Proc. Natl. Acad. Sci. USAVol. 90, pp. 4304-4308, May 1993Genetics

General involvement of hypoxia-inducible factor 1 in transcriptionalresponse to hypoxiaGUANG L. WANG AND GREGG L. SEMENZA*Center for Medical Genetics, Departments of Pediatrics and Medicine, The Johns Hopkins University School of Medicine, Baltimore, MD 21205

Communicated by Victor A. McKusick, February 11, 1993

ABSTRACT Transcription of the human erythropoietin(EPO) gene is activated in Hep3B cells exposed to hypoxia.Hypoxia-inducible factor 1 (HIF-1) is a nuclear factor whoseDNA binding activity is induced by hypoxia in Hep3B cells, andHIF-1 binds at a site in the EPO gene enhancer that is requiredfor hypoxic activation of transcription. In this paper, wedemonstrate that HIF-1 DNA binding activity is also induced byhypoxia in a variety of mammalian cell lines in which the EPOgene is not transcribed. The composition of the HIF-1 DNAbinding complex and its isolated DNA binding subunit and themechanism ofHIF-1 activation appear to be similar or identicalin EPO-producing and non-EPO-producing cells. Transcrip-tion of reporter genes containing the EPO gene enhancer isinduced by hypoxia in non-EPO-producing cells and mutationsthat eliminate HIF-1 binding eliminate inducibility. Theseresults provide evidence that HIF-1 and its recognition se-quence are common components of a general mammaliancellular response to hypoxia.

Cells sense and respond to extracellular and intracellularstimuli to maintain homeostasis, and hypoxia is one of themost fundamental of all environmental stimuli. A majorphysiologic mechanism by which mammals respond to tissuehypoxia is through stimulation of erythropoiesis resulting inan increased blood 02-carrying capacity. Erythropoietin(EPO) is the primary humoral regulator of mammalian eryth-ropoiesis. EPO RNA levels increase several hundredfold inrodent liver and kidney in response to hypoxia or anemia (forreview, see refs. 1 and 2). Human EPO RNA levels showsimilar increases in transgenic mouse liver and kidney (3-6).Hypoxia also induces EPO RNA transcription in Hep3Bhuman hepatoblastoma cells (7), demonstrating that the samecell type can sense hypoxia and respond by increasing EPORNA levels. The production ofEPO and subsequent increasein erythropoiesis thus provide a major homeostatic mecha-nism for maintaining tissue oxygenation.A major research objective in our laboratory has been to

identify the cis-acting DNA sequences and trans-acting pro-tein factors that regulate hypoxia-inducible human EPO geneexpression. Liver-specific DNase I-hypersensitive sites havebeen identified in the human EPO gene 3'-flanking sequence(8). In addition, a 256-nt fragment that encompasses thehypersensitive sites functioned as a hypoxia-inducible en-hancer when cloned 3' to a simian virus 40 (SV40) promoter-chloramphenicol acetyltransferase (CAT) reporter gene andtransiently expressed in Hep3B cells (8). Deletion and scan-ning-mutagenesis studies further delimited the enhancer to a50-nt sequence that was functionally tripartite: site 1 (nt 4-12)and site 2 (nt 19-23) were absolutely required for hypoxicinduction, whereas site 3 (nt 26-48), a putative thyroid/steroid hormone receptor binding site, functioned to amplifythe induction signal (9). We identified (9) in Hep3B nuclearextracts hypoxia-inducible factor 1 (HIF-1), a DNA binding

activity induced by hypoxia that binds specifically to theenhancer site 1 sequence but not to a mutant sequencecontaining a 3-nt substitution that eliminated enhancer func-tion in transient expression assays. The HIF-1 binding site,as established by transient expression and electrophoretic-mobility-shift assays (EMSAs) (9), is completely conservedin the enhancer of the mouse EPO gene (10). Treatment ofHep3B cells with cycloheximide inhibited the hypoxic induc-tion of both EPO RNA (7) and HIF-1 DNA binding activity(9). These results provided further evidence for the impor-tance of HIF-1 in EPO gene transcriptional activation byhypoxia and suggested that induction of HIF-1 activity re-quires de novo protein synthesis.A growing number of other mammalian genes have been

