Title: Hypoxia-Inducible Factors, Stem Cells, and Cancer
Abstract: Regions of severe oxygen deprivation (hypoxia) arise in tumors due to rapid cell division and aberrant blood vessel formation. The hypoxia-inducible factors (HIFs) mediate transcriptional responses to localized hypoxia in normal tissues and in cancers and can promote tumor progression by altering cellular metabolism and stimulating angiogenesis. Recently, HIFs have been shown to activate specific signaling pathways such as Notch and the expression of transcription factors such as Oct4 that control stem cell self renewal and multipotency. As many cancers are thought to develop from a small number of transformed, self-renewing, and multipotent “cancer stem cells,” these results suggest new roles for HIFs in tumor progression. Regions of severe oxygen deprivation (hypoxia) arise in tumors due to rapid cell division and aberrant blood vessel formation. The hypoxia-inducible factors (HIFs) mediate transcriptional responses to localized hypoxia in normal tissues and in cancers and can promote tumor progression by altering cellular metabolism and stimulating angiogenesis. Recently, HIFs have been shown to activate specific signaling pathways such as Notch and the expression of transcription factors such as Oct4 that control stem cell self renewal and multipotency. As many cancers are thought to develop from a small number of transformed, self-renewing, and multipotent “cancer stem cells,” these results suggest new roles for HIFs in tumor progression. Solid tumors are known to contain poorly vascularized regions characterized by severe hypoxia (oxygen deprivation), low pH, and nutrient starvation (Carmeliet and Jain, 2000Carmeliet P. Jain R.K. Angiogenesis in cancer and other diseases.Nature. 2000; 407: 249-257Crossref PubMed Scopus (7002) Google Scholar, Pouyssegur et al., 2006Pouyssegur J. Dayan F. Mazure N.M. Hypoxia signalling in cancer and approaches to enforce tumour regression.Nature. 2006; 441: 437-443Crossref PubMed Scopus (1258) Google Scholar). Tumor hypoxia is typically associated with poor patient prognosis, partly because low oxygen levels reduce the effectiveness of radiation therapy, which kills tumor cells by generating reactive oxygen species. Over the past decade, work from many laboratories has indicated that hypoxic microenvironments contribute to cancer progression by activating adaptive transcriptional programs that promote cell survival, motility, and tumor angiogenesis. Recent reports describing molecular connections between oxygen-regulated transcription factors and pathways known to control stem cell function suggest a new mechanism whereby hypoxia-induced transcription factors may drive tumor growth through the generation or expansion of tumor-initiating cells or cancer stem cells. Here, we discuss how these results add an important new facet to our traditional view of hypoxia and cancer. Many of the cellular responses to hypoxia are mediated through changes in gene expression. The transcription factors primarily responsible for these changes are the hypoxia-inducible factors (HIFs), the biology of which has been reviewed elsewhere (Pouyssegur et al., 2006Pouyssegur J. Dayan F. Mazure N.M. Hypoxia signalling in cancer and approaches to enforce tumour regression.Nature. 2006; 441: 437-443Crossref PubMed Scopus (1258) Google Scholar, Semenza, 2003Semenza G.L. Targeting HIF-1 for cancer therapy.Nat. Rev. Cancer. 