Identification of Mouse TRAP100: a Transcriptional Coregulatory Factor for Thyroid Hormone and Vitamin D Receptors

Jiachang Zhang and Joseph D. Fondell

Department of Physiology University of Maryland School of Medicine Baltimore, Maryland 21201


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Nuclear hormone receptors (NRs) regulate transcription in part by recruiting distinct transcriptional coregulatory complexes to target gene promoters. The thyroid hormone receptor (TR) was recently purified from thyroid hormone-cultured HeLa cells in association with a complex of novel nuclear proteins termed TRAPs (thyroid hormone receptor-associated proteins) ranging in size from 20 to 240 kDa. The TRAP complex markedly enhances TR-mediated transcription in vitro, suggesting a coactivator role for one or more of the TRAP components. Here we present the mouse cDNA for the 100-kDa component of the TRAP complex (mTRAP100). The mTRAP100 protein contains seven LxxLL motifs thought to be potential binding surfaces for liganded NRs, yet surprisingly fails to interact with TR and other NRs in vitro. By contrast, mTRAP100 coprecipitates in vivo with another component of the TRAP complex (TRAP220), which directly contacts TR and the vitamin D receptor in a ligand-dependent manner. Our findings thus suggest that TRAP100 is targeted to NRs in association with TRAP complexes specifically containing TRAP220. Transient overexpression of mTRAP100 in mammalian cells further enhances ligand-dependent transcription by both TR and the vitamin D receptor, revealing a functional role for mTRAP100 in NR-mediated transactivation. The presence of an intrinsic mTRAP100 transactivation function is suggested by the ability of mTRAP100 to activate transcription constituitively when tethered to the GAL4 DNA-binding domain. Collectively, these findings suggest that TRAP100, in concert with other TRAPs, plays an important functional role in mediating transactivation by specific NRs.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Members of the nuclear hormone receptor (NR) superfamily are potent regulators of gene expression in response to their cognate physiological ligands which include steroids, retinoids, thyroid hormone, and vitamin D3 (1, 2, 3). NRs have a modular structure consisting of three separable domains: 1) a highly conserved DNA-binding domain containing two zinc finger motifs (2, 3); 2) a poorly conserved amino-terminal domain which, at least for some NRs, contains an autonomous activation function (AF-1) (2, 3, 4, 5); and 3) a carboxy-terminal ligand-binding domain containing a dimerization surface and an additional activation function (AF-2), which is essential for ligand-dependent activation (2, 3, 6, 7, 8, 9). Importantly, the AF-2 core domain comprises a highly conserved amphipathic {alpha}-helical motif (2, 3) that is present in all known transcriptionally active members of the NR superfamily (3, 6, 7, 8, 9). Both AF-1 and AF-2 activities depend on promoter and tissue type (4, 5), and both activate transcription in yeast (which do not express endogenous NRs), thus indicating that the molecular mechanisms of NR gene regulation have been conserved during evolution (10, 11).

A great wealth of evidence implicates the involvement of specific coregulatory factors in NR-mediated gene regulation (for reviews see Refs. 12, 13). Remarkably, yeast two-hybrid screens using NR-AF-2 regions as bait have led to the identification of a wide array of putative NR-coactivators including TRIP1 (14), SRC-1 (15), ARA70 (16), TIF1 (17), GRIP1 (18), RAC3/ACTR/AIB1/TRAM-1 (19, 20, 21, 22), p120 (23), TRIP230 (24), PGC-1 (25), and PBP (26). Similarly, conventional expression library screens have identified other presumptive coactivators including p160 (renamed NCoA-1) (27), RIP140 (28), TIF2 (29), RAP46 (30), and p/CIP (31). Most of these proteins display one or more copies of a consensus {alpha}-helical motif of repeated leucines (LxxLL), which are thought to be necessary for ligand-dependent interactions with the AF-2 domain of NRs (31, 32). Of note, SRC-1/p160, TIF2/GRIP1, RAC3/ACTR/AIB1/TRAM-1, and p/CIP all share sequence homologies suggesting a novel family of SRC-like NR-coactivators.

Recent studies demonstrated that the pleiotropic coactivator CBP/p300 forms a ternary complex with NRs and various SRC-like cofactors and is functionally required for ligand-dependent activation (20, 27, 33). Given that CBP/p300 is a histone acetyltransferase (HAT) (34), and a known correlation exists between hyperacetylated histones and derepressed chromatin (35), these findings are consistent with a NR role in chromatin remodeling. Interestingly, intrinsic HAT activity has also been attributed to some of the SRC-like coactivators (20, 33). On the basis of these and other studies, a model has been proposed in which liganded NRs are thought to activate gene expression by promoter-specific recruitment of HAT activity (20, 27, 31, 33). Other studies, however, using chromatin-based transcription assays indicate that ligand-induced disruption of chromatin by NRs may be insufficient for transcriptional activation (36, 37). The chromatin studies thus suggest that additional accessory factors and additional steps may be necessary for the liganded-NR to productively interface with the basal transcription apparatus and activate gene transcription.

