(Received for publication, January 6, 1997, and in revised form, February 24, 1997)
From the Erythropoietin Group, Institute of Molecular Medicine, John Radcliffe Hospital, Oxford OX3 9DU, United Kingdom
Hypoxia-inducible factor-1 (HIF-1), a
heterodimeric DNA binding complex composed of two
basic-helix-loop-helix Per-AHR-ARNT-Sim proteins (HIF-1 and -1
),
is a key component of a widely operative transcriptional response
activated by hypoxia, cobaltous ions, and iron chelation. To identify
regions of HIF-1 subunits responsible for oxygen-regulated activity, we
constructed chimeric genes in which portions of coding sequence from
HIF-1 genes were either linked to a heterologous DNA binding domain or
encoded between such a DNA binding domain and a constitutive activation
domain. Sequences from HIF-1
but not HIF-1
conferred
oxygen-regulated activity. Two minimal domains within HIF-1
(amino
acids 549-582 and amino acids 775-826) were defined by deletional
analysis, each of which could act independently to convey inducible
responses. Both these regions confer transcriptional activation, and in
both cases adjacent sequences appeared functionally repressive in
transactivation assays. The inducible operation of the first domain,
but not the second, involved major changes in the level of the
activator fusion protein in transfected cells, inclusion of this
sequence being associated with a marked reduction of expressed protein
level in normoxic cells, which was relieved by stimulation with
hypoxia, cobaltous ions, or iron chelation. These results lead us to
propose a dual mechanism of activation in which the operation of an
inducible activation domain is amplified by regulation of transcription factor abundance, most likely occurring through changes in protein stability.
Hypoxia-inducible factor-1, a DNA binding complex first identified
as a factor critical for the inducible activity of the erythropoietin
3 enhancer (1), is now recognized to be a key component of a widely
operative transcriptional control system responding to physiological
levels of cellular hypoxia (2-5). Deletional and mutational analysis
of cis-acting sequences has demonstrated functionally
critical HIF-11 binding sites in many
oxygen-regulated promoters and enhancers (6-12). The importance of
HIF-1 in the regulation of such genes has been confirmed by the
reduction or abrogation of hypoxia-inducible expression in mutant cells
(13, 14) that are unable to form a functional HIF complex (15-19).
Together these studies have provided strong evidence for a critical
role for HIF-1 in the regulation of genes involved in a variety of
important biological processes that include glucose transport and
metabolism, vascular growth, vasomotor regulation, erythropoiesis, iron
metabolism, and catecholamine synthesis (reviewed in Ref. 5).
As is observed for HIF-1-responsive genes (20-22), the HIF-1 complex
is inducible by particular transition elements such as cobaltous ions
and by iron chelating agents such as desferrioxamine (DFO) but not by
inhibitors of mitochondrial respiration such as cyanide or azide (3, 8,
23). These distinctive features have led to the proposal of a specific
oxygen sensing mechanism underlying these responses, most probably
involving the operation of a ferroprotein sensor (20). Recent affinity
purification and molecular cloning of HIF-1 (24) has revealed that the
DNA binding complex consists of a heterodimer of two
basic-helix-loop-helix Per-AHR-ARNT-Sim proteins HIF-1 and HIF-1
,
a molecule that is identical to the aryl hydrocarbon receptor nuclear
translocator (ARNT) (25). An important goal is now to define the
regions of the HIF-1 molecules that are responsible for their regulated activity and to understand the mechanism by which the complex is
induced and activated in hypoxic cells.
Since in the majority of studies, HIF-1 activation does not appear to be mediated through regulation of its mRNAs (15, 17, 26), we focused our analysis on other possible mechanisms of regulation. As with other transcription factors, studies of the regulatory mechanisms are potentially complicated by the ultimate dependence of transcriptional activation on a series of interrelated events which may include nuclear accumulation, dimerization, DNA binding, co-factor recruitment, and transactivation. For HIF-1, a further difficulty in this analysis lies in the operation of the native system in all cells so far tested (2, 3, 27, 28). For these reasons we used the construction of chimeric genes to define regions of the HIF-1 genes that could confer oxygen-regulated behavior on heterologous transcription factors. Two types of chimeric gene were produced, those in which the heterologous transcription factor encoded nuclear localization and DNA binding, but lacked intrinsic transactivation potential, and others in which an activation domain was either intrinsic to the heterologous gene or added to the chimeric gene from a second heterologous gene. This allowed for both the definition of activation domains of HIF-1 genes and analysis of regulatory domains that did not necessarily contain intrinsic transactivation potential.
Sequences from HIF-1 but not HIF-1
/ARNT conveyed inducible
activity on heterologous transcription factors, and two regions within
the C-terminal portion of the HIF-1
molecule were defined, each of
which possessed transactivation potential and each of which could act
independently to convey inducible properties. Both domains were
responsive to cobaltous ions and iron chelation as well as hypoxia. The
inducible activity of one regulatory domain, but not the other,
appeared to be closely connected with modulation of the level of the
encoded fusion protein, most probably arising from an effect on protein
stability. These studies therefore define the existence of more than
one regulatory domain in the HIF-1
subunit and strongly suggest the
operation of more than one mechanism of regulation.
HeLa
and Hep3B cells were grown in minimal essential medium with Earle's
salts supplemented with 10% fetal calf serum, glutamine (2 mM), penicillin (50 IU/ml), and streptomycin sulfate (50 µg/ml). Hepa-1 (Hepa1c1c7) cells and the ARNT (HIF-1) -deficient
mutant derivative, c4 cells (13, 14), were grown in minimal essential medium-
without nucleosides, with the same supplements.
