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Molecular Endocrinology 20 (6): 1276-1286
Copyright © 2006 by The Endocrine Society

Transcriptional Intermediary Factor 1{alpha} Mediates Physical Interaction and Functional Synergy between the Coactivator-Associated Arginine Methyltransferase 1 and Glucocorticoid Receptor-Interacting Protein 1 Nuclear Receptor Coactivators

Catherine Teyssier, Chen-Yin Ou, Konstantin Khetchoumian, Régine Losson and Michael R. Stallcup

Department of Biochemistry and Molecular Biology (C.T., C.-Y.O., M.R.S.), University of Southern California, Los Angeles, California 90089; and Institut de Génétique et de Biologie Moléculaire et Cellulaire (K.K., R.L.), Centre National de la Recherche Scientifique/Institut National de la Santé et de la Recherche Médicale/Université Louis Pasteur/Collège de France, 67404 Illkirch-Cedex, France

Address all correspondence and requests for reprints to: Michael R. Stallcup, Department of Biochemistry and Molecular Biology, University of Southern California, 1333 San Pablo Avenue, MCA 51A, Los Angeles, California 90089-9151. E-mail: stallcup{at}usc.edu.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
In previous studies transcriptional intermediary factor 1{alpha} (TIF1{alpha}) was identified as a direct binding partner and potential transcriptional coactivator for nuclear receptors (NRs) but its overexpression inhibited, rather than enhanced, transcriptional activation by NRs. Here we show that TIF1{alpha} bound to and enhanced the function of the C-terminal activation domain (AD) of coactivator associated arginine methyltransferase 1 (CARM1) and the N-terminal AD of glucocorticoid receptor-interacting protein 1 (GRIP1). Furthermore, although TIF1{alpha} had little or no NR coactivator activity by itself, it cooperated synergistically with GRIP1 and CARM1 to enhance NR-mediated transcription. Inhibition of endogenous TIF1{alpha} expression reduced transcriptional activation by the GRIP1 N-terminal domain but not by the CARM1 C-terminal domain, suggesting that TIF1{alpha} may be more important for mediating the activity of the former than the latter. Reduction of endogenous TIF1{alpha} levels also compromised the androgen-dependent induction of an endogenous target gene of the androgen receptor. Finally, TIF1{alpha} formed a ternary complex with the GRIP1 N-terminal and CARM1 C-terminal domains. Thus, we conclude that TIF1{alpha} cooperates with NR coactivators GRIP1 and CARM1 by forming a stable ternary complex with them and enhancing the AD function of one or both of them.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
THE NUCLEAR RECEPTORS (NR) belong to a family of transcriptional activator proteins, many of which are receptors for specific hormones or metabolites, including steroid and thyroid hormones, vitamin D, and retinoic acid (1, 2). Thus, many NRs regulate transcription in a hormone-dependent manner. NRs bind to specific enhancer elements associated with their target gene promoters and activate transcription by recruiting a large array of coactivator proteins, which remodel chromatin structure in the promoter region and recruit RNA polymerase II and its associated transcription machinery (3, 4, 5). Some coactivators, such as the p160 coactivators steroid receptor coactivator 1 (SRC-1), glucocorticoid receptor-interacting protein 1 (GRIP1), and activator of the thyroid and retinoic acid receptors (ACTR), bind directly to the NRs and have been designated as primary coactivators. Other coactivators have been designated as secondary coactivators for NRs, because their physical association with the promoter of the NR target gene and their functions as coactivators depend on their binding to the p160 coactivators or other primary coactivators (6).

Among the most well-characterized secondary coactivators are the protein acetyltransferases, cAMP response element-binding protein (CREB)-binding protein (CBP) and p300 (7), and the protein arginine methyltransferases (PRMT), PRMT1 and coactivator-associated arginine methyltransferase 1 (CARM1) (8, 9). CBP and p300 acetylate histones and other protein components of the transcription machinery (10, 11, 12). PRMT1 methylates histone H4 on Arg-3, whereas CARM1 methylates histone H3 on Arg-2, Arg-17, and Arg-26 (9). All of these enzymes and the histone modifications they catalyze are associated with steroid hormone-regulated promoters in a hormone-dependent manner (12, 13, 14, 15), suggesting their physiological relevance to the transcriptional activation process. Furthermore, the importance of these histone modifications to transcriptional activation by a different transcription factor, p53, has been demonstrated in a cell-free transcription system, using reconstituted chromatin templates (16). As suggested by their different mechanisms of action, CARM1 and PRMT1 function synergistically with each other and with p300/CBP as coactivators for NRs and p53 (11, 16, 17, 18).

CARM1 shares homology with the other members of the PRMT family in the highly conserved core region of approximately 310 amino acids, which contains the Ado-Met binding site and the methyltransferase activity (19, 20). The same conserved domain is responsible for homodimerization or homooligomerization and for binding to GRIP1 (21). Point mutational analysis showed that the CARM1 methyltransferase activity is necessary for its coactivator function (11). In addition to the conserved methyltransferase domain, each PRMT member has a unique N-terminal region that varies widely in length, and CARM1 also has a unique C terminus (CARM1-C) (19). Although this C-terminal domain is not involved in the methyltransferase, oligomerization, or GRIP1 binding activities, its deletion severely compromised coactivator function (21). CARM1-C contains a strong autonomous activation domain (AD), suggesting that it may bind to or otherwise activate downstream factors that are involved in the transcriptional activation process (21). In the current study we identified transcriptional intermediary factor 1{alpha} (TIF1{alpha}) (22) as a CARM1-C binding protein and tested its ability to cooperate with CARM1 and GRIP1 as coactivators for NRs.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Identification of TIF1{alpha} as a CARM1-C-Interacting Protein
To investigate the mechanism of action of the CARM1 C-terminal AD, we used it as bait in a yeast two-hybrid screen. A fragment of TIF1{alpha} (amino acids 193–607), previously characterized as a NR-binding protein and putative coactivator for NRs (22, 23), was represented by two of the confirmed positive clones (Fig. 1AGo).


