(Received for publication, August 18, 1994; and in revised form, November 10, 1994)
From the
Hypoxia-inducible factor 1 (HIF-1) is a DNA-binding protein that
activates erythropoietin (Epo) gene transcription in Hep3B cells
subjected to hypoxia or cobalt chloride treatment. HIF-1 DNA binding
activity is also induced by hypoxia or cobalt in non-Epo-producing
cells, suggesting a general role for HIF-1 in hypoxia signal
transduction and transcriptional regulation. Here we report the
biochemical purification of HIF-1 from Epo-producing Hep3B cells and
non-Epo-producing HeLa S3 cells. HIF-1 protein was purified 11,250-fold
by DEAE ion-exchange and DNA affinity chromatography. Analysis of HIF-1
isolated from a preparative gel shift assay revealed four polypeptides.
Peptide mapping of these HIF-1 components demonstrated that 91-, 93-,
and 94-kDa polypeptides had similar tryptic maps, whereas the 120-kDa
polypeptide had a distinct profile. Glycerol gradient sedimentation
analysis suggested that HIF-1 exists predominantly in a heterodimeric
form and to a lesser extent as a heterotetramer. Partially purified
HIF-1 bound specifically to the wild-type HIF-1 binding site from the EPO enhancer but not to a mutant sequence that lacks
hypoxia-inducible enhancer activity. UV cross-linking analysis with
purified HIF-1 indicated that both subunits of HIF-1 contact DNA
directly. We conclude that in both cobalt chloride-treated HeLa cells
and hypoxic Hep3B cells HIF-1 is composed of two different subunits:
120-kDa HIF-1 and 91-94-kDa HIF-1
.
Gene transcription is regulated by transcription factors that
can recognize specific DNA sequences and modulate the assembly of an
active transcription initiation complex in response to various
extracellular and intracellular signals(1) . Oxygen
(O) is the final electron acceptor in respiratory redox
reactions in mammalian cells, and hypoxia provokes acute as well as
long term physiological responses. The expression of many genes is
known to be induced by hypoxia, including the genes encoding tyrosine
hydroxylase(2) , endothelin(3) , glycolytic enzymes (4, 5) and growth factors such as platelet-derived
growth factor B(6) , vascular endothelial growth
factor(7) , and erythropoietin
(Epo)(
)(8, 9) . The molecular mechanisms
that underlie hypoxia signal transduction in mammalian cells, however,
remain undefined.
Epo is a glycoprotein growth factor that
stimulates proliferation and differentiation of erythroid progenitor
cells. Tissue hypoxia resulting from reduced ambient O levels or reduced blood O
carrying capacity (anemia)
activates EPO gene expression, which is regulated primarily at
the level of transcription (10, 11) . A
hypoxia-inducible enhancer was defined in the 3`-flanking sequence of
the EPO gene(12, 13, 14, 15) . The
hypoxia-inducible enhancer is functionally tripartite, with the first
two sites essential for hypoxia inducibility and a third site
functioning to amplify the induction
signal(14, 15, 16, 17, 18) .
A hypoxia-induced DNA-binding protein (HIF-1) that binds specifically
to site 1 of the hypoxia-inducible enhancer was
identified(15) . The induction of HIF-1 DNA binding activity in
hypoxic cells requires on-going protein and RNA synthesis (15, 19) . Protein phosphorylation is also required
for hypoxia signal transduction and HIF-1 DNA binding
activity(19) . HIF-1 recognizes an 8-base pair DNA sequence
5`-TACGTGCT-3` in the EPO enhancer and contacts both DNA
strands in the major groove(19) . UV cross-linking analysis
indicated that HIF-1 contains a DNA-binding subunit of
120 kDa as
well as a subunit of
94 kDa that was cross-linked less
efficiently(20) . The divalent cation Co
and
the iron chelator desferrioxamine also induce EPO gene
expression (10, 21, 22) as well as HIF-1 DNA
binding activity(20, 23) .
The potential importance of HIF-1 as a transcriptional activator was underscored by the finding that it was induced by hypoxia in all cell types tested (20) and that the EPO enhancer can mediate hypoxia-inducible reporter gene expression in non-Epo-producing cells (20, 24) . Furthermore, HIF-1 binding sites are present in several hypoxia-inducible genes encoding glycolytic enzymes, and DNA sequences encompassing these sites can mediate hypoxia-inducible reporter gene transcription(5) . These results suggest the existence of a common hypoxia signal transduction pathway and a central role for HIF-1 in the transcriptional regulation of hypoxia-responsive genes. Here we describe the biochemical purification of human HIF-1 and the characterization of its DNA binding activity and molecular composition.