For transactivation assays cells were transfected by electroporation
using a 1-millifarad capacitor array charged at 375 V. For each
transfection, approximately 107 cells were resuspended in 1 ml of RPMI 1640 containing a mixture of activator plasmid (5 or 10 µg), reporter plasmid (10 or 50 µg), and the transfection control
plasmid pSVGal (40 µg) (Promega, Madison, WI). In experiments
where amplification of the activator plasmid was desired, this was
achieved by additional co-transfection with 2.5 µg of a plasmid
expressing the SV40 large T antigen, pCMV-TAg (29). After discharge of
the capacitor, cells were left on ice for 10 min before being
resuspended in the appropriate culture medium. Aliquots of this
suspension were then used for parallel incubations. Conditions used for
normoxic and hypoxic incubation were 5% CO2, balance air
and 1% O2, 5% CO2, balance N2,
respectively. Chemicals were used at the following final
concentrations: cobaltous chloride, 100 µM;
desferrioxamine mesylate, 100 µM; potassium cyanide, 1 mM; sodium azide, 2 mM. Unless otherwise stated
experimental incubations were for 16 h. All activator plasmids were tested in at least three independent transfection experiments. Results are presented either as mean ± S.D. or as a typical
result from a set of transfections performed in parallel.
Two different chimeric
activator/reporter systems were used in transactivation assays. The
first system was based on pGR, a plasmid encoding the N-terminal 500 amino acids of the human glucocorticoid receptor, and the
glucocorticoid responsive reporter pMMTV-luc (Fig.
1A). pGR was constructed by cloning the 1.6-kb KpnI to EcoRI fragment from pRShGR (30) into the
corresponding sites in the polylinker of pcDNA3 (Invitrogen, San
Diego, CA). MMTV-luc was made by inserting a 1.4-kb BamHI
fragment from pMMTV-CAT (31) containing the glucocorticoid responsive
mouse mammary tumor virus promoter into the BglII site of
pGL2 basic (Promega, Madison, WI). Derivative plasmids based on pGR
were made by inserting the following restriction fragments 3 to the
glucocorticoid receptor sequence using appropriate linkers to preserve
the reading frame: pGR/AHR5-805, a 2.6-kb
NarI-XbaI fragment of murine aryl hydrocarbon receptor (AHR) from pcDNA3/
AHR (32); pGR/
1-789, a 2.6-kb
NcoI-NotI fragment of human ARNT from
pBM5/Neo/hARNT (14); pGR/
28-826, a 3.1-kb
BglII-NotI fragment of human HIF-1
from
pBluescript/HIF-1
3.2-3T7 (24). Further glucocorticoid receptor
HIF-1
fusions were all derived as restriction fragments from
pBluescript/HIF-1
3.2-3T7 as follows: pGR/
166-826,
HindIII-NotI; pGR/
244-826,
SalI-NotI; pGR/
329-826,
EcoRI(partial)-NotI; pGR/
530-826,
EcoRI-NotI; pGR/
652-826, SpeI-SpeI; pGR/
28-652,
BglII-SpeI; pGR/
28-329,
BglII-EcoRI; pGR/
28-825,
BglII-HpaI.
The second system was based on pCOTG (a plasmid containing an SV40
origin of replication and a cytomegalovirus-promoted truncated Gal4
gene encoding amino acids 1-147), and the Gal4-responsive luciferase
reporter pUAS-tk-luc (consisting of two copies of a 17-base pair Gal4
upstream activating site and the tk promoter, 105 to +50 (33),
inserted into the HindIII site of pA3LUC (34)) (Fig.
1B). To analyze the function of sequences from the
C-terminal region of HIF-1
, derivative plasmids based on pCOTG were
made by inserting, 3
to the Gal4 sequence, the following restriction fragments from pBluescript/HIF-1
3.2-3T7 using appropriate linkers to
preserve the reading frame: pGal/
530-826, a 1.6-kb
EcoRI-XbaI fragment; pGal/
652-826, a 1-kb
SpeI-SpeI fragment; pGal/
530-652, a 369-base
pair EcoRI-SpeI fragment.
To analyze the regulatory function of HIF-1 amino acids 530-652 on
the operation of heterologous activation domains, sequences coding for
the human aryl hydrocarbon receptor nuclear translocator (amino acids
696-789) (ARNT-ta) and herpes simplex virus protein 16 (amino acids
410-490) (VP16) were generated by PCR using Pfu polymerase
with priming oligonucleotides incorporating in frame SpeI
and XbaI restriction sites and inserted 3
to the HIF-1
sequence in pGal/
530-652 to generate pGal/
530-652/ARNT-ta and pGal/
530-652/VP16, respectively. Control plasmids pGal/ARNT-ta and
pGal/VP16 were derived by deletion of the HIF-1
sequence from these
plasmids and insertion of appropriate linkers to preserve the reading
frame. Further derivatives of pGal/ARNT-ta, containing subsequences of
HIF-1
, were generated by PCR amplification of pBluescript/HIF-1
3.2-3T7 using Pfu polymerase and
priming oligonucleotides incorporating EcoRI and/or
SpeI sites to permit in frame insertion into pGal/ARNT-ta.
