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• W1964918262 abstract "Hypoxia-inducible factor-1 (HIF-1), a heterodimeric DNA binding complex composed of two basic-helix-loop-helix Per-AHR-ARNT-Sim proteins (HIF-1α and −1β), 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-1α but not HIF-1β conferred oxygen-regulated activity. Two minimal domains within HIF-1α (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-inducible factor-1 (HIF-1), a heterodimeric DNA binding complex composed of two basic-helix-loop-helix Per-AHR-ARNT-Sim proteins (HIF-1α and −1β), 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-1α but not HIF-1β conferred oxygen-regulated activity. Two minimal domains within HIF-1α (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. INTRODUCTIONHypoxia-inducible factor-1, a DNA binding complex first identified as a factor critical for the inducible activity of the erythropoietin 3′ enhancer (1Semenza G.L. Wang G.L. Mol. Cell. Biol. 1992; 12: 5447-5454Google Scholar), is now recognized to be a key component of a widely operative transcriptional control system responding to physiological levels of cellular hypoxia (2Maxwell P.H. Pugh C.W. Ratcliffe P.J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 2423-2427Google Scholar, 3Wang G.L. Semenza G.L. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 4304-4308Google Scholar, 4Jiang B.-H. Semenza G.L. Bauer C. Marti H.H. Am. J. Physiol. 1996; 271: C1172-C1180Google Scholar, 5Bunn H.F. Poyton R.O. Physiol. Rev. 1996; 76: 839-885Google Scholar). Deletional and mutational analysis of cis-acting sequences has demonstrated functionally critical HIF-1 1The abbreviations used are: HIF-1hypoxia-inducible factor-1DFOdesferrioxamineARNTaryl hydrocarbon receptor nuclear translocator (identical to HIF-1β)AHRaryl hydrocarbon receptorARNT-tatrans-activation domain from the human aryl hydrocarbon receptor nuclear translocator (amino acids 696-789)VP16trans-activation domain from the herpes simplex virus protein 16 (amino acids 410-490)GRthe N-terminal 500 amino acids of the human glucocorticoid receptorGalthe N-terminal 147 amino acids of the yeast Gal4 transcription factorMMTVmouse mammary tumor virus promoterEMSAelectrophoretic mobility shift assay(s). binding sites in many oxygen-regulated promoters and enhancers (6Firth J.D. Ebert B.L. Pugh C.W. Ratcliffe P.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6496-6500Google Scholar, 7Semenza G.L. Roth P.H. Fang H.-M. Wang G.L. J. Biol. Chem. 1994; 269: 23757-23763Google Scholar, 8Ebert B.L. Firth J.D. Ratcliffe P.J. J. Biol. Chem. 1995; 270: 29083-29089Google Scholar, 9Levy A.P. Levy N.S. Wegner S. Goldberg M.A. J. Biol. Chem. 1995; 270: 13333-13340Google Scholar, 10Liu Y. Cox S.R. Morita T. Kourembanas S. Circ. Res. 1995; 77: 638-643Google Scholar, 11Norris M.L. Millhorn D.E. J. Biol. Chem. 1995; 270: 23774-23779Google Scholar, 12Estes S.C. Stoler D.L. Anderson G.R. J. Virol. 1995; 69: 6335-6341Google Scholar). The importance of HIF-1 in the regulation of such genes has been confirmed by the reduction or abrogation of hypoxia-inducible expression in mutant cells (13Hankinson O. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 373-376Google Scholar, 14Hoffman E.C. Reyes H. Chu F.-F. Sander F. Conley L.H. Brooks B.A. Hankinson O. Science. 1991; 252: 954-958Google Scholar) that are unable to form a functional HIF complex (15Wood S.M. Gleadle J.M. Pugh C.W. Hankinson O. Ratcliffe P.J. J. Biol. Chem. 1996; 271: 15117-15123Google Scholar, 16Li H. Ko H.P. Whitlock Jr., J.P. J. Biol. Chem. 1996; 271: 21262-21267Google Scholar, 17Gradin K. McGuire J. Wenger R.H. Kvietikova I. Whitelaw M.L. Toftgard R. Tora L. Gassmann M. Poellinger L. Mol. Cell. Biol. 1996; 16: 5221-5231Google Scholar, 18Forsythe J.A. Jiang B.H. Iyer N.V. Agani F. Leung S.W. Koos R.D. Semenza G.L. Mol. Cell. Biol. 1996; 16: 4604-4613Google Scholar, 19Salceda S. Beck I. Caro J. Arch. Biochem. Biophys. 1996; 334: 389-394Google Scholar). Together these studies have provided strong evidence for a critical role for HIF-1 in the regulation of genes involved in a variety of important biological processes that include glucose transport and metabolism, vascular growth, vasomotor regulation, erythropoiesis, iron metabolism, and catecholamine synthesis (reviewed in Ref. 5Bunn H.F. Poyton R.O. Physiol. Rev. 1996; 76: 839-885Google Scholar).As is observed for HIF-1-responsive genes (20Goldberg M.A. Dunning S.P. Bunn H.F. Science. 