 |
INTRODUCTION |
The hepatocyte nuclear factor-4
(HNF-4)1 is a member of the
superfamily of nuclear hormone receptor (NHR) proteins that control transcription in diverse metabolic pathways. Originally identified by
its importance in the regulation of liver-specific genes, it is also
expressed in the pancreas, kidney, stomach, skin, and intestine (1-3).
There are currently three family members HNF-4
, -
, and -
(4,
5), and seven splice variants of HNF-4
have been identified from
human, rat, and mouse cDNAs (6-11). HNF-4 is a crucial regulator
of several metabolic pathways, including those for glucose and lipid
homeostasis. Mutations in HNF-4
impair insulin secretion and cause
type 2 diabetes (12). In addition, the promoters of apolipoproteins
apoAI, apoAII, apoAIV, apoB, apoCII, and apoCIII all contain binding
sequences for HNF-4 (13-17). HNF-4
also plays a vital role in
development. In mice, HNF-4
transcripts have been detected as early
as day 4.5 and its knock-out impairs gastrulation and is
embryonic-lethal (3, 18).
In a recent study, we performed a systematic analysis of the functional
domains of HNF-4 that are involved in DNA binding, dimerization, and
transactivation (19). We have found that HNF-4 contains two activation
functions (AFs), designated AF-1 and AF-2, that are located in the A/B
and D/E regions, respectively, and activate transcription in a cell
type-independent manner. In most cell types examined, AF-1 and AF-2
synergize for full HNF-4 activity. The AF-1 consists of the extreme
N-terminal 24 amino acids and functions as a constitutive autonomous
activator of transcription. The AF-2 transactivator is more complex,
spanning the region between amino acids 128 and 366. The 360-366
region of HNF-4 (AF-2 AD) contains a motif that is highly conserved
among transcriptionally active nuclear receptors and is essential for
AF-2 activity. Unlike the AF-2 domains of retinoid X receptor, retinoic
acid receptor, and thyroid receptor, the corresponding AF-2 AD region
does not exhibit an autonomous function, although it can adopt a
similar amphipathic
-helical conformation (20-24). For AF-2
activity in HNF-4, the entire 128-366 region is required, representing
a new subclass of activation domains defined by the requirement of an intact D/E region for activity. The F region, which is unique to HNF-4,
has a negative effect on AF-2 activity. It was recently shown that long
chain fatty acids directly modulate the transcriptional activity of
HNF-4
by binding as their acyl-CoA thioesters to HNF-4. Depending on
the chain length and the degree of saturation of the fatty acyl-CoA
ligand, HNF-4 can either activate or repress transcription of the
target genes (25). However, the involvement of the different HNF-4
domains in ligand-mediated transcriptional activation has yet to be defined.
Recent studies have shown that the ligand binding domains (LBDs) of
certain nuclear hormone receptors interact strongly with the
coactivator CBP and its functional homolog p300 in a
ligand-dependent manner (26, 27). CBP is a multi-faceted
protein involved in multiple signal transduction pathways with
different activators and in interactions with components of the basal
transcription machinery like TATA-binding protein, TFIIB, and RNA
helicase (28-31). It has intrinsic histone acetylase (HAT) activity
(32, 33) and interacts with the nuclear hormone receptor coactivators
SRC-1, P/CAF, and p/CIP, which have their own HAT activity (27,
34-39). Together these proteins form a putative coactivator complex,
the components of which appear to be specific for different
transcriptional activators (40). Current theory suggests that
transcription factors bound to their DNA response elements associate
with CBP or other coactivators and direct acetylation of histones in
their vicinity. The consequent restructuring of the chromatin leads to
enhanced assembly of the basal transcription machinery, possibly recruited by CBP, to form a stable preinitiation complex. However, chromatin disruption by histone acetylation is not sufficient for
NHR-dependent transactivation (41). In addition, p300 can acetylate the general transcription factors TFIIE
and TFIIF (42) suggesting that modification of core proteins by CBP or other coactivators may have a role in transcriptional activation.
In the present study, we show that HNF-4 interacts in vitro
with CBP and that HNF-4 can recruit CBP while bound to the DNA. CBP
targets both the AF-1 and the AF-2 domains of HNF-4. The AF-1 domain
interacts weakly only with the N-terminal region of CBP, and the AF-2
domain of HNF-4 interacts with both the N- and C-terminal regions of
CBP, and this interaction is both AF-2 AD and ligand-independent. In
cotransfection assays, CBP functions as a coactivator and stimulates the transcriptional activity of HNF-4. CBP does not activate gene expression in the absence of HNF-4, and dominant negative forms of
HNF-4 prevent transcriptional activation by CBP. Although both the N-
and C-terminal regions of CBP associate with HNF-4, transcriptional coactivation may be mediated by the C-terminal region. These findings demonstrate that CBP acts as a transcriptional coactivator for HNF-4
and provide new insights into the regulatory function of HNF-4.
 |
MATERIALS AND METHODS |
Plasmids--
The wild type apoCIII promoter
[apoCIII(
890/+24)CAT] plasmid has been described previously (43).
