(Received for publication, November 6, 1996, and in revised form, April 25, 1997)
From the Center for Medical Genetics, Departments of Pediatrics and Medicine, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21287-3914
Hypoxia-inducible factor 1 (HIF-1) binds to
cis-acting hypoxia-response elements within the erythropoietin,
vascular endothelial growth factor, and other genes to activate
transcription in hypoxic cells. HIF-1 is a basic helix-loop-helix
transcription factor composed of HIF-1 and HIF-1
subunits. Here,
we demonstrate that HIF-1
contains two transactivation domains
located between amino acids 531 and 826. When expressed as GAL4 fusion
proteins, the transcriptional activity of these domains increased in
response to hypoxia. Fusion protein levels were unaffected by changes
in cellular O2 tension. Two minimal transactivation
domains were localized to amino acid residues 531-575 and 786-826.
The transcriptional activation domains were separated by amino acid
sequences that inhibited transactivation. Deletion analysis
demonstrated that the gradual removal of inhibitory domain sequences
(amino acids 576-785) was associated with progressively increased
transcriptional activity of the fusion proteins, especially in cells
cultured at 20% O2. Transcriptional activity of
GAL4/HIF-1
fusion proteins was increased in cells exposed to 1%
O2, cobalt chloride, or desferrioxamine, each of which also
increased levels of endogenous HIF-1
protein but did not affect
fusion protein levels. These results indicate that increased
transcriptional activity mediated by HIF-1 in hypoxic cells results
from both increased HIF-1
protein levels and increased activity of
HIF-1
transactivation domains.
Human cells require O2 for essential metabolic processes, most notably oxidative phosphorylation. Hypoxia is a significant pathophysiologic component of many cardiovascular, hematologic, and pulmonary disorders (reviewed in Ref. 1). Systemic hypoxia, e.g. due to anemia, evokes systemic responses such as increased synthesis of erythropoietin (EPO),1 the primary humoral regulator of erythropoiesis (reviewed in Ref. 2). Local hypoxia, e.g. due to vascular disease, evokes local responses such as increased synthesis of vascular endothelial growth factor (VEGF) which stimulates angiogenesis (reviewed in Ref. 3). Regardless of whether hypoxia is systemic or local in etiology, intracellular responses also occur such as the transition from oxidative phosphorylation to glycolysis (4).
These systemic, local, and intracellular responses to hypoxia all
involve changes in gene expression that occur, at least in part, at the
level of transcription and are coordinately regulated by
hypoxia-inducible factor 1 (HIF-1), a basic helix-loop-helix transcription factor (5, 6). HIF-1 DNA binding activity is induced when
mammalian cells are subjected to hypoxia (5, 7). Cis-acting
hypoxia-response elements (HREs) have been identified in promoter or
enhancer elements of human and mouse genes encoding EPO (8-10), VEGF
(11-14), the glycolytic enzymes aldolase A, enolase 1, lactate
dehydrogenase A, phosphoglycerate kinase 1 (15-18), and glucose
transporter 1 (19), as well as inducible nitric oxide synthase (20) and
heme oxygenase 1 (21) which control production of the vasoactive
molecules NO and CO, respectively. All of these HREs are sequences of
50 base pairs or less that mediate transcriptional activation of
reporter genes in response to hypoxia and contain functionally
essential HIF-1-binding sites of the consensus sequence 5-RCGTG-3
(18).
These studies suggest that HIF-1 plays a major role in mediating
homeostatic responses to hypoxia at the transcriptional level. Therefore, to understand the molecular control of these fundamental physiologic processes, it will be necessary to determine the mechanisms by which HIF-1 transcriptional activity is regulated. In addition to
hypoxia, HIF-1 DNA binding activity and expression of downstream target
genes such as EPO and VEGF can be induced by
exposing cells to cobalt chloride or iron chelators such as
desferrioxamine, findings which are consistent with the involvement of
a hemoprotein in hypoxia signal transduction (17, 22-25). HIF-1 DNA
binding activity is regulated by dramatic changes in the steady-state protein levels of the HIF-1 and (to a lesser extent) HIF-1
subunits (6, 26).
