From the Division of Hematology, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115
Received for publication, October 18, 2000
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ABSTRACT |
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Hypoxia-inducible factor 1 Hypoxia-inducible factor 1 (HIF1)1, a heterodimeric
basic helix-loop-helix-Per-AhR-Sim transcription factor, has
emerged as a key player in response to low oxygen tension among various
biological processes including embryogenesis, angiogenesis, and
tumorigenesis (1, 2). HIF1 is composed of two subunits, HIF1 The primary regulatory step of HIF1 activity is the accumulation of
HIF1 p300/CBP contains a cysteine- and histidine-rich 1 (C/H1) domain
encompassing two zinc-binding modules (33). Although C/H1 is known to
bind to the C-terminal activation domain (CAD) of HIF1 RAMSY--
The interaction between LexA-CAD and B42-C/H1 was
first determined in the matchmaker LexA two-hybrid system
(CLONTECH) after cotransformation into yeast strain
EGY48[p8op-lacZ] according to the manufacturer's instructions. To
create overhangs for in vivo homologous recombination, PCR
primers for random mutagenesis were designed such that the upstream
primer (5'-GCGAGTTTAAACCAATTGTCGTAGA-3') started 92 base pairs upstream
of the EcoRI site of pLexA, whereas the downstream primer
(5'-CAGGAAAGAGTTACTCAAGAACAAGAATT-3') was 157 base pairs downstream of
the XhoI site of the vector. The mutagenic PCR reaction was
performed in a total volume of 25 µl with 1 mM
dCTP, dGTP, and dTTP and 0.2 mM dATP in the presence of 3 mM MgCl2 and 0.3 mM
MnCl2 using pLexA-CAD as a template. The linearized vector
was prepared by digestion of pLexA-CAD with EcoRI and
XhoI to remove the CAD, followed by gel purification. Subsequently, 20 µl of mutagenic PCR fragments together with 100 ng
of digested pLexA vector were cotransformed into the yeast strain
EGY48[p8op-lacZ, pB42-C/H1] to amend the linearized vector through
in vivo homologous recombination. The transformed cells were
streaked on plates containing appropriate nutrition selection (CLONTECH) plus X-gal. White colonies were gathered
onto a fresh X-gal plate for verification of loss of the
lacZ gene expression and were subsequently replicated onto a
In Vitro HIF1 Plasmids and Site-directed Mutagenesis--
pB42-C/H1 was
constructed by insertion of the p300 C/H1 domain into
EcoRI/XhoI-digested pB42-AD
(CLONTECH). Similarly, the C/H1 domain was also
cloned into EcoRI/XbaI-digested
p(His)VP162 or
BamHI/XbaI-digested pEYFP-Nuc
(CLONTECH), resulting in p(His)VP16-C/H1 and
pEYFP-C/H1, respectively. pLexA-CAD and pGal4-CAD were constructed individually by insertion of HIF1 Transfection--
FuGene 6 transfection reagent (Roche
Molecular Biochemicals) was used for transfection of Hep3B (in a 6-well
plate) and COS7 (in a 12-well plate) according to the manufacturer's
instructions. Six h later, cells were either maintained under normoxia
or subjected to hypoxia overnight and harvested accordingly 24 h
after transfection. Luciferase and RAMSY--
In search of the molecular determinants of HIF1
Next, the CAD region was PCR-amplified under a mutagenic condition in
which the dATP concentration was reduced to one-fifth to generate
random mutations (37, 38). The mutant products contained a flanking
region at both ends that were identical to the corresponding ends of
the gapped backbone vector (pLexA), thereby enabling in vivo
recombination upon cotransformation (Fig. 1c). With
appropriate nutritional selections, colonies with potential loss of
C/H1 interaction, appearing white on X-gal plates, were collected.
Approximately 10-15% of the total transformants were gathered and
tested further for inability to transactivate the LEU2 gene.
Only those that were unable to activate both reporters were selected
for PCR amplification and DNA sequencing of the mutagenized region.
