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INTRODUCTION |
Nuclear receptors regulate target gene expression by binding to
their cognate DNA response elements in the control region of target
genes and recruiting associate proteins to the transcription machinery
(1-3). The associate proteins of nuclear receptors can be
coactivators, corepressors, or coregulators. A number of coactivators,
mainly the p160 family, have been identified, including SRC-1/NCoA-1,
TIF2/GRIP1/NCoA-2, and p/CIP/RAC3/ACTR/AIB1 (4-12), several of which
have been shown to encode intrinsic histone acetyltransferase activities (4, 5, 12). On the contrary, corepressors are found to
interact with histone deacetylases. For example, corepressors N-CoR/SMRT recruit histone deacetylases
(HDACs)1 to remove specific
acetyl groups from histone proteins of specific gene-regulatory
regions. As a result, chromatin is packed, and gene activity is
repressed (13, 14). Upon ligand binding to receptors, the AF-2 domain
(helix 12) is repositioned, corepressors are released, and coactivators
are recruited to activate target gene expression. One mechanism of gene
activation is believed to be mediated by relaxation of chromatin due to
the action of acetyltransferase encoded by the coactivator complexes.
It was first suggested that the molecular basis underlying nuclear
receptor interaction with coactivators involved a signature motif
LXXLL (L is a leucine, and X is any amino acid)
present in many coactivators (7, 15-17). By studying the x-ray
crystal structure of a ternary complex formed by peroxisome
proliferator-activated receptor-
, the ligand, and an 88-amino
acid peptide of coactivator SRC-1, it was found that a charge clamp was
formed on the ligand-binding domain of peroxisome
proliferator-activated receptor that made a direct contact with
the backbone atoms of the LXXLL helices of SRC-1 (19-21).
Later, it was shown that aporeceptor interaction with corepressor
involved a CoRNR box (L/IXXI/VI) (L is a leucine, I is an
isoleucine, V is a valine, and X is any amino acid) found in
corepressors such as N-CoR and SMRT. However, in competition experiments, CoRNR peptides were able to block both corepressor and
coactivator interaction with nuclear receptors (22), suggesting that a
similar and probably overlapping receptor-interaction motif is present
in coactivators and corepressors. Furthermore, by studying mutant
receptors and corepressors, it was found that mutations in nuclear
receptor residues that directly participated in coactivator binding
disrupted their interaction with corepressors (23, 24). It was then
suggested that a consensus LXXI/HIXXXI/L sequence of corepressors is an extended helix compared with the LXXLL
helix found in coactivators, and both helices were able to interact with nuclear receptors in the same receptor pocket (24).
The human receptor-interacting protein 140 (RIP140) was first
identified as a coactivator for a chimeric estrogen receptor (25).
However, the mouse RIP140 was characterized in this laboratory as a
potent corepressor for orphan nuclear receptor TR2 in the absence of
putative ligands (26). Later, we demonstrated a strong ligand-dependent interaction of RIP140 with retinoic acid
receptor (RAR) and retinoid X receptor (RXR), mediated by a C-terminal segment of RIP140 (27). Two unique features of RIP140 are:
(a) in contrast to classical coactivators that interact with
ligand-bound hormone receptors to activate target gene expression,
RIP140 suppressed gene activation by interacting with ligand-bound
nuclear receptors in most reported studies (28-31), and (b)
the ligand-dependent receptor-interacting motif of RIP140
does not involve any of its nine copies of the LXXLL motif,
rather it utilizes its C-terminal domain for holo-RAR/RXR interaction
and N-terminal amino acids (AA) 154-350 for Ah receptor interaction
(26, 32).
This first aim of this study was to dissect the LXXLL-less
motif of RIP140 that interacts with ligand-bound RAR/RXR. This motif
was determined by using different molecular approaches, including
two-hybrid interaction, coimmunoprecipitation, and GST pull-down
assays. The functional role of this motif in the suppressive effects of
RIP140 on RA induction of target gene expression was demonstrated in
mutation studies. Finally, association of HDAC3, RIP140, and RAR/RXR
was demonstrated to be ligand-dependent in coimmunoprecipitation experiments, and histone acetylation decreased on
the promoter region carrying a RA response element in the presence of
RIP140. These data provided a molecular explanation for the action of
RIP140 as a novel ligand-dependent negative modulator of
RA-regulated gene expression.
