From the Erythropoietin Group, Room 425, Institute of Molecular Medicine, John Radcliffe Hospital, Oxford, OX3 9DS, United Kingdom
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ABSTRACT |
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Endothelial PAS protein 1 (EPAS1) is a basic
helix-loop-helix Per-AHR-ARNT-Sim transcription factor related to
hypoxia-inducible factor-1 Hypoxia-inducible factor-1
(HIF-1)1 is a transcriptional
complex that plays a central role in oxygen-regulated gene expression (reviewed in Refs. 1-3). Affinity purification and molecular cloning
of HIF-1 has revealed that the DNA binding complex consists of a
heterodimer of proteins, HIF-1 HIF-1 activation is mediated predominantly by post-translational
processes affecting the Recent cloning experiments have identified several new members of the
basic helix-loop-helix PAS family, the most similar to HIF-1 Here we show that fusion of EPAS1 sequences with Gal can confer
hypoxia-inducible activity on a GAL-responsive reporter. Our analysis
defines two transactivation domains for EPAS1 (a C-terminal transactivation domain and an internal transactivation domain), which
are interspersed with sequences that possess repressive and regulatory
properties, some of which can confer regulation of fusion protein
levels. Overall these findings demonstrate a similar domain
architecture to HIF-1 Cell Culture--
Hep3B cells were grown in minimal essential
medium supplemented with 10% fetal calf serum, glutamine (2 mM), penicillin (50 IU/ml) and streptomycin sulfate (50 µg/ml).
Recombinant Plasmids Used in Mammalian Cells--
The plasmids
used are shown schematically in Fig. 1.
The chimeric activator/reporter system used in transactivation assays was based on pGal (a plasmid based on pcDNA3, which contains an SV40 origin of replication and a cytomegalovirus promoted, truncated GAL4 gene encoding amino acids 1-147 followed by a polylinker bearing
the rare restriction endonuclease sites, SacII,
AscI, NotI), and the GAL4-responsive luciferase
reporter pUAS-tk-Luc (consisting of two copies of a 17-base pair Gal4
DNA binding site and the thymidine kinase promoter,
To analyze the regulatory function of EPAS1 or HIF-1
Plasmids bearing mutations were generated using a commercially
available site-directed mutagenesis kit (QuikChange; Stratagene) and
mutagenic oligonucleotides designed according to the manufacturer's recommendations. Mutations in HIF-1
A cytomegalovirus-promoted plasmid constitutively expressing
Transient Transfection for Functional Assays--
For
transactivation assays, cells were transfected by electroporation using
a 1 mF 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 (ranging between 50 ng
and 20 µg), reporter plasmid (50 µg) and the transfection control
plasmid pCMV Luciferase and Analysis of Fusion Protein Levels in Whole Cell Extracts--
To
achieve fusion protein levels sufficient for detection of certain
activator plasmids, plasmids were amplified by co-transfection with a
plasmid expressing the SV40 large T antigen, pCMV-TAg (20). Transfections were performed as above using doses of activator and
amplifier plasmids ranging between 0.02 and 7 µg and 0.05 and 10 µg, respectively, to identify the range in which protein levels were
detectable. After transfection cells were incubated for 48 h to
allow plasmid amplification and expression. Cells were incubated in
normoxia throughout or stimulated by addition of 100 µM
desferrioxamine to the medium for the final 16 h. At harvest,
cells were cooled rapidly by rinsing with ice-cold phosphate-buffered saline, and removed by scraping with a rubber policeman. An ice-cold 7 M urea, 10% glycerol, 1% SDS, 10 mM Tris, pH
6.8, buffer containing 5 mM dithiothreitol, 50 µM phenylmethylsulfonyl fluoride, and leupeptin,
pepstatin, and aprotinin all at 0.1 µg/ml was added to the cell
pellet, which was then disrupted using a hand-held homogenizer
(Ultra-Turrax T8 with 5G dispersing tool; Janke & Kunkel GmbH, Staufen,
Germany) for 20 s and then allowed to stand on ice for 5 min.
Extract was either snap frozen on dry ice for storage or mixed with an
equal volume of 2× Laemmli sample buffer before 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 before indirect immunostaining. Proteins were
labeled with mouse monoclonal antibodies directed against either the
GAL4 DNA binding domain (RK5C1; Santa Cruz Biotechnology) or EPAS1
amino acids 535-631 (190b) (17) followed by peroxidase-conjugated
swine anti-mouse immunoglobulin (DAKO). Peroxidase activity was
detected by enhanced chemiluminescence (Super Signal Ultra; Pierce).
EPAS1 Protein Sequences Confer Hypoxically Regulated
Transactivation When Fused to a GAL4 DNA Binding Domain (Gal)--
As
a first step in the definition of regulatory domains and
transactivation domains in EPAS1, a plasmid expressing a fusion protein
consisting of Gal linked to EPAS1 amino acids 19-870 was constructed
(pGal/EPAS19-870), and activity was tested by co-transfection with a
Gal4-responsive reporter gene (pUAS-tk-Luc) into Hep3B cells. For
comparison, the activity of a similar fusion between Gal and HIF-1 Definition of Transactivation Domains in EPAS1--
To define the
regions of the EPAS1 gene that were responsible for transactivation and
those that conveyed inducible responses on the GAL4 DNA binding domain,
deletions were made, which removed either N-terminal or C-terminal
sequence from EPAS1. A series of five C terminus deletions of EPAS1 was
first tested in Hep3B cells co-transfected with pUAS-tk-luc (Fig.
