Oxygen-regulated and Transactivating Domains in Endothelial PAS Protein 1: Comparison with Hypoxia-inducible Factor-1α
1999; Elsevier BV; Volume: 274; Issue: 4 Linguagem: Inglês
10.1074/jbc.274.4.2060
ISSN1083-351X
AutoresJohn F. O’Rourke, Ya‐Min Tian, Peter J. Ratcliffe, Christopher W. Pugh,
Tópico(s)Nitric Oxide and Endothelin Effects
ResumoEndothelial PAS protein 1 (EPAS1) is a basic helix-loop-helix Per-AHR-ARNT-Sim transcription factor related to hypoxia-inducible factor-1α (HIF-1α). 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-1α 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-1α, and inducibility was strikingly reduced as their expression level was increased. Despite these quantitative differences, EPAS1 regulation appeared similar to HIF-1α, conforming to a model involving the modulation of both protein level and activity, through distinct internal and C-terminal domains. Endothelial PAS protein 1 (EPAS1) is a basic helix-loop-helix Per-AHR-ARNT-Sim transcription factor related to hypoxia-inducible factor-1α (HIF-1α). 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-1α 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-1α, and inducibility was strikingly reduced as their expression level was increased. Despite these quantitative differences, EPAS1 regulation appeared similar to HIF-1α, conforming to a model involving the modulation of both protein level and activity, through distinct internal and C-terminal domains. hypoxia-inducible factor-1 the α subunit of HIF-1 Per-AHR-ARNT-Sim endothelial PAS protein, also known as member of PAS superfamily 2 (MOP2), HIF-like factor (HLF), and HIF-related factor (HRF) aryl hydrocarbon receptor nuclear translocator (identical to HIF-1β) aryl hydrocarbon receptor transactivation domain from the herpes simplex virus protein 16 (amino acids 410–490) the N-terminal 147 amino acids of the yeast transcription factor, GAL4. Hypoxia-inducible factor-1 (HIF-1)1 is a transcriptional complex that plays a central role in oxygen-regulated gene expression (reviewed in Refs. 1Bunn H.F. Poyton R.O. Physiol. Rev. 1996; 76: 839-885Crossref PubMed Scopus (1045) Google Scholar, 2Semenza G.L. J. Lab. Clin. Med. 1998; 131: 207-214Abstract Full Text PDF PubMed Scopus (185) Google Scholar, 3Gleadle J.M. Ratcliffe P.J. Mol. Med. Today. 1998; 4: 122-129Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). Affinity purification and molecular cloning of HIF-1 has revealed that the DNA binding complex consists of a heterodimer of proteins, HIF-1α and the aryl hydrocarbon receptor nuclear translocator (ARNT) (4Wang G.L. Jiang B.-H. Rue E.A. Semenza G.L. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5510-5514Crossref PubMed Scopus (5030) Google Scholar). Both are members of the rapidly expanding PAS superfamily of basic helix-loop-helix proteins defined by the presence of two regions containing repeated sequences that share homology with the prototypical members from which the family's name is derived, drosophila periodic, the aryl hydrocarbon receptor, the aryl hydrocarbon receptor nuclear translocator and drosophila single minded (5Huang Z.J. Edery I. Rosbash M. Nature. 1993; 364: 259-262Crossref PubMed Scopus (414) Google Scholar). HIF-1 binds to hypoxia response elements containing the consensus BRCGTGV and activates the transcription of a wide variety of genes that encode products involved in hematopoiesis (erythropoietin), angiogenesis, and vasomotor control (vascular endothelial growth factor, nitric oxide synthases, and endothelins), energy metabolism (glycolytic enzymes and glucose transporters), catecholamine synthesis (tyrosine hydroxylase), and iron metabolism (transferrin) (for reviews see Refs. 1Bunn H.F. Poyton R.O. Physiol. Rev. 1996; 76: 839-885Crossref PubMed Scopus (1045) Google Scholar, 2Semenza G.L. J. Lab. Clin. Med. 1998; 131: 207-214Abstract Full Text PDF PubMed Scopus (185) Google Scholar, 3Gleadle J.M. Ratcliffe P.J. Mol. Med. Today. 