Equal amounts of complementary oligonucleotides were annealed, phosphorylated, and ligated. Ligated oligonucleotides (60-500 base pairs) were extracted with phenol/chloroform, ethanol precipitated, resuspended in deionized water, and coupled to CNBr-activated Sepharose 4B as instructed by the manufacturer (Pharmacia Biotech Inc.). Approximately 50 µg of ligated double-stranded oligonucleotides were coupled per ml of Sepharose.
For purification of HIF-1 from hypoxia-treated
Hep3B cells, nuclear extracts (95 mg) were fractionated by the use of a
4-ml DEAESepharose CL-6B column as described above. 0.25 M KCl
elute fractions were dialyzed against buffer Z-100 and applied onto a
Sephacryl S-300 gel filtration column (50 ml, 1.5 30 cm). The
fractions containing HIF-1 activity were pooled and applied to a 2-ml
calf thymus DNA column (0.8 mg of calf thymus DNA/ml of Sepharose)
prepared by coupling single-stranded calf thymus DNA to CNBr-activated
Sepharose 4B. The flowthrough was collected and applied to a 0.4-ml W18
column as described above after incubation with calf thymus DNA (2.2
µg/ml) for 10 min followed by another 0.2-ml W18 column after
dialysis against buffer Z-100.
Figure 1:
Analysis of HIF-1 induced by CoCl treatment of HeLa cells. A, dose-dependent induction of
HIF-1 DNA binding activity by CoCl
treatment. Nuclear
extracts, prepared from HeLa cells cultured in the presence of the
indicated concentration of CoCl
for 4 h at 37 °C, were
incubated with W18 probe and analyzed by gel shift assay. Lanes
1-8 and 9-12 represent extracts prepared in
two separate experiments. Arrows indicate HIF-1, constitutive
DNA binding activity (C), nonspecific activity (NS),
and free probe (F). B, methylation interference
analysis with nuclear extracts from CoCl
-treated HeLa
cells. W18 was 5`-end labeled on the coding or noncoding strand,
partially methylated, and incubated with nuclear extracts. DNA-protein
complexes corresponding to HIF-1, constitutive DNA binding activities (C1 and C2), and nonspecific binding activity (NS) were isolated from a preparative gel shift assay (lower) in addition to free probe (F) (not shown).
DNA was purified, cleaved with piperidine, and analyzed on a 15%
denaturing polyacrylamide gel (upper). Results are summarized
at left for coding strand and at right for noncoding
strand. The guanine residues are numbered according to their locations
in the W18 probe. The HIF-1 binding site is boxed. Complete
methylation interference with HIF-1 binding is indicated by closed
circles. Partial and complete methylation interference with
constitutive DNA binding activity are indicated by open and closed squares, respectively.
To provide evidence that HIF-1 DNA
binding activity in crude nuclear extracts from hypoxic Hep3B cells and
CoCl-treated HeLa cells has the same DNA binding
properties, methylation interference analysis was performed. Crude
nuclear extract from CoCl
-treated HeLa cells was incubated
with partially methylated W18 probe, labeled at the 5` end of either
coding or noncoding strand, and the reaction mixtures were resolved on
a native polyacrylamide gel (Fig. 1B, lower).
Probe DNA from the HIF-1, constitutive, and nonspecific bands as well
as free probe was isolated, cleaved with piperidine, and analyzed by
denaturing polyacrylamide gel electrophoresis (Fig. 1B, upper). On the coding strand, methylation of G
or
G
eliminated or greatly reduced HIF-1 binding,
respectively (Fig. 1B, lane 2). Methylation of
G
only partially interfered with the binding of
constitutive factors (Fig. 1B, lanes 3 and 4). On the noncoding strand, methylation of G
or
G
blocked HIF-1 binding to the probe (Fig. 1B, lane 8). Only the methylation of
G
interfered with binding of constitutive factors (Fig. 1B, lanes 9 and 10). The
nonspecific binding activity was unaffected by DNA methylation on
either strand (Fig. 1B, lanes 5 and 11). These results are identical to those obtained with crude
nuclear extract from hypoxic Hep3B cells(19) . In both cases,
the results indicate that (i) HIF-1 closely contacts G
and
G
on the coding strand and G
and G
on the noncoding strand through the major groove of the DNA helix
and (ii) HIF-1 and the constitutive DNA binding factors can be
distinguished by the nature of their DNA binding site contacts.