As an alternative method of identifying specific nuclear factors that interact with NRs and possibly potentiate their function, a HeLa-derived cell line was generated that stably expresses an epitope-tagged thyroid hormone receptor (TR) (38). When immunopurified from T3-treated cells, TR is specifically associated with a distinct set of novel nuclear proteins termed TRAPs (TR-associated proteins) ranging in size from 20 to 240 kDa. Cell-free transcription assays demonstrated that the TRAP complex dramatically enhances TR-mediated transcription from thyroid response element (TRE)-linked promoter templates (38). It was therefore hypothesized that specific TRAPs, either collectively or individually, might function as TR-specific transcriptional coactivators. Evidence supporting a more common TRAP coactivator role for other NRs comes from the recent purification of an apparently identical complex of proteins (termed DRIPs) that specifically interact with the vitamin D receptor (VDR) and enhance VDR transactivation in vitro (39). Cognate human cDNAs for the 220-kDa and 100-kDa components of the TRAP complex (hTRAP220 and hTRAP100) have been reported (40). Sequence homologies show that hTRAP100 is nearly identical to the 100-kDa component of the human DRIP complex (DRIP100) (39) and that TRAP220 is the probable human ortholog of the mouse-derived PPAR-binding protein (26). On the basis of its ability to interact with TR and other NRs in an avid ligand-dependent fashion (40), TRAP220 has been proposed to act as an anchoring factor, possibly serving to target other TRAP components to a liganded NR during TRAP complex-mediated coactivation. In contrast, the functional role played by the TRAP100 component is ill understood.

In this study, we present the cDNA sequence of the mouse TRAP100 (mTRAP100) gene. We provide evidence that mTRAP100 enhances ligand-dependent transcriptional activation by both TR and VDR when transiently overexpressed in cultured mammalian cells. Although mTRAP100 fails to exhibit strong direct interactions with various NRs, the findings presented here indicate that TRAP100 is targeted to a liganded NR through TRAP protein complexes specifically containing the NR-interacting factor TRAP220. When tethered to a heterologous DNA-binding domain, mTRAP100 activates transcription constituitively, possibly revealing intrinsic transactivation functions. We suggest that the TRAP100 component of the TRAP coactivator complex may play an important functional role in facilitating TRAP-mediated enhancement of NR- signaling pathways.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Identification and Structure of the mTRAP100 Gene
The recent cloning of the mouse homolog for hTRAP220 demonstrated that specific components of the TRAP coactivator complex are conserved among mammals (26). To determine whether the TRAP100 component was also conserved in mice, we screened a mouse-expressed sequence tag (EST) data base (41) with random peptide sequences derived from the hTRAP100 protein (40). Several mouse cDNA clones with significant homology were identified, obtained (from American Type Culture Collection, Manassas, VA) and completely sequenced. The largest cDNA clone contains an open reading frame of 2868 bp flanked by 5'- and 3'-untranslated regions and encodes a polypeptide of 956 amino acids with a predicted molecular mass of 106 kDa (Fig. 1AGo). The ATG start codon was defined by the presence of several upstream in-frame stop codons within the putative 5'-untranslated sequence. In vitro translation of the open reading frame containing or lacking the 5'-untranslated region generates proteins of equal molecular mass, thus confirming the coding sequence start site (data not shown). The relatively short 3'-untranslated region (298 bp) contains a consensus AAUAAA sequence 12 bp upstream of the poly(A) tail, further defining the 3'-end of the cDNA (data not shown). Sequence comparison of the translated open reading frame reveals a 91% identity and 95% similarity to the hTRAP100 protein (Fig. 1BGo). We have thus termed the protein encoded by this cDNA as mTRAP100. To verify that the cloned mTRAP100 cDNA corresponds to the 100-kDa component of the TRAP coactivator complex, polyclonal antibodies were raised against a portion of the expressed mTRAP100 protein and used to probe the purified hTRAP complex by Western blot. As shown in Fig. 1CGo, anti-mTRAP100 antibodies specifically recognize a 100-kDa protein in a crude Hela cell nuclear extract as well as in the purified TR/TRAP coactivator complex.



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Figure 1. Sequence and Expression of mTRAP100

A, Amino acid sequence of mTRAP100. The underlined sequences indicate the LxxLL motifs. B, Schematic comparison of the human and mouse TRAP100 proteins. The relative positions of the conserved ATP/GTP binding site motif or P-loop and the leucine-zipper region are indicated. The six conserved LxxLL motifs are indicated by black vertical bars. The seventh additional LxxLL motif found in the mTRAP100 protein is indicated by an asterisk. The horizontal line in the hTRAP100 protein represents a 19-amino acid deletion relative to the mTRAP100 sequence. C, Anti-mTRAP100 antibodies specifically recognize the TRAP100 component of the purified TR/TRAP coactivator complex. Fifty micrograms of HeLa nuclear extract or 660 ng of the f:TR{alpha}/TRAP coactivator complex immunopurified from {alpha}-2 cells (38 ) were fractionated by SDS/PAGE, transferred to a nitrocellulose membrane, and probed with rabbit polyclonal antisera raised against the amino-terminal 52 amino acids of mTRAP100 ({alpha}-mTRAP100) by Western blot. Preimmune serum obtained from rabbit before immunization with mTRAP100 is shown as a control. D, Northern blot analysis of mTRAP100 expression in different mouse tissues. A membrane containing 2 µg of poly(A)+ RNA from the indicated tissues was probed with a 417-bp 32P-labeled DNA fragment spanning the carboxy-terminal 139 amino acids of the mTRAP100.

