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Molecular Endocrinology, doi:10.1210/me.2005-0529
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Molecular Endocrinology 21 (8): 1822-1834
Copyright © 2007 by The Endocrine Society

Nuclear Receptor Coregulator (NRC): Mapping of the Dimerization Domain, Activation of p53 and STAT-2, and Identification of the Activation Domain AD2 Necessary for Nuclear Receptor Signaling

Muktar A. Mahajan, Audrey Murray, David Levy and Herbert H. Samuels

Department of Pharmacology (M.A.M., A.M., H.H.S.) and Department of Pathology (D.L.), New York University School of Medicine, New York, New York 10016

Address all correspondence and requests for reprints to: Muktar A. Mahajan or Herbert H. Samuels, Department of Pharmacology, New York University School of Medicine, 550 First Avenue, New York, New York 10016. E-mail: muktar.mahajan{at}med.nyu.edu or herbert.samuels{at}med.nyu.edu.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Nuclear receptor coregulator (NRC) is a 250-kDa nuclear protein involved in transcriptional activation of nuclear hormone receptors, nuclear factor-{kappa}B, c-Jun, c-Fos, and cAMP response element-binding protein. NRC is organized into a modular structure consisting of two activation domains (AD1 and AD2), two nuclear hormone receptor-interacting motifs, LxxLL-1 and LxxLL-2, and a C-terminal regulatory region rich in serines, threonines, and leucines. The LxxLL-1 motif of NRC binds to a broad spectrum of nuclear hormone receptors with high affinity whereas LxxLL-2 interacts with a very limited number of receptors. In this study we present further evidence that NRC can act as a dimer and have identified a dimerization region of 146 amino acids including LxxLL-1. Mutation of the core LxxLL-1 motif, however, indicates that it is not involved in the dimerization of NRC. AD2, just C-terminal of LxxLL-1, was found to play a central role in ligand-dependent activation by nuclear receptors even though AD1 exhibits more potent intrinsic activity. Thus, a short region of approximately 300 amino acids including and flanking LxxLL-1 plays an important role in NRC dimerization and nuclear receptor binding and transcriptional activation. In addition, consistent with its role as a cointegrator for transcriptional activation, NRC also functions as a coactivator for signal transducer and activator of transcription 2 (STAT-2) and p53. Activation of p53 by NRC appears to involve a novel mechanism where NRC interacts indirectly with p53 through Trap80, a member of the mediator complex, which binds NRC interacting factor-1 (NIF-1), which interacts with and potentiates the effect of NRC.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
NUCLEAR HORMONE RECEPTORS make up a superfamily of ligand-dependent transcription factors that are involved in regulating the expression of wide array of target genes necessary for cellular homeostasis, differentiation, development, and drug detoxification (1). The nuclear receptor superfamily can be broadly classified into three subfamilies containing receptors for steroid hormones, nonsteroid hormones and ligands, and orphan receptors. In the presence of their cognate ligands, the steroid receptors such as glucocorticoid receptor (GR), progesterone receptor (PR), and estrogen receptors (ERs) activate transcription predominantly as homodimers.In contrast, nonsteroid members such as the thyroid hormone receptors (TRs), retinoic acid receptors (RARs), vitamin D receptor (VDR), liver X receptors, and the peroxisome proliferator activator receptors (PPARs) predominately function as heterodimers with the retinoid X receptors (RXRs). The RXRs are also involved in heterodimerization with other nonsteroid receptors and with some orphan nuclear hormone receptors (1). In general, members of the nuclear receptor family share a common signature structure consisting of a unique variable N-terminal A/B domain which can harbor an autonomous activation function (AF-1), a central zinc finger DNA binding C domain, followed by a hinge D region and a C-terminal ligand binding domain (LBD) containing the EF region. For some receptors the LBD involves domains D, E, and F. For certain receptors the LBD contains a dimerization region, and all the receptors exhibit a ligand-dependent activation function referred to as AF-2 (1, 2).

Nuclear receptors bind to their target DNA sequences and activate transcription through mechanisms involving both AF-1 and AF-2. Typically, a nuclear receptor upon ligand binding undergoes a conformational change from a transcriptionally repressive or inactive form to an active conformational form. Ligand binding results in reorientation of helix 12 located at the C terminus of the LBD, which contributes to the formation of a hydrophobic groove that allows for physical interaction of the receptor with coactivators. AF-2-dependent coactivators contain signature LxxLL motif/s (NR box/es) involved in interaction with the hydrophobic groove formed in the LBD, which leads to the AF-2 activity of the receptors (3, 4, 5, 6, 7).

Coactivators serve to transmit signals from activated receptors to facilitate transcription through a number of mechanisms. Members of p160/steroid receptor coactivator (SRC) family [SRC-1, GRIP-1/TIF-2 (SRC-2), ACTR/AIB1/TRAM/NCoA2 (SRC-3)], DRIP205/TRAP220/PBP, RIP140, and NRC/ASC-2/TRBP/RAP250 (8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25) are well-characterized coactivators that contain LxxLL motifs. Different coactivators enhance transcription through distinct mechanisms. For example, coactivators such as CREB-binding protein (CBP)/p300 and some members of p160 family with histone acetyl transferase activity may facilitate transcription via histone acetylation and, thus, modulate local chromatin architecture (13, 26, 27, 28). The p160 co-activator GRIP-1 contains two activation domains, AD1 and AD2. AD1 functions through an interaction with CBP/p300, whereas AD2 interacts with coactivator-associated arginine methyltransferase 1 (CARM1), which harbors methyl transferase activity, which methylates arginine residues in histones. The C-terminal region of GRIP-1 also interacts with AF-1 region found in the N terminus of some of the nuclear receptors (29).