HIF-1 is an 826-amino acid protein that is unique to HIF-1, whereas
HIF-1
is identical to the aryl hydrocarbon nuclear translocator (ARNT) protein and is a common subunit for a family of heterodimeric transcription factors that are characterized by the presence of a
300-amino acid PAS domain immediately after the basic helix-loop-helix domain (6, 27). The basic helix-loop-helix and PAS domains comprise the
amino-terminal half of HIF-1
and HIF-1
(ARNT) and are required
for dimerization and DNA binding, whereas the carboxyl-terminal half of
each protein is required for transactivation (28-30). Forced expression of HIF-1
was sufficient to activate transcription of
reporter genes containing an HRE in non-hypoxic cells, indicating that
HIF-1
(ARNT) was present in excess (18). Hypoxia and forced expression of HIF-1 had synergistic effects on reporter gene
expression, indicating that in addition to increasing the steady-state
levels of HIF-1
protein, other hypoxia-induced events may be
required for maximal transcription (11, 28). Deletion of the
carboxyl-terminal half of HIF-1
resulted in a loss of
hypoxia-induced transactivation, suggesting an additional level at
which HIF-1 activity may be regulated (28). In this paper we define two
transactivation domains in the carboxyl-terminal half of HIF-1
and
demonstrate that their transcriptional activity is modulated by
cellular O2 tension.
A series of GAL4 fusion protein
expression plasmids were constructed using polymerase chain reaction
and primers that contained a BamHI site and were designed to
amplify fragments of HIF-1 cDNA. Polymerase chain reaction was
performed with Pfu polymerase (Stratagene) and pBluescript/HIF-1
3.2-3 (6) template DNA under the following conditions: 6 min at
94 °C; 94 °C for 40 s, 50 °C for 1 min, 72 °C for 1 min for 20 cycles; and 10 min at 72 °C. Polymerase chain reaction
products were ligated into the BamHI site of pGal0 (kindly
provided by C. V. Dang, The Johns Hopkins University) to make in-frame
fusions with the GAL4(1-147) DNA-binding domain (31). The reporter
plasmid, GAL4E1bLuc, containing five GAL4-binding sites upstream of a
minimal E1b TATA sequence and the luciferase gene (32), was kindly
provided by R. A. Maurer, Oregon Health Sciences University. pSV
gal
plasmid (Promega) containing the simian virus 40 (SV40) promoter and
-galactosidase gene, 2×WT33 reporter containing two copies of a
33-base pair HRE from the EPO gene, and P11w reporter
containing a 47-base pair HRE from the VEGF gene, were
described previously (5, 7, 11). The human HIF-1
expression vector
pCEP4/HIF-1
was described previously (11). Plasmid p1-390 is
identical to the previously described pCEP4/HIF-1
AflII
(28). Plasmid p1-390TAD was constructed by Pfu polymerase
amplification of HIF-1
cDNA sequences encoding amino acids
786-826 (using oligonucleotides containing an AflII site)
and ligation of the polymerase chain reaction product into the
AflII site of p1-390.
Hep3B cells were maintained
in culture as described previously (5) and transfected by
electroporation with a Gene Pulser (Bio-Rad) at 260 V and 960 microfarads (11, 28) using 6 µg of GAL4E1bLuc, 3 µg of pSVgal,
and 9 µg of each GAL4 fusion plasmid; or using 6 µg of 2×WT33 or
P11w reporter, 3 µg of pSV
gal, and 2 µg of pCEP4/HIF-1
or
pCEP4. Cells were incubated at 20% O2 for 24 h,
followed by 24 h at 1 or 20% O2 either untreated or in the presence of 75 µM cobalt chloride
(CoCl2) or 130 µM desferrioxamine. 293 cells
were transfected by calcium phosphate precipitation (33) with 2 µg of
GAL4E1bLuc, 1 µg of pSV
gal, and 3 µg of GAL4 fusion plasmid.