Figure 2:
Purification of HIF-1 from
CoCl-treated HeLa S3 cells. A, gel shift assay of
column fractions for HIF-1 DNA binding activity. Nuclear extracts were
fractionated by DEAE-Sepharose chromatography, and fractions containing
HIF-1 activity were applied to a W18 DNA affinity column. 5 µg of
protein were incubated with 0.1 µg of calf thymus DNA for gel shift
analysis of crude nuclear extract (Crude NE, lane 1)
and HIF-1 active fractions from DEAE-Sepharose columns (DEAE, lane 2). For fractions from the W18 column (lanes
3-13), 1-µl aliquots were incubated with 5 ng of calf
thymus DNA. The positions of the two HIF-1 bands, constitutive activity (C), nonspecific activity (NS), and free probe (F) are indicated. FT, flowthrough. 0.25 M, 0.5 M, 1 M, and 2 M are fractions eluted with indicated concentration
of KCl in buffer Z. B, sequence-specific DNA binding of the
partially purified fractions. Aliquots (5 µg) of fractions from the
DEAE-Sepharose column were incubated with W18 probe in the presence of
no competitor (lane 1), 10-fold (lanes 2 and 5), 50-fold (lanes 3 and 6), or 250-fold (lanes 4 and 7) molar excess of unlabeled W18 (W; lanes 2-4) or M18 (M; lanes
5-7) oligonucleotide.
Partially purified
HIF-1 fractions were then incubated with nonspecific competitor calf
thymus DNA at concentrations that allowed optimal detection of HIF-1
DNA binding activity by gel shift assays and applied to a W18 DNA
affinity column. HIF-1 was eluted in 0.5 M and 1 M KCl (Fig. 2A, lanes 10 and 11).
Fractions containing HIF-1 were pooled and dialyzed against buffer
Z-100. To eliminate nonspecific DNA-binding proteins that were not
removed by calf thymus DNA competitor, the dialysate was applied to an
M18 DNA column. HIF-1 DNA binding activity was detected in the
flowthrough, which was then applied directly onto second W18 column.
HIF-1 activity was detected exclusively in 0.5 M KCl fractions
(data not shown). Two rounds of W18 and one round of M18 column
chromatography resulted in a purification of 2,800-fold.
The results of the final large scale purification are summarized in Table 1. From 120 liters of HeLa cells, approximately 60 µg of highly purified HIF-1 were obtained (Table 1). The total purification was 11,250-fold and yielded approximately 22% of the starting of HIF-1 DNA binding activity (Table 1). Our objective was to identify HIF-1 subunits and isolate HIF-1 components for the purpose of peptide mapping and protein microsequencing analysis. Since additional steps of purification resulted in markedly lower yield, we did not purify HIF-1 further to homogeneity. Aliquots from flowthrough of the M18 column (Fig. 3A, Load) as well as the 0.25 M KCl wash and 0.5 M KCl elute fractions of the second W18 column were analyzed by 6% SDS-PAGE and silver staining. Four polypeptides of 90-120 kDa were highly enriched in the 0.5 M KCl fraction (lane 3, arrows), which had high HIF-1 DNA binding activity compared with the 0.25 M KCl fraction (lane 2), which had very little HIF-1 activity. The 0.5 M KCl fraction, however, still had many of the contaminant proteins found in the 0.25 M KCl fraction.
Figure 3:
Analysis of affinity-purified HIF-1 by
SDS-PAGE. A, purification of HIF-1 from
CoCl-treated HeLa S3 cells. Flowthrough fraction from the
M18 DNA column (Load, lane 1) and 0.25 M KCl
and 0.5 M KCl fractions from the second W18 DNA affinity
column (lanes 2 and 3) were analyzed. An aliquot of
each fraction (5 µg of load or 1 µg of affinity column
fractions) was resolved by 6% SDS-PAGE and silver stained. B,
purification of HIF-1 from hypoxic Hep3B cells. HIF-1 fractions from
the first W18 column (Load, lane 1) and 0.25 M KCl and 0.5 M KCl fractions from the second W18 column (lanes 2 and 3) were analyzed. An aliquot of each
fraction (50 µl) was resolved by 7% SDS-PAGE and silver stained.
