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Originally published In Press as doi:10.1074/jbc.M307621200 on October 27, 2003

J. Biol. Chem., Vol. 279, Issue 1, 319-325, January 2, 2004
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Suppressive Effect of Receptor-interacting Protein 140 on Coregulator Binding to Retinoic Acid Receptor Complexes, Histone-modifying Enzyme Activity, and Gene Activation*

Xinli Hu{ddagger}, Yixin Chen{ddagger}, Mariya Farooqui{ddagger}, Mary C. Thomas§, Cheng-Ming Chiang§, and Li-Na Wei{ddagger}

From the {ddagger}Department of Pharmacology, University of Minnesota Medical School, Minneapolis, Minnesota 55455 and the §Department of Biochemistry, Case Western Reserve University, Cleveland, Ohio 44106

Received for publication, July 15, 2003 , and in revised form, October 14, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Gene induction by retinoic acid (RA) is suppressed by overexpression of receptor-interacting protein 140 (RIP140). RIP140-mediated suppression was reversed most effectively by overexpressing the coactivator p300/CREB-binding protein-associated factor (P/CAF). Immunoprecipitation demonstrated coexistence of holoreceptors complexed with RIP140 or P/CAF. Chromatin immunoprecipitation revealed rapid RA-enhanced recruitment of RIP140, but delayed P/CAF recruitment, to an RA-targeted promoter in COS-1 cells supplemented with RIP140. In RA-induced P19 cells, endogenous RIP140 was rapidly (within 4 h) and significantly recruited to both the RAR{beta}2 and TR2 genes, whereas the peak of endogenous P/CAF recruitment occurred much later (48 h) and to a lesser degree. Consistent with these observations, significant histone acetylation of endogenous RA receptor (RAR) targets was only observed 48 h following RA treatment. In vitro experiments confirmed RA-induced transcription from a chromatin template, which was reduced by adding RIP140. This study presents evidence for coexistence of multiple RAR-coregulator complexes and a preferential RA-induced recruitment of RIP140 to endogenous RAR-targeted promoters after short term RA treatment, which correlates with suppressed induction of RA-regulated gene expression in the presence of RIP140.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Eukaryotic gene regulation is achieved by a complex network of events that results in alteration of chromatin structures and coregulator recruitment to the transcription machinery (1, 2). These changes are achieved by the coordinated action of complexes such as ATP-dependent chromatin-remodeling and histone-modifying complexes (3). The histone-modifying complexes act on the tails of histones to produce covalent alterations such as methylation of specific arginine or lysine residues and acetylation of specific arginine residues (4, 5). Coordinated modification has been observed for several model genes, such as the chicken {beta}-globin locus (6). These highly specific modifications are the result of an ordered recruitment of various factors (7–9). Although the "on/off" switch of transcription regulation by hormones can be explained by recruitment of different types of coregulators, the question of how hormones activate genes at different levels remains to be addressed.

Current models propose that transcription activation is stimulated when hormones occupy their receptors, inducing conformational changes that result in alteration of coregulator recruitment. Recruitment of coregulators, called coactivators or corepressors, is critical for nuclear receptor-mediated gene regulation (10–12). Without hormones, aporeceptors are primarily associated with corepressors, whereas hormone-occupied receptors are usually associated with coactivators. These coregulators modulate gene transcription mainly by regulating enzyme activities, such as those involved in histone acetylation, e.g. histone acetyltransferase and histone deacetylase for coactivators and corepressors, respectively. Recently, other protein modification pathways such as methylation (13) have also been identified.

Receptor-interacting protein 140 (RIP140)1 is a coregulator for a wide variety of transcription factors including nuclear hormone receptors (14–19). Although originally shown to be a coactivator for the estrogen receptor in a chimeric reporter system (14), it was later shown to exert primarily a suppressive activity on gene transcription (17–21). For hormone-regulated genes, induction of gene expression is attenuated by RIP140 expression, which itself occurs in a hormone-dependent manner. Our recent studies on retinoic acid (RA)-inducible systems suggested a model for the novel action of RIP140 in hormonal regulation of gene expression (22–24). This involves two features of RIP140: its interaction with nuclear hormone receptors in a hormone-dependent manner and its ability to recruit histone deacetylase to the nuclear receptors and to their target DNAs in the presence of hormones. However, it is unknown how RIP140-receptor complexes operate in the presence of other coactivator complexes also recruited by hormone-bound receptors. Preliminary examination of the receptor-interacting domains of RIP140 originally revealed nine copies of the typical receptor-interacting motif LXXLL (where L is a leucine and X is any amino acid) (25) found in many coactivators. Our recent studies identified a novel atypical receptor-interacting motif in the carboxyl terminus of RIP140 (amino acids 1063–1076, LTKTNPILYYMLQK) that exhibits a high affinity (2 nM) toward hormone-occupied RA receptors (RARs) and retinoid X receptors (RXRs) and is responsible for the hormone-dependent suppression of gene activation (20, 22, 24).