Cells were incubated at 20% O2 for 12 h, followed by
36 h at 1 or 20% O2 either untreated or in the
presence of 75 µM CoCl2 or 130 µM desferrioxamine. Luciferase activity was determined
using 20 µg of cell extract as described (11).
COS cells cultured to 50% confluence were transfected with GAL4 fusion plasmids using the DEAE-dextran method (35). The cells were washed twice in phosphate-buffered saline, incubated 5 h at 37 °C with 10 µg of GAL4 fusion protein plasmid and 10 µg of carrier plasmid pGEM4 in Dulbecco's modified Eagle's medium (Mediatech) supplemented with 4.5 g/liter glucose, 0.3 mg/ml DEAE-dextran (Pharmacia), and 0.1 mM chloroquine (Sigma). Plates were then washed twice in phosphate-buffered saline and incubated in fresh Dulbecco's modified Eagle's medium with 4.5 g/liter glucose and 10% fetal calf serum at 20% O2 for 24 h, followed by 24 h at 1% O2 or 20% O2 in the absence or presence of 75 µM CoCl2 or 130 µM desferrioxamine.
Nuclear Extract Preparation, Immunoblot Assay, and Electrophoretic Mobility Shift Assay (EMSA)Confluent Hep3B and
293 cells were incubated in fresh medium at 1 or 20% O2 in
the absence or presence of 75 µM CoCl2 or 130 µM desferrioxamine for 4 h. Nuclear extracts were
prepared as described (5, 34). HIF-1 and HIF-1
proteins were
detected by immunoblot analysis using affinity-purified anti-HIF-1
or anti-HIF-1
antibodies as described (26). Nuclear extracts were prepared from transfected COS cells, aliquots were fractionated in
SDS-10% polyacrylamide gels, and immunoblot assays were performed using 1 µg/ml rabbit polyclonal anti-GAL4 antibody (Santa Cruz), followed by incubation with a 1:2000 dilution of goat anti-rabbit antiserum (Amersham), and detection by ECL reagents (Amersham). Nuclear
extracts were prepared from transfected 293 cells and EMSA was
performed with oligonucleotide probe W18, containing nucleotides 1-18
of the EPO HRE, as described previously (5, 34).
We demonstrated previously
that deletion of the COOH-terminal half of HIF-1 did not affect the
dimerization of HIF-1
and HIF-1
, nor HIF-1 DNA binding activity,
but decreased transactivation of a reporter gene containing an HRE from
the EPO gene (28). An extensive series of experiments were
performed to determine whether the COOH-terminal half of HIF-1
contained one or more transactivation domains. To identify HIF-1
amino acid sequences that mediate transcriptional activation, plasmids
were constructed encoding the GAL4 DNA-binding domain fused in-frame
with HIF-1
coding sequences and expressed under the control of the
SV40 promoter. Human Hep3B hepatoblastoma or 293 embryonic kidney cells
were co-transfected with: a GAL4/HIF-1
expression plasmid; reporter plasmid GAL4E1bLuc, which contained five GAL4-binding sites and a TATA
box from the adenovirus E1b gene upstream of luciferase coding
sequences (32); and control plasmid pSV
gal, which contained E. coli
-galactosidase coding sequences under the control of the
SV40 promoter. Transfected cells were exposed to 20% O2
for 24 h and then exposed to 20 or 1% O2 for 24 h.
-Galactosidase and luciferase activity were measured in fixed
amounts of cellular protein, and the luciferase/
-galactosidase ratio
was determined. Luciferase/
-galactosidase activity obtained in the
transfected cells was normalized to the values obtained for the
parental vector, pGal0 at 20% O2 to determine the relative
luciferase activity.