Compilation of sequencing data from a total of 32 clones revealed that
codons Leu-795, Cys-800, Leu-818, and Leu-822 of HIF1 Essential Role of the Identified Residues for CAD
Transactivation--
Initially, CAD and its mutants were fused to a
Gal4 DNA-binding domain and tested for transcriptional activation of a
Gal4-luc reporter in Hep3B cells. As shown previously (16, 17), CAD exhibited strong hypoxia-induced transcriptional activity (Fig. 2a). By contrast, none of
these mutants (L795P, C800R, L818S, and L822S) did so, even though
their protein expression levels were similar to that of the wild type
(Fig. 2a, inset). Because these mutants were
originally identified to be critical for interaction with C/H1 in
yeast, the above result implied that CAD transactivation relies on
interaction with endogenous p300/CBP. To test this hypothesis, we
cotransfected a vector that either expresses C/H1 fused to a VP16
activation domain (VP16-C/H1) or C/H1 fused to a yellow fluorescent
protein (EYFP-C/H1) as a tag in the above setting. As shown in
Fig. 2b, the C/H1 domain, when fused to EYFP, apparently competed against endogenous p300/CBP for CAD binding, thereby inhibiting transcription. In contrast, VP16-C/H1 further enhanced transcriptional activity of the wild-type CAD but not that of the
mutants (Fig. 2c). These results confirm that the residues identified by RAMSY are functionally critical for CAD transcriptional activity in mammalian cells. To provide evidence that this observation is a result of loss of C/H1 binding, we performed GST pull-down experiments in which GST-C/H1 or GST- Stringent Necessity of Leucines and Hydrophobic Cysteine for CAD
Activity--
It appears that CAD requires hydrophobic leucines for
C/H1 binding; replacement with the hydrophilic residues abolished its transcriptional activity. To test the stringency of these leucines, we
asked whether different hydrophobic residues such as valine and alanine
could mimic leucine. Interestingly, individual replacement of Leu-795
and Leu-822 with valine abrogated CAD transcription (Fig.
3, a and c),
whereas L818V substitution showed only modest reduction. However,
alanine substitutions of these leucines abolished CAD function. These
findings argue against the loss of CAD activity as a result of global
structural disruption but rather suggest that these leucines engage in
direct contact with the C/H1 domain. In keeping with this notion, the
impairment of CAD function was in good agreement with loss of C/H1
binding in vitro, with the exception of L795V (Fig. 3,
b and d). Taken together, these results suggest
the stringent requirement of these leucines for CAD transcriptional activity.
Cys-800 has been suggested to be a target of redox modulation of HIF1
transactivation because serine substitution inhibited CAD activity and
p300/CBP binding (25), but this notion is apparently not supported by
alanine replacement, showing a modest effect (36). To gain a definitive
understanding of the functional role of Cys-800, we mutated Cys-800 to
structurally close but biochemically distinct residues including
serine, threonine, aspartate, asparagine, alanine, and valine.
Remarkably, substitutions with the first four hydrophilic residues
exhibited invariable loss of CAD transcriptional activity (Fig. 3,
e and f) and C/H1 binding (data not shown). However, hydrophobic residue substitutions not only retained CAD function, but valine replacement further increased CAD activity (Fig.
3f), whereas C/H1 binding activity and protein expression levels were equivalent to those of the wild type (data not shown). Thus, we conclude that HIF1 Molecular Determinants of HIF1
The effectiveness of RAMSY warranted investigation of the C/H1 domain.
Following random mutagenesis of the C/H1 domain in B42-C/H1, compiled
sequencing data from a total of 57 clones revealed that codons
Leu-344, Leu-345, Cys-388, and Cys-393 of p300 were among the most
frequently mutated (Table I, bottom). Consistently, an
independent study based on sequence alignment also demonstrated recently that these two cysteines are required for HIF1 We have successfully employed the RAMSY technique to identify the
molecular determinants of the HIF1 It is noteworthy that valine substitution markedly increased normoxic
CAD activity, thereby decreasing hypoxic inducibility of CAD. However,
the hypoxic induction still remained (Fig. 3f), indicating
the possibility of additional mechanisms contributing to
hypoxia-induced CAD-C/H1 interaction or CAD transcriptional activity.