These priming oligonucleotides were designed to amplify the appropriate
codons to generate pGal/
530-634/ARNT-ta, pGal/
530-611/ARNT-ta,
pGal/
530-582/ARNT-ta, pGal/
549-652/ARNT-ta, pGal/
549-634/ARNT-ta, pGal/
549-611/ARNT-ta,
pGal/
549-582/ARNT-ta, pGal/
572-652/ARNT-ta, and
pGal/
572-634/ARNT-ta.
To generate plasmids bearing C-terminal subsequences from HIF-1
similar PCR amplifications were used to create products with EcoRI and SpeI linkers suitable for in frame
insertion into pCOTG to create pGal/
668-826, pGal/
708-826,
pGal/
741-826, pGal/
767-826, and pGal/
775-826. The C-terminal
deletions pGal/
652-794 and pGal/
652-813 were made by insertion of
SpeI-PvuII and SpeI-PstI (after repair using Klenow) restriction fragments from
pBluescript/HIF-1
3.2-3T7 into pCOTG.
Constructs bearing mutations altering individual amino acids in the
HIF-1 component of pGal/
530-652/ARNT-ta were generated using a
commercially available site-directed mutagenesis kit (QuikChange; Stratagene, La Jolla, CA) and the following mutagenic oligonucleotides with their complementary sequences; Y565 to F,
5
GATGTTAGCTCCCTTTATCCCAATGGATG3
; S551,T552,T555 all to A,
5
GAACCCATTTGCTGCTCAGGACGCAGATTTAGAC3
; S577 to A,
5
CTTCCAGTTACGTGCCTTCGATCAG3
; S581 to A,
5
CTTCGATCAGTTGGCACCATTAGAAAG3
. S551,T552,T555,S577 all to A was
created by the sequential use of the corresponding oligonucleotides.
All PCR products and mutations were sequenced by the dideoxy method to
confirm veracity.
Luciferase
activities in cell lysates were determined at room temperature using a
commercially available luciferase assay system (Promega, Madison, WI),
according to the manufacturer's instructions and a TD-20e luminometer
(Turner Designs, Sunnyvale, CA). Relative -galactosidase activity in
lysates was measured using
o-nitrophenyl-
-D-galactopyranoside (0.67 mg/ml) as substrate in a 0.1 M phosphate buffer (pH 7.0)
containing 10 mM KCl, 1 mM MgSO4,
and 30 mM
-mercaptoethanol incubated at 30 °C for
45-90 min. The A420 was determined after
stopping the reaction by the addition of 1 M sodium
carbonate.
Cells were cooled
rapidly by rinsing with ice-cold phosphate-buffered saline and
harvested by scraping with a rubber policeman. The cell pellet was
subjected to a single freeze-thaw cycle, and subsequent steps were
performed at 4 °C. Cells were disrupted by passage 10 times through
a 25-gauge needle in 2.5 volumes of extraction buffer (20 mM HEPES (pH 7.9), 1 mM EDTA, 1 mM
EGTA, 0.4 M NaCl, 20% glycerol, 0.5% Nonidet P-40, 5 mM dithiothreitol, 1 mM phenylmethylsulfonyl
fluoride, supplemented with leupeptin, pepstatin, and aprotinin all at
10 µg/ml). Proteins were eluted by mixing on a shaking platform for
15 min. Supernatant was prepared by centrifugation at 13,000 × g for 10 min, mixed with an equal volume of 2 × Laemmli sample buffer, and denatured at 90 °C for 5 min prior to
SDS-polyacrylamide gel electrophoresis. Proteins were transferred onto
Immobilon-P membrane (Millipore, Bedford, MA) by electrophoresis
overnight at 20 V in Towbin buffer containing 10% methanol and 0.005%
SDS. Membranes were blocked using phosphate-buffered saline
supplemented with 5% dry milk powder and 0.1% Tween 20 prior to
indirect immunostaining. Proteins were labeled with mouse monoclonal
antibodies directed against either the C-terminal 19 amino acids of
human ARNT (HIF-1) (2B.10) (35) or the Gal4 DNA binding domain
(RK5C1; Santa Cruz Biotechnology) followed by peroxidase-conjugated
swine anti-mouse immunoglobulin (DAKO). Peroxidase activity was
detected by enhanced chemiluminescence (Super Signal Ultra;
Pierce).
Nuclear extract was prepared using a modification of the
protocol described by Schreiber et al. (36). For EMSA, a
Gal4 binding oligonucleotide, 5-CCGGAGTACTGTCCTCCG-3
, was labeled
using [
-32P]ATP (3000 Ci/mmol) (Amersham, UK) and T4
polynucleotide kinase and then annealed with 4 × molar excess of
the complementary strand. Binding reactions were performed in a 20-µl
volume containing (final concentration) 20 mM HEPES-KOH (pH
7.9), 2.4 mM EDTA, 0.3 mM dithiothreitol, 2 mM spermidine, 0.1 µg/µl poly(dI)-poly(dC), 10 mg/ml
bovine serum albumin, and 100 mM NaCl. Nuclear extract (5 µg) was incubated with this mixture for 5 min at room temperature before probe (approximately 0.1 ng) was added. Incubation was continued
for a further 30 min prior to electrophoresis (12.5 V/cm) at 4 °C
using 5% polyacrylamide in 0.3 × TBE (30 mM Tris, 30 mM boric acid, 0.06 mM EDTA (pH 7.3), at
20 °C). Gels were vacuum dried prior to autoradiography.
