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Molecular and Cellular Biology, January 2000, p. 402-415, Vol. 20, No. 1
0270-7306/0/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Redox-Regulated Recruitment of the Transcriptional
Coactivators CREB-Binding Protein and SRC-1 to Hypoxia-Inducible
Factor 1
Pilar
Carrero,1
Kensaku
Okamoto,1,2
Pascal
Coumailleau,1,
Sallyann
O'Brien,1
Hirotoshi
Tanaka,3 and
Lorenz
Poellinger1,*
Department of Cell and Molecular Biology,
Karolinska Institutet, S-171 77 Stockholm,
Sweden,1 and Second Department of
Internal Medicine, Asahikawa Medical College, Asahikawa
078-8510,2 and Department of
Clinical Immunology and AIDS Research Center Institute of Medical
Science, University of Tokyo, Minato-ku, Tokyo
108-8639,3 Japan
Received 29 March 1999/Returned for modification 20 May
1999/Accepted 14 September 1999
 |
ABSTRACT |
Hypoxia-inducible factor 1
(HIF-1
) functions as a
transcription factor that is activated by decreased cellular oxygen
concentrations to induce expression of a network of genes involved in
angiogenesis, erythropoiesis, and glucose homeostasis. Here we
demonstrate that two members of the SRC-1/p160 family of
transcriptional coactivators harboring histone acetyltransferase
activity, SRC-1 and transcription intermediary factor 2 (TIF2), are
able to interact with HIF-1
and enhance its transactivation
potential in a hypoxia-dependent manner. HIF-1
contains within its C
terminus two transactivation domains. The hypoxia-inducible activity of
both these domains was enhanced by either SRC-1 or the CREB-binding
protein (CBP)/p300 coactivator. Moreover, at limiting concentrations,
SRC-1 produced this effect in synergy with CBP. Interestingly, this
effect was strongly potentiated by the redox regulatory protein Ref-1,
a dual-function protein harboring DNA repair endonuclease and cysteine reducing activities. These data indicate that all three proteins, CBP,
SRC-1, and Ref-1, are important components of the hypoxia signaling
pathway and have a common function in regulation of HIF-1
function
in hypoxic cells. Given the absence of cysteine residues in one of the
Ref-1-regulated transactivation domains of HIF-1
, it is thus
possible that Ref-1 functions in hypoxic cells by targeting critical
steps in the recruitment of the CBP-SRC-1 coactivator complex.
 |
INTRODUCTION |
Reduced tissue oxygen tension levels
(hypoxia) play a major role in the regulation of many physiological and
pathological processes (for reviews, see references
8 and 55). Adaptive physiological
responses to hypoxia include induced expression of genes encoding
erythropoietin and vascular endothelial growth factor and activation of
genes involved in glucose transport and metabolism (reviewed in
references 8 and 55). Moreover,
hypoxia is a critical determinant of the pathogenesis of many
disorders, including tumor angiogenesis, ischemic heart disease, and
stroke. Under hypoxic conditions, the diverse target genes described
above are all transcriptionally up-regulated by the transcription
factor hypoxia-inducible factor 1 (HIF-1).
HIF-1 is a heterodimeric complex of two basic helix-loop-helix
(bHLH)/Per-Arnt-Sim domain (PAS) proteins, HIF-1
and Arnt (54). The HIF-1
protein is rapidly degraded in normoxic
cells by the ubiquitin-proteasome pathway but is stabilized under
hypoxic conditions (20, 21, 25, 27, 44) and shows
hypoxia-induced import into the nucleus (26), thus allowing
heterodimerization with the nuclear factor Arnt (12). The
active HIF-1 heterodimer binds DNA at conserved hypoxia response
elements (HREs) to activate transcription of target genes
(55). The mechanism of hypoxia-dependent activation of
HIF-1
function is not yet understood. We and others have recently
demonstrated that the mechanism of signal transduction by HIF-1
is a
multistep process since nuclear translocation of the protein per se is
not sufficient for transcriptional activation by HIF-1
(26). In addition, within the nucleus HIF-1
requires hypoxia-dependent activation to enable it to recruit the CREB-binding protein (CBP)/p300 coactivator protein (26), which has
previously been shown to play a role in activation of the
erythropoietin and vascular endothelial growth factor genes in response
to hypoxia (5).
CBP and p300 are ubiquitous, evolutionarily conserved, nuclear
phosphoproteins that function, at least in part, by linking several
different signal-responsive transcriptional activators to the basal
transcription apparatus (28, 29, 32). It has been shown that
CBP/p300 can form a complex with SRC-1, a protein originally identified
as a coactivator of steroid hormone receptors (15, 28, 42,
60). SRC-1 belongs to a family of 160-kDa proteins that interact
with several members of the nuclear receptor family in a
hormone-dependent manner and thereby enhance transcriptional activation
(50). To date, several structurally and functionally related
proteins have been identified, including transcription intermidiary
factor 2 (TIF2) (52) (also known as GRIP1
[17]), p/CIP (49) (also known as ACTR
[10]), RAC3 (33), AIB1 (2), and
TRAM-1 (48). The finding that the p160 coactivators can bind
to other type of coactivators, such as CBP/p300 and p/CAF (10, 28,
60), suggests that p160 proteins play an important role in
bridging the interactions of receptor activation functions with the
basal transcription machinery to stimulate transactivation. Moreover,
both the CBP/p300 and SRC-1/p160 classes of coactivators possess
intrinsic histone acetyltransferase (HAT) activity which may contribute
to locally remodel chromatin structures for better accessibility of the
transcription machinery to DNA (6, 10, 41, 47).
The mechanism underlying hypoxia-dependent transcriptional activation
by HIF-1
remains unclear. Understanding how functional activity of
HIF-1
is triggered by low oxygen tension and how the activated
protein is able to mediate transcriptional activation is necessary to
elucidate the mechanism of signal transduction by HIF-1
. In this
study, we demonstrate that two p160 proteins, SRC-1 and TIF2, interact
with HIF-1
to enhance its hypoxia-dependent transactivation
function. We also show that SRC-1 and CBP/p300 synergistically enhance
HIF-1
-mediated transcriptional regulation under hypoxic conditions.
Moreover, the redox-dependent enzyme Ref-1 appears to be critical in
linking the effects of the two coactivator proteins CBP/p300 and SRC-1
to HIF-1
.
 |
MATERIALS AND METHODS |
Plasmids and fusion proteins.
Plasmids containing
full-length HIF-1
(pCMV4/HIF-1
), full-length Arnt (pCMV4/Arnt),
and the GAL4-driven luciferase reporter gene have been previously
described (14, 26). The Rc/RSV-mCBP-HA construct expressing
full-length mouse CBP was obtained from R. H. Goodman (Vollum
Institute, Portland, Oreg.), and plasmids pCMV
-p300-HA and
pCMV
-p300
1254-1376 were a kind gift from D. M. Livingston (Dana-Farber Cancer Institute and Harvard Medical School, Boston, Mass.). Plasmids containing human full-length SRC-1 and SRC-1
PAS cDNAs in pSG5 vector (42) were kindly provided by B. W. O'Malley (Baylor College of Medicine, Houston, Tex.). pSG5/SRC-1 M1234 (16) was obtained from M. G. Parker (Imperial Cancer
Research Fund, London, United Kingdom). pCMV5/Ref-1 was produced by
inserting the 1.45-kb EcoRI fragment of pBS/Ref-1
(57) (kindly provided by T. Curran, St. Jude's Children
Hospital, Memphis, Tenn.) into the EcoRI site of pCMV5.
pSG5/TIF2 was a kind gift from P. Chambon (Institut de
Génétique et de Biologie Moléculaire et Cellulaire, Strasbourg, France). pT81/HRE-luc contains three tandem copies of the
erythropoietin HRE in front of the herpes simplex thymidine kinase
promoter and the luciferase gene (P. Coumailleau, P. Carrero, and L. Poellinger, unpublished data).
GAL4 DNA binding domain (DBD) fusion proteins encoding HIF-1
subfragments (corresponding to amino acids 1 to 826, 1 to 813, 526 to
642, 526 to 826, and 776 to 826) were subcloned into the pCMX
expression vector (51). The green fluorescent protein (GFP) fusion protein containing full-length HIF-1
has been described elsewhere (26). pGFP/Ref-1 was produced by inserting the
BamHI-SpeI fragment of pCRII/Ref-1 into the
BamHI-NheI sites of pGFP. A GAL4 DBD fusion
protein carrying the
bHLH/HIF-1
sequence (residues 71 to 826) was
generated by amplifying the equivalent DNA sequence by PCR using
Pfu DNA polymerase (Stratagene) together with primer pairs
carrying KpnI or BamHI ends. The resulting
product was inserted in frame to KpnI-BamHI sites
of pCMX. GAL4/HIF 776-813 was generated by cleaving the C-terminal 13 amino acids from GAL4/HIF 776-826 by PstI digestion.
GAL4/HIF 531-584 was produced by inserting the
XhoI-BamHI fragment of CMV4/HIF-1
531-584
(obtained from T. Pereira) into the XhoI-BamHI
sites of pCMX. Nucleotide sequences were confirmed by sequencing. All
GAL4 fusion constructs show similar levels of expression under hypoxic
conditions (27). A glutathione S-transferase
(GST)-HIF-1
fusion protein was generated by excision of full-length
HIF-1
from a corresponding bacterial GST fusion expression vector (a
kind gift from I. Pongratz) and insertion in frame into the
BglII-XhoI sites of the pBC expression vector
(9).
Cell culture.
COS7 cells (from the American Type Culture
Collection) were routinely maintained in high-glucose Dulbecco's
modified Eagle's medium (DMEM) containing 10% fetal calf serum (FCS),
100 IU of penicillin per ml, 100 µg of streptomycin sulfate per ml, 2 mM L-glutamine, and 1× minimal essential medium containing
nonessential amino acids. The human embryonic kidney cell line (293 cells) was cultured in 1:1 mixture of DMEM and F-12 medium supplemented with 10 and 5% FCS, respectively, penicillin-streptomycin sulfate, and
nonessential amino acids as described above. All media and growth
factors were purchased from Life Technologies, Inc.
Transfection and transient expression assays.
One day before
transfection, cells were seeded in six-well plates, and the medium was
changed to OPTI-MEM medium lacking phenol red (Life Technologies)
before transfection. A DNA mixture containing 0.5 µg of luciferase
reporter, 0.2 µg of a combination of expression vectors for HIF-1
and Arnt, 0.75 to 1.5 µg of expression vectors for different
coactivators, and 0.2 µg of internal control plasmid pRSV-AF together
with carrier DNA pCMV4 was prepared and mixed with Lipofectamine (Life
Technologies) according to the manufacturer's recommendations. DNA
precipitates were allowed to form at room temperature for 30 min and
added to the culture. After 6 h of transfection, the medium was
replaced with DMEM-10% FCS. Hypoxic induction was achieved by
incubation of the cells with 1% O2, or 21% O2
for normoxia. After 36 h, aliquots of medium were collected and
analyzed for alkaline phosphatase activity, and cells were harvested
and extracts were analyzed for luciferase activity. Alkaline
phosphatase activity was used to correct for differences in
transfection efficiency. All transfections were done in duplicate; results are presented as mean ± standard error (SE).
For analysis of nuclear translocation of HIF-1