We previously cloned and characterized NRC (nuclear receptor coregulator) in detail (22). NRC harbors two LxxLL motifs, LxxLL-1 and LxxLL-2 (see Fig. 1Go). LxxLL-1 is a high-affinity interaction motif that binds most nuclear hormone receptors, and receptor binding mediates a conformational change in NRC, resulting in the enhanced activity of the coactivator. LxxLL-2 exhibits a limited specificity and has been shown to associate with the liver X receptors (30) and with lower affinity to ER{alpha} (22). NRC also contains a potent N-terminal AD (AD1) and a centrally localized AD (AD2), which is just C terminal to LxxLL-1. An inhibitory serine-threonine-leucine domain at the C terminus appears to modulate the overall transcriptional output of NRC. CBP associates with NRC with high affinity in vivo (22) and in vitro (24), and the transcriptional activity of NRC was also shown to be inhibited by the viral protein, E1A (22).


Figure 1
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  		Fig. 1. Two-Hybrid Interaction Assay in Yeast with Various Regions of NRC

The domain structure of NRC is shown at the top. Full-length LexA-NRC (LN) and LexA-NRC deletions, L2, L3, and L4 as shown were tested for interaction in yeast with B1, B2, B3, and B4 representing various regions of NRC expressed as B42 fusions. The interactions were quantified by measuring the activity of ß-galactosidase in yeast extracts from duplicate samples. The line diagram at the top depicts various fusions with numbers that correspond to the amino acids of NRC. For details refer to Materials and Methods. (–) Depicts no interaction, whereas (+) depicts positive interaction. The figure depicts a representative study of several experiments each giving similar results. STL, Serine-threonine-leucine-rich region.

 
We recently reported the cloning and characterization of NIF-1 (NRC interacting factor-1), a zinc-finger leucine-zipper BED domain protein (31). The BED finger in the BED domain is about a 50- to 60-amino acid region, which has been named after the Drosophila proteins boundary element-associated factor (BEAF) DNA replication-related element-binding factor (DREF) and is found in plants, fungi, and animals (32). BED domain proteins have been suggested to modulate nucleosome structure and activate transcription (32, 33, 34). NIF-1 interacts in vitro and in vivo with NRC and activates nuclear receptors as well as c-Fos and c-Jun in a similar fashion as NRC (31). A number of RNA binding factors such as PPAR-interacting protein (PRIP)-interacting methyltransferase (PIMT) (35), CAPER (36), and coactivator activator (CoAA) (37) have also been isolated and characterized as interacting proteins for NRC. Purification of an ASC-2 (NRC) complex (ASCOM) suggests that at least eight different proteins form a complex with NRC (ASC-2) (38). The gene for NRC has been deleted in mice, and studies with these mice suggest that the NRC is necessary for embryogenesis, placental function, and cardiac development (39, 40, 41, 42). Our recent studies with NRC MEF–/– cells and NRC+/– newborn and adult mice indicates that NRC plays a role in cell survival, apoptosis, reproduction, and wound healing (42).

Although NRC contains two LxxLL motifs, only LxxLL-1 displays high-affinity ligand-dependent interaction with TR, RAR, RXR, VDR, GR, and PPAR. These receptors function primarily as dimers and members of p160 family, DRIP205/TRAP220/ PPAR-binding protein and receptor-interacting protein 140 contain multiple LxxLL motifs capable of interacting with nuclear receptor dimers (1, 43, 44). In contrast with the p160 family, NRC contains a single LxxLL-1 interacting motif. The strong interaction of NRC with receptor dimers appears to be facilitated through the formation of NRC dimers, which contributes two LxxLL-1 motifs to interact with receptor dimers (22, 33). In the present study we have further localized the dimerization region of NRC and also show that of the two ADs of NRC, AD2 is specifically involved in nuclear receptor signaling. We have also extended our studies on NRC to show that NRC plays an important role in transcriptional activation by signal transducer and activator of transcription 2 (Stat2), as well as p53 through a novel mechanism involving the association of NIF-1 with Trap80 of the Mediator complex, which is known to play a role in p53-mediated transcriptional activation.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
NRC Dimerizes through a Region that Contains the LxxLL-1 Domain
Yeast two-hybrid assays were used to map the NRC dimerization region. Various regions spanning the N terminus or C terminus of NRC were expressed in yeast as LexA and B42 fusions. B42 is a prokaryotic acidic AD. As shown in Fig. 1Go, a LexA fusion expressing amino acids 1153–2063 of NRC (L3) did not interact with either the full-length or any other B42 fusions of NRC (B1, B2, B3, and B4). Similarly, the N terminus of NRC (amino acids 1–848), when conditionally expressed as a B42 fusion (B1), failed to interact with either the full-length or the various LexA fusions of NRC (L1, L2, L3, and L4) (Fig. 1Go). These findings suggest that the N-terminal region (amino acids 1–848) and the C-terminal region (amino acids 1153–2063) do not play a role in the dimerization of NRC. We next examined the interaction of the B42 fusions NRC-B2 (amino acids 849-1153), NRC-B4 (amino acids 849-2063) with the LexA fusion NRC-L4 containing amino acids 848-2063 and NRC-L2 (amino acids 849-1153). Although NRC-L2(849–1153) is modestly intrinsically active in yeast as a LexA fusion (22), the results of Fig. 2AGo indicate that NRC-B2 strongly interacts with NRC-L4 and NRC-L2. NRC-B4 also showed a similar interaction with NRC-L2 as did NRC-L4 and NRC-B4. These yeast interaction results suggest that a region within amino acids 849-1153 is involved in NRC dimerization.