2003; 3: 721-732Crossref PubMed Scopus (4846) Google Scholar). Briefly, HIFs are members of the bHLH-PAS family of proteins and bind to canonical DNA sequences (hypoxia regulated elements, or HREs) in the promoters or enhancers of target genes. They consist of an α (HIF-α) and a β (HIF-β, or ARNT) subunit and activate the expression of at least 150 genes encoding proteins that regulate cell metabolism, survival, motility, basement membrane integrity, angiogenesis, hematopoiesis, and other functions. Regulation of HIF activity is mediated primarily through the stability of the α subunit: under conditions of abundant oxygen (>8%–10%), HIF-α proteins are translated but rapidly degraded. HIF-α degradation is triggered by the hydroxylation of two key proline residues in its highly conserved oxygen-dependent degradation domain (ODD). These hydroxylation events, catalyzed by specific proline hydroxylase (PHD) enzymes, are necessary and sufficient for binding to the Von Hippel-Lindau tumor suppressor protein (pVHL), the recognition component of an E3 ubiquitin ligase that targets the HIFs to the 26S proteasome for destruction. As oxygen levels decrease below 8%–10%, HIF-α proteins become increasingly stabilized, although the nature of the oxygen-sensing mechanisms regulating these events remains controversial. Once stabilized, HIF-α proteins bind to constitutively expressed ARNT (HIF-β) subunits in the nucleus, bind to DNA, and activate transcription through interactions with coactivators, including CBP/p300. Interestingly, binding to CBP/p300 is regulated by hydroxylation of a conserved asparagine residue in the HIF-α C-terminal domain (Pouyssegur et al., 2006Pouyssegur J. Dayan F. Mazure N.M. Hypoxia signalling in cancer and approaches to enforce tumour regression.Nature. 2006; 441: 437-443Crossref PubMed Scopus (1258) Google Scholar). HIF-1α and HIF-2α share a high degree of sequence identity, underscored by their shared ability to heterodimerize with ARNT and bind to HREs to activate transcription of common, as well as some unique, target genes (Raval et al., 2005Raval R.R. Lau K.W. Tran M.G. Sowter H.M. Mandriota S.J. Li J.L. Pugh C.W. Maxwell P.H. Harris A.L. Ratcliffe P.J. Contrasting properties of hypoxia-inducible factor 1 (HIF-1) and HIF-2 in von Hippel-Lindau-associated renal cell carcinoma.Mol. Cell. Biol. 2005; 25: 5675-5686Crossref PubMed Scopus (707) Google Scholar and references therein). Whereas HIF-1α is expressed in an apparently ubiquitous fashion, HIF-2α expression is restricted to particular cell types, including vascular endothelial cells, neural crest cell derivatives, lung type II pneumocytes, liver parenchyma, cardiomyocytes, and interstitial cells in the kidney (Wiesener et al., 2003Wiesener M.S. Jurgensen J.S. Rosenberger C. Scholze C.K. Horstrup J.H. Warnecke C. Mandriota S. Bechmann I. Frei U.A. Pugh C.W. et al.Widespread hypoxia-inducible expression of HIF-2alpha in distinct cell populations of different organs.FASEB J. 2003; 17: 271-273Crossref PubMed Scopus (510) Google Scholar). Genetic ablation experiments in mice have demonstrated that all HIF subunits tested to date are essential for embryonic development and survival. These analyses have led to the view that oxygen gradients develop as a function of limited diffusion in rapidly growing tissues. The inability to mount proper transcriptional responses to physiological hypoxia in HIF-deficient embryos results in developmental arrest and death. The specific phenotypes observed in mutant embryos differ depending on which HIF subunit is mutated, but alterations in cell survival, differentiation, and tissue angiogenesis have been reported in mice lacking ARNT, HIF-1α, or HIF-2α (Ramirez-Bergeron and Simon, 2001Ramirez-Bergeron D.L. Simon M.C. Hypoxia-inducible factor and the development of stem cells of the cardiovascular system.Stem Cells. 