As a first step in studying the mechanism
of oxygen-regulated transcriptional activation by HIF-1, plasmids
expressing fusion proteins consisting of the N-terminal DNA binding
domain (amino acids 1-500) of the human glucocorticoid receptor (pGR)
linked to the complete coding sequence or near complete C-terminal
sequence of HIF-1 (pGR/
28-826) or HIF-1
/ARNT (pGR/
1-789)
were constructed. For comparison, a similar plasmid expressing the
truncated glucocorticoid receptor fused to the related transcription
factor, the mouse aryl hydrocarbon receptor (pGR/AHR5-805), was made.
The plasmids were transfected with the reporter plasmid pMMTV-luc into
HeLa and Hep3B cells. Results are given in Table I. The
activator plasmid pGR was constitutively active in both cell types,
increasing reporter gene expression 10.2- and 6.5-fold in HeLa and
Hep3B cells, respectively. In normoxic cells the fusions with HIF-1
and the AHR both reduced activity; in contrast, the fusion with HIF-1
/ARNT showed increased transactivation in comparison with pGR.
In hypoxic cells, a 5-10-fold increase in activity was observed for
pGR/
28-826, whereas only a small increase in activity was observed
in hypoxic cells with pGR, pGR/
1-789, or pGR/AHR5-805, which was
similar to that observed after transfection of the luciferase reporter
gene alone. Thus HIF-1
but not HIF-1
/ARNT could convey oxygen-regulated expression in this assay. When tested in Hepa-1 cells
and the HIF-1
/ARNT-deficient derivative, c4, essentially similar
results were obtained (data not shown), indicating that this property
of HIF-1
sequences was independent of any interaction between
HIF-1
and HIF-1
/ARNT.
|
To determine the extent to which regulation of the truncated
glucocorticoid receptor/HIF-1 fusion resembled that of endogenous HIF-1, we tested the response to a number of chemical agents. HeLa
cells were co-transfected with pMMTV-luc and pGR/
28-826 or pGR,
split into aliquots, and exposed in parallel to hypoxia, cobaltous
chloride (100 µM), DFO (100 µM), azide (2 mM), or cyanide (1 mM). Results are given in
Table II. The response characteristics were identical to
those previously reported for HIF-1. Similar responses were induced by
hypoxia, cobaltous ions, and DFO but not azide or cyanide.
|
To define the
regions of the HIF-1 gene that conveyed induction by hypoxia,
cobaltous ions, and DFO on the activity of the truncated glucocorticoid
receptor, deletions were made which removed either the 3
-untranslated
region or portions of coding sequence from HIF-1
. These experiments
were first performed in HeLa cells that were co-transfected with
plasmids expressing the chimeric genes and pMMTV-luc. To enable
comparison of the regulatory properties of the chimera with those of
HIF-1, transfected cells were again exposed to cobalt and
desferrioxamine as well as hypoxia. Deletion of the HIF-1
3
-untranslated region together with the C-terminal amino acid
(pGR500/
28-825) did not affect the properties of the chimera
indicating that the regulation was dependent on HIF-1
coding
sequences (data not shown). A series of 5 N terminus deletions of
HIF-1
was tested (Fig. 2). Chimeric genes containing
the first 2 deletions (pGR500/
166-826 and pGR500/
244-826) showed
very little activity in normoxic cells; some increase in activity in response to stimulation was observed but was difficult to quantify because of the low overall level of activity. pGR500/
329-826 had
slightly higher activity which was clearly induced by cobalt, desferrioxamine, and hypoxia. pGR500/
530-826 had activity that was
comparable to pGR in normoxic cells and that was induced by all three
stimuli. The chimeric gene bearing the final deletion, pGR/
652-826
showed a further increase in activity in normoxic cells, but its
activity was less inducible by any of the stimuli.
Two 3 deletions of HIF-1
coding sequence were tested in a similar
manner. pGR500/
28-329 had little activity in normoxic cells, and
induction by cobalt, DFO, and hypoxia was unimpressive. pGR500/
28-652 showed a higher level of activity, and induction by
all three stimuli was observed but was of lower magnitude than that
observed for the near full-length fusion protein pGR500/
28-826 or
for pGR500/
530-826.
When this series of fusion genes was tested in Hep3B cells, similar
behavior was observed except that, in these cells, the fusion
containing the C terminus of HIF-1, (pGR500/
652-826) also showed
clear inducible activity.
These results demonstrate that amino acids 530-826 of HIF-1 mediate
transactivating function and are sufficient to convey an inducible
response similar in amplitude to that observed with the near
full-length sequence, 28-826. Although levels of protein expression
were not defined in these experiments, sequences 5
to amino acid 530 appeared to be capable of a repressive action. The difference in
inducible behavior between pGR/
530-826 and pGR/
652-826 suggested
that amino acids 530-652 contained one or more sequences that were
responsive to hypoxic stimulation. In Hep3B cells, the inducible
activity of pGR/
652-826 suggested that other sequences within the
C-terminal domain might also be responsive to regulation. Furthermore,
since pGR/
530-826 showed both a reduction in normoxic activity and
an increase in hypoxic activity when compared with pGR/
652-826, the
interaction between sequences 530-652 and 652-826 appeared to be
complex.
Since the N-terminal 500 amino acids of the glucocorticoid receptor
themselves contain sequences with transactivating potential, we
considered the possibility that the inducible transcriptional activity
of the GR/HIF-1 fusion genes might be dependent on interactions with
this region of the glucocorticoid receptor or might be specific for
this type of chimeric gene. We therefore fused sequences derived from
HIF-1
to the N terminus (amino acids 1-147) of the transcription factor Gal4. Following the results presented above this analysis was
focused on the amino acids 530-826 of HIF-1
.