in living cells, we
transiently expressed GFP-tagged full-length HIF-1

and
pSG5/TIF2,
pSG5/SRC-1, or pSG5/SRC-1 M1234 in COS7 cells as described
previously
(
26). Briefly, a plasmid mixture containing 6 µg
of the
expression vector for GFP-HIF-1

, 5 µg of pSG5/TIF2, pSG5/SRC-1,
or pSG5/SRC-1 M1234 plasmid when indicated, and carrier DNA pGEM-3Z
to
keep the total amount of DNA constant at 11 µg was mixed with
20 µl
of TransIT-LT1 reagent (Panvera Corp., Madison, Wis.) and
added to the
culture. After 6 h of incubation, the medium was
replaced as
described above, and cells were induced 24 h later
with the
hypoxia-mimicking agent 2,2'-dipyridyl (100 µM) or treated
with
vehicle only. Two hours after treatment, the medium was withdrawn
and
cells were refed fresh DMEM containing 10% FCS. Visualization
of
intracellular trafficking of GFP-tagged proteins was performed
as
described elsewhere (
26).
For protein-protein interaction assays, COS7 cells were transfected by
using Lipofectamine (Life Technologies) with 10 µg
of the
GST-HIF-1

expression vector and pCMV4 as carrier DNA.
After 6 h of transfection, the medium was replaced with DMEM-10%
FCS. Hypoxic
induction was achieved by incubation of the cells
with the hypoxia
mimic CoCl
2 (100 µM) or with vehicle only. After
24 h, cells were harvested in ice-cold phosphate-buffered saline
(PBS).
Whole-cell extracts were prepared in buffer containing
0.4 M KCl, 20 mM
HEPES (pH 7.4), 1 mM dithiothreitol, and 20%
glycerol. The protein
content of cell extracts was determined
by a colorimetric method
(Bio-Rad).
Assays of GST protein-protein interaction.
The analysis of
interactions between SRC-1 and HIF-1
bound to glutathione-Sepharose
was carried out by the method of Chatton et al. (9), with
minor modifications. Approximately 250 µg of whole-cell extracts from
COS7 cells transiently expressing GST-HIF-1
was incubated with
[35S]methionine-labeled in vitro-translated SRC-1 protein
in 250 µl of a low-stringency buffer (50 mM Tris-HCl [pH 7.8],
0.1% NP-40, 250 mM NaCl). The complexes were immobilized on 60 µl of
a 50% suspension of glutathione-Sepharose beads (Pharmacia Biotech). After washing, the radiolabeled proteins were eluted, separated by
sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and detected by fluorography.
Bacterially expressed GST-Ref-1 fusion protein (1 µg) or GST (0.5 µg) was incubated with 45 µg of protein A-Sepharose CL-4B
(Pharmacia Biotech) in 250 µl of PBS with 0.4 µg of GST antibody
for 1 h at 4°C. After washing, the beads were incubated with
[
35S]methionine-labeled in vitro-translated proteins in
PBS containing
3% bovine serum albumin and 1 mM dithiothreitol, with
or without
5 mM diamide, at room temperature for 30 min. The beads were
then
washed five times, and bound proteins were eluted, separated by
SDS-PAGE, and visualized by
autoradiography.
 |
RESULTS |
TIF2 enhances HIF-1
activity and interacts with HIF-1
in a
hypoxia-dependent manner in mammalian cells.
To gain a better
understanding of the mechanism of transcriptional activation by
HIF-1
, we tested the effect of transient expression of the
transcriptional coactivator TIF2 on the functional activity of
HIF-1
. TIF2 belongs to the p160 family of coactivators which have
been shown to be required for nuclear receptor-mediated transcriptional
activation (17, 48, 52). Transient transfection experiments
were initially performed with COS7 cells. As shown in Fig.
1A, the reporter gene activity was not
significantly altered by hypoxic treatment (1% O2) in the
absence of coexpressed proteins, most probably due to the low levels of
endogenous HIF-1 activity in COS7 cells (unpublished observations).
Upon transient coexpression of HIF-1
and Arnt, pT81/HRE-luc reporter
gene activity was stimulated around 10-fold under normoxic conditions,
possibly due to a small but detectable pool of HIF-1
localized in
the nucleus under these conditions (26). This reporter gene
activity showed a modest (~1.5-fold) increase by hypoxia (Fig. 1A).
However, as expected (5, 26), ectopic expression of CBP
further enhanced hypoxia-dependent activation by HIF-1
in a
dose-dependent manner compared with hypoxia-stimulated activity in the
absence of exogenous coactivators (two- to threefold). Although
expression of CBP slightly stimulated HIF-1
activity already at
normoxia, its overall activity was almost entirely dependent on the
exposure to hypoxia as has been described previously (5,
26). The effect was most likely limited to threefold due to the
large quantities of endogenous CBP already present in the cell prior to
transfection. Coexpression of HIF-1
together with TIF2 resulted in
potent (around 12-fold) activation of the reporter gene over the levels
observed at normoxia, thereby establishing that both TIF2 and CBP
support transcription by the HIF-1
-Arnt complex (Fig. 1A). In
conclusion, TIF2 which was originally identified as a coactivator
supporting hormone-dependent transcriptional activation by members of
the steroid hormone receptor family, also functions as a conditionally
regulated coactivator in hypoxia-dependent transcriptional activation
by the HIF-1
-Arnt complex.