Interestingly, among all the clones of the CAD mutants sequenced, none
of the mutations occurred at codons 781-783 of the conserved
RLL sequence that was reported to be critical for hypoxic induction in
HIF2 We have shown that RAMSY, similar to the reverse two-hybrid system
(41), is a simple, efficient, and reliable approach by which molecular
determinants of two interacting mammalian proteins can be uncovered
readily in yeast. We demonstrated its efficiency to pinpoint the most
critical (if not all) residues involving protein-protein interactions;
single (instead of multiple) mutations of the identified residues
abrogate the protein function, indicating a critical role for these
residues. As the yeast two-hybrid system has been widely employed in
the last decade, RAMSY should provide a broadly applicable means for
the efficient revelation of the molecular basis of a wide range of
protein-protein interactions and in turn lead to a better understanding
of the mechanisms underlying various biological processes.
(HIF1
) plays a
pivotal role in embryogenesis, angiogenesis, and tumorigenesis.
HIF1
-mediated transcription requires the coactivator p300, at
least in part, through interaction with the cysteine- and
histidine-rich 1 domain of p300. To understand the molecular
basis of this interaction, we have developed a random mutagenesis
screen in yeast approach for efficient identification of residues that
are functionally critical for protein interactions. As a result, four
residues (Leu-795, Cys-800, Leu-818, and Leu-822) in the
C-terminal activation domain of HIF1
have been identified as crucial
for HIF1 transactivation in mammalian systems. Moreover, data from
residue substitution experiments indicate the stringent necessity of
leucine and hydrophobic cysteine for C-terminal activation domain
function. Likewise, Leu-344, Leu-345, Cys-388, and Cys-393 in the
cysteine- and histidine-rich 1 domain of p300 have also been shown to
be essential for the functional interaction. We propose that
hypoxia-induced HIF1
-p300 interaction relies upon a leucine-rich
hydrophobic interface that is regulated by the hydrophilic and
hydrophobic sulfhydryls of HIF1
Cys-800.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and
arylhydrocarbon receptor nuclear translocator, and is required
for transcriptional up-regulation of a growing number of
hypoxia-responsive genes, such as erythropoietin, vascular endothelial
growth factor, and inducible nitric-oxide synthase (3, 4). During
embryonic development, HIF1 is responsible for vascular development and expression of genes encoding glucose transporter and glycolytic enzymes
(5-7). Given that hypoxia is associated with tumor angiogenesis, progression, and metastasis (8, 9), the role of HIF1 in angiogenesis
and tumor progression has been studied (7, 10). Furthermore, HIF1
has been shown to be necessary for hypoxic stabilization of wild-type
p53 (11), consistent with the notion that HIF1
is involved in
hypoxia-mediated apoptosis and cell proliferation (12).
protein (13). Activation of HIF1 involves a multi-step temporal
process including hypoxia-induced post-translational stabilization (13,
14), nuclear translocation (15), and transcriptional activation (16,
17). HIF1
stabilization is a result of inhibition of the
ubiquitin-proteasomal degradation pathway (18-23) that targets the
oxygen-dependent degradation domain of HIF1
(18,
21-23). Stabilized HIF1
exerts its transcriptional activity by
recruiting the transcriptional coactivator and integrator p300/CBP (15, 24, 25). p300 (26) and its closely related family
member CBP (27) mediate a multitude of signal-dependent transcriptional events by acting as molecular scaffolds through linking
various transcription factors to the basal transcription apparatus (28,
29). Moreover, p300/CBP facilitates transcription through chromosome
remodeling and acetylation of transcription factors (30-32).
(24, 34), the
molecular basis underlying this interaction is unclear. A redox
mechanism has been proposed primarily based upon the evidence that the
reducing factor Ref-1 enhances transcriptional activity of HIF1 (13)
and CAD (25, 35). In keeping with this notion, serine substitution of a
highly conserved cysteine (Cys-800) within the CAD abrogated the
transcriptional activity as well as p300/CBP binding (25). However,
substitution with alanine showed an insignificant effect (36),
apparently arguing against a redox role for Cys-800. To gain a
definitive understanding of HIF1
-p300 interaction, we developed a
random mutagenesis screen in
yeast (RAMSY) approach for rapid and systematic
identification of critical residues that engage in CAD-C/H1
interaction. Our data indicate that the HIF1
-p300 interaction
requires a leucine-rich interface that is regulated by a single
cysteine residue.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Leu plate to confirm their inability to activate the
LEU2 gene. Only those clones that were white on X-gal plates
and unable to grow on
Leu plates were collected for plasmid
isolation. The CAD region was amplified with PCR using an upstream
primer (5'-GGATGGTGACTTGCTGGCAGTG-3') and a downstream primer
(5'-GGCGACCACACCCGTCCTGT-3'). The PCR products were then subjected to
cycle sequencing with the fmol DNA sequencing system
(Promega) using an internal primer (5'-GATCTTCGTCAGCAGAGCTTCACCA-3'). The sequencing data were analyzed with FileMaker Pro (Claris) to
quickly identify residues with a high frequency of mutation. Likewise,
random mutagenesis of the C/H1 domain with pB42-C/H1 as DNA template
was performed in a similar way using an upstream primer
(5'-CCGCCGATCCAGCCTGAC-3') and a downstream primer
(5'-ACCTGAGAAAGCAACCTGACCTACA-3'). The C/H1 mutants were sequenced in
the DNA Sequencing Facility at the Brigham and Women's Hospital.