Figure 2
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  		Fig. 2. The LxxLL-1 Motif Is Not Essential for Dimerization of NRC

The line diagram at the top depicts various fusions with numbers that correspond to the amino acids of NRC. As depicted, various LexA and B42 NRC fusions harboring the dimerization region were tested in yeast two-hybrid assays. L2, L4 are LexA-NRC fusions that show interactions with the homologous B42-NRC fusions, B2 and B4. The relative strength of interactions was determined by measuring the ß-galactosidase activity from duplicate yeast sample extracts. B, Same type of experiment as panel A. L2m and B2m are identical to L2 and B2 constructs except for a change of leucines to alanines in LxxLL-1 as described in Materials and Methods.

 
Interestingly, amino acids 849-1153 of NRC also harbor LxxLL-1 (LVNLL), necessary for interaction of NRC with TR, RAR, RXR, VDR, GR, and ER (22). To assess whether the LxxLL-1 motif plays a role in dimerization of NRC, we introduced mutations in LxxLL-1 to change the leucines to alanines (AVNAA) and expressed the mutated protein (amino acids 849-1153) either as a LexA or B42 fusion in yeast two-hybrid assays. Figure 2 shows the results obtained with the wild-type and the LxxLL-1 mutant NRC. The mutant LexA-NRC(849–1153) (L2m) interacted with the LxxLL mutant B42 fusion construct of NRC(849–1153) (B2m) as strongly as the corresponding wild-type fusions (L2 and B2), suggesting that LxxLL-1 is not directly involved in dimerization. To further map the dimerization region contained within amino acids 849-1153 of NRC, we studied polypeptides containing amino acids 849–995 and 995-1153 in yeast two-hybrid assays. As shown in Fig. 3Go, LexA-NRC(849–995) (L5) interacted strongly with the corresponding B42-NRC(849–995) (B5) and B42-NRC(849–1153) (B2) fusions, but failed to interact with the B42-NRC(995–1153)(B6). In addition, the LexA fusion NRC-L6 failed to interact with the corresponding B42-NRC fusion (995–1153)(B6) and B42-NRC(849–1153)(B2). The B42 LxxLL-1 mutant containing the dimerization region interacted similarly to the B42 wild-type fusion as shown (B2m vs. L5). Taken together, these results indicate that the dimerization domain of NRC is contained within a 146-amino acid region (amino acids 849–995) of the protein containing the LxxLL-1 motif. However, the LxxLL-1 motif does not appear to be essential for NRC dimerization. We also expressed shorter regions of NRC to further map the NRC dimerization domain. However, the results were not conclusive, possibly due to disruption of the tertiary structure of the dimerization region.


Figure 3
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  		Fig. 3. Mapping of the NRC Dimerization Domain in Yeast

L5 and L6 are LexA fusions whereas B5 and B6 are B42 fusions of short regions of NRC as depicted in the line diagram with the amino acid numbers. The yeast interaction assay was carried out as in Figs. 1Go and 2Go.

 
NRC Forms Dimers in Vitro
We previously reported that glutathione-S-transferase (GST)-NRC(849–1153) containing LxxLL-1 could bind 35S-labeled NRC(849–1153) in vitro. The yeast two-hybrid assays localized the dimerization domain to a 146-amino acid region contained within residues 849–995. To confirm this interaction in vitro, we expressed amino acids 849–995 of NRC as a GST fusion in bacteria followed by its purification and in vitro binding with 35S-labeled NRC(849–1153). GST or GST-NRC(849–995) immobilized on glutathione agarose beads was incubated with 35S-labeled NRC(849–1153). After washing the beads, the 35S-labeled NRC(849–1153), which bound to GST or GST-NRC(849–995) in vitro, was visualized by SDS-PAGE gel electrophoresis followed by autoradiography (Fig. 4Go). The GST control showed no binding of 35S-labeled NRC(849–1153) whereas GST-NRC(849–995) interacts with 35S-labeled NRC(849–1153) in vitro, which is consistent with our findings of the NRC dimerization data in yeast.


Figure 4
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  		Fig. 4. NRC Forms Dimers in Vitro

An NRC region corresponding to amino acids 848–995 was expressed as a GST fusion in bacteria and purified using glutathione agarose beads. The GST-fusion protein was then tested for in vitro binding with 35S-labeled NRC(849–1153) followed by autoradiography (lane 3). Incubation of 35S-labeled NRC(849–1153) with GST alone as a control (lane 2). Lane 1 shows the input of 35S-labeled NRC(849–1153) (one fifth of the total). The details are given in Materials and Methods.

 
NRC Forms Dimers in Mammalian Cells
Because NRC dimerizes in yeast and in vitro, we tested the ability of NRC to dimerize in mammalian cells by employing a mammalian two-hybrid recruitment assay, which has been used to assess protein-protein interactions in vivo. Gal4-DNA-binding domain (DBD) fusions of NRC carrying the dimerization region were coexpressed in HeLa cells along with a Gal4-responsive reporter plasmid, G5-pPBL-CAT2 (22). As shown in Fig. 5Go, the Gal4-DBD alone exhibited very little activity whereas Gal4-NRC(849–1153) was 2.5-fold greater and Gal4-NRC was nearly 3-fold greater than the Gal4-DBD. As previously described, these Gal4-NRC proteins show intrinsic levels of basal activity (22, 31). The activities of the Gal4-NRC fusions can be enhanced further upon coexpression of an interacting protein containing an AD(s). Whereas the activity of the Gal4-DBD was not enhanced by expression of full-length wild-type NRC, the activities of Gal4-NRC(849–1153) and Gal4-NRC were increased about 2.5-fold when coexpressed with NRC (Fig. 5Go, lanes 4 and 6). The further increase in activity suggests dimerization and activation mediated by the ADs of NRC. We also examined whether Gal4-NRCm(849–1153), which contains mutations in LxxLL-1, could interact with NRC in vivo. As previously reported, the intrinsic basal activity of Gal4-NRCm(849–1153) was lower than Gal4-NRC(849–1153) (22). However, expression of NRC enhanced the activity of Gal4-NRCm(849–1153) and wild-type Gal4-NRC(849–1153) to the same extent, further indicating, as found in yeast, that LxxLL-1 is not involved in the dimerization of NRC.