We propose that RIP140 regulates the level of hormonal induction of gene activation by antagonizing or attenuating the recruitment of coactivators to nuclear receptors such as RAR. Multiple coregulatory complexes with antagonistic biological functions could be recruited to the same target in a mutually exclusive manner. Differences in the composition of transcription factor complexes on the target gene at specific time points might translate to quantitative regulation of gene expression. In this study, results using RA-regulated gene systems are presented to support this hypothesis. Several issues are addressed, including altered levels of RA-induced receptor-mediated gene activation as a result of changing levels of coregulators, the dynamics of RIP140 and p300/CREB-binding protein-associated factor (P/CAF) recruitment to an RA reporter gene and two endogenous RA-regulated gene promoters, and the direct effect of RIP140 on the in vitro transcription efficiency of RA-responsive genes assembled into chromatin templates.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Transfection and Reporter Assays—The reporter IR0-tK-luciferase (IR0-tK-Luc), RAR, RXR, RIP140, and FLAG-P/CAF expression vectors as well as the transfection procedure for COS-1 have been described previously (26). RA (0.5 µM each of all-trans- and 9-cis-RA) was added for 30 h in induction experiments.

Protein Purification—For in vitro transcription, FLAG-p300, FLAG-P/CAF, FLAG-RAR, and His-RXR were each expressed and purified from Sf21 insect cells. FLAG-tagged proteins were purified with anti-FLAG-M2 affinity resin (Sigma) and eluted with 0.2 mg/ml FLAG peptides (27). Heterodimers of FLAG-RAR·His-RXR were copurified with a Talon metal affinity resin (Clontech, BD Biosciences).

Coimmunoprecipitation—COS-1 cells were transfected with IR0-tK-Luc and different combinations of expression vectors for RAR, RXR, FLAG-P/CAF, and RIP140. Transfected cells were treated with vehicle or 0.5 µM all-trans- and 9-cis-RA and harvested 48 h later. The holoreceptor complexes were immunoprecipitated by anti-RAR (Affinity BioReagents) or anti-FLAG (Sigma), and the coregulators or RAR was detected on Western blots using anti-RIP140 (24), anti-RAR, or anti-FLAG (for P/CAF).

Chromatin Immunoprecipitation (ChIP) Assays—COS-1 cells were transfected with IR0-tK-Luc and different combinations of expression vectors for RAR, RXR, and RIP140. Samples were harvested at 15-min intervals following RA treatment over a 2-h period. ChIP assays were performed as described previously (21). The primers used to amplify the precipitated promoter region were 5'-AGC GTC TTG TCA TTG GCG-3' and 5'-GTT AAG CGG GTC GCT GCA G-3'. The expression levels of introduced RA receptors and RIP140 were monitored by Western blot analyses.

To determine the ability of two endogenous RA-regulated genes (RAR{beta}2 and TR2) to recruit endogenous RIP140 and P/CAF, P19 cells were induced with 0.5 µM all-trans- and 9-cis-RA. Cells were collected at 15-min intervals over a 2-h period or 4 and 48 h for immunoprecipitation with anti-RIP140, anti-P/CAF (Upstate Biotechnology), and anti-RAR. Samples were also immunoprecipitated with anti-acetylated H4 (Upstate Biotechnology) to determine whether the promoters were acetylated. The primers 5'-GAT GTC AGA CTA GTT GGG TCA TTT-3' and 5'-TGC GTT CCG GAT CCT ACC CCG-3' were used to amplify the endogenous RAR{beta}2 promoter region (containing DR5). The primers 5'-CCG CCG CTG GAC TCC GGG GCC CTC-3' and 5'-CGC TGG GCT GGA GAG AGC AGA GGC-3' were used to amplify the TR2 promoter region (containing IR0).