3A). Whereas chimeric genes
containing deletions to amino acids 819 and 682 (pGal/EPAS19-819 and
pGal/EPAS19-682) showed inducible transactivation comparable with that
of the entire EPAS1 fusion (pGal/EPAS19-870), further C-terminal
deletions to amino acids 551, 495, and 416 removed most or all
activity. This indicated the existence of a powerful transactivation
domain lying N-terminal to amino acid 682, and that sequences between
amino acids 551 and 682 were necessary for this function. The inducible
activity of pGal/EPAS19-682 indicated the presence of at least one
domain capable of conveying inducible responses that lies N-terminal to
amino acid 682, but given the lack of transactivator function in the
C-terminal deletions to amino acids 551, 495, and 416, these
experiments did not define the domains responsible for this regulation
further.
Seven N-terminal deletions of EPAS1 coding sequence were tested in a
similar manner (Fig. 3B). Striking differences in the activity of these constructs were observed. Deletion of amino acids 19 to 495 produced a large increase in activity, particularly in normoxic
cells, suggesting that this region contains sequences capable of
exerting a functionally suppressive effect (compare pGal/EPAS19-870
and pGal/EPAS495-870). Whereas further deletion to amino acid
517 caused only a small effect on activity, deletion to amino acid 551 (pGal/EPAS517-870) produced a complete loss of this transactivating
function, suggesting that the N-terminal boundary of this
transactivating region resides between 517 and 551. With further
N-terminal deletions an increase in activity was observed indicating
the existence of a second transactivation domain capable of operation
in isolation, at the C terminus of EPAS1. Comparison of the activities
of pGal/EPAS551-870, pGal/EPAS682-870, pGal/EPAS724-870,
pGal/EPAS819-870, and pGal/EPAS828-870 indicated that this
transactivating function was contained within amino acids 828-870,
whereas amino acids 682-827 were functionally repressive. An inducible
response was observed with both plasmids pGal/EPAS724-870 and
pGal/EPAS819-870 but not plasmid pGal/EPAS828-870 locating a minimal
sequence, which could confer inducible behavior on this transactivator
between amino acids 819 and 828. In view of this evidence for
regulatory C-terminal sequences, the low amplitude of induction
observed for highly active plasmids pGal/EPAS495-870 and
pGal/EPAS517-870 appeared surprising. We therefore retested pGal/EPAS495-870 using low doses as described above, and noted that as
overall activity was reduced there was a substantial increase in the
amplitude of the inducible response (Table
I).
The combined results of the N-terminal and C-terminal deletions of
EPAS1 sequence suggested the presence of two domains in EPAS possessing
both regulatory and transactivating potential; a powerful internal
domain contained within exon 11 (amino acids 517-682) and a weaker
domain contained within the C-terminal exon (amino acids 819-870).
These experiments also demonstrated the presence of two regions capable
of exerting functionally suppressive effects on transactivation,
sequences N-terminal to amino acid 495 and sequences lying between
amino acids 682 and 819.
To test whether the internal transactivation domain could function
independently and to define the functional sequences in more detail, a
further series of plasmids was constructed and tested in Hep3B cells.
pGal/EPAS517-682 showed powerful inducible transactivation, confirming
that this internal transactivation domain could function independently
and indicating that it also contained regulatory sequences. Deletions
from the C terminus and N terminus of this region both produced a
decrease in activity, indicating that this entire domain was necessary
for maximal activity (Fig. 4).
Interestingly, the N-terminal deletion (pGal/EPAS534-682) showed
enhanced transactivation in normoxic cells and complete loss of the
inducible response, defining a short sequence (amino acids 517-534) as
critical for the regulatory property.
Domains from EPAS1 Can Confer Hypoxic Regulation on the
Heterologous VP16 Transactivator--
Though experiments described
above indicated that the N-terminal 495 amino acids of EPAS1 sequence
had functionally suppressive effects on the internal and C-terminal
transactivation domains and might contribute to regulation, such a
function could not be demonstrated independently because deletion of
the native transactivation domains of EPAS1 removed all activity. To
enable further analysis of this possibility, a second set of fusions
was created in which the powerful C-terminal 80 amino acid
transactivator from herpes simplex virus protein 16 (VP16) was linked
to the C terminus of the Gal/EPAS1 fusion proteins.
First, the N-terminal sequence of EPAS1 (amino acids 9 to 517) was
tested by creation of pGal/EPAS9-517/VP16. When compared with the
activity of a plasmid encoding the Gal/VP16 fusion alone, inclusion of
EPAS1 amino acids 9 to 517 had a profoundly suppressive effect on
transactivation in normoxic conditions. On stimulation by hypoxia,
cobaltous ions, or desferrioxamine the extent of repression was reduced
(Fig. 5A), indicating that
this sequence could convey regulatory effects in isolation and that
these effects could be conveyed on a heterologous transactivator.
Other portions of the EPAS1 molecule were assayed for regulatory
activity in a similar way by inserting three portions of EPAS1 sequence
(corresponding to exons 2-6, 7-11, and 12-15, and covering all
except the N-terminal 8 amino acids) between the Gal and VP16 domains
(pGal/EPAS9-295/VP16, pGal/EPAS295-682/VP16, and
pGal/EPAS682-870/VP16). Results are shown in Fig. 5B. In
comparison with pGal/VP16, both pGal/EPAS9-295/VP16 and
pGal/EPAS682-870/VP16 had rather similar activity, which was not
inducible. In contrast, pGal/EPAS295-682/VP16 had much reduced
activity in normoxic cells and showed induction by all three stimuli.
These results suggested that repressive and regulatory properties of
the internal domain of EPAS1 could be conferred on a heterologous
transactivator. In contrast, comparison of the activity of
pGal/EPAS682-870/VP16 with the simple Gal/EPAS fusions (Fig.
3B) indicated that sequences from the C-terminal domain of
EPAS1, which exerted powerfully repressive and regulatory effects on
the native C-terminal transactivation domain, had little or no such
effects on the VP16 transactivator.
In an attempt to pin-point shorter sequences with regulatory potential,
sequences corresponding to exons 7-11 were individually inserted
in-frame into the Gal/VP16 fusion and tested in an analogous manner. To
maximize the chance of observing subsequences with the ability to
convey regulation in isolation, plasmids were tested at several doses.
