3, 4, 13). Under homeostatic conditions, Nrf2 remains in an inactive cytoplasmic form through association with the bricabrac, tramtrack, and broad complex (BTB) domain-containing protein Keap1 (17). In response to endoplasmic reticulum stress or oxidative stress, Nrf2-Keap1 dissociation is triggered and Nrf2 accumulates in the nucleus, where it forms an active heterodimeric transcription factor, inducing the transcription of target genes involved in redox homeostasis (5,6,14,15,17).Although it is clear that Keap1 can maintain Nrf2 in the cytoplasm, accumulation of many transcriptional regulators is also suppressed through the action of the 26S proteasome. Although it was initially thought that Nrf2 activation was strictly regulated through inhibition of nuclear import, increasing evidence indicates that Nrf2 protein levels are maintained at low levels through proteasome-mediated degradation (18,20,21,24,28). The fact that Keap1 has been implicated in both Nrf2 cytoplasmic sequestration and proteolysis suggests a model in which the regulation of Nrf2 activity is tightly regulated by proteolysis in the cytoplasmic compartment. Similar modes of regulation have been documented for other critical cellular regulators, such as p53 (9, 22) and cyclin D1 (8).In general, proteins are targeted to the 26S proteasome through the covalent attachment of polyubiquitin chains. Ubiquitin conjugation is mediated by the sequential activities of an E1 enzyme, which mediates the ATP-dependent activation of ubiquitin, an E2 ubiquitin-conjugating enzyme (Ubc), and an E3 ubiquitin ligase; E2 and E3 function to coordinate the transfer of ubiquitin to the substrate protein. In addition to functioning in ubiquitin transfer, E3 generally drives substrate specificity and has thus been of intense interest.The SCF liga...

HIF-2alpha promotes von Hippel-Lindau (VHL)-deficient renal clear cell carcinoma (RCC) tumorigenesis, while HIF-1alpha inhibits RCC growth. As HIF-1alpha antagonizes c-Myc function, we hypothesized that HIF-2alpha might enhance c-Myc activity. We demonstrate here that HIF-2alpha promotes cell-cycle progression in hypoxic RCCs and multiple other cell lines. This correlates with enhanced c-Myc promoter binding, transcriptional effects on both activated and repressed target genes, and interactions with Sp1, Miz1, and Max. Finally, HIF-2alpha augments c-Myc transformation of primary mouse embryo fibroblasts (MEFs). Enhanced c-Myc activity likely contributes to HIF-2alpha-mediated neoplastic progression following loss of the VHL tumor suppressor and influences the behavior of hypoxic tumor cells.

The division, differentiation, and function of stem cells and multipotent progenitors are influenced by complex signals in the microenvironment, including oxygen availability. Using a genetic "knock-in" strategy, we demonstrate that targeted replacement of the oxygen-regulated transcription factor HIF-1␣ with HIF-2␣ results in expanded expression of HIF-2␣-specific target genes including Oct-4, a transcription factor essential for maintaining stem cell pluripotency. We show that HIF-2␣, but not HIF-1␣, binds to the Oct-4 promoter and induces Oct-4 expression and transcriptional activity, thereby contributing to impaired development in homozygous Hif-2␣ KI/KI embryos, defective hematopoietic stem cell differentiation in embryoid bodies, and large embryonic stem cell (ES)-derived tumors characterized by altered cellular differentiation. Furthermore, loss of HIF-2␣ severely reduces the number of embryonic primordial germ cells, which require Oct-4 expression for survival and/or maintenance. These results identify Oct-4 as a HIF-2␣-specific target gene and indicate that HIF-2␣ can regulate stem cell function and/or differentiation through activation of Oct-4, which in turn contributes to HIF-2␣'s tumor promoting activity.[Keywords: HIF; hypoxia; HIF-2␣; Oct-4; VEGF; TGF-␣; stem cells; cancer] Supplemental material is available at http://www.genesdev.org.