Figure 1
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  		Fig. 1. TIF1{alpha} Stimulates Transcriptional Activation by the CARM1-C AD

A, Domains of TIF1{alpha} are shown, along with the fragment identified by yeast two-hybrid (Y2H) screen (amino acids 193–607). B, CV-1 cells were transfected with 250 ng of GK1 reporter plasmid; 125 ng of pM vector encoding Gal4 DBD or Gal4 DBD fused to CARM1 full length (C1), CARM1-N (C1N, amino acids 3–460), or CARM1-C (C1C, amino acids 461–608); and 400 ng of pSG5 empty vector (white bars) or pSG5.TIF1{alpha} (black bars). Luciferase activity was measured 48 h after transfection. Each data point represents the mean and range of variation of two transfected cell cultures. Results shown are from a single experiment, which is representative of four independent experiments. RLU, Relative light units; PHD, plant homeo domain.

 
Enhancement of CARM1 Coactivator Function and C-Terminal Activation Function by TIF1{alpha}
To confirm the interaction between TIF1{alpha} and CARM1 and investigate whether this interaction is relevant to the coactivator function of CARM1, we tested the effect of TIF1{alpha} overexpression on the transcriptional activation activity of CARM1 or its fragments fused to the Gal4 DNA binding domain (DBD). As observed previously (21) the CARM1 C-terminal domain (residues 461–608) had a much stronger transactivation activity than full-length CARM1 (Fig. 1BGo, white bars), although CARM1 and its C-terminal fragment were expressed at similar levels in COS7 cells (21). The activity of full-length CARM1 and CARM1-C (residues 461–608), but not that of the CARM1 N-terminal domain (residues 3–460), was enhanced in the presence of TIF1{alpha} (Fig. 1BGo). The Gal4-CARM1 (3–460) and Gal4-CARM1 (full length) fusion proteins interacted with VP16-CARM1 equally in a mammalian two-hybrid homodimerization assay (21), indicating a similar expression level of these two CARM1 fusion proteins.

The ability of TIF1{alpha} and CARM1 to cooperate as NR coactivators was tested directly in a transient reporter gene assay system optimized to observe synergistic cooperation of three different NR coactivators (11). In this system, the combined overexpression of GRIP1 and CARM1 or GRIP1 and TIF1{alpha} resulted in a modest enhancement of reporter gene activation by four different hormone-stimulated NRs: glucocorticoid receptor (GR), estrogen receptor (ER) {alpha}, androgen receptor (AR), and thyroid hormone receptor (TR) (Fig. 2Go). Coexpression of all three coactivators produced a synergistic enhancement of the activity of these NRs, which was hormone dependent (Fig. 2Go and data not shown). In contrast, TIF1{alpha} and CARM1 did not cooperate as coactivators for NRs in the absence of GRIP1. Thus, although TIF1{alpha} can bind directly to NRs (22), it apparently does not serve as a primary coactivator for NRs. Instead, the coactivator function of TIF1{alpha} and its ability to cooperate synergistically with CARM1 as a coactivator depends on the presence of the primary p160 coactivator GRIP1. Thus, in this assay system TIF1{alpha} apparently functions as a secondary coactivator within the context of a larger coactivator complex.


Figure 2
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  		Fig. 2. Transcriptional Synergy between the Three Coactivators: GRIP1, CARM1, and TIF1{alpha}

CV-1 cells were transiently transfected with 250 ng of luciferase reporter plasmid controlled by an appropriate hormone response element; expression vector for GR (0.1 ng), ER (0.1 ng), TR (0.1 ng), or AR (0.5 ng); and expression vectors for GRIP1 (50 ng), CARM1 (250 ng), and TIF1{alpha} (250 ng), as indicated. Transfected cells were grown without hormone (white bars in panel D) or with (black bars in panels A–D) 20 nM dex for GR, E2 for ER, T3 for TR, or DHT for AR; after 48 h, cell extracts were assayed for luciferase activity. Each data point represents the mean and range of variation of two transfected cell cultures. Results shown are from a single experiment, which is representative of four separate experiments for GR, six experiments for ER, three experiments for TR, and two experiments for AR. RLU, Relative light units; dex, dexamethasone.

 
Requirement of Specific Functional Domains for GRIP1-CARM1-TIF1{alpha} Coactivator Synergy
To investigate the mechanism of the synergy between the three coactivators, we tested the effects of deleting their various functional domains on their synergistic coactivator function. CV-1 cells were transfected with plasmids encoding ER (Fig. 3Go, A and D) or GR (Fig. 3Go, B and C) and a suitable luciferase reporter gene, along with plasmids encoding GRIP1, CARM1, and/or TIF1{alpha} or mutant forms of these proteins. TIF1{alpha} contains an LXXLL motif that is responsible for binding NR ligand binding domains in the presence of the appropriate hormone; mutation of the two tandem leucines in this motif to alanines eliminated binding of TIF1{alpha} to NR ligand-binding domains (23). However, this TIF1{alpha} mutant (TIF1{alpha} L/A) cooperated as effectively as wild-type TIF1{alpha} with GRIP1 and CARM1 to produce a synergistic enhancement of NR function (Fig. 3AGo), indicating again that the effect of TIF1{alpha} in our assay was independent of its ability to bind to NRs.