reported whose expression is induced by hypoxia (11-15).The molecular basis for induction has not been establishedfor any of these genes, although induction of vascular endo-thelial growth factorRNA is inhibited by cycloheximide (14).To determine whether HIF-1 plays a general role in thehypoxic activation of gene transcription, we have analyzedmammalian cell lines in which the EPO gene is not expressed.HIF-1 was induced by hypoxia in all cell lines tested,including human 293 embryonic kidney and HeLa cervicalcarcinoma cells, mouse Ltk- fibroblasts, and C2C12 skeletalmyoblasts, Ratl fibroblasts, and Chinese hamster ovary(CHO) cells. The physiological relevance of HIF-1 inductionwas supported by our demonstration that the hypoxia-inducible enhancer was functional in CHO and 293 cells,whereas the 3-nt mutation that eliminated HIF-1 bindingrendered the enhancer noninducible. These results implicateHIF-1 in the transcriptional regulation of gene expression byhypoxia in a variety of mammalian cell types.

MATERIALS AND METHODSCell Culture and Nuclear Extract Preparation. Hep3B and

HeLa cells were maintained in minimal essential mediumwith Earle's salts, 1 mM sodium pyruvate, 2mM L-glutamine,and 1 mM nonessential amino acids. 293, Ratl, C2C12, andLtk- cells were maintained in Dulbecco's modified Eagle'smedium. CHO cells were grown in a-modified minimumessential medium. Media were supplemented with 10% (vol/vol) heat-inactivated fetal calf serum, 50 x 10-3 unit ofpenicillin per ml, and 50 pkg of streptomycin per ml (allpurchased from GIBCO). Hypoxia was induced by placingcells in a modular incubator chamber flushed with 1% 02/5%C02/balance N2 for 4 h at 37°C. When used, cycloheximide(Sigma) was added to a final concentration of 100 uM,starting 2 h prior to hypoxia treatment. For heat shocktreatment, cells were incubated for 1 h at 42.5°C in a standard

Abbreviations: CAT, chloramphenicol acetyltransferase; EPO,erythropoietin; HIF-1, hypoxia-inducible factor 1; SV40, simianvirus 40; EMSA, electrophoretic-mobility-shift assay.*To whom reprint requests should be addressed at: CMSC-1004, TheJohns Hopkins Hospital, 600 North Wolfe Street, Baltimore, MD21205.

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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5% C02/95% air incubator. Cells were harvested and nuclearextracts were prepared as described (9).EMSA. DNA probe was prepared and EMSAs were per-

formed as described (9). For analysis of cobalt chloride-treated HeLa cells, 10 ,g of nuclear extract was incubatedwith 0.2 ,g of denatured calf thymus DNA per bindingreaction mixture. In all other cases, 5 ug of nuclear extractand 0.1 ,ug of denatured calf thymus DNA were used. Forcompetition experiments, 20- to 500-fold molar excess ofunlabeled oligonucleotide was added to the binding reactionmixture 5 min prior to probe addition. Sequences of the W18oligonucleotides were 5'-agcttGCCCTACGTGCTGTCT-CAg-3' and 5'-aattcTGAGACAGCACGTAGGGCa-3' inwhich EPO gene sequences are in uppercase type, underlinednucleotides are replaced with AAA and TTT, respectively, inthe M18 oligonucleotides (9), and artificial cloning sequencesare in lowercase type.

