Oxygen-regulated and transactivating domains in endothelial PAS protein 1: comparison with hypoxia-inducible factor-1alpha.

Endothelial PAS protein 1 (EPAS1) is a basic helix-loop-helix Per-AHR-ARNT-Sim transcription factor related to hypoxia-inducible factor-1alpha (HIF-1alpha). To analyze EPAS1 domains responsible for transactivation and oxygen-regulated function, we constructed chimeric fusions of EPAS1 with a GAL4 DNA binding domain, plus or minus the VP16 activation domain. Two transactivation domains were defined in EPAS1; a C-terminal domain (amino acids 828-870), and a larger internal domain (amino acids 517-682). These activation domains were interspersed by functionally repressive sequences, several of which independently conveyed oxygen-regulated activity. Two types of activity were defined. Sequences lying N-terminal to and overlapping the internal transactivation domain conferred regulated repression on the VP16 transactivator. Sequences lying C-terminal to this internal domain conveyed repression and oxygen-regulated activity on the native EPAS1 C-terminal activation domain, but not the Gal/VP16 fusion. Fusions containing internal but not C-terminal regulatory domains manifested regulation of fusion protein level. Comparison of EPAS1 with HIF-1alpha demonstrated a similar organization for both proteins, and for the C terminus defined a conserved RLL motif critical for inducibility. Overall, EPAS1 sequences were less inducible than those of HIF-1alpha, and inducibility was strikingly reduced as their expression level was increased. Despite these quantitative differences, EPAS1 regulation appeared similar to HIF-1alpha, conforming to a model involving the modulation of both protein level and activity, through distinct internal and C-terminal domains.

Regulation of the Drosophila bHLH-PAS protein Sima by hypoxia: functional evidence for homology with mammalian HIF-1 alpha.

Hypoxia inducible factor-1 (HIF-1) is a heterodimeric complex of two basic-helix-loop-helix proteins of the PAS family which is critical for oxygen-dependent expression of many mammalian genes. Regulation is mediated by the alpha subunit (HIF-1 alpha) and sequences from HIF-1 alpha can confer hypoxia-inducible activity on a Ga14 fusion protein. To analyse conservation of this system of gene regulation between Drosophila and mammalian cells we constructed Ga14 fusions with a series of Drosophila basic-helix-loop-helix PAS (bHLH-PAS) proteins and tested for hypoxia inducibility in transfected Hep3B cells. We found that Ga14 functions with Similar (Sima) but not other Drosophila bHLH-PAS proteins showed inducible activity following exposure to stimuli which classically activate mammalian HIF-1:hypoxia, cobaltous ions, and desferrioxamine. We also found that Sima protein accumulated in Drosophila SL2 cells following hypoxia. Together these findings indicate the existence of functional homologies between Sima and HIF-1 alpha, and that conservation is such as to enable Sima to interact with the hypoxia signal transduction system in mammalian cells.

Oxygen sensing, hypoxia-inducible factor-1 and the regulation of mammalian gene expression.

A great many aspects of the anatomy and physiology of large animals are constrained by the need to match oxygen supply to cellular metabolism and appear likely to involve the regulation of gene expression by oxygen. Some insight into possible underlying mechanisms has been provided by studies of erythropoietin, a haemopoietic growth factor which stimulates red cell production in response to hypoxia. Studies of hypoxia-inducible cis-acting sequences from the erythropoietin gene have led to the recognition of a widespread transcriptional response to hypoxia based on the activation of a DNA-binding complex termed hypoxia-inducible factor-1 (HIF-1). Perturbation of the transcriptional response by particular transition metal ions, iron chelators and certain redox-active agents have suggested a specific oxygen sensing mechanism, perhaps involving a haem protein in a flavoprotein/cytochrome system. In addition to erythropoietin, HIF-1-responsive genes include examples with functions in cellular energy metabolism, iron metabolism, catecholamine metabolism, vasomotor control and angiogenesis, suggesting an important role in the coordination of oxygen supply and cellular metabolism. In support of this, we have demonstrated an important role for HIF-1 in tumour angiogenesis. HIF-1 itself consists of a heterodimer of two basic-helix-loop-helix proteins of the PAS family, termed HIF-1alpha and HIF-1beta, although other closely related members of this family may also contribute to the response to hypoxia. We have fused domains of HIF-1 genes to heterologous transcription factors to assay for regulatory function. These experiments have defined several domains in HIF-1alpha which can independently confer the hypoxia-inducible property, and they suggest a mechanism of HIF-1 activation in which post-translational activation/derepression of HIF-1alpha is amplified by changes in HIF-1alpha abundance most probably arising from suppression of proteolytic breakdown. Pursuit of the mechanism(s) underlying these processes should ultimately lead to better definition of the oxygen-sensing process.

Activation of hypoxia-inducible factor-1; definition of regulatory domains within the alpha subunit.

Hypoxia-inducible factor-1 (HIF-1), a heterodimeric DNA binding complex composed of two basic-helix-loop-helix Per-AHR-ARNT-Sim proteins (HIF-1alpha and -1beta), is a key component of a widely operative transcriptional response activated by hypoxia, cobaltous ions, and iron chelation. To identify regions of HIF-1 subunits responsible for oxygen-regulated activity, we constructed chimeric genes in which portions of coding sequence from HIF-1 genes were either linked to a heterologous DNA binding domain or encoded between such a DNA binding domain and a constitutive activation domain. Sequences from HIF-1alpha but not HIF-1beta conferred oxygen-regulated activity. Two minimal domains within HIF-1alpha (amino acids 549-582 and amino acids 775-826) were defined by deletional analysis, each of which could act independently to convey inducible responses. Both these regions confer transcriptional activation, and in both cases adjacent sequences appeared functionally repressive in transactivation assays. The inducible operation of the first domain, but not the second, involved major changes in the level of the activator fusion protein in transfected cells, inclusion of this sequence being associated with a marked reduction of expressed protein level in normoxic cells, which was relieved by stimulation with hypoxia, cobaltous ions, or iron chelation. These results lead us to propose a dual mechanism of activation in which the operation of an inducible activation domain is amplified by regulation of transcription factor abundance, most likely occurring through changes in protein stability.