Results are shown for cells transfected with 100 ng in Fig.
5C; identical results were obtained with 10 ng. Though
sequences corresponding to exon 7 were suppressive in this assay, no
individual exon conveyed regulation at any dose with the exception of
exon 11 (amino acids 517-682).
Finally, the effect of removing individual exons from the internal
domain (exons 7-11) was tested (Fig. 6).
Removal of exon 11 (creating pGal/EPAS295-517/VP16) led to much higher
activity in normoxic cells and greatly reduced inducibility. Removal of successive exons from the N terminus led to a more progressive increase
in activity in normoxic cells and a progressive reduction in the extent
of induction. The results therefore suggest that multiple sequences
within exons 7-11 contribute to regulatory properties of this domain.
Sequences lying both N-terminal and C-terminal to amino acid 517 were
able to confer the inducible property in isolation, though the effect
was only modest for the N-terminal portion.
These data indicate an organization for EPAS1 in which two
transactivation domains are surrounded by sequences that have
repressive effects and that confer regulation. Previous analyses of the
C-terminal domain (amino acids 530-826) of HIF-1 Comparison with Regulatory Domains in HIF-1
Sequence comparison indicated a high degree of conservation in the
intron-exon boundaries of the two genes. However, the sequence of
several portions of the internal region is poorly conserved between
HIF-1
The main contrast between EPAS1 and HIF-1 Regulated Fusion Protein Levels Contribute to the Inducible
Activity Conveyed by Internal EPAS1 Domains--
Whole cell extracts
were prepared from Hep3B cells transfected with plasmids that express
chimeric proteins containing domains from EPAS1 fused to Gal and
subjected to Western blot analysis. Although a clear signal was
obtained from the Gal protein in cells transfected with pGal, products
of cells transfected with the majority of fusion proteins were below
the limit of detection, precluding an assessment of whether levels were
regulated in response to exposure to hypoxia, cobalt, or
desferrioxamine. To increase expressed fusion protein levels into a
range that could be detected by Western analysis, Hep3B cells were
co-transfected with the activator plasmid and an amplifying plasmid,
pCMV-TAg, after which the cells were split for parallel incubations in
normoxia and stimulating conditions. The expression of plasmids
containing either the N-terminal portion (amino acids 9 to 517) or the
C-terminal portion (amino acids 517 to 870) of EPAS1 fused to Gal were
first tested (Fig. 8). Regulation of Gal
fusion protein level was observed with pGal/EPAS517-870, but not
pGal/EPAS9-517. To analyze this further, a series of Gal/EPAS1 fusions
with different EPAS1 N-terminal deletions was examined. Three plasmids
expressing fusion proteins containing successive N-terminal deletions
to EPAS1 amino acids 345, 495, and 517 all manifested regulated fusion
protein level, whereas two plasmids expressing further deletions to
amino acids 682 and 819 expressed unregulated levels of fusion protein
(Fig. 8). We also conducted titrations in which plasmids were expressed at varying levels. The same results were obtained even when plasmid concentrations used in transfections were adjusted such that expression levels were at the limit of detection by Western analysis (data not
shown). Using higher plasmid doses, a dependence of inducible expression on overall level of expression was again observed. An
example of such a titration for the plasmid pGal/EPAS495-870 is shown
in the bottom panel of Fig. 8. The fusion protein level is seen to be
inducible by desferrioxamine only at the lower levels of
expression.
Definition of Amino Acids Critical to the Regulated Function of the
C Terminus of EPAS1 and HIF-1 In these experiments, we have analyzed the activation and
regulatory domains of EPAS1, and compared their function with those of
HIF-1 Analysis of transactivation defined two domains in EPAS1, one lying at
the C terminus within exon 15 and the other in an internal region
corresponding broadly to exon 11. A similar arrangement exists in
HIF-1 Analysis of regulation defined at least three EPAS1 sequences, which
could independently convey oxygen-regulated activity. These were
located within and N-terminal to the internal activation domain and
between the activation domains. Two types of activity were
distinguished. Sequences overlapping and extending N-terminal to the
internal transactivation domain conveyed regulation on a Gal/VP16
fusion, indicating that their operation was not dependent on a specific
interaction with EPAS1 transactivation domains. In contrast, sequences
lying adjacent to and conveying regulation on the C-terminal EPAS1
transactivation domain had no action on the heterologous Gal/VP16
system, indicating a different mode of operation.
Comparison with regulatory domains of HIF-1 The comparative analysis also highlighted differences between the
molecules. For instance a striking difference was observed in the
dose-dependent activity of Gal fusions to EPAS1 (amino acids 19-870) or HIF-1 Analysis of individual sequences also showed lower inducibility in both
the C-terminal domain and the internal regulatory domains of EPAS1.
This was most striking for the N-terminal portion of the internal
domain. We considered whether the poor conservation in EPAS1 of
HIF-1 The exon analysis did reveal one unexpected but striking finding. EPAS1
exon 7 was very strongly repressive in these assays although it did not
confer regulation. Similar results were obtained with HIF-1 Overall, when the functional analysis was compared with the extent of
sequence similarity, a good correlation was observed (Fig.
10). Outside the basic helix-loop-helix
PAS domains (which are known to possess similar dimerization and DNA
binding properties) (13, 15), the highest levels of sequence similarity
are seen in the N-terminal portion of EPAS1 exon 11 (HIF-1 (HIF-1
). To analyze EPAS1 domains
responsible for transactivation and oxygen-regulated function, we
constructed chimeric fusions of EPAS1 with a GAL4 DNA binding domain,
plus or minus the VP16 activation domain. Two transactivation domains
were defined in EPAS1; a C-terminal domain (amino acids 828-870), and
a larger internal domain (amino acids 517-682). These activation
domains were interspersed by functionally repressive sequences, several of which independently conveyed oxygen-regulated activity. Two types of
activity were defined. Sequences lying N-terminal to and overlapping
the internal transactivation domain conferred regulated repression on
the VP16 transactivator. Sequences lying C-terminal to this internal
domain conveyed repression and oxygen-regulated activity on the native
EPAS1 C-terminal activation domain, but not the Gal/VP16 fusion.