Plasmid Construction and Transient Expression Assay. Theconstruction of pSVcat reporter plasmids containing WT50,MUT50, 2xWT33, and 2xMUT33 has been described (9).2xWT50 and 4xWT50 were constructed by ligation ofmono-mers and cloning in the BamHI site of pSVcat as described(9). Transfection with 2 ,ug of pSVpgal and 15 ug of pSVcatreporter plasmids was performed by electroporation of 3.9 x106 CHO cells in 0.3 ml of OptiMEM I (GIBCO) with aGenePulser apparatus (Bio-Rad) at 250 V/960 ,uF. 293 cellswere transfected by calcium phosphate coprecipitation (16).Duplicate plates of transfected cells were cultured in 1% or20% 02 for 40 h, harvested, and assayed for ,B-galactosidaseactivity and CAT protein (9).UV Crosslinking. Nuclear extract (100 ,g) was preincu-

bated at 22°C for S min in 60 ,ul of gel-shift binding buffercontaining 2 ,ug of denatured calf thymus DNA. 32P-labeledprobe (2 ng, 2 x 105 cpm) was added and the mixture wasincubated for an additional 15 min. The tops ofthe open tubeswere covered with plastic wrap and exposed to a UV lamp(312 nm; Fisher Scientific) at 5 cm for 30 or 60 min at 4°C.Samples were mixed with an equal volume of 2x Laemmlisample buffer, boiled for 5 min, and fractionated by SDS/PAGE on a 5% gel (17). For the isolation of DNA-proteincomplexes, nuclear extract was incubated with probe asdescribed above and UV-irradiated for 30 min, and EMSAswere performed. After electrophoresis, the gel was subjectedto UV-irradiation for an additional 30 min and exposed toX-Omat AR film (Kodak) at 4°C, and regions of the gelcorresponding to autoradiographic bands were excised. Gelslices were crushed, mixed with 2 vol of2x Laemmli samplebuffer, boiled for 5 min, pelleted by microcentrifugation, andthe supernatants were fractionated by SDS/PAGE on 5%gels. The dried gel was exposed to film with intensifyingscreens at -80°C.

RESULTSHIF-1 DNA Binding Activity Is Induced by Hypoxia in Many

Mammalian Cell Types. We have detected (9) HIF-1 DNAbinding activity in nuclear extracts prepared from Hep3Bcells exposed to 1% 02 for 4 h. HIF-1 binds specifically to adouble-stranded oligonucleotide probe (W18) containing nt1-18 of the 50-nt hypoxia-inducible enhancer ofthe EPO genebut not to a probe (M18) in which nt 7-9 have been mutated(9). This 3-nt substitution eliminated hypoxia-inducible func-tion of the 50-nt enhancer in Hep3B transient expressionassays (9). We have now analyzed six mammalian cell linesin which the EPO gene is not expressed. Nuclear extractswere prepared from cells exposed to 1% or 20% 02 for 4 h andtested for HIF-1 DNA binding activity by EMSAs. HIF-1activity was present in nuclear extracts from hypoxic, but notfrom nonhypoxic, cells of all lines (Fig. 1A). In addition toHep3B, the lines tested included mouse Ltk- fibroblasts,

CELL LINE: Ltk CHO RatlHep 293 HL Hep C2HYPOXIA: + + + -++ + + +

HLF-1I w

NS _ W we

B~..

12 34 5 67

*.0*. ." *ow

.-~' .I.

FIG. 1. EMSAs of HIF-1 DNA binding activity. (A) Induction ofHIF-1 by hypoxia. Nuclear extracts were prepared from hypoxic (+)and nonhypoxic (-) cells of the following lines: Ltk, mouse Ltk-fibroblasts; CHO, Chinese hamster ovary cells; Ratl, rat fibroblasts;Hep, human Hep3B hepatoblastoma cells; 293, human embryonickidney cells; HL, human HeLa cervical carcinoma cells; C2, mouseC2C12 myoblasts. Aliquots (5 ,Lg) of nuclear extract were incubatedwith W18 probe and analyzed by nondenaturing PAGE. Migration ofDNA-protein complexes containing HIF-1 and constitutive (bandsC) and nonspecific (bands NS) DNA binding activities is indicated.Radioactivity at bottom of gel represents free probe. Two sets ofHep3B nuclear extracts are included because the figure is a com-posite of two experiments. (B) Sequence specificity of HIF-1 DNAbinding activity in hypoxic HeLa cells. Aliquots (5 ,ug) of nuclearextract were incubated with W18 probe in the presence of nocompetitor (lane 1), 20-fold (lanes 2 and 5), 100-fold (lanes 3 and 6),or 500-fold (lanes 4 and 7) molar excess of unlabeled W18 (w; lanes2-4) or M18 (m; lanes 5-7) oligonucleotide.