1988; 242: 1412-1415Google Scholar, 21Goldberg M.A. Schneider T.J. J. Biol. Chem. 1994; 269: 4355-4359Google Scholar, 22Gleadle J.M. Ebert B.L. Firth J.D. Ratcliffe P.J. Am. J. Physiol. 1995; 268: C1362-C1368Google Scholar), the HIF-1 complex is inducible by particular transition elements such as cobaltous ions and by iron chelating agents such as desferrioxamine (DFO) but not by inhibitors of mitochondrial respiration such as cyanide or azide (3Wang G.L. Semenza G.L. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 4304-4308Google Scholar, 8Ebert B.L. Firth J.D. Ratcliffe P.J. J. Biol. Chem. 1995; 270: 29083-29089Google Scholar, 23Wang G.L. Semenza G.L. Blood. 1993; 82: 3610-3615Google Scholar). These distinctive features have led to the proposal of a specific oxygen sensing mechanism underlying these responses, most probably involving the operation of a ferroprotein sensor (20Goldberg M.A. Dunning S.P. Bunn H.F. Science. 1988; 242: 1412-1415Google Scholar). Recent affinity purification and molecular cloning of HIF-1 (24Wang G.L. Jiang B.-H. Rue E.A. Semenza G.L. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5510-5514Google Scholar) has revealed that the DNA binding complex consists of a heterodimer of two basic-helix-loop-helix Per-AHR-ARNT-Sim proteins HIF-1α and HIF-1β, a molecule that is identical to the aryl hydrocarbon receptor nuclear translocator (ARNT) (25Reyes H. Reisz-Porszasz S. Hankinson O. Science. 1992; 256: 1193-1195Google Scholar). An important goal is now to define the regions of the HIF-1 molecules that are responsible for their regulated activity and to understand the mechanism by which the complex is induced and activated in hypoxic cells.Since in the majority of studies, HIF-1 activation does not appear to be mediated through regulation of its mRNAs (15Wood S.M. Gleadle J.M. Pugh C.W. Hankinson O. Ratcliffe P.J. J. Biol. Chem. 1996; 271: 15117-15123Google Scholar, 17Gradin K. McGuire J. Wenger R.H. Kvietikova I. Whitelaw M.L. Toftgard R. Tora L. Gassmann M. Poellinger L. Mol. Cell. Biol. 1996; 16: 5221-5231Google Scholar, 26Huang L.E. Arany Z. Livingston D.M. Bunn H.F. J. Biol. Chem. 1996; 271: 32253-32259Google Scholar), we focused our analysis on other possible mechanisms of regulation. As with other transcription factors, studies of the regulatory mechanisms are potentially complicated by the ultimate dependence of transcriptional activation on a series of interrelated events which may include nuclear accumulation, dimerization, DNA binding, co-factor recruitment, and transactivation. For HIF-1, a further difficulty in this analysis lies in the operation of the native system in all cells so far tested (2Maxwell P.H. Pugh C.W. Ratcliffe P.J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 2423-2427Google Scholar, 3Wang G.L. Semenza G.L. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 4304-4308Google Scholar, 27Beck I. Weinmann R. Caro J. Blood. 1993; 82: 704-711Google Scholar, 28Pugh C.W. Ebert B.L. Ebrahim O. Ratcliffe P.J. Biochim. Biophys. Acta. 1994; 1217: 297-306Google Scholar). For these reasons we used the construction of chimeric genes to define regions of the HIF-1 genes that could confer oxygen-regulated behavior on heterologous transcription factors. Two types of chimeric gene were produced, those in which the heterologous transcription factor encoded nuclear localization and DNA binding, but lacked intrinsic transactivation potential, and others in which an activation domain was either intrinsic to the heterologous gene or added to the chimeric gene from a second heterologous gene. This allowed for both the definition of activation domains of HIF-1 genes and analysis of regulatory domains that did not necessarily contain intrinsic transactivation potential.Sequences from HIF-1α but not HIF-1β/ARNT conveyed inducible activity on heterologous transcription factors, and two regions within the C-terminal portion of the HIF-1α molecule were defined, each of which possessed transactivation potential and each of which could act independently to convey inducible properties. Both domains were responsive to cobaltous ions and iron chelation as well as hypoxia. The inducible activity of one regulatory domain, but not the other, appeared to be closely connected with modulation of the level of the encoded fusion protein, most probably arising from an effect on protein stability. These