Reverse Transcription-PCR—Total RNA was extracted from P19 cells treated with vehicle or 0.5 µM all-trans- and 9-cis-RA for 4 and 48 h. Five µg of total RNA was used to synthesize cDNA using Superscript II reverse transcriptase (Invitrogen). One hundredth of reverse transcription products was used as template in PCR reaction using primer pairs 5'-ATG ATC ATT TGG ATC-3' and 5'-CTG CAA AAG TGC TTA TCC-3' to amplify about 270 bp of RAR{beta}2 cDNA. Primers for the mouse TR2 transcripts are 5'-GTG CTG GCA AGT GAT GAA CG-3' and 5'-AGT TGT AAT CTG CAG GCT CC-3', which allowed TR2 transcripts to be amplified in fragments of 450 bp in size.

Chromatin Assembly and in Vitro Transcription—An RA-responding G-less template, X121, was constructed by inserting four copies of an inverted repeat with zero nucleotide in the spacer (IR0) derived from the TR2 promoter region (26) into the EcoRI and SmaI site of pML{Delta}53 (28) located upstream of the adenovirus major late core promoter and 280-bp G-less cassette. The chromatin was reconstituted on X121 using Drosophila S190 chromatin assembly extract (29) and purified HeLa core histones (30) as described previously (27). In vitro transcription was performed by incubating chromatin (containing ~100 ng of DNA) in the presence or absence of 0.5 µM all-trans- and 9-cis-RA, 2 nM RAR/RXR heterodimers, 1 nM p300, 1 nM RIP140 carboxyl-terminal domain, and 1 nM P/CAF at 27 °C for 30 min. To deplete endogenous P/CAF, HeLa cell nuclear extracts were incubated with anti-P/CAF for 60 min at 4 °C and immunoprecipitated by addition of protein G-agarose at 4 °C for another 30 min. Native or P/CAF-depleted nuclear extracts were then added, and samples were incubated at 30 °C for 20 min prior to the addition of transcription mixture including nucleoside triphosphates as described previously (31). Transcription was conducted at 30 °C for 45 min and terminated by the addition of 90 µl of a stop solution (0.5 M EDTA, 3 M NaOAc, pH 5.2, 10% SDS, 2 µl of 10 mg/ml glycogen). The samples were then processed as described previously (31), and the transcription signals were quantified using a PhosphorImager and ImageQuan software (Molecular Dynamics).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Suppression of RA Induction by RIP140 —Several major RAR·RXR coactivators have been reported, including the p300/CREB-binding protein-P/CAF complex and members of the p160 family such as TIF2, its mouse homolog GRIP1, and Src-1. The effects of these coregulators were analyzed in transient transfection experiments conducted in COS-1 cells (Fig. 1A). As predicted, the activation of reporter gene activity by introduction of RAR·RXR and RA could be further enhanced by overexpression of TIF2, p300, and P/CAF (Fig. 1A, columns 3, 5, and 6 versus column 2). To identify the most likely target of RIP140 suppressive effect among the known coactivators, RIP140 was cotransfected into COS-1 cells together with each known individual coactivator (Fig. 1B). The suppressive effect of RIP140 (Fig. 1B, column 10) on RA-induced reporter expression (column 8) was most effectively reversed by P/CAF (column 14), although it was not a full reversal under these experimental conditions. This may be due to the possibility of RIP140 acting toward several coactivators, which would be able to be reversed by adding back a single coactivator. In the control experiment when no additional RAR·RXR was added, RIP140 had no effect on this reporter (Fig. 1B, column 9). To further confirm the RAR·RXR-specific suppressive effect of RIP140, an identical reporter with the RARE deleted was used to test the effect of RIP140 on this reporter system. As shown in Fig. 1C, this reporter was not affected by RA, exogenous RAR·RXR, or RIP140 expression, confirming that the suppressive effect of RIP140 was through RAR·RXR and the RARE.



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  		FIG. 1.
Suppression of RA induction by RIP140 and the rescuing effects of various RAR coactivators. The percentage of RA-induced expression of the reporter IR0-tK-Luc relative to control levels was determined in COS-1 cells in the presence of different expression vectors as indicated. Three independent experiments were conducted to obtain these results. A, the effect of different individual coactivators on RA induction. B, the rescuing effects of individual coactivators on RIP140-mediated suppression. Student's t test was performed to compare the difference between RIP140 (column 10) and columns 11, 12, 13, and 14.*, p < 0.05; #, p > 0.2. C, the effect of RAR·RXR and RIP140 on the reporter containing no RARE. RLU, relative luciferase activity. atRA, all-trans-RA. 9c RA, 9-cis-RA.