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EXPERIMENTAL PROCEDURES |
Construction of Expression Vectors for Mammalian Two-hybrid
Interaction Tests--
RIP140 and its deletions were each fused to the
Gal4 DNA-binding domain of pM vector (CLONTECH) to
dissect the interacting domain by either restriction digestion or
polymerase chain reaction (PCR) of RIP140 cDNA. Full-length RIP
(RIP-F), N-terminal domain AA 1-496 (RIP-N), central portion AA
496-1006 (RIP-cent), and C-terminal domain AA 977-1118 (RIP-C) were
described previously (26). R-18 was made by HindIII
digestion of RIP-C, R-19 was made by SmaI digestion of R-18,
and R-20 to R-32 were made by PCR cloning. The ligand-binding domain of
RAR and RXR was fused to the pVP16 vector
(CLONTECH) for the two-hybrid test, and the reporter (Gal4-luc) and techniques for culturing COS-1 cells, transfection, and luciferase and lacZ assays were as described previously (26). Cultures were maintained in Dulbecco's modified Eagle's medium containing dextran charcoal-treated serum.
all-trans-RA (at-RA) and 9-cis-RA were each added
at a final concentration of 5 × 10
7
M. Each experiment was carried out in triplicate. At least
three independent experiments were conducted to obtain the mean and the
S.E.
Coimmunoprecipitation Tests--
COS-1 cells were cotransfected
with RIP140 (wild type mutant L-384 as described later or an empty
vector), RAR
, RXR
, and FLAG-tagged HDAC3 (33). Cells were treated
with vehicle or at-RA (1 µM) 24 h after transfection
and harvested 24 h later for resuspension in lysis buffer (50 mM Tris-Cl, pH 8.0, 150 mM NaCl, 10% glycerol, 0.5% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, and
a protease inhibitor mixture). Cells were sonicated twice in 20-s
pulses on ice, and lysates were clarified by centrifugation at
10,000 × g for 10 min. For immunoprecipiation,
150-200 µl of total lysate was incubated with anti-FLAG antibody
(Sigma) at 4 °C for 3 h, followed by the addition of 20 µl of
protein G-agarose resin (Sigma), continued incubation at 4 °C for
1 h, and washing with 0.1% Nonidet P-40 in phosphate-buffered
saline. Beads were resuspended in loading buffer for separation on 10%
SDS-polyacrylamide gel electrophoresis. Proteins were transferred to
polyvinylidene difluoride membranes and incubated with anti-RIP140
(Santa Cruz Biotechnology, Santa Cruz, CA) or anti-RAR
or
anti-RXR
(Affinity Bioreagents, Golden, CO). After washing, blots
were incubated with a secondary antibody against the species from which
the primary antibody was derived, followed by extensive washing and
detection with ECL (Amersham Pharmacia Biotech).
In Vitro Protein Interaction Test--
GST pull-down assay was
conducted as described previously (24). Various RIP140 segments as
shown in Fig. 2A were cloned by transferring each
corresponding fragment of RIP140 dissected from the pM fusions to a GST
vector (26). R-33, R-34, R-35, R-36, and R-37 were derived from R-18,
R-20, R-21, R-28, and R-31, respectively. Full-length RAR and RXR were
expressed from T7 promoter and labeled with
[35S]methionine where indicated using a TNT kit
(Promega, Madison, WI). Escherichia coli BL21 transformed
with the GST-fusion vectors was induced with 0.1 mM
isopropyl-1-thio-
-D-galactopyranoside for 4 h, and
fusion proteins were purified from glutathione-Sepharose columns. The
partially purified GST-RIP fusion protein was incubated with
[35S]methionine-labeled RAR or RXR in the presence of
unlabeled receptor partner. RA was added at a concentration of
10
6 M. Peptide LTKTNPILYYMLQK
(RIP140 amino acid 1063-1076) of either L- or
D-amino acids was synthesized and purified by the
microchemical facility of the University of Minnesota.
Determination of Biological Activities of RIP140 and Its
Mutants on RA Induction of Target Genes--
The expression vectors
for RAR, RXR, and full-length RIP140 were as described previously (26).