Figure 5
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  		Fig. 5. Dimerization of NRC Occurs in Mammalian Cells

HeLa cells were transfected with the Gal4 reporter, G5-pBL-CAT2, along with vectors expressing the various Gal4-DBD chimeras indicated with and without the expression of wild-type NRC. Expression of wild-type NRC enhances the activity of the Gal4-NRC chimeras but not Gal4 alone. The experiment was carried out in duplicate and was repeated three times with similar results. CAT, Chloramphenicol acetyltransferase.

 
Nuclear Hormone Receptors Signal through AD2
We previously reported that NRC contains two ADs, AD1 and AD2. AD1, located at the N terminus, is as potent as the herpes simplex virus VP16 AD, whereas AD2, located just C terminal of the LxxLL-1 module, exhibits less intrinsic activity (22). Studies using LexA-NRC (full length) in yeast indicated that the binding of liganded RXR markedly increases the transcriptional activation potential of NRC, suggesting that when nuclear receptors bind to NRC, the coactivator undergoes a conformational change leading to activation (22). In previous studies in HeLa cells, we found that the activity of Gal4-NRC(849–1153) was enhanced in a ligand-dependent manner by endogenous RAR/RXR, suggesting that nuclear hormone receptors can signal through AD2 (22). To further clarify the role of AD1 and AD2 in nuclear receptor signaling, we generated a series of Gal4-NRC fusions expressing AD1, LxxLL-1, and AD2 (Gal4-Ad1-L-AD2), Ad1 and LxxLL-1 without AD2 (Gal4-AD1-L), and compared the activation of these NRC proteins to Gal4-NRC (full length), Gal4-NRC(849–1153), and Gal4-NRCm(849–1153) containing mutations in LxxLL-1 (Fig. 6Go). Ligand-dependent activation by 9-cis-RA was found with Gal4-NRC (full length), Gal4-NRC(849–1153), and Gal4-Ad1-L-AD2 but not Gal4-AD1-L. As expected, no 9-cis-RA-mediated activation was found with Gal4-NRCm(849–1153)-containing mutations in LxxLL-1. We obtained similar results using the NRC proteins shown in Fig. 6Go (Ad1-L-Ad2, Ad1-L) (not Gal4 fusions) with the {Delta}MTV-IR-CAT reporter using endogenous RAR/RXR or by expressing TR{alpha} (data not shown). These findings support the notion that AD2, rather than AD1, is necessary for NRC to enhance transcriptional activation by nuclear hormone receptors.


Figure 6
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  		Fig. 6. AD2 Is Necessary for Nuclear Receptor Signaling

HeLa cells were transfected with G5-pBL-CAT2 and the Gal4-NRC chimeras as depicted in the figure. Gal4-AD1-L-AD2 contains both ADs as well as LxxLL-1. Gal4-AD1-L lacks AD2. Gal4-NRC(849–1153) lacks AD1 but contains LxxLL-1 and AD2 whereas Gal4-NRCm(849–1153) has leucine to alanine substitutions in LxxLL-1. After transfection, the cells were incubated with and without 9-cis-RA which activates endogenous RAR/RXR in these cells. Although each of the Gal4-NRC chimeras show intrinsic activity, only those that contain AD2 and an intact LxxLL-1 motif show ligand-dependent enhancement. The various plasmids are described in detail in Materials and Methods. CAT, Chloramphenicol acetyltransferase; 9-cisRA, 9-cis-retinoic acid.

 
NRC Activates p53 and Stat2
Although NRC was cloned and characterized as a coactivator for nuclear hormone receptors, it has been shown by a number of laboratories that NRC enhances the activity of a variety of transcription factors including CREB, nuclear factor-{kappa}B, c-Jun, and c-Fos (22, 24, 31, 51). In our effort to identify other transcription factors regulated by NRC, we identified p53 and Stat2 as target factors that are regulated by NRC.

Stats are SH2 and SH3 domain-containing transcription factors that mediate the effects of interferon (INF) as well as many cytokines and growth factors upon phosphorylation and nuclear translocation. Response to INF{alpha} and -ß requires Stat2 and the INF-regulatory factor p48, which form a heterotrimeric transcription complex (ISGF3) with Stat1. The heterotrimeric complex then activates INF-regulated target sequence present in large number of cytokine-regulated genes. Stat1 plays an important role in DNA binding of the complex whereas Stat2 contributes to activation through a potent transactivation domain found in its C-terminal region. We tested the ability of NRC to activate Stat1 and Stat2 in HeLa cells using the INF-regulated target sequence reporter, pFluc-54 (49, 52). The reporter alone showed a significant level of activity in HeLa cells without incubation with INF. This likely reflects cytokine activity in the serum component of the culture medium. Expression of NRC further enhanced the activity of the Stat reporter approximately 4-fold whereas transfection with the control pEX vector had no effect (Fig. 7AGo). The ADs of Stat1 and Stat2 are localized at the C terminus of the proteins, and the AD of Stat2 is more potent than that of Stat1 (49). We examined the intrinsic activities of the C-terminal ADs of Stat1 and Stat2 as Gal4-DBD fusions (Gal4-Stat1c and Gal4-Stat2c) using G5-pBL-CAT2 (Fig. 7BGo). The intrinsic activity of Gal-Stat2c was greater than that of Gal-Stat1c, consistent with the idea that Stat2 contains a more potent AD (49). Expression of NRC enhanced the activity of Gal4-Stat2c approximately 4-fold whereas the activities of the Gal4-DBD alone and GalStat1c were not significantly affected by NRC (Fig. 7BGo). These findings suggest that the AD of Stat2 is targeted by NRC, which acts to modulate the Stat2 activity.