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FIG. 1.
TIF2 enhances the HIF-1 -mediated transactivation
function and interacts with HIF-1 in vivo. (A) COS7 cells were
transiently cotransfected as described in Materials and Methods with
0.5 µg of pT81/HRE-luc reporter construct, hHIF1- expression
vector (pCMV4/HIF-1 ; 0.2 µg), Arnt expression vector (pCMV4/Arnt;
0.2 µg), and 0.75 to 1.5 µg of either TIF2 (pSG5/TIF2) or CBP
(Rc/RSV-CBP-HA), as indicated. The total amount of DNA was kept
constant by the addition of parental pCMV4 when appropriate. Following
transfection cells were incubated either under normoxic (21%
O2) or hypoxic (1% O2) conditions. Luciferase
activities were normalized for transfection efficiency by
cotransfection of alkaline phosphatase-expressing pRSV-AF. Data are
presented as luciferase activity relative to cells transfected with
pCMV4 and reporter gene only and incubated at normoxia. Values
represent the mean ± SE of two independent experiments. (B)
Intranuclear redistribution of the GFP-HIF-1 fusion protein in the
presence of TIF2. GFP-HIF-1 was transfected into COS7 cells in the
absence or presence of exogenous TIF2 (pSG5/TIF2). After 24 h of
expression, either 100 µM 2,2'-dipyridyl or vehicle (H2O)
was added to the culture medium and incubated for 2 h before
observation. Subsequently, 2,2'-dipyridyl was withdrawn by washing the
cells and changing the medium, and the cells were thereafter incubated
for an additional 24 h. Cells were observed microscopically at
different time points as indicated by arrows. Photographs were taken
with a Zeiss fluorescence microscope.
|
|
In response to hypoxia, HIF-1

is imported to the nucleus and shows a
strictly nuclear localization (
26). To characterize
the
mechanism of TIF2-dependent enhancement of transcriptional
activation
by HIF-1

in living cells, we next examined whether
overexpression of
TIF2 would affect the intracellular localization
of HIF-1

. To this
end, we transiently transfected COS7 cells
with an expression vector
encoding an in-frame fusion of GFP with
full-length HIF-1

. As shown
previously (
26), fluorescence of
this GFP-HIF-1

construct was uniformly distributed throughout
the cell under normoxic
conditions, and treatment of the transfected
cells with the
hypoxia-mimicking agent 2,2'-dipyridyl for 2 h
led to a complete
nuclear accumulation of the GFP-HIF-1

protein
(
26) (Fig.
1B). Transient expression of the TIF2 coactivator
protein in the
presence of GFP-HIF-1

had no effect on either
the intensity or the
subcellular distribution of fluorescence
activity at normoxia. However,
upon exposure of cells coexpressing
GFP-HIF-1

and TIF2 to
2,2'-dipyridyl, GFP-HIF-1

-dependent fluorescence
activity was
detected in dot-like structures throughout the nucleus
(Fig.
1B). It
has previously been described that TIF2 is a nuclear
protein mainly
associated with such dot-like discrete bodies within
the nucleus
(
52). These observations strongly suggest that TIF2
induced
relocalization of GFP-HIF-1

within the nucleus, indicating
that
GFP-HIF-1

and TIF2 interacted with the one another in vivo
and that
the dot-like fluorescence pattern might represent transcriptionally
active
chromatin.
We have previously observed that return of cells to normoxia following
exposure to hypoxia or withdrawal of hypoxia-mimicking
chemicals
results in an export of the nuclear pool of HIF-1

(
26).
To address whether the interaction between GFP-HIF-1

and TIF2
in
vivo would be dependent on the hypoxic stimulus, we therefore
initially
exposed the transfected cells to 2,2'-dipyridyl and
then further
incubated them at normal levels of atmospheric oxygen
tension after
withdrawal of 2,2'-dipyridyl by washing of the cells
in medium (see
schematic representation in Fig.
1B). As expected
(
26),
after 24 h of incubation under normoxic conditions, we
observed a
distribution of GFP-HIF-1

in both the nuclear and
cytoplasmic
compartments that was indistinguishable from the distribution
of
fluorescence activity prior to exposure to the hypoxic signal
(Fig.
1B). Transient expression of TIF2 did not induce any quantitative
or
qualitative differences in the export of GFP-HIF-1

(Fig.
1B),
demonstrating that the hypoxia-induced nuclear relocalization
of
HIF-1

by TIF2 could be reversed following withdrawal of the
hypoxic
signal.
Regulation of the hypoxia-inducible transactivation function of
HIF-1
by SRC-1.
As outlined above, TIF2 belongs to a growing
family of the p160 family of coactivators including SRC-1. TIF2 and
SRC-1 appear to have similar activities as coactivators to enhance the
transcriptional potential of many members of the ligand-activated
nuclear hormone receptors (18, 28, 42, 52). SRC-1 has been
demonstrated to directly and constitutively interact with CBP (28,
60). Moreover, in analogy to CBP (6, 41), SRC-1 has
been shown to possess intrinsic HAT activity (10, 47).
Against this background we also tested the ability of SRC-1 to modulate
HIF-1
-dependent transcriptional activation of the minimal HRE-driven
reporter gene. In transient transfection experiments using COS7 cells, HRE-dependent reporter gene activity was stimulated from two- to
eightfold in a dose-dependent manner by SRC-1, compared with hypoxia-stimulated activity in the absence of exogenous coactivators (Fig. 2A). Transient expression of SRC-1
also enhanced the transcriptional activity of human HIF-1
(hHIF-1
) under normoxic conditions, albeit to a lesser extent
(around threefold [Fig. 2A]). Thus, SRC-1 and TIF2 have similar
properties in enhancing the transcriptional potential of hHIF-1
. To
determine whether regulation of HIF-1
function by members of the
p160 family of coactivators was cell type specific, we next carried out
similar transfection experiments with the human 293 embryonic kidney
cell line. In these experiments we observed that both SRC-1 (Fig. 2B)
and TIF2 (data not shown) can significantly enhance hHIF-1
-dependent
transcriptional activation in 293 cells.

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FIG. 2.
SRC-1 stimulates HIF-1 activity in a
hypoxia-dependent manner and interacts in vitro with HIF-1 . COS7 (A)
and human embryonic kidney 293 (B) cells were cotransfected with
pT81/HRE-luc (0.5 µg), 0.2 µg of pCMV4/HIF-1 (hHIF1- ), 0.2 µg of pCMV4/Arnt (Arnt), and 0.75 to 1.5 µg of SRC-1 (pSG5/SRC-1),
as indicated. Six hours after transfection, cells were exposed to
either 21 or 1% O2 for 36 h before harvest.
Luciferase values are presented as relative luciferase activity as
described in the legend to Fig. 1. The results of three independent
experiments performed in duplicate ± SE are shown. (C) The SRC-1
PAS domain is not required for functional interaction with HIF-1 .
(Top) Schematic representation of full-length SRC-1 and SRC-1 PAS.
(Bottom) pGAL4/HIF 71-826 was cotransfected into COS7 cells together
with a reporter plasmid expressing the luciferase gene driven by the
thymidine kinase minimal promoter under the control of five copies of
GAL4 binding sites and 1.5 µg of an expression vector encoding either
full-length SRC-1 or a deletion mutant of SRC-1, SRC-1 PAS, lacking
the PAS domain. Cells were exposed to 21 or 1% O2 for
36 h before harvest and reporter gene assays. Luciferase values
are presented as relative luciferase activity as described in the
legend to Fig. 1. The results of two independent experiments performed
in duplicate ± SE are shown. (D) In vitro interaction between
SRC-1 and HIF-1 . COS7 cells were transfected with 10 µg of the
expression plasmid pGST-HIF-1 (GST-HIF) or empty GST expression
vector (GST). Cells were exposed to either 100 µM CoCl2
or vehicle (H2O) for 24 h. Cell extracts were prepared
and incubated with [35S]methionine-labeled in
vitro-translated SRC-1 protein. The complexes were immobilized on
glutathione-agarose beads for 2 h and eluted with the sample
buffer by boiling. The eluted material was analyzed by SDS-PAGE (5%
gel) and visualized by fluorography. Lane 1 represents one-fifth of the
amount of [35S]methionine-labeled SRC-1 used in the
binding reactions. Positions of molecular mass markers are shown on the
left in kilodaltons.
|
|
Interestingly, in analogy to HIF-1

, the p160 family of coactivators
belongs to the larger family of bHLH/PAS factors (schematically
represented in Fig.
2C). SRC-1 was originally cloned as an N-terminally
truncated fragment (
42), here termed SRC-1