Mutant RIP140 (L-384) with only the receptor-interacting motif (AA
1063-1076) deleted was made by ligating the AA 1077-1118
fragment to AA 1-1063 of RIP140. Reporter carrying a direct repeat 5 (DR5)-tk-luc and tests of RA induction in the COS-1 system were as
described previously (26).
Chromatin Immunoprecipitation (ChIP) Assay--
COS-1 cells were
transfected with the DR5-tk reporter, RAR, and RXR expression vectors,
and either a CMV-driven wild type RIP140 expression vector, the L-384
mutant, or an empty vector. The ChIP assay (33) was performed according
to the manufacturer's recommendations (Upstate Biotechnology, Lake
Placid, NY). After transfection for 24 h, cellular histone was
cross-linked to DNA by adding formaldehyde to a final concentration of
1%. Precipitated chromatin was incubated with an anti-acetylated
histone 3 antibody (Upstate Biotechnology) overnight at 4 °C,
treated with proteinase K, and purified by phenol extraction. For PCR
detection of precipitated chromatin DNA, primers for the tk promoter
region (130 base pairs) following the DR5 site were
5'-AGCGTCTTGTCATTGGCG-3' and 5'-TTAAGCGGGTCGCTGCAG-3'. Control PCRs to
amplify the CMV promoter were conducted by using primers
5'-CTGACCGCCCAACGAC-3' and 5'-GACTAATACGTAGATG-3', which allowed the
CMV promoter region to be amplified in the size of 255 base pairs.
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RESULTS |
In Vivo Ligand-dependent RAR/RXR Interaction of RIP140
Detected by Two-hybrid Interaction Tests--
Previously, we have
confirmed that RIP140 interaction with RAR and RXR depends upon the
presence of ligands and utilizes a small C-terminal segment of RIP140
that lacks a typical LXXLL motif (27). To determine the
molecular basis of this ligand-dependent RIP140 interaction
with RAR and RXR, we first utilized mammalian two-hybrid interaction
tests. As shown in Fig. 1, pM-RIP140
interaction with pVP-RAR or pVP-RXR + RAR is mediated by the C-terminal
segment between AA 977 and AA 1161, and this interaction is dependent upon the presence of ligand (at-RA for pM-RAR and at-RA + 9-cis-RA for pM-RXR + RAR (Fig. 1B, columns
1-3)). The C-terminal segment was further deleted from AA 1118 to
AA 1161, resulting in R-18, which maintained a very similar pattern of
interaction (Fig. 1B, column 4). R-18 was further
deleted to retain only AA 977-1006 (R-19), 977-1033 (R-21), and
977-1076 (R-20). Among these deletions, only R-20 was able to interact
with RAR/RXR (Fig. 1B, column 6), indicating that the
interacting motif was located between AA 1033 and AA 1076. To confirm
this result, 5' deletions were made from R-18 to generate R-22 and
R-23, which retained AA 1023-1118 and AA 1084-1118, respectively. As
predicted, R-22 (Fig. 1B, column 8) but not R-23 was able to
interact with RAR/RXR, suggesting that the interacting motif resides in
the central portion of R-18. This was supported by the positive result
for construct R-24, which contained only AA 1023-1076 (Fig. 1B,
column 10). Further deletions from the 3'-end to generate R-25 (AA
1023-1063) and R-27 (AA 977-1063) abolished interaction. However, 5'
deletions of R-24 to generate R-26 (AA 1047-1076) and R-28 (AA
1063-1076) did not affect the interaction (Fig. 1B, columns
12 and 14). R-28 was the smallest clone that remained
fully functional to interact with RAR/RXR. This was confirmed by the
positive results of two constructs containing this region (R-29 and
R-30) and the negative results of further deletions (R-31 (AA
1069-1076; Fig. 1B, column 17) and R-32 (AA 1069-1076)).
The pattern of interaction with pM-RXR + RAR in the presence of
9-cis-RA alone was very similar to that of at-RA + 9-cis-RA (data not shown).

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Fig. 1.
Mammalian two-hybrid interaction tests to
dissect the receptor-interacting motif of RIP140. A,
RIP140 constructs generated in the pM vector. Numbers denote
AA positions of RIP140. Restriction enzymes used are: R,
EcoRI; S, SmaI; and H,
HindIII. Results of the interactions tests are shown in the
right column. B, data of two-hybrid interaction
tests from three independent experiments.