 
To assess further the potential antagonism between P/CAF and RIP140, the concentration dependence of the effects of both coregulators on RA-induced expression was examined (Fig. 2). Panel A of Fig. 2 shows the dose dependence of the activation mediated by P/CAF. Without RIP140, P/CAF enhanced RA activation of the reporter in a dose-dependent manner (Fig. 2A, line of filled squares). However, in the presence of RIP140, the activating effect of P/CAF was dramatically reduced, although a negligible dose dependence of P/CAF was still observed (Fig. 2A, line of open squares). Panel B of Fig. 2 shows the dose dependence of suppression mediated by RIP140. Without additional P/CAF, RIP140 suppressed RA activation of the reporter gene in a dose-dependent manner (Fig. 2B, line of filled triangles). With additional P/CAF expressed, RA activation apparently was to a much higher degree. As predicted, RIP140 still suppressed RA activation of the reporter in a dose-dependent manner even in the presence of additional P/CAF (Fig. 2B, line of open triangles). If there was only the endogenous P/CAF, the RA induction level was lower with a different amount of RIP140 (Fig. 2B). Thus, the specific antagonistic effect of RIP140 toward P/CAF was also confirmed in these experiments where clear dose dependence of both the coactivating activity of P/CAF and the suppressive activity of RIP140 was demonstrated.



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  		FIG. 2.
Dose-dependent antagonism between RIP140 and P/CAF. The percentage of RA-induced expression of the reporter IR0-tK-Luc relative to control levels in COS-1 cells was determined in the presence of various amounts of RIP140 and/or P/CAF as indicated. Three independent experiments were conducted to obtain these results. A, dose dependence of coactivating activity of P/CAF in the presence (open squares) or absence (filled squares) of 50 ng of RIP140. B, dose dependence of suppressive activity of RIP140 in the presence (open triangles) or absence (filled triangles) of 50 ng of P/CAF.

 
Coexistence of P/CAF- and RIP140-containing Receptor Complexes in the Presence of RA—The strong antagonism of RIP140 toward P/CAF with regards to RA induction prompted us to examine the RIP140- and P/CAF-containing receptor complexes in vivo. A competitive immunoprecipitation experiment was conducted using COS-1 cells (Fig. 3). Neither RIP140 nor P/CAF could be detected in the absence of RA (Fig. 3, lane A, columns 2–5), which confirmed the ligand dependence of the recruitment of RIP140 and P/CAF. With the introduction of ligand, RIP140 was detected efficiently in cells expressing RIP140 and receptors (Fig. 3, lane A, column 8) but was detected at a much lower level in cells also harvesting additional P/CAF (column 9). As expected in the negative control, RIP140 was not detected in cells harvesting only receptors or together with P/CAF (Fig. 3, lane A, columns 6 and 7). The same Western blot was probed with anti-FLAG (Fig. 3, lane B). FLAG-P/CAF was efficiently detected in cells harvesting receptors and P/CAF (Fig. 3, column 7). Interestingly, the FLAG-P/CAF signal was also dramatically reduced in cells harvesting additional RIP140 (Fig. 3, column 9). In negative controls, FLAG-P/CAF was not detected in cells harvesting only receptors or together with RIP140 (Fig. 3, columns 8 and 9). The input of each experiment was shown in Fig. 3, lanes C and D. Thus, both RIP140 and P/CAF efficiently formed complexes with RAR·RXR in the presence of hormones. However, when both RIP140 and P/CAF were present, although both complexes existed, they were clearly detected at much lower levels, suggesting mutually exclusive recruitment of these two coregulatory complexes to the RAR-targeted promoter.



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  		FIG. 3.
Competition between RIP140 and P/CAF for receptor immunocomplex formation. COS-1 cells were transfected with expression vectors for RIP140, FLAG-P/CAF, and RAR·RXR in the presence or absence of RA as indicated. Immunocomplexes were precipitated with anti-RAR, and Western blots were probed with anti-RIP140 or anti-FLAG (for P/CAF). For testing interaction between RIP140 and P/CAF, immunocomplexes were precipitated with anti-FLAG, and Western blots were probed with anti-RIP140 or anti-RAR. The Input panels show Western blots performed on COS-1 extracts prior to immunoprecipitation (IP) with anti-RAR or anti-FLAG.

 
To examine whether the detection of both RIP140 and P/CAF in the RAR complexes was caused by the direct interaction of RIP140 with P/CAF, immunoprecipitation was conducted with {alpha}-FLAG followed by detecting the complexes with {alpha}-RIP140. As shown in Fig. 3, lane E, no RIP140 was detected in the P/CAF precipitates with or without ligand, suggesting that RIP140 and P/CAF did not interact directly. In the positive controls, RAR was detected in these complexes in the presence of RA (Fig. 3, lane F, columns 7 and 9), but less RAR was associated with P/CAF in the presence of RIP140 (comparing column 9 to column 7), further confirming the mutually exclusive nature of receptor complexes recruiting P/CAF or RIP140. The input controls clearly showed that RIP140 and RAR were expressed in comparable levels (Fig. 3, lanes G and H).