Figure 7
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  		Fig. 7. NRC Enhances the Activity of Stat2 via its C-Terminal AD

A, The reporter plasmid pFluc-54 was transfected in HeLa cells along with pEX-NRC or a control pEX vector. The amount of the various plasmids used are described in Materials and Methods. B, The Gal4-reporter G5-pBL-CAT2 was transfected into HeLa cells along with Gal4-Stat-2c or Gal4-Stat-1c, which express Gal4-fusions with the C-terminal ADs of the Stat proteins. Cells were also transfected as indicated to express Gal4 alone. Wild-type NRC was expressed from a pEX vector, whereas transfection with pEX alone served as a control. The experiment was repeated at least two times with similar results. For details of the plasmids used refer to Materials and Methods. CAT, Chloramphenicol acetyltransferase.

 
p53 is involved in the cellular response to stress and mediates either growth arrest or apoptosis in a cell type-specific manner and functions as a transcriptional activator of a number of genes including its own negative regulator, mdm2 (46, 53, 54). We previously studied the activity of p53 in cells using two p53-responsive reporters, CosX1-CAT and {Delta}MTV-59-CAT (46). Both contain a p53-responsive sequence from the first intron of the mdm2 gene. To study the effect of NRC on the activity of p53, HeLa cells were transfected with a p53 expression vector alone and with an NRC expression vector or an equal molar amount of a control pEX vector. Although HeLa cells have low levels of p53 as a result of its increased degradation, we have found that HeLa is a useful cell line to study p53 transcriptional activation (46). As shown in Fig. 8AGo, low levels of expression of p53 stimulated {Delta}MTV-59-CAT about 2-fold (lanes 1 and 2), and this activity was enhanced approximately 7-fold further by expression of NRC. Interestingly, the activity of {Delta}MTV-59-CAT was stimulated approximately 4-fold by NRC alone (lane 4), which is consistent with the notion that HeLa cells we used (46) contain low levels of functional p53. Similar results were obtained using CosX1-CAT (data not shown). The potentiation of p53-mediated transactivation by NRC was also assessed in HeLa cells using a Gal4-DBD-p53 chimera and G5-pBL-CAT2. Gal4-p53 stimulated the activity of the G5-pBL-CAT2 Gal4 reporter 4-fold compared with the Gal4 control. Activation of the reporter by Gal4-p53 was enhanced 3-fold further by expression of NRC (Fig. 8BGo). The control pEX vector did not affect the transactivation potentiation of p53. Taken together, our results indicate that NRC acts to enhance the transcriptional activity of p53.


Figure 8
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  		Fig. 8. NRC Enhances the Transcriptional Activity of p53

A, HeLa cells were transfected with the p53-responsive reporter {Delta}MTV-59-CAT with either pEX-NRC or a control vector pEX vector and/or p53. The samples were taken in duplicate, and the experiment was repeated at least twice with similar results. For concentrations of the various plasmids refer to Materials and Methods. B, The Gal4-responsive reporter plasmid G5-pBL-CAT2 was transfected with vectors expressing either Gal4, Gal4-p53 and with pEX-NRC or the control pEX vector as indicated. The results are the average of at least two independent experiments. For other details refer to Materials and Methods. CAT, Chloramphenicol acetyltransferase.

 
To further provide evidence for a role for NRC in p53 action, we use a small interfering RNA (siRNA) against NRC mRNA to knock down NRC protein expression to assess whether this affects human mdm2 (hmdm2) expression. U2OS cells were used for this study because they express NRC, hmdm2, and p53. Figure 9AGo shows that knockdown of NRC leads to a marked reduction of hmdm2 expression. This occurred without reduction in the level of p53 in the cell. Although hmdm2 may be modulated independently of p53 activation (55), these results further support a role for NRC in p53 action. To further document a role for NRC on the regulation of endogenous genes, we examined the effect of NRC knockdown on the all-trans-retinoic acid (ATRA) induction of the Sox9 gene. We previously showed that ATRA markedly enhances the expression of the Sox9 gene in MCF-7 cells (50). Figure 9BGo shows that knockdown of NRC leads to a marked reduction in Sox9 gene expression. Thus, these studies indicate that NRC not only affects gene expression in transient transfection assays but is also important for regulated expression of endogenous genes.


Figure 9
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  		Fig. 9. NRC Regulates Expression of the Endogenous p53 Target Gene, hmdm2, and Retinoic Acid-Induced Sox-9 Gene

A, p53 expressing U2OS cells were transfected with an siRNA that targets NRC mRNA or a control siRNA. After 50 h, cell extracts were prepared from duplicate samples and analyzed by Western blotting using specific antibodies for NRC, hmdm2, p53, and ß-actin. Equal amounts of protein were loaded in each lane. ß-Actin served as a control for equal loading and for nonspecific expression effects of siRNA. The experiment was repeated three times with similar results. B, MCF-7 cells were transfected with siRNAs as in panel A. After 36 h, cells were treated with 1 µM RA for 18–20 h before cell extracts were prepared from duplicate samples and analyzed by Western blotting using specific antibodies for NRC, Sox-9, and ß-actin. Con., Control.

 
To begin studying how the activity of p53 is enhanced by NRC, we examined whether NRC associates with p53 using both in vitro binding studies and yeast two-hybrid assays. However, these studies identified no direct interaction between NRC and p53 (data not shown). Therefore, we examined whether NRC associates with a known p53 interactor. Because p53 is known to associate directly with Trap80 (47), a component of the Mediator complex, we examined whether NRC might associate with p53 via Trap80 in yeast two-hybrid assays. As expected, p53 showed significant interaction with Trap80 in yeast (Fig. 10AGo). However, NRC did not show any significant interaction with Trap80 (data not shown). Because NIF-1 interacts with and enhances the activity of NRC, we examined whether NIF-1 interacted with p53 and Trap80. As shown in Fig. 10AGo, Trap80 associates with NIF-1, and the interaction is comparable to that found for Trap80 and p53. These findings suggest that the enhancement of p53 activity by NRC is mediated through protein-protein interactions involving Trap80 and NIF-1 (Fig. 10BGo).