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Based on these results, it is concluded that RIP140 interacts with RAR
and RXR in a ligand-dependent manner, and the interaction with liganded RAR and RXR is mediated by a small C-terminal peptide sequence (LTKTNPILYYMLQK) from AA 1063 to AA 1076.
In Vitro Ligand-dependent Interaction of RIP140
Detected by GST Pull-down Assays--
To confirm the
ligand-dependent RAR/RXR-interacting motif of RIP140,
in vitro interaction tests based on GST pull-down assays were performed. In these tests, RIP140 portions were expressed as GST
fusions, and RAR/RXR was expressed in TNT, with either one labeled with
[35S]methionine. Fig.
2A shows the maps of the
representative clones, Fig. 2B shows the GST pull-down
experiments that utilized labeled RAR, and Fig. 2C shows the
Coomassie Blue-stained gel separating partially purified RIP fragments
(labeled with asterisks on the right). As shown
in Fig. 2B, all the clones that contain AA 1063-1076, i.e. R-33, R-34, and R-36, interacted with RAR/RXR in the
presence of at-RA, whereas clones in which this motif was
deleted, i.e. R-35 and R-37, failed the test. This
result further supports the notion that RIP140 interacts with
holo-RAR/RXR through C-terminal AA sequence 1063-1076 and that the
interaction requires ligand binding to one molecule of the receptor
dimer.

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Fig. 2.
GST pull-down assay to confirm RIP140
interaction with RAR and RXR in vitro.
A, RIP140 constructs generated as GST-fusion proteins.
Results from the pull-down assays are shown in the right
column. B, GST pull-down assay performed by using
labeled RAR and unlabeled RXR. C, a protein gel
demonstrating the presence of GST-fusion protein in each construct, as
indicated by the asterisks.
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To confirm the specificity of this peptide sequence in mediating RIP140
interaction with RAR, peptide competition was conducted in GST
pull-down assays using the smallest RIP140 clone (R-36), as shown in
Fig. 3A. Peptide was added at
a concentration range of 2-20 µM, and either RAR
(top) or RXR (bottom) was labeled in the receptor
input. The specific bands pulled-down by R-36 for either RAR-labeled or
RXR-labeled receptor dimer were effectively competed out by the
addition of 2 µM peptide. At a concentration of 20 µM, this peptide was able to compete with R-36 for more than 90%. To further substantiate the specificity in the
competition experiment, a peptide with the same sequence of
D-amino acids was tested in parallel, as shown in Fig.
3B. The L-peptide successfully competed in this
experiment (Fig. 3B, lane 3), whereas the
D-peptide failed to compete even at a concentration as high
as 100 µM (Fig. 3B, lane 4).
Therefore, it is concluded that the C-terminal AA 1063-1076 sequence
of RIP140 is a ligand-dependent, specific
RAR/RXR-interacting motif.

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Fig. 3.
Peptide competition experiment to demonstrate
specific interaction of the dissected receptor-interacting motif
carried on the smallest clone, R-36, in GST pull-down assays.
A, a dose-dependent competition of GST pull-down
assay of R-36 interaction with RAR (top) and RXR
(bottom) by the L-peptide. B,
competition with L-peptide (lane 3) but not with
D-peptide (lane 4). GST pull-down assay of R-36
interaction with RAR (top) and RXR (bottom) was
conducted using 100 µM peptide in the reactions.
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Functional Role of the Receptor-interacting Motif of RIP140 in
Suppression of RA Induction--
The biological activity of RIP140 in
hormone receptor actions has been controversial. In the RAR/RXR system,
we have consistently observed a ligand-dependent
interaction of RIP140 with RAR and RXR heterodimers, which resulted in
strongly suppressed RA induction of reporter activities (27). To
determine whether the interaction of RAR/RXR is required for the
biological activity of RIP140, represented as suppression of RA-induced
reporter activity, a mutant RIP140 (L-384) was constructed in
which region AA 1063-1076 was specifically deleted. Transfection
experiments were conducted to determine RA induction of reporter
activities in the presence of RIP140 or this mutant, as shown in Fig.