RA-dependent Recruitment of Coregulator Complexes to an RA Reporter Gene Promoter—Previous studies have revealed that the suppressive activity of RIP140 is due to its ability to recruit histone deacetylase to receptor-bound promoters of the target genes (21). P/CAF, on the other hand, has been reported to have intrinsic histone acetyltransferase activity (32, 33). Therefore, complexes containing RIP140 or P/CAF could possess opposing enzyme activities and potentially exert antagonistic effects on chromatin remodeling. To examine this possibility, ChIP assays were performed to determine the time dependence of receptor/coregulator recruitment to a reporter gene promoter (IR0-tK-Luc) as well as histone acetylation of the promoter.

In COS-1 cells containing sufficient endogenous P/CAF and exogenous RIP140, it appeared that RAR (regardless of the presence or absence of RIP140) was expressed at constant levels (Fig. 4E) and associated consistently with the targeted promoter without any obvious cycling (Fig. 4A). In contrast, both P/CAF and RIP140 exhibited more complex binding patterns over time (Fig. 4B). RIP140 was recruited as early as 15 min following RA treatment and remained strongly associated with receptor DNA until 30 min later when it dissociated from the receptor DNA complex. RIP140 subsequently reassociated with receptor DNA at 90–105 min following RA treatment (Fig. 4B, top).



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  		FIG. 4.
Time course of recruitment of receptors and coregulators to an RA reporter gene promoter in COS-1 and an endogenous RAR{beta}2 promoter. A, COS-1 cells were transfected with RAR, the IR0-tK-Luc reporter, and either a control (–) or an RIP140 (+) vector. ChIP assay was performed with anti-acetylated H4 ({alpha}-AcH4) to determine the acetylation status of the IR0-containing promoter at different time points of RA induction as indicated. B, time-dependent recruitment of exogenously provided RIP140 (top) and endogenous P/CAF to the IR0-containing promoter in COS-1 cells transfected with RAR and IR0-tK-Luc in absence (center, –) or presence (bottom, +) of RIP140. C, constitutive recruitment of RAR to the RA reporter in COS-1 cells expressing RAR and either a control (–) or an RIP140 (+) expression vector. D, control aliquots of non-immunoprecipitated DNA at each time point of analysis. E, Western blot analyses of the expression vectors introduced into COS-1 cells. RAR expression in COS-1 expressing either a control (–) or an RIP140 (+) vector is shown in the top two lanes. RIP140 expression is shown in the bottom lane. F, ChIP assay performed on the endogenous RAR{beta}2 promoter of P19 cells using anti-RIP140, anti-P/CAF, anti-acetylated H4, and anti-RAR.

 
Without exogenous RIP140, P/CAF was clearly recruited to the targeted promoter 45 min following RA treatment with a cycle lasting ~60 min. Interestingly, P/CAF recruitment was dramatically reduced when RIP140 was present (Fig. 4B, bottom). Note that RIP140 was not recruited during the same period as P/CAF. However, RIP140 apparently delayed P/CAF recruitment as well as reduced the amount of P/CAF associated with the receptor DNA complexes.

Finally, we examined the pattern of histone acetylation induced by RA on the target gene promoter with or without additional RIP140 expression (Fig. 4C). RA-induced hyperacetylation occurred in a cyclic fashion, with the first cycle peaking at 30 min following RA treatment (Fig. 4, column 3). The subsequent cycles each spanned ~40–50 min. With the addition of RIP140, the same pattern occurred with a similar time course for each cycle, but the level of acetylation was dramatically reduced. Furthermore, the first reduction in histone acetylation (30–60 min) occurred shortly after the first peak of RIP140 recruitment to the promoter (15–30 min), whereas a dramatic reduction in histone acetylation during the second cycle coincided with the second peak of RIP140 recruitment (90–105 min) (Fig. 4, B and C, columns 7 and 8). Conversely, the second and third cycles of histone acetylation roughly coincided with the recruitment of P/CAF to the promoter (Fig. 4, columns 6 and 9).