Figure 3
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  		Fig. 3. Coactivator Domains Required for GRIP1-CARM1-TIF1{alpha} Synergy

A, CV-1 cells were transiently transfected with MMTV(ERE)-LUC reporter plasmid (250 ng) and expression vectors encoding ER (0.01 ng), GRIP1 (50 ng), CARM1 (100 ng), and TIF1{alpha} wild type or L/A mutant (100 ng), as indicated. B, CV-1 cells were transiently transfected with MMTV-LUC reporter plasmid (250 ng) and expression vectors encoding GR (0.01 ng), CARM1 (200 ng), TIF1{alpha} (200 ng), and GRIP1 wild type or mutants GRIP1{Delta}N (deletion of amino acids 5–563) or GRIP1{Delta}C (lacking amino acids 1122–1462) (25 ng). C, CV-1 cells were transiently transfected with MMTV-LUC reporter plasmid (250 ng) and expression vectors encoding GR (0.01 ng), GRIP1 (25 ng), TIF1{alpha} (200 ng), and CARM1 wild type, CARM1-N (amino acids 3–460), or CARM1-C (amino acids 461–608) (200 ng). D, CV-1 cells were transiently transfected with MMTV(ERE)-LUC reporter plasmid (250 ng) and expression vectors encoding ER (0.01 ng), GRIP1 (125 ng), TIF1{alpha} (250 ng), and CARM1 wild type or CARM1 methyltransferase-deficient mutants E267Q or VLD (250 ng). In panels A–D, cells were grown for 48 h with dexamethasone or E2. Each data point represents the mean and range of variation of two transfected cell cultures. The results presented are from a single experiment representative of three independent experiments. RLU, Relative light units; WT, wild type.

 
This result further suggested that the cooperation of TIF1{alpha} with GRIP1 and CARM1 resulted from protein-protein interactions with them, rather than direct binding of TIF1{alpha} to NRs. We therefore investigated which domains of GRIP1 were important for synergy with TIF1{alpha} (Fig. 3BGo). Deletion of the N-terminal part of GRIP1 (deletion of amino acids 5–563) abolished the synergistic cooperation among GRIP1, CARM1, and TIF1{alpha}. Because the N-terminal domain of GRIP1 (GRIP1-N) is not required for binding NRs or CARM1 (24, 25), this result indicated that GRIP1-N provided some other function (possibly binding to TIF1{alpha}) that is important for the cooperation with TIF1{alpha}. Deletion of the GRIP1 C-terminal AD2 region also eliminated the synergy between the three coactivators, consistent with the fact that the deleted region contains the binding site for CARM1 (25, 26).

In tests using CARM1 deletion mutants, a mutant lacking CARM1-C (deletion of amino acids 461–608) failed to act synergistically with GRIP1 and TIF1{alpha} (Fig. 3CGo). Similarly, although our yeast two-hybrid data (data not shown) and mammalian one-hybrid results (Fig. 1BGo) demonstrated that CARM1-C can bind to TIF1{alpha}, CARM1-C alone (amino acids 461–608) did not function synergistically with GRIP1 and TIF1{alpha} (Fig. 3CGo), presumably because the central methyltransferase domain of CARM1 provides several essential functions, including the methyltransferase, homooligomerization, and GRIP1-binding activities (21). Thus, CARM1 regions required for binding to both GRIP1 and TIF1{alpha} were necessary for the synergistic effect. To investigate the importance of CARM1 methyltransferase activity in the three-coactivator synergy, we tested two different methyltransferase-deficient mutants of CARM1, which are expressed at wild-type levels. In the presence of GRIP1 and TIF1{alpha}, CARM1(E267Q) (11) and CARM1(VLD) (amino acids VLD189–191 changed to alanines) (25) retained partial activity compared with wild-type CARM1 (Fig. 3DGo). This result indicates that the methyltransferase activity contributes to, but is not absolutely essential for, the GRIP1-CARM1-TIF1{alpha} synergy. This result suggests that CARM1 coactivator function involved not only the methyltransferase activity but also the activity of the C-terminal AD, which presumably contributes to transcriptional activation via protein-protein interactions. It is worth noting that in a similar assay system, the coactivator synergy between GRIP1, CARM1, and p300 depends much more heavily on the methyltransferase activity of CARM1 (11). Thus, the C-terminal AD of CARM1 is especially important for the synergy of this particular combination of coactivators. The fact that TIF1{alpha} binds to and enhances the activity of the C-terminal AD of CARM1 may provide an explanation of the enhanced importance of the CARM1 C-terminal domain in mediating CARM1 synergy with TIF1{alpha}.

A Functional Ternary Coactivator Complex of GRIP1, CARM1, and TIF1{alpha}
The fact that GRIP1-CARM1-TIF1{alpha} synergy depends on the N-terminal domain of GRIP1 (Fig. 3BGo) suggests that TIF1{alpha} may be able to bind to GRIP1-N. In a mammalian one-hybrid assay TIF1{alpha} enhanced transcriptional activation by Gal4 DBD fused to full length GRIP1 or to GRIP1-N (amino acids 5–765), but had little or no effect on Gal4 DBD fused to the GRIP1 C-terminal domain (amino acids 1121–1462) (Fig. 4AGo). This result suggested that TIF1{alpha} preferentially interacted with the N-terminal part of GRIP1. We tested this possible interaction directly by coimmunoprecipitation from transiently transfected COS7 cells (Fig. 4BGo). An antibody against TIF1{alpha} specifically precipitated hemagglutinin (HA)-tagged GRIP1 (5–479), and the detection of HA-GRIP1-N depended on the coexpression of TIF1{alpha}.