4. Consistent with our previous
observations, RA induced reporter activities >50-fold (Fig. 4,
columns 1 and 2). In the presence of wild type
RIP140, the basal level reporter activity remained the same in the
absence of RA (column 3), but RA-induced reporter activity
decreased >20-fold (column 4). In contrast, the
mutant, L-384, failed to effectively suppress RA induction
(columns 5 and 6). This result confirmed that the
suppressive effect of RIP140 on RA reporters was mediated by its direct
interaction with RAR/RXR through the C-terminal AA 1063-1076 sequence
in a ligand-dependent manner.

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Fig. 4.
Demonstration of biological activity of
RIP140 and its mutants. RA induction of DR5-tk-luc was examined in
the presence of a control expression vector (Cont,
lanes 1 and 2), a wild type RIP140
(RIP-F, lanes 3 and 4), or a mutant
RIP in which the receptor-interacting motif is deleted
(L-384, lanes 5 and 6).
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Ligand-dependent Complex Formation of RAR/RXR, RIP140,
and HDAC Detected by Coimmunoprecipitation--
Previously, we have
demonstrated a direct interaction of RIP140 with HDAC3 and that the
immunoprecipitates of anti-RIP140 encode HDAC activity (33). We have
therefore hypothesized that RIP140 could function as a novel
ligand-dependent corepressor for nuclear receptor actions
by recruiting HDAC to nuclear receptors in a
ligand-dependent manner. To test this hypothesis, we
examined whether HDAC3 can form immunocomplexes with RIP140 and RAR/RXR in vivo, and whether the formation of these complexes is
ligand-dependent. COS-1 cells were transfected with
expression vectors for RIP140 (wild type mutant L-384 or an empty
vector), FLAG-HDAC3, RAR
, and RXR
. Anti-FLAG antibody was used to
precipitate proteins complexed with HDAC3. As shown in Fig.
5, RAR
(Fig. 5A, lane 4),
RXR
(Fig. 5B, lane 4), and RIP140 (Fig. 5C, lane
4) were all detected in immunocomplexes in the presence of at-RA.
In the absence of RA, these proteins were either absent or detected at a negligible level (lane 6 of Fig. 5, A-C). To
confirm the specificity of RIP140 action in facilitating the formation
of these immunocomplexes, two control experiments were conducted by
using either the L-384 mutant RIP140 that had a deletion in its
C-terminal motif (lane 5) or an empty vector (no RIP140,
lane 8). Under either condition, immunocomplex formation was
much less efficient, as shown in the much reduced level of RAR (Fig.
5A), RXR (Fig. 5B), and RIP140 (Fig.
5C) in the precipitates. To monitor the efficiency of
protein expression in these cultures, total lysates were examined on
the same blots for the expression of each component as shown on
lanes 1-3 and 7. This result strongly supports
our hypothesis that RIP140 facilitates immunocomplex formation of HDAC3
with RAR/RXR in the presence of RA and that the C-terminal
receptor-interacting motif of RIP140 is required for this activity.

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Fig. 5.
Coimmunoprecipitation experiments demonstrate
RA-dependent complex formation of HDAC3 with RIP140 and
RAR/RXR. COS-1 cells were cotransfected with FLAG-HDAC3, RIP140
(wild type, lanes 1, 3, 4, and 6; mutant,
lanes 2 and 5; or empty vector, lanes
7 and 8), and RAR and RXR in the presence
(lanes 1, 2, 4, 5, 7, and 8) or absence
(lanes 3 and 6) of RA. Total lysate was monitored
on Western blot (lanes 1-3 and 7) for expression
of these components. The lysate was precipitated with anti-FLAG, and
the immunocomplexes were resolved by polyacrylamide gel electrophoresis
and detected with anti-RAR (A), anti-RXR
(B), and anti-RIP140 (C).
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Deacetylation of RA-responsive Promoter by RA in the Presence of
RIP140--
Our demonstrations of HDAC activity in the
immunoprecipitates of anti-RIP140 (33) and RA-induced complex formation
of RIP140, HDAC3, and holo-RAR/RXR (Fig. 5) predict decreased
acetylation of chromatin histones around the promoter region of RAR/RXR
target genes in the presence of RIP140 and RA. To test this
possibility, we employed ChIP assays by using a classical DR5-tk
reporter as a model, which was also used to examine the biological
activity of RIP140 on RA-regulated target gene expression (Fig. 4). In this assay, acetylated chromatin can be precipitated with
anti-acetylated histone 3, and the precipitated DNA fragments
can be detected by PCR. On the contrary, hypoacetylated chromatin is
precipitated less efficiently; therefore, less DNA is amplified. As
shown in the top panel of Fig.