Hypoxia response elements.

Hypoxia-inducible factor-1 (HIF-1) has been shown to mediate the transcriptional activation of its target genes in response to oxygen concentration, most likely via a pathway involving a specific oxygen sensor. Molecular cloning of HIF-1 has shown that this widely expressed, DNA binding transcription factor is a heterodimer of two proteins, HIF-1 alpha and HIF-1 beta. A major control of HIF-1 activity by oxygen tension is achieved by changes in the level of the HIF-1 alpha subunit, which complexes with the constitutively expressed HIF-1 beta subunit. Such changes in HIF-1 alpha abundance occur via regulated stability, probably involving proteolysis, rather than at the level of transcription or translation. Further analysis has shown the existence of two separate regulatory domains in the C-terminus of the alpha subunit. Thus, a mechanism of oxygen-regulated HIF-1 activation is proposed, which involves the operation of one inducible domain being amplified by changes in protein level conferred by a second regulatory domain. Evidence for a critical role of HIF-1 in the response of diverse target genes involved in cellular growth and metabolism comes from studies on cultured, mutant mouse cells that lack a functional HIF-1 beta subunit. Furthermore, studies on tumor xenografts derived from the mutant and wild-type cells show that HIF-1 is activated in vivo, and has major effects on gene expression in response to tumor hypoxia. Thus, HIF-1 is a critical component of the oxygen-signaling pathway, and is a prime candidate regulator molecule for the role of coordinating vascular oxygen supply with cellular growth and energy metabolism.

Identification of hypoxically inducible mRNAs in HeLa cells using differential-display PCR. Role of hypoxia-inducible factor-1.

Oxygen is an important regulator of gene expression in mammalian cells, though the extent of operation and the organization of the inducible mechanisms involved are still largely undetermined. To define better the response to hypoxia, we have used differential display PCR to identify genes whose expression is induced in HeLa cells exposed to 1% oxygen. Among six genes whose induction by hypoxia was newly defined in this way, three were of known function, encoding the glucose transporter isoform 3 (Glut-3), adenylate kinase isoenzyme 3 (AK-3), and tissue factor, two were expressed sequence tags (ESTs), and one corresponded to a new sequence. One regulator of the transcriptional response to hypoxia has recently been identified as a heterodimeric DNA-binding complex termed hypoxia-inducible factor-1 (HIF-1), which is also inducible by the iron chelator, desferrioxamine. Of the six hypoxically regulated genes, at least four were also induced by exposure of the cells to desferrioxamine. To analyse further the mechanisms underlying induction of the genes identified in the differential display, inducible expression was compared in wild-type mouse hepatoma cells (Hepa-1), and mutant derivatives (c4) which fail to generate HIF-1, due to a functional defect in one component, HIF-1 beta. Two types of response were defined. For Glut-3 and AK-3, mutant (c4) cells showed almost complete loss of the inducible response to both hypoxia and desferrioxamine. In contrast, tissue factor mRNA was more inducible by both stimuli in c4 than wild-type cells. These studies demonstrate the critical importance of HIF-1 beta in newly recognized responses to hypoxia, and provide further evidence of the importance of this system of gene regulation in mammalian cells; they also demonstrate responses to both hypoxia and desferrioxamine which are independent of HIF-1 beta and which appear exaggerated in HIF-1 beta-deficient cells.

Isoenzyme-specific regulation of genes involved in energy metabolism by hypoxia: similarities with the regulation of erythropoietin.

Recent studies have indicated that regulatory mechanisms underlying the oxygen-dependent expression of the haematopoietic growth factor erythropoietin are widely operative in non-erythropoietin-producing cells and are involved in the regulation of other genes. An important characteristic of this system is that the inducible response to hypoxia is mimicked by exposure to particular transition metals such as cobaltous ions, and by iron chelation. We have investigated the extent of operation of this system in the regulation of a range of genes concerned with energy metabolism. The effects of hypoxia (1% oxygen), cobaltous ions and desferrioxamine on gene expression in tissue-culture cells was studied using RNase protection assays. Hypoxia induced the expression of glucose transporters in an isoform-specific manner; GLUT-1 and GLUT-3 were induced by hypoxia, whereas expression of GLUT-2 was decreased. Isoenzyme-specific regulation by hypoxia was also observed for genes encoding phosphofructokinase, aldolase and lactate dehydrogenase. For all of these genes, responses to cobaltous ions and desferrioxamine correlated in both direction and magnitude with the response to hypoxia. In contrast, a reduction in mitochondrial transcripts was observed in hypoxia, but these changes were not mimicked by either cobaltous ions or desferrioxamine. These findings indicate that similarities with erythropoietin regulation extend to the oxygen-dependent regulation of genes encoding glucose transporters and glycolytic enzymes but not to the regulation of mitochondrial transcripts, and they show that in glucose metabolism regulation by this system is isoenzyme- or isoform-specific.