 
Although highly homologous to its human counterpart, mTRAP100 lacks the amino-terminal 50 amino acids found on the hTRAP100 protein (Fig. 1BGo). Interestingly, mTRAP100 contains seven LxxLL motifs (Fig. 1AGo) previously implicated as signature NR-interaction protein surfaces (31, 32). Six of the signature motifs are conserved in both mTRAP100 and hTRAP100 in the same relative locations (Fig. 1BGo), whereas the seventh additional LxxLL motif found in mTRAP100 is located at positions 699–703 (Fig. 1Go, A and B). The mTRAP100 protein also contains a consensus ATP/GTP-binding site motif or P-loop at positions 407 to 414 and a presumptive leucine zipper motif at positions 734 to 755 (Fig. 1Go, A and B). Both the P-loop and leucine zipper are highly conserved within the hTRAP100 protein (Fig. 1BGo), possibly indicating that these motifs facilitate important functional or regulatory roles for the TRAP100 proteins. To examine whether expression of the mTRAP100 gene exhibits tissue specificity, we performed Northern blot analysis using poly-A+ RNA from eight different mouse tissues (Fig. 1DGo). The mTRAP100 mRNA displays relatively low expression in the spleen, lung, and skeletal muscle, whereas the mRNA is abundantly expressed in the testes, heart, and brain (Fig. 1DGo).

mTRAP100 and TRAP220 Interactions with Nuclear Receptors
The simple LxxLL signature motif is thought to provide a binding surface for liganded NRs, and its presence within some transcriptional coactivators is both necessary and sufficient for functional NR interactions (31, 32). The presence of seven such signature motifs within the mTRAP100 protein (Fig. 1AGo) therefore suggested that mTRAP100 might be a direct target for NR interactions. To test this hypothesis, we expressed full-length mTRAP100 as glutathione S-transferase (GST)-fusion protein in Escherichia coli and used the purified protein in GST pull-down assays. Surprisingly, mTRAP100 failed to bind either TR{alpha} or TRß (Fig. 2AGo) in either the presence or absence of T3. Furthermore, mTRAP100 failed to bind the retinoic acid receptor-{alpha} (RAR{alpha}), the rat vitamin D receptor (rVDR), and the progesterone receptor (PR) (Fig. 2AGo) regardless of the presence or absence of ligand. These findings suggest that mTRAP100 is not a direct target for ligand-dependent interactions with NRs and that recruitment of mTRAP100 to NR-regulated promoters presumably requires other TRAP components.



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Figure 2. mTRAP100 and TRAP220 Interactions with Nuclear Receptors in Vitro

A, mTRAP100 fails to interact with liganded nuclear receptors in vitro. GST-mTRAP100 fusion protein (1 µg) was incubated with [35S]methionine-labeled hTR{alpha}, hTRß, rVDR, hRAR{alpha}, and hPR in the presence or absence of ligand as indicated (see Materials and Methods). B, TRAP220 strongly interacts with TR and VDR in a hormone-dependent manner. GST (1 µg) or GST-TRAP220-RBD fusion protein (1 µg) containing the TRAP220 receptor-binding domain (a.a. 622–701) (40 ) was incubated with [35S] methionine-labeled hTR{alpha}, hTRß, hVDR, and rVDR in the presence or absence of ligand as indicated. In each panel, 25% of the labeled input is indicated for each reaction.

 
LxxLL motifs have also been identified within the TRAP220 component of the TRAP complex and were recently implicated in mediating the observed hormone-dependent interactions between TRAP220 and various NRs (40). In stark contrast to our results with mTRAP100, we found that a GST fusion protein containing only one TRAP220 LxxLL motif effectively binds to both TR and VDR in a convincing ligand-dependent manner (Fig. 2BGo). This finding is consistent with TRAP220’s proposed role as an intermediary factor that presumably targets other TRAPs to liganded NRs. Moreover, and in light of mTRAP100’s inability to contact NRs despite the presence of multiple consensus LxxLL motifs, these data support the notion that specific amino acid residues flanking the consensus LxxLL motifs of NR-coregulatory factors are critical determinants in modulating the affinity of the NR interaction (42, 43).