The reporter plasmids containing the apoCIII(
890/+24) promoter
mutated in the regulatory element B (BM5) and I4
(I4M) have been described previously (14, 43). Plasmid
(BA1)5CAT contains five copies of an HNF-4 binding regulatory element from the apoB gene (element BA1) in front of the
minimal apoB promoter (15). Plasmid pG5CAT contains five GAL4-binding sites upstream of the
-globin promoter and the CAT gene
(19). The construction of the various HNF-4 mutants cloned in the
vector pcDNAI/Amp (Invitrogen), as well as the GAL-HNF-4 chimeras
have been previously described (19). The HNF-4 mutants HNF-4-(45-142),
HNF-4-(45-370)68/71G, and HNF-4-(45-370)103/106G were generated by
polymerase chain reaction (PCR). The N-terminal primer was HNF-4
45F,
5'-ACTCGGGATCCGCCGCCGCCATGCTGGGTGTCAGTGCCCTGTGTC. The C-terminal primers were HNF-4 142R,
5'-GATGTCGAATTCTCAGGAGGGTAGGCTGCTGTCCTC, and HNF-4 370R,
5'-TCTAGAGAATTCTCAGGCAGACCCTCCAAGCAGCATC. Mutations were
generated using the following primers: 68/71G F,
5'-TCAAGCGGTGACGGCGGCAAGGGA; 68/71G R, 5'-TCCCTTGCCGCCGTCACCGCTTGA;
103/106G F, 5'-AACCAGGGTCGTTACGGCAGGCTC; and 103/106G R,
5'-GAGCCTGCCGTAACGACCCTGGTT. The underlined nucleotides indicate the
restriction sites for BamHI and EcoRI as well as the initiator codon, ATG, and the terminator codon, TGA (TCA in reverse
orientation). The bold nucleotides indicate the Kozak sequence adjacent
to the initiator codon for optimal translation. The HNF-4-(45-142)
clone was generated using HNF-4 45F and HNF-4 142R as primers. The
HNF-4-(45-370)68/71G and HNF-4-(45-370)103/106G clones were obtained
by a two-step PCR strategy. First, two separate PCR reactions were
carried out, one containing HNF-4 45F and the relevant reverse primer
for mutation (68/71G R or 103/106G R), and the other containing HNF-4
370R and the relevant forward primer for mutation (68/71G F or 103/106G
F). Aliquots containing 2% of each of these initial PCR reactions were
mixed and used for another round of PCR containing the HNF-4 45F and
HNF-4 370R primers. The PCR products were digested with
BamHI and EcoRI and cloned into the expression
vector pcDNAI/amp.
GST-CBP fusion constructs in pGEX-3T (Amersham Pharmacia Biotech) and
the various CBP mutants cloned in vector pRc/RSV (Invitrogen) (44) were
a generous gift from Dr. Dimitris Thanos (Columbia University).
In Vitro Transcription and
Translation--
[35S]Methionine-labeled HNF-4 proteins
were produced from cDNA templates cloned downstream of T7 RNA
polymerase promoter using the TNT T7 Quick-coupled reticulocyte lysate
system (Promega).
Preparation and Expression of GST Fusion
Proteins--
Escherichia coli strain BL21 was transformed
with the GST-CBP fusion proteins GST-CBP1, GST-CBP2, GST-CBP3, and
GST-CBP4. Fresh transformants were grown in LB media supplemented with
ampicillin (50 µg/ml) at 37 °C until an
A550 = 0.5 was reached.
Isopropyl-
-D-thiogalactopyranoside was added to a final
concentration of 0.5 mM. After incubation for 3 h
cells were harvested by centrifugation at 4,000 × g
for 10 min. Cell pellets were resuspended in 1/20 of the original culture volume in ice-cold phosphate resuspension buffer
(phosphate-buffered saline containing 20% glycerol, 1% Nonidet P-40,
100 mM EDTA, pH 8.0, 1 mM DTT, 1 mM
phenylmethylsulfonyl fluoride, 0.1 mM benzamidine, 2 µg/ml aprotinin, 1 µg/ml pepstatin, and 1 µg/ml leupeptin) and were broken by sonication on ice. Cell debris was removed by
centrifugation at 10,000 rpm for 20 min at 4 °C, and the supernatant
was incubated for 2 h with glutathione-agarose beads (SIGMA G4510)
equilibrated to the same buffer. The coupled proteins were washed
several times with the phosphate resuspension buffer and were stored at
4 °C covered by an equal volume of buffer. The purity of proteins
bound to the GST-agarose beads was identified by SDS-PAGE. When
necessary, the pure GST-CBP proteins were eluted from the agarose beads
by resuspending the pelleted beads in a half-volume of cold phosphate resuspension buffer containing 2.5 mM glutathione.
The suspension was incubated for 2-3 min on ice, the beads pelleted by
centrifugation, and the eluate reserved. The beads were washed 2-3
more times, and the protein content in each faction of the eluate was
observed by SDS-PAGE.