Figure 10
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  		Fig. 10. Trap80 Interacts with p53 and NIF-1 in Yeast

A, Yeast were transformed with vectors expressing LexA or LexA-Trap80 along with the vectors expressing B42-NIF-1, B42-p53, or B42 alone as indicated. ß-Galactosidase activity was detected in duplicate from yeast cell lysates as described in Materials and Methods. B, The diagram depicts a putative model for p53 activation by a coactivator complex involving various protein-protein interaction among NRC, NIF-1, and Trap80, a component of the DRIP/TRAP mediator complex. Trap 80 is known to associate with p53 (47 ). Med-Trap Complex, Mediator, TR-associated proteins.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Nuclear hormone receptors are ligand-dependent transcription factors that bind to target genes and activate gene expression through two activation functions, AF-1 and AF-2. AF-1 is localized to the N-terminal A/B domain of certain nuclear receptors (e.g. GR, PR, ER) whereas AF-2 is localized to the LBD. AF-1 can activate transcription in the absence of ligand (e.g. when expressed as a Gal4-DBD fusion) whereas AF-2 is primarily ligand dependent. Abundant evidence indicates that activation through AF-2 occurs through the ligand-dependent recruitment of coactivators to the LBD. In general, the AF-2-dependent coactivators contain conserved AF-2-interaction motifs consisting of LxxLL motifs, referred to as NR boxes. Site-directed mutagenesis, transfection, in vitro binding, and crystallography studies have shown that the leucines in the LxxLL motif play a critical and essential role in binding to the LBD hydrophobic groove contributed by helices 3, 5, and 12 with helix 12 playing a critical role. Known coactivators such as SRC-1, SRC-2, and SRC-3 (p160 family) contain three or more LxxLL motifs. Although these motifs seem to display receptor-specific differential utilization, combinations of two appropriately spaced motifs are required for activation by TR, RAR, AR, and PPAR, and this specificity seems to be dictated by residues C-terminal to the core LxxLL motifs (56). Thus, various combinations of LxxLL motifs in the p160 family facilitate interaction of the coactivator with a variety of nuclear receptor LBD-AF-2 regions. Similarly, TRAP220/DRIP205 contains two LxxLL motifs with one of the motifs displaying preference for RXR, whereas the other motif interacts with either TR or VDR (44, 57).

The crystal structures for several nuclear receptor LBDs bound to coactivator peptides containing LxxLL motifs have been solved. These include the PPAR{gamma} LBD and an SRC-1 peptide containing two LxxLL motifs and the LBDs of TRß and ER{alpha} with a peptide containing LxxLL motifs derived from GRIP-1 (SRC-2) (1, 43). These crystal structures revealed that the leucines in the LxxLL motif form a hydrophobic surface on one face of an amphipathic {alpha}-helix, which interacts with the hydrophobic groove of the LBD. More importantly, the crystal structure also indicates that two LxxLL motifs from a single coactivator molecule bind to the two LBDs of a receptor dimer, indicating that a homo- or heterodimer can cooperatively recruit one molecule of a coactivator. Thus, except for NRC, other coactivators (e.g. the p160 family and Trap220/DRIP205) contain multiple LxxLL motifs capable of docking to the receptor dimer.

An interesting question relates as to how NRC, with a single functional LxxLL-1 motif, binds to a receptor dimer? We previously provided evidence that NRC formed dimers, which facilitates a high-affinity interaction of NRC with receptor dimers and explains how receptor dimers efficiently interact with NRC through a single LxxLL-1 motif (22). In our present studies we have further mapped the NRC dimerization domain. The dimerization domain is localized to 146 amino acids, which also contain the LxxLL-1 motif necessary for interaction with a broad array of nuclear receptors. Dimerization of NRC was not affected when LxxLL-1 (LVNLL) was mutated to AVNAA, suggesting that the LxxLL-1 motif is not involved in NRC dimerization. An analysis of secondary structure and potential phosphorylation sites at high stringency (http://scansite.mit.edu/) identified no phosphorylation sites or known dimerization motifs. Secondary structure prediction indicated that the 146-residue dimerization region contains a central hydrophilic region that is flanked by more hydrophobic residues. Analysis at low stringency identified that the 146-amino acid dimerization region contains seven putative SH3 domain homologies. Whether such SH3 domain homologies play any role in the dimerization of NRC will require more detailed study. We want to point out that although NRC interacts with itself through what we refer to as a dimerization region, it remains possible that NRC may form an oligomer(s) rather than a dimer. This would not alter the interpretation that NRC contains a self-associating interaction region.

It has been shown that certain coactivators activate nuclear receptors through interactions with the components of RNA polymerase II complex or through recruitment of histone acetyl transferases and/or histone methyl transferase to gene promoters. The intrinsic histone acetyl transferase activity of CBP has been shown to play a key role in transcriptional activation of some transcription factors. Recently methylation of arginine residues of CBP/p300 by CARM1 has been shown to inhibit CREB activation whereas methylation of acetylated nucleosomes by CARM1 has been shown to activate nuclear hormone receptors when coexpressed with p300 (58). Thus both methylation and acetylation of nucleosomes appear to enhance transcription activation. NRC was previously shown to strongly interact with CBP in vivo, and the activity of NRC was inhibited by E1A (22). Thus, collaboration of NRC with CBP, and possibly other factors, may be necessary for transcriptional activation by NRC.