CHO cells, Ratl fibroblasts, human embryonic kidney 293cells, human HeLa cervical carcinoma cells, and C2C12mouse myoblasts. As in Hep3B cells, a constitutively ex-pressed sequence-specific DNA binding activity and a non-specific DNA binding activity were also detected in nuclearextracts from each cell line. All three of these DNA bindingactivities appeared as doublet bands (9). There were varia-tions among extracts in the mobility of individual bands thatmay reflect differences in post-translational modification.Bands with mobility greater than the nonspecific DNA bind-ing activity appeared variably in extract preparations andmay be due to protein degradation.The specificity of the binding activities in nuclear extract

from hypoxic HeLa cells was analyzed by adding increasingamounts of unlabeled wild-type or mutant competitor oligo-nucleotides to reaction mixtures (Fig. 1B). Binding of HIF-1and the constitutive factor to the probe was inhibited byexcess wild-type (lanes 2-4), but not mutant (lanes 5-7),oligonucleotide, whereas the nonspecific binding activity wasinhibited by either oligonucleotide. These results are identi-cal to the pattern of competition obtained with Hep3Bnuclear extracts (9). Similar results were observed withnuclear extract from hypoxic Ltk- cells (data not shown).From the results shown in Fig. 1, we conclude that in manycell types hypoxia induces a DNA binding activity with thesame electrophoretic mobility and sequence specificity as theHIF-1 DNA binding activity previously characterized inHep3B cells.

Evidence for Induction of HIF-1 in Different Cell Types bya Common Mechanism. EPO gene transcription can be acti-vated by exposure of Hep3B cells to either hypoxia or cobaltchloride (7). HIF-1 binding activity was induced after 1 h andwas maximal after 4-h treatment of Hep3B cells with 75 ,Mcobalt chloride (Fig. 2A), which is similar to the kinetics ofHIF-1 induction by hypoxia (data not shown). Exposure ofHeLa cells to cobalt chloride for 4 h also induced HIF-1activity. In contrast to hypoxia, which induced a doubletband corresponding to HIF-1 in EMSAs, cobalt chlorideinduced a single band of HIF-1 activity in both Hep3B and

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ACELL LINE: HL HepSTIMULUS: 0 4 0 1 4

HIF- I *

NS ---*0

B CHep CHO - + CHX- +- + - +- + STIMULUS

Zi X s.*.

Proc. Natl. Acad. Sci. USA 90 (1993)

A 30' 60' EMSA- + - + I1 I2 C1C2 NS

205 -.

116.5

80

49.5

FIG. 2. Stimulus specificity of HIF-1 induction. (A) Effect ofcobalt chloride treatment. Nuclear extracts were prepared fromHeLa (HL) or Hep3B (Hep) cells treated with 75 ,uM cobalt chloridefor 0, 1, or 4 h. (B) Effect of heat shock. Nuclear extracts wereprepared from Hep3B or CHO cells that were either untreated (-) orsubjected to heat shock (+) for 1 h at 42.5°C. (C) Effect ofcycloheximide. Nuclear extracts were prepared from CHO cells thatwere treated with 100 AM cycloheximide (CHX) or solvent aloneunder hypoxic (+) or nonhypoxic (-) conditions. All extracts wereanalyzed by EMSA using the W18 probe.

HeLa cells (compare Figs. 1A and 2A). We have not deter-mined the basis for this reproducible difference in responseto stimulation by hypoxia as compared to cobalt chloride.To demonstrate stimulus specificity, we analyzed the ef-

fect of heat shock on HIF-1 activity. EPO RNA levels inHep3B cells are unaffected by heat shock (18). Hep3B andCHO cells were exposed to 42.5°C for 1 h as a heat shockstimulus. Nuclear extracts, prepared from treated and un-treated cells, were analyzed by an EMSA with the W18probe. The constitutive and nonspecific DNA binding activ-ities in Fig. 2B comigrate with the corresponding bands inFig. 2A. The Hep3B nuclear extracts in Fig. 2B generatedhigher-mobility bands (probably due to degradation) that are

only faintly visible in Fig. 2A. Most importantly, HIF-1 wasnot induced by heat shock in either Hep3B orCHO cells (Fig.2B). Control experiments using an oligonucleotide probecontaining the heat shock element (19) demonstrated appro-priate induction of heat shock factor in both Hep3B and CHOcell nuclear extracts (data not shown).The hypoxic induction ofboth HIF-1 DNA binding activity

