1 Department of Pharmacology and Heart Research Institute, BK21 Human Life Sciences, Seoul National University College of Medicine, 28 Yongon-dong, Chongno-gu, Seoul 110-799, Korea
2 Institute of Animal Science and Technology, College of Agriculture and Life Sciences, Seoul National University, Suwon 441-744, Korea
*Author for correspondence (e-mail: parkjw{at}plaza.snu.ac.kr)
Accepted July 13, 2001
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SUMMARY |
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Key words: HIF-1, ARNT, Zinc ion, Dominant-negative isoform
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INTRODUCTION |
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HIF-1, an 826 amino acid protein, is unique to HIF-1, whereas ARNT is a common partner and is able to dimerize with the aryl hydrocarbon receptor and other bHLH-PAS proteins (Wang et al., 1995). The bHLH and PAS domains comprise the N-terminal halves of both HIF-1
and ARNT, which are required for dimerization and DNA binding (Jiang et al., 1996). The C-terminal half of both proteins is required for transactivation. In the case of HIF-1
, its transactivation domains are localized to two amino acid residues 531-575 (N-terminal TAD) and 786-826 (C-terminal TAD), which are separated by an inhibitory domain (Jiang et al., 1997). Two nuclear localization signals (NLSs) are localized to the N-terminal (amino acids 17-74) and the C-terminal parts (amino acids 718-721). The C-terminal NLS motif of HIF-1
plays a crucial role in mediating hypoxia-inducible nuclear import of the protein, whereas the N-terminal NLS motif may be less important (Kallio et al., 1998). In addition, HIF-1
contains an oxygen-dependent degradation (ODD) domain, which is localized to amino acid residues 401-603 (Huang et al., 1998). The ODD domain is suggested to control HIF-1
degradation by the ubiquitin-proteasome pathway because its deletion makes HIF-1
stable even under normoxic conditions (Huang et al., 1998) (see the HIF-1
structure in Fig. 2).
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Here, we identify for the first time a new alternative splicing variant of human HIF-1 that is induced by zinc treatment. The difference between the new variant and wild-type HIF-1
is its lack of the 12th exon, which produces a frame-shift and a shorter form of HIF-1
. In the corresponding protein, the bHLH-PAS structure and the N-terminal NLS motif are conserved, but a part of the ODD domain, both the TADs and the C-terminal NLS motif are removed. The expressed HIF-1
variant loses transactivation activity and hypoxia-inducible nuclear import, as might be expected from its protein structure. We have also demonstrated that the HIF-1
variant functions as a dominant-negative isoform and inhibits HIF-1-regulated gene induction under hypoxic conditions.
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MATERIALS AND METHODS |
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Cloning and sequencing of a human HIF-1 cDNA isoform
The full length HIF-1 cDNA isoform was determined by RT-PCR. RNAs were extracted from HEK 293 cells treated with 500 µM ZnCl2 for 4 hours and reverse-transcribed using the avian myeloblastosis virus reverse transcriptase system (Promega). This template was amplified using two primers located 24 bases upstream from the start codon and at the stop codon of the human HIF-1
coding sequence (sense, 5-AGACATCGCGGGGACCGATT-3; antisense, 5-TCAG-TTAACTTGATCCAAAGCTCT-3). The PCR products of the full-length HIF-1
cDNAs were cloned using a pCR2.1-TOPO cloning kit (Invitrogen). To select a colony of E. coli transformed with the full-length HIF-1
cDNA isoform, colony PCRs using the specific primers (5-CCCCAGATTCAGGATCAGACA-3 and 5-CCATCATGTTCCATTTTTCGC-3) were performed. The plasmid containing the full-length HIF-1
cDNA isoform was amplified and purified, and the insert DNA sequence analyzed.
Expression plasmids and the establishment of a transfected cell line
Haemagglutinin (HA)-tagged HIF-1 and ARNT expression plasmids (pcDNA3) were generous gifts from Eric Huang and Jie Gu. HA-tagged HIF-1
variant expression plasmid was made using a PCR-based mutagenesis kit (Stratagene). Mutagenesis with specific oligonucleotides (sense, 5-AACTACAGTTCCTGAGGAAGA-3; antisense, 5-CTGAGTAGAAAATGGGTTCTT-3) was employed to delete the nucleotides derived from the 12th exon. The fidelity of the PCR and identity of the construct were confirmed by sequencing the insert.