2001; 19: 279-286Crossref PubMed Scopus (85) Google Scholar). In contrast to the exquisitely regulated HIF activation observed in embryos, the highly disorganized vascular supply of solid tumors typically produces regions of severe hypoxia or anoxia closely abutting well-oxygenated areas (Pouyssegur et al., 2006Pouyssegur J. Dayan F. Mazure N.M. Hypoxia signalling in cancer and approaches to enforce tumour regression.Nature. 2006; 441: 437-443Crossref PubMed Scopus (1258) Google Scholar). The consequent stabilization of HIF proteins in hypoxic cancer cells is thought to promote tumor progression, in large part by inducing the localized expression of specific target genes encoding vascular endothelial growth factor (VEGF), glycolytic enzymes (PGK, ALDA), glucose transporters (GLUT1), and proteins regulating motility (lysl oxidase) and metastasis (CXCR4, E-cadherin), among others (Figure 1) (Semenza, 2003Semenza G.L. Targeting HIF-1 for cancer therapy.Nat. Rev. Cancer. 2003; 3: 721-732Crossref PubMed Scopus (4846) Google Scholar). Many tumor studies support this view: for example, subcutaneous fibrosarcomas generated from HIF-1α deficient, Ras-transformed murine embryonic fibroblasts (MEFs) grew more slowly than their HIF-replete controls (Ryan et al., 2000Ryan H.E. Poloni M. McNulty W. Elson D. Gassmann M. Arbeit J.M. Johnson R.S. Hypoxia-inducible factor-1alpha is a positive factor in solid tumor growth.Cancer Res. 2000; 60: 4010-4015PubMed Google Scholar). Similar xenograft experiments with ARNT-deficient hepatoma cells also showed a clear decrease in tumor growth compared to ARNT-expressing counterparts (Maxwell et al., 1997Maxwell P.H. Dachs G.U. Gleadle J.M. Nicholls L.G. Harris A.L. Stratford I.J. Hankinson O. Pugh C.W. Ratcliffe P.J. Hypoxia-inducible factor-1 modulates gene expression in solid tumors and influences both angiogenesis and tumor growth.Proc. Natl. Acad. Sci. USA. 1997; 94: 8104-8109Crossref PubMed Scopus (904) Google Scholar). HIF activity can also be induced or enhanced in some transformed cells through oxygen-independent oncogenic signaling pathways, including those regulated by IGF2/IGF1R, TGF-α/EGFR, and PI3K/Akt (Semenza, 2003Semenza G.L. Targeting HIF-1 for cancer therapy.Nat. Rev. Cancer. 2003; 3: 721-732Crossref PubMed Scopus (4846) Google Scholar). Expression of the HIF-α proteins in human tumor cells often correlates with a poor prognosis: for example, high-grade pediatric astrocytomas display greater HIF-2α expression than low-grade astrocytomas (Khatua et al., 2003Khatua S. Peterson K.M. Brown K.M. Lawlor C. Santi M.R. LaFleur B. Dressman D. Stephan D.A. MacDonald T.J. Overexpression of the EGFR/FKBP12/HIF-2alpha pathway identified in childhood astrocytomas by angiogenesis gene profiling.Cancer Res. 2003; 63: 1865-1870PubMed Google Scholar). Interestingly, HIF-1α and HIF-2α share some target genes, including those encoding VEGF, GLUT1, and ADM-1; in contrast, genes encoding glycolytic enzymes (PGK1, ALDA) are unique HIF-1α targets, and those encoding TGF-α and cyclin D1 appear to be unique HIF-2α targets, at least in certain cell types (Figure 1) (Raval et al., 2005Raval R.R. Lau K.W. Tran M.G. Sowter H.M. Mandriota S.J. Li J.L. Pugh C.W. Maxwell P.H. Harris A.L. Ratcliffe P.J. Contrasting properties of hypoxia-inducible factor 1 (HIF-1) and HIF-2 in von Hippel-Lindau-associated renal cell carcinoma.Mol. Cell. Biol. 2005; 25: 5675-5686Crossref PubMed Scopus (707) Google Scholar). Although much remains to be determined, extensive analyses have solidified the central concept that HIF activity in cancer cells drives tumor progression by inducing the expression of genes that promote adaptation to hypoxia. The degree to which HIF activation in tumor stromal cells, such as infiltrating macrophages and leukocytes, contributes to tumor angiogenesis and progression is an important question currently under