(9) and EPO gene transcription (7) is blocked when Hep3Bcells are cultured in the presence of cycloheximide, aninhibitor of protein synthesis. We prepared nuclear extractsfrom CHO cells that were exposed to 1% or 20% 02 in thepresence or absence of 100 ,uM cycloheximide and testedthem for HIF-1 activity (Fig. 2C). HIF-1 induction wasblocked by cycloheximide treatment, suggesting that in CHOcells, as in Hep3B cells, de novo protein synthesis is requiredfor hypoxic induction of HIF-1 DNA binding activity.

Detection of the HIF-1 DNA Binding Subunit in HypoxicNuclear Extracts. UV crosslinking was performed to charac-terize the DNA binding protein(s) of HIF-1. Binding reac-tions with nonhypoxic and hypoxic Hep3B nuclear extractsand the W18 probe were followed by UV-irradiation of thesamples for 30 (Fig. 3A, lanes 1 and 2) or 60 (lanes 3 and 4)min. Proteins that had been UV-crosslinked to the probewere identified by denaturing SDS/PAGE. A DNA-proteincomplex corresponding to a molecular mass of -148 kDa(arrow; lanes 2 and 4) was detected only when the probe wasincubated with hypoxic nuclear extract.To determine the relationship between this induced UV-

crosslinked complex and the HIF-1 activity detected byEMSA, W18 probe was incubated with nuclear extract fromhypoxic Hep3B cells for 15 min and the reaction mixture wasUV-irradiated for 30 min. An EMSA was performed (Fig. 3ALower), the gel was UV-irradiated for an additional 30 min,

12 3 4 5 6 7 8 9

...... ...t,. ...

I I I_

12 12 NSI C

B CELL LINE: Hep Ltk HLHYPOXIA: - + - + - +

205

HIF- a-116.5-

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FIG. 3. Detection of HIF-1 DNA binding subunit by UVcrosslinking. (A) Analysis of Hep3B cells. In lanes 1-4, nuclearextracts from nonhypoxic (-) and hypoxic (+) Hep3B cells wereincubated with W18 probe, UV-irradiated for 30 min (lanes 1 and 2)or 60 min (lanes 3 and 4), and analyzed by SDS/PAGE. In lanes 5-9,nuclear extract from hypoxic Hep3B cells was incubated with W18probe, preparative EMSA was performed (Lower), individual bandsrepresenting induced (I), constitutive (C), and nonspecific (NS)DNAbinding activities were excised and analyzed by SDS/PAGE. Mi-gration of protein standards (molecular mass in kDa) is indicated.Arrow, hypoxia-induced DNA-protein complex. (B) Analysis ofnon-EPO-producing cells. Nuclear extracts from hypoxic (+) ornonhypoxic (-) Hep3B (Hep), Ltk- (Ltk), or HeLa (HL) cells wereincubated with W18 probe, UV-irradiated for 60 min, and fraction-ated by SDS/PAGE.

and individual bands were excised from the gel for proteinanalysis by denaturing SDS/PAGE. The two mobility-shiftbands corresponding to HIF-1 generated the same 148-kDaDNA-protein complex seen with whole nuclear extract (com-pare lanes 5 and 6 with lanes 2 and 4). Size estimation of thiscomplex on different gels ranged between 138 and 148 kDa.Two additional less-prominent complexes of 110 kDa and>205 kDa were also detected (lanes 5 and 6). The nonspecificmobility-shift band generated several prominent DNA-protein complexes (lane 9) and the constitutive bands gen-erated four very faint complexes (lanes 7 and 8) of 46-56 kDathat were seen only upon longer autoradiographic exposure.The induced and constitutive binding activities were of equalintensity by EMSA but not by crosslinking, indicating a lowerefficiency of UV-crosslinking of the constitutive bindingproteins to DNA. The size of the DNA probe was 16 kDa,