HEK 293 cells were stably transfected with the pcDNA3 or pcDNA3-HA/HIF-1 variant using the calcium phosphate method. Transfected cells were incubated for 36 hours in supplemented Dulbeccos modified Eagles medium and then 0.45 mg/ml of G418 was added to select the transfected cells. Stable transfectants from three different transfections were pooled after 30 days to avoid bias in gene expression due to variable sites of chromosomal integration.
Reporter assays
The EPO and VEGF enhancer-driven luciferase reporter genes were constructed as previously described (Chun et al, 2000a). The synthetic DNAs coding the HIF-1-binding enhancer regions (EPO, 5-GGTACCGGCCCTACGTGCTGTCTCACACAGCCTGTCTGACCTCTCGACCTACCGGCCAGATCT-3; VEGF, 5-GTACCCACAGTGCATACGTGGGCTCCAACAGGTCCTCTTGATC-3) were inserted into the pGL3 promoter plasmid (Promega). To assay the HIF-1 activity, HEK 293 cells were cotransfected with the luciferase reporter genes and 10 µg of plasmid cytomegalovirus-ß-gal for each 100 mm dish, using the calcium phosphate method. Transfected cells were split into nine aliquots and incubated for 42 hours. After stabilizing, the cells were incubated for 16 hours at 20% or 1% O2. They were then lysed and assayed for luciferase activity using a Biocounter M1500 luminometer (Lumac). ß-gal assays were performed for normalization of transfection efficiency.
RT-PCR for HIF-1 and ß-actin
Total RNAs were isolated from HEK 293 cells by TRIZOL (GIBCO/BRL) and qualified on a denaturing agarose gel. RT-PCR was performed using a PCR-Access kit (Promega) as previously described (Chun et al., 2000b). The RT-PCR conditions used were: one cycle of reverse transcription at 48°C for 1 hour and 25 cycles of denaturation at 94°C for 30 seconds, annealing at 56°C for 30 seconds, and elongation at 68°C for 1 minute. The resulting PCR fragments (5 µl) were electrophoresed on a 2% agarose gel at 100 V in 0.5xTAE, and the gels were then stained with ethidium bromide. The nucleotide sequences of the primers are summarized in Table 1.
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Extractions of nuclear and cytosolic proteins
Nuclear proteins were extracted as previously described (Chun et al., 2000b). Cells were quickly cooled by placing the plates on ice and then washing twice in ice-cold PBS. Scraped cells were quickly cooled by placing the plates on ice and washing twice in ice-cold phosphate-buffered saline before the cells were removed by scraping. Cells were centrifuged at 1000 g for 5 minutes at 4°C and then washed twice with ice-cold phosphate-buffered saline. Cells were then resuspended in three packed cell volumes of lysis buffer consisting of 10 mM Tris, pH 7.8, 10 mM KCl, 0.1 mM EDTA, 1.5 mM MgCl2, and 0.2% Nonidet P-40, 0.5 mM dithiothreitol, 1 mM Na3VO4, and 0.4 mM phenylmethylsulfonyl fluoride. Cells were then vortexed at medium speed for 10 seconds and incubated on ice for 5 minutes. Nuclei were then pelleted at 1000 g for 5 minutes at 4°C, and the supernatant was collected for the cytosolic fraction. One packed volume of extract buffer, consisting of 20 mM Tris, pH 7.8, 420 mM NaCl, 0.1 mM EDTA, 1.5 mM MgCl2, 20% glycerol, 0.5 mM dithiothreitol, 1 mM Na3VO4, and 0.4 mM phenylmethylsulfonyl fluoride, was added to the nuclei and the whole vortexed at medium speed for 5 seconds per minute for 10 minutes. The nuclear extracts were then centrifuged at 20,000 g at 4°C for 5 minutes, aliquoted into chilled tubes, frozen quickly in liquid nitrogen and stored at 70°C. The cytosolic fraction was centrifuged at 80,000 g at 4°C for 1 hour and the resulting supernatant was stored at 70°C. Protein concentration was measured using BCA (bicinchoninic acid) method (Bio-Rad) with bovine serum albumin as a standard.