NRC contains two ADs, AD1 and AD2. Although NRC contains a potent N-terminal AD (AD1), our results suggest that the Q/P-rich region, AD2, and not the AD1 AD of NRC plays an important role in nuclear receptor signaling. It is possible that AD2 of NRC associates with some nuclear cofactors necessary for assembly, modification, or association with transcription components. Recently, a number of factors (PIMT, CoAA, CAPER, and NIF-1) have been identified that bind NRC and enhance nuclear receptor function, and it is noteworthy that NIF-1, PIMT, and CoAA associate with the region of NRC that is just N terminal to AD2. Whether any of these factors including CBP/p300 or any of the components of the ASCOM complex specifically play a role in nuclear receptor signaling via AD2 remains to be elucidated. Because NRC activates a number of different transcription factors, it is possible that some of these factors utilize AD1 for transcriptional activation. Our present data strongly suggests that AD2 of NRC plays an essential role in nuclear receptor signaling. It is interesting to note that AD2 is juxtaposed C terminal to the nuclear receptor interaction domain, LxxLL-l, and it is possible that the liganded receptor modifies the structure of AD2 to facilitate the interaction of cofactors necessary for assembly, modification, or association with transcription components.

A number of known AF-2-dependent coactivators (SRCs, DRIPS/TRAPS) including NRC activate a broad spectrum of transcription factors (e.g. cJun, cFos, CREB, NF-{kappa}B). The underlying mechanism for this broad activity is unknown but may reflect an interaction of NRC with CBP, which is known to mediate transcriptional activation by a wide variety of factors. In our present study we have shown that NRC enhances the transcriptional activity of Stat2 and p53. Stat2 is unique in that it is the only Stat that does not homodimerize and instead forms a transcription factor trimeric complex (ISGF3) containing Stat1, Stat2, and p48 that regulates a number of important genes in response to interferon, growth factors, and various cytokines (52). The transactivation potential of ISGF3 is contributed primarily by Stat2 because it contains a much stronger AD than other Stat proteins (e.g. Stat1 and Stat 3). The C-terminal region of Stat2 contains a potent AD, and the last 40 amino acids of Stat2 are absolutely required for ISGF3 activity. Interestingly, the C-terminal Stat2 AD is also known to associate with CBP/p300 (48). Because CBP is also known to associate in vivo with NRC (22), NRC may cooperate with CBP in activating Stat2. In our present study we show that expression of NRC enhances the activity of the pFLuc54 reporter that is activated by the ISGF3 Stat transcription factor complex. Importantly, the activity of the C-terminal AD of Stat2 was greatly enhanced (~6 fold) by expression of NRC. In contrast, the activities of the less active C-terminal domains of Stat1 and Stat3 were not affected by NRC. These studies suggest that NRC targets and activates the ISGF3 complex via the C-terminal AD of Stat2.

p53 plays important functions in regulating cell growth, proliferation, development, and oncogenesis. p53 is a tetrameric sequence-specific DNA binding transcriptional activator that is necessary for normal cell growth, DNA repair, apoptosis, and cell proliferation. Using two different reporters containing sequences from the mdm2 gene that are activated by p53 ({Delta}MTV-59-CAT and CosX1-CAT), we found that NRC markedly enhances (~7-fold) the activation potential of wild-type p53 in cells. In addition, the activity of the Gal4-p53 (full length) chimera was enhanced 2- to 3-fold further by NRC. Furthermore, knockdown of endogenous NRC by siRNA leads to a reduction in expression of the p53-responsive hmdm2 gene. Interestingly, in vitro binding and yeast two-hybrid interaction data did not support a direct association between NRC and p53. Our findings, however, support a mechanism by which p53 activation by NRC is mediated through Trap80 and NIF-1. Trap80 is a component of the Mediator DRIP/TRAP complex that is known to bind in vitro with p53 (47). In yeast two-hybrid assays we found that Trap80 interacted avidly with NIF-1, suggesting that NRC activates p53 via NIF-1 and Trap80 interactions as depicted in Fig. 10BGo.

In conclusion, in this study we have mapped the dimerization domain of NRC and have documented an important role for AD2 of NRC in nuclear hormone receptor signaling. In addition, our studies support a role for NRC in transcriptional activation by Stat2, and the activation of p53 through a novel mechanism involving Trap80 and NIF-1. In future studies we hope to clarify the detailed mechanism(s) by which NRC enhances the activation of c-Fos, c-Jun, and Stat2 and to examine the role of the AD1 and AD2 domains in the activation of diverse factors by NRC.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Plasmids
We have previously described the cloning and expression of various human and rat NRC deletion constructs in yeast pJG4–5 (B42) and pEG202 or pEG{Delta}PL (LexA) plasmids (22, 31) including the following: B42-NRC(849–1153 (B2); LexA-NRC(849–1153) (L2) and the corresponding LxxLL-1 mutant (L2m); B42-NRC(849–1153) and the corresponding LxxLL-1 mutant (B2m); B42-NRC (1–848) (B1); B42-NRC(1153–2063) (B3); LexA-NRC(1153–2063) (L3); LexA-NRC(1–2063)(LN); B42-NRC(1–2063) (BN); B42-NRC(849–2063) (B4); and LexA-NRC(849–2063 (L4). The following NRC deletion clones were generated by PCR and cloned into pJG4–5 and pEG{Delta}PL followed by sequencing and expression studies: LexA-NRC (849–995) (L5); B42-NRC(849–995) (B5); LexA-NRC(995–1153) (L6); and B42-NRC (849–995) (B6). GST-NRC(849–995) was generated by cloning the corresponding DNA fragment of NRC into pGEX-4T followed by expression in Escherichia coli. NRC expressing AD1 and the LxxLL-1 region (Gal4-Ad1-L) (amino acids 1–929) was generated by subcloning a HindIII-BglII fragment of NRC into the Gal4 DBD vector pSG424 (22). Gal4-Ad1-L-Ad2 (amino acids 1–1153) was constructed by cloning a HindIII-AflIII fragment of NRC into pSG424. The following vectors have been described previously: Gal4-NRC (full length); Gal4-NRC(849–1153); Gal4-NRCm(849–1153) containing mutations in LxxLL-1; pEX-NRC expressing full-length NRC (22); and pEX-cTR{alpha} (45) along with the reporter plasmids {Delta}MTV-IR-CAT and G5-pBL-CAT2 (45). pEX-NIF-1, B42-NIF-1 (31), CMV-p53, Gal4-p53, and the p53 reporter plasmids CosX1-CAT and {Delta}MTV-59-CAT have been described previously (46). B42-p53 was a generous gift from Arnold Levine (UMDNJ, New Brunswick, NJ). LexA-Trap80 was generated by cloning a full-length insert released from CMV-Trap80 (kindly provided by Robert Roeder (47) into pEG{Delta}PL (22). The Gal4-plasmids expressing the C-terminal regions of Stat1 and Stat2 (Gal4-Stat1c and Gal4-Stat2c and the pFLuc54 Stat reporter) (48, 49) were from David Levy (New York University School of Medicine).