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suggesting that the UV-crosslinked DNA binding protein ofthe HIF-1 complex has a molecular mass of 122-132 kDa,although the DNA-protein complex may migrate differentlythan the isolated protein, and the estimation of its molecularmass can, therefore, only be considered tentative.Nuclear extracts from hypoxic Ltk- and HeLa cells gen-

erated a DNA-protein complex that comigrated with thecomplex generated from hypoxic Hep3B nuclear extracts(Fig. 3B). The Ltk- nuclear proteins crosslinked to the probedisplayed a greater degree of size heterogeneity, possibly dueto degradation or post-translational modification. Analysis ofCHO cell nuclear extracts gave results that were similar tothe patterns seen with Hep3B and HeLa cells (data notshown). The composition ofthe HIF-1 DNA binding complexand its isolated DNA binding subunit and the mechanism ofHIF-1 activation thus appear to be similar or identical inEPO-producing and non-EPO-producing cell lines.The EPO Gene Hypoxia-Inducible Enhancer Is Functional in

Non-EPO-Producing Cells. To establish the functional signif-icance of HIF-1 induction in non-EPO-producing cells, tran-sient expression assays were performed using an SV40 pro-moter-CAT reporter gene construct (pSVcat). The wild-type50-nt hypoxia-inducible enhancer (WT50) and a mutant se-quence (MUT50) containing the 3-nt substitution that elimi-nated HIF-1 binding were cloned, in one to four copies, 3' tothe CAT coding sequence, as were the first 33 nt of theenhancer (WT33) and a mutant sequence (MUT33) with a 5-ntsubstitution that eliminated hypoxia inducibility in Hep3Bcells (Fig. 4A). These pSVcat constructs were each cotrans-fected into CHO cells by electroporation with an SV40promoter-,l-galactosidase fusion gene. Transfected cellswere split onto two plates, one ofwhich was incubated in 20%02 and the other was incubated in 1% 02 for 40 h. CATprotein and ,3-galactosidase activity in cellular extracts werequantitated and the CAT/p8-galactosidase ratios for hypoxic

A

4 C.GCCCTACGTGCTGTCTCACACAGCCTGTCTGACCICTCGACCTACCGGCC WI50-----AAAJ--- -- 50

----------------- .---- 7WT33- - ATGCC---- MT33

B

WT50

WT50

WT50

MUT500

Mean SEM0.72

3.44 0.72

4.25 0.66

2.86 0.46

1.71 0.19

0.96 0.02

0.90 0.11

0 1 2 3 4

Fold induction by hypoxia

FIG. 4. Functional analysis of the EPO gene hypoxia-inducibleenhancer in CHO cells. (A) Structure of the enhancer. Sequence ofthe 50-nt enhancer is shown with putative transcription factor bindingsites overlined (9). Mutation and deletion constructs are shownbelow. Dashes, identity with the 50-nt sequence; SHR, steroidhormone receptor superfamily member; ?, unknown. (B) Transientexpression assays. Indicated constructs were cloned 3' to pSVcattranscription unit, cotransfected into CHO cells with pSV,3gal, andequal numbers of cells were incubated at 1% and 20% 02. Foldinduction by hypoxia represents CAT/,B-galactosidase expression incells at 1% 02 divided by CAT/,8-galactosidase expression in cells at20% 02 (mean and SEM of data from four experiments; 2xMUT33data is from a single experiment).