6A, an expected 130-base pair
fragment can be amplified efficiently from cells transfected with the
control vector (lane 1) but not from cells cotransfected
with RIP140 (lane 2), indicating a decrease in acetylation
on the tk promoter region regulated by the DR5 element. The negative
controls in which a nonspecific rabbit antiserum was used are shown in
lanes 3 and 4. Two positive controls of input DNA
are shown in lanes 5 and 6. Lane 7 shows a negative control of water, and lane 8 shows a
positive control of plasmid DNA. For an internal control of this assay,
the acetylation status of the CMV promoter used in the expression
vector was monitored in parallel experiments as shown in the
bottom panel of Fig. 6B. Because no DR5 is
present in the CMV promoter-driven expression vector, this promoter is highly acetylated and is therefore amplified efficiently, regardless of
the presence or absence of RIP140 (lanes 1 and
2).

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Fig. 6.
ChIP assay to demonstrate specific changes in
histone acetylation status on the tk promoter as a result of expression
of RIP140 and RAR/RXR in the presence of RA. A,
deacetylation of the tk promoter containing DR5 (top panel, lane
2) but not a nonspecific promoter CMV (bottom panel, lane
2) in the presence of RIP140. Positive controls are the input
(lanes 5 and 6) and plasmid DNA (lane
8). Negative control reactions with a nonspecific rabbit antiserum
are shown on lanes 3 and 4. Lane 7 shows a negative control of water. In the immunoprecipitated chromatin,
histone acetylation decreases as a result of expression RIP140
(lane 2) as compared with expression of a control vector
(lane 1). The top panel shows specific changes on
the tk promoter, and the bottom panel shows the results of
an internal control, CMV promoter, where acetylation status remains the
same regardless of the presence of absence of RIP140. B,
specificity of RIP140-triggered deacetylation of the tk promoter. ChIP
was conducted as described in A in the presence of the
indicated amounts of RIP140 (lanes 1-4), or the L-384
RIP140 mutant (lane 5). The top panel shows that
acetylation is gradually reduced as a result of expressing more wild
type RIP140, the second panel shows the input plasmid
control, the third panel shows a nonspecific IgG control,
and the bottom panel shows a Western blot of RIP140
expressed in these cultures.
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To confirm the specificity of RIP140 effects, ChIP assay was conducted
by using various concentrations of RIP140 expression vector and the
L-384 mutant as shown in Fig. 6B. Deacetylation of the
promoter (top panel) occurs in the presence of wild type RIP140 in a dose-dependent manner (lanes 1-4),
whereas the promoter remains acetylated at the same level in the
presence of mutant RIP140 (lane 5) and the control
(lane 1). The second panel shows input DNA, the
third panel shows nonspecific IgG control, and the
bottom panel shows a Western blot of transfected RIP140
expression in these cultures. These results clearly show the
specificity of hypoacetylation on the promoter containing the DR5
element as a result of expression of wild type RIP140/RAR/RXR in the
presence of RA, but not in the presence of mutant RIP140 with the
C-terminal receptor-interacting motif deleted. It is concluded that the
coimmunoprecipitated complex of RIP140, holo-RAR/RXR, and HDAC3 is
correlated with decreased histone acetylation on the promoter driven by
the RAR/RXR target element DR5 and that the C-terminal
receptor-interacting motif of RIP140 is required for this activity.
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DISCUSSION |
This study demonstrates a novel holo-RAR/RXR-interacting motif
(LTKTNPILYYMLQK) of RIP140, which diverts from the reported LXXLL box found in coactivators or the CoRNR box
(L/IXXI/VI) found in corepressors. This motif mediates
strong ligand-dependent interaction of RIP140 with RAR/RXR
as demonstrated in both in vitro and in vivo
protein interaction tests. Interaction of RIP140 with RAR/RXR results
in suppressed RA induction of target gene expression, and the presence
of this motif in RIP140 is essential for this biological activity.