In contrast with the truncated glucocorticoid receptor, the truncated
Gal4 gene (Gal) had negligible intrinsic transcriptional activity,
increasing reporter gene expression only 1.4-fold. Results for the
first series of chimeric activator genes are shown in Fig. 3. When the C-terminal sequences of HIF-1 were
fused to Gal 1-147 (pGal/530-826) substantial activity was observed
which was induced a further 12-60-fold by hypoxia, cobalt, and
desferrioxamine, confirming that amino acids 530-826 of HIF-1
are
sufficient to convey an inducible transcriptional response to these
stimuli in a heterologous system. When the N-terminal portion of this sequence was tested (pGal/
530-652), little activity was observed in
normoxic cells but some inducible activity (albeit at a much lower
level) was retained, confirming that at least one of the sites able to
confer induction was contained within amino acids 530-652. We next
determined if this domain of HIF-1
could confer regulation on an
otherwise constitutive activation domain. A chimeric gene was
constructed in which the Gal4 N terminus was linked to the C-terminal
activation domain of HIF-1
/ARNT (amino acids 697-789). As expected
from the first series of experiments, this fusion protein showed
considerable activity which was not inducible (Fig. 3). A fusion gene
was then constructed in which amino acids 530-652 from HIF-1
were
encoded between the Gal4 N terminus and the HIF-1
/ARNT C terminus
(pGal/
530-652/ARNT-ta). This gene showed a low level of activity in
normoxic cells but was very strongly inducible by all three stimuli,
demonstrating that this 123-amino acid sequence from HIF-1
was able
to confer a high amplitude modulation on a constitutive activation
domain. The interactions with the HIF-1
/ARNT activation domain were
both negative and positive involving a 10-15-fold repression in
normoxic cells and a 4-5-fold activation in cells treated with
DFO.
Finally we tested the C-terminal portion of the HIF-1 gene, amino
acids 652-826, for the ability to confer inducible responses in this
system. pGal/
652-826 showed a low level of activity in normoxic
cells, which was more strikingly inducible than the equivalent glucocorticoid receptor fusion. Thus, two portions of the HIF-1
gene
were defined, each of which could act independently to confer inducible
responses to these three stimuli. Further experiments were performed to
analyze these sequences in more detail.
A series of deletions was made from both the 5 and 3
ends of this sequence to define the minimal region which could confer regulation by hypoxia on the constitutively active Gal/ARNT-ta fusion
gene. As before, sequences from HIF-1
were linked in frame between
the DNA binding domain of Gal4 and the C-terminal activation domain of
ARNT.
Results of a series of experiments in which Hep3B cells were
co-transfected with this series of activator plasmids and pUAS-tk-luc are shown in Fig. 4. Consideration of the 3 deletions
shows that whereas deletion to amino acid 634 had little effect on
activity, further deletions of HIF-1
sequence to 611 and 582 were
associated with marked increases in constitutive activity in normoxic
cells. In comparison with pGal/
530-632/ARNT-ta, similar plasmids
bearing HIF-1
sequences 530-611 and sequences 530-582 showed
approximately 10- and 20-fold increases in normoxic expression (Fig.
4). Although the ratio of stimulated to unstimulated activity was
reduced, inducibility was clearly retained. Similar increases in
constitutive expression were observed using independently constructed
plasmids bearing these 3
deletions in the context of a 5
deletion of the HIF-1
sequence to amino acid 549.
When the series of 5 deletions of HIF-1
sequence was considered two
different effects were observed. The deletion of amino acids 530-549
also increased normoxic activity, with the remaining sequence retaining
inducible activity. In contrast deletion of the HIF-1
sequence to
amino acid 572 caused complete loss of inducible activity. Again this
effect was observed in independently constructed plasmids bearing this
deletion in the context of different 3
termini; for instance, compare
plasmids expressing amino acids 549-652 with 572-652 and 549-634
with 572-634 (Fig. 4).
Taken together these findings indicate that subsequences within amino
acids 530 and 652 have the potential to generate high levels of
transcriptional activation in this context, with amino acids 549-572
being essential for this effect. Sequences 582-652 and 530-549
contain elements that effectively reduce the activity of the chimeric
transcription factor. Although the amplitude of induction was less than
with sequences 530-652, inducible activity could clearly be conferred
on the Gal/ARNT-ta fusion protein by the 33-amino acid HIF-1
sequence 549-582.