Expression of HIF-1 residues 531-826 fused to the GAL4 DNA-binding
domain, GalA(531-826), resulted in dramatically higher levels of
relative luciferase activity both in Hep3B and 293 cells compared with
Gal0 (Fig. 1A). Compared with 20%
O2, luciferase activity was 5- and 66-fold higher in
GalA-expressing Hep3B and 293 cells, respectively, at 1%
O2. To further localize the transcriptional activation
domain(s), we deleted 46 and 122 amino acids from the NH2
terminus of the HIF-1
sequences to construct GalB(577-826) and
GalC(653-826), respectively (Fig. 1A). Compared with GalA, transcriptional activation mediated by GalB(577-826) and
GalC(653-826) was lower in Hep3B cells. In 293 cells, GalA, GalB, and
GalC had similar activity. The lower activity of GalB(577-826) and
GalC(653-826) compared with GalA(531-826) in Hep3B cells suggested
that the deleted sequences included a transactivation domain.
GalD(531-653) also activated reporter gene transcription in both Hep3B
and 293 cells. The transcriptional activities of GalD(531-653) and
GalC(653-826) were 4-40-fold higher in cells at 1% compared with
20% O2. Thus, even though deletion of amino acids 531-653
had no apparent effect on transactivation by GalB and GalC in 293 cells, this region functioned as an independent transactivation domain
in 293 as well as Hep3B cells. From these results we conclude that
there are two transactivation domains within the COOH-terminal half of
HIF-1
which appear to have redundant effects in 293 cells and
non-redundant effects in Hep3B cells.
To determine whether the effects on transcriptional activation were due
to different expression levels of fusion proteins, we compared GAL4
fusion protein expression and reporter gene transcription in
transiently-transfected COS cells. We used COS cells because all of the
GAL4 fusion proteins were expressed at detectable levels in COS cells
whereas many of these proteins were not expressed at detectable levels
in Hep3B or 293 cells. Each GAL4 fusion protein was expressed at
similar levels in cells cultured at 1 and 20% O2 (Fig.
2A and data not shown). Therefore, the much
higher transactivation mediated by GalA(531-826), GalC(653-826), and
GalD(531-653) in cells at 1% O2 relative to 20%
O2 was not due to higher levels of fusion protein at 1%
O2, but rather was due to higher specific transcriptional
activity. When comparing the transcriptional activity of these three
constructs in COS cells, GalD(531-653) had the lowest fusion protein
levels but activated transcription as well as or better than GalA and
GalC (in contrast to Hep3B and 293 cells). The transcriptional activity
of Gal0 in hypoxic cells was 2 orders of magnitude lower than GalD
despite higher protein levels (data not shown). Taken together, these
results demonstrate that: (i) HIF-1 contains two separate and
independent transactivation domains; and (ii) the transcriptional
activity of these domains increased when cells were exposed to 1%
O2, an effect that was independent of protein expression
levels.
Distinct Inhibitory and Transactivation Domains
To further
analyze the hypoxia-inducible transactivation domain in GalC(653-826),
an additional series of deletion constructs was generated (Fig.
1B). Compared with GalC(653-826), GalE(692-826), GalF(726-826), and GalG(757-826) exhibited a progressive increase in
transcriptional activity, especially at 20% O2.
Strikingly, GalH(786-826) mediated the highest level of reporter gene
transcription of all the fusion constructs. Compared with Gal0 at 20%
O2, the transcriptional activity of GalH(786-826) was
90,000-fold higher in Hep3B cells and 4,000-fold higher in 293 cells
(Fig. 1B). In addition, the transcriptional activity of
GalH(786-826) was not increased in hypoxic Hep3B and 293 cells, but
was still hypoxia-induced in COS cells (Fig. 2B). These data
indicate that multiple inhibitory sequences are present within HIF-1
amino acids 653-785 which have a negative effect on transcriptional
activation especially at 20% O2. Comparison of the results
for GalC(653-826) and GalH(786-826) in the three cell lines indicate
that amino acids 653-785 inhibited transactivation 3-42-fold at 1%
O2 and 88-473-fold at 20% O2. Further
deletion of HIF-1
sequences in GalI(807-826) resulted in a loss of
transcriptional activity in all cell types despite continued protein
expression (Figs. 1B and 2B). Taken together, these results indicate that (i) a transcriptional activation domain is
located between amino acids 786 and 826; (ii) amino acids 653-785 constitute an inhibitory domain which represses transactivation mediated by amino acids 786-826 especially at 20% O2;
(iii) transcriptional activity mediated by amino acids 653-826 may
therefore be derepressed in cells cultured at 1% O2; and
(iv) transactivation mediated by the fusion proteins was not a
reflection of protein expression levels, but instead was indicative of
the specific transcriptional activity of each construct.