The cycling of receptor and coregulators on the endogenous RAR{beta}2 promoters was also examined in P19 cells (Fig. 4F). Because this cell line expressed all these components, the experiments were designed to examine the behavior of endogenous factors on the endogenous target gene. Similar to the cycling pattern observed on the artificial promoter in the COS-1 cell system (Fig. 4, A–E), RIP140 was recruited to the endogenous RAR target promoter earlier than P/CAF, with the first peak appearing at 15 min after RA treatment (Fig. 4, column 2). The first peak of P/CAF occurred at 30 min following the addition of RA, coinciding with the first peak of histone acetylation (Fig. 4, column 3). Interestingly, the cycling time of the cofactor recruitment and the acetylation status was about 45 min, slightly shorter than that of the artificial promoter. As seen in the reporter system, RAR was also constantly associated with the DR5 element of the endogenous RAR{beta}2 promoter.

RA-dependent Recruitment of Endogenous Coregulator Complexes to the Endogenous RA-regulated Gene Promoters in P19 Cells—To examine the temporal dynamics of coregulator recruitment to different endogenous RA target promoters, we examined the recruitment of endogenous RIP140 and P/CAF to two previously reported RA-regulated genes, a rapidly induced gene RAR{beta}2 and a slowly induced gene TR2 in P19 cells. Similar to what we observed in COS-1 cells, endogenous RAR was associated with both the RAR{beta}2 and TR2 promoters constitutively in P19 (Fig. 5, ChIP lanes, columns 10–12). There was a dramatic increase of RIP140 association to both RAR{beta}2 (Fig. 5, ChIP lanes, bottom) and TR2 (ChIP lanes, top) at 4 h following RA treatment (column 2). RIP140 recruitment to both promoters was decreased by 48 h (Fig. 5, column 3), corresponding to the decreased expression level of RIP140 after long term RA treatment in P19 cells (Western lane) (34). P/CAF recruitment to the TR2 promoter occurred to a much lesser degree at both 4 and 48 h (Fig. 5, ChIP lanes, columns 5 and 6). P/CAF recruitment to the RAR{beta}2 promoter was more significant, particularly at 48 h (Fig. 5, column 6). The status of histone acetylation of both promoters was also examined (Fig. 5, columns 7–9). RAR{beta}2 clearly exhibited a higher level of histone acetylation, in particular at 48 h. Histone acetylation of the TR2 promoter was much weaker. Consistently, an early increase of RAR{beta}2 mRNA was readily detected at 4 h of RA induction (Fig. 5, RT-PCR lane, column 2 versus column 1), whereas the mRNA level of TR2 remained largely unchanged at this early time (RT-PCR lane, column 4 versus column 5). At 48 h of RA treatment, both RAR{beta}2 and TR2 transcripts were more dramatically increased (Fig. 5, RT-PCR lane, columns 3 and 6).



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  		FIG. 5.
Recruitment of endogenous RIP140, P/CAF, and RAR to two endogenous RA-regulated genes and the histone acetylation status of these gene promoters in P19 cells. P19 cells without (–) or with RA treatment for 4 or 48 h were collected for ChIP assays of the endogenous TR2 (IR0) or RAR{beta}2 (DR5) with the specific antibody depicted above each column as shown at the top. The expression of endogenous RIP140 was detected on a Western blot as shown in the center. Total RNA was also prepared from these cells to examine the change of the mRNA level of RAR{beta}2 and TR2, as well as an internal control actin, in response to RA treatment by reverse transcription (RT)-PCR (bottom). {alpha}-Ac H4, anti-acetylated H4.

 
Suppression of Gene Transcription on in Vitro-reconstituted Chromatin Templates by RIP140—To determine the effect of RIP140 on transcriptional activation by RAR·RXR, we generated a template containing four copies of an IR0 element fused to a G-less cassette (28), which could be transcribed into RNA of 280 nucleotides in length. The template was assembled into chromatin, and in vitro transcription was conducted as described under "Materials and Methods." Without RA, RAR·RXR heterodimers could not activate transcription (Fig. 6, bottom, column 2). The addition of RA alone activated low levels of transcription (Fig. 6, bottom, column 3), possibly due to the presence of endogenous receptors in the nuclear extract. By providing additional receptors, RA induction was more significant (Fig. 6, bottom, column 4). Addition of P/CAF triggered a slightly stronger activation (Fig. 6, bottom, column 5). In contrast, the addition of RIP140 reduced transcription (Fig. 6, bottom, column 6). Adding both RIP140 and P/CAF returned transcription efficiency to levels comparable with those without additional coregulators (Fig. 6, bottom, compare columns 7 and 4), suggesting the presence of sufficient endogenous coactivators. To determine whether P/CAF was one of these factors, nuclear extracts were depleted of P/CAF with an anti-P/CAF antibody and used in transcription assays (Fig. 6, bottom, columns 8–10). Indeed, P/CAF-depleted nuclear extract was much less efficient in promoting transcription (Fig. 6, bottom, compare columns 9 and 4), which could be partially recovered by addition of P/CAF to the transcription reaction (column 8).