-p300 Interaction--
Full-length HIF1
or
Gal4-CAD was translated in vitro using the TNT T7
quick-coupled transcription/translation system (Promega) in the
presence of [35S]methionine (Amersham Pharmacia Biotech).
5 µl of translated products were incubated with 2 µg of
Sepharose-conjugated GST-C/H1 or GST-
C/H1 in a final volume of 200 µl in NETN buffer (100 mM NaCl, 1 mM EDTA, 20 mM Tris (pH 8.0), and 0.5% Nonidet P-40). The incubation
was performed at room temperature for 3 h or at 4 °C overnight.
The reaction mixture was washed five times with NETN buffer before
SDS-polyacrylamide gel electrophoresis. The gel was stained with
Coomassie Blue to visualize equivalent usage of GST fusions.
CAD into
EcoRI/BamHI-digested pLexA
(CLONTECH) and pCMX-G4(N). pGST-C/H1 and
pGST-
C/H1 were gifts of the Livingston Laboratory (Dana-Farber
Cancer Institute). All of the pGal4-CAD mutants were generated with
PCR-mediated site-directed mutagenesis. p(HA)HIF1
,
p(HA)HIF1
(
ODD), and pEpoE-luc were described previously (13, 18).
Residue substitutions in full-length HIF1
were performed with the
altered sites in vitro mutagenesis system (Promega).
All the constructs were confirmed by DNA sequencing.
-galactosidase activities were
measured essentially as described previously (18). All the results
represent three or four independent experiments. For Western blot
analysis 293 cells were transfected and lysed as described previously
(18).
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-p300
interaction, we developed a RAMSY technique that couples PCR-mediated random mutagenesis (37, 38) with a LexA yeast two-hybrid system (Fig.
1). We began the two-hybrid assay by
constructing two fusion plasmids; HIF1
CAD (amino acids 776-826)
(16, 17) was fused to a LexA DNA-binding domain (LexA-CAD), whereas
p300 C/H1 (amino acids 311-528) (24) was fused to an activation domain
(B42-C/H1). Despite its transcriptional activity in mammalian cells,
CAD fusion alone failed to activate the lacZ and
LEU2 reporter genes in yeast, in agreement with a previous
report (25). This is presumably due to a lack of necessary factor(s) in
yeast. Accordingly, cotransformation with pB42-C/H1 resulted in marked
activation of both reporter genes in a galactose-dependent
manner (data not shown).
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Fig. 1.
Schematic illustration of RAMSY.
a, a yeast two-hybrid assay was established to demonstrate
CAD-C/H1 interaction. b, the CAD region was mutagenized by
PCR with a biased concentration of dATP. c, the mutagenized
products were cotransformed with a gapped pLexA vector into the yeast
two-hybrid strain, resulting in in vivo homologous
recombination. d, after appropriate nutritional selection,
white colonies indicating loss of CAD-C/H1 interaction were selected
and confirmed with their inability to activate the
LEU2 gene. e, plasmids were isolated from
these clones, and the CAD region was PCR-amplified for DNA sequencing
(f). g, compilation of sequencing data revealed
the most frequently mutated residues.
were
among the most frequently mutated (Table
I, top), indicating the
importance of these residues for C/H1 interaction. Of note, in addition
to the above clones, 16 more clones contained either premature stop
codons or reading frameshifts, and 3 more clones showed no
mutations within the CAD presumably because nonsense mutations
occurred in the upstream homologous recombination region. Silent
mutations were also observed in some clones (data not shown). It is
noteworthy that all of these four residues are conserved across various
species of cloned HIF1
as well as HIF2
including human, bovine,
rat, mouse, chicken, Xenopus, and quail (data not shown).