To further localize the transactivation domain between
amino acids 531 and 653, we deleted NH2-terminal or
COOH-terminal HIF-1 sequences from GalD(531-653) to construct
in-frame GAL4 fusion proteins. Deletion of amino acids 531-576 in
GalJ(577-656) abolished transactivation function (Fig. 1C).
GalK(531-614) and GalL(531-575) maintained or exceeded the
transcriptional activity of GalD at 1 and 20% O2 in both
Hep3B and 293 cells (Fig. 1C). Transcriptional activity of
GalL in cells exposed to 1% O2 was induced 27-fold in 293 cells and 3-fold in Hep3B cells. In COS cells, transfection of pGalD
resulted in the lowest level of fusion protein expression (Fig.
2C). Therefore, the higher degree of transactivation
mediated by GalK and GalL may reflect higher levels of fusion protein
expression or higher specific transcriptional activity associated with
the deletion mutants. GalJ(577-653) was expressed at higher levels than GalD in cells at 1 and 20% O2, but GalJ had no
transcriptional activity (Fig. 2C). These results indicate
that the NH2-terminal transactivation domain is located
between amino acids 531 and 575. In contrast to the 41-amino acid
GalH(786-826) transactivation domain, which was constitutively active
in Hep3B and 293 cells, activity of the 45-amino acid GalL(531-575)
transactivation domain was modulated by cellular O2
concentration in all three cell types.
To test whether inhibitory sequences
affect the activity of the transactivation domain present in
GalL(531-575), we analyzed the transcriptional activity of fusion
protein GalM(531-784) which contained both inhibitory and
transactivation domains. Compared with GalL, the transcriptional
activity of GalM was greatly reduced (Fig. 1D,
2D). GalN(577-784) with the deletion of both
NH2- and COOH-terminal transactivation domains had no
increased transcriptional activity relative to Gal0. Immunoblot
analysis demonstrated that GalA, GalL, GalM, and GalN were expressed at
similar levels at both 20 and 1% O2 (Fig. 2D).
Therefore, the presence of inhibitory sequences did not decrease the
expression of the fusion proteins, but rather decreased the
transcriptional activity of the HIF-1 NH2-terminal
transactivation domain.
It has
previously been demonstrated that EPO and VEGF mRNA, and HIF-1 DNA
binding activity are induced in cells exposed to 1% O2,
CoCl2, or desferrioxamine (7, 22-25). To determine whether CoCl2 and desferrioxamine also modulate HIF-1
transactivation domain function, cells were cotransfected with
pSV
gal, GAL4E1bLuc and Gal0, GalC(653-826), or GalD(531-653).
Transfected cells were cultured at 20% O2 or exposed to
1% O2, 75 µM CoCl2, or 130 µM desferrioxamine. As in the case of 1% O2,
treatment with CoCl2 or desferrioxamine resulted in much
higher transcriptional activity mediated by GalC or GalD (Fig.
3A). Protein expression levels in COS cells
did not correlate with transcriptional activity (Fig. 3B).
These results indicate that the activity of both transactivation domains of HIF-1
can be stimulated by exposing cells to 1%
O2, CoCl2, or desferrioxamine, suggesting that
similar hypoxia signal-transduction pathways result in the induction of
HIF-1 DNA binding activity and HIF-1
transactivation domain
function.