1998; 4: 122-129Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). HIF-1 activation is mediated predominantly by post-translational processes affecting the α subunit (6Huang L.E. Arany Z. Livingston D.M. Bunn H.F. J. Biol. Chem. 1996; 271: 32253-32259Abstract Full Text Full Text PDF PubMed Scopus (1021) Google Scholar, 7Pugh C.W. O'Rourke J.F. Nagao M. Gleadle J.M. Ratcliffe P.J. J. Biol. Chem. 1997; 272: 11205-11214Abstract Full Text Full Text PDF PubMed Scopus (447) Google Scholar, 8Salceda S. Caro J. J. Biol. Chem. 1997; 272: 22642-22647Abstract Full Text Full Text PDF PubMed Scopus (1403) Google Scholar, 9Jiang B.-H. Zheng J.Z. Leung S.W. Roe R. Semenza G.L. J. Biol. Chem. 1997; 272: 19253-19260Abstract Full Text Full Text PDF PubMed Scopus (544) Google Scholar, 10Kallio P.J. Pongratz I. Gradin K. McGuire J. Poellinger L. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5667-5672Crossref PubMed Scopus (338) Google Scholar, 11Huang L.E. Gu J. Schau M. Bunn H.F. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7987-7992Crossref PubMed Scopus (1845) Google Scholar). Understanding the interactions of these processes with the sensing and/or signal transduction processes is an important but potentially complex issue in which primary points of interaction need to be defined and distinguished from processes that are downstream consequences of such interactions. A key step in such analyses is the definition of functional domains within the molecules, in particular, regions that can independently confer the regulatory characteristic on a heterologous system. Several groups have now analyzed aspects of HIF-1α regulation and defined domains that can independently convey oxygen-regulated properties onto heterologous transcription factors such as the yeast GAL4 DNA binding domain (Gal) (7Pugh C.W. O'Rourke J.F. Nagao M. Gleadle J.M. Ratcliffe P.J. J. Biol. Chem. 1997; 272: 11205-11214Abstract Full Text Full Text PDF PubMed Scopus (447) Google Scholar, 9Jiang B.-H. Zheng J.Z. Leung S.W. Roe R. Semenza G.L. J. Biol. Chem. 1997; 272: 19253-19260Abstract Full Text Full Text PDF PubMed Scopus (544) Google Scholar, 11Huang L.E. Gu J. Schau M. Bunn H.F. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7987-7992Crossref PubMed Scopus (1845) Google Scholar, 12Li H. Ko H.P. Whitlock Jr., J.P. J. Biol. Chem. 1996; 271: 21262-21267Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). Recent cloning experiments have identified several new members of the basic helix-loop-helix PAS family, the most similar to HIF-1α being a molecule first described as endothelial PAS protein 1 (EPAS1) (13Tian H. McKnight S.L. Russell D.W. Genes Dev. 1997; 11: 72-82Crossref PubMed Scopus (1064) Google Scholar), but also independently identified by other groups and termed member of PAS superfamily 2 (MOP2) (14Hogenesch J.B. Chan W.K. Jackiw V.H. Brown R.C. Gu Y.-Z. Pray-Grant M. Perdew G.H. Bradfield C.A. J. Biol. Chem. 1997; 272: 8581-8593Abstract Full Text Full Text PDF PubMed Scopus (392) Google Scholar), HIF-like factor (HLF) (15Ema M. Taya S. Yokotani N. Sogawa K. Matsuda Y. Fujii-Kuriyama Y. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4273-4278Crossref PubMed Scopus (841) Google Scholar), and HIF-related factor (HRF) (16Flamme I. Fröhlich T. von Reutern M. Kappel A. Damert A. Risau W. Mech. Dev. 1997; 63: 51-60Crossref PubMed Scopus (296) Google Scholar). The protein shares 48% sequence identity with HIF-1α, forms heterodimers with ARNT, and can activate transcription from a hypoxia response element (13Tian H. McKnight S.L. Russell D.W. Genes Dev. 1997; 11: 72-82Crossref PubMed Scopus (1064) Google Scholar). In hypoxic cells, EPAS1 protein levels are greatly up-regulated (17Wiesener M.S. Turley H. Allen W.E. Willam C. Eckardt K.-U. Talks K.L. Wood S.M. Gatter K.C. Harris A.L. Pugh C.W. Ratcliffe P.J. Maxwell P.H. Blood. 1998; 92: 2260-2268Crossref PubMed Google Scholar). Moreover, responses to chemical and pharmacological probes with known effects on HIF-1 activation are very similar (17Wiesener M.S. Turley H. Allen W.E. Willam C. Eckardt K.