Protein Binding Assays Using GST Fusion Proteins--
For the
binding assays 5-50 µl of glutathione beads containing approximately
0.5 µg of the fusion GST-CBP protein were incubated at 4 °C in
0.35 ml of 150 mM sodium acetate, pH 7.0, 25 mM
HEPES, pH 7.2, 2 mM EDTA, 0.25% BSA, 0.1% Nonidet P-40, 5 mM DTT, and 1 mM phenylmethylsulfonyl fluoride
(GST-CBP1, GST-CBP2, and GST-CBP3), or of 50 mM NaCl, 20 mM Tris-HCl, pH 8.0, 0.05% Nonidet P-40, 0.25% BSA, 1 mM DTT, and 1 mM phenylmethylsulfonyl fluoride
(GST-CBP4). Equal amounts of in vitro translated and
[35S]methionine-labeled HNF-4 proteins were added, and
binding was allowed to proceed for 2 h at 4 °C. The beads were
washed three times with the interaction buffer and once with the same
buffer lacking BSA. The bound proteins were eluted with 50 µl of 2×
SDS-PAGE loading buffer and were resolved by electrophoresis. The
proteins were visualized by autoradiography.
Electrophoretic Mobility Shift Assay (EMSA)--
HNF-4 cDNA
was cloned in the bacterial pET15b vector under the control of the T7
promoter and was expressed in E. coli BL21(DE3) strain. The
expression and purification of HNF-4 protein has been previously
described (45). A double-stranded oligonucleotide corresponding to the
B regulatory element of the apoCIII promoter (CIIIB) which binds HNF-4
was used as a probe (15). Bacterially expressed HNF-4 protein was
incubated with the 32P-labeled probe for 15 min at 4 °C
in the presence of 25 mM Hepes, pH 7.6, 40 mM
KCl, 1 mM DTT, 5 mM MgCl2, 0.1%
Nonidet P-40, 30 µg of BSA, and 50 ng of purified HNF-4. Where
indicated, the reactions also included 0.25 µg of either GST-CBP1,
GST-CBP2, or GST-CBP4 proteins that had been eluted from the agarose beads.
Cell Culture and DNA Transfection--
HepG2, HeLa, or CV1 cells
were maintained as stocks in Dulbecco's modified Eagle's medium
supplemented with 10% fetal calf serum. Fifty to 60% confluent 30-mm
dishes were transfected using the calcium-phosphate coprecipitation
method, as described previously (46). The transfection mixture
contained 3 µg of the CAT reporter plasmid, 50 ng of the HNF-4 or
HNF-4 mutant expression plasmids (shown in Fig. 5A), or 100 ng of GAL-HNF-4 fusion plasmid, 1 µg of CMV-
-galactosidase
plasmid, and where appropriate, 1.25 µg of CBP or CBP mutant plasmid.
In every case vector DNA (pcDNAI/Amp) was added as necessary to
achieve a constant amount of transfected DNA (5.75 µg). Forty hours
post-transfection the cells were washed with phosphate-buffered saline
and were collected in TEN solution (0.04 M Tris-HCl, pH
7.4, 1 mM EDTA, pH 8.0, 0.15 M NaCl). Whole cell extracts were prepared in 0.25 M Tris-HCl, pH 7.8, by
three sequential freeze-thaw cycles. CAT activities were determined using [14C]chloramphenicol and acetyl-CoA as described
previously (47). CAT enzyme levels that exhibited more than 60%
conversion of acetylated product were diluted and reassayed for CAT
activity in the linear range. The CAT values were not normalized for
-galactosidase, since CBP appeared to increase the expression of
-galactosidase. The results represent the mean of at least three
independent transfection experiments each carried out in duplicate.
 |
RESULTS |
HNF-4 Interacts with CBP in Vitro--
To investigate whether
there is an interaction between HNF-4 and CBP, we used a
protein-protein interaction assay with glutathione S-transferase (GST)-CBP fusion proteins. Several fragments
of CBP cloned in frame with the GST gene (Fig.
1A) were expressed, purified,
and immobilized on glutathione-agarose beads before incubation with
in vitro translated, [35S]methionine-labeled
HNF-4. As shown in Fig. 1B, HNF-4 associates with CBP
in vitro. Both the N-terminal fragment of CBP (GST-CBP1, lane 3) and the C-terminal fragment of CBP (GST-CBP4,
lane 6) were found to interact with HNF-4. These
interactions were specific as CBP regions 2 and 3 (lanes 4 and 5, respectively) did not associate with HNF-4 nor did
GST alone (lane 2). The interaction of HNF-4 with the C
terminus of CBP appeared to be weaker than that with the N terminus,
since they retained approximately 10 and 20% of the HNF-4 input
(lane 1), respectively.

View larger version (32K):
[in this window]
[in a new window]
|
Fig. 1.