PAS (Fig.
2C),
lacking
the bHLH and PAS motifs. Both the bHLH and PAS domains
represent
potent protein-protein interaction interfaces (
34,
35) and
are critical for dimerization between HIF-1

and Arnt
(
14,
54).
To investigate the role of the bHLH and PAS motifs
of SRC-1 in
hypoxia-dependent transactivation by HIF-1

, we examined
the effect
of overexpression of SRC-1

PAS or full-length SRC-1 on the
transcriptional
activity of a fusion protein, GAL4/HIF 71-826, containing the
GAL4 DBD fused in frame to a fragment of hHIF-1

spanning amino
acids 71 to 826. Hypoxia-dependent activation of this
construct
was monitored by using a GAL4-responsive reporter gene. As
shown
in Fig.
2C, the chimeric protein mediated ~4-fold
hypoxia-dependent
activation of reporter gene activity. In agreement
with the effect
of SRC-1 on transcriptional activation mediated by the
HIF-1

-Arnt
complex, coexpression of full-length SRC-1 coactivator
further
enhanced the activity of GAL4/HIF 71-826 about 12-fold under
hypoxic
conditions, resulting in ~45-fold stimulation of the reporter
gene over the levels observed at normoxia (Fig.
2C). SRC-1 showed
no
detectable effect on the transcriptional activity by the GAL4
DBD alone
(data not shown). These results demonstrate that SRC-1
targets the
HIF-1

protein. Next, we examined the effect of the
SRC-1

PAS
mutant on GAL4/HIF 71-826-mediated transactivation.
This truncated
coactivator fragment showed activity very similar
to if not more potent
than that of full-length SRC-1 in enhancing
the hypoxia-dependent
activation response (Fig.
2C). Thus, in
excellent agreement with the
high activity of SRC-1

PAS in enhancing
steroid hormone
receptor-dependent transcriptional regulation
(
42), the bHLH
and PAS motifs of SRC-1 were not important to
support HIF-1
function.
The present experiments indicated the functional importance of both CBP
and SRC-1 for enhancing the transcriptional potential
by HIF-1

in
hypoxic cells. CBP has previously been demonstrated
to physically
interact with HIF-1

(
5). To investigate whether
SRC-1 was
able to interact with HIF-1

, we performed GST precipitation
assays
using cell extracts prepared from COS7 cells transiently
expressing
GST-tagged HIF-1

in the presence and absence of 100
µM
CoCl
2, a chemical known to mimic hypoxic induction of
target
gene expression and to activate HIF-1

(
8,
55).
This material
was subsequently incubated with in vitro-translated,
35S-labeled SRC-1. As shown in Fig.
2D, in the presence of
CoCl
2,
35S-labeled SRC-1 was specifically
retained by the GST-HIF-1

fusion
protein immobilized on
glutathione-Sepharose beads (lane 4), whereas
we observed no
significant binding to GST-HIF-1

extracted from
nontreated cells
(lane 3) or to GST alone (lane 2). Thus, SRC-1
interacted with HIF-1

in a hypoxia-dependent manner in
vitro.
Role of LXXLL motifs in HIF-1
-SRC-1 interaction.
SRC-1
contains four copies of the short sequence motif LXXLL. Three of these
motifs were identified in the central domain of the protein, and the
fourth motif was found in the eight most C-terminal amino acids of
human SRC-1 (16). The LXXLL motifs have been shown to be
necessary to mediate the binding of SRC-1 to ligand-occupied nuclear
receptors (16, 38, 56). To investigate the role of the LXXLL
motifs of SRC-1 in hypoxia-dependent transactivation by HIF-1
, we
compared the activities of wild-type SRC-1 and SRC-1 M1234, a mutant
protein in which the conserved leucine couplets were changed to alanine
at residues 636/7, 693/4, 752/3, and 1438/9 (16) in
transient transfection experiments. As shown in Fig. 3A,
the mutant protein in which all four functional binding motifs are
disabled enhanced the activity of GAL4/HIF 71-826 to the same extent as
the wild-type SRC-1 under hypoxic conditions. Moreover, transient
expression of either wild-type SRC-1 or SRC-1 M1234 together with
GFP-HIF-1
protein in COS7 cells had no effect on the subcellular
distribution of fluorescence activity at normoxia. Under hypoxic
conditions, SRC-1 induced relocalization of GFP-HIF-1
from a
uniform nuclear distribution to discrete dot-like structures within the
nucleus (Fig. 3B). This effect was also produced by the mutant SRC-1
M1234 protein (Fig. 3B), indicating that both proteins interact with
HIF-1
in a hypoxia-dependent manner in vivo. Taken together, these
results suggest that in contrast to nuclear receptor signaling, the
LXXLL motif is not required for HIF-1
-SRC-1 interaction.

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FIG. 3.
LXXLL motifs of SRC-1 are not required for
HIF-1 -mediated transactivation function and interaction with
HIF-1 in vivo. (A) (Top) Schematic representation of full-length
SRC-1 and mutant SRC-1 M1234. Black bars represent the approximate
locations of the LXXLL motifs in the linear SRC-1 sequence; circles
with numbers indicate sites of mutation of LXXLL motifs in which
conserved leucine residues are replaced by alanines. (Bottom) pGAL4/HIF
71-826 was cotransfected into COS7 cells together with a
GAL4-responsive reporter plasmid and 1.5 µg of either full-length
SRC-1 or mutated SRC-1, SRC-1 M1234 expression vectors. Cells were
exposed to 21 or 1% O2 for 36 h before harvest and
reporter gene assays. Luciferase values are presented as relative
luciferase activity as described in the legend to Fig. 1. The results
of two independent experiments performed in duplicate ± SE are
shown. (B) Intranuclear redistribution of the GFP-HIF-1 fusion
protein in the presence of SRC-1 and SRC-1 M1234. pGFP-HIF-1 was
transfected into COS7 cells with or without SRC-1 or SRC-1 M1234 as
indicated. After 24 h of expression, either 100 µM
2,2'-dipyridyl or vehicle (H2O) was added to the culture
medium and incubated for 2 h. Cells were observed using a
fluorescence microscope. Photographs of several cells for each
condition were taken and representative cells are shown. Bar = 10 µm.
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Definition of the functional domains of HIF-1
which interact
with the coactivators CBP and SRC-1.
To identify HIF-1
structures which could serve as interaction surfaces with the SRC-1 and
CBP coactivator proteins, we constructed a series of GAL4 DBD fusion
proteins containing HIF-1
fragments of various lengths (as
schematically represented in Fig. 4A). These constructs were transiently transfected into COS7 cells together
with a GAL4-responsive reporter gene. Fusion of full-length HIF-1
to
the GAL4 DBD produced significant hypoxia-dependent induction of
reporter gene activity in the absence of ectopically expressed
coactivator proteins (26) (Fig. 4A). Coexpression of either
CBP or SRC-1 strongly (around 8- to 10-fold) enhanced the
transcriptional potential of HIF-1
in hypoxic cells. These coactivators also increased to a lesser extent the transcriptional activity of GAL4/HIF 1-826 under normoxic conditions. Neither CBP nor
SRC-1 enhanced the transcriptional activity of the GAL4 DBD alone (data
not shown). GAL4/HIF 71-826, which lacks the bHLH domain of HIF-1
,
induced relative luciferase activity about 3.5-fold over background
levels (Fig. 4A). In the presence of coexpressed CBP or SRC-1,
luciferase activity was induced under hypoxic conditions by GAL4/HIF
71-826 to a 30- or 34-fold-higher level, respectively, in comparison
with the activity observed at hypoxia in the absence of exogenous
coactivators.