-U. Talks K.L. Wood S.M. Gatter K.C. Harris A.L. Pugh C.W. Ratcliffe P.J. Maxwell P.H. Blood. 1998; 92: 2260-2268Crossref PubMed Google Scholar), suggesting that one or more regulatory mechanisms are shared, and indicating that it should be informative to define regulatory and activation domains in EPAS1, and to compare their function with those in HIF-1α. Here we show that fusion of EPAS1 sequences with Gal can confer hypoxia-inducible activity on a GAL-responsive reporter. Our analysis defines two transactivation domains for EPAS1 (a C-terminal transactivation domain and an internal transactivation domain), which are interspersed with sequences that possess repressive and regulatory properties, some of which can confer regulation of fusion protein levels. Overall these findings demonstrate a similar domain architecture to HIF-1α. However, some Gal/EPAS1 fusions showed higher activity in normoxic cells and a lower amplitude of induction, particularly at higher levels of expression, indicating that there are quantitative differences in the activation characteristics of these molecules. Hep3B cells were grown in minimal essential medium supplemented with 10% fetal calf serum, glutamine (2 mm), penicillin (50 IU/ml) and streptomycin sulfate (50 μg/ml). The plasmids used are shown schematically in Fig. 1. The chimeric activator/reporter system used in transactivation assays was based on pGal (a plasmid based on pcDNA3, which contains an SV40 origin of replication and a cytomegalovirus promoted, truncated GAL4 gene encoding amino acids 1–147 followed by a polylinker bearing the rare restriction endonuclease sites, SacII,AscI, NotI), and the GAL4-responsive luciferase reporter pUAS-tk-Luc (consisting of two copies of a 17-base pair Gal4 DNA binding site and the thymidine kinase promoter, −105 to +50 (18Webster N. Jin J.R. Green S. Hollis M. Chambon P. Cell. 1988; 52: 169-178Abstract Full Text PDF PubMed Scopus (229) Google Scholar), inserted into the HindIII site of pA3LUC) (19Wood W.M. Kao M.Y. Gordon D. Ridgway E.C. J. Biol. Chem. 1989; 264: 14840-14847Abstract Full Text PDF PubMed Google Scholar). To analyze the function of sequences from EPAS1 or HIF-1α, they were amplified by polymerase chain reaction using Pfu polymerase (Stratagene, La Jolla, CA) and forward oligonucleotides containing aSacII recognition sequence in the appropriate reading frame and reverse oligonucleotides containing an AscI recognition site and cloned into pGal. phEP-1 was used as template for EPAS1 (13Tian H. McKnight S.L. Russell D.W. Genes Dev. 1997; 11: 72-82Crossref PubMed Scopus (1064) Google Scholar) and pBluescript/HIF-1α 3.2–3T7 (4Wang G.L. Jiang B.-H. Rue E.A. Semenza G.L. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5510-5514Crossref PubMed Scopus (5030) Google Scholar) as template for HIF-1α sequences. To analyze the regulatory function of EPAS1 or HIF-1α amino acids on the operation of a heterologous activation domain, sequences coding for the herpes simplex virus protein 16 amino acids 410–490 (VP16) were generated by polymerase chain reaction using Pfu polymerase with priming oligonucleotides incorporating in frame AscI and NotI restriction sites, and inserted 3′ to the EPAS1 sequence. The control plasmid pGal/VP16 was produced by insertion of this polymerase chain reaction product directly into pGal, preserving the reading frame. All plasmids were subjected to in vitrotranscription/translation reactions in the presence of35S-methionine and products analyzed by SDS-polyacrylamide gel electrophoresis to confirm the production of an appropriate fusion protein. Plasmids bearing mutations were generated using a commercially available site-directed mutagenesis kit (QuikChange; Stratagene) and mutagenic oligonucleotides designed according to the manufacturer's recommendations. Mutations in HIF-1α and EPAS1 were made in the context of pCOTG/α775–826 (7Pugh C.W. O'Rourke J.F. Nagao M. Gleadle J.M. Ratcliffe P.J. J. Biol. Chem. 1997; 272: 