Yeast Two-Hybrid Assays
About 1 µg each of the various B42 and LexA fusion plasmids were transformed into the yeast strain EGY48 harboring the reporter plasmid pSH18–34 by Li/polyethylene glycol method as described previously (22). The interaction studies were carried out both on X-gal plates (SD-dextrose and SD-Gal/Raf) and by liquid ß-galactosidase assays using yeast extracts as described previously in detail (22, 31). Figure 1Go depicts interactions as + and – where + indicates significant interaction (30 ß-galactosidase units or greater), whereas – indicates no significant interaction (<2 ß-galactosidase units).

Mammalian Transfections
HeLa cells were transfected by the calcium-BES method as described previously (31). In general, HeLa cells (100,000–200,000 cells per well) in six-well plates or (40,000–60,000 cells per well) in 12-well plates were transfected in duplicate or triplicate with the various plasmids (10–500 ng/well). The Gal4 reporter plasmid G5-pBL-CAT2 was used at 50 ng/well whereas various Gal4 fusions were used at 50–600 ng/well). The {Delta}MTV-IR-CAT, {Delta}MTV-59-CAT, CosX-CAT, and pFluc-54 reporters were used at 500 ng/well. The Gal4-Stat plasmids were used at 20–50 ng/well.

In Vitro Binding
GST-NRC(849–995) protein was expressed in E. coli after inducing the protein with isopropyl-ß-D-thiogalactopyranoside followed by its purification on glutathione-agarose beads. Coupled in vitro transcription/translation was used to synthesize 35S-labeled NRC(849–2253) in reticulocyte lysates (Promega Corp., Madison, WI) was carried out as described previously (22, 45). The in vitro binding assay was carried out using approximately 200 ng of purified GST-NRC(849–995) protein and 2 µl of reticulocyte lysate labeled 35S-labeled NRC(849–1153) as described previously (22, 45).

Knockdown of NRC Expression by siRNA
An siRNA against human NRC mRNA from QIAGEN (Chatsworth, CA) (catalog no. SI02655485) was used at 25 nM and 40 nM in different experiments. A control siRNA was the same as used earlier (42). U2OS osteosarcoma cells and MCF-7 cells were transfected with siRNA in 12-well or six-well tissue culture plates using HiPerfect (QIAGEN) according to the manufacturer’s instructions. Forty eight hours after siRNA transfection, U2OS cells were harvested and examined for the levels of NRC, hmdm2, and p53 by Western blotting. Forty eight hours after the introduction of the siRNAs in MCF-7 cells, ATRA was added to half of the wells at 1 µM for 18 h before cell harvesting to study induction of Sox-9 by RAR (50). Western blotting with various antibodies was carried out using equal amounts of protein extracts, which were prepared as described previously (22). Sox-9 rabbit polyclonal antibody was obtained from Chemicon (Temecula, CA), mdm2 (2A10) antibody from Calbiochem (La Jolla, CA), ß-actin monoclonal antibody from Abcam, and p53 antibody (DO-1) was obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).


   ACKNOWLEDGMENTS
 
We thank Robert Roeder for providing us with CMV-Trap80 and Arnold Levine for the yeast p53 plasmid expression vectors.


   FOOTNOTES
 
This work was supported by National Institutes of Health Grant DK16636 and a grant from the Entertainment Industries Foundation.

Disclosure: The authors have nothing to disclose.

First Published Online May 29, 2007

Abbreviations: AD, Activation domain; ASC-2, activating signal cointegrator-2; ATRA, all-trans-retinoic acid; CARM1, coactivator-associated arginine methyltransferase 1; CBP, CREB-binding protein; CoAA, coactivator activator; CREB, cAMP response element binding protein; DBD, DNA-binding domain; DRIP205, vitamin D receptor-interacting protein 205; ER, estrogen receptor; GR, glucocorticoid receptor; GRIP, GR-interacting protein; GST, glutathione-S-transferase; INF, interferon; LBD, ligand-binding domain; NIF-1, NRC interacting factor-1; NRC, nuclear receptor coactivator; PIMT, PPAR-interacting protein (PRIP)-interacting methyltransferase; PPAR, peroxisome proliferator activator receptor; PR, progesterone receptor; RAR, retinoic acid receptor; RXR, retinoid X receptor; siRNA, small interfering RNA; SRC, steroid receptor coactivator; Stat, signal transducer and activator of transcription; TR, thyroid hormone receptor; TRAP, TR-associated protein; VDR, vitamin D receptor.

Received for publication December 22, 2005. Accepted for publication May 21, 2007.


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NURSA Molecule Pages Link:

Coregulators:   NIF-1  |  ASC-2
Ligands:   all-trans-Retinoic acid  |  9-cis-Retinoic acid



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