studies therefore define the existence of more than one regulatory domain in the HIF-1α subunit and strongly suggest the operation of more than one mechanism of regulation.DISCUSSIONWe have demonstrated that sequences from the α subunit of HIF-1 convey hypoxia-inducible activity when fused to the DNA binding domain of heterologous transcription factors. As has been established for the activation of HIF-1, the chimeric transcription factors also responded to cobaltous ions and desferrioxamine but not to mitochondrial inhibitors. Such responses were not observed for the HIF-1β/ARNT subunit, defining a regulatory function for HIF-1α.In our initial analysis of HIF-1α, we fused sequences from this gene to the N-terminal 500 amino acids of the glucocorticoid receptor, a sequence which itself possesses transactivation activity localized to the N terminus. This strategy was designed to enable sequences from HIF-1α to be assayed for regulatory properties independently of their own transactivation capability. When the activity of such fusion proteins was tested, an inducible response was observed for fusions containing C-terminal sequences lying distal to the portions of the molecule known to be involved in DNA binding and dimerization (37Jiang B.-H. Rue E. Wang G.L. Roe R. Semenza G.L. J. Biol. Chem. 1996; 271: 17771-17778Google Scholar). When fused to the truncated glucocorticoid receptor, HIF-1α sequences 530-826 and 28-826 conferred similar inducible behavior. Since, despite the intrinsic transcriptional activity of the truncated glucocorticoid receptor, several fusions containing N-terminal sequences had very low activities which made induction difficult to assess (Fig. 2, and data not shown), this result does not necessarily exclude inducible properties within HIF-1α sequences lying N-terminal to amino acid 530. However, the findings did indicate that sequences lying distal to amino acid 530 were sufficient to convey highly inducible activity and focused our detailed analysis on this portion of the molecule.This analysis defined two regions within these sequences which could independently confer inducible characteristics on heterologous transcription factors. One region was defined within HIF-1α amino acids 530-652. That this region was responsive to the inducing stimuli was first suggested by comparison of the activity of different glucocorticoid receptor/HIF-1α fusion proteins in HeLa cells (Fig. 2) and confirmed by its action on constitutively active chimeric transcription factors constructed from the DNA binding domain of Gal4 and activation domains from HIF-1β/ARNT or the herpes simplex virus protein VP16 (Fig. 3, Fig. 4, Fig. 5, Fig. 6). The second region was defined within amino acids 652-826. Although inducible activity was clearly observed when this sequence was tested as a Gal4 fusion in Hep3B cells, the sequence had only constitutive activity when tested as a glucocorticoid receptor fusion in HeLa cells. This difference could not be assigned to reporter system or cell type specificity, since the relevant glucocorticoid receptor/HIF-1α fusion showed inducible activity in Hep3B cells, and the relevant Gal4/HIF-1α fusion showed activity in HeLa cells albeit of lower amplitude. Whatever the reason for the differences, these experiments did define two regulatory domains of HIF-1α that were capable of independent action. Deletional analysis demonstrated that in each case the property was located within a relatively short amino acid sequence (amino acids 549-582 for the first domain and amino acids 775-826 for the second), and functional analysis demonstrated that in each case the inducible characteristic included stimulation by cobaltous ions and DFO as well as hypoxia. Somewhat surprisingly, in Hep3B cells, stimulation by cobaltous ions and DFO was more effective than hypoxia, a difference that is not generally observed in the regulation of endogenous HIF-1-dependent genes (20Goldberg M.A. Dunning S.P. Bunn H.F. Science. 1988; 242: 1412-1415Google Scholar, 22Gleadle J.M. Ebert B.L. Firth J.D. Ratcliffe P.J. Am. J. Physiol. 1995; 268: C1362-C1368Google Scholar, 23Wang G.L. Semenza G.L. Blood. 1993; 82: 3610-3615Google Scholar) and that was not observed in the transactivation assays performed in HeLa cells.Amino acids in both the critical regions are 100% conserved between human and mouse genes although, overall, amino acids 530-826 of the human sequence are only 83% conserved in the mouse (16Li H. Ko H.P. Whitlock Jr., J.P. J. Biol. Chem. 1996; 271: 21262-21267Google Scholar, 24Wang G.L. Jiang B.