11205-11214Abstract Full Text Full Text PDF PubMed Scopus (447) Google Scholar) and pGal/EPAS819–870, respectively. These mutations were sequenced by the dideoxy method to confirm veracity. A cytomegalovirus-promoted plasmid constitutively expressing β-galactosidase (pCMVβGal) was used as a transfection control. For transactivation assays, cells were transfected by electroporation using a 1 mF capacitor array charged at 375 V. For each transfection, approximately 107 cells were resuspended in 1 ml of RPMI 1640 containing a mixture of activator plasmid (ranging between 50 ng and 20 μg), reporter plasmid (50 μg) and the transfection control plasmid pCMVβGal (15 μg). After discharge of the capacitor, cells were left on ice for 10 min before being resuspended in the appropriate culture medium. Aliquots of this suspension were then used for parallel incubations. Conditions used for normoxic and hypoxic incubation were 5% CO2, balance air, and 1% O2, 5% CO2, balance N2, respectively. Chemicals were used at the following final concentrations: cobaltous chloride, 100 μm; desferrioxamine mesylate, 100 μm; potassium cyanide, 1 mm; and sodium azide, 2 mm. Experimental incubations were for 16–18 h. All activator plasmids were tested in at least three independent transfection experiments. Luciferase activities in cell lysates were determined at room temperature using a commercially available luciferase assay system (Promega, Madison, WI), according to the manufacturer's instructions, and a TD-20e luminometer (Turner Designs, Sunnyvale, CA). Relative β-galactosidase activity in lysates was measured usingo-nitrophenyl-β-d-galactopyranoside (0.67 mg/ml) as substrate in a 0.1 m phosphate buffer (pH 7.0) containing 10 mm KCl, 1 mm MgSO4, and 30 mm β-mercaptoethanol incubated at 30 °C for 15–45 min. The A 420 was determined after stopping the reaction by the addition of 0.3 m sodium carbonate (final concentration). To achieve fusion protein levels sufficient for detection of certain activator plasmids, plasmids were amplified by co-transfection with a plasmid expressing the SV40 large T antigen, pCMV-TAg (20de Chasseval R. de Villartay J.-P. Nucleic Acids Res. 1992; 20: 245-250Crossref PubMed Scopus (24) Google Scholar). Transfections were performed as above using doses of activator and amplifier plasmids ranging between 0.02 and 7 μg and 0.05 and 10 μg, respectively, to identify the range in which protein levels were detectable. After transfection cells were incubated for 48 h to allow plasmid amplification and expression. Cells were incubated in normoxia throughout or stimulated by addition of 100 μmdesferrioxamine to the medium for the final 16 h. At harvest, cells were cooled rapidly by rinsing with ice-cold phosphate-buffered saline, and removed by scraping with a rubber policeman. An ice-cold 7m urea, 10% glycerol, 1% SDS, 10 mm Tris, pH 6.8, buffer containing 5 mm dithiothreitol, 50 μm phenylmethylsulfonyl fluoride, and leupeptin, pepstatin, and aprotinin all at 0.1 μg/ml was added to the cell pellet, which was then disrupted using a hand-held homogenizer (Ultra-Turrax T8 with 5G dispersing tool; Janke & Kunkel GmbH, Staufen, Germany) for 20 s and then allowed to stand on ice for 5 min. Extract was either snap frozen on dry ice for storage or mixed with an equal volume of 2× Laemmli sample buffer before SDS-polyacrylamide gel electrophoresis. Proteins were transferred onto Immobilon-P membrane (Millipore, Bedford, MA) by electrophoresis overnight at 20 V in Towbin buffer containing 10% methanol and 0.005% SDS. Membranes were blocked using phosphate-buffered saline supplemented with 5% dry milk powder and 0.1% Tween 20 before indirect immunostaining. Proteins were labeled with mouse monoclonal antibodies directed against either the GAL4 DNA binding domain (RK5C1; Santa Cruz Biotechnology) or EPAS1 amino acids 535–631 (190b) (17Wiesener M.S. Turley H. Allen W.E. Willam C. Eckardt K.-U. Talks K.L. Wood S.M. Gatter K.C. Harris A.L. Pugh C.W. Ratcliffe P.J. Maxwell P.H. Blood. 