Hence we decided to focus on these four residues for in-depth analysis
of their role in transcriptional activation in mammalian systems.
Critical codons identified by RAMSY
C/H1 (24) was incubated with
in vitro translated Gal4-CAD or the mutants. As expected, wild-type Gal4-CAD interacted with GST-C/H1 but not with GST-
C/H1, and furthermore none of the mutants bound to C/H1 (Fig. 2d).
Therefore, we conclude that Leu-795, Cys-800, Leu-818, and Leu-822 of
HIF1
are functionally indispensable for CAD transcriptional activity in mammalian cells.
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Fig. 2.
Residues identified by RAMSY are functionally
critical in human cells. a, pGal4-CAD (CAD) and its
mutants (L795P, C800R, L818S, and L822S) were transfected into Hep3B
cells for testing their ability to transactivate Gal4-luc. Luciferase
activity from normoxic (N) and hypoxic (H) cells
is plotted in relative light units (RLU). Expression levels
of Gal4-CAD and the mutants in 293 cells are shown in the
Western blot (inset). b, coexpression of
EYFP-C/H1 (+) but not EYFP ( ) inhibited
Gal4-CAD transcriptional activity. c, coexpression of VP-16
(+) enhanced wild-type but not the mutant Gal4-CAD
transcriptional activity. d, GST-C/H1 bound to in
vitro translated Gal4-CAD but not to the five indicated Gal4-CAD
mutants. The GST fusion lacking C/H1 is marked as
C/H1.
The top panel shows 20% of input; the middle
panel shows C/H1 bound Gal4-CAD; and the bottom
panel shows the amount of GST fusions in each sample.
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Fig. 3.
The stringency of leucines and hydrophobicity
of cysteine favor CAD transcriptional activity. a and
c, transcriptional activity of Gal4-CAD with hydrophobic
residue (Val and Ala) substitutions at codons Leu-795, Leu-818,
and Leu-822 was tested with pGal4-luc. b and d,
the C/H1 binding activity of these hydrophobic substitutions was
analyzed in a GST pull-down assay. e and f,
distinct effects of hydrophobic (Val and Ala) versus
hydrophilic (Ser, Asp, Asn, and Thr) replacement of Cys-800 on
hypoxia-induced transactivation. RLU, relative light
units; N, normoxic; H, hypoxic.
Cys-800 plays a regulatory role
for C/H1 binding and HIF1 transactivation by the reversible change between -SH (hydrophobic) and -S
(hydrophilic) groups.
-p300 Interaction--
To verify
the role of these identified residues in HIF1-mediated transcription,
we introduced mutations to a full-length HIF1
and tested their
effects on a HIF1-responsive luciferase reporter. Consistent with the
results obtained from Gal4 fusions, all of the individual mutations
resulted in a significant decrease in HIF1 transcription in COS7 cells
(Fig. 4b). Furthermore, unlike wild-type HIF1
, these mutants failed to bind to C/H1 in a GST pull-down assay (data not shown). It is noteworthy that in addition to
CAD, HIF1
possesses another activation domain upstream of CAD,
namely N-terminal activation domain (16, 17) (Fig. 4a), but
its transcriptional activity appears to be less potent in the absence
of CAD (39). Moreover, we previously showed that CAD is sufficient for
HIF-1-mediated transcription in the absence of N-terminal activation
domain (18, 40). Consistently, introduction of L795V and L822V
mutations to an N-terminal activation domain-deleted HIF1
significantly reduced HIF1 transcriptional activity (Fig. 4c). These results lend further support to the notion
that Leu-795, Cys-800, Leu-818, and Leu-822 of HIF1
are
essential for HIF1 transactivation.
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Fig. 4.