mTRAP100 Coprecipitates with TRAP220 in Vivo
Given that TRAP100 was first identified as a component of the multimeric TR/TRAP complex (Fig. 1CGo) (38, 40) yet fails to directly contact TR (Fig. 2AGo) (40), it is conceivable that TRAP100 is targeted to TR (and possibly other NRs) indirectly through interactions with other components of the TRAP coactivator complex. TRAP220 would appear to be the most plausible candidate to fulfill this role in view of its strong ligand-dependent interactions with various NRs. In support of this supposition, we did find that mTRAP100 directly (albeit weakly) contacts the full-length hTRAP220 protein in vitro as determined by the GST pull-down assay (data not shown). However, further attempts to elucidate direct mTRAP100/TRAP220 interactions in vitro using other protein-binding assays were largely unsuccessful, possibly implying that the mTRAP100/TRAP220 association is indirect or that additional TRAP components are required to effectively facilitate or stabilize this association.

To examine the association of mTRAP100 and TRAP220 under more physiologically relevant conditions, both TRAP220 and a FLAG-tagged mTRAP100 were overexpressed in COS cells and assayed for association by coimmunoprecipitation. Protein complexes containing mTRAP100 were immunoprecipitated from transfected cells with anti-FLAG antibodies coupled to agarose beads, fractionated by SDS/PAGE, and subsequently probed by Western blot with antibodies generated against TRAP220. Figure 3Go (lane 10) clearly shows TRAP220 associates with mTRAP100 when both proteins are overexpressed in COS cells. This result suggests that TRAP220 and mTRAP100 are in the same protein complex that ultimately binds to TR rather than each protein forming a separate complex with TR. Taking into account the likely presence of other TRAP components endogenously expressed in COS cells, these experiments also support the notion that other TRAP subunits may interact with TRAP100 and TRAP220 and possibly stabilize or facilitate formation of TRAP100/TRAP220-containing protein complexes. Taken collectively, the findings in Figs. 2Go and 3Go are consistent with a TRAP complex recruitment model in which TRAP220 functions as an intermediary protein, interacting with other TRAP components as well as TRAP100 and subsequently targeting them to a NR in a ligand-dependent fashion. The ability of a liganded NR to recognize specific hormone response elements would ultimately target the entire TRAP coactivator complex to gene-specific regulatory regions where TRAPs presumably interface with the basal transcription apparatus.



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Figure 3. mTRAP100 Associates with TRAP220 in Vivo

COS cells were transfected with pSG5-FLAG-mTRAP100 or pSG5-TRAP220, either separately or together as indicated above the lanes. To verify TRAP100 and TRAP220 coexpression, whole-cell extract from transfected cells (lanes 2, 4, and 5) or mock transfected cells (lanes 1 and 3) was fractionated by SDS/PAGE, transferred to a nitrocellulose membrane, and probed by Western blot with rabbit polyclonal antisera against TRAP220 ({alpha}-220), mTRAP100 ({alpha}-100), or mouse monoclonal antisera against the FLAG epitope ({alpha}-FLAG). The weak 220-kDa and 100-kDa bands in lanes 1 and 3 presumably reveal endogenous TRAP proteins. In lanes 7–10, whole-cell extract prepared from transfected and mock transfected cells was first incubated with anti-FLAG antibodies coupled to a resin (M2-affinity resin; Eastman Kodak Co.). The resulting immuno-protein complexes were then precipitated and washed, fractionated by SDS/PAGE, transferred to a nitrocellulose membrane, and probed by Western blot with {alpha}-220. The portion of membrane corresponding to lane 10 was stripped and reprobed with {alpha}-100 (lane 11) to verify the presence of TRAP100 in the immunoprecipitated complex. HeLa nuclear extract (30 µg) was used in lane 6 as a positive control for the {alpha}-220 antisera.

 
mTRAP100 Enhances Ligand-Dependent Transcription by both TR and VDR
We next sought to determine the functional significance of TRAP100 in affecting T3-dependent transcription by TR. Toward this end, CV-1 cells were transiently cotransfected with mTRAP100 and TR expression vectors and grown in either the presence or absence of T3. Transcription was then measured from a luciferase reporter containing two T3-response elements (2xT3RE-tk-Luc) (Fig. 4AGo). In the absence of T3, mTRAP100 modestly reduced the basal level of expression from the TRE-containing reporter. In the presence of T3, however, mTRAP100 enhances TR-mediated activation 3-fold (Fig. 4AGo) consistent with a transcriptional coactivator function for the mTRAP100 protein. Given the presence of the hTRAP100 homolog (DRIP100) in a VDR-associated coactivator complex (39), we further asked whether mTRAP100 might enhance VDR-mediated transcription. Accordingly, NIH3T3 cells were transiently transfected with both mTRAP100 and VDR expression vectors, and transcription was measured from a cotransfected luciferase reporter gene containing four vitamin D response elements (4xVDRE-Ld-Luc) (Fig. 4BGo). Similar to the case with TR, mTRAP100 overexpression enhances VDR-mediated activation greater than 3-fold in the presence of vitamin D3 (Fig. 4BGo). The ability of overexpressed mTRAP100 to modestly inhibit basal transcription from both the 2xT3RE-tk-Luc and 4xVDRE-Ld-Luc reporters in the absence of ligand may reflect mTRAP100 sequestration of other commonly required nuclear cofactors. Nonetheless, these findings suggest that the TRAP100 component of the TRAP coactivator complex plays an important functional role in facilitating TRAP-mediated enhancement of ligand-dependent transactivation by both TR and VDR.