Electrophoretic Mobility Gel Shift (EMSA) assay
The oligonucleotide probe used in the gel shift assay for HIF-1 consisted of the sequence 5-ACCGGCCCTACGTGCTGTCTCAC-3. The 32P-labelled double-stranded probe was prepared and EMSA assay was performed as previously described (Chun et al., 2000b). DNA-protein binding reactions were carried out for 20 minutes at 4°C and run on a 5% non-denaturing polyacrylamide gel. For supershift analysis, 1 µl of rat HIF-1 antiserum was added to the completed EMSA reaction mixture and incubated for 2 hours at 4°C prior to loading.
Immunoblotting and immunoprecipitation
For the immunoblotting of HIF-1 and its variant protein, 20 µg of cell extract was separated on a 6% and 10% SDS/polyacrylamide gel, respectively, and transferred to an Immobilon-P membrane (Millipore). Immobilized proteins were incubated overnight at 4°C with rabbit anti-HIF-1
antibody, diluted 1:5000 in 5% nonfat milk in TBS/0.1% Tween-20 (TTBS). Horseradish peroxidase-conjugated anti-rabbit antiserum (Amersham Pharmacia Biotech) was used as a secondary antibody (1:5000 dilution in 5% nonfat milk in TTBS). After extensive washing with TTBS, the complexes were visualized by enhanced chemiluminescence plus (Amersham Pharmacia Biotech). HIF-1 antiserum was generated in rabbits against a bacterially expressed fragment encompassing amino acids 418-698 of human HIF-1
, as previously described (Chun et al., 2000a). For immunoprecipitation, HEK 293 cells were cotransfected with the pHA/HIF-1
variant and pARNT. Forty-two hours after transfection, the cells were solubilized, and the cell lysates incubated with rabbit anti-HA (SantaCruz Biotechnology) or goat anti-ARNT antibodies (SantaCruz Biotechnology), followed by incubation with protein A-Sepharose beads (Amersham Pharmacia Biotech). After washing, the immunocomplexes were eluted by boiling for 3 minutes in the SDS sample buffer containing 10 mM DTT and subjected to SDS-PAGE and immunoblotting.
Immunofluorescence microscopy
Transfected HEK 293 cells were subjected to normoxia or hypoxia (4 hours), and fixed with 4% paraformaldehyde in phosphate buffered saline (PBS) at room temperature for 15 minutes. The cells were washed three times with 100 mM glycine in PBS, permeabilized with 0.1% Triton X-100 in PBS for 1 minute and washed three times with PBS. After blocking nonspecific binding with 5% normal goat serum in PBS for 1 hour, the cells were incubated overnight at 4°C with rabbit anti-HIF-1 antibody (1:1000), rabbit anti-HA antibody (1:250) purchased from SantaCruz Biotechnology, or the mouse monoclonal anti-ARNT 2B10 antibody (1:500) purchased from Affinity BioReagents. The primary antibodies were stained by incubation with FITC-conjugated goat anti-rabbit antibody (Jackson ImmunoResearch Laboratories), FITC-conjugated goat anti-rabbit antibody (Jackson ImmunoResearch Laboratories), or FITC-conjugated goat anti-mouse antibody (Zymed Laboratories) at room temperature for 1 hour, respectively. After washing in PBS, the cells were analyzed by confocal laser scanning microscopy (BioRad MRC1024).
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RESULTS |
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Structure of HIF-1 cDNA isoform
The HIF-1 cDNA isoform was cloned and sequenced as described in Materials and Methods, and its structure is summarized in Fig. 2. The cloned cDNA was 2047 bp in size and its nucleotide sequence was identical to the 2481 bp HIF-1
cDNA, except for a 434 bp deletion in the middle. A comparison with the genomic organization of HIF-1
(Iyer et al., 1998) showed that the cloned cDNA was derived from an alternatively spliced mRNA that lacked the 12th exon of the HIF-1
gene (Fig. 2). In this mRNA, the 11th exon and the 13th exon were directly joined, which generated an immediate termination codon and a new frame in the 13th exon. Consequently, this alternative splicing introduces four new amino acids, Asn-Tyr-Ser-Ser, as a new C-terminus following the Gln553 of the wild-type 826 amino acid HIF-1
protein. This novel form of HIF-1
mRNA translates into a 557 amino acid polypeptide, which we designated HIF-1
Z. HIF-1
Z conserves both the bHLH and PAS domains of HIF-1
, which are essential for binding with ARNT. However, it loses a part of the ODD domain, TAD and NLS motif, which suggests that it may be partly regulated by oxygen tension and may have no transactivation activity and an impaired ability to translocate into the nucleus.