Expression of Endogenous HIF-1 and Transcriptional Activation of EPO and VEGF Reporter Genes in Response to Inducers of Hypoxia Signal Transduction
It has been well documented that HIF-1 activates
human EPO and VEGF gene transcription in response
to hypoxia (5, 11, 14, 28). We noted previously that the combination of
forced expression of HIF-1 and exposure of cells to 1% O2
had synergistic effects on transcriptional activation of EPO
and VEGF reporters (11, 28). As shown for Hep3B cells
transfected with luciferase reporter plasmids containing HREs from the
EPO (Fig. 4A) or VEGF (Fig. 4B) genes, there was a synergistic effect of forced
expression of HIF-1 and exposure to CoCl2 or
desferrioxamine, as previously demonstrated for 1% O2.
Similar results were obtained using 293 cells (data not shown). The
expression of endogenous HIF-1
and HIF-1
protein was also
analyzed. HIF-1
and, to a lesser extent, HIF-1
protein levels
were greatly induced in the nuclei of Hep3B and 293 cells exposed to
1% O2, CoCl2, or desferrioxamine (Fig. 4C). We have previously demonstrated that HIF-1
protein
accumulates in nuclear extracts of hypoxic Hep3B cells and that
HIF-1
cannot be detected in cytoplasmic extracts of hypoxic or
non-hypoxic cells (6). Note that the immunoblot assays shown in Fig. 4C also demonstrate irrelevant cross-reacting bands of higher molecular weight that show no difference between the various experimental conditions and thus serve as internal controls for the specificity of
the responses. The results shown in Fig. 4 thus indicate that 1%
O2, CoCl2, and desferrioxamine share in common
the ability to dramatically increase the steady-state level of HIF-1
protein and to increase the specific activity of the HIF-1
transactivation domains, which may account, at least in part, for the
observed synergistic effects of hypoxia (or CoCl2 or
desferrioxamine) and recombinant HIF-1 on reporter gene expression.
Analysis of Transactivation Domain Function in the Context of the HIF-1 Heterodimer
The use of GAL4 fusion constructs allowed us to
analyze the transcriptional activity of HIF-1 sequences in a manner
that was independent of the HIF-1
transactivation domain(s), and
independent of the effects of hypoxia on HIF-1
protein levels.
However, it was also important to demonstrate transactivation domain
function in the context of the HIF-1
/HIF-1
heterodimer. We
therefore co-transfected cells with a reporter plasmid containing the
VEGF HRE and cytomegalovirus promoter-based expression
vectors (Fig. 5A). In the presence of empty
expression vector pCEP4, reporter gene expression was induced 5-fold in
hypoxic compared with non-hypoxic cells (Fig. 5B), due to
induction of endogenous HIF-1 activity as demonstrated by EMSA (Fig.
5C). Cells were then transfected with p1-390, which
expressed the first 390 amino acids of HIF-1
. HIF-1
(1-390) was
expressed at high levels at both 20 and 1% O2 (Fig.
5C) as previously reported (28). The 2-fold-increased reporter gene transcription at 20% O2 may reflect activity
of the HIF-1
transactivation domain(s) as previously reported (28). In transfected cells at 1% O2, reporter transcription was
repressed (Fig. 5B), indicating that overexpression of
HIF-1
(1-390) had a dominant-negative effect by competing with
endogenous HIF-1
for heterodimerization with HIF-1
and binding to
the VEGF HRE. Amino acid sequences 786-826, representing
the COOH-terminal transactivation domain, were then fused immediately
downstream of amino acid 390 (Fig. 5A). Expression of
HIF-1
(1-390/TAD) resulted in 4- and 12-fold increased reporter gene
transcription at 20 and 1% O2, respectively (Fig.
5B). HIF-1
(1-390) and HIF-1
(1-390/TAD) were constitutively expressed at similar levels (Fig. 5C).
Immunoblot analysis using an antibody directed against amino acids
807-826 revealed that levels of HIF-1
and HIF-1
(1-390/TAD)
protein paralleled the levels of HIF-1 and HIF-1*,
respectively, as demonstrated by EMSA (data not shown). Taken together,
these results indicate that the presence of amino acids 786-826
specifically increased the transcriptional activity of HIF-1
(1-390/TAD) without affecting steady-state protein levels.