investigation. Evidence from a variety of experimental systems has shown that hypoxia also regulates the proliferation and differentiation of different stem cell populations, including embryonic stem (ES) cells, neuronal and neural crest stem cells, hematopoietic stem cells (HSCs), and trophoblast stem cells. The direct role of HIFs in controlling these effects has been demonstrated in some, but as yet not all, of these stem cell types. The hypoxic responses of different stem cell populations is consistent with the idea that oxygen levels may be an important component of particular stem cell “niches” and that HIF activity can regulate the defining features of stem cells, including self renewal and multipotency. In the following sections, we will discuss the mechanisms by which hypoxia and HIFs mediate these effects and also discuss their implications for cancer biology. Finally, we propose a molecular model for how HIFs may promote the adoption of stem cell characteristics by differentiated hypoxic tumor cells. Stem cell niches are defined as particular locations or microenvironments that maintain the combined properties of stem cell self renewal and multipotency. In the fruit fly Drosophila melanogaster and the nematode Caenorhabditis elegans, germ stem cell niches have been described in remarkable detail. Germ stem cells in the Drosophila ovariole and testis require physical interaction with supporting cap or hub cells, respectively, to retain stem cell identity (Ohlstein et al., 2004Ohlstein B. Kai T. Decotto E. Spradling A. The stem cell niche: theme and variations.Curr. Opin. Cell Biol. 2004; 16: 693-699Crossref PubMed Scopus (273) Google Scholar). In the C. elegans gonad, the niche consists primarily of a single distal tip cell whose long cytoplasmic processes make extensive physical contact with germ stem cells (Morrison and Kimble, 2006Morrison S.J. Kimble J. Asymmetric and symmetric stem-cell divisions in development and cancer.Nature. 2006; 441: 1068-1074Crossref PubMed Scopus (968) Google Scholar). In mammals, spatially defined stem cell niches have also been identified in multiple tissues, including the gonad (Seydoux and Braun, 2006Seydoux G. Braun R.E. Pathway to totipotency: lessons from germ cells.Cell. 2006; 27: 891-904Abstract Full Text Full Text PDF Scopus (288) Google Scholar), skin (Fuchs, 2007Fuchs E. Scratching the surface of skin development.Nature. 2007; 445: 834-842Crossref PubMed Scopus (582) Google Scholar), intestine (Sancho et al., 2004Sancho E. Batlle E. Clevers H. Signaling pathways in intestinal development and cancer.Annu. Rev. Cell Dev. Biol. 2004; 20: 695-723Crossref PubMed Scopus (411) Google Scholar), and brain (Merkle and Alvarez-Buylla, 2006Merkle F.T. Alvarez-Buylla A. Neural stem cells in mammalian development.Curr. Opin. Cell Biol. 2006; 18: 704-709Crossref PubMed Scopus (250) Google Scholar), although in some cases the cells comprising the niche have not yet been explicitly identified (Joseph and Morrison, 2005Joseph N.M. Morrison S.J. Toward an understanding of the physiological function of Mammalian stem cells.Dev. Cell. 2005; 9: 173-183Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). A combination of genetic and molecular analyses has identified a number of molecular factors—typically supplied by the supporting cells of the niche—that control stem cell identity. These factors include components of the BMP, Notch, Wnt, JAK-STAT, and Sonic hedgehog (Shh) signaling pathways, which provide intercellular cues that regulate stem cell identity and differentiation (Sancho et al., 2004Sancho E. Batlle E. Clevers H. Signaling pathways in intestinal development and cancer.Annu. Rev. Cell Dev. Biol. 2004; 20: 695-723Crossref PubMed Scopus (411) Google Scholar, Joseph and Morrison, 2005Joseph N.M. Morrison S.J. Toward an understanding of the physiological function of Mammalian stem cells.Dev. Cell. 2005; 9: 173-183Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, Ohlstein et al., 2004Ohlstein B. Kai T. Decotto E. Spradling A. The stem cell niche: theme and variations.Curr. Opin. Cell Biol. 2004; 16: 693-699Crossref PubMed Scopus (273) Google Scholar, Fuchs, 2007Fuchs E. Scratching the surface of skin development.Nature. 