To define further the function of this domain of HIF-1, we wished to
determine if the sequence could convey changes in the level of
activator plasmid product. Whole cell extracts were prepared from
transfected Hep3B cells and were subject to Western analysis. Of
several antibodies and antisera tested, a monoclonal antibody (2B.10)
that recognizes an epitope in the C-terminal 19 amino acids of ARNT
(HIF-1
) (35) was found to give the best sensitivity. This antibody
also permitted the use of the endogenous HIF-1
/ARNT signal as an
internal control for comparison between Western blots and was therefore
used in preference to the Gal antibody (RK5C1) for experiments using
the Gal/ARNT-ta fusions, although similar results were obtained with
both reagents. Although a clear signal was obtained from the Gal4-ARNT
fusion protein in cells transfected with pGal/ARNT-ta, products of
cells transfected with pGal/
530-652/ARNT-ta were below the limit of
detection. This indicated that amino acids 530-652 of HIF-1
could
greatly reduce the level of the fusion protein but precluded an
assessment of whether levels were regulated in response to exposure to
hypoxia, cobalt, or DFO. To increase these levels into a range that
could be detected by Western analysis, Hep3B cells were co-transfected
with activator plasmid and amplifying plasmid pCMV-TAg, after which the
cells were split for parallel 48-h cultures in normoxia and stimulating
conditions. Whereas levels of the Gal4-ARNT fusion protein were high
and not regulated by the applied stimuli, the products of
pGal/
530-652/ARNT-ta and pGal/
530-634/ARNT-ta were very much
lower in normoxic cells and regulated by each of the stimuli, levels
being raised strikingly by DFO and cobalt and increased to a lesser
extent by hypoxia (Fig. 5A). This indicated
that amino acids 530-634 of HIF-1
also contained sequences that
affected the level of expressed activator protein and that might
contribute to the functional response through that mechanism. To
explore that possibility further we studied the products of other
activator plasmids in the same way. Fig. 5B shows results
for similar activator plasmids bearing sequences 549-652 and 572-652.
Whereas the product from the former was highly inducible with a low
level in normoxic cells, the latter manifested a constitutively high
level of expression. This indicated that sequences between 549 and 572 were critical for the regulated effects on protein level as well as for
transactivation, the striking finding being that deletion of this
sequence led to a high level of product with much reduced
transcriptional activity. Since we had observed that removal of
surrounding sequences 532-549 and 582-652 markedly increased the
activity of this series of plasmids, we also compared the level of
product from the activator plasmid pGal/
549-582/ARNT-ta. This fusion
protein was expressed at a slightly higher level in normoxic cells,
although the difference was much less than the difference in activity,
particularly under stimulated conditions (compare Figs. 4 and 5).
Two further experiments were performed on this modulatory sequence.
First we tested whether modulation could be conveyed on an activator
function that was not derived from the HIF-1 complex. In this
experiment we replaced the HIF-1/ARNT transactivation domain with
amino acids 410-490 from the herpes simplex virus protein VP16. In
keeping with the known properties of the VP16 activation domain, this
plasmid (pGal/VP16) showed powerful constitutive transactivation.
Inclusion of the HIF-1
domain (pGal/
530-652/VP16) conveyed
modulation on this fusion protein, activity being increased approximately 30-fold by exposure to DFO, from an activity in normoxic
cells which was almost 80-fold less than that of pGal/VP16 (Fig. 6). The effect of the HIF-1
sequence was
therefore negative under all conditions. This action was different from
that of HIF-1
sequence on the Gal/ARNT-ta fusions where both
positive and negative effects were observed. Since we were concerned
that the high activities had saturated the reporter system, we tested a
lower concentration of the Gal/VP16 plasmids. Similar results were
obtained, inducible activity being observed from a much reduced
normoxic base line.
Finally we tested the effect of a number of point mutations. Results
are shown in Table III. Mutations of phosphoacceptor
amino acids within and close by the critical amino acids 549-572
had no discernible effect on the function of
pGal/530-652/ARNT-ta.
To determine the sequences that were critical for the
inducible activity demonstrated in Gal4 chimeras containing the
C-terminal portion of HIF-1 (amino acids, 652-826), a further set
of fusions was made containing progressive deletions of this sequence.
Results of co-transfection experiments are shown in Fig.
7. The first three deletions from the 5
end of this sequence had
little effect on activity so that pGal/
652-826, pGal/
668-826,
pGal/
708-826, and pGal/
741-826 all showed similar basal and
inducible activity. In contrast, pGal/
767-826 and pGal/
775-826
showed an increase in basal expression but retained inducibility by
hypoxia, cobalt, and desferrioxamine from this higher normoxic
activity.
When deletions of the 3 end of this sequence were made, it was found
that both the basal activity and inducible property were almost
entirely ablated by removal of the C-terminal 13 amino acids. These
experiments therefore defined a second domain of HIF-1
that conveyed
responses to hypoxia, cobalt, and DFO.
We next determined the extent to which these functional results
reflected differences in the levels of fusion protein generated by the
activator plasmid. When pGal/652-826 was co-transfected with
pCMV-TAg in a manner analogous to that used to assess the previous set
of plasmids, a very high level of protein product was observed which
was not inducible. This suggested that the behavior of sequences
530-652 and 652-826 was different. To explore this pGal/
530-826
and pGal/
652-826 were compared directly (Fig. 8A). In contrast with the high level of expression of
pGal/
652-826, pGal/
530-826 showed a much lower level of
expression that was inducible, indicating that the addition of
sequences 530-652 markedly reduced and modulated the protein level in
this context as well as the previously assayed fusion proteins.
It also appeared that the 15-30-fold induction observed for the
functional activity of the HIF-1 C-terminal fusions was not reflected in changes in protein level. To consider this further we
performed additional experiments in which no amplifying plasmid was
used. Protein levels were much lower but clearly within the detectable
range. Results for the most active Gal/HIF-1
fusion pGal/
775-826
are shown in Fig. 8B; again no regulation of expressed protein level was observed.