RIP140 and RAR/RXR can be efficiently coimmunoprecipitated with HDAC3
in the presence of RA but are less efficiently coimmunoprecipitated with HDAC3 in its absence. RAR/RXR cannot be coprecipitated efficiently with HDAC3 with expression of the C-terminal motif-deleted RIP140 or
without expression of RIP140, suggesting a role of this C-terminal motif of RIP140 in enhancing RA-induced molecular interactions among
these proteins. Finally, expression of wild type RIP140 but not mutant
RIP140 dose-dependently renders hypoacetylation of the
promoter region of RA target gene in the presence of RA, suggesting
recruitment of HDAC3 by the RIP140/RAR/RXR complex to the RA-responsive
promoter in a ligand-dependent manner. However, it remains
to be determined whether the recruitment of HDAC3 to the RA-responsive
promoter and the repression of gene expression as a result of RIP140
expression also occur for endogenous gene promoters and whether other
HDACs can also be recruited by RIP140/RAR/RXR complexes.
RIP140 is able to interact with numerous nuclear receptors in a
ligand-dependent manner in the case of hormone receptors
and in a ligand-independent manner in the case of orphan receptors. However, the receptor-interacting domain of RIP140 varies among different receptor systems. For instance, its interaction with orphan
receptor TR2 utilizes various portions of the molecule that contain the
LXXLL motif (26), whereas its interaction with holo-RAR/RXR
utilizes the novel motif present in its C terminus. Whereas it has been
demonstrated that its interaction with other hormone receptors can be
mediated by its nine LXXLL motifs, the evidence for this
type of interaction is less compelling. In our two-hybrid interaction
tests, we occasionally detected a very low level of interaction in the
absence of ligand; however, this detection system can be complicated by
the nuclear environment of the cell types used and the activity of
reporter. The ligand-dependent enhancement of RIP140
interaction with RAR/RXR through the LXXLL-less motif is
significant and represents a novel example of holo-receptor interaction
with its coregulator. Structural studies are required to resolve the
molecular basis of this interaction.
The biological role of RIP140 has been debated because variable results
have been presented in different studies. Whereas the initial study
suggested a coactivator function of RIP140 in a chimeric estrogen
receptor system (25), many later studies from different laboratories
have demonstrated RIP140 as a corepressor or negative coregulator
(28-31). We first demonstrated that RIP140 suppressed TR2 target gene
expression (26). Later, we found that RIP140 expression also suppressed
RA induction of target gene, despite its ligand-dependent
interaction with RAR/RXR (27). More recently, we demonstrated a direct
association of RIP140 with HDAC3 through its N-terminal domain and that
the immunoprecipitated complexes pulled out with anti-RIP140 encode
HDAC activity (33). The current study extends the findings of
these previous studies and provides strong evidence for a suppressive
role of RIP140 in RA-mediated gene expression. A molecular explanation
for the negative role of RIP140 in RA signaling pathways is presented, i.e. the recruitment of HDAC3 by RIP140/RAR/RXR complex to
RA-responsive promoters in a RA-dependent manner. This view
contradicts the central dogma that ligand induces association of holo
nuclear receptors with coactivators that encode histone
acetyltransferase activity. The fact that the novel
receptor-interacting motif of RIP140 diverts from the classical
LXXLL box found in coactivators and the CoRNR box found in
corepressors may explain the unique feature of RIP140. It will be
interesting to examine how RIP140, as compared with other coactivators,
interacts with holo-RAR/RXR. RIP140 represents the first negative
cofactor for nuclear receptors that acts in a
ligand-dependent manner. Exactly how this observation can
be translated into a specific biological event remains to be explored.
It is noted that a number of studies have reported negative regulation
of gene expression by a direct effect of RA on certain RA response
elements found in the parathyroid hormone-related protein gene, the
thyrotropin-
gene, and the mouse Oct-3/4 gene (18, 34, 35), etc. It
is tempting to speculate a role of RIP140 on specific gene suppression
mediated by the direct action of RA-bound RAR/RXR in certain cell types
or under specific conditions. It would be interesting to examine the
difference in receptor conformation when complexed with a typical
corepressor, coactivator, or a novel coregulator like RIP140.