Figure 4
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  		Fig. 4. TIF1{alpha} Interacts with the GRIP1 N-Terminal AD3

A, CV-1 cells were transfected with 250 ng of GK1 reporter plasmid; 125 ng of pM vector encoding Gal4 DBD or Gal4 DBD fused to GRIP1 full length (G1), GRIP1-N (G1N, amino acids 5–765), or GRIP1-C (G1C, amino acids 1121–1462); and 400 ng of pSG5 empty vector (white bars) or pSG5.TIF1{alpha} (black bars). Luciferase results shown are the mean and range of variation of two transfected cell cultures and are from a single experiment representative of three independent experiments. B, COS7 cells were transfected with expression vectors for HA-tagged GRIP1-N (amino acids 5–765) (2 µg) and TIF1{alpha} (3 µg), as indicated. Coimmunoprecipitation (IP) and immunoblots (WB) were conducted with the indicated antibodies. RLU, Relative light units.

 
Because TIF1{alpha} binds to CARM1-C as well as to GRIP1-N, and because the central region of CARM1 also binds to the C-terminal region of GRIP1, we tested whether these three coactivators can form a ternary complex. To simplify the analysis and to test the specific importance of the TIF1{alpha} interactions with CARM1-C and GRIP1-N in such a complex, we used only those regions of GRIP1 and CARM1. As shown previously (Fig. 4AGo), the transcriptional activation activity of Gal4DBD-GRIP1-N (amino acids 5–765) was enhanced by TIF1{alpha} (Fig. 5AGo). In contrast, Gal4DBD-GRIP1-N activity was only slightly increased by VP16-CARM1-C (amino acids 461–608), reflecting the fact that GRIP1-N and CARM1-C do not bind each other (25, 27). However, the coexpression of TIF1{alpha} with VP16-CARM1-C caused a synergistic enhancement of Gal4DBD-GRIP1-N activity, suggesting that TIF1{alpha} acts as a bridge between GRIP1-N and CARM1-C. The formation of a ternary complex was further tested by coimmunoprecipitation. When HA epitope-tagged GRIP1-N and CARM1-C were coexpressed in COS7 cells, and immunoprecipitation was performed with antibodies against CARM1-C, a strong GRIP1-N signal was detected in the pellet only if TIF1{alpha} was coexpressed (Fig. 5BGo, right panel, lane 4). In the absence of coexpressed TIF1{alpha}, the GRIP1-N signal was much weaker (lane 1). The weak GRIP1-N band in lane 2 is presumably due to the presence of endogenous CARM1 in COS7 cells. Thus, GRIP1, CARM1, and TIF1{alpha} may form a ternary coactivator complex associated with NRs (Fig. 5CGo).


Figure 5
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  		Fig. 5. TIF1{alpha} Stabilizes a Complex Containing GRIP1-N and CARM1-C

A, CV-1 cells were transfected with 250 ng of GK1 reporter plasmid, 125 ng of pM vector encoding Gal4DBD or Gal4DBD fused to GRIP1-N (amino acids 5–765), 125 ng of pVP16 vector encoding VP16 AD or VP16-CARM1-C (amino acids 461–608), and 400 ng of pSG5 empty vector (white bars) or pSG5.TIF1{alpha} (black bars). Each data point represents the mean and range of variation of two transfected cell cultures. The results presented are from a single experiment representative of three independent experiments. B, COS7 cells were transfected with expression vectors for HA-tagged GRIP1-N (amino acids 5–479) (1 µg), HA-tagged CARM1-C (amino acids 461–608) (1 µg), and TIF1{alpha} (3 µg), as indicated. Coimmunoprecipitation (IP) and immunoblots (WB) were conducted with the indicated antibodies. C, Model for a functional ternary complex between GRIP1, CARM1, and TIF1{alpha}. N and C within protein diagrams represent N-terminal and C-terminal domains. NRs bind to a hormone response element (HRE) and recruit coactivators. GRIP1, CARM1, and TIF1{alpha} form a ternary complex. GRIP1 binds to NRs with its LXXLL motifs, to TIF1{alpha} with its N-terminal AD3, and to CARM1 with its C-terminal AD2. CARM1 binds to GRIP1 with its central methyltransferase domain and to TIF1{alpha} with its C-terminal AD. TIF1{alpha} binds to CARM1-C and to GRIP1-N using unknown domains, and it may interact with NRs through its LXXLL motif. Some coactivators contribute to the assembly of the RNA polymerase II transcription initiation complex (Pol II TIC) by catalyzing posttranslational modifications of histones and other proteins, using cofactors S-adenosylmethionine (SAM) or acetyl CoA (AcCoA). Other coactivators contribute through direct or indirect protein-protein interactions with the transcription machinery. RLU, Relative light units.