2007; 445: 834-842Crossref PubMed Scopus (582) Google Scholar). These signaling functions have been highly conserved through evolution. For example, altered Notch signaling affects the function of a variety of mammalian stem cells (hematopoietic, intestinal, and skin), as well as intestinal stem cells in Drosophila and germ stem cells in C. elegans (Joseph and Morrison, 2005Joseph N.M. Morrison S.J. Toward an understanding of the physiological function of Mammalian stem cells.Dev. Cell. 2005; 9: 173-183Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, Ohlstein et al., 2004Ohlstein B. Kai T. Decotto E. Spradling A. The stem cell niche: theme and variations.Curr. Opin. Cell Biol. 2004; 16: 693-699Crossref PubMed Scopus (273) Google Scholar, Ohlstein and Spradling, 2006Ohlstein B. Spradling A. The adult Drosophila posterior midgut is maintained by pluripotent stem cells.Nature. 2006; 439: 470-474Crossref PubMed Scopus (700) Google Scholar). Coordinate regulation of genes controlling stem cell function is achieved in part through the activity of chromatin remodeling factors, including Bmi-1 and PRC2 (Joseph and Morrison, 2005Joseph N.M. Morrison S.J. Toward an understanding of the physiological function of Mammalian stem cells.Dev. Cell. 2005; 9: 173-183Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, Lee et al., 2006Lee T.I. Jenner R.G. Boyer L.A. Guenther M.G. Levine S.S. Kumar R.M. Chevalier B. Johnstone S.E. Cole M.F. Isono K. et al.Control of developmental regulators by Polycomb in human embryonic stem cells.Cell. 2006; 125: 301-313Abstract Full Text Full Text PDF PubMed Scopus (1791) Google Scholar). Local oxygen concentrations can directly influence stem cell self renewal and differentiation. One attractive hypothesis is that stem cells, particularly in long-lived animals, might benefit from residing in hypoxic niches where oxidative DNA damage may be reduced. Direct measurement of oxygen levels has revealed that bone marrow is, in general, quite hypoxic (∼1%–2% O2) (Cipolleschi et al., 1993Cipolleschi M.G. Dello Sbarba P. Olivotto M. The role of hypoxia in the maintenance of hematopoietic stem cells.Blood. 1993; 82: 2031-2037Crossref PubMed Google Scholar). Given the ongoing debate as to whether HSCs in bone marrow are associated with osteoblasts or sinusoidal endothelial cells (or both) (Kiel et al., 2005Kiel M.J. Yilmaz O.H. Iwashita T. Yilmaz O.H. Terhorst C. Morrison S.J. SLAM family receptors distinguish hematopoietic stem and progenitor cells and reveal endothelial niches for stem cells.Cell. 2005; 121: 1109-1121Abstract Full Text Full Text PDF PubMed Scopus (2255) Google Scholar), it will be interesting to determine the oxygen concentrations in specific bone marrow subdomains, although such an experiment remains technically challenging. Wherever HSCs reside, their proliferation and function is clearly affected by oxygen. Danet et al., 2003Danet G.H. Pan Y. Luongo J.L. Bonnet D.A. Simon M.C. Expansion of human SCID-repopulating cells under hypoxic conditions.J. Clin. Invest. 