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FIG. 4.
Definition of HIF-1 structures which are regulated by
the CBP and SRC-1 coactivators. (A) Different subregions of HIF-1
were fused to the GAL4 DBD and transfected into COS7 cells together
with a GAL4-responsive reporter plasmid in the absence or presence of
1.5 µg of either CBP or SRC-1 expression plasmid. Cells were exposed
to 21 or 1% O2 for 36 h before harvest. N-TAD and
C-TAD, N- and C-terminal transactivation domains. (B) The C-terminal
transactivation domain of Arnt mediates transcriptional activation by
CBP and SRC-1. COS7 cells were cotransfected with the indicated
GAL4-Arnt fusion proteins and 1.5 µg of CBP or SRC-1 expression
vectors together with a GAL4-responsive reporter plasmid. Six hours
after transfection, cells were exposed to either 21 or 1%
O2 for 36 h before harvest and reporter gene assays.
(C) HIF-1 prevents Arnt from functionally interacting with CBP and
SRC-1. GAL4/Arnt 128-774 and a deletion mutant (GAL4/Arnt 128-603) were
transfected into COS7 cells together with 0.2 µg of hHIF-1
expression vector and 1.5 µg of CBP and SRC-1 expression vectors, as
indicated. Luciferase activity was measured following 36 h of
exposure to either 21 or 1% O2. Normalized reporter gene
activities are expressed relative to that of nonfusion GAL4 in
normoxia. The results of three independent experiments performed in
duplicate ± SE are shown.
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We next examined the effect of CBP and SRC-1 on the activity of GAL4
fusion proteins containing sequences lying C terminal
to the DNA
binding and dimerization domains of HIF-1

. A hypoxia-inducible
activation response was produced by GAL4/HIF 526-826 alone, in
excellent agreement with the presence within this HIF-1

fragment
of
two distinct transactivation domains, labeled N-TAD and C-TAD
in Fig.
4A, which function in a hypoxia-dependent fashion when
fused to the
GAL4 DBD (
23,
43). Upon transient expression
of CBP or
SRC-1, the transcriptional activity of GAL4/HIF 526-826
was greatly
(about seven- to ninefold) stimulated, demonstrating
that these
coactivators target the C terminus of HIF-1

. Moreover,
these results
further establish that the enhancing effect of both
CBP and SRC-1 on
HIF-1

function is independent of the presence
of the bHLH and PAS
domains of HIF-1

.
To further investigate whether one individual or both transcriptional
activation domains of HIF-1

were targeted by the coactivators,
we
next deleted residues 813 to 826, a region that is contained
within the
C-terminal transactivation domain of HIF-1

(
23,
43) and
has been identified as a point of interaction with CBP
(
5,
7). Interestingly, GAL4/HIF 1-813 maintained a hypoxia-inducible
response that was significantly enhanced (14- to 13-fold) by
overexpression
of CBP and SRC-1 (Fig.
4A), indicating that the
hypoxia-dependent
function of the N-terminal transactivation domain of
HIF-1

may
also be regulated by the coactivators. Against this
background,
we were interested in examining the regulatory properties
of the
individual transactivation domains of HIF-1

. As shown in Fig.
4A, GAL4/HIF 531-584, a chimeric protein spanning the N-terminal
transactivation domain of HIF-1

(
21,
23), showed
hypoxia-dependent
induction of reporter gene activity, albeit with a
lower potency
than the constructs spanning both transactivation
domains. In
the presence of CBP, however, an 11-fold stimulation of
reporter
gene activity was observed in hypoxic cells, whereas
overexpression
of SRC-1 only slightly (1.5-fold) enhanced the activity
of GAL4/HIF
531-584 under identical conditions. We next examined the
transactivation
capacity of GAL4/HIF 526-641, which harbors both the
N-terminal
transactivation domain and a structure which has been
proposed
to function as an inhibitory sequence (
23). In
comparison with
GAL4/HIF 531-584, the transcriptional activity of this
chimeric
protein was greatly reduced and showed complete abrogation of
the effect of CBP and SRC-1 on its transactivation function (Fig.
4A),
indicating either that an inhibitory region that may serve
to modulate
HIF-1

function under certain as yet unidentified
conditions is
contained between residues 584 and 641 (
23) or,
alternatively, that the fusion of the N-terminal transactivation
domain
to this sequence produces a malfolded domain when expressed
outside the
context of the full-length protein. In comparison,
a fusion protein
containing the carboxy-terminal transactivation
domain of HIF-1

,
GAL4/HIF 776-826, produced a very modest (about
twofold)
hypoxia-dependent induction response. However, its transcriptional
activity was dramatically enhanced in the presence of either CBP
(29-fold increase) or SRC-1 (17-fold increase). Upon deletion
of the 13 most carboxy-terminal amino acids, both basal activity
and inducible
properties of GAL4/HIF 776-813 were completely abrogated
independently
of the absence or presence of coactivators (Fig.
4A). Taken together,
these results suggest that (i) CBP mediates
the hypoxia-inducible
transcriptional activation by HIF-1

by
targeting both of the
transactivation domains of HIF-1

and (ii)
the 13 most C-terminal
amino acids of HIF-1

are crucial for stimulation
of the activity of
the C-terminal transactivation domain by CBP
or SRC-1. However, in the
context of the full-length protein,
this C-terminal portion plays only
a minor part in determining
hypoxia-dependent functional activity in
the presence of either
CBP or SRC-1.
Since Arnt, the functional partner factor of HIF-1

, has recently
been shown to interact with CBP/p300 (
30), we also examined
whether SRC-1 might functionally interact with Arnt. In normoxic
cells,
Arnt functions as a constitutively active transcription
factor on
E-box-driven promoters (
1,
46). We transiently
expressed in
COS7 cells Arnt fused to the GAL4 DBD together with
either CBP or
SRC-1. In these experiments, we observed that constitutive
activation
of the reporter gene by GAL4/Arnt 128-774 was potently
further enhanced
by coexpression of CBP or SRC-1 (Fig.
4B). Deletion
of the C-terminal
Q-rich transactivation domain of Arnt (schematically
represented in
Fig.
4B) completely abolished the effect of these
coactivators on
transcription by GAL4/Arnt 128-603 (Fig.
4B).
These observations
suggest that the subunits of the HIF-1 heterodimer,
HIF-1

and Arnt,
interact independently with CBP and SRC-1. Interestingly,
whereas Arnt
interacted constitutively with CBP and SRC-1, the
functional
communication of the HIF-1

-Arnt heterodimeric complex
with either
CBP or SRC-1 was hypoxia dependent. Thus, dimerization
with
HIF-1

appears to impose hypoxia-dependent regulation on
the ability
of Arnt to functionally interact with these coactivator
proteins. To
further examine this question, we analyzed the effect
of overexpression
of HIF-1

on the activity of GAL4-Arnt fusion
proteins at normoxia.
Cotransfection of COS7 cells with HIF-1
and GAL4/Arnt 128-774 resulted in ~4-fold activation of the reporter
gene compared to cells
transfected with the GAL4 fusion protein
alone (Fig.
4C), probably due
to the presence of small amounts
of HIF-1

in the nucleus already at
normoxia (
26). However,
constitutive activation of the
reporter gene was completely abolished
by coexpression of HIF-1

together with CBP or SRC-1 (Fig.
4C).
This effect was indistinguishable
from the one observed with GAL4/Arnt
128-603 in the presence of either
of the coactivators (Fig.
4B).
This deletion mutant, however, when
cotransfected with HIF-1
resulted in further ~5-fold stimulation
of reporter gene activity
(Fig.
4C), in agreement with its ability to
functionally interact
with HIF-1

via the PAS domain (
14,
54). These results are
consistent with the model that
dimerization with HIF-1

impairs
the ability of Arnt to functionally
interact with CBP or SRC-1.
However, the mechanism of this putative
negative regulatory effect
of HIF-1

needs to be further
investigated.
SRC-1 and CBP act synergistically to enhance HIF-1
-dependent
transcription.
The ability of either CBP or SRC-1 to enhance the
transcriptional activity of HIF-1
prompted us to investigate the
effect of coexpression of both coactivators on HIF-1
function. We
initially investigated the effects of transient expression of SRC-1 and CBP individually and in combination with one another on
hypoxia-dependent activation by HIF-1
of a minimal HRE-driven
reporter gene. As observed in Fig. 2A and B, the effect of SRC-1 on
HIF-1
-dependent activation of the reporter gene was strictly dose
dependent in COS7 and 293 cells, respectively. For instance, at a low
concentration of SRC-1 expression vector (0.75 µg), no significant
effect on HIF-1
-dependent activation of the reporter gene was
detected (Fig. 2A, 2B, and 5A). In a
similar fashion, an identically low concentration of CBP expression
vector produced no enhancement of the transactivation function of
HIF-1
(Fig. 5A). Under all of these conditions, poor
hypoxia-dependent inducibility of the reporter gene was observed.
However, a potent, hypoxia-dependent activation response could be
reconstituted upon transient coexpression of SRC-1 and CBP at doses
which yielded no significant effect individually (Fig. 5A), indicating
strong synergy between these two coactivators in enhancing HIF-1
function.