 
Role of Endogenous TIF1{alpha} in Mediating Transcriptional Activation by CARM1-C, GRIP1-N, and AR
To assess the role of endogenous TIF1{alpha} in transcriptional activation by CARM1-C and GRIP1-N, we reduced TIF1{alpha} expression in COS7 cells by using a specific short interfering RNA (siRNA). The siRNA against TIF1{alpha}, but not the scrambled-sequence control siRNA, specifically reduced the level of endogenous TIF1{alpha} protein but not ß-actin protein (Fig. 6AGo, top panel). Reduction of TIF1{alpha} levels in COS-7 cells also reduced reporter gene activation by a Gal4DBD-GRIP1-N fusion protein by about half but surprisingly had no effect on the activity of the Gal4DBD-CARM1-C fusion protein (Fig. 6AGo, lower panels). Thus, endogenous TIF1{alpha} is required for efficient transcriptional activation by the GRIP1-N. In contrast, although overexpression of exogenous TIF1{alpha} enhanced transcription mediated by CARM1-C fused to Gal4 DBD (Fig. 1BGo), inhibition of endogenous TIF1{alpha} expression had little or no effect on transcriptional activation by CARM1-C, suggesting that the strong activation activity harbored by CARM1-C could be mediated by factors other than TIF1{alpha}.


Figure 6
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  		Fig. 6. Requirement of Endogenous TIF1{alpha} for Mediating Transcriptional Activation by GRIP1-N, CARM1-C, and AR

A, COS7 cells in 24-well plates were transfected with no siRNA (white bars), 90 pmol of the TIF1{alpha}-specific siRNA duplex (siTIF1{alpha}) (black bars), or 90 pmol of scrambled-sequence control siRNA duplex (siSC) (gray bars). After 1 d, cells were transfected with pG5-Luc reporter plasmid (200 ng) and expression vector encoding Gal4DBD-GRIP1-N (400 ng) or Gal4DBD-CARM1-C (100 ng). Luciferase activity was quantified 24 h after DNA transfection. Each data point represents the mean and range of variation of three transfected cell cultures. The results presented are from a single experiment representative of three independent experiments. Top panel, Immunoblots of extracts from COS7 cells subjected to the same siRNAs treatment and analyzed with anti-TIF1{alpha} and anti-ß-actin antibodies. B, LNCaP cells were transfected with the indicated amounts of siRNA against TIF1{alpha} or control scrambled-sequence siRNA. After 72 h, cells were treated with DHT and then harvested after an additional 24 h. Total RNA was used for reverse transcription, and the resulting cDNA was analyzed by real-time PCR to measure levels of ß-actin, TIF1{alpha}, and PSA mRNA. Each sample was run in duplicate, and average CT values (with range of variation) for TIF1{alpha} and PSA mRNA were normalized to that of ß-actin. Results shown are from a single experiment, which is representative of four independent experiments. RLU, Relative light units.

 
To test the role of endogenous TIF1{alpha} in NR function, the same siRNAs were used to reduce TIF1{alpha} expression in the LNCaP prostate cancer cell line. The siRNA against TIF1{alpha} reduced the level of TIF1{alpha} mRNA by about 75% relative to levels of ß-actin mRNA (Fig. 6BGo, top panel). Treatment of LNCaP cells with dihydrotestosterone (DHT) strongly induced the expression of the prostate-specific antigen (PSA) gene (lower panel), which is a known target of the hormone-activated AR (28), but had a modest or no effect on the levels of TIF1{alpha} mRNA (upper panel) or ß-actin mRNA (data not shown). The TIF1{alpha}-specific siRNA reduced the hormone-induced PSA mRNA level by up to 45%, whereas the scrambled-sequence control siRNA had no effect (Fig. 6BGo, bottom panel). Thus, endogenous TIF1{alpha} is required for efficient induction of endogenous target gene expression by the hormone-activated AR.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
The p160 Coactivator Pathway
Coactivator complexes function as signal transduction pathways that transmit the activating signal from the DNA-bound NR to the transcription machinery. Each component of a coactivator complex has specific domains for interacting with upstream and downstream components of the signaling pathway (6). The p160 coactivators, which are primary coactivators because they bind directly to NRs, contain intrinsic ADs to recruit secondary coactivators as downstream components of the signaling pathway (Fig. 5CGo). AD1, located near amino acid 1000, binds p300 and CBP (29, 30), two related proteins that serve as coactivators for many different families of DNA-binding transcriptional activator proteins, including NRs (7). The C-terminal AD2 recruits the methyltransferases CARM1 and PRMT1 (17, 25, 26, 30). AD3, recently identified in the N-terminal region of the p160 proteins (31), recruits secondary coactivators coiled-coil coactivator (CoCoA) (31), flightless-I (Fli-I) (32), and GRIP1-associated coactivator 63 (GAC63) (33). The N-terminal domain also binds to other proteins that have been characterized as coactivators, including hMMS19 (34), BAF57 (35), and cyclin T1 (36). Because the sequence of the 350 N-terminal amino acids is 60% identical among the three p160 family members (37), it is perhaps not surprising that many different proteins can bind to this large, highly conserved domain. The present study adds TIF1{alpha} to the growing list of proteins that cooperate with p160 coactivators by binding to the N-terminal domain (Fig. 4BGo). TIF1{alpha} also enhances transcriptional activation by AD3 (Fig. 4AGo). Moreover, inhibition of endogenous TIF1{alpha} expression by siRNA reduced the ability of AD3 to activate transcription of a transient reporter gene and also specifically compromised the transcriptional activation of an endogenous target gene by hormone-activated AR (Fig. 6Go). Whether endogenous TIF1{alpha} is also required for efficient transcriptional activation by all, or only some, NRs remains to be investigated. Thus, our data indicate that TIF1{alpha} has a physiologically relevant role in transcriptional activation by at least some NRs and possibly by other transcription factors that collaborate with p160 coactivators and CARM1.