Molecular determinants of
HIF1 -p300 interaction. a, a
schematic illustration of HIF1
in full-length and N-terminal
activation domain-deleted forms. b, the role of these
HIF1
residues was examined further in a full-length context for
hypoxia-induced transactivation of a HIF1-mediated luciferase reporter
(EpoE-luc). c, deletion of the N-terminal activation domain
(
ODD) did not affect HIF1 transcription, but additional
CAD mutations (L795V, L822V) reduced HIF1 activity. d,
transfection with the C/H1 mutants (L344P, L345P, C388R, and C393R)
failed to suppress CAD activity. bHLH, basic
helix-loop-helix; PAS, per-AhR-Sim; ODD, oxygen-dependent
degradation; NAD, N-terminal activation domain;
RLU, relative light unit; N, normoxic;
H, hypoxic.
interaction and for the integrity of a zinc bundle structure within the
-helical C/H1 domain (33). The biological function of the four residues was
subsequently evaluated by testing whether mutations of these residues
would interfere with endogenous p300/CBP binding to CAD in Hep3B cells,
as shown in Fig. 2b. In contrast to the wild-type EYFP-C/H1,
these mutant fusions failed to inhibit Gal4-CAD activity (Fig.
4d), implying that the identified p300/CBP residues are required for HIF1
binding and transactivation. Consistently, because
the C/H1 domain utilizes distinct residues to interact with a variety
of factors (33, 34), over-expression of the C/H1 mutants might compete
for binding to the competitive inhibitors of HIF1
transactivation,
e.g. p35srj, thereby releasing more endogenous p300/CBP and
in turn stimulating Gal4-CAD transcriptional activity.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-p300 interaction, which provides
a molecular basis for understanding the mechanisms underlying HIF1
activation. As a result, we propose that hypoxia-induced HIF1
-p300
interaction requires a leucine-rich hydrophobic interface that is
regulated by the reversible change between hydrophobic and hydrophilic
Cys-800 of HIF1
. This hypothesis is in part based upon the results
from Cys-800 replacement with structurally similar but biochemically
distinct residues including valine, and is consistent with the
functional role of the reducing factor Ref-1 in CAD transcriptional activation (25, 35). Our results have also ruled out the possibility that CAD transcriptional activity requires Cys-800 to form a disulfide bond with the C/H1 domain of p300/CBP.
(36). This difference might be resolved by sequencing a larger
population of CAD mutants even though the result could be
unpredictable. In theory, the RLL sequence had the same mutation
probability as the identified residues because of random mutagenesis
(37). Alternatively, it is possible that functionally defective mutants
in mammalian systems are functional in yeast and therefore cannot be
detected by RAMSY. In addition, p300/CBP binds the nuclear hormone
receptor coactivator SRC-1, which has been shown recently to be an
active part of the HIF1 transcriptional complex (35). The involvement
of SRC-1 might explain why the L795V mutation did not affect C/H1
binding in vitro but significantly inhibited CAD
transactivation (Fig. 3a), because such mutation might pose
a steric hindrance to SRC-1 binding in vivo, thereby
interfering with CAD function. Likewise, it is conceivable that the
"superactive" nature of the C800V mutation is a result of favored
SRC-1 binding in addition to the unaffected p300/CBP binding.
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ACKNOWLEDGEMENTS |
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We are indebted to H. Franklin Bunn for his generous support of this work. We are grateful to H. Franklin Bunn, William G. Kaelin, and Carl Wu for critical reading of the manuscript. We thank Mark Hochstrasser for advice on random mutagenesis, Wen-Fang Wang for advice on yeast work, and David Livingston for DNA reagents.
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FOOTNOTES |
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* This work was supported by Grant RO1 DK41234 (to H. Franklin Bunn) from NIDDK, National Institutes of Health and by National Research Service Award F32 DK09856 (to J. G.).The 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.
Current address: Laboratory of Human Carcinogenesis, National
Cancer Institute, Bldg. 37, Room 2C08, 37 Convent Dr., MSC 4255, Bethesda, MD 20892-4255.
§ To whom correspondence should be addressed. Tel.: 301-402-8785; Fax: 301-480-1264; E-mail: huange@mail.nih.gov.
Published, JBC Papers in Press, November 3, 2000, DOI 10.1074/jbc.M009522200
2 L. E. Huang, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are:
HIF1, hypoxia-inducible factor 1;
CREB, cAMP-response element-binding
protein;
CBP, CREB-binding protein;
C/H1, cysteine- and histidine-rich
1;
CAD, C-terminal activation domain;
RAMSY, random mutagenesis screen
in yeast;
PCR, polymerase chain reaction;
X-gal, 5-bromo-4-chloro-3-indolyl -D-galactopyranoside;
GST, glutathione S-transferase.
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