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Figure 4. mTRAP100 Enhances Ligand-Dependent Transcription by TR and VDR

A, Transient overexpression of mTRAP100 enhances TR-mediated transactivation from a TRE-reporter. CV-1 cells were transfected with 0.33 µg p2xT3RE-tk-Luc reporter, 0.33 µg pRSV-TRß, 0.15 µg of the internal control plasmid pSV-ß-gal, and 1 µg of either pSG5-mTRAP100 or the empty pSG5 vector. TRIAC (10-7 M final) was added as indicated. B, Transient overexpression of mTRAP100 enhances VDR-mediated transactivation from a VDRE-reporter. NIH3T3 cells were transfected with 0.66 µg p4xVDRE-Ld-Luc, 0.33 µg pSG5-hVDR, 0.15 µg of the internal control plasmid pSV-ß-gal, and 1.33 µg of either pSG5-mTRAP100 or the empty pSG5 vector. 1,25-(OH)2D3 (2.5 x 10-8 M final) was added as indicated. Relative luciferase activities were determined from three independent transfections in the absence (open bars) or presence of ligand (hatched bars), although similar results were obtained with multiple transfections. The data are presented as the mean ± SD of the triplicated transfections.

 
Autonomous Transactivation by mTRAP100
To examine whether mTRAP100 might directly activate transcription when recruited to a specific promoter, we generated a chimeric protein vector in which the full-length mTRAP100 protein is fused to the DNA-binding domain of the yeast transcription factor GAL4 (GAL4-mTRAP100). The GAL4-mTRAP100 vector was then transfected into COS cells, and transcription was measured from a luciferase reporter containing five GAL4-binding sites (Fig. 5Go). While GAL4 alone does not efficiently activate transcription, the GAL4-mTRAP100 protein exhibits moderate transcriptional stimulation from the luciferase reporter (Fig. 5Go). These results indicate that mTRAP100 may contain an intrinsic transactivation domain that functions autonomously in mammalian cells and thus suggest a transcription-enhancing functional role for the TRAP100 protein during TRAP-mediated coactivation. Presumably, higher levels of transcriptional activation are achieved when TRAP100 functions in concert with the other components of the TRAP coactivator complex.



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Figure 5. Autonomous Transactivation by mTRAP100

COS cells were transiently transfected with 0.15 µg p5xGAL4-tk-Luc, 0.15 µg pSV-ß-gal, and 0.33 µg of either pSG5 (empty expression vector), pSG424 (GAL4 expression vector), or pSG424-mTRAP100 (GAL4-full-length mTRAP100 fusion protein expression vector) as indicated. Relative luciferase activities were determined from three independent transfections. The data are presented as the mean ± SD of the triplicated transfections.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The TRAP coactivator complex was first identified as a large multimeric complex of novel proteins that copurify with TR from T3-cultured HeLa cells (38). Subsequent T3-dependent transcription assays clearly demonstrated a pronounced TRAP complex enhancement of TR-mediated activation in vitro (38), thereby revealing a novel NR-coactivator pathway or activation step (see below) apparently distinct from those involving SRC-family members and CBP/p300. In this study we have identified the mouse cDNA for the 100-kDa component of the TRAP complex (mTRAP100) and, in an effort to better understand how the TRAP coactivator complex works, we have functionally characterized the mTRAP100 protein. We have shown that overexpression of mTRAP100 in cultured mammalian cells significantly enhances ligand-dependent transcription by both TR and VDR and that mTRAP100 can activate transcription autonomously when tethered to a heterologous DNA-binding domain. Based on these data, we suggest that TRAP100 facilitates an important functional role during TRAP complex-mediated enhancement of ligand-dependent transcription by specific NRs.

A striking feature of the mTRAP100 protein is the presence of seven LxxLL signature motifs previously shown in other NR coactivators to be binding surfaces for liganded NRs (32). In spite of the multiple signature motifs, however, mTRAP100 failed to interact in vitro with any of the NRs tested here (VDR, RAR, PR, and TR), thus suggesting that TRAP100 is not recruited to a hormone-responsive promoter via direct contacts with a liganded NR. The question then arises as to how TRAP100 is specifically targeted to NR-regulated genes. In this report, we have demonstrated that mTRAP100 associates with the 220-kDa component of the TRAP complex (termed TRAP220) in vivo. Importantly, TRAP220 is the only TRAP subunit demonstrated thus far to directly and convincingly contact TR and VDR (as well as other NRs) in a ligand-dependent manner. Hence, our data are consistent with a TRAP complex recruitment model in which TRAP220 acts as nucleation surface for the assembly of TRAP100 and other TRAP components into a holo-complex and subsequently targets their respective transactivation functions to specific NRs in a ligand-dependent fashion. Whether TRAP100 directly contacts TRAP220 or alternatively requires other TRAP components to indirectly associate with TRAP220 is currently unresolved. However, given the sheer size of the TRAP complex (~1.5 MDa), the number of putative subunits, and the potential for multiple protein-protein interactions, it appears highly probable that other TRAP components are involved in facilitating this association.