A recent study found that reducing the endogenous level of CARM1 or of any one of the three p160 coactivators [steroid receptor coactivator-1 (SRC-1), GRIP1/TIF2, or amplified in breast cancer 1 (AIB 1)] in LNCaP cells had little or no effect on transcriptional activation of the PSA gene by AR. The authors concluded that CARM1 may not be required for PSA gene activation and that loss of one p160 coactivator is compensated by the other two p160 coactivators (38). Our results demonstrate a physiologically relevant role for TIF1{alpha} in transcriptional activation by NRs and suggest a specific molecular mechanism by which TIF1{alpha} cooperates with GRIP1 and CARM1 in transient transfection assays. However, the specific molecular mechanism of TIF1{alpha} coactivator function and cooperation with the many other coactivators involved in transcriptional activation of endogenous genes remains to be determined. In addition, our current state of knowledge suggests that specific coactivator requirements will vary for different promoters (39, 40). Because the N-terminal region of the p160 coactivators is very highly conserved, we assume that TIF1{alpha} can bind to all three p160 family members. Thus, TIF1{alpha} could theoretically be recruited to endogenous promoters by interaction with any of the p160 coactivators or with CARM1.

Multiple Roles for CARM1 in the Coactivator Signaling Pathway
In response to the appropriate hormone, CARM1 is recruited to the promoters of NR target genes through its interaction with the C-terminal AD2 of p160 coactivators (25, 27). Its association with hormone-dependent promoters coincides with methylation of histone H3 on Arg-2, Arg-17, and Arg-26 in the vicinity of the promoter (13, 14, 15). A requirement for the methyltransferase activity of CARM1 in transcriptional activation was observed in transient transfection assays (11), and the importance of histone methylation by CARM1 has been demonstrated in a cell-free transcription system using reconstituted chromatin templates (16). However, the specific mechanism by which histone methylation contributes to transcriptional activation is unknown. CARM1 also methylates p300 (41, 42, 43) and presumably other nonhistone components of the transcription machinery; the mechanistic contributions of arginine-specific methylation of nonhistone proteins is also still under investigation.

In addition to the methyltransferase activity of CARM1, the strong autonomous AD in the C-terminal region of CARM1 is also required for CARM1 coactivator activity (21). Because this C-terminal domain is not required for binding to p160 coactivators, homooligomerization, or methyltransferase activity, it presumably contributes to the coactivator function of CARM1 by binding to another protein, which is a downstream component of the p160 coactivator signaling pathway. The present study characterized TIF1{alpha} as a CARM1-C-interacting protein. Although CARM1-C has a very powerful autonomous activation activity, overexpression of TIF1{alpha} further enhanced CARM1-C activity (Fig. 1BGo). In addition, TIF1{alpha} cooperated synergistically with CARM1 and GRIP1 to enhance transcriptional activation by several different NRs (Fig. 2Go), and the C-terminal AD of CARM1 was critical for that synergy (Fig. 3CGo). Although these results implicate TIF1{alpha} as a mediator of the function of the CARM1 C-terminal AD, inhibition of endogenous TIF1{alpha} did not reduce CARM1 C-mediated transcription (Fig. 6AGo). These results leave the physiological role of TIF1{alpha} in mediating transcriptional activation by CARM1-C unresolved and suggest that factors other than TIF1{alpha} participate in mediating the strong autonomous activation activity of CARM1-C. However, in spite of this uncertainty, our results clearly show a synergistic functional relationship between TIF1{alpha}, CARM1, and GRIP1; the ability of TIF1{alpha} to nucleate the formation of a ternary complex among these three coactivators; and a requirement for endogenous TIF1{alpha} for efficient transcriptional activation of transient and endogenous target genes by GRIP1-N and by AR.

TIF1{alpha}, a Secondary Coactivator for NRs
TIF1{alpha}, which was initially identified in a yeast genetic screen for proteins that enhance transcriptional activation by retinoid X receptors, interacts selectively with NRs (retinoic acid receptor, retinoic X receptor, ER) in an agonist-dependent fashion in vitro, in yeast, and in mammalian cells (22), using an LXXLL motif similar to those found in other coactivators that bind to the AF-2 activation functions of NRs (23, 44). Although these findings suggested that TIF1{alpha} would prove to be a transcriptional coactivator for NRs in mammalian cells, enhancement of NR activity by TIF1{alpha} has been difficult to demonstrate (22, 23). Instead, TIF1{alpha} exhibited a transcriptional repression activity when fused to a heterologous DBD (23, 45) or when overexpressed with NRs, suggesting that it was a corepressor or was sequestering important components of the transcription machinery (22). Our results may explain why TIF1{alpha} coactivator function was not observed in these earlier experiments. In our experimental system, TIF1{alpha} did not function as a coactivator by itself, but rather only in cooperation with GRIP1 and CARM1 (Figs. 2Go and 3Go). Furthermore, although TIF1{alpha} can use its LXXLL motif to bind directly to the NR AF-2 region (23), our results indicate that the LXXLL motif is not required for the synergistic coactivator function of TIF1{alpha} with GRIP1 and CARM1 (Fig. 3AGo). Although it had no direct functional effect on NRs, TIF1{alpha} bound to and enhanced the activity of both the C-terminal AD of CARM1 and the N-terminal AD of GRIP1. Thus, in spite of its direct binding to NRs, TIF1{alpha} functions as a secondary coactivator for NRs. The previously observed squelching activity of overexpressed TIF1{alpha} (22) may have been due to sequestration of GRIP1 or CARM1, both of which bind to TIF1{alpha}.