Interaction of HNF-4 with CBP in
vitro. A, schematic representation of CBP
showing the domains (gray) for nuclear hormone receptor
interaction (receptor interacting domains, RID) and CREB
binding (kinase-inducible interaction domain, KIX), the
cysteine/histidine-rich domains 1, 2, and 3 (CH1, CH2, and
CH3), the glutamine-rich domain (Q-rich), the
bromodomain (Br), and the region associated with histone
acetyltransferase (HAT) activity. The asterisks
indicate three LXXLL motifs (nuclear receptor boxes), and
the numbers represent amino acid residues. The bars
below the diagram show the fragments of CBP cloned in frame with
the GST gene to form GST fusion proteins. B, interaction of
HNF-4 with various GST-CBP fragments. The GST-CBP proteins and GST
alone were expressed, purified on glutathione-agarose beads, and
incubated with in vitro translated,
[35S]methionine-labeled HNF-4. The interacting proteins
were eluted in SDS-PAGE sample buffer, resolved by electrophoresis on a
12% gel, and autoradiographed. Lane 1 contains 40% input
of HNF-4 used in each reaction.
|
|
To investigate whether HNF-4 can recruit CBP to the promoter, we
carried out EMSA experiments using recombinant HNF-4 along with the
GST-CBP fusion proteins. The CIIIB element of the apoCIII promoter that
binds HNF-4 with strong affinity (15) was used as a probe. Bacterially
expressed purified HNF-4 binds strongly to CIIIB (Fig.
2, lane 2). Interestingly, the
same N-terminal fragment of CBP-(1-771) that specifically interacts
with HNF-4 forms a ternary complex on the DNA with HNF-4 (Fig. 2,
lane 3). GST-CBP4-(1892-2441) does not appear to bind to
the DNA·HNF-4 complex (Fig. 2, lane 5). This is in
agreement with the observation from the protein-protein interaction
assay that the N terminus of CBP forms a stronger complex with HNF-4
than the C terminus (Fig. 1B, lane 3 versus
lane 6). The specificity of the HNF-4·CBP complex formed
in the EMSA was demonstrated by the inability of GST-CBP2-(706-1069)
to supershift the HNF-4·DNA complex (Fig. 2, lane 4),
confirming the result (Fig. 1, lane 4) that this region of
CBP does not associate with HNF-4.

View larger version (49K):
[in this window]
[in a new window]
|
Fig. 2.
CBP associates with HNF-4 on DNA
elements. The N-terminal region of CBP (GST-CBP1) is recruited to
the DNA by HNF-4. Shown is an EMSA experiment using bacterially
expressed, purified HNF-4 (50 ng) bound to the CIIIB element of the
apoCIII promoter, either alone (lane 2) or in the presence
(+) of GST-CBP fusion proteins (lanes 3-5) that had been
eluted from the glutathione-agarose beads (0.25 µg). The GST-CBP
derivatives used are shown schematically in Fig. 1A.
|
|
CBP Is a Transcriptional Coactivator for HNF-4--
Based on the
interaction between HNF-4 and CBP, we sought next to evaluate the role
of CBP in HNF-4-dependent transcription. Although CBP is
widely expressed, its low levels are rate-limiting (27, 30), which
permitted the use of cotransfection experiments. To test for
transcriptional coactivation by CBP, we utilized the apoCIII promoter
in combination with HNF-4. We showed previously that HNF-4 binds with
high affinity to regulatory elements CIIIB and CIII-I4
present in the proximal promoter and in the enhancer region of the
apoCIII gene, respectively (14, 15), and that it strongly
activates the transcription of the apoCIII promoter (19). To examine
the role of CBP in the HNF-4-dependent transactivation of
the apoCIII gene, we performed transient transfection
experiments in three different cell types (HepG2, HeLa, and CV1) using
the wild type apoCIII promoter along with mammalian expression vectors for HNF-4 and/or CBP (Fig.
3A). We chose HepG2 cells,
because they support the expression of liver-specific genes like
apoCIII, and HeLa and CV1 cells in which the expression of
apoCIII gene is totally dependent on the expression of
cotransfected HNF-4. In HepG2 cells, HNF-4 enhanced apoCIII promoter
activity by 4.5-fold and CBP by 6.5-fold in the absence of transfected
HNF-4 (Fig. 3A). However, when the CBP expression vector was
cotransfected along with HNF-4, apoCIII promoter activity levels were
increased from 4.5- to 6.5-25-fold. The increase in apoCIII promoter
activity by CBP alone observed in HepG2 cells is probably attributable to the interaction of transfected CBP with endogenous HNF-4.

View larger version (23K):
[in this window]
[in a new window]
|
Fig. 3.
CBP is a transcriptional coactivator for
HNF-4. A, transient transfection experiments were
performed in HepG2, HeLa, and CV1 cells with the apoCIII promoter
construct (shown at the top of the panel)
and plasmids HNF-4 and CBP expressing their corresponding
cDNAs. Typical CAT assays for each cell type are shown.
The CAT activity achieved with the reporter alone was set to 1, and other activities are presented relative to this value.
B, transient transfection experiments in HeLa and HepG2
cells were performed with the (BA1)5CAT reporter plasmid
(shown in the schematic diagram) and plasmids HNF-4 and CBP expressing
their corresponding cDNAs. The bar graphs are mean
values of CAT activity of at least three independent transfections,
each carried out in duplicate, and show the relative transcription of
the reporter in the absence ( ) and presence (+) of expression
plasmids CBP and HNF-4. C, transient transfection
experiments in HepG2 cells were performed with the pG5CAT
reporter (shown schematically at the top of the
panel) and expression plasmids for CBP and/or a fusion of
GAL4 DNA binding domain (residues 1-147) with HNF-4 (GAL-HNF-4). The
bar graphs are mean values of CAT activity of at least three
independent transfections, each carried out in duplicate, and show the
relative transcription of the reporter in the absence ( ) and presence
(+) of expression plasmids CBP and HNF-4.
|
|
Therefore, CBP functions as a coactivator for HNF-4-induced
apoCIII gene transcription. This enhancement of HNF-4
transactivation by CBP is more profound in HeLa and CV1 cells where the
expression of CBP alone had no effect on the levels of activation of
the reporter in the absence of cotransfected HNF-4 (Fig.