2003; 112: 126-135Crossref PubMed Scopus (276) Google Scholar demonstrated that culturing human bone marrow HSCs under hypoxic conditions (1.5% O2) promoted their ability to engraft and repopulate the hematopoietic compartment of immunodeficient NOD/SCID mice (Danet et al., 2003Danet G.H. Pan Y. Luongo J.L. Bonnet D.A. Simon M.C. Expansion of human SCID-repopulating cells under hypoxic conditions.J. Clin. Invest. 2003; 112: 126-135Crossref PubMed Scopus (276) Google Scholar). Similar results were obtained for hematopoietic progenitors isolated from embryonic yolk sacs or generated from ES cells grown in three-dimensional embryoid bodies in vitro (Ramirez-Bergeron and Simon, 2001Ramirez-Bergeron D.L. Simon M.C. Hypoxia-inducible factor and the development of stem cells of the cardiovascular system.Stem Cells. 2001; 19: 279-286Crossref PubMed Scopus (85) Google Scholar). These oxygen-mediated effects are not unique to HSCs: culturing neural crest stem cells or neuronal stem cells under hypoxic conditions (∼5% O2) promotes their proliferation and skews cellular differentiation toward specific fates (Morrison et al., 2000aMorrison S.J. Csete M. Groves A.K. Melega W. Wold B. Anderson D.J. Culture in reduced levels of oxygen promotes clonogenic sympathoadrenal differentiation by isolated neural crest stem cells.J. Neurosci. 2000; 20: 7370-7376Crossref PubMed Google Scholar, Studer et al., 2000Studer L. Csete M. Lee S.H. Kabbani N. Walikonis J. Wold B. McKay R. Enhanced proliferation, survival, and dopaminergic differentiation of CNS precursors in lowered oxygen.J. Neurosci. 2000; 20: 7377-7383Crossref PubMed Google Scholar). Differentiation of human placental cytotrophoblast cells is also directly influenced by hypoxia (Genbacev et al., 1997Genbacev O. Zhou Y. Ludlow J.W. Fisher S.J. Regulation of human placental development by oxygen tension.Science. 1997; 277: 1669-1672Crossref PubMed Scopus (724) Google Scholar). Finally, Pahlman and colleagues have demonstrated that hypoxic conditions confer a more immature phenotype on human neuroblastoma and breast cancer cell lines (Axelson et al., 2005Axelson H. Fredlund E. Ovenberger M. Landberg G. Pahlman S. Hypoxia-induced dedifferentiation of tumor cells–a mechanism behind heterogeneity and aggressiveness of solid tumors.Semin. Cell Dev. Biol. 2005; 16: 554-563Crossref PubMed Scopus (220) Google Scholar). Some of the effects of hypoxia on stem cell function are directly mediated by the HIF proteins. Targeted mutation of the ARNT subunit eliminates both HIF-1α and HIF-2α function and results in a decreased number of progenitor cells of all hematopoietic lineages in the embryonic yolk sac of Arnt−/− mouse embryos. This phenotype is recapitulated when Arnt−/− ES cells are induced to form hematopoietic progenitor cells in embryoid bodies in vitro (Ramirez-Bergeron and Simon, 2001Ramirez-Bergeron D.L. Simon M.C. Hypoxia-inducible factor and the development of stem cells of the cardiovascular system.Stem Cells. 2001; 19: 279-286Crossref PubMed Scopus (85) Google Scholar). Although Arnt-deficient mouse embryos display a variety of developmental abnormalities, they die at embryonic day (E) 9.5–E10.5 due to a defective placenta. Analysis of placentas from Arnt−/− (or HIF-1α−/−, HIF-2α−/− double) mutant mouse embryos revealed that HIF activity influences the differentiation of trophoblastic stem cells into either spongiotrophoblasts, which occupy a particularly hypoxic zone, or into trophoblast giant cells, which lie close to the oxygen-rich maternal spiral arteries (Cowden Dahl et al., 2005Cowden Dahl K.D. Fryer B.H. Mack F.A. Compernolle V. Maltepe E. Adelman D.M. Carmeliet P. Simon M.C. Hypoxia-inducible factors 1alpha and 2alpha regulate trophoblast differentiation.Mol. Cell. Biol. 2005; 25: 10479-10491Crossref PubMed Scopus (164) Google Scholar). The effects of HIF activity on trophoblast cell-fate determination have also been recapitulated using