Detection of HIF-1Z protein in Zn-treated cells and its expression
To demonstrate the presence of HIF-1Z mRNA translation products, an immunoblotting experiment was carried out using anti-HIF-1
antibody to recognize the remaining ODD domain in HIF-1
Z. After cells were incubated with zinc ion for 8 hours, the cells were harvested and total proteins extracted with a denaturing sample buffer for SDS-PAGE. An immunoreactive protein was detected in the lysate, whereas the protein was not expressed in the control cells (Fig. 3A). When the zinc-treated cells were incubated with cycloheximide (10 µg/ml), a protein synthesis inhibitor, the protein expression diminished, indicating that the protein was newly synthesized after zinc treatment. On SDS-polyacrylamide gel, the molecular mass of HIF-1
Z was determined to be 62 kDa and matched the theoretical molecular weight (63.2 kDa) calculated using the compute pI/Mw program in ExPASy Proteomics tools, which suggests that the protein is translated from HIF-1
Z. To characterize the HIF-1
Z protein, we constructed a HA-tagged HIF-1
Z expression plasmid and transfected HEK 293 cells. The expressed HIF-1
Z protein showed the same migration pattern as the zinc-induced protein (Fig. 3B), which confirmed that the zinc-induced protein is the product of HIF-1
Z mRNA. The majority of the expressed HIF-1
Z protein was present in the cytosolic fraction; a small amount was observed in the nuclear fraction. HIF-1
Z protein was induced slightly by hypoxia, whereas wild-type HIF-1
was dramatically induced only in the nuclear fraction. To re-examine the subcellular localization and oxygen-independent expression of HIF-1
Z, we stained HIF-1
Z under normoxic or hypoxic conditions using immunofluorescence and confocal laser scanning microscopy. Expressed HIF-1
Z was not localized to the nuclei of HEK 293 cells under either normoxic or hypoxic conditions (Fig. 4, first and second rows). By contrast, HIF-1
was detected only in hypoxic cells and localized to the nuclei. The immunofluorescence images observed in a higher magnification showed that HIF-1
Z was localized to the cytoplasm, whereas HIF-1
was to the nuclei (Fig. 4, third row). These properties of HIF-1
Z represent the loss of the ODD domain and the NLS motif as expected from its protein structure, which is shown in Fig. 2.
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Recovery effect of ARNT on the HIF-1 inhibition by HIF-1Z
How does HIF-1Z inhibit the HIF-1 formation? We thought of two possible answers for this question: first, it substitutes for wild-type HIF-1
in the HIF-1 complex, and second, it inhibits the accumulation of HIF-1
or ARNT in the nucleus. Because HIF-1
Z conserves intact basic regions for DNA-binding and bHLH/PAS domains for ARNT-binding, HIF-1
Z might retain the ability to DNA-bind and dimerize with ARNT. Therefore, HIF-1
Z can substitute for HIF-1
in the HIF-1 complex in the nucleus, and a new band for the HIF-1
Z-ARNT complex might be searched for by EMSA. However, no new band was found in the EMSA of the pHIF-1
Z-transfected cell line (Fig. 6C). This finding suggests that HIF-1
Z does not compete with endogenous HIF-1
in the formation of HIF-1 in the nucleus. To examine further whether HIF-1
Z competes with HIF-1
, increasing amounts of pHIF-1
were cotransfected into a pHIF-1
Z-tranfected cell line with EPO-enhancer reporter plasmid. Although the HIF-1 activity suppressed by HIF-1
Z was somehow increased by HIF-1
, it did not fully recover (Fig. 7A). Surprisingly, however, ARNT fully restored the HIF-1 activity in hypoxic pHIF-1
Z-transfected cells (Fig. 7B). This result suggests that HIF-1
Z inhibits ARNT, but not HIF-1
. Therefore, we propose that HIF-1
Z, by sequestering ARNT, works against HIF-1 as a dominant-negative isoform.