To determine whether inducible functional activity was reflected in
changes in DNA binding activity, nuclear extracts were analyzed by
electrophoretic mobility shift assays (EMSA) using Gal4 binding
oligonucleotides. Cells were transfected and exposed to stimuli using
conditions identical to the functional assays. Fig. 9
shows an EMSA using nuclear extracts prepared from cells transfected
with the plasmid containing the GAL4 DNA binding domain alone (pCOTG),
pGal/530-826, and pGal/
652-826. In keeping with the results of
the Western analysis of protein levels, the Gal4 DNA binding activity
was low and showed increases upon stimulation in cells transfected with
pGal/
530-826. In contrast, cells transfected with pGal/
652-826
showed much higher levels of DNA binding activity, which were not
regulated by the applied stimuli, and resembled those from cells
transfected with the plasmid encoding the DNA binding domain of Gal4
alone.
We have demonstrated that sequences from the subunit of HIF-1
convey hypoxia-inducible activity when fused to the DNA binding domain
of heterologous transcription factors. As has been established for the
activation of HIF-1, the chimeric transcription factors also responded
to cobaltous ions and desferrioxamine but not to mitochondrial
inhibitors. Such responses were not observed for the HIF-1
/ARNT
subunit, defining a regulatory function for HIF-1
.
In our initial analysis of HIF-1, we fused sequences from this gene
to the N-terminal 500 amino acids of the glucocorticoid receptor, a
sequence which itself possesses transactivation activity localized to
the N terminus. This strategy was designed to enable sequences from
HIF-1
to be assayed for regulatory properties independently of their
own transactivation capability. When the activity of such fusion
proteins was tested, an inducible response was observed for fusions
containing C-terminal sequences lying distal to the portions of the
molecule known to be involved in DNA binding and dimerization (37).
When fused to the truncated glucocorticoid receptor, HIF-1
sequences
530-826 and 28-826 conferred similar inducible behavior. Since,
despite the intrinsic transcriptional activity of the truncated
glucocorticoid receptor, several fusions containing N-terminal
sequences had very low activities which made induction difficult to
assess (Fig. 2, and data not shown), this result does not necessarily
exclude inducible properties within HIF-1
sequences lying N-terminal
to amino acid 530. However, the findings did indicate that sequences
lying distal to amino acid 530 were sufficient to convey highly
inducible activity and focused our detailed analysis on this portion of
the molecule.
This analysis defined two regions within these sequences which could
independently confer inducible characteristics on heterologous transcription factors. One region was defined within HIF-1 amino acids 530-652. That this region was responsive to the inducing stimuli
was first suggested by comparison of the activity of different glucocorticoid receptor/HIF-1
fusion proteins in HeLa cells (Fig. 2)
and confirmed by its action on constitutively active chimeric transcription factors constructed from the DNA binding domain of Gal4
and activation domains from HIF-1
/ARNT or the herpes simplex virus
protein VP16 (Figs. 3 and 6). The second region was defined within
amino acids 652-826. Although inducible activity was clearly observed
when this sequence was tested as a Gal4 fusion in Hep3B cells, the
sequence had only constitutive activity when tested as a glucocorticoid
receptor fusion in HeLa cells. This difference could not be assigned to
reporter system or cell type specificity, since the relevant
glucocorticoid receptor/HIF-1
fusion showed inducible activity in
Hep3B cells, and the relevant Gal4/HIF-1
fusion showed activity in
HeLa cells albeit of lower amplitude. Whatever the reason for the
differences, these experiments did define two regulatory domains of
HIF-1
that were capable of independent action. Deletional analysis
demonstrated that in each case the property was located within a
relatively short amino acid sequence (amino acids 549-582 for the
first domain and amino acids 775-826 for the second), and functional
analysis demonstrated that in each case the inducible characteristic
included stimulation by cobaltous ions and DFO as well as hypoxia.
Somewhat surprisingly, in Hep3B cells, stimulation by cobaltous ions
and DFO was more effective than hypoxia, a difference that is not
generally observed in the regulation of endogenous
HIF-1-dependent genes (20, 22, 23) and that was not
observed in the transactivation assays performed in HeLa cells.
Amino acids in both the critical regions are 100% conserved between
human and mouse genes although, overall, amino acids 530-826 of the
human sequence are only 83% conserved in the mouse (16, 24, 38). In
keeping with the functional significance of this conservation, the
C-terminal 52-amino acid domain that we have defined in the human
HIF-1 lies within the region homologous to an 83-amino
acid-inducible activation domain defined in studies of mouse HIF-1
published during the course of this work (16). Our finding of a second
regulatory domain lying N-terminal to this region does not imply a
species difference in the mode of regulation since in the analysis of
the mouse gene that domain was not analyzed independently or in the
context of a heterologous activation domain.
An important aspect of our analysis of these regulatory domains was the
finding that sequences within amino acids 530-652 of HIF-1 had a
striking effect on the levels of fusion protein in the transfected
cells. In normoxic cells, the level of the Gal/ARNT fusion was
dramatically reduced by this sequence, the reduction being relieved by
exposure of cells to hypoxia, cobalt, and DFO in a manner that
correlated with the functional results. Studies of the regulation of
endogenous HIF-1 have demonstrated large increases in HIF-1
protein
level in hypoxic cells (24, 26) despite relative stability of HIF-1
mRNA levels (15, 17, 26). Based on the apparent stability of
HIF-1
protein in hypoxic cells and its rapid degradation when cells
are re-oxygenated, it has been proposed that the regulation of HIF-1
involves changes in stability of the
subunit (26). Although similar
measurements are difficult in transiently transfected cells, and we
have not formally addressed the mechanism by which the fusion protein
levels are regulated, our results are most consistent with this
proposal and with the regulatory domain we have defined containing a
regulated determinant of protein stability. First, the effect of the
HIF-1
sequences was always to reduce protein levels, reduction being profound in normoxic cells and relieved to a greater or lesser extent
in stimulated cells. Second, these effects were observed on gene
products expressed using the powerful constitutive cytomegalovirus promoter and optimized translational initiation sequences from different heterologous genes. Third, this region of HIF-1
is rich in
proline, glutamic acid/aspartic acid, serine, and threonine residues
that have been implicated as signals directing protein degradation
(39).