and nonhypoxic transfectants were compared to determinethe fold induction by hypoxia.The data from four transfection experiments are presented

in Fig. 4B. pSVcat plasmids with no enhancer and the mutant50-nt enhancer (MUT50) were uninducible by hypoxia. Asingle copy of the wild-type 50-nt enhancer (WT50) mediateda 1.71-fold induction by hypoxia, which was statisticallysignificant compared to the enhancerless and MUT50 con-structs (P < 0.05; Student's t test). Furthermore, two andfour copies of WT50 mediated 2.86- and 4.25-fold inductionby hypoxia, respectively. Thus, there was a direct correlationbetween the number of copies of WT50 and the degree ofhypoxic induction. Two copies of the 33-nt element (WT33)containing only binding sites 1 and 2 also mediated a statis-tically significant (P < 0.05) 3.44-fold induction, while twocopies of the mutant element (MUT33) provided no induciblefunction. As previously demonstrated in Hep3B cells (9), thehypoxic induction mediated by these sequences was due toincreased expression at 1% 02 and was, therefore, due totranscriptional activation rather than derepression. Similarresults were obtained in two experiments in which humanembryonic kidney 293 cells were transfected by calciumphosphate coprecipitation: 2xWT50 provided a 3.03-foldinduction compared to a 0.95-fold induction for pSVcat alone(P < 0.05). These results suggest that HIF-1 and its bindingsite sequence can mediate hypoxic activation oftranscriptionin a variety of mammalian cell types.

DISCUSSIONMany Mammalian Cell Types Can Sense and Respond to

Changes in Oxygen Tension. Studies of human EPO geneexpression in our laboratory (8, 9) and elsewhere (10, 20, 21)have provided data regarding the cis-acting DNA sequencesand trans-acting protein factors that mediate hypoxia-inducible transcription of the EPO gene in Hep3B cells. Theinitial steps of the hypoxia signal-transduction pathway arethe sensing of02 tension and the presently undefined primaryresponse to hypoxia. A model, in which the 02 sensor is aheme-containing protein that exists in different conforma-tional states depending upon the presence or absence ofbound ligand (02), has been proposed based upon severallines of supporting data (18): (i) Cobalt chloride and nickelchloride stimulated EPO production and both divalent cat-ions are known to substitute for ferrous iron in heme,effectively locking hemoglobin in the deoxy conformation.(ii) Carbon monoxide, which is known to lock hemoglobin inthe oxy conformation, reduced the hypoxic induction ofEPO. (iii) Treatment of cells with desferrioxamine, an ironchelator, or dioxoheptanoic acid, an inhibitor of heme syn-thesis, reduced hypoxic induction of EPO. This hypothesiswas supported by the identification of FixL, an 02 sensor inthe bacterium Rhizobium melliloti, as a heme-containingprotein kinase that is activated by hypoxia, resulting in thetranscription of nifA and fixK, whose gene products in turnactivate transcription of at least 14 nifandfix genes (22-24).We have provided several lines of experimental evidence

suggesting that HIF-1 plays a crucial role in the hypoxicactivation ofEPO gene transcription: (i) Hypoxia and cobaltchloride each stimulatedEPO gene transcription and inducedHIF-1 DNA binding activity. (ii) Mutations of the HIF-1binding site eliminated hypoxia-inducible enhancer activity(9). (iii) Cycloheximide inhibited EPO gene transcription andHIF-1 induction (9). In this paper, we also demonstrate thatHIF-1 is induced by hypoxia in many mammalian cell types,including all seven tissue-culture cell lines we have tested todate. Compared to the HIF-1 activity characterized in EPO-producing Hep3B cells, the HIF-1 activity induced in the sixnon-EPO-producing cell lines has the same mobility in non-

denaturing gels, the same sequence specificity, and its DNA

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binding subunit has the same mobility on denaturing gels. Asin Hep3B cells, HIF-1 is induced by hypoxia or cobaltchloride, but not by heat shock, and the induction is blockedby the protein synthesis inhibitor cycloheximide. Theseresults indicate that all cell types tested are able to sense 02tension and respond to hypoxia by induction of HIF-1 via asignal-transduction pathway that probably requires de novoprotein synthesis.