Molecular mass markers are myosin (200 kDa),
-galactosidase (116
kDa), phosphorylase (97 kDa), BSA (66 kDa), and ovalbumin (45 kDa).
HIF-1 polypeptides in lanes3 are indicated by arrows at right of each
figure.
In an initial
pilot purification of HIF-1 from hypoxia-induced Hep3B cells, a
different purification protocol was used. Gel filtration over a
Sephacryl S-300 column was also found to be effective in separating
HIF-1 from constitutive DNA binding activity (data not shown). In
addition, a calf thymus DNA column was used to remove nonspecific
DNA-binding proteins prior to two rounds of W18 DNA affinity
chromatography. HIF-1 activity was detected in 0.5 M KCl
fractions from both DNA affinity columns. An aliquot from the 0.5 M KCl elute fraction of the first W18 column (Fig. 3B, Load) as well as the 0.25 M KCl wash and 0.5 M KCl elute fractions of the second W18
column were analyzed by 7% SDS-PAGE and silver staining. Four
polypeptides of similar molecular mass to those that co-purified with
HIF-1 DNA binding activity in CoCl-treated HeLa cells were
present in the affinity-purified preparation from hypoxic Hep3B cells (lane 3, arrows), indicating that HIF-1 from the two
different cell types is composed of the same polypeptide subunits.
Affinity-purified HIF-1 from both CoCl
-treated HeLa cells
and hypoxic Hep3B cells bound specifically to the W18 probe in gel
shift assays (data not shown).
Figure 4:
Identification of HIF-1 polypeptides. A, identification of HIF-1 components on a 6%
SDS-polyacrylamide gel with 3.2% cross-linking. An aliquot of
affinity-purified HIF-1 (A) was resolved on a 6%
SDS-polyacrylamide gel along with the HIF-1 protein complex isolated by
a preparative native gel shift assay (HIF-1). MW,
molecular mass markers with size (kDa) indicated at left.
Numbers to the right indicate the apparent molecular mass (in
kDa) of HIF-1 polypeptides. B, identification of HIF-1
components on a 6% SDS-polyacrylamide gel with 5% cross-linking. An
aliquot of affinity-purified HIF-1 was resolved on a 6%
SDS-polyacrylamide gel along with the HIF-1 protein complex isolated by
a preparative gel shift assay. The 120-kDa polypeptide, 94/93/91-kDa
polypeptides, and two contaminant proteins (1 and
2) are indicated. C, alignment of HIF-1 components
identified on two gel systems with different degrees of cross-linking.
Gel slices isolated from a 6% SDS-polyacrylamide gel with 5%
cross-linking corresponding to 120-kDa HIF-1 polypeptide (120), 94/93/91-kDa HIF-1 polypeptides (94/93/91),
and two contaminant proteins (
1 and
2) were
resolved on a 6% SDS-polyacrylamide gel with 3.2% cross-linking in
parallel with an aliquot (30 µl) of affinity-purified HIF-1 (A).
On a 6% SDS-polyacrylamide gel with
3.2% cross-linking, the 120 kDa HIF-1 polypeptide migrated very close
to a contaminant polypeptide of slightly greater apparent molecular
weight (Fig. 4A, lane A), making isolation of
the 120 kDa polypeptide difficult. This problem was resolved by
separating the HIF-1 polypeptides on a 6% SDS-polyacrylamide gel with
5% cross-linking. The 120-kDa polypeptide migrated much faster on the
more highly cross-linked gel relative to the migration of the 116-kDa
molecular mass marker, whereas migration of the contaminant band (1) was unchanged (Fig. 4B, lane
A). Under these conditions, however, the 91-kDa polypeptide ran
very close to another contaminant band (
2) below it. Two
polyacrylamide gel systems with different degrees of cross-linking were
therefore required for the isolation of the 91-94-kDa and the
120-kDa HIF-1 polypeptides, respectively.
To confirm that the HIF-1 polypeptides identified by the two gel systems were identical, two dimensional denaturing gel electrophoresis was performed. Affinity-purified HIF-1 was first resolved on a 6% SDS-polyacrylamide gel with 5% cross-linking (as in Fig. 4B, lane A). Regions of the gel containing the 120-kDa, 94/93/91-kDa HIF-1 polypeptides, as well as the two contaminant bands, were isolated and analyzed by electrophoresis on a 6% SDS-polyacrylamide gel with 3.2% cross-linking in parallel with an aliquot of the affinity-purified HIF-1. As shown in Fig. 4C, the isolated HIF-1 and contaminant polypeptides co-migrate with the corresponding bands in the control sample, indicating that the differences in their migration were due to different degrees of cross-linking of the SDS-polyacrylamide gels.