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  		FIG. 6.
In vitro transcription of an RA-responsive chromatin template. The IR0-containing G-less DNA (top diagram) was assembled into chromatin and subjected to in vitro transcription as described under "Materials and Methods." The nuclear extract used (native or P/CAF-depleted), as well as any factors added prior to transcription, are depicted above and to the left of each lane, respectively. PG5MLT is a G-less in vitro transcription construct (second diagram) (27) with five copies of the Gal4 binding site upstream of adenovirus major late core promoter, and its transcript is 380 nucleotides. This was used as the internal control. The transcription efficiency of three independent experiments was quantified, and the relative activity was shown at the bottom of the figure.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Current models of hormonal action describe the on/off switches triggered by the recruitment of either coactivators in the presence of hormones or corepressors in their absence. RIP140 has been shown to be a novel coregulator for a number of hormone receptor systems, but its actions seemed contrary to those of either coactivators or corepressors. Like most coactivators, RIP140 is recruited to holoreceptors. However, this recruitment usually leads to a suppression of hormone target genes, consistent with the activity of a corepressor. By using an RA reporter system in our previous studies (22–24), we formulated a model to explain the action of RIP140 and attributed this novel action to its high affinity toward holoreceptors and its associated histone deacetylase activity.

The in vitro transcription assays on reconstituted chromatin templates presented in the current study confirm the suppressive effect of RIP140 on transcription. This suppressive effect may in part be due to a novel receptor-interacting motif, LYYML, in its carboxyl terminus (23, 24). However, because this motif is not capable of recruiting histone deacetylase, it might repress activation only by interfering with the recruitment of coactivators in the in vitro transcription assays. This might explain why the suppressive effect of RIP140 in the in vitro transcription assay was not as pronounced as that observed in the reporter assay.

P/CAF has been shown to be a coactivator for a wide variety of transcription factors (35–39). By using in vivo systems, the current study identifies P/CAF as an important antagonistic target of RIP140 for RAR-targeted genes. Both P/CAF and RIP140 can interact with receptors directly. However, the results of coimmunoprecipitation and ChIP assays suggest that the receptor complexes containing RIP140 or P/CAF might be mutually exclusive. It is unknown whether these two coregulators can interact with the same receptor simultaneously. It also remains to be determined whether the antagonistic nature of RIP140 toward P/CAF recruitment applies to other RA-regulated genes.

ChIP assays revealed an interesting pattern of RIP140 and P/CAF recruitment to the target DNA in COS-1 cells. As predicted, RA receptors are constantly associated with the DNA, whereas the coregulators associate with receptor DNA in a differentially cyclic fashion. On the artificial promoter of RARE-tK, RIP140 recruitment occurs in the very early phase of RA induction (as early as 15 min), and its association peaks at 30 min in the first cycle and 90–105 min in the second cycle. P/CAF recruitment occurs much later, with the first cycle peaking at 60 min and the second cycle beginning at 120 min. Interestingly, despite variations in the recruitment of different coregulators, RA-induced histone hyperacetylation of the gene promoter cycles constantly. Indeed, the initial cycle of histone acetylation occurred despite the recruitment of RIP140 expression, suggesting the involvement of other histone acetyltransferase-containing coactivators in the initial step of histone acetylation. The second and third peaks of acetylation occur at 75 and 120 min, roughly corresponding to the time of P/CAF recruitment. This suggests a role for P/CAF recruitment in the RA-induced acetylation of the promoter, at least for the second and third cycles. On the endogenous RAR{beta}2 promoter in P19, the cycling pattern of the coregulators is similar to that of the artificial promoter in COS-1 cells. However, one cycle took only 45 min, and the cycling of P/CAF parallels closely gene acetylation status. Regardless of the slight difference in the duration of factors cycling in the two cell lines and different target gene systems, the mutually exclusive nature of RIP140 and P/CAF recruitment to the RAR-targeted promoter was consistent.