Fusions containing internal but not C-terminal regulatory domains
manifested regulation of fusion protein level. Comparison of EPAS1 with
HIF-1
demonstrated a similar organization for both proteins, and for
the C terminus defined a conserved RLL motif critical for inducibility.
Overall, EPAS1 sequences were less inducible than those of HIF-1
,
and inducibility was strikingly reduced as their expression level was
increased. Despite these quantitative differences, EPAS1 regulation
appeared similar to HIF-1
, conforming to a model involving the
modulation of both protein level and activity, through distinct
internal and C-terminal domains.
INTRODUCTION
Top
Abstract
Introduction
References
and the aryl hydrocarbon receptor
nuclear translocator (ARNT) (4). Both are members of the rapidly
expanding PAS superfamily of basic helix-loop-helix proteins defined by
the presence of two regions containing repeated sequences that share
homology with the prototypical members from which the family's name is
derived, drosophila periodic, the aryl
hydrocarbon receptor, the aryl hydrocarbon receptor nuclear
translocator and drosophila single minded (5). HIF-1 binds
to hypoxia response elements containing the consensus BRCGTGV and
activates the transcription of a wide variety of genes that encode
products involved in hematopoiesis (erythropoietin), angiogenesis, and
vasomotor control (vascular endothelial growth factor, nitric oxide
synthases, and endothelins), energy metabolism (glycolytic enzymes and
glucose transporters), catecholamine synthesis (tyrosine hydroxylase),
and iron metabolism (transferrin) (for reviews see Refs. 1-3).
subunit (6-11). Understanding the interactions of these processes with the sensing and/or signal transduction processes is an important but potentially complex issue in
which primary points of interaction need to be defined and
distinguished from processes that are downstream consequences of such
interactions. A key step in such analyses is the definition of
functional domains within the molecules, in particular, regions that
can independently confer the regulatory characteristic on a
heterologous system. Several groups have now analyzed aspects of
HIF-1
regulation and defined domains that can independently convey
oxygen-regulated properties onto heterologous transcription factors
such as the yeast GAL4 DNA binding domain (Gal) (7, 9, 11, 12).
being a
molecule first described as endothelial PAS protein 1 (EPAS1) (13), but
also independently identified by other groups and termed member of PAS
superfamily 2 (MOP2) (14), HIF-like factor (HLF) (15), and HIF-related
factor (HRF) (16). The protein shares 48% sequence identity with
HIF-1
, forms heterodimers with ARNT, and can activate transcription
from a hypoxia response element (13). In hypoxic cells, EPAS1 protein
levels are greatly up-regulated (17). Moreover, responses to chemical
and pharmacological probes with known effects on HIF-1 activation are
very similar (17), suggesting that one or more regulatory mechanisms
are shared, and indicating that it should be informative to define regulatory and activation domains in EPAS1, and to compare their function with those in HIF-1
.
. However, some Gal/EPAS1 fusions showed higher
activity in normoxic cells and a lower amplitude of induction,
particularly at higher levels of expression, indicating that there are
quantitative differences in the activation characteristics of these molecules.
EXPERIMENTAL PROCEDURES
105 to +50 (18),
inserted into the HindIII site of pA3LUC) (19). To analyze
the function of sequences from EPAS1 or HIF-1
, they were amplified
by polymerase chain reaction using Pfu polymerase
(Stratagene, La Jolla, CA) and forward oligonucleotides containing a
SacII recognition sequence in the appropriate reading frame
and reverse oligonucleotides containing an AscI recognition
site and cloned into pGal. phEP-1 was used as template for EPAS1 (13)
and pBluescript/HIF-1
3.2-3T7 (4) as template for HIF-1
sequences.
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Fig. 1.
Schematic representation of the chimeric
activator and reporter plasmids used. The restriction sites used
in construction are indicated (see Experimental Procedures).
amino acids on
the operation of a heterologous activation domain, sequences coding for
the herpes simplex virus protein 16 amino acids 410-490 (VP16) were
generated by polymerase chain reaction using Pfu polymerase with priming oligonucleotides incorporating in frame AscI
and NotI restriction sites, and inserted 3' to the EPAS1
sequence. The control plasmid pGal/VP16 was produced by insertion of
this polymerase chain reaction product directly into pGal, preserving the reading frame. All plasmids were subjected to in vitro
transcription/translation reactions in the presence of
35S-methionine and products analyzed by SDS-polyacrylamide
gel electrophoresis to confirm the production of an appropriate fusion protein.
and EPAS1 were made in the context of pCOTG/
775-826 (7) and pGal/EPAS819-870, respectively. These mutations were sequenced by the dideoxy method to confirm veracity.
-galactosidase (pCMV
Gal) was used as a transfection control.
Gal (15 µg). 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; and sodium azide, 2 mM. Experimental incubations were for 16-18 h. All
activator plasmids were tested in at least three independent
transfection experiments.
-Galactosidase Assays on Mammalian Cell
Extracts--
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
15-45 min. The A420 was determined after
stopping the reaction by the addition of 0.3 M sodium
carbonate (final concentration).