We demonstrated previously that COOH-terminal deletions
of HIF-1 markedly decreased transactivation of an EPO
reporter gene in hypoxic cells (28). We demonstrate directly here that
there are two independent transactivation domains present in the
COOH-terminal half of HIF-1
which we designate as the
NH2-terminal (amino acids 531-575) and COOH-terminal
(amino acids 786-826) transactivation domains (TAD-N and TAD-C,
respectively). The activities of these transactivation domains within
the context of the intact COOH-terminal region (amino acids 531-826)
were repressed in cells at 20% O2 and increased in
response to hypoxia.
We identified an inhibitory domain (ID; amino acids 576-785) between the two transactivation domains which repressed the transcriptional activity of TAD-N and TAD-C particularly in cells at 20% O2. In cells at 20% O2, the activity of TAD-N present in GalL and TAD-C present in GalH was more than 1 and 2 orders of magnitude higher, respectively, than the activity of ID/TAD-N and ID/TAD-C sequences present in GalM (Fig. 1D) and GalC (Fig. 1B). Both TAD-C and TAD-N are rich in acidic residues (17 and 27%, respectively) and hydrophobic residues (27% in each), which are present in many previously described transactivation domains (reviewed in Ref. 37). There is, however, no specific sequence similarity between these two regions.
Deletion analysis suggested that hypoxia-inducible transcriptional
activity of GalC may result from derepression of TAD-C by ID in
response to 1% O2. Negative regulatory domains have also been identified in c-Myb (38-40), C/EBP (41, 42), and heat shock
factor 1 (43). In the case of C/EBP
, interaction between the
inhibitory and transactivation domains was demonstrated in a yeast
two-hybrid system and repression could be eliminated either by deletion
or phosphorylation of the inhibitory domain (41, 42). We were unable to
demonstrate a functional interaction between the ID and TAD-C sequences
of HIF-1
in the yeast two-hybrid system (data not shown). Our data
indicated that transcriptional activation of HIF-1
was repressed in
cells at 20% O2, and removal of ID sequences or exposure
to hypoxia (1% O2) activated the TAD-C present in GalH.
The regulation of HIF-1
transcriptional activity may involve changes
in protein phosphorylation, conformation, and/or induction of
co-activator(s) in response to hypoxia.
While this work was in progress, Li et al. (44) reported
that (i) a single mouse HIF-1 transactivation domain was located within the COOH-terminal 83 amino acids; (ii) the transactivation and
hypoxia-regulatory domains were not separable; and (iii) the COOH-terminal 45 amino acids (778-822) did not contain a functional transactivation domain. Our results differ from those reported in
several important respects: (i) we have identified two
hypoxia-inducible transactivation domains in the COOH-terminal half of
human HIF-1
. (ii) Deletion analysis of HIF-1
revealed that
removal of ID sequences resulted in a potent transactivation domain
that was constitutively active in Hep3B and 293 cells, establishing
that ID and TAD-C are distinct domains. (iii) Two minimal
transactivation domains were localized to residues 531-575 and
residues 786-826, and shown to be highly potent activators in Hep3B,
293, and COS cells. The amino acid sequences of TAD-N and TAD-C are
identical in human and mouse HIF-1
proteins (6, 44) (Fig.
6). (iv) We demonstrated that all of the GAL4 fusion
constructs were expressed as proteins of the predicted molecular weight
in COS cells. Although some of the differences between studies may
be due to the use of different cell types, it is also possible that the
negative results reported elsewhere were due to lack of expression of
GAL4 fusion constructs, since fusion protein expression levels were not
determined (44). We have demonstrated that TAD-C (residues 786-826)
functioned as a potent transcriptional activation domain in Hep3B, 293, and COS cells. When fused to the GAL4 DNA-binding domain, TAD-C also activated reporter gene transcription in Saccharomyces
cerevisiae (data not shown), indicating that amino acids 786-826
of HIF-1
constitute a transactivation domain that is active in both
mammalian and yeast cells. In contrast, a GAL4 fusion construct
containing mouse HIF-1
amino acids 778-822, which are identical to
human HIF-1
amino acids 782-826, was reported to lack
transcriptional activity, but its protein expression was not verified
(44).