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FIG. 5.
CBP and SRC-1 cooperatively enhance HIF-1 -mediated
transcriptional activation. (A) COS7 cells were transiently
cotransfected with pCMV4/HIF-1 and 0.75 to 1.5 µg of SRC-1 and/or
0.75 to 1.5 µg of CBP expression plasmids together with a
hypoxia-responsive reporter gene (pT81/HRE-luc) and subsequently
exposed to 21 or 1% O2. Luciferase values are presented as
relative luciferase activity as described in the legend to Fig. 1. The
results of three independent experiments performed in duplicate ± SE are shown. (B) Cooperative activation by CBP and SRC-1 of
GAL4-HIF-1 fusion proteins. COS7 cells were cotransfected with
GAL4-HIF-1 fusion constructs together with a GAL4-responsive
reporter plasmid in the absence or presence of CBP and/or SRC-1, as
indicated (in micrograms). Cells were exposed to 21 or 1%
O2 before harvest. After normalization for transfection
efficiency using alkaline phosphatase activity, reporter gene
activities are expressed as relative to that of GAL4 in normoxia. The
results of two independent experiments performed in duplicate ± SE are shown. (C) p300 protein stimulates the activity of the two
minimal transactivation domains of HIF-1 in a hypoxia-dependent
manner. (Right) Schematic representation of full-length p300
(pCMV -p300-HA) and p300 (pCMV -p300 1254-1376). (Left) The
two minimal transactivation domains of HIF-1 were fused to GAL4 DBD
and transfected into COS7 cells together with a GAL4-responsive
reporter plasmid in the absence or presence of p300, p300 , and/or
SRC-1 expression vectors, as indicated. Cells were exposed to 21 or 1%
O2 before harvest. Normalized reporter gene activities are
expressed as relative to that of GAL4 in normoxia. The results of a
representative experiment performed in duplicate are shown.
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To further substantiate this synergistic effect, we next examined the
transactivation potential of a number of GAL4 DBD fusion
proteins
containing distinct subregions of HIF-1

. Low concentrations
of SRC-1
and CBP produced modest stimulation of the transcriptional
activity by
GAL4/HIF 71-826, spanning HIF-1

lacking the N-terminal
bHLH domain
(Fig.
5B). In contrast, coexpression of SRC-1 and
CBP generated strong
enhancement of the GAL4/HIF 71-826-mediated
activation response in
hypoxic cells. This effect was more than
additive relative to the
effects detected in the presence of CBP
or SRC-1 alone, and it was also
observed with the GAL4/HIF 526-826
fusion protein, which contains both
N-terminal and C-terminal
transactivation domains of HIF-1

(Fig.
5B). Moreover, coexpression
of low concentrations of SRC-1 and CBP
potently stimulated hypoxia-inducible
transcriptional activation by the
individual transactivation domains
of HIF-1

contained within
GAL4/HIF 531-584 and GAL4/HIF 776-826,
respectively. These results are
in excellent agreement with the
data demonstrating that the two
transactivation domains of HIF-1
independently can functionally
interact with either SRC-1 or CBP
(Fig.
4A). These data also are
consistent with the notion that
CBP and SRC-1 constitutively interact
with one another (
28,
60) and, in analogy to the mechanism
of transcriptional activation
by members of the steroid hormone
receptor family (
45), that
recruitment of both coactivators
may be necessary to trigger the
full activity of HIF-1

in hypoxic
cells. It remains to be established
whether the recruitment of both of
these classes of coactivators
to HIF-1

occurs simultaneously or in a
temporally regulated fashion.
Furthermore, it is unclear whether CBP,
SRC-1, or both coactivators
provide the physical contact points with
the transactivation domains
of HIF-1

upon hypoxic activation.
Interestingly, the functional
interaction between HIF-1

and the two
coactivator proteins was
synergistic when both transactivation domains
of HIF-1

were contained
within the analyzed GAL4-HIF-1

fusion
proteins. In contrast,
GAL4 fusion proteins spanning the isolated
transactivation domains
showed an additive mode of regulation in the
presence of both
SRC-1 and CBP, strongly suggesting that synergistic
regulation
by these two coactivators may require the presence and
integrity
of both transactivation domains of HIF-1

.
CBP and p300 are closely related proteins that exhibit strong sequence
similarity and similar functions with respect to their
roles as
coactivators (
3,
4,
11,
36). In analogy to
CBP, p300
enhanced in the presence of SRC-1 hypoxia-dependent
transcriptional
activation of reporter genes by GAL4 fusion proteins
containing either
one of the two individual transactivation domains
of HIF-1

(Fig.
5C). As observed in experiments using CBP (Fig.
5B), the CBP/p300 and
SRC-1 classes of coactivators produced together
rather modest (3- to
4-fold) hypoxic activation of the N-terminal
transactivation domain
located between residues 531 and 584 of
HIF-1

(Fig.
5C), whereas in
the presence of SRC-1, CBP or p300
more potently (around 11-fold)
stimulated hypoxic activation by
the C-terminal transactivation domain
of HIF-1

(Fig.
5C).
Although the mechanism of action of the CBP/p300 class of coactivators
remains largely unclear, recent observations have suggested
that these
proteins contribute to transcriptional regulation not
only by acting as
simple adaptors between DNA binding factors
and transcription
initiation factors but also by harboring intrinsic
HAT activity,
linking their effect to regulation of chromatin
structure and/or
function (
6,
31,
41). To investigate the
potential role of
the acetyltransferase activity of p300 in supporting
HIF-1

-dependent
transcription, we transfected COS7 cells with
a p300 deletion mutant,
p300

HAT, lacking the HAT domain which
is centrally located in the
protein (schematically represented
in the right panel of Fig.
5C). In
the absence of SRC-1, transient
expression of p300

HAT resulted in
only very moderate (twofold)
stimulation of the hypoxia-dependent
activation response produced
by GAL4/HIF 531-584, containing the
N-terminal transactivation
domain of HIF-1

, whereas it did not
produce any significant effect
on the hypoxia-induced transcriptional
activity of GAL4/HIF 776-826,
spanning the minimal C-terminal
transactivation domain (Fig.
5C).
In the presence of SRC-1, p300

HAT
enhanced reporter gene activation,
most notably when coexpressed in
hypoxic cells together with GAL4/HIF
776-826, containing the C-terminal
transactivation domain of HIF-1

.
However, p300

HAT was about
twofold less potent than wild-type
p300 in producing this response
(Fig.
5C), indicating that the
acetyltransferase catalytic activity of
p300 may contribute to
trigger the full activity of the
HIF-1

-mediated transcriptional
activation response. However, given
the observed synergy between
CBP/p300 and SRC-1 in enhancing activation
potency by HIF-1

,
and the fact that SRC-1 also harbors intrinsic HAT
activity (
47),
the effect of the deletion of the HAT domain
of p300 may be masked
by corresponding activities of CBP-associated
proteins, possibly
that of SRC-1
itself.
Ref-1 potentiates HIF-1
function in the presence of CBP and
SRC-1.
The nuclear redox regulator Ref-1 is known to stabilize the
DNA binding activity of AP-1 by reduction of a conserved cysteine residue of Fos and Jun. Ref-1 is a bifunctional enzyme: it harbors both
redox and endonuclease DNA repair activities (57) and has been implicated in up-regulation of HIF-1
-dependent induction of
gene expression under hypoxic conditions (13, 20). Against this background, we wanted to further investigate the effect of Ref-1
on HIF-1
-dependent transcriptional activation and to examine its
effect on the functional interaction of HIF-1
with the coactivators CBP and SRC-1. To investigate whether HIF-1
is a target of
regulation by Ref-1, we initially used the different GAL4 DBD fusion
proteins containing distinct subfragments of HIF-1
. As shown in Fig.
6A, transient overexpression of Ref-1
markedly potentiated hypoxic induction of reporter gene expression by
the fusion proteins GAL4/HIF 71-826 and GAL4/HIF 526-826, spanning both
transactivation domains of HIF-1
. The transcriptional activation
function of GAL4/HIF 531-584, containing the N-terminal transactivation
domain of HIF-1
, was only very moderately but significantly
stimulated by Ref-1 in hypoxic cells, whereas Ref-1 produced more
potent regulation of hypoxia-inducible transactivation by GAL4/HIF
776-826, containing the C-terminal transactivation domain (Fig. 6A).