RESULTS
(pGal/
28-826) was tested. Both EPAS1 and HIF-1
sequences
conferred transcriptional activity on the GAL4 DNA binding domain, and
in each case, activity was inducible by hypoxia. Induction was also
obtained by exposure of transfected cells to cobaltous ions or
desferrioxamine (Fig. 2) but not with
exposure to the mitochondrial inhibitors, azide and cyanide (not
shown), a pattern that is in keeping with the known characteristics of
HIF-1 and HIF-1 target gene activation (21-24). Despite these
similarities, there were substantial differences in the amplitude of
induction shown by the fusion proteins, with the EPAS1 fusion showing
higher normoxic activity than the HIF-1
fusion, but less potent
induction by all three stimuli. Because we had noted in previous
experiments that the amplitude of induction by hypoxia of certain
Gal/HIF-1
fusions varied with the level of expression and was less
in cells transfected either with large or very small amounts of
plasmid, we compared the inducible activity of both plasmids over a
wide range of transfection doses. Results illustrated in Fig. 2 show that for the EPAS1 fusion, reporter activity was maximal and appeared to saturate at plasmid doses from 1 µg upward. The maximum amplitude of induction occurred at 0.25 µg and was much reduced when the highest dose of plasmid was used. In contrast, the HIF-1
fusion was
not responsive at the lowest plasmid dose, manifested highly inducible
activity over the remainder of the dose range, and showed signs of
saturation with a reduction in the amplitude of induction only at the
highest dose tested.
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Fig. 2.
Comparison of the transcriptional activity of
Gal/EPAS1 and Gal/HIF-1 fusion genes in Hep3B cells. Activator
plasmids encoded the indicated amino acids of EPAS1 or HIF-1
fused
3' to the 147-amino acid GAL4 DNA binding domain. Cells were
co-transfected with an activator plasmid, the GAL4-luciferase reporter
plasmid (pUAS-tk-Luc) (50 µg), and pCMV
Gal (15 µg) (to provide a
control to correct for variation in transfection efficiency) and
harvested after 16 h incubation in the presence of normoxia,
hypoxia, 100 µM cobaltous ions, or 100 µM
desferrioxamine. Different doses of each activator plasmid between 0.05 and 20 µg were used as indicated. Bars indicate the corrected
luciferase activity (arbitrary units). Results obtained when 5 µg
pGal was used are shown for comparison.
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Fig. 3.
Transcriptional activity of different
Gal/EPAS1 fusion genes. A, C-terminal deletions and
B, N-terminal deletions. Activator plasmids encoding the
indicated amino acids of EPAS1 fused 3' to the 147-amino acid GAL4 DNA
binding domain or the GAL4 DNA binding domain alone. Hep3B cells were
co-transfected with an activator plasmid (1 µg), the GAL4-luciferase
reporter plasmid (pUAS-tk-Luc) (50 µg) and pCMV Gal (15 µg)
(transfection control) and harvested after 16 h incubation in the
presence of normoxia, hypoxia, 100 µM cobaltous ions, or
100 µM desferrioxamine. Bars show corrected luciferase
activity (arbitrary units).
Dependence of the amplitude of induction of a Gal fusion protein
containing EPAS495-870 on the dose of activator plasmid used
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Fig. 4.
Definition of an internal transactivation
domain in EPAS1. Hep3B cells were co-transfected with 200 ng of
chimeric Gal activator plasmids expressing the indicated amino acids
from exon 11 of EPAS1 fused to the GAL4 DNA binding domain and 50 µg
of the GAL4 responsive reporter plasmid (pUAS-tk-Luc). pCMV Gal (15 µg) was used to provide a control for transfection efficiency.
Bars show the corrected luciferase activity (arbitrary units)
after 16 h incubation in the presence of normoxia, hypoxia, 100 µM cobaltous ions, or 100 µM
desferrioxamine.
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Fig. 5.
Action of EPAS1 sequences on the
transcriptional activity of a Gal/VP16 fusion gene. Panel A,
transfection of Hep3B cells was as outlined in Fig. 2 except that 100 ng of activator plasmid was used. Comparison is made between
transfection with pGal/VP16 and pGal/EPAS9-517/VP16. After
transfection cells were incubated in the presence of normoxia, hypoxia,
100 µM cobaltous ions, or 100 µM
desferrioxamine for 16 h before preparation of extracts and
determination of corrected luciferase activity. Panels B and
C, transfections of Hep3B cells were performed and analyzed
as in panel A using plasmids encoding the indicated EPAS1
sequences between the GAL4 DNA binding domain and amino acids 410-490
of the herpes simplex virus protein 16. The control plasmid was
included in each set of transfection experiments and bars show the
relative luciferase activity expressed as a percentage of the normoxic
activity of the Gal/VP16 control activator plasmid. The amplitude of
induction (stimulated/normoxic activity) is indicated to the
left of the relevant bar. The EPAS1 exons included in the
test plasmids are indicated in parentheses.
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Fig. 6.
Actions of different portions of the EPAS1
internal regulatory domain on the transcriptional activity of Gal/VP16
fusion genes. Transfections using Gal/VP16 activator plasmids
containing various combinations of exons from within the internal
regulatory domain of EPAS1 were performed as described in Fig. 5.
Columns show the relative luciferase activity expressed as a percentage
of the normoxic activity when the Gal/VP16 control activator plasmid
was used. The amplitude of induction (stimulated/normoxic activity) is
indicated to the left of the relevant bar. The EPAS1 exons
included in the test plasmids are indicated in
parentheses.
have defined a
similar organization (7, 9). In our analysis of HIF-1
we also showed that amino acids 28-530 confer a suppressive effect on the endogenous transactivating capacity (t1) of the human glucocorticoid receptor DNA
binding domain (amino acids 1-500) (7). The power of this suppressive
effect almost totally obliterated the activity of this relatively weak
transactivator making it difficult to ascribe any regulatory function
to this region of HIF-1
. We therefore sought to extend the current
comparison of EPAS1 with HIF-1
by analyzing further the functional
effects of HIF-1
sequences on the Gal/VP16 fusion.