1998; 92: 2260-2268Crossref PubMed Google Scholar) followed by peroxidase-conjugated swine anti-mouse immunoglobulin (DAKO). Peroxidase activity was detected by enhanced chemiluminescence (Super Signal Ultra; Pierce). As a first step in the definition of regulatory domains and transactivation domains in EPAS1, a plasmid expressing a fusion protein consisting of Gal linked to EPAS1 amino acids 19–870 was constructed (pGal/EPAS19–870), and activity was tested by co-transfection with a Gal4-responsive reporter gene (pUAS-tk-Luc) into Hep3B cells. For comparison, the activity of a similar fusion between Gal and HIF-1α (pGal/α28–826) was tested. Both EPAS1 and HIF-1α sequences conferred transcriptional activity on the GAL4 DNA binding domain, and in each case, activity was inducible by hypoxia. Induction was also obtained by exposure of transfected cells to cobaltous ions or desferrioxamine (Fig. 2) but not with exposure to the mitochondrial inhibitors, azide and cyanide (not shown), a pattern that is in keeping with the known characteristics of HIF-1 and HIF-1 target gene activation (21Goldberg M.A. Dunning S.P. Bunn H.F. Science. 1988; 242: 1412-1415Crossref PubMed Scopus (874) Google Scholar, 22Gleadle J.M. Ebert B.L. Firth J.D. Ratcliffe P.J. Am. J. Physiol. 1995; 268: C1362-C1368Crossref PubMed Google Scholar, 23Wang G.L. Semenza G.L. Blood. 1993; 82: 3610-3615Crossref PubMed Google Scholar, 24Ebert B.L. Firth J.D. Ratcliffe P.J. J. Biol. Chem. 1995; 270: 29083-29089Abstract Full Text Full Text PDF PubMed Scopus (447) Google Scholar). Despite these similarities, there were substantial differences in the amplitude of induction shown by the fusion proteins, with the EPAS1 fusion showing higher normoxic activity than the HIF-1α fusion, but less potent induction by all three stimuli. Because we had noted in previous experiments that the amplitude of induction by hypoxia of certain Gal/HIF-1α fusions varied with the level of expression and was less in cells transfected either with large or very small amounts of plasmid, we compared the inducible activity of both plasmids over a wide range of transfection doses. Results illustrated in Fig. 2 show that for the EPAS1 fusion, reporter activity was maximal and appeared to saturate at plasmid doses from 1 μg upward. The maximum amplitude of induction occurred at 0.25 μg and was much reduced when the highest dose of plasmid was used. In contrast, the HIF-1α fusion was not responsive at the lowest plasmid dose, manifested highly inducible activity over the remainder of the dose range, and showed signs of saturation with a reduction in the amplitude of induction only at the highest dose tested. To define the regions of the EPAS1 gene that were responsible for transactivation and those that conveyed inducible responses on the GAL4 DNA binding domain, deletions were made, which removed either N-terminal or C-terminal sequence from EPAS1. A series of five C terminus deletions of EPAS1 was first tested in Hep3B cells co-transfected with pUAS-tk-luc (Fig.3 A). Whereas chimeric genes containing deletions to amino acids 819 and 682 (pGal/EPAS19–819 and pGal/EPAS19–682) showed inducible transactivation comparable with that of the entire EPAS1 fusion (pGal/EPAS19–870), further C-terminal deletions to amino acids 551, 495, and 416 removed most or all activity. This indicated the existence of a powerful transactivation domain lying N-terminal to amino acid 682, and that sequences between amino acids 551 and 682 were necessary for this function. The inducible activity of pGal/EPAS19–682 indicated the presence of at least one domain capable of conveying inducible responses that lies N-terminal to amino acid 682, but given the lack of transactivator function in the C-terminal deletions to amino acids 551, 495, and 416, these experiments did not define the domains responsible for this regulation further. Seven N-terminal deletions of EPAS1 coding sequence were tested in a similar manner (Fig. 3 B). Striking differences in the