3A). Remarkably, a significant stimulation of the reporter
above the levels observed with HNF-4 alone was induced by coexpression
of full-length CBP. Specifically, cotransfection of CBP and HNF-4
increased apoCIII promoter activity by 4-fold (HeLa cells) and 2-fold
(CV1 cells) over the activation by HNF-4 alone (Fig. 3A),
implying that the synergism occurs through HNF-4.
The apoCIII promoter is complex, containing binding sites for various
transcriptional activators. To discern whether the synergism between
HNF-4 and CBP observed is entirely due to HNF-4, two simpler promoter
plasmids were used in transfection experiments, one containing five
HNF-4-binding sites [(BA1)5CAT] and the other with a
heterologous promoter [pG5CAT] (19). As seen in Fig.
3B, the transcriptional activity of (BA1)5CAT
was increased approximately 2-fold by overexpression of CBP and HNF-4
as compared with HNF-4 alone, both in HeLa and HepG2 cells. In
agreement with the results presented in Fig. 3A, this effect
appears to be due to the interplay between CBP and HNF-4 rather than
merely the presence of CBP, since the expression of CBP alone does not
increase basal activity of the reporter. The 2-fold increase observed
in HepG2 cells is probably due to the interaction of CBP with
endogenous HNF-4. Similarly, cotransfection with GAL-HNF-4 and CBP
resulted in a 3-fold enhancement in the transcriptional activity of the
reporter pG5CAT over its activity in the presence of
GAL-HNF-4 alone (Fig. 3C).
These results clearly indicate that CBP-mediated transcriptional
enhancement is dependent on the presence of HNF-4, since in the absence
of HNF-4 (HeLa and CV1 cells), CBP has no effect on basal transcription.
CBP Recruitment to the ApoCIII Promoter Is Mediated by HNF-4
Binding Either to the Enhancer or the Proximal Promoter Site--
To
examine whether recruitment of CBP to the apoCIII promoter requires
synergism between the two HNF-4 sites, we carried out cotransfection
experiments in HepG2 cells. HNF-4 and CBP responsiveness was studied by
analyzing two promoter constructs with mutations in the proximal
HNF-4-binding site, apoCIII(
890/+24)BM5CAT, or the distal
HNF-4-binding site, apoCIII(
890/+24)I4MCAT. We have previously shown that mutations in the proximal and enhancer
HNF-4-binding sites severely compromise the apoCIII promoter strength
to 36 and 20%, respectively (43, 48). As seen in Fig.
4, transfection by HNF-4 or CBP alone
results in a modest 2-4-fold transactivation of the mutant apoCIII
promoter templates. However, the simultaneous expression of CBP and
HNF-4 led to a 12-20-fold stimulation of transcription, corresponding
to a 2-4-fold synergism (Fig. 4). Therefore, we conclude that CBP can
act in synergy with HNF-4 at either of these elements.

View larger version (49K):
[in this window]
[in a new window]
|
Fig. 4.
CBP recruitment to the apoCIII promoter is
mediated by HNF-4 binding either to the enhancer or the proximal
promoter site. Transient transfection experiments were performed
in HepG2 cells with mutant apoCIII promoter templates (shown at the
top of the panel) and plasmids HNF-4 and CBP
expressing their corresponding cDNAs. Typical CAT assays for each
promoter mutant are shown. The CAT activity achieved with the reporter
alone was set to 1, and other activities are presented relative to this
value and are the means of at least three independent experiments each
carried out in duplicate.
|
|
CBP Targets Both the AF-1 and the AF-2 Domains of HNF-4--
To
identify the regions of HNF-4 involved in protein-protein interactions
with CBP, we carried out in vitro binding experiments using
the N- (CBP1) and C (CBP4)-terminal regions of CBP, immobilized on
glutathione-agarose beads and several in vitro translated
and [35S]methionine-labeled HNF-4 deletion mutants. As
seen in Fig. 5A, both the N-
and C-terminal fragments of CBP form complexes with mutants
HNF-4-(1-370) and HNF-4-(1-360), lacking the F domain of HNF-4 or the
core AF-2 AD motif, respectively. In addition, removal of the AF-1
domain in mutants HNF-4-(48-455) or HNF-4-(48-370) does not affect
the interaction of CBP and HNF-4. Interestingly, deletion of the entire
AF-2 domain in mutants HNF-4-(1-174) or HNF-4-(1-128)does not abolish
the interaction with HNF-4, thus unmasking a second region in HNF-4
that interacts with CBP. This region encompasses the AF-1 domain of
HNF-4, which appears to interact with CBP weakly, since the amount of
AF-1 retained by the GST-CBP1 decreases from approximately 20 to 10%
of the input. Furthermore, the association between AF-1 and GST-CBP4 is
abolished when the AF-2 domain is deleted. Therefore the AF-2 domain,
in particular the E region between 174 and 360, is essential for interaction between HNF-4 and the C terminus of CBP. Surprisingly, interaction with both the N- and C-terminal regions of CBP is completely lost with mutants HNF-4-(128-370) and HNF-4-(174-370) encompassing the entire AF-2 domain or lacking the D domain of HNF-4,
respectively (Fig. 5A). Since removal of the AF-1 does not
affect the CBP interactions, and the AF-2 alone does not form a complex
with either GST-CBP1 or GST-CBP4, it seemed possible that the DNA
binding domain (DBD) of HNF-4 might be involved in these interactions.