--
To permit a
direct comparison with the current analysis of EPAS1, we made similar
Gal/VP16 fusions bearing HIF-1
amino acids and tested their function
in Hep3B cells. During the course of this work, the intron-exon
structure of HIF-1
was published (25). Constructs were first
designed to follow from our previous analysis of HIF-1
.
Subsequently, as with EPAS1, the HIF-1
sequences tested were based
on the known intron-exon structure. Fig.
7A shows the effect of
N-terminal HIF-1
sequences (amino acids 13-553). These sequences
had a profoundly suppressive effect in the system and conferred high
levels of regulation. Though this effect was similar to that observed
for EPAS1, the HIF-1
sequence was associated with a higher degree of
suppression and a higher amplitude of regulation. Fig. 7B
shows a similar analysis for other regions of HIF-1
. As reported
previously, we found that pGal/
530-652/VP16 showed regulated
activity. Such regulated activity was again observed with sequences
lying N-terminal to this region (pGal/
28-530/VP16) but not
C-terminal to this region (pGal/
652-813/VP16). Interestingly, HIF-1
C-terminal sequences, which confer repression and high level
regulation on the native transactivation domains (but excluding a
subdomain, amino acids 530-572, necessary for regulation of protein
level of a Gal/HIF-1
fusion) (7), had little or no effect on the
Gal/VP16 fusion (pGal/
572-774/VP16, Fig. 7B).
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Fig. 7.
Action of HIF-1 sequences on the
transcriptional activity of a Gal/VP16 fusion gene. Transfections
of Hep3B cells were as described in Fig. 5 except that activator
plasmids encoded DNA for the indicated sequences of HIF-1
expressed
between the DNA binding domain of GAL4 (amino acids 1-147) and amino
acids 410-490 of the herpes simplex virus protein VP16. Panels
A-D illustrate the results of three series of transfection
experiments. In each set of experiments the control plasmid, pGal/VP16
was included (though its activity is only illustrated in panel
A). Columns show the relative luciferase activity expressed as a
percentage of the normoxic activity of Gal/VP16. The amplitude of
induction (stimulated/normoxic activity) is indicated to the
left of the relevant bar. The EPAS1 exons included in the
test plasmids are indicated in parentheses. The
asterisk denotes the fact that the actual exon 10/11
boundary is at amino acids 512/513 (see Fig. 10).
and EPAS1 (Fig. 10), and in particular, HIF-1
contains a
number of nonconserved sequences inserted into the N-terminal portion
of exon 10. In an attempt to define sequences that might contribute to
the enhanced inducibility observed for the HIF-1
fusions, we
extended the comparison by testing individually subsequences covering
HIF-1
amino acids 294 to 698 (corresponding to exons 8-12). As with
EPAS1 exon 11, the corresponding HIF-1
exon 12 conferred regulation
in isolation (Fig. 7C). However, none of the other
individual subsequences (including that containing the nonconserved
portion of exon 10) conferred high level regulation in isolation,
though HIF-1
exon 8 was (like EPAS1 exon 7) profoundly suppressive,
and HIF-1
exon 9 showed modest inducible activity. Nevertheless when
considered together, HIF-1
sequences 345-553, corresponding to
exons 8-11, conferred substantially greater inducibility than the
corresponding portion of EPAS1 (compare Fig. 7D with Fig.
6).
was therefore in the
overall level of inducibility. Nevertheless, there were many similarities in the organization of the two molecules that involved transactivation domains interspersed by sequences that have repressive effects and that confer regulation. In each case, these repressive sequences appeared to be of two types; internal sequences whose action
was clearly evident on the heterologous Gal/VP16 fusions, and sequences
lying C-terminal to the internal transactivation domain whose
repressive action could not be transferred in that way. Our previous
analysis of the C-terminal domain (amino acids 530-826) of HIF-1
had defined two types of regulatory mechanism. Sequences lying
C-terminal to the internal transactivation domain had an action that
was not dependent on changes in protein level, whereas those
overlapping the internal transactivation domain could also confer
regulation of fusion protein level. We therefore tested the ability of
different regulatory sequences from EPAS1 to confer regulation on Gal
fusion protein levels.
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Fig. 8.
Expression of Gal/EPAS1 fusion proteins in
transfected Hep3B cells. Cells were transfected with activator
plasmid and pCMV-TAg and incubated for 48 h in normoxia with or
without 100 µM desferrioxamine applied for the last
16 h (N and D, respectively). Results from
untransfected cells (U) are also shown. Whole cell extracts
(50 µg of protein) were analyzed by SDS-polyacrylamide gel
electrophoresis and expressed fusion proteins were detected by enhanced
chemiluminescence after indirect immunostaining with the specified
mouse monoclonal antibody followed by peroxidase-conjugated goat
anti-mouse immunoglobulin. The four panels at the
top show results obtained after transfection with the
indicated constructs, using monoclonal antibody RK5C1 directed against
the GAL4 DNA binding domain. Results for pGal/EPAS517-870 and
pGal/EPAS345-870 identified with monoclonal antibody 190b against
human EPAS1 are shown in the two middle panels. The effects
of overall expression on the amplitude of regulation are illustrated in
the bottom panel. Transfections were performed with
increasing doses of the pGal/EPAS495-870 activator plasmid and a fixed
dose of pCMV-TAg. In each panel, the position of the
relevant fusion protein is indicated by a closed arrow; in
the middle panels, open arrows indicate the
position of the endogenous EPAS1 protein.
--
Our functional analysis of EPAS1
and the comparison with HIF-1
, indicates that in certain regions
sequence comparison should give clues as to functionally critical
residues. Nevertheless, the large size of some of the regulatory
domains together with a level of redundancy among subsequences makes
this a relatively difficult task for the internal regulatory domains.
However, at the C terminus of EPAS1 our experiments showed that
addition of nine amino acids was sufficient to confer regulation on an
otherwise constitutive transactivation domain (Fig. 3B).