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DISCUSSION |
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The incorporation of transition metals other than iron, such as cobalt and nickel, in a putative oxygen-sensing ferroprotein is supposed to reduce oxygen binding to the ferroprotein, resulting in HIF-1 accumulation and EPO induction (Goldberg et al., 1988; Huang et al., 1999). Although zinc can also substitute for iron in haem moiety (Shibayama et al., 1986), it does not induce EPO under normoxic conditions (Goldberg et al., 1988). On the contrary, it has been reported that zinc suppresses EPO production in hypoxic cells (Gopfert et al., 1995; Dittmer and Bauer, 1992). Previously, we discussed this reciprocal behaviour of zinc on the oxygen-sensing pathway (Chun et al., 2000a). Zinc induced the accumulation and nuclear translocation of HIF-1
in the same manner as cobalt and nickel. However, it also blocked the nuclear translocation of ARNT under hypoxic conditions, which inhibited the formation of HIF-1 in the nucleus and suppressed EPO mRNA induction. We believe that the generation of HIF-1
Z seems to be responsible for the inhibitory effects of zinc ion on HIF-1-mediated hypoxic responses, because HIF-1
Z was expressed by the zinc ion and its recombinant protein showed the same effect as zinc on HIF-1 activity and ARNT translocation.
Recently, Gothie et al. (Gothie et al., 2000) reported two isoforms of human HIF-1 that result from alternative splicing of mRNA. One has an additional 3 bp at the junction of the 1st and the 2nd exons without a frameshift. The other loses the 14th exon resulting in a frameshift and an immediate termination of translation. The latter translates into a 736 amino acid polypeptide (HIF-1
736). Because HIF-1
736 conserves most of the essential domains of HIF-1
, it is regulated by oxygen tension and transactivates VEGF promoter. They also demonstrated that HIF-1
736 can compete with endogenous HIF-1
, using a reporter assay, and that it inhibits the hypoxic induction of luciferase by up to 50%. This HIF-1
736 is comparable to HIF-1
Z in terms of mRNA and protein structure. Both proteins are generated by the deletion of one exon, producing shorter isoforms. However, there are big differences between them. HIF-1
Z lacked a part of ODD, both TADs, and NLS because of deletion of the 12th exon. HIF-1
Z was also more stable under normoxia and did not transactivate EPO and VEGF promoters, which suggests that HIF-1
Z is a better dominant-negative isoform at competing with HIF-1
. In fact, HIF-1
Z almost completely suppressed the tranactivation activities of HIF-1
as shown in Fig. 6A.
Endothelial PAS domain protein 1 (EPAS1) is homologous with HIF-1 and preferentially expressed in vascular endothelial cells. Like HIF-1
, it also dimerizes with ARNT and binds to the HIF-1-binding site. Maemura et al. (Maemura et al., 1999) found that a recombinant EPAS1485 lacking the TAD functions as a dominant-negative mutant of EPAS1870 and also competed with HIF-1
in the VEGF promoter assay. However, they did not investigate the association with ARNT and specifically its nuclear import. Although, as compared to HIF-1
Z, EPAS1485 is not a natural protein and has a different amino acid sequence, its protein structure is comparable with that of HIF-1
Z. It has intact bHLH and PAS domains like HIF-1
Z, and it might be able to bind with ARNT. Thus, we can speculate that it may share a common mechanism with HIF-1
Z, which is summarized in Fig. 11.
In zinc-treated cells, full-length HIF-1 mRNA was reduced and a new spliced variant emerged. The zinc ion probably changes the splicing process of the pre-mRNA, but does not affect the transcription of HIF-1
gene. To our knowledge, it has never been reported that the zinc ion can induce a newly spliced variant, even in other gene transcripts. How does the zinc ion change the splicing process of mRNA? The question remains to be answered. One possible explanation is that the zinc ion affects a putative zinc-dependent RNA-splicing process. The splicing of nuclear pre-mRNA occurs in a multicomponent complex containing nuclear ribonucleoproteins and splicing factors. In higher eukaryotes, serine/arginine (SR) splicing factors are involved in the first steps of splice site recognition (Smith and Valcarcel, 2000). Human 9G8 SR factor contains a specific sequence in the median region, which is a zinc knuckle motif (CCHC motif) (Cavaloc et al., 1994). Moreover, splicing factors containing the zinc knuckle motif have also been found in an insect (Mazroui et al., 1999) and a plant (Lopato et al., 1999). Cavaloc et al. (Cavaloc et al., 1999) demonstrated that the zinc knuckle is involved in the RNA recognition specificity of 9G8. Therefore, because zinc is essential for the binding between RNA and the zinc knuckle motif, a large zinc concentration change may affect the RNA recognition specificity of splicing factor, and alter the splicing process. However, given that the zinc-induced alteration of RNA splicing occurs specifically at exon 12 of the HIF-1
gene, general RNA-splicing machinary like 9G8 SR factor may not be a key molecule to produce HIF-1
Z. Other putative molecule but 9G8 SR factor might be involved in the zinc effect and remains to be identified.
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ACKNOWLEDGMENTS |
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REFERENCES |
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Bunn, H. F. and Poyton, R. O. (1996). Oxygen sensing and molecular adaptation to hypoxia. Physiol. Rev. 76, 839-885.