-H. Rue E.A. Semenza G.L. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5510-5514Google Scholar, 38Wenger R.H. Rolfs A. Marti H.H. Guénet J.-L. Gassmann M. Biochem. Biophys. Res. Commun. 1996; 223: 54-59Google Scholar). In keeping with the functional significance of this conservation, the C-terminal 52-amino acid domain that we have defined in the human HIF-1α lies within the region homologous to an 83-amino acid-inducible activation domain defined in studies of mouse HIF-1α published during the course of this work (16Li H. Ko H.P. Whitlock Jr., J.P. J. Biol. Chem. 1996; 271: 21262-21267Google Scholar). Our finding of a second regulatory domain lying N-terminal to this region does not imply a species difference in the mode of regulation since in the analysis of the mouse gene that domain was not analyzed independently or in the context of a heterologous activation domain.An important aspect of our analysis of these regulatory domains was the finding that sequences within amino acids 530-652 of HIF-1α had a striking effect on the levels of fusion protein in the transfected cells. In normoxic cells, the level of the Gal/ARNT fusion was dramatically reduced by this sequence, the reduction being relieved by exposure of cells to hypoxia, cobalt, and DFO in a manner that correlated with the functional results. Studies of the regulation of endogenous HIF-1 have demonstrated large increases in HIF-1α protein level in hypoxic cells (24Wang G.L. Jiang B.-H. Rue E.A. Semenza G.L. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5510-5514Google Scholar, 26Huang L.E. Arany Z. Livingston D.M. Bunn H.F. J. Biol. Chem. 1996; 271: 32253-32259Google Scholar) despite relative stability of HIF-1α mRNA levels (15Wood S.M. Gleadle J.M. Pugh C.W. Hankinson O. Ratcliffe P.J. J. Biol. Chem. 1996; 271: 15117-15123Google Scholar, 17Gradin K. McGuire J. Wenger R.H. Kvietikova I. Whitelaw M.L. Toftgard R. Tora L. Gassmann M. Poellinger L. Mol. Cell. Biol. 1996; 16: 5221-5231Google Scholar, 26Huang L.E. Arany Z. Livingston D.M. Bunn H.F. J. Biol. Chem. 1996; 271: 32253-32259Google Scholar). Based on the apparent stability of HIF-1α protein in hypoxic cells and its rapid degradation when cells are re-oxygenated, it has been proposed that the regulation of HIF-1 involves changes in stability of the α subunit (26Huang L.E. Arany Z. Livingston D.M. Bunn H.F. J. Biol. Chem. 1996; 271: 32253-32259Google Scholar). Although similar measurements are difficult in transiently transfected cells, and we have not formally addressed the mechanism by which the fusion protein levels are regulated, our results are most consistent with this proposal and with the regulatory domain we have defined containing a regulated determinant of protein stability. First, the effect of the HIF-1α sequences was always to reduce protein levels, reduction being profound in normoxic cells and relieved to a greater or lesser extent in stimulated cells. Second, these effects were observed on gene products expressed using the powerful constitutive cytomegalovirus promoter and optimized translational initiation sequences from different heterologous genes. Third, this region of HIF-1α is rich in proline, glutamic acid/aspartic acid, serine, and threonine residues that have been implicated as signals directing protein degradation (39Rechsteiner M. Rogers S.W. Trends Biol. Sci. 1996; 21: 267-271Google Scholar).Further analysis indicated that sequences 549-572 were critically important for the effect on protein level. Deletion of sequences surrounding this region also had effects; for instance, deletion of amino acids 582-634 increased fusion protein levels in normoxic cells, suggesting that these sequences might also contribute to the mechanism of regulation. In the functional analysis, successive deletions of amino acids 582-634 led to a progressive increase in the activity of the chimeric transcription factor in normoxic cells and a progressive reduction in the amplitude of the inducible response. Although in the overall analysis of HIF-1α sequences 530-652 correlation was clearly present between the functional effects and protein levels, we cannot be sure whether regulation of protein level could fully account for the effects on activity. Substantial quantitative discrepancies were apparent