TIF1{alpha} contains an N-terminal RBCC (Ring finger, B boxes, coiled coil) motif, a poorly conserved central region, an NR box, and a C-terminal region with a PHD finger and a bromodomain (Fig. 1AGo) (22, 45). The latter two domains are also found in other transcriptional cofactors and chromatin-remodeling proteins. TIF1{alpha} belongs to a family of nuclear proteins that also includes TIF1ß, TIF1{gamma}, and TIF1{delta}, all of which are believed to regulate chromatin structure (23, 45, 46, 47). TIF1{alpha} is associated with highly accessible euchromatic regions (48), whereas TIF1ß and TIF1{delta} associate with both euchromatin and heterochromatin (45, 46). TIF1{alpha} as well as TIF1ß and TIF1{delta} interact with members of the heterochromatin protein (HP1) family (23, 45, 46). However, although the HP1-binding domain of TIF1ß is critical for silencing of transcription (45) and association with heterochromatin (49, 50), Gal4 fusions of TIF1{alpha} repress transcription independently of HP1 binding through a mechanism involving histone deacetylation (45). Note also that with respect to our present data supporting a role for TIF1{alpha} in transcriptional activation, there is now an indication that HP1 can mediate either positive or negative transcriptional regulation (Refs. 51 and 52 and references therein). TIF1{alpha} is a phosphoprotein that undergoes ligand-dependent hyperphosphorylation upon NR binding in vivo, and it also has a kinase activity that can phosphorylate basal transcription factors (TFIIE{alpha}, TAFII28, and TAFII55) (53) and also the HP1 proteins (45) in vitro.

TIF1{alpha} can associate simultaneously with GRIP1-N and CARM1-C (Fig. 5Go). Deletion analysis showed that the synergy observed between GRIP1, CARM1, and TIF1{alpha} required GRIP1-N and CARM1-C, which are both binding sites for TIF1{alpha} (Fig. 3Go, B and C). The fact that each of these three coactivators can bind to the other two suggests that TIF1{alpha} forms a stable ternary complex with CARM1 and a p160 coactivator, which presumably contributes to the observed coactivator synergy of these three coactivators. In addition, because TIF1{alpha} also enhances the activities of the GRIP1-N and CARM1-C ADs, TIF1{alpha} may also make contact with and activate currently unknown downstream target(s) that are important for transcriptional activation. The downstream signaling by TIF1{alpha} could be through protein-protein interactions or by phosphorylation of a downstream protein by the intrinsic TIF1{alpha} kinase activity. We therefore propose a new role for TIF1{alpha} as a stabilizing member of the p160 coactivator complex and a mediator of essential GRIP1 and CARM1 ADs.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Plasmids
Proteins with N-terminal hemagglutinin A (HA) epitope tags were expressed in mammalian cells from vector pSG5.HA, which has simian virus 40 and T7 promoters (25). pSG5.HA-GRIP1, pSG5.HA-GRIP1({Delta}N) (encoding amino acids 563–1462), pSG5.HA-GRIP1({Delta}C) (encoding amino acids 5–1121) (26), pSG5.HA-GRIP1-N (encoding amino acids 5–479) (31), pSG5.HA-CARM1 (wild-type or VLD mutant) (25), pSG5.HA-CARM1(E267Q) mutant (11), pSG5.HA-CARM1-N (encoding amino acids 3–460), and pSG5.HA-CARM1-C (encoding amino acids 461–608) (21) were previously described. Other previously described mammalian expression vectors were: pHE0, encoding human ER{alpha} (54); pKSX encoding mouse GR (55); pCMXhTRß1 encoding human thyroid receptor (TR) ß1 (56); pCMV-AR encoding human AR (57); pSG5.TIF1{alpha} and pSG5.TIF1{alpha}(L/A) encoding TIF1{alpha} wild type and a TIF1{alpha} mutant with the LXXLL motif altered to LXXAA (23); the luciferase reporter plasmids mouse mammary tumor virus (MMTV)(ERE)-LUC for ER, MMTV-LUC for GR and AR (58), MMTV(TRE)-LUC for TR, and GK1 (59) or pG5-Luc (Promega Corp., Madison, WI) for Gal4 DBD; pM.CARM1 encoding Gal4DBD fused to full-length CARM1, pM.CARM1-N encoding Gal4DBD-CARM1 (3–460); pM.CARM1-C encoding Gal4DBD-CARM1 (461–608) (21); pM.GRIP1 encoding Gal4DBD fused to full-length GRIP1 and pM.GRIP1-C encoding Gal4DBD-GRIP1 (1121–1462) (26); and pM.GRIP1-N encoding Gal4DBD-GRIP1 (5–765) (27). pVP16-CARM1-C was constructed by inserting PCR-amplified cDNA encoding CARM1-C (amino acids 461–608) into EcoRI and BamHI sites of pVP16 (CLONTECH Laboratories, Inc., Palo Alto, CA).

Yeast Two-Hybrid Screen for Proteins that Interact with CARM1-C
The yeast two-hybrid system was employed essentially as described previously (31). cDNA encoding CARM1-C (amino acids 461–608) was cloned into the EcoRI and BamHI restriction sites downstream from the Gal4 DBD coding sequences in pGBT9 (CLONTECH) to generate a bait plasmid. The yeast strain HF7c (containing his3 and lacZ genes controlled by Gal4-responsive elements) was sequentially transformed with the pGBT9-CARM1-C plasmid and a 17-d mouse embryo cDNA library in pGAD10 (CLONTECH). A histidine jump-start procedure was performed to improve the chances of detecting weak interactions. Transformants (106) were first plated on synthetic complete media plates lacking leucine and tryptophan, incubated until colonies appeared, and harvested. The amplified transformants (~107) were plated onto synthetic complete media plates lacking histidine, leucine, and tryptophan, and containing 50 mM 3-amino-1,2,4-triazole to suppress low-level expression of His3 due to autonomous transcriptional activation activity of the CARM1-C bait. This selection resulted in the growth of 48 colonies, of which 25 were subsequently confirmed as positive by testing for the expression of ß-galactosidase.