trophoblast stem cell lines cultured in vitro. These experiments implicate the HIF proteins in the control of HSC and trophoblast stem cell function; future work will be necessary to determine their specific functions in other stem cell populations. To date, only a few HIF target genes that might confer these effects have been identified. Expression of the VEGF gene, in particular, accounted for many of the HIF-mediated effects on hematopoietic progenitors (Ramirez-Bergeron and Simon, 2001Ramirez-Bergeron D.L. Simon M.C. Hypoxia-inducible factor and the development of stem cells of the cardiovascular system.Stem Cells. 2001; 19: 279-286Crossref PubMed Scopus (85) Google Scholar), but there is little doubt that other factors and signaling pathways are involved. Recent reports have identified new molecular mechanisms by which HIFs directly modify cellular differentiation and stem cell function. Lendahl, Poellinger, and colleagues reported that hypoxia blocked the differentiation of myogenic satellite cells, a myogenic cell line (C2C12), and primary neural stem cells in a Notch-dependent manner (Gustafsson et al., 2005Gustafsson M.V. Zheng X. Pereira T. Gradin K. Jin S. Lundkvist J. Ruas J.L. Poellinger L. Lendahl U. Bondesson M. Hypoxia requires notch signaling to maintain the undifferentiated cell state.Dev. Cell. 2005; 9: 617-628Abstract Full Text Full Text PDF PubMed Scopus (840) Google Scholar). When Notch receptors interact with the Jagged or Delta family of ligands, two proteolytic cleavage events result in the release of the Notch intracellular domain from the plasma membrane and its transport to the nucleus, where it forms a DNA-binding complex with other coactivators including MAML, CSL, and p300, and activates target-gene expression (Bray, 2006Bray S.J. Notch signalling: a simple pathway becomes complex.Nat. Rev. Mol. Cell Biol. 2006; 7: 678-689Crossref PubMed Scopus (1815) Google Scholar). Using neurogenic rat embryonic carcinoma cells, the authors demonstrated that hypoxic treatment increased stabilization of the transcriptionally active Notch intracellular domain and stimulation of Notch target genes Hes-1 and Hey-2. Chromatin immunoprecipitation (ChIP) experiments revealed that HIF-1α was physically recruited to a DNA-binding complex containing the Notch intracellular domain in hypoxic cells. Hypoxic induction of Notch target genes was dependent on the Notch intracellular domain and also required the functional C-terminal transactivation domain of HIF-1α, which interacts directly with p300/CBP. Moreover, it appears that this property was not unique to HIF-1α, as HIF-2α also augmented Notch target-gene expression in hypoxic A-498 renal carcinoma cells. Exactly how HIF-α proteins integrate into the Notch intracellular domain:MAML:CSL complex is not yet understood, nor is it known whether this response modulates the expression of all Notch target genes, or only a subset (Gustafsson et al., 2005Gustafsson M.V. Zheng X. Pereira T. Gradin K. Jin S. Lundkvist J. Ruas J.L. Poellinger L. Lendahl U. Bondesson M. Hypoxia requires notch signaling to maintain the undifferentiated cell state.Dev. Cell. 2005; 9: 617-628Abstract Full Text Full Text PDF PubMed Scopus (840) Google Scholar). Notch pathway signaling (Bray, 2006Bray S.J. Notch signalling: a simple pathway becomes complex.Nat. Rev. Mol. Cell Biol. 2006; 7: 678-689Crossref PubMed Scopus (1815) Google Scholar) has profound effects on cellular differentiation in Drosophila, C. elegans, and mammals, making the direct connection to HIF factors particularly intriguing. The results from Gustafsson et al., 2005Gustafsson M.V. Zheng X. Pereira T. Gradin K. Jin S. Lundkvist J. Ruas J.L. Poellinger L. Lendahl U. Bondesson M. Hypoxia