Comparison of published data suggested that this might also be true of
HIF-1
(7, 9). To test this directly, we tested the activity of
pGal/
775-826 and pGal/
786-826 (Fig.
9A). Results indicated that,
as with EPAS1, addition of this short sequence to the N terminus of the
transactivation domain conveyed regulation on an otherwise
constitutively active region. The effects of scanning mutations through
this region and the effect of mutating a single conserved cysteine
lying close to this region (HIF-1
, Cys-800) are illustrated in Fig.
9C. Mutation of the cysteine residue, Cys-800, to alanine
produced a modest reduction in activity but did not ablate
inducibility. Relatively modest effects were also observed with
mutations affecting conserved serines (HIF-1
, PSD775-777;
GQS784-786), and a nonconserved cysteine (HIF-1
, LAC778-780). In
contrast, a much greater effect was observed with mutation of the
conserved sequence arginine-leucine-leucine. Mutation of this sequence
in the context of either the HIF-1
C terminus or the EPAS1 C
terminus (HIF-1
, RLL781-783; EPAS1, RLL825-827) greatly increased
normoxic activity and essentially recreated constitutive
transactivation.
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Fig. 9.
Definition of functionally critical amino
acids in the C termini of EPAS1 and HIF-1 . A, HIF-1
amino acids 775-785 are critical for regulated function. Hep3B cells
were co-transfected with 1 µg of an activator plasmid (encoding the
indicated amino acids of HIF-1
fused 3' to the 147 amino acid GAL4
DNA binding domain or the GAL4 DNA binding domain alone), the
GAL4-luciferase reporter plasmid (pUAS-tk-Luc) (50 µg), and
pCMV
Gal (15 µg) (transfection control) and harvested after 16 h incubation in the presence of normoxia, hypoxia, 100 µM
cobaltous ions, or 100 µM desferrioxamine. Bars show the
corrected luciferase activity (arbitrary units). B, sequence
comparison of HIF-1
amino acids 775-786 and EPAS1 amino acids
819-828. C and D, mutational analysis of the
C-terminal amino acids of HIF-1
and EPAS1. Hep3B cells were
transfected with 500 ng of pCOTG/
775-826 (7) or with similar
plasmids encoding the indicated amino acid mutations in the HIF-1
sequence in combination with the GAL4-luciferase reporter plasmid
(pUAS-tk-Luc) (50 µg) and pCMV
Gal (15 µg) as described
previously. Columns indicate the -fold induction (stimulated/normoxic
activity) in response to hypoxia, cobaltous ions, and desferrioxamine.
The constitutive activity, normalized to the activity of equivalent
plasmids expressing Gal alone in normoxic cells is indicated. Data for
pGal/EPAS819-870 with and without the RLL-AAA mutation is also
shown.
DISCUSSION
.
(7, 9). For the C-terminal domain, conservation between the
genes was striking with activity residing in a very similar sequence of
around 40 amino acids. In contrast, the internal activation domain of
EPAS1 was not so similar to that identified in HIF-1
. A sequence of
165 amino acids was necessary for maximal activity of the internal
activation domain of EPAS1, whereas for HIF-1
the highest level of
activity was observed with a short 30-40-amino acid region
corresponding to the N-terminal portion of the EPAS1 domain. Thus for
EPAS1, a proline rich domain in the C-terminal portion of exon 11 (which is not present in the corresponding exon 12 of HIF-1
)
contributes to transactivation.
showed striking
similarities. First, HIF-1
regulatory domains also manifest a dual
pattern of activity where the internal regulatory sequences conveyed
regulation on the Gal/VP16 fusion but those at the C terminus did not.
Second, the position of C-terminal and internal regulatory domains was
similar, with internal regulatory sequences overlapping and extending
N-terminal to the internal transactivation domain. In a previous
analysis of HIF-1
, we focused our analysis on the function of
residues C-terminal to amino acid 530 and defined regulatory sequences
that overlapped the internal transactivation domain (7). In those
experiments we found that sequences lying N-terminal to amino acid 530 greatly reduced activity when fused to a truncated glucocorticoid
receptor, but levels of activity were so low that regulatory effects
could not be assayed. Using the Gal/VP16 system, the regulatory effects
of these sequences were clearly demonstrated (Fig. 7). These data are
consistent with a recently published analysis of HIF-1
in which an
extensive domain responsible for oxygen-regulated proteolytic
degradation was defined between amino acids 401 and 603 (11).
Interestingly, in the context of the native molecule, portions of this
domain were shown to act independently to confer partial instability (11). The present analysis of the independent operation of subsequences is consistent with that observation, and establishes a third point of
similarity between HIF-1
and EPAS1. In each case, independently operating subsequences within the internal regulatory domain could be
defined, which were capable of conveying regulatory activity on the
heterologous Gal/VP16 fusion.
(amino acids 28-826) (Fig. 2). The extent of induction varied markedly with the dose of transfected Gal/EPAS1 plasmid, such that at high plasmid doses the fusion protein displayed essentially constitutive activity, whereas at low doses it showed clear
inducible activity. In contrast, the HIF-1
fusion showed higher
levels of inducibility than EPAS1 which were maintained at all doses
tested. Such behavior suggests that interaction of EPAS1 with the
sensing/signal transduction system involves some readily saturable
process. This could explain why inconsistent degrees of induction by
hypoxia are seen when forced overexpression of EPAS1 drives high levels
of hypoxia response element-dependent reporter gene
expression (13, 14, 17). It might also explain patterns of gene
expression seen in vivo. For instance, several groups have
reported in situ mRNA studies in which high levels of
EPAS1 expression appear to be associated with high levels of mRNA
for the EPAS1 target gene vascular endothelial growth factor, even in
situations where the cells are not obviously hypoxic (15, 26).
exon 10, which encodes a substantial portion of the oxygen
degradation domain (11), might account for their different behavior.