activity of these constructs were observed. Deletion of amino acids 19 to 495 produced a large increase in activity, particularly in normoxic cells, suggesting that this region contains sequences capable of exerting a functionally suppressive effect (compare pGal/EPAS19–870 and pGal/EPAS495–870). Whereas further deletion to amino acid 517 caused only a small effect on activity, deletion to amino acid 551 (pGal/EPAS517–870) produced a complete loss of this transactivating function, suggesting that the N-terminal boundary of this transactivating region resides between 517 and 551. With further N-terminal deletions an increase in activity was observed indicating the existence of a second transactivation domain capable of operation in isolation, at the C terminus of EPAS1. Comparison of the activities of pGal/EPAS551–870, pGal/EPAS682–870, pGal/EPAS724–870, pGal/EPAS819–870, and pGal/EPAS828–870 indicated that this transactivating function was contained within amino acids 828–870, whereas amino acids 682–827 were functionally repressive. An inducible response was observed with both plasmids pGal/EPAS724–870 and pGal/EPAS819–870 but not plasmid pGal/EPAS828–870 locating a minimal sequence, which could confer inducible behavior on this transactivator between amino acids 819 and 828. In view of this evidence for regulatory C-terminal sequences, the low amplitude of induction observed for highly active plasmids pGal/EPAS495–870 and pGal/EPAS517–870 appeared surprising. We therefore retested pGal/EPAS495–870 using low doses as described above, and noted that as overall activity was reduced there was a substantial increase in the amplitude of the inducible response (TableI).Table IDependence of the amplitude of induction of a Gal fusion protein containing EPAS495–870 on the dose of activator plasmid usedPlasmid DoseNormoxic ActivityFold induction byHypoxiaCobaltDesferrioxamineμg0.0516.04.44.711.30.2553.54.95.015.21.02312.72.26.75.05341.60.72.8204971.20.41.6A co-transfection was performed as described in Fig. 3 using the indicated doses of the activator plasmid expressing amino acids 495–870 of EPAS1 fused to the GAL4 DNA binding domain. The corrected, relative normoxic activity is shown in column 2. Fold induction was calculated as a ratio of the corrected luciferase counts produced by cells stimulated with hypoxia, 100 μm cobaltous ions or 100 μm desferrioxamine to those obtained when the cells were cultured without stimulation. Open table in a new tab A co-transfection was performed as described in Fig. 3 using the indicated doses of the activator plasmid expressing amino acids 495–870 of EPAS1 fused to the GAL4 DNA binding domain. The corrected, relative normoxic activity is shown in column 2. Fold induction was calculated as a ratio of the corrected luciferase counts produced by cells stimulated with hypoxia, 100 μm cobaltous ions or 100 μm desferrioxamine to those obtained when the cells were cultured without stimulation. The combined results of the N-terminal and C-terminal deletions of EPAS1 sequence suggested the presence of two domains in EPAS possessing both regulatory and transactivating potential; a powerful internal domain contained within exon 11 (amino acids 517–682) and a weaker domain contained within the C-terminal exon (amino acids 819–870). These experiments also demonstrated the presence of two regions capable of exerting functionally suppressive effects on transactivation, sequences N-terminal to amino acid 495 and sequences lying between amino acids 682 and 819. To test whether the internal transactivation domain could function independently and to define the functional sequences in more detail, a further series of plasmids was constructed and tested in Hep3B cells. pGal/EPAS517–682 showed powerful inducible transactivation, confirming that this internal transactivation domain could function independently and indicating that it also contained regulatory sequences. Deletions from the C terminus and N terminus of this region both produced a decrease