In light of the myriad of putative NR accessory proteins recently discovered (12, 13), it is puzzling as to why a highly conserved superfamily of ligand-induced transcription factors would functionally require such a large and diverse group of accessory factors including the TRAP coactivator complex. One explanation might be that different NR cofactors represent alternative transcriptional regulatory pathways available to a given NR within different cell types. In this manner, type of ligand (agonist, antagonist, or none) might dictate differential usage of NR cofactors within different tissues. A second possibility is that multiple NR-coregulatory factors may be a conserved mechanism of integrating multiple cellular signal pathways into hormone-induced NR pathways. Third, different NR cofactors may reflect different (and perhaps sequential) transcriptional regulatory steps during a hormone-induced NR pathway (35, 36). For instance, NRs may initially require chromatin remodeling factors (i.e. the HAT activity associated with SRC-related factors and CBP/p300) to penetrate target genes within condensed chromatin. Subsequently, other coregulatory factors (i.e. TRAPs/DRIPs) may be required in order for the NR to effectively communicate with the basal transcription apparatus.

The presence of TRAP100 within two apparently identical coactivator complexes specifically associated with TR and VDR suggests that TRAP-mediated transcriptional coactivation may be commonly used by other members of the NR superfamily, particularly those that heterodimerize with RXR (44). Indeed, the recent cloning of the mTRAP220 homolog as a ligand-dependent PPAR-interacting protein suggests that the TRAP coactivator complex may be involved in PPAR pathways (26). In the context of TRAP220’s proposed role as a bridging factor between other TRAP components and liganded NRs, the ability of TRAP220 to interact with a diverse array of NRs suggests that TRAPs may be involved in other pathways as well. A more complete understanding of how the holocomplex operates and the functional roles fulfilled by the other TRAP components await the identification and characterization of their respective cDNAs.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Identification of the mTRAP100 cDNA
Mouse cDNAs encoding putative mTRAP100 sequences were identified by screening short polypeptide sequences derived from the hTRAP100 protein (40) against the data base of mouse expressed sequence tags (dbEST) at the NCBI (http://www.ncbi.nlm.nih.gov/dbEST). cDNA clones with significant homology were identified, obtained from the American Type Culture Collection (ATCC, Manassas, VA) and sequenced (University of Maryland School of Medicine Biopolymer Laboratory). The mouse EST clone encoding the full-length mTRAP100 cDNA was deposited by the I.M.A.G.E. Consortium (I.M.A.G.E. ID 615136) and distributed by the ATCC (ATCC ID 773544). The mTRAP100 sequence has been deposited in Genbank (accession number AF126543).

Northern Blot Analysis
A mouse multiple tissue Northern blot (CLONTECH Laboratories, Inc., Palo Alto, CA) was probed with a 32P-labeled DNA fragment corresponding to amino acid residues 817–956 of the mTRAP100 cDNA. Northern blot hybridization and membrane washes were performed as recommended by the manufacturer.