However, our analysis of individual exons did not define a particular
exon as responsible for this difference.
exon 8, which lies outside the degradation domain. We have not yet defined the
mechanism of action of these exons, though in isolation they do not
affect fusion protein level (data not shown). Interestingly these
regions align with, and show similarity to, a region of AHR that is
also strongly repressive, and corresponds to the ligand and HSP90
binding domain (27, 28).
exon 12),
and in exon 15 (both molecules), which are the regions that show the clearest functional resemblance in these studies. The less well conserved region corresponding to HIF-1
exons 9-11 and EPAS1 exons
8-10 showed a greater difference in function.
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Fig. 10.
Graph showing the amino acid similarity of
EPAS1 residues aligned to HIF-1 . Global alignment of the
complete amino acid sequences of EPAS1 and HIF-1
was performed using
the gap program of Genetics Computer Group, Inc. Gap creation and
extension penalties were used at their default settings. The percentage
similarity of EPAS1 sequences to HIF-1
was calculated in consecutive
decapeptide windows and plotted versus the HIF-1
amino
acid number. Percentage similarity was calculated by counting the
number of amino acid identities and conservative substitutions (as
defined by the default amino acid scoring matrix of the Genetics
Computer Group, Inc. program) in each window. Regions of EPAS1 aligning
with gaps in the HIF-1
sequence were ignored in the calculation.
bHLH, basic helix-loop-helix.
The detailed deletional analysis of the two molecules highlighted short
conserved sequences lying immediately adjacent to the activation
domains that contained minimal elements necessary for regulated
activity. In a previous analysis of HIF-1 (7), we mutated all
phosphoacceptor amino acids in an internal regulatory region close to
the internal activation domain and found no functional effects. In the
current analysis, we focused our attention on the sequences lying
adjacent to the C-terminal activation domain. The addition of a further
nine amino acids in EPAS1 or eleven amino acids in HIF-1
was found
to confer regulation on the otherwise constitutive domains (compare
EPAS1 sequences 819-870 and 828-870, and HIF-1
sequences 775-826
and 786-826). In the context of these minimal sequences, we found that
neither of two conserved serine residues was necessary for the
inducible property. Because protein oxidation, possibly involving
sulfhydryl chemistry, is an attractive candidate for HIF-1 regulation
(6, 8, 29-31), we mutated each of two cysteine residues in or adjacent
to the region and again found little effect on the inducible
characteristics of this domain. In contrast however a conserved RLL
sequence was critical for induction. At present we are unable to
determine the molecular basis for this finding, though it is of
interest that the C-terminal activation domain contains several
dileucine repeats, that leucine-rich regions are known to be important
in the interaction of co-activators such as p300 with transcription factors (32), and that p300 can interact with this domain of HIF-1
and EPAS1.2
Existing data on HIF-1 support a model in which
oxygen-dependent regulation of HIF-1
abundance, mediated
by the proteasome acting on an internal degradation domain, serves to
amplify a further oxygen-regulated activity at the C terminus (7, 8, 11). The overall similarity between EPAS1 and HIF-1
regulatory sequences, in which internal regulatory sequences conveyed inducible characteristics on Gal/VP16, but C-terminal regulatory sequences did
not, led us to consider a similar model for EPAS1 regulation. We
therefore tested for oxygen-dependent regulation of
Gal/EPAS fusion protein abundance. We found that Gal/EPAS fusions
containing N-terminal EPAS deletions to amino acids 345, 495, and 517 showed a regulated level of protein product. This was not seen with
further deletions to amino acids 682 or 819 suggesting that sequences lying N-terminal to amino acid 682 were necessary for this effect. The
extent of regulation was lower than has been described for HIF-1
fusions (11) and, as described for functional assays, appeared to
saturate as levels of expression were increased. We did not observe
such regulation with a Gal fusion to amino acids 9-517. The inability
to detect fusion proteins at low levels of expression, coupled with the
demonstration of reduced regulation at high levels of expression
indicates that regulatory effects occurring through changes in protein
level might easily be missed and illustrates a common problem in
transfection studies of inducible systems. Overall, however our results
indicate that internal sequences in EPAS1 can support regulation
through changes in protein level. Taken together with the different
activities of internal and C-terminal regulatory sequences on the
Gal/VP16 fusion, they are consistent with the dual activation model
proposed above, and suggest quantitative rather than qualitative
differences in the mechanisms of activation for EPAS1 and HIF-1
.
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ACKNOWLEDGEMENTS |
---|
We thank Liz Rose for secretarial assistance,
Sylvia Bartlett, Neil Bacon, and Patrick Maxwell for their
contributions to this work, and the following for donation of
experimental materials; Krishna Chatterjee (pUAS-tk-Luc), Stephen
Goodbourn (pCOTG), Steven McKnight (phEP-1), Gregg Semenza
(pBluescript/HIF-1 3.2-3T7), and Dave Simmons (pCMV-TAg).
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FOOTNOTES |
---|
* This work was supported by the Wellcome Trust and the Medical Research Council, UKThe costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 44-1865-222382;
Fax: 44-1865-222500; E-mail: cwpugh{at}molbiol.ox.ac.uk.
The abbreviations used are:
HIF-1, hypoxia-inducible factor-1; HIF-1, the
subunit of HIF-1; PAS, Per-AHR-ARNT-Sim; EPAS1, endothelial PAS protein, also known as member
of PAS superfamily 2 (MOP2), HIF-like factor (HLF), and HIF-related
factor (HRF); ARNT, aryl hydrocarbon receptor nuclear translocator
(identical to HIF-1
); AHR, aryl hydrocarbon receptor; VP16, transactivation domain from the herpes simplex virus protein 16 (amino
acids 410-490); Gal, the N-terminal 147 amino acids of the yeast
transcription factor, GAL4.
2 Shoumo Bhattacharya, personal communication.
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REFERENCES |
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