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FIG. 6.
Ref-1 enhances HIF-1 function. (A) COS7 cells were
cotransfected with different GAL4-HIF-1 fusion constructs together
with a GAL4-responsive reporter plasmid in the absence or presence of
1.5 µg of Ref-1 (pCMV5/Ref-1), as indicated. Cells were exposed to 21 or 1% O2 before harvest. After normalization for
transfection efficiency using alkaline phosphatase activity, reporter
gene activities were expressed as relative to that of GAL4 in normoxia.
The results of two independent experiments performed in duplicate ± SE are shown. (B) Ref-1 potentiates CBP and SRC-1 activation of
HIF-1 . The same GAL4- HIF-1 fusion proteins as shown in panel A were
cotransfected into COS7 cells together with a GAL4-responsive reporter
plasmid in the absence or presence of different combinations of Ref-1
(0.75 µg), CBP (0.75 µg), and/or SRC-1 (0.75 µg) expression
vector, as indicated. The bottom panel shows an enlargement of the area
marked with dots. (C) Effect of hypoxia treatment on subcellular
localization of Ref-1. COS7 cells grown on coverslips were transiently
transfected with 3 µg of pGFP/Ref-1. After 6 h of incubation,
the medium was changed to fresh DMEM supplemented with 10% FCS and
incubated for 24 h. Cells were then exposed to 21 or 1%
O2 for 4 h. After being washed three times with PBS,
cells were fixed with 4% paraformaldehyde in PBS for 2 h at room
temperature, subsequently washed three times with PBS, and mounted.
Cells were observed with a fluorescence microscope. Representative
cells are shown. Bar = 10 µm.
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Given the striking potentiation of HIF-1

function by the
coactivators CBP and SRC-1, we next examined the effect of Ref-1
on transcriptional activation by the GAL4-HIF-1

fusion proteins
in
combination with CBP, SRC-1, or both proteins. Following transient
expression of Ref-1 together with either CBP or SRC-1,
hypoxia-inducible
transcriptional activation by the tested
GAL4-HIF-1

fusion proteins
was not significantly altered in
comparison to the results obtained
in the presence of the fusion
proteins and Ref-1 alone (Fig.
6B).
Remarkably, however, the
hypoxia-inducible transcriptional potencies
of all fusion proteins
containing either both or one of the HIF-1
transactivation domains
were dramatically (10- to 53-fold) enhanced
by coexpression of Ref-1 in
the presence of CBP and SRC-1 (Fig.
6B). Under these conditions, the
fusion proteins spanning the
N- or C-terminal transactivation domains
produced 12- or 53-fold
hypoxia-dependent activation responses,
respectively, whereas
the fusion protein containing the C-terminal
transactivation domain
lacking the CBP interaction interface, GAL4/HIF
776-813, showed
no regulation by Ref-1 and the coactivators. Thus,
these data
indicate that these two coactivators may have been limiting
under
these conditions for the Ref-1-mediated effect on
HIF-1

-dependent
transcription. Moreover, these results clearly
demonstrate that
both the N-terminal and C-terminal transactivation
domains of
HIF-1

are targets of regulation by Ref-1, implying that
Ref-1
together with the CBP and SRC-1 classes of transcriptional
coactivators
plays a key role in regulation of HIF-1

function.
Moreover, this
is the first example of a transactivation domain
representing
a target of regulation by Ref-1; conversely, these data
represent
the first example of a noncovalent protein modifier of the
HIF-1
transactivation domains identified in mammalian
cells.
The precise mechanism by which Ref-1 activates HIF-1

is not known.
Interestingly, a GFP-tagged Ref-1 fusion protein was exclusively
localized in the nucleus both under normoxic and hypoxic conditions
(Fig.
6C), indicating that nuclear translocation of HIF-1

is
required for functional interaction with Ref-1. This redox regulator
protein has been shown to form complexes with Jun in vitro
(
58).
To further investigate whether Ref-1 could interact
with HIF-1

,
we tried to trap the interaction by using a
cross-linking reagent
such as diamide, which oxidizes cysteine
sulfhydryls to disulfides.
A series of experiments was performed with
GST-tagged purified
Ref-1 (Fig.
7).
[
35S]methionine-labeled in vitro-translated GAL4/HIF
1-826 was incubated
with GST-Ref-1 in the presence or absence of
diamide (Fig.
7A).
GAL4/HIF 1-826 was found to weakly bind GST-Ref-1
(Fig.
7A, lane
1), and the cross-linking agent stabilized this
interaction between
Ref-1 and GAL4-HIF (compare lanes 2 and 3). No
binding of [
35S]methionine-labeled GAL4-HIF was observed
when it was incubated
with the anti-GST affinity gel alone in the
absence of GST-Ref-1
(Fig.
7A, control lane 4) or when it was
incubated with purified
GST (Fig.
7A, lane 5), indicating that there
was no significant
background binding.

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FIG. 7.
Ref-1 interacts with HIF-1 in vitro.
[35S]methionine-labeled in vitro-translated GAL4/HIF
1-826 (A) and GAL4/HIF 531-584 (N-TAD) or GAL4/HIF 776-826 (C-TAD) (B)
were incubated for 30 min at room temperature with recombinant
GST/Ref-1 or GST in the presence or absence of diamide as indicated.
Bound proteins were eluted in SDS sample buffer, run on SDS-7.5% (A)
and 12.5% (B) polyacrylamide gels, and visualized by fluorography.
Lane 6 in panel A represents 1/10 of the amount of the
[35S]methionine-labeled HIF-1 protein used in the
binding reactions; lane 4 represents
[35S]methionine-labeled HIF-1 incubated with anti-GST
affinity gel alone in the absence of GST/Ref-1. (C) Anti-GST Western
blot. One-tenth input of proteins used in GST pull-down assays followed
by elution and SDS-PAGE was analyzed by Western blotting using anti-GST
antibodies. Positions of molecular mass markers are shown on the left
in kilodaltons.
|
|
Next, we tried a series of pull-down experiments with
[
35S]methionine-labeled in vitro-translated GAL4/HIF
776-826 and GAL4/HIF
531-584. As shown in Fig.
7B, GAL4/HIF 531-584 bound GST-Ref-1,
and no effect of diamide was observed (lanes 1 and
2), consistent
with the absence of cysteine residues in the N-terminal
transactivation
domain of HIF-1

. An interaction between GAL4/HIF
726-826 and
GST-Ref-1 was also detected, and this interaction was
enhanced
in the presence of diamide (Fig.
7B, lanes 3 and 4). In
excellent
agreement with this observation, the C-terminal
transactivation
domain contains two cysteine residues. Low levels of
background
binding of the labeled proteins to GST were observed in the
presence
or absence of diamide (Fig.
7B, lanes 5 to 8). Immunoblot
analysis
verified that the input concentrations of GST-Ref-1 and GST
alone
were similar (Fig.
7C).
The experiments above established that Ref-1 potentiates the effect of
both the CBP and SRC-1 coactivators on hypoxia-inducible
promoter
activation by HIF-1

. Moreover, Ref-1 functionally and
physically
interacted with the distinct N- and C-terminal transactivation
domains
of HIF-1

. However, only the C-terminal and not the N-terminal
transactivation domain of HIF-1

contains cysteine residues through
which Ref-1 regulation occurs (
57). These data indicate that
an auxiliary factor, possibly a coactivator, may mediate interaction
of
Ref-1 with at least the N-terminal transactivation domain of
HIF-1

.
 |
DISCUSSION |
In this report we demonstrate that two members of the p160 family,
SRC-1 and TIF2, are able to interact with HIF-1
in a
hypoxia-dependent manner and enhance its hypoxia-inducible
transactivation potential. Moreover, low concentrations of SRC-1 can
produce this effect in synergy with CBP, and importantly, this effect
is greatly potentiated by the redox regulatory protein Ref-1,
indicating that these three proteins are important components of the
hypoxia signaling pathway.
Conditional recruitment of the SRC-1 and CBP classes of
coactivators to HIF-1
is an important step in the hypoxia signaling
pathway.
TIF2 and SRC-1 are related proteins that have originally
been identified as coactivators of nuclear hormone receptors and are
expressed in most tissues (17-19, 28, 42, 52, 53). The two
proteins could have redundant functions, or they could serve as
coactivators for different or overlapping subsets of transcription
factors. Our results indicate that both SRC-1 and TIF2 are able to
functionally interact in very similar manners with HIF-1
, a protein
belonging to the bHLH/PAS family of transcription factors. In fact,
SRC-1 and TIF2 are members of the same family of factors harboring a
bHLH/PAS motif in their N termini. Whereas both the HLH (35)
and PAS (34) motifs are potent dimerization interfaces
mediating, for instance, HIF-1
-Arnt heterodimerization (14,
54), the N-terminal bHLH/PAS motif of SRC-1 is not important for
functional interaction with HIF-1
. In agreement with this observation, the bHLH/PAS region of SRC-1 is irrelevant for enhancing steroid hormone receptor-dependent transcription or mediating physical
interaction with the receptors (28, 42, 60). In the case of
various members of these receptors, a conserved helix (helix 12) of the
ligand binding domain is establishing a physical contact with the SRC-1
class of coactivators, and in turn, there are multiple motifs within
the SRC-1 family of proteins mediating this interaction with receptors
(19, 24, 38, 53, 56). These interaction interfaces appear to
be characterized by the integrity of the short signature motif LXXLL,
where L is leucine and X is any amino acid (16).
Interestingly, our results indicate that an SRC-1 mutant protein with
mutated LXXLL motifs that is unable to support estrogen
receptor-dependent transcription (16) functionally interacts
with HIF-1
. Thus, HIF-1
may employ a mechanism of coactivator
recruitment that is different from that of steroid receptors. In a
similar fashion, the interaction between SRC-1 and the p50 subunit of
NF-
B occurs via a region that does not harbor an LXXLL motif
(39). It will now be interesting to identify the structural
motif(s) of SRC-1 which mediates interaction with the hypoxia-activated
form of HIF-1
.
Here we have identified the two transactivation domains localized in
the C terminus of HIF-1