Further analysis indicated that sequences 549-572 were critically
important for the effect on protein level. Deletion of sequences surrounding this region also had effects; for instance, deletion of
amino acids 582-634 increased fusion protein levels in normoxic cells,
suggesting that these sequences might also contribute to the mechanism
of regulation. In the functional analysis, successive deletions of
amino acids 582-634 led to a progressive increase in the activity of
the chimeric transcription factor in normoxic cells and a progressive
reduction in the amplitude of the inducible response. Although in the
overall analysis of HIF-1 sequences 530-652 correlation was clearly
present between the functional effects and protein levels, we cannot be
sure whether regulation of protein level could fully account for the
effects on activity. Substantial quantitative discrepancies were
apparent between the two measurements, but given the likelihood of a
nonlinear relationship in the process of transcriptional activation,
and the fact that we used a plasmid amplification system in these
experiments, it is difficult to know whether such differences are
evidence for additional mechanisms of transcriptional regulation at
this site.
In the analysis of the C-terminal domain of HIF-1 much more
convincing support for this possibility was obtained. Gal4/HIF-1
fusion proteins containing C-terminal sequences were expressed at a
much higher level than fusion proteins containing amino acids 530-652,
and irrespective of whether the plasmid amplification system was used,
their levels were not regulated by the inducing stimuli. Moreover, when
Gal4 DNA binding activity was assayed by EMSA, activity in cells
transfected with the Gal/HIF-1
C-terminal fusion was similar to that
obtained in cells transfected with the plasmid encoding the Gal4 DNA
binding domain alone. Thus the inducible activation associated with
this domain did not appear to result from changes in protein level or
DNA binding activity. Together with the analysis of amino acids
530-652 our results strongly suggest the operation of at least two
different mechanisms of transcriptional regulation for HIF-1
, one
based on post-translational enhancement of activation and the other
involving regulation of transcription factor levels, most probably
through changes in protein stability.
One further point in relation to the analysis of amino acid sequences
530-652 is the co-localization of a potential stability determinant
and activation domain, which raises an issue as to the relation between
the two processes. Both processes may be separately and actively
regulated or one may occur as a default consequence of lack of
activation of the other. These possibilities are difficult to
distinguish, although the increased protein level and loss of
functional activity observed when the critical amino acids 549-572
were deleted shows that increases in protein level can be independent
of the process of transactivation. Interestingly, transcriptional
activation was only modest when amino acids 530-652 were considered in
isolation (Fig. 3), even when the highly active core sequence 549-582
was assessed as a simple Gal4 fusion (data not shown), nor was a
positive interaction observed when the sequences were placed adjacent
to the VP16 activation domain (Fig. 5). In contrast, in stimulated
cells, a strongly positive interaction was observed with both the
constitutive C-terminal activation domain of HIF-1/ARNT and the
inducible C-terminal activation domain of HIF-1
, allowing the
possibility that such interactions in cis or in
trans could be important in the function of the native HIF-1
heterodimer. In considering the functional data alone, the power of
this interaction is disguised. Thus, in stimulated cells the activity
of chimeric genes expressing HIF-1
sequences 530-826 was only a
little greater than those expressing sequences 652-826, but when the
differences in protein level and DNA binding activity are considered
(Figs. 8 and 9), it can be seen that the specific activation potential
of the product containing the additional amino acids 530-652 must be
very much greater.
Overall, our results suggest a model in which the function of an inducible activation domain is amplified by modulation of protein level, most probably occurring through changes in protein stability. There are many precedents for post-translational modifications that enhance transactivation through phosphorylation, ligand-dependent conformational change, or co-factor recruitment (40). Less well recognized is the regulation of transcription through changes in the stability of transcription factors, although several examples have recently been described, dependent either on the action of a specific protease or on the inducible targeting of the protein to the ubiquitin-dependent proteosomal system of degradation (39, 41). Aside from the preponderance of proline, glutamic acid/aspartic acid, serine, and threonine residues, examination of the critical sequences defined in the deletional analysis did not reveal any known recognition motifs for such systems nor did mutation of phosphoacceptor sites at residues 551, 552, 555, 565, 576, and 581 affect the operation of this regulatory domain. Nevertheless detailed analysis of these sequences should now permit important new insights to be gained into this mechanism of transcriptional regulation and the underlying processes of oxygen sensing.
We thank Sylvia Bartlett for her expert
technical assistance in this work and the following for donation of
experimental materials: Krishna Chatterjee (pRShGR, pMMTV-CAT,
pUAS-tk-luc), Stephen Goodbourn (pCOTG), Oliver Hankinson
(pBM5/Neo/hARNT, pcDNA3/AHR, Hepa 1 cells, and their
ARNT-deficient mutant derivative, c4), Gary Perdew (mouse
monoclonal antibody 2B.10), Gregg Semenza (pBluescript/HIF-1
3.2-3T7), and Dave Simmons (pCMV-TAg).