requires notch signaling to maintain the undifferentiated cell state.Dev. Cell. 2005; 9: 617-628Abstract Full Text Full Text PDF PubMed Scopus (840) Google Scholar suggest that altered Notch signaling may underlie some of the developmental defects observed in HIF-deficient embryos, and in adult cells and tissues (such as the chondrocyte growth plate) from which HIF-1α has been selectively deleted (Schipani et al., 2001Schipani E. Ryan H.E. Didrickson S. Kobayashi T. Knight M. Johnson R.S. Hypoxia in cartilage: HIF-1alpha is essential for chondrocyte growth arrest and survival.Genes Dev. 2001; 15: 2865-2876PubMed Google Scholar). It is also striking that a primary effect of hypoxia, acting through Notch, was to inhibit the differentiation of a variety of cell types. Notch signaling is critical for the maintenance of undifferentiated stem and progenitor cell populations in the mammalian intestinal crypt and also influences differentiation of mature enterocytes (Wilson and Radtke, 2006Wilson A. Radtke F. Multiple functions of Notch signaling in self-renewing organs and cancer.FEBS Lett. 2006; 580: 2860-2868Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar). Forced Notch activation in hematopoietic bone marrow or T cell progenitor cells also blocks differentiation and results in T cell acute lymphoblastic leukemia (Pear and Aster, 2004Pear W.S. Aster J.C. T cell acute lymphoblastic leukemia/lymphoma: a human cancer commonly associated with aberrant NOTCH1 signaling.Curr. Opin. Hematol. 2004; 11: 426-433Crossref PubMed Scopus (83) Google Scholar). It is interesting to note, however, that bone marrow-specific deletion of Jagged1 and Notch1 function does not deplete HSCs or disrupt hematopoiesis (Mancini et al., 2005Mancini S.J. Mantei N. Dumortier A. Suter U. MacDonald H.R. Radtke F. Jagged1-dependent Notch signaling is dispensable for hematopoietic stem cell self-renewal and differentiation.Blood. 2005; 105: 2340-2342Crossref PubMed Scopus (224) Google Scholar), raising the possibility that other Notch receptors and/or ligands are active in these cells. It is tempting to speculate that a stem cell residing in a hypoxic niche may require HIF-α proteins to fully activate Notch target genes that inhibit differentiation, thereby contributing to stem cell self renewal and multipotency. Testing this hypothesis directly will entail selective inactivation of HIF-α or Notch in specific stem cell populations in vivo. It is important to remember, however, that the effects of Notch signaling can be cell-type dependent, as Notch activation actually promotes terminal differentiation in epidermal keratinocytes and certain neural stem cells (Morrison et al., 2000bMorrison S.J. Perez S.E. Qiao Z. Verdi J.M. Hicks C. Weinmaster G. Anderson D.J. Transient Notch activation initiates an irreversible switch from neurogenesis to gliogenesis by neural crest stem cells.Cell. 2000; 101: 499-510Abstract Full Text Full Text PDF PubMed Scopus (559) Google Scholar, Wilson and Radtke, 2006Wilson A. Radtke F. Multiple functions of Notch signaling in self-renewing organs and cancer.FEBS Lett. 2006; 580: 2860-2868Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar). Our laboratory recently reported that hypoxia regulates stem cell function through direct activation of specific HIF target genes. To determine the functional redundancy between HIF-1α and HIF-2α in embryonic development, we targeted a HIF-2α cDNA into the HIF-1α locus in murine ES cells, thereby replacing HIF-1α expression with HIF-2α. This “knockin” allele was designed to test the degree to which expanded HIF-2α expression, under the regulatory control of the HIF-1α locus, could complement a HIF-1α null mutation (Covello et al., 2006Covello K.L.