To examine the role of HNF-4 DBD in interactions with CBP, we prepared
mutant HNF-4-(45-142), which encompasses the DBD of HNF-4 alone, and
two other mutants containing regions C, D, and E of HNF-4, but with
mutations in one or the other of the two zinc fingers, in an attempt to
disrupt the structure of that region. This involved a double mutation
changing C68G/C71G and or C103G/C106G. As seen in Fig. 5B,
there is no interaction between the HNF-4 DBD and GST-CBP1.
Furthermore, mutation of either zinc finger does not affect the
interaction of GST-CBP1 and GST-CBP4 with HNF-4. Assuming that the
mutations to the zinc fingers do disrupt the DNA binding domain, it
would seem that this region does not mediate the interaction of HNF-4
with CBP per se.

View larger version (56K):
[in this window]
[in a new window]
|
Fig. 5.
CBP targets both the AF-1 and the AF-2
domains of HNF-4. In vitro translated and
[35S]methionine-labeled HNF-4 mutant proteins were
incubated with GST or GST-CBP1 or GST-CBP4 immobilized on the
glutathione beads. Labeled HNF-4 proteins retained on the beads after
extensive washing were analyzed by SDS-PAGE and autoradiography,
together with 40% of the translated products used in each incubation
(INPUT). At the top of the figure is a schematic
representation of the functional domains of HNF-4. The
numbers represent amino acid residues. The
letters indicate the domains of HNF-4, and functional
domains are marked above. The mutant HNF-4 proteins used in
the protein-protein interaction assay are shown schematically on the
left of the figure.
|
|
This analysis identified two domains of HNF-4 that interact with CBP.
The AF-1 domain interacts only weakly with the N-terminal region of
CBP, and the AF-2 domain interacts with both the N- and C-terminal
regions of CBP, and this interaction is both AF-2 AD and
ligand-independent.
HNF-4 Mutated in the AF-2 Domain Does Not Allow CBP to Activate
Transcription--
To examine whether there is a correlation between
the interacting domains of HNF-4 that associate with CBP and the
ability of CBP to enhance transcription, we carried out cotransfection experiments using the (BA1)5CAT reporter construct. The
deletion mutants of HNF-4 that were used are shown in Fig.
5A, with the exception of HNF-4-(
128-175), containing an
internal deletion from amino acids 128 to 175. Wild type HNF-4 and its
deletion mutants were shown to express in comparable amounts with
insufficient variations to account for the observed differences in
their transcriptional activation potential (19). Fig.
6 shows the transcriptional activity of
the (BA1)5CAT reporter transfected into HepG2 and HeLa
cells with expression vectors for the HNF-4 deletion mutants in the
presence and absence of CBP.

View larger version (23K):
[in this window]
[in a new window]
|
Fig. 6.
The effect of mutation of HNF-4 on
synergistic transcriptional activation by CBP. Transient
transfection experiments were performed in HeLa and HepG2 cells using
the (BA1)5CAT reporter plasmid and effector plasmids
expressing HNF-4 and the indicated deletion mutants. Transfections were
carried out with (black bars) or without (gray
bars) an expression vector for CBP. The bar graphs are
mean values of CAT activity of at least three independent
transfections, each carried out in duplicate and show the relative
transcription of the reporter in the absence ( ) and presence (+) of
expression plasmids CBP and HNF-4.
|
|
HNF-4 with deletion of the F domain (HNF-4-(1-370)) activated the
promoter in both HepG2 and HeLa cells 12- and 70-fold, respectively (Fig. 6). However, addition of CBP increased the (BA1)5
promoter activity to approximately 24- and 125-fold, respectively,
indicating that the absence of the F domain does not interfere with the
functional interaction between HNF-4 and CBP and that CBP enhances
HNF-4 transactivation by 2-fold. Mutant HNF-4-(48-455) lacking the
AF-1 domain activated the (BA1)5 promoter by 2-fold in
HepG2 and by 18-fold in HeLa cells. In each of these cases, in both
cells types, the presence of CBP increased HNF-4 transactivation by
2-fold. As shown previously (19), the dominant negative HNF-4 mutants, HNF-4-(1-360) and HNF-4-(
128-175), abolished transcriptional activation of the (BA1)5 promoter. These mutants also
showed very little or no increase in transactivation potential in the
presence of CBP (Fig. 6). These results indicate that CBP does not
activate gene expression in the absence of HNF-4, and HNF-4 mutated in the AF-2 domain does not allow CBP to activate expression, suggesting that the mere recruitment of CBP is not sufficient for enhancement of
gene expression.