between the two measurements, but given the likelihood of a nonlinear relationship in the process of transcriptional activation, and the fact that we used a plasmid amplification system in these experiments, it is difficult to know whether such differences are evidence for additional mechanisms of transcriptional regulation at this site.In the analysis of the C-terminal domain of HIF-1α much more convincing support for this possibility was obtained. Gal4/HIF-1α fusion proteins containing C-terminal sequences were expressed at a much higher level than fusion proteins containing amino acids 530-652, and irrespective of whether the plasmid amplification system was used, their levels were not regulated by the inducing stimuli. Moreover, when Gal4 DNA binding activity was assayed by EMSA, activity in cells transfected with the Gal/HIF-1α C-terminal fusion was similar to that obtained in cells transfected with the plasmid encoding the Gal4 DNA binding domain alone. Thus the inducible activation associated with this domain did not appear to result from changes in protein level or DNA binding activity. Together with the analysis of amino acids 530-652 our results strongly suggest the operation of at least two different mechanisms of transcriptional regulation for HIF-1α, one based on post-translational enhancement of activation and the other involving regulation of transcription factor levels, most probably through changes in protein stability.One further point in relation to the analysis of amino acid sequences 530-652 is the co-localization of a potential stability determinant and activation domain, which raises an issue as to the relation between the two processes. Both processes may be separately and actively regulated or one may occur as a default consequence of lack of activation of the other. These possibilities are difficult to distinguish, although the increased protein level and loss of functional activity observed when the critical amino acids 549-572 were deleted shows that increases in protein level can be independent of the process of transactivation. Interestingly, transcriptional activation was only modest when amino acids 530-652 were considered in isolation (Fig. 3), even when the highly active core sequence 549-582 was assessed as a simple Gal4 fusion (data not shown), nor was a positive interaction observed when the sequences were placed adjacent to the VP16 activation domain (Fig. 5). In contrast, in stimulated cells, a strongly positive interaction was observed with both the constitutive C-terminal activation domain of HIF-1β/ARNT and the inducible C-terminal activation domain of HIF-1α, allowing the possibility that such interactions in cis or in trans could be important in the function of the native HIF-1 heterodimer. In considering the functional data alone, the power of this interaction is disguised. Thus, in stimulated cells the activity of chimeric genes expressing HIF-1α sequences 530-826 was only a little greater than those expressing sequences 652-826, but when the differences in protein level and DNA binding activity are considered (Fig. 8, Fig. 9), it can be seen that the specific activation potential of the product containing the additional amino acids 530-652 must be very much greater.Overall, our results suggest a model in which the function of an inducible activation domain is amplified by modulation of protein level, most probably occurring through changes in protein stability. There are many precedents for post-translational modifications that enhance transactivation through phosphorylation, ligand-dependent conformational change, or co-factor recruitment (40Calkhoven C.F. Geert A.B. Biochem. J. 1996; 317: 329-342Google Scholar). Less well recognized is the regulation of transcription through changes in the stability of transcription factors, although several examples have recently been described, dependent either on the action of a specific protease or on the inducible targeting of the protein to the ubiquitin-dependent proteosomal system of degradation (39Rechsteiner M. Rogers S.W. Trends Biol. Sci. 1996; 21: 267-271Google Scholar, 41Pahl H.L. Baeuerle P.A. Curr. Opin. Cell Biol. 1996; 8: 340-347Google Scholar). Aside from the preponderance of proline, glutamic acid/aspartic acid, serine, and threonine residues, examination of the critical sequences defined in the deletional analysis did not reveal any known recognition motifs