Significantly, in the presence of RIP140, the degree of hyperacetylation is dramatically reduced, mostly between the peaks of acetylation. Moreover, on the artificial promoter, introduction of RIP140 delays and reduces the amount of P/CAF recruited. Apparently, both coactivator (P/CAF) and corepressor (RIP140) complexes can associate with the receptor-targeted promoter following RA treatment, but RIP140 is preferentially recruited and seems to antagonize or attenuate P/CAF recruitment. This effect of RIP140 on the dynamics of P/CAF recruitment could explain, at least partially, its suppressive activity on RA induction of gene activation.

One apparent discrepancy in the ChIP assays conducted on COS-1 cells is the significant drop in acetylation between the first and the second peaks despite the lack of significant RIP140 recruitment during this period. It is possible that other unidentified factors are present in these cells to deacetylate histones between the first and the second peaks of acetylation. Alternatively, this may be an effect of the first peak of RIP140 recruitment (30 min), which occurs just prior to hypoacetylation of the promoter. Similar cycles of histone acetylation have been observed in other hormone-inducible gene systems in which the mechanism of acetylation is well studied but the deacetylation between the peaks of acetylation is not as well understood (8, 9). Because RIP140 is recruited in a cyclic fashion to hormone receptor-targeted genes, presumably it could exert its activity repeatedly as long as the levels of hormones and RIP140 are both sufficient.

In the ChIP assays conducted to examine endogenous RA-regulated genes in P19 cells, RIP140 was rapidly and effectively recruited to the promoters for both the RAR{beta}2 and TR2 genes. The subsequent decline in association of RIP140 with receptor DNA after long term RA treatment may be due to decreased RIP140 levels in P19 cells, possibly caused by negative feedback on its own expression (34). In contrast, RA-dependent P/CAF recruitment to the RAR{beta}2 promoter seemed to be greater than its recruitment to the TR2 promoter, suggesting gene-dependent variations in cofactor recruitment. This is also reflected by the more obvious induction of histone hyperacetylation on the RAR{beta}2 promoter at 48 h of RA treatment and is in agreement with the much stronger effect of RA induction on RAR{beta}2 than on TR2 (26). Likewise, reduced P/CAF (i.e. coactivator) recruitment coupled with enhanced RIP140 (corepressor) recruitment to the TR2 promoter correlates with the much weaker induction of TR2 by RA in P19 cells.

In currently accepted models of hormone actions, the hormone-induced "on" switch for gene transcription elicits an extremely efficient and rapid response in the mammalian genome from a silenced state to an activated state. In contrast, the "off" switch remains somewhat passive and inefficient because it requires the termination of hormone production and, possibly, the degradation of receptors and/or the coactivator complexes. We propose that RIP140 serves as a negative modulator, or a "brake," for the stimulatory effect of hormones. By rapidly forming complexes with receptors, RIP140 could reduce rapidly and efficiently the levels of hormone-induced expression of certain target genes at an early stage, enabling a smooth transition from a silent to an activated state of transcription instead of an abrupt change in the level of target gene expression. That is, already activated transcription factors could be altered in terms of their enzymatic activities or capacities to accommodate other coregulators. If this model were correct, the ultimate control would reside with the dynamic and kinetic behavior of these coregulator complexes. This may be particularly relevant in a physiological setting where different kinds of complexes are formed in the same cell.

It remains unclear whether RIP140 plays a role in transcription initiation. The current study demonstrates a unique role for RIP140 in hormone receptor-mediated events caused by its antagonistic activity toward other coregulators, especially P/CAF. However, RIP140 may exert other functions in other gene activation systems. Indeed, with nine copies of the LXXLL motif scattered throughout the molecule, RIP140 has great potential to interact with numerous receptors. Future challenges include determining how these specific domains interact with other transcription factors, defining their binding affinity and the time course of their association with specific promoters, as well as examining whether the associated histone deacetylase activity provides a common mechanism for the gene-suppressive activity of RIP140.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grants DK54733, DK60521, K02 DA13926, DA11190, and DA11806 (to L.-N. W.) and National Institutes of Health Grants GM59643 and CA81017 (to C.-M. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

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

1 The abbreviations used are: RIP140, receptor-interacting protein 140; RA, retinoic acid; RAR, retinoic acid receptor; RXR, retinoid X receptor; CREB, cAMP-response element-binding protein; P/CAF, p300/CREB-binding protein-associated factor; Luc, luciferase; ChIP, chromatin immunoprecipitation; RARE, retinoic acid response element. Back


   ACKNOWLEDGMENTS
 
We thank Drs. Y. Nakatani and H. Gronemeyer for expression vectors of p300 and P/CAF and of TIF2, respectively.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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