Figure 5:
Analysis of HIF-1 subunits. A,
reverse phase HPLC of tryptic peptides derived from 91- and 93/94-kDa
polypeptides of HIF-1. Absorbance profile at 215 nm of tryptic peptides
derived from 91-kDa HIF-1 polypeptide (top), 93/94-kDa
polypeptides (middle), and trypsin (bottom). B, HIF-1 and HIF-1
contact DNA directly. UV
cross-linking analysis with affinity-purified HIF-1 and probe W18 in
the absence (lane 1) or presence of 250-fold molar excess of
unlabeled W18 (lane 2) or M18 (lane 3)
oligonucleotide. The binding reaction mixtures were UV-irradiated and
analyzed on a 6% SDS-polyacrylamide gel. Molecular mass standards are
indicated at left.
Figure 6:
Glycerol gradient sedimentation analysis.
Nuclear extract prepared from Hep3B cells exposed to 1% O for 4 h (Load) was sedimented through a 10-30%
linear glycerol gradient. Aliquots (10 µl) from each fraction were
analyzed by gel shift assay. Arrows at top indicate
the peak migration for ferritin (440 kDa), catalase (232 kDa), aldolase
(158 kDa), and BSA (67 kDa).
HIF-1 was identified as a nuclear protein that binds to the
hypoxia-inducible enhancer in the 3`-flanking region of the EPO gene(15) . Several lines of evidence support the proposed
role of HIF-1 as a physiological regulator of EPO transcription in response to hypoxia: (i) induction of both HIF-1
activity (15) and EPO transcription in hypoxic cells (22) requires de novo protein synthesis; (ii)
treatment of hypoxic cells with the protein kinase inhibitor
2-aminopurine inhibits induction of both HIF-1 DNA binding activity and EPO RNA (19) ; (iii) the kinetics of HIF-1 induction
by hypoxia and of HIF-1 decay after return to normoxia parallel EPO transcriptional activity(19) ; (iv) CoCl and
desferrioxamine induce EPO expression and HIF-1
activity(19, 20) . In addition to regulating EPO transcription, HIF-1 appears to play a more general role in
regulating other hypoxia-inducible genes as
well(5, 20) .
In order to investigate the
biochemical properties of HIF-1 and the role of HIF-1 in the hypoxia
signal transduction pathway, it is necessary to clone the genes
encoding HIF-1 subunits. Our initial attempt to clone the DNA-binding
subunit of HIF-1 by screening cDNA expression libraries with
oligonucleotide W18 was unsuccessful. This may be in part due to the
fact that HIF-1 binds DNA as a heterodimer and that the DNA binding
activity of HIF-1 requires protein phosphorylation(19) . Many
transcription factors have been purified biochemically by DNA affinity
chromatography(27, 32) . However, HIF-1 has a very
rapid dissociation rate from DNA with a half-time of less than 1
min(19) , a factor that probably reduced the yield obtained by
affinity chromatography. The binding of HIF-1 to DNA may have been
stabilized by using concatenated oligonucleotides containing the HIF-1
binding site (but note HIF-1 in flowthrough fractions of the affinity
column in Fig. 2A). For the purpose of large scale
protein purification, CoCl-treated HeLa S3 cells were used.
The mechanism of HIF-1 induction by CoCl
is unknown.
Co
may substitute for ferrous iron in the heme moiety
of a putative hemoprotein oxygen sensor, resulting in a fixed deoxy
conformation(22) . Induction of HIF-1 and EPO RNA in
Hep3B cells by hypoxia and CoCl
have similar
kinetics(10, 11, 20) , and the effects of
these stimuli are not additive(23) , suggesting a common
mechanism of HIF-1 induction by these two stimuli. HIF-1 activity
induced by hypoxia and CoCl
are indistinguishable with
respect to DNA binding specificity and contacts with target DNA
sequences. Furthermore, the four polypeptides identified as HIF-1
components in CoCl
-treated HeLa cells also co-purified with
HIF-1 DNA binding activity in hypoxic Hep3B cells (Fig. 3).