in activity, indicating that this entire domain was necessary for maximal activity (Fig. 4). Interestingly, the N-terminal deletion (pGal/EPAS534–682) showed enhanced transactivation in normoxic cells and complete loss of the inducible response, defining a short sequence (amino acids 517–534) as critical for the regulatory property. Though experiments described above indicated that the N-terminal 495 amino acids of EPAS1 sequence had functionally suppressive effects on the internal and C-terminal transactivation domains and might contribute to regulation, such a function could not be demonstrated independently because deletion of the native transactivation domains of EPAS1 removed all activity. To enable further analysis of this possibility, a second set of fusions was created in which the powerful C-terminal 80 amino acid transactivator from herpes simplex virus protein 16 (VP16) was linked to the C terminus of the Gal/EPAS1 fusion proteins. First, the N-terminal sequence of EPAS1 (amino acids 9 to 517) was tested by creation of pGal/EPAS9–517/VP16. When compared with the activity of a plasmid encoding the Gal/VP16 fusion alone, inclusion of EPAS1 amino acids 9 to 517 had a profoundly suppressive effect on transactivation in normoxic conditions. On stimulation by hypoxia, cobaltous ions, or desferrioxamine the extent of repression was reduced (Fig. 5 A), indicating that this sequence could convey regulatory effects in isolation and that these effects could be conveyed on a heterologous transactivator. Other portions of the EPAS1 molecule were assayed for regulatory activity in a similar way by inserting three portions of EPAS1 sequence (corresponding to exons 2–6, 7–11, and 12–15, and covering all except the N-terminal 8 amino acids) between the Gal and VP16 domains (pGal/EPAS9–295/VP16, pGal/EPAS295–682/VP16, and pGal/EPAS682–870/VP16). Results are shown in Fig. 5 B. In comparison with pGal/VP16, both pGal/EPAS9–295/VP16 and pGal/EPAS682–870/VP16 had rather similar activity, which was not inducible. In contrast, pGal/EPAS295–682/VP16 had much reduced activity in normoxic cells and showed induction by all three stimuli. These results suggested that repressive and regulatory properties of the internal domain of EPAS1 could be conferred on a heterologous transactivator. In contrast, comparison of the activity of pGal/EPAS682–870/VP16 with the simple Gal/EPAS fusions (Fig.3 B) indicated that sequences from the C-terminal domain of EPAS1, which exerted powerfully repressive and regulatory effects on the native C-terminal transactivation domain, had little or no such effects on the VP16 transactivator. In an attempt to pin-point shorter sequences with regulatory potential, sequences corresponding to exons 7–11 were individually inserted in-frame into the Gal/VP16 fusion and tested in an analogous manner. To maximize the chance of observing subsequences with the ability to convey regulation in isolation, plasmids were tested at several doses. Results are shown for cells transfected with 100 ng in Fig.5 C; identical results were obtained with 10 ng. Though sequences corresponding to exon 7 were suppressive in this assay, no individual exon conveyed regulation at any dose with the exception of exon 11 (amino acids 517–682). Finally, the effect of removing individual exons from the internal domain (exons 7–11) was tested (Fig. 6). Removal of exon 11 (creating pGal/EPAS295–517/VP16) led to much higher activity in normoxic cells and greatly reduced inducibility. Removal of successive exons from the N terminus led to a more progressive increase in activity in normoxic cells and a progressive reduction in the extent of induction. The results therefore suggest that multiple sequences within exons 7–11 contribute to regulatory properties of this domain. Sequences lying both N-terminal and C-terminal to amino acid 517 were able to confer the inducible property in isolation, though the effect was only modest for
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