Effect of N- and C-terminal Mutants of CBP on the Transcriptional
Potential of HNF-4--
The two regions of CBP at the N and C termini
that interact with HNF-4 in vitro were tested in
cotransfection experiments to determine whether these regions alone can
coactivate HNF-4. HeLa cells were transfected with the reporter
(BA1)5CAT and expression vectors coding for HNF-4, CBP, or
one of the CBP mutants depicted on the left of Fig.
7. The results indicate that only the
mutants that contain the C-terminal region of CBP can coactivate HNF-4 (Fig. 7). Specifically, the C-terminal mutants CBP-(468-2441) and
CBP-(1892-2441) were able to coactivate at a level equal to that
induced by the wild type CBP. Remarkably, the N-terminal mutant
CBP-(1-771) was not able to coactivate, although this region of CBP
interacts strongly with HNF-4 in in vitro assays. Finally, the mutant CBP-(1-771 + 1892-2441) that contains both of the HNF-4 interacting domains is able to coactivate at a level similar to that
elicited by CBP, indicating that coactivation may be mediated through
the C domain (Fig. 7).

View larger version (20K):
[in this window]
[in a new window]
|
Fig. 7.
Effect of N- and C-terminal mutants of CBP on
the transcriptional potential of HNF-4. HeLa cells were
transiently transfected with the (BA1)5CAT reporter
construct and expression vectors for HNF-4 and CBP. The bar
graphs show the relative transcription of the
(BA1)5CAT reporter transfected with HNF-4 in the absence
( ) and presence (+) of CBP. The values depicted are mean values of
CAT activity of at least three independent transfections, each carried
out in duplicate. The relative CAT activity of the reporter in the
presence of HNF-4 alone is significantly different from that with HNF-4 + CBP-(468-2441), HNF-4 + CBP-(1892-2441), and HNF-4 + CBP-(1-771 + 1892-2441) ( = 0.012, 0.023, and 0.032, respectively, as determined
by a Student's t test). Also the relative CAT activity with
HNF-4 alone is not significantly different from that with HNF-4 + CBP-(1-771) ( >0.1).
|
|
 |
DISCUSSION |
This work describes the functional association of HNF-4 with CBP
and the recruitment of CBP to the HNF-4·DNA complex. The ability of
HNF-4 to recruit CBP appears to be essential for enhancement of its
function and is consistent with previous studies demonstrating the
importance of CBP in nuclear receptor function (27).
We observed a physical association of HNF-4 with CBP in protein-protein
binding assays (Fig. 1), and we have also shown that CBP can complex
with HNF-4 whereas the HNF-4 is bound to DNA (Fig. 2), implying a role
for CBP in HNF-4-mediated transcriptional activation. HNF-4 binds to
the N-terminal and also the C-terminal regions of CBP. Both CBP and
SRC-1 family members contain the nuclear receptor (NR) box, a short
leucine-rich motif (LXXLL, where L is leucine and
X is any amino acid) that appears to mediate the
interactions between these proteins and the NHRs. It has been suggested
that the amino acids within this motif may determine the particular
interactions between nuclear hormone receptors and cointegrators (37,
49). The three leucine-rich motifs of CBP are located at amino acids
65, 356, and 2964 (shown as asterisks in Fig.
1A), and interestingly they are contained within the two
GST-CBP fusion proteins that were identified in our study as
interacting with HNF-4. The CBP N terminus between amino acids 1 and
771 encompasses the two receptor interacting domains that were
previously described to interact in a ligand-dependent
manner with retinoic acid receptor-
and other nuclear receptors
(27). The C terminus between amino acids 1892 and 2441 includes the glutamine-rich region and the NR box shown previously to interact with
the transcriptional coactivators SRC-1 and p/CIP (35, 37, 39).
Transient transfection analyses show transcriptional enhancement of
HNF-4 by CBP using the apoCIII promoter and HNF-4 homopolymeric templates (Fig. 3). In these systems, CBP functions cooperatively with
HNF-4 to enhance transcription. CBP does not activate gene expression
in the absence of HNF-4, implying that HNF-4 functions as a high
affinity site for the entry of CBP to the transcription initiation
complex. Previously CBP has been shown to interact with a number of
nuclear hormone receptors in a ligand-dependent and
dose-dependent manner (26, 27, 35). In our studies, CBP
enhanced HNF-4 transactivation by 2-5- fold, which is in good agreement with the previous studies. The natural apoCIII promoter was approximately twice as responsive to CBP as the heterologous promoter pG5CAT and the homopolymeric promoter
(BA1)5CAT (Fig. 3). This may be due to the presence of two
HNF-4-binding sites in the apoCIII promoter that are known to act
synergistically together and with SP1 and other transcription factors
to activate the transcription of the apoCIII gene (14, 19,
50).