Involvement of HIF-1 in Hypoxic Activation of Transcrip-tion. Mutations of the HIF-1 binding site result in loss ofhypoxia-inducible enhancer function whereas basal expres-sion is unaffected (9), suggesting that HIF-1 is an activator ofEPO gene transcription in hypoxic Hep3B cells. In CHO and293 cells, transcription of the endogenous EPO gene is notinduced by hypoxia. However, in these cells, transient ex-pression of an SV40 promoter-CAT reporter gene carryingone or more copies of the EPO gene hypoxia-inducibleenhancer element was activated by hypoxia only when theHIF-1 binding site was intact. These results indicate that, asin Hep3B cells, the HIF-1 binding site was crucial fortranscriptional activation of reporter gene expression in CHOand 293 cells and that the observed induction of HIF-1 DNAbinding activity may, therefore, be functionally relevant totranscriptional regulation within many different cell types.The hypoxia-inducible function of the 50-nt enhancer in

CHO and 293 cells must be reconciled with the restrictedexpression of the EPO gene. The level of reporter geneinduction was much less in the non-EPO-producing CHO and293 cells than in EPO-producing Hep3B cells. A single copyofWT50 provided a 7-fold induction in Hep3B cells (9), while4xWT50 provided a 4-fold induction in CHO cells. Tran-scriptional activation mediated by HIF-1 binding at nt 4-12is absolutely dependent upon binding of a second factor at nt19-23 in both Hep3B cells (9) and CHO cells (Fig. 4B;compare 2xWT33 and 2xMUT33). We have not detectedbinding of an induced factor at this site, suggesting that aconstitutive factor binding at nt 19-23 may facilitate HIF-1binding or its interaction with the transcription initiationcomplex. Qualitative and/or quantitative differences in thecomposition of this constitutive factor may exist in differentcell types and may be responsible for the lower level ofhypoxic activation mediated by the enhancer in non-EPO-producing cells.Another means by which EPO gene expression may be

restricted is through interactions between factors binding tothe promoter and enhancer. Compared to a heterologouspromoter, the EPO promoter increased the degree ofhypoxicinduction of reporter genes mediated by the enhancer, pri-marily by decreasing the basal level of transcription (21).A sequence-specific promoter-binding factor, detected inkidney nuclear extracts, was down-regulated in cobalt-stimulated mice (25). Factors binding to the promoter may,therefore, negatively regulate EPO gene transcription. Ourstudies of human EPO gene expression in transgenic micealso identified distal sequences located between 0.4 and 6 kb5' to the gene that restrict EPO gene expression in vivo tospecific cell types in liver and kidney (4, 5).Based upon the results presented in this paper, it appears

that many mammalian cell types can sense 02 tension andrespond to hypoxia by changes in gene expression. Inductionof HIF-1 DNA binding activity by hypoxia in all cell linestested suggests that this factor plays a general role in theregulation of transcription by 02 tension. The data presentedhere are consistent with this hypothesis, but confirmation willrequire additional experimentation. Of greatest importancewill be the isolation of nucleic acid and protein sequences

encoding HIF-1 to determine its subunit structure, regulationof DNA binding activity, and interaction with other tran-scription factors; and the identification of other genes whosetranscriptional activation by hypoxia, like that of the EPOgene, is mediated by the binding of HIF-1 to cis-acting DNAregulatory sequences.

Note Added in Proof. The enhancer of the mouse EPO gene can alsomediate hypoxia-inducible reporter gene transcription in multiplecell lines (26).

We thank Carl Wu for providing the heat shock element oligonu-cleotide and Chi Dang, Eduardo Marban, Shawn Robinson, and RuoXu for providing cell lines. We are grateful to Chi Dang and HaigKazazian, Jr., for helpful discussions and comments on the manu-script. G.L.S. is a Lucille P. Markey Scholar and this work wassupported in part by grants from the Lucille P. Markey CharitableTrust and the National Institutes of Health.

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J. D. & Antonarakis, S. E. (1990) Mol. Cell. Biol. 10, 930-988.5. Semenza, G. L., Koury, S. T., Nejfelt, M. K., Gearhart, J. D.

& Antonarakis, S. E. (1991) Proc. Natl. Acad. Sci. USA 88,8725-8729.

6. Koury, S. T., Bondurant, M. C., Koury, M. J. & Semenza,G. L. (1991) Blood 77, 2497-2503.

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