Plasmid Construction
The GST-mTRAP100 (full) and GST-mTRAP100 (1–52) plasmids were constructed by first PCR amplifying either full-length mTRAP100 or mTRAP100 amino acids 1–52 with primers creating BamHI and EcoRI restriction sites at the 5'- and 3'-ends of the cDNA, respectively. The PCR fragments were then ligated into pGEX-2TK (Pharmacia Biotech, Piscataway, NJ) predigested with BamHI and EcoRI, thus generating the in-frame GST fusion protein expression vectors. The GST-TRAP220-receptor binding domain (GST-220-RBD[622–701]) (40) and GST-TRAP220 (1401–1484) plasmids were generated in a similar fashion by first PCR amplifying hTRAP220 amino acids 622–701 and 1401–1484, respectively, with primers creating BamHI and EcoRI restiction sites at the 5'- and 3'-ends followed by ligation into the BamHI/EcoRI sites of pGEX-2TK. The pFLAG-hTR{alpha} expression plasmid has been described previously (38). The pSG5-mTRAP100 mammalian expression vector was generated by subcloning the EcoRI/BamHI fragment of IMAGE clone 615136 (containing the full-length mTRAP100 cDNA) into the EcoRI/BamHI sites of pSG5 (Stratagene, La Jolla, CA). The pSG5-FLAG-mTRAP100 expression vector was created by first generating BglII and BamHI sites at the 5'- and 3'-ends of the mTRAP100 cDNA by PCR and then cloning the fragment into the BamHI site of pFLAG-(s). Subsequently, the FLAG-mTRAP100 fragment was liberated by EcoRI/BamHI digestion and subcloned into the EcoRI/BamHI sites of pSG5. pSG5-TRAP220 was generated by liberating the SmaI/SacI TRAP220 cDNA from pGEM-HA-TRAP220 (provided by R. G. Roeder, Rockefeller University, New York, NY) and subsequently blunt-end ligating the full-length hTRAP220 cDNA (40) into the BamHI site of pSG5. The pSG424-mTRAP100 (GAL4-mTRAP100) mammalian expression vector was generated by first creating EcoRI sites on both the 5'- and 3'-ends of the mTRAP100 cDNA by PCR followed by an in-frame ligation into the EcoRI site of the plasmid pSG424 (45). The pBK-CMV-FLAG-hTR{alpha} expression vector was generated by subcloning the BglII/EcoRI fragment of pFLAG-hTR{alpha} into the BamHI/EcoRI sites of pBK-CMV (Stratagene). The pFLAG-hTRß plasmid was generated by first PCR-creating an NdeI site at the translation start codon of the hTRß cDNA within the pRSV-TRß expression vector (provided by H. Samuels, New York University, New York, NY). Subsequently, the NdeI-BamHI fragment spanning the full-length hTRß cDNA was inserted into the NdeI/BamHI sites of pFLAG(s)-7 (46). pGEM-rVDR was created by EcoRI digestion of pSV40-rVDR (provided by H. DeLuca, University of Wisconsin, Madison, WI) and subcloning the full-length rVDR fragment into the EcoRI site of pGEM-4Z (Promega Corp., Madison, WI). The NR expression vectors, pCMX-hRAR{alpha}, pT7ß-hPRA, and pSG5-hVDR, were kindly provided by R. Evans (Salk Institute, San Diego, CA), M.-J. Tsai (Baylor College of Medicine, Houston, TX), and K. Ozato (The National Institute of Child Health and Human Development, Bethesda, MD), respectively. The luciferase reporter plasmids, p2xT3RE-tk-Luc, p4xVDRE-Ld-Luc, and p5xGAL4-tk-Luc, were kindly provided by C. Glass (University of California, San Diego, CA), K. Ozato, and M. Privalsky (University of California, Davis, CA), respectively.

Antibody Production
The GST-mTRAP100 (1–52) and GST-TRAP220 (1401–1484) proteins were expressed in and purified from E. coli strain BL21(DE3)pLysS and injected into rabbits (Covance Research Products, Inc., Denver, PA). Anti-FLAG epitope mouse monoclonal antibodies and anti-FLAG antibodies coupled to agarose (M2-affinity resin) were obtained commercially (Eastman Kodak Co., Rochester, NY).

GST Pull-Down Assay
The GST-mTRAP100 and GST-TRAP220-RBD (622–701) proteins were expressed in and purified from E. coli strain BL21(DE3)pLysS. In general, 20 µl of a 50% GST-protein/glutathione Sepharose (Pharmacia Biotech) bead slurry were resuspended in 200 µl binding buffer [20 mM HEPES (pH 7.9), 100 mM KCl, 0.5 mM EDTA, 5 mM MgCl2, 1 mM dithiothreitol, 10% glycerol, 0.05% NP-40, 0.5% milk] together with 5 µl of in vitro translated, [35S]methionine-labeled NRs generated from the pFLAG-hTR{alpha}, pFLAG-hTRß, pCMX-hRAR{alpha}, pT7ß-hPRA, pCMX-mERß, pGEM-rVDR, and pSG5-hVDR templates using a kit (TNT, Promega Corp.). The reactions were incubated for 1 h at 4 C on a rocker. Protein complexes were isolated by pelleting the beads and washing three times in binding buffer followed by resuspension in SDS-sample loading buffer. After SDS-PAGE fractionation, bound [35S]-labeled NRs were visualized by autoradiography. The ligands TRIAC (1 µM final), retinoic acid (1 µM final), 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] (0.5 µM final), and progesterone (1 µM final) were added to the reactions as indicated in Fig. 2Go.