It was previously shown that CBP synergistically activates the human
interferon-
promoter and that transcriptional synergy requires
recruitment of CBP via a novel surface assembled from the activation
domains of all the activators of the enhanceosome (44). Our studies
suggest that although CBP can act in synergy with HNF-4 (Fig. 4),
recruitment of CBP to the apoCIII promoter is mediated by either of the
HNF-4-binding sites and so does not appear to require such an assembly.
We have also shown that CBP targets both the AF-1 and the AF-2 domains
of HNF-4. The N terminus of CBP-(1-771) interacts strongly with the
A/B/C and C/D/E regions of HNF-4, but not with the C (DBD) region alone
(Fig. 5). This suggests that the N terminus of CBP can contact both the
AF-1 and AF-2 domains of HNF-4 and can bind, with varying strength, to
each in the absence of the other. This fits with previous data showing
that, when tethered to a promoter via a GAL4 DBD, both the AF-1 (amino
acids 1-24) and the AF-2 (amino acids 128-370) domains of HNF-4 are
capable of activating transcription to similar extent (19). If contact with a coactivator complex is the major route by which transcription is
activated, then it is necessary for both of these regions to be able to
interact with coactivators independently. CBP has also been observed to
enhance the ligand-dependent interaction between the N- and
C-terminal activation domains of the androgen receptor (AR) (51), and
the coactivator SRC-1 also acts to enhance cooperativity between
the AF-1 and AF-2 functions of the progesterone receptor (52).
The AF-2 domain of HNF-4 is very complex spanning residues 128-366
(19). It contains a short
-helical motif at residues 360-366 termed
AF-2 AD, which is conserved between nuclear hormone receptors and is
required for ligand-dependent transactivation (20-22). The
association of CBP with HNF-4 AF-2 occurs outside the AF-2 AD region
(Fig. 5A), and none of the studies presented here included a
ligand. This is in agreement with yeast two-hybrid system where CBP
interacted with HNF-4 in the absence of ligand (53). We conclude that
the interaction of HNF-4 AF-2 and CBP is AF-2 AD and
ligand-independent. Deletion or mutation of this region in HNF-4 causes
complete loss of transcriptional activity (19, 54), and addition of CBP
fails to activate transcription (Fig. 6). The mere recruitment of CBP
by HNF-4, therefore, is not sufficient for transcriptional enhancement
of gene expression.
In vitro, the C-terminal region of CBP associates only with
the C/D/E region of HNF-4 and does not interact with the A/B/C region
(Fig. 5A). Hence, the interaction domain appears to be located within the AF-2 region of HNF-4. However, HNF-4-(128-370) and
HNF-4-(174-370) were not capable of interacting with the C-terminal region of CBP, implying that the presence of HNF-4 DBD is necessary for
this interaction to occur. Mutagenesis studies have localized dimerization domains in both the DBD and the LBD of NHRs (55-57). The
dimerization domain in the HNF-4 DBD is required for high affinity,
cooperative, specific binding to an HNF-4 response element (58). The
LBD dimerization domain in HNF-4 appears to be important for
dimerization of the receptor in solution (59-61). We propose that the
interaction of the C terminus of CBP with HNF-4 occurs at a surface
formed when HNF-4 dimerizes. The HNF-4-(128-370) and HNF-4-(174-370)
mutants that cannot interact with the C terminus of CBP exclude part,
or all, of the DBD dimerization domain and therefore may not be able to
form dimers in solution. The mutation of the zinc fingers of the HNF-4
DBD does not affect the interaction with CBP, suggesting that the
region that is important for HNF-4/CBP association lies outside this
area. It is interesting to note that studies on the interaction of CBP
with other nuclear receptors have either used a heterologous
dimerization domain or constructs that included the DBD and/or hinge
regions and, therefore, the DBD dimerization domain of the receptors in
addition to the LBD (27, 51). Moreover, the association of the
corepressors SMRT and NCoR with certain receptors is dependent on
dimerization of the receptor (62).
In transient transfection analysis deletion of the 468 amino acids from
the N terminus did not affect the ability of CBP to act synergistically
with HNF-4 (Fig. 7, CBP-(468-2441)). In addition, although a minimal
transactivation domain has been defined between aa 344 and 451 of CBP
(63), cotransfection of CBP-(1-771) failed to enhance transcriptional
activation by HNF-4. Therefore, we concluded that the interaction of
HNF-4 with the N terminus of CBP is not sufficient to direct
transcriptional activation. Interestingly, in the presence of HNF-4 CBP
mutants containing only aa 1892-2441 or a deletion of aa 772-1891 are
both capable of activating transcription to levels approximating those
of full- length CBP. The HAT activity of CBP is associated with aa
1099-1758 (32, 33) and is not contained within either of these
deletion mutants. However, SRC-1 has been shown to interact with CBP in
the region between aa 2058 and 2163 (35, 39). We propose that although
HNF-4 associates with CBP, it can also associate with other
coactivator(s) and can utilize the HAT activity from this putative
coactivator. This is supported by our finding that recruitment of CBP
by HNF-4 is not sufficient to activate transcription. In addition,
since the AF-2 AD helix is not involved in interactions with CBP, it
would be available to contact other coactivators. In this scenario, CBP
may be functioning as a platform for the assembly of a coactivator complex.