Regulation of HIF-1
When Hep3B cells were subjected to
hypoxia, the steady-state levels of HIF-1 and HIF-1
protein and
HIF-1 DNA binding activity increased dramatically (5, 6, 26). We have
previously shown that forced expression of full-length HIF-1
resulted in much lower protein levels at 20% O2 than at
1% O2 (28), whereas COOH-terminal-truncated HIF-1
protein (amino acids 1-390) was expressed at equally high levels in
transfected cells at 20 or 1% O2 (28), suggesting that
sequences COOH-terminal to amino acid 390 were required for regulation
of HIF-1
protein levels by cellular O2 tension. In
contrast, we have demonstrated in this study that all the GAL4 fusion
proteins containing COOH-terminal HIF-1
sequences were expressed at
similar levels in cells at 20 and 1% O2 (Fig. 2),
suggesting that HIF-1
sequences NH2-terminal to amino
acid 531 are required for the regulation of HIF-1
protein levels by
cellular O2 tension. Whether sequences COOH-terminal to
amino acid 531 are also necessary, but not sufficient, remains to be
established.
We demonstrated previously that forced expression of HIF-1 and exposure
to 1% O2 had synergistic effects on EPO and
VEGF reporter gene transcription (11, 28). In this study, we
provide evidence that the synergistic effects are due at least in part
to increased activity of the HIF-1 transactivation domains in
response to hypoxia. HIF-1
transactivation domains were also
activated by exposure of cells to CoCl2 and desferrioxamine
independently of protein expression levels. In the same experiment,
endogenous HIF-1
protein levels were induced in cells exposed to 1%
O2, CoCl2, or desferrioxamine, similar to the
effects of these agents on EPO and VEGF mRNA
expression that have previously been reported (22-25). These data
suggest that hypoxia signal-transduction pathways are triggered in
cells exposed to 1% O2, CoCl2, or
desferrioxamine, possibly by interacting with one or more hemoproteins
involved in O2 sensing. These pathways lead to increased
steady-state levels of HIF-1
as well as increased transcriptional
activity mediated by TAD-C and TAD-N of HIF-1
, which may together
have a synergistic effect in activating transcription of HIF-1 target
genes such as EPO and VEGF. The components of the
signal transduction pathway(s) involved in these two regulatory
mechanisms remain undefined.
These studies demonstrate that amino
acids 531-826 of HIF-1 contain two transactivation domains, TAD-N
(amino acids 531-575) and TAD-C (amino acids 786-826). Our data
suggest that the transcriptional activity of TAD-C may be repressed
under non-hypoxic conditions by the inhibitory domain, ID (amino acids
576-785), and derepressed in response to hypoxia. However, we were
unable to directly demonstrate repression of TAD-C by ID in
trans (data not shown) and whether ID participates in the
physiological regulation of TAD-C function remains to be determined.
TAD-N also appears to be repressed by ID, but in addition, when
isolated from ID, the activity of TAD-N is still hypoxia-inducible in
all three cell types, suggesting the existence of a second mechanism by
which hypoxia induces HIF-1
transcriptional activity. Several
additional observations provide further evidence for considerable
complexity in the regulation of HIF-1
transcriptional activity.
First, deletion of ID sequences resulted in a graded, rather than
single-step, increase in transcriptional activity. Second, differences
were observed between cell types with respect to whether TAD-C function
was hypoxia-inducible or constitutively active. These results suggest
that cell types may differ with respect to the array of expressed
kinases and/or co-activators that can modulate HIF-1
transcriptional
activity. Nevertheless, it is clear from studies involving all three
cell types that transactivation mediated by HIF-1
is dramatically
increased in response to hypoxia. The modulation of both HIF-1
protein expression and transactivation domain function may ensure that
the biological activity of HIF-1
is very tightly regulated by
O2 tension in mammalian cells.
We thank C. V. Dang for providing COS cells and pGal0 and R. A. Maurer for providing GAL4E1BLuc.