Cavaloc, Y., Popielarz, M., Fuchs, J. P., Gattoni, R. and Stevenin, J. (1994). Characterization and cloning of the human splicing factor 9G8: a novel 35 kDa factor of the serine/arginine protein family. EMBO J. 13, 2639-2649.[Abstract]
Cavaloc, Y., Bourgeois, C. F., Kister, L. and Stevenin, J. (1999). The splicing factors 9G8 and SRp20 transactivate splicing through different and specific enhancers. RNA 5, 468-483.
Chilov, D., Camenisch, G., Kvietikova, I., Ziegler, U., Gassmann, M. and Wenger, R. H. (1999). Induction and nuclear translocation of hypoxia-inducible factor-1 (HIF-1): heterodimerization with ARNT is not necessary for nuclear accumulation of HIF-1. J. Cell Sci. 112, 1203-1212.
Chun, Y. S., Choi, E., Kim, G. T., Choi, H., Kim, C. H., Lee, M. J., Kim, M. S. and Park, J. W. (2000a). Cadmium blocks hypoxia-inducible factor (HIF)-1-mediated response to hypoxia by stimulating the proteasome-dependent degradation of HIF-1alpha. Eur. J. Biochem. 267, 4198-4204.
Chun, Y. S., Choi, E., Kim, G. T., Lee, M. J., Lee, M. J., Lee, S. E., Kim, M. S. and Park, J. W. (2000b). Zinc induces the accumulation of hypoxia-inducible factor (HIF)-1alpha, but inhibits the nuclear translocation of HIF-1beta, causing HIF-1 inactivation. Biochem. Biophys. Res. Commun. 268, 652-656.[Medline]
Dittmer, J. and Bauer, C. (1992). Inhibitory effect of zinc on stimulated erythropoietin synthesis in HepG2 cells. Biochem. J. 285, 113-116.[Medline]
Fagotto, F., Funayama, N., Gluck, U. and Gumbiner, B. M. (1996). Binding to cadherins antagonizes the signaling activity of beta-catenin during axis formation in Xenopus. J. Cell Biol. 132, 1105-1114.[Abstract]
Goldberg, M. A., Dunning, S. P. and Bunn, H. F. (1988). Regulation of the erythropoietin gene: evidence that the oxygen sensor is a heme protein. Science 242, 1412-1415.[Medline]
Gopfert, T., Eckardt, K. U., Gess, B. and Kurtz, A. (1995). Cobalt exerts opposite effects on erythropoietin gene expression in rat hepatocytes in vivo and in vitro. Am. J. Physiol. 269, R995-1001.
Gothie, E., Richard, D. E., Berra, E., Pages, G. and Pouyssegur, J. (2000). Identification of alternative spliced variants of human hypoxia-inducible factor-1alpha. J. Biol. Chem. 275, 6922-6927.
Huang, L. E., Arany, Z., Livingston, D. M. and Bunn, H. F. (1996). Activation of hypoxia-inducible transcription factor depends primarily upon redox-sensitive stabilization of its alpha subunit. J. Biol. Chem. 271, 32253-32259.
Huang, L. E., Gu, J., Schau, M. and Bunn H. F. (1998). Regulation of hypoxia-inducible factor 1alpha is mediated by an O2-dependent degradation domain via the ubiquitin-proteasome pathway. Proc. Natl. Acad. Sci. USA 95, 7987-7992.
Huang, L. E., Willmore, W. G., Gu, J., Goldberg, M. A. and Bunn, H. F. (1999). Inhibition of hypoxia-inducible factor 1 activation by carbon monoxide and nitric oxide. Implications for oxygen sensing and signaling. J. Biol. Chem. 274, 9038-9044.
Iyer, N. V., Leung, S. W. and Semenza, G. L. (1998). The human hypoxia-inducible factor 1alpha gene: HIF1A structure and evolutionary conservation. Genomics 52, 159-165.[Medline]
Jiang, B. H., Rue, E., Wang, G. L., Roe, R. and Semenza, G. L. (1996). Dimerization, DNA binding, and transactivation properties of hypoxia-inducible factor 1. J. Biol. Chem. 271, 17771-17778.