Transient Transfections
CV-1, COS, and NIH3T3 cells were routinely maintained in DMEM (high glucose) supplemented with 10% FBS. One day before transfection, cells were seeded in 12-well plates: NIH3T3 cells at a density of 8 x 104 cells per well in DMEM containing 10% charcoal/dextran-stripped FBS (HyClone Laboratories, Inc., Logan, UT); CV-1 cells at a density of 8 x 104 cells per well in DMEM containing 10% dialyzed FBS (Gibco BRL, Gaithersburg, MD); COS cells at a density of 5 x 104 cells per well in DMEM containing 10% FBS. For NIH3T3 cells, a DNA mixture containing 0.66 µg p4xVDRE-Ld-Luc, 0.33 µg pSG5-hVDR, 0.15 µg of the internal control plasmid pSV-ß-gal (Promega Corp.), 1.66 µg sonicated salmon sperm DNA, and 1.33 µg of either pSG5-mTRAP100 or the empty pSG5 vector was added to each well using the calcium phosphate transfection method. For CV-1 cells, a DNA mixture containing 0.33 µg p2xT3RE-tk-Luc, 0.33 µg pRSV-TRß, 0.15 µg pSV-ß-gal, 2 µg sonicated salmon sperm DNA, and 1 µg of either pSG5-mTRAP100 or the empty pSG5 vector was added to each well as above. For COS cells, a DNA mixture containing 0.15 µg p5xGAL4-tk-Luc, 0.15 µg pSV-ß-gal, 1.66 µg sonicated salmon sperm DNA, and 0.33 µg of either pSG5 (empty expression vector), pSG424 (GAL4 alone), or pSG424-mTRAP100 (GAL4-mTRAP100) were added to each well. The precipitate from each set of transfections was removed after 16 h with PBS and replaced with fresh DMEM containing either 10% charcoal/dextran-stripped FBS (NIH3T3), 10% dialyzed FBS (CV-1), or 10% FBS (COS) together with vehicle alone or vehicle plus the ligands TRIAC (10-7 M final) or 1,25-(OH)2D3 (2.5 x 10-8 M final) as stated in the figure legends. After 36–48 h, transfected cells in each well were harvested with a cell lysis buffer supplied in a kit (Luciferase Assay System, Promega Corp.), and luciferase activity was determined by adding a commercial assay solution according to the manufacturers instructions (Promega Corp.) and then measuring in a Lumat LB 9507 luminometer (EG&G Wallac, Inc., Gaithersburg, MD). The ß-galactosidase activity of the lysed transfected cells (as above) was determined using a kit (ß-galactosidase Enzyme Assay System, Promega Corp.) according to the manufacturer’s instructions. The luciferase activity was normalized to the ß-gal activity and expressed as relative luciferase light units. For coimmunoprecipitation experiments, COS cells were seeded in 10-cm plates containing DMEM/10% FBS at a cell density of 5 x 105 cells per plate 1 day before transfection. The plates were transfected essentially as above with 5 µg pSG5-FLAG-mTRAP100 or 5 µg pSG5-TRAP220, either separately or together in subsets (see legend to Fig. 3Go) along with pBS-KSII (Stratagene) as carrier DNA to a total of 40 µg. Nontransfected COS cells were used as negative controls for the immunoprecipitation assays (see below).

Coimmunoprecipitation
COS cells in 10-cm culture dishes were collected for harvesting by gentle scraping in 1 ml ice-cold PBS and pelleting by centrifigution at 1200 rpm at 4 C. The PBS was aspirated and the cell pellet (106 cells) resuspended in 0.5 ml ice-cold buffer A [50 mM Tris-Cl (pH 7.4), 150 mM NaCl, 5 mM EDTA, 0.5% NP-40, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 10 µg/ml leupeptin]. The lysis mixture was rotated 360o for 30 min at 4 C and then cleared by centrifugation at 12,000 x g for 10 min at 4 C. Anti-FLAG antibodies (20 µl) coupled to agarose beads (M2 Affinity Resin; Eastman Kodak Co.) were added per 1 ml of lysate, and the mixture was rotated slowly overnight at 4 C. The beads were then pelleted by gentle centrifugation and washed three times with 1 ml buffer A. After the final wash, the precipitated protein complexes were resuspended in SDS-sample loading buffer, fractionated by SDS-PAGE, and transferred to a nitrocellulose membrane by Western blot. Membranes were screened with relevant antibodies and developed by enhanced chemiluminescence (ECL system, Amersham, Arlington Heights, IL) according to the manufacturers instructions.

Nuclear Extract Preparation and Purification of the TR/TRAP Complex
HeLa cells and the HeLa-derived constituitively expressing FLAG-hTR{alpha} cell line {alpha}-2 (38) were routinely maintained in DMEM/10% FBS and DMEM/10% dialyzed FBS, respectively. Preparation of nuclear extract from both cell lines and the subsequent immunopurification of the TR/TRAP coactivator complex from {alpha}-2 cell nuclear extracts was essentially as described (38).


    ACKNOWLEDGMENTS
 
The authors wish to thank Hector DeLuca, Ron Evans, Chris Glass, Bert O’Malley, Keiko Ozato, Martin Privalsky, Herb Samuels, and Ming-Jer Tsai for plasmids. The authors acknowledge R. G. Roeder for plasmids originated in his laboratory and Zhao-jun Ren for the construction of some of the expression vectors used here. We also thank Dhan Kalvakolanu for critically reading the manuscript and helpful discussions.


    FOOTNOTES
 
Address requests for reprints to: Dr. Joseph D. Fondell, Department of Physiology, University of Maryland School of Medicine, 655 West Baltimore Street, Baltimore, Maryland 21201-1559.

This work was supported in part by Grant IRG97–153-01 from the American Cancer Society.

Received for publication December 28, 1998. Revision received February 12, 1999. Accepted for publication March 1, 1999.


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