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J. Biol. Chem., Vol. 276, Issue 19, 16107-16112, May 11, 2001

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Ligand-dependent Formation of Retinoid Receptors, Receptor-interacting Protein 140 (RIP140), and Histone Deacetylase Complex Is Mediated by a Novel Receptor-interacting Motif of RIP140^*

Li-Na Wei, Maria Farooqui, and Xinli Hu

From the Department of Pharmacology, University of Minnesota Medical School, Minneapolis, Minnesota 55455

Received for publication, November 8, 2000, and in revised form, February 7, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Receptor-interacting protein 140 (RIP140) interacts with retinoic acid receptor and retinoid X receptor in a ligand-dependent manner and suppresses retinoic acid (RA) induction of its target genes. The receptor-interacting motif is mapped to a C-terminal peptide sequence (LTKTNPILYYMLQK) of RIP140. The functional role of this motif in mediating the suppressive effects of RIP140 on RA induction is demonstrated in mutation studies. RA induces coimmunoprecipitation of histone deacetylase 3 with retinoic acid receptor/retinoid X receptor in the presence of wild type RIP140, but not in the presence of the C-terminal motif-deleted RIP140. A decrease in histone acetylation on the promoter region that carries a RA response element is associated with the expression of wild type RIP140, but not with expression of the mutant RIP140, in a dose-dependent manner. These data provide a molecular explanation for RIP140 acting as a novel ligand-dependent, negative modulator of RA-regulated gene expression.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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 × 10⁷ 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 10⁶ 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.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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).

View larger version (38K): [in this window] [in a new window]   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.

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.

View larger version (36K): [in this window] [in a new window]   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.

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.

View larger version (67K): [in this window] [in a new window]   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.

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.

View larger version (45K): [in this window] [in a new window]   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).

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.

View larger version (64K): [in this window] [in a new window]   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).

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).

View larger version (45K): [in this window] [in a new window]   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.

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.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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.

	FOOTNOTES

^* This work was supported by National Institutes of Health Grant DK54733, American Cancer Society Grant RPG-99-237-010CNE, and United States Department of Agriculture Grant 98-35200-6264 (to L.-N. W.).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.

To whom correspondence should be addressed: Dept. of Pharmacology, University of Minnesota, 6-120 Jackson Hall, 321 Church St. SE, Minneapolis, MN 55455-0217. Tel.: 612-625-9402; Fax: 612-625-8408; E-mail: weixx009@tc.umn.edu.

Published, JBC Papers in Press, March 5, 2001, DOI 10.1074/jbc.M010185200

	ABBREVIATIONS

The abbreviations used are: HDAC, histone deacetylase; RIP140, receptor-interacting protein 140; RAR, retinoic acid receptor; RXR, retinoid X receptor; RA, retinoic acid; AA, amino acid(s); GST, glutathione S-transferase; PCR, polymerase chain reaction; at-RA, all-trans-RA; DR5, direct repeat 5; tk, thymidine kinase; ChIP, chromatin immunoprecipitation; CMV, cytomegalovirus.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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