Coimmunoprecipitation and Immunoblot Experiments
The procedures were performed as described previously (11) with anti-TIF1{alpha} (Chemicon, Temecula, CA), anti-ß-actin (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), anti-CARM1 (Upstate Biotechnology, Inc., Lake Placid, NY), and anti-HA (Roche Clinical Laboratories, Indianapolis, IN) antibodies (1 µg of antibody for immunoprecipitation and 1:1000 dilution for immunoblots).

Cell Culture, Transfection, and siRNA Treatment
COS7 and CV-1 cells (60) were maintained in DMEM supplemented with 10% fetal bovine serum. Transient transfections were performed as described previously (11). Empty vectors were added to all transfections to balance the total amount of DNA used for each transfection. When hormone was required, transfected cells were grown during the last 30 h before harvest in medium supplemented with 5% charcoal/dextran-stripped fetal bovine serum and 20 nM of the appropriate hormone for each NR: estradiol (E2) for ER, dexamethasone for GR, T3 for TR, and DHT for AR. Luciferase assays were performed with the Promega Luciferase Assay Kit according to manufacturer’s protocols. The siRNA oligonucleotides for human TIF1{alpha} were designed using the Target Finder program (Ambion, Inc., Austin, TX) and chemically synthesized by the USC Norris Comprehensive Cancer Center Microchemical Core: siRNA sense, 5'-UGGGUUUUGUGUAGAGUGUTT-3'; siRNA antisense, 5'-ACACUCUACACAAAACCCATT-3'. The annealed siRNAs were transfected into COS7 cells with lipofectamine 2000 (Invitrogen, San Diego, CA) 24 h before DNA transfection.

LNCaP cells were seeded into six-well plates with RPMI 1640 containing 5% charcoal-dextran-stripped FBS and grown until reaching 50% confluence at the day of siRNA transfection. The transfection complex of siRNA and 1.25 µl siLentFect (Bio-Rad Laboratories, Inc., Hercules, CA) was added into each well, and after 3 d, some wells were treated with 20 nM DHT. After an additional day, total RNA were extracted using Trizol reagent (Invitrogen), and first-strand cDNA was synthesized by reverse transcribing 0.4 µg of total RNA using iScript cDNA Synthesis Kit (Bio-Rad). Out of 20 µl total volume of the reverse transcription reaction, 2 µl were used as template in quantitative real-time PCR, using a Stratagene Mx3000P instrument. SYBR Green QPCR Master Mix (Stratagene, La Jolla, CA) and 150 nM of forward and reverse primers were used in 40 PCR cycles as follows: 95 C for 15 sec, 55 C for 2 min, and 72 C for 30 sec. After amplification, a melting curve analysis was performed to confirm the homogeneity of products from each reaction. The primers used were as follows: TIF1{alpha}, 5'-AGTCATTCGTTGCCCAGTTTGCAG (forward) and 5'-TCTGCGTTGTCCTCACAGCTTGTA (reverse); PSA, 5'-TCACAGCTACCCACTGCATCA-3' (forward) and 5'-AGGTCGTGGCTGGAGTCATC-3' (reverse); ß-actin, 5'-ACCCCATCGAGCACGGCATCG-3' (forward) and 5'-GTCACCGGAGTCCATCACGATG-3' (reverse). Each sample was run in duplicate to obtain average comparative threshold (CT) values for TIF1{alpha}, PSA, and ß-actin mRNA. TIF1{alpha} and PSA mRNA values were normalized to that of ß-actin. The CT method was used with standard curves to determine the relative amount of PSA and TIF1{alpha} mRNA.


   ACKNOWLEDGMENTS
 
We thank Dan Gerke (University of Southern California) for technical help.


   FOOTNOTES
 
This work was supported by National Institutes of Health Grant DK55274 (to M.R.S).

Disclosure of potential conflicts: C.T., C.-Y.O., K.K., R.L. have nothing to declare. M.R.S. receives royalties from Upstate Biotechnology, Inc., and received lecture fees from Bristol Myers-Squibb and Wyeth Pharmaceuticals.

First Published Online December 1, 2005

Abbreviations: AD, Activation domain; AR, androgen receptor; CARM1, coactivator-associated arginine methyltransferase 1; CBP, CREB-binding protein; DBD, DNA-binding domain; DHT, dihydrotestosterone; ER, estrogen receptor; GR, glucocorticoid receptor; GRIP1, GR-interacting protein 1; GRIP1-N, N-terminal domain of GRIP1; MMTV, mouse mammary tumor virus; NR, nuclear receptor; PRMT, protein arginine methyltransferase; PSA, prostate-specific antigen; siRNA, short interfering RNA; TIF1{alpha}, transcriptional intermediary factor 1{alpha}; TR, thyroid hormone receptor.

Received for publication September 23, 2005. Accepted for publication November 21, 2005.


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NURSA Molecule Pages Link:

Nuclear Receptors:   TRβ  |  ERα  |  GR  |  AR
Coregulators:   TIF1α  |  TIF1β  |  CARM1  |  GRIP1
Ligands:   Dexamethasone  |  17β-Estradiol  |  Dihydrotestosterone  |  Thyroid hormone



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