Jiang, B. H., Zheng, J. Z., Leung, S. W., Roe, R. and Semenza, G. L. (1997). Transactivation and inhibitory domains of hypoxia-inducible factor 1alpha. Modulation of transcriptional activity by oxygen tension. J. Biol. Chem. 272, 19253-19260.
Kallio, P. J., Okamoto, K., OBrien, S., Carrero, P., Makino, Y., Tanaka, H. and Poellinger, L. (1998). Signal transduction in hypoxic cells: inducible nuclear translocation and recruitment of the CBP/p300 coactivator by the hypoxia-inducible factor-1alpha. EMBO J. 17, 6573-6586.
Kallio, P. j., Wilson, W. J., OBrien, S., Makino, Y. and Poellinger, L. (1999). Regulation of the hypoxia-inducible transcription factor 1alpha by the ubiquitin-proteasome pathway. J. Biol. Chem. 274, 6519-6525.
Lopato, S., Gattoni, R., Fabini, G., Stevenin, J. and Barta, A. (1999). A novel family of plant splicing factors with a Zn knuckle motif: examination of RNA binding and splicing activities. Plant Mol. Biol. 39, 761-773.[Medline]
Maemura, K., Hsieh, C. M., Jain, M. K., Fukumoto, S., Layne, M. D., Liu, Y., Kourembanas, S., Yet, S. F., Perrella, M. A. and Lee, M. E. (1999). Generation of a dominant-negative mutant of endothelial PAS domain protein 1 by deletion of a potent C-terminal transactivation domain. J. Biol. Chem. 274, 31565-31570.
Maxwell, P. H., Wiesener, M. S., Chang, G. W., Clifford, S. C., Vaux, E. C., Cockman, M. E., Wykoff, C. C., Pugh, C. W., Maher, E. R. and Ratcliffe, P. J. (1999). The tumour suppressor protein VHL targets hypoxia-inducible factors for oxygen-dependent proteolysis. Nature 399, 271-275.[Medline]
Mazroui, R., Puoti, A. and Kramer, A. (1999). Splicing factor SF1 from Drosophila and Caenorhabditis: presence of an N-terminal RS domain and requirement for viability. RNA 5, 1615-1631.
Minopoli, G., de Candia, P., Bonetti, A., Faraonio, R., Zambrano, N. and Russo, T. (2001). The beta-amyloid precursor protein functions as a cytosolic anchoring site that prevents Fe65 nuclear translocation. J. Biol. Chem. 276, 6545-6550.
Sadek, C. M., Jalaguier, S., Feeney, E. P., Aitola, M., Damdimopoulos, A. E., Pelto-Huikko, M. and Gustafsson, J. A. (2000). Isolation and characterization of AINT: a novel ARNT interacting protein expressed during murine embryonic development. Mech. Dev. 97, 13-26.[Medline]
Semenza, G. L. (2000a). HIF-1 and human disease: one highly involved factor. Genes Dev. 14, 1983-1991.
Semenza, G. L. (2000b). HIF-1: mediator of physiological and pathophysiological responses to hypoxia. J. Appl. Physiol. 88, 1474-1480.
Semenza, G. L., Nejfelt, M. K., Chi, S. M. and Antonarakis, S. E. (1991). Hypoxia-inducible nuclear factors bind to an enhancer element located 3' to the human erythropoietin gene. Proc. Natl. Acad. Sci. USA 88, 5680-5684.[Abstract]
Shibayama, N., Morimoto, H. and Miyazaki, G. (1986). Oxygen equilibrium study and light absorption spectra of Ni(II)-Fe(II) hybrid hemoglobins. J. Mol. Biol. 192, 323-329.[Medline]
Smith, C. W. and Valcarcel, J. (2000). Alternative pre-mRNA splicing: the logic of combinatorial control. Trends Biochem. Sci. 25, 381-388.[Medline]
Wang, G. L., Jiang, B. H., Rue, E. A. and Semenza, G. L. (1995). Hypoxia-inducible factor 1 is a basic-helix-loop-helix-PAS heterodimer regulated by cellular O2 tension. Proc. Natl. Acad. Sci. USA 92, 5510-5514.[Abstract]