for such systems nor did mutation of phosphoacceptor sites at residues 551, 552, 555, 565, 576, and 581 affect the operation of this regulatory domain. Nevertheless detailed analysis of these sequences should now permit important new insights to be gained into this mechanism of transcriptional regulation and the underlying processes of oxygen sensing. INTRODUCTIONHypoxia-inducible factor-1, a DNA binding complex first identified as a factor critical for the inducible activity of the erythropoietin 3′ enhancer (1Semenza G.L. Wang G.L. Mol. Cell. Biol. 1992; 12: 5447-5454Google Scholar), is now recognized to be a key component of a widely operative transcriptional control system responding to physiological levels of cellular hypoxia (2Maxwell P.H. Pugh C.W. Ratcliffe P.J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 2423-2427Google Scholar, 3Wang G.L. Semenza G.L. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 4304-4308Google Scholar, 4Jiang B.-H. Semenza G.L. Bauer C. Marti H.H. Am. J. Physiol. 1996; 271: C1172-C1180Google Scholar, 5Bunn H.F. Poyton R.O. Physiol. Rev. 1996; 76: 839-885Google Scholar). Deletional and mutational analysis of cis-acting sequences has demonstrated functionally critical HIF-1 1The abbreviations used are: HIF-1hypoxia-inducible factor-1DFOdesferrioxamineARNTaryl hydrocarbon receptor nuclear translocator (identical to HIF-1β)AHRaryl hydrocarbon receptorARNT-tatrans-activation domain from the human aryl hydrocarbon receptor nuclear translocator (amino acids 696-789)VP16trans-activation domain from the herpes simplex virus protein 16 (amino acids 410-490)GRthe N-terminal 500 amino acids of the human glucocorticoid receptorGalthe N-terminal 147 amino acids of the yeast Gal4 transcription factorMMTVmouse mammary tumor virus promoterEMSAelectrophoretic mobility shift assay(s). binding sites in many oxygen-regulated promoters and enhancers (6Firth J.D. Ebert B.L. Pugh C.W. Ratcliffe P.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6496-6500Google Scholar, 7Semenza G.L. Roth P.H. Fang H.-M. Wang G.L. J. Biol. Chem. 1994; 269: 23757-23763Google Scholar, 8Ebert B.L. Firth J.D. Ratcliffe P.J. J. Biol. Chem. 1995; 270: 29083-29089Google Scholar, 9Levy A.P. Levy N.S. Wegner S. Goldberg M.A. J. Biol. Chem. 1995; 270: 13333-13340Google Scholar, 10Liu Y. Cox S.R. Morita T. Kourembanas S. Circ. Res. 1995; 77: 638-643Google Scholar, 11Norris M.L. Millhorn D.E. J. Biol. Chem. 1995; 270: 23774-23779Google Scholar, 12Estes S.C. Stoler D.L. Anderson G.R. J. Virol. 1995; 69: 6335-6341Google Scholar). The importance of HIF-1 in the regulation of such genes has been confirmed by the reduction or abrogation of hypoxia-inducible expression in mutant cells (13Hankinson O. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 373-376Google Scholar, 14Hoffman E.C. Reyes H. Chu F.-F. Sander F. Conley L.H. Brooks B.A. Hankinson O. Science. 1991; 252: 954-958Google Scholar) that are unable to form a functional HIF complex (15Wood S.M. Gleadle J.M. Pugh C.W. Hankinson O. Ratcliffe P.J. J. Biol. Chem. 1996; 271: 15117-15123Google Scholar, 16Li H. Ko H.P. Whitlock Jr., J.P. J. Biol. Chem. 1996; 271: 21262-21267Google Scholar, 17Gradin K. McGuire J. Wenger R.H. Kvietikova I. Whitelaw M.L. Toftgard R. Tora L. Gassmann M. Poellinger L. Mol. Cell. Biol. 1996; 16: 5221-5231Google Scholar, 18Forsythe J.A. Jiang B.H. Iyer N.V. Agani F. Leung S.W. Koos R.D. Semenza G.L. Mol. Cell. Biol. 1996; 16: 4604-4613Google Scholar, 19Salceda S. Beck I. Caro J. Arch. Biochem. Biophys. 1996; 334: 389-394Google Scholar). Together these studies have provided strong evidence for a critical role for HIF-1 in the regulation of genes involved in a variety of important biological processes that include glucose transport and metabolism, vascular growth, vasomotor regulation, erythropoiesis, iron metabolism, and catecholamine synthesis (reviewed in Ref. 5Bunn H.F. Poyton R.O. Physiol. Rev. 1996; 76: 839-885Google Scholar).As is observed for HIF-1-responsive genes (20Goldberg M.A. Dunning S.P. Bunn H.F. Science. 1988; 242: 1412-1415Google Scholar, 21Goldberg M.A. Schneider T.J. J. Biol. Chem. 1994; 269: 4355-4359Google Scholar, 22Gleadle J.M. Ebert B.L. Firth J.D. Ratcliffe P.J. Am. J. Physiol. 1995; 268: C1362-C1368Google Scholar), the HIF-1 complex is inducible by particular transition elements such as cobaltous ions and by iron chelating agents such as desferrioxamine (DFO) but not by inhibitors of mitochondrial respiration such as cyanide or azide (3Wang G.L. Semenza G.L. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 4304-4308Google Scholar, 8Ebert B.L. Firth J.D. Ratcliffe P.J. J. Biol. Chem. 1995; 270: 29083-29089Google Scholar, 23Wang G.L. Semenza G.L. Blood. 1993; 82: 3610-3615Google Scholar). These distinctive features have led to the proposal of a specific oxygen sensing mechanism underlying these responses, most probably involving the operation of a ferroprotein sensor (20Goldberg M.A. Dunning S.P. Bunn H.F. Science. 1988; 242: 1412-1415Google Scholar). Recent affinity purification and molecular cloning of HIF-1 (24Wang G.L. Jiang B.-H. Rue E.A. Semenza G.L. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5510-5514Google Scholar) has revealed that the DNA binding complex consists of a heterodimer of two basic-helix-loop-helix Per-AHR-ARNT-Sim proteins HIF-1α and HIF-1β, a molecule that is identical to the aryl hydrocarbon receptor nuclear translocator (ARNT) (25Reyes H. Reisz-Porszasz S. Hankinson O. Science. 1992; 256: 1193-1195Google Scholar). An important goal is now to define the regions of the HIF-1 molecules that are responsible for their regulated activity and to understand the mechanism by which the complex is induced and activated in hypoxic cells.Since in the majority of studies, HIF-1 activation does not appear to be mediated through regulation of its mRNAs (15Wood S.M. Gleadle J.M. Pugh C.W. Hankinson O. Ratcliffe P.J. J. Biol. Chem. 1996; 271: 15117-15123Google Scholar, 17Gradin K. McGuire J. Wenger R.H. Kvietikova I. Whitelaw M.L. Toftgard R. Tora L. Gassmann M. Poellinger L. Mol. Cell. Biol. 1996; 16: 5221-5231Google Scholar, 26Huang L.E. Arany Z. Livingston D.M. Bunn H.F. J. Biol. Chem. 1996; 271: 32253-32259Google Scholar), we focused our analysis on other possible mechanisms of regulation. As with other transcription factors, studies of the regulatory mechanisms are potentially complicated by the ultimate dependence of transcriptional activation on a series of interrelated events which may include nuclear accumulation, dimerization, DNA binding, co-factor recruitment, and transactivation. For HIF-1, a further difficulty in this analysis lies in the operation of the native system in all cells so far tested (2Maxwell P.H. Pugh C.W. Ratcliffe P.J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 2423-2427Google Scholar, 3Wang G.L. Semenza G.L. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 4304-4308Google Scholar, 27Beck I. Weinmann R. Caro J. Blood. 1993; 82: 704-711Google Scholar, 28Pugh C.W. Ebert B.L. Ebrahim O. Ratcliffe P.J. Biochim. Biophys. Acta. 1994; 1217: 297-306Google Scholar). For these reasons we used the construction of chimeric genes to define regions of the HIF-1 genes that could confer oxygen-regulated behavior on heterologous transcription factors. Two types of chimeric gene were produced, those in which the heterologous transcription factor encoded nuclear localization and DNA binding, but lacked intrinsic transactivation potential, and others in which an activation domain was either intrinsic to the heterologous gene or added to the chimeric gene from a second heterologous gene. This allowed for both the definition of activation domains of HIF-1 genes and analysis of regulatory domains that did not necessarily contain intrinsic transactivation potential.Sequences from HIF-1α but not HIF-1β/ARNT conveyed inducible activity on heterologous transcription factors, and two regions within the C-terminal portion of the HIF-1α molecule were defined, each of which possessed transactivation potential and each of which could act independently to convey inducible properties. Both domains were responsive to cobaltous ions and iron chelation as well as hypoxia. The inducible activity of one regulatory domain, but not the other, appeared to be closely connected with modulation of the level of the encoded fusion protein, most probably arising from an effect on protein stability. These studies therefore define the existence of more than one regulatory domain in the HIF-1α subunit and strongly suggest the operation of more than one mechanism of regulation." @default.
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• W1964918262 title "Activation of Hypoxia-inducible Factor-1; Definition of Regulatory Domains within the α Subunit" @default.
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