Purification of HIF-1 required separation of HIF-1 DNA binding activity from a constitutive activity since both factors specifically bind to the EPO enhancer. Several lines of evidence suggested that the constitutive DNA binding activity is not part of the HIF-1 complex: (i) UV cross-linking analysis identified DNA-binding subunits of different molecular weights in the HIF-1 and constitutive activities isolated from a preparative gel shift assay(20) ; (ii) HIF-1 is highly sensitive to heat treatment, whereas the constitutive activity remained mostly intact when extracts were incubated at 65 °C for 15 min or at 80 °C for 2 min (data not shown); (iii) HIF-1 and the constitutive activity bind an overlapping DNA sequence but have distinct methylation interference patterns ((19) ; Fig. 1B); (iv) HIF-1 and constitutive activity can be separated according to their biochemical properties by ion-exchange, glycerol gradient sedimentation, and gel filtration chromatography; (v) they have a different sensitivity to the alkylation reagent N-ethylmaleimide (data not shown).
In this report we
provide evidence to support the conclusion that the 120-, 94-, 93-, and
91-kDa polypeptides are components of HIF-1. First, all four
polypeptides co-purified with HIF-1 DNA binding activity in both
Epo-producing Hep3B cells and non-Epo-producing HeLa cells, in which
HIF-1 was induced by the physiological stimulus hypoxia and the
chemical inducer CoCl, respectively (Fig. 3).
Second, the HIF-1
DNA complex isolated from native polyacrylamide
gels contained these four polypeptides (Fig. 4). Finally, the
molecular sizes of these polypeptides are consistent with the size of
DNA-binding proteins detected by UV cross-linking analysis with either
crude nuclear extracts or affinity-purified HIF-1 ((20) , Fig. 5B). The 94-, 93-, and 91-kDa polypeptides have a
similar profile of tryptic peptides (Fig. 4A),
suggesting that they represent related sequences. The difference in
their apparent molecular masses could be due to differences in
post-translational modification. To determine whether HIF-1 is a
glycoprotein as is the case for several other transcription
factors(33, 34) , we applied nuclear extracts from
CoCl
-treated HeLa cells onto a wheat germ agglutinin
affinity column. HIF-1 was found in the flowthrough (data not shown),
suggesting that HIF-1 is unlikely to be modified by glycosylation. The
possibility of protein phosphorylation can be investigated when
antibodies against HIF-1 are available. Alternatively, the
91-94-kDa species could represent proteolytic products generated
during purification or the differential usage of small exons not
detected by tryptic peptide mapping.
Glycerol gradient sedimentation
analysis indicated that HIF-1 is present in solution as stable protein
complexes. The estimation of native sizes of the HIF-1 complexes
suggested that HIF-1 exists either as a heterodimer, consisting of one
120-kDa HIF-1 subunit and one of the 91-94-kDa HIF-1
subunits or as a heterotetramer, which corresponds to the lower and
upper bands of the HIF-1 doublet detected by gel shift assay,
respectively. The proteins in each band of the HIF-1 doublet have same
DNA binding specificity (15) and make identical binding site
contacts(19) . At least one 120-kDa HIF-1
and one
91-94-kDa HIF-1
are also present in the larger protein
complex since UV cross-linking analysis of the isolated slower
migrating band identified both subunits(20) . The exact
stoichiometry of the larger form of HIF-1, however, is unknown. It is
possible that protein component(s) other than HIF-1 subunits are also
part of the complex or contribute to the stabilization of the complex.
The larger form of HIF-1 is more abundant in nuclear extracts of
hypoxic Hep3B cells than in nuclear extracts of
CoCl
-treated Hep3B cells, hypoxic HeLa cells, or
CoCl
-treated HeLa cells(20) .
To study the role of HIF-1 in the hypoxia signal transduction pathway, it is necessary to clone the genes encoding HIF-1 subunits in order to express recombinant HIF-1 proteins and to generate antibodies against HIF-1 subunits. As a first step, we have biochemically purified HIF-1 from HeLa cells. Since the hypoxic induction of HIF-1 DNA binding activity requires on-going transcription and translation, it is possible that genes encoding HIF-1 subunits are hypoxia-inducible. Alternatively, the newly synthesized component could be a modifying enzyme of HIF-1, such as a protein kinase or phosphatase. Phosphorylation is known to be involved in the hypoxic induction of HIF-1 DNA binding activity (19) and EPO gene expression(35, 36) . Protein microsequence analysis of purified HIF-1 subunits will allow the isolation of cDNA sequences necessary to establish the precise mechanisms of HIF-1 induction.