as targets of regulation by
SRC-1. These two
functional domains of HIF-1

are contained within
54- or
38-residue-long stretches of amino acids. Interestingly,
the identical
regions of HIF-1

were also targeted for regulation
by CBP, Ref-1,
and, most notably, a combination of Ref-1 together
with SRC-1 and CBP.
This striking interdigitation in regulatory
potential between these
three proteins indicates a common link
in their mechanisms of action.
SRC-1 and CBP constitutively interact
with one another (
15,
28,
60), and both proteins appear
to potentiate steroid hormone
receptor-mediated transactivation
as a complex (reviewed in reference
50). Furthermore, both proteins
contain intrinsic
HAT activity (
6,
41,
47). It is thus
reasonable to expect
functional redundancy within the CBP-SRC-1
complex with regard to
acetylation activity. Consistent with this
notion, we observed only
partial reduction of the effect on HIF-1

-mediated
transcriptional
activation by using the p300

HAT mutant lacking
the domain harboring
HAT activity (
37) but, as schematically
represented in Fig.
5C, maintaining intact HIF-1

and SRC-1 interaction
domains.
It is unclear whether the transactivation domain of HIF-1

preferentially interacts with any specific component of the CBP-SRC-1
complex. It has been shown that CBP interacts with HIF-1

via
its
first cysteine/histidine-rich region (CH1 [
5]). In
contrast,
interaction of CBP with Arnt has been reported to be mediated
by the CREB-binding site of CBP (
30). These observations
suggest
that HIF-1

and Arnt within the hypoxia-activated
heterodimeric
complex may interact independently with two distinct
regions of
CBP/p300. Interestingly, it has been reported that it is not
possible
to detect any interaction between CBP and Arnt in an in vitro
protein-protein interaction assay, whereas the interaction was
demonstrated using an in vivo assay (
30). These data
indicate
that the interaction could be mediated or strengthened by a
factor(s)
missing in the in vitro system (
5,
30). For
instance, in
analogy to the mechanism of coactivator assembly on
nuclear receptors,
SRC-1 is a plausible candidate to facilitate this
interaction.
Regulation of HIF-1
function by the redox regulatory protein
Ref-1.
What is the mechanism of hypoxia-inducible recruitment of
the coactivators to HIF-1
? We and others have previously observed that hypoxia-dependent activation of HIF-1
function is a multistep mechanism including massive up-regulation of HIF-1
protein levels (20, 21, 25, 44) by inhibition of ubiquitination of HIF-1
(27), nuclear translocation (26), dimerization
with the constitutively nuclear factor Arnt (14, 54), and
recruitment of CBP (5, 26). Moreover, we have recently
demonstrated that GAL4-HIF-1
fusion proteins which show
constitutive nuclear compartmentalization due to the nuclear
localization signal contained within the GAL4 DBD require the hypoxic
signal for functional interaction with CBP (26). In analogy
to the steroid receptor system, it is an attractive scenario that the
hypoxic signal determines a conformational change in HIF-1
which is
critical for recruitment of the coactivators. This model of
conformational regulation of HIF-1
function is supported by the
present experiments which show that the integrity of the C terminus of
HIF-1
containing both transactivation domains is important for
cooperative regulation by SRC-1 and CBP. Moreover, the transactivation
domain of Arnt is also able to functionally interact with both CBP and
SRC-1 (this study and reference 30). Thus,
conformational regulation may also provide a mechanism of conditional
coactivator assembly on the individual transactivation domains within
the heterodimeric HIF-1
-Arnt complex. To understand the hypoxia
signaling pathway, it is critical to identify what determines
recruitment of the coactivators within the nucleus of hypoxic cells. It
is possible that noncovalent modification of the C terminus of HIF-1
plays a role in determining this regulatory effect. Although this is a
plausible mechanism, a protein kinase mediating such a modification has
not yet been demonstrated. In the present study we observed that Ref-1
enhances the effects of SRC-1 and CBP on the transactivation potential
of HIF-1
. Ref-1 is known to stimulate the DNA binding activity of a
number of transcription factors, including Fos, Jun, and p53, by a
redox-dependent mechanism (22, 57). The stimulatory effect
on Fos and Jun is elicited by reduction of a conserved cysteine residue
located in the DNA binding domain of each protein (reference
57 and references therein). What is the target of
regulation by Ref-1 in the hypoxia signaling pathway? Intriguingly,
only the C-terminal transactivation domain of HIF-1
contains
cysteine residues, whereas the N-terminal one located between amino
acid residues 531 and 584 lacks cysteines. Transient overexpression of
Ref-1 alone resulted in significant stimulation of the
hypoxia-inducible activity of the C-terminal transactivation domain of
HIF-1
. Under identical conditions, Ref-1 produced only a subtle
effect on the function of the N-terminal hypoxia-responsive
transactivation domain, indicating preferential regulation of the
C-terminal transactivation domain containing the cysteine residues.
However, in combination with transient expression of both SRC-1 and
CBP, Ref-1 dramatically stimulated the hypoxia-dependent activities of
both transactivation domains. Moreover, Ref-1 protein interacts in
vitro with both N- and C-terminal transactivation domains of HIF-1
.
These data demonstrate that both domains are individually regulated by
Ref-1, providing the first example of a transactivation domain as a
target of Ref-1 function.
Given the absence of cysteine residues in the N-terminal
transactivation domain of HIF-1

, the molecular target of Ref-1 in
the hypoxia signaling pathway may not be restricted to HIF-1
itself.
In line with this model, recent experiments have indicated
hypoxia-inducible enhancement by Ref-1 on the ligand-dependent
functional activities of either the retinoic acid receptor

2
or the
dioxin receptor, a bHLH/PAS transcription factor (P. Carrero,
K. Okamoto, and L. Poellinger, unpublished data). Thus, the effect
of
Ref-1 on CBP- and SRC-1-regulated transcriptional activities
appears
not to be restricted to HIF-1

but also detected in a
hypoxia-dependent fashion among other conditionally regulated
transcription factors (
59). This suggests that Ref-1 may be
involved in regulation of coactivator assembly on these conditionally
regulated transcription factors by, for instance, affecting the
stability of the coactivator complex tethered to the transactivation
domains or by enhancing recruitment of the coactivators. We are
currently investigating this possibility. To understand this mechanism
of regulation, it will now be critical to examine whether cysteine
residues within any of the components of the coactivator complex
are
targeted for regulation by Ref-1. If so, Ref-1 may represent
under
hypoxic conditions an important regulator of transcriptional
activation
processes that depend on inducible recruitment of the
CBP-SRC-1
coactivator
complex.
 |
ACKNOWLEDGMENTS |
We thank M. G. Parker for the SRC-1 M1234 construct. We also
thank Anders Berkenstam and Yuichi Makino for stimulating discussions and helpful advice.
This study was supported by grants from the Swedish Medical Research
Council, Pharmacia and Upjohn, Akiyama Foundation, and NOVARTIS
Foundation (Japan) for the Promotion of Science.
 |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Cell and Molecular Biology, Karolinska Institutet, S-171 77 Stockholm, Sweden. Phone: 46-8 728 7330. Fax: 46-8 34 88 19. E-mail:
Lorenz.Poellinger{at}cmb.ki.se.
Present address: Department of Molecular and Cellular Biology of
Development, Genes and Development Laboratory, UMR CNRS 7622, Universite Pierre et Marie Curie, 75252 Paris Cedex 05, France.
 |
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