Protein-Protein Interactions and Transcriptional Antagonism between the Subfamily of NGFI-B/Nur77 Orphan Nuclear Receptors and Glucocorticoid Receptor

Christine Martens, Steve Bilodeau, Mario Maira, Yves Gauthier and Jacques Drouin

Laboratoire de Génétique Moléculaire, Institut de Recherches Cliniques de Montréal, Montréal, Quebec, Canada H2W 1R7

Address all correspondence and requests for reprints to: Dr Jacques Drouin, Laboratoire de génétique moléculaire, Institut de Recherches Cliniques de Montréal (IRCM), 110, Avenue des Pins Ouest, Montréal, Quebec, Canada H2W 1R7. E-mail: jacques.drouin{at}ircm.qc.ca.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Glucocorticoids (Gc) act through the glucocorticoid receptor (GR) to enhance or repress transcription of glucocorticoid-responsive genes depending on the promoter and cellular context. Repression of proopiomelanocortin (POMC) gene expression by Gc was proposed to use different mechanisms. We described the POMC promoter Nur response element (NurRE) as a target for Gc repression. NGFI-B (Nur77), an orphan nuclear receptor, and two related factors, Nurr1 and NOR1, bind the NurRE as homo- or heterodimers to enhance POMC gene expression in response to CRH. Gc antagonize CRH-stimulated as well as NGFI-B-dependent transcription. We now show that GR antagonizes NurRE-dependent transcription induced by all members of the Nur77 subfamily and that these nuclear receptors can all interact directly with GR. Transcriptional antagonism as well as direct protein-protein interaction between NGFI-B and GR take place primarily via their respective DNA binding domains, although DNA binding itself and the GR homodimerization interface are not involved. In vivo, GR and Nur factors can be coimmunoprecipitated whereas GR is recruited to the POMC promoter upon glucocorticoid action. Thus, our data suggest a mechanism for transrepression between two nuclear receptors, GR and NGFI-B, that is unique, although quite similar to that proposed for transrepression between GR and activator protein 1 (AP-1) or nuclear factor-{kappa}B (NF{kappa}B).


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
GLUCOCORTICOIDS (Gc) MEDIATE their effects by binding to the intracellular glucocorticoid receptor (GR), which, like other members of the steroid/thyroid hormone receptor superfamily, functions as a ligand-activated transcriptional regulator. GR can enhance gene transcription by binding with high affinity to the Gc response element (GRE) as a homodimer and by contacting the basal transcription machinery, coactivators, and other transcription factors (1, 2). This process is fairly well understood, but the mechanisms for transcriptional repression by GR are not as clearly defined. GR can repress gene expression either by binding to negative response elements (nGRE) or indirectly via protein-protein interaction (2). The best examples of the latter were described for the control of the inflammatory response. Indeed, the response to inflammation is partly mediated through activation of the transcription factors AP-1 (activator protein 1) and NF{kappa}B (nuclear factor {kappa}B)/Rel (3). The activation of AP-1 and NF{kappa}B target genes is repressed by Gc via a mechanism that may involve physical interaction between GR DNA binding domain (DBD) and AP-1 or RelA (4, 5, 6, 7, 8, 9, 10). Alternatively or in addition, these factors may antagonize each other’s action by sequestration of a common target such as cAMP response element binding protein (CREB)-binding protein (CBP)/p300 (11), although the implication of CBP itself is controversial (12).

The hypothalamo-pituitary-adrenal (HPA) axis is another system in which repression of transcription by Gc serves an essential function. In this case, Gc (and GR) provide a negative feedback loop after activation of the axis, e.g. in response to stress. Activation of the HPA axis starts with the secretion of hypothalamic CRH, the activation of pituitary proopiomelanocortin (POMC) gene transcription, and ACTH secretion in response to CRH, resulting in ACTH-induced stimulation of adrenal Gc synthesis. At the hypothalamic level, Gc represses CRH gene transcription (13, 14) and CRH secretion (15) and, similarly in the anterior pituitary, Gc repress POMC transcription (14, 16, 17, 18) as well as ACTH secretion (15). Different mechanisms have been proposed for Gc repression of POMC transcription. We have previously localized a nGRE in the proximal region of the POMC gene promoter (18, 19). The action of the nGRE appeared to be promoter context dependent (18) and required the cooperative binding of three GR molecules (19). Repression through the nGRE would thus be impaired with a GR mutation that prevents dimerization. Such GR mutation was introduced in mice by targeted gene replacement (knock-in), and it was found to hamper pituitary, but not hypothalamic, Gc feedback (20). Thus, within the specific context of these mice, the nGRE is a likely target to account for deficiency of dimerization-dependent GR repression. However, Gc repression of POMC transcription may also involve other promoter targets (21), in particular promoter targets that are dependent on cell-specific promoter recognition. We have identified such a target in the distal POMC promoter (22). This novel target, the NurRE (Nur response element), is a binding site for dimers of NGFI-B (Nur77), but it is not a GR binding site (23). We have proposed that GR antagonism of NurRE-dependent transcription occurs through protein-protein interactions, although the precise nature of this putative interaction remained to be defined. NGFI-B distinguished itself from other orphan nuclear receptors (24, 25) because it was the first nuclear receptor found to be active on transcription as a monomer (26). The binding site for NGFI-B monomers is the NGFI-B response element (NBRE) (26, 27) but, in contrast, the much more potent NurRE bound by NGFI-B dimers is a palindrome of two NBRE sequences separated by 6 bp (23, 28). There are two nuclear receptors related to NGFI-B, Nurr1 (Nur-related 1) (29) and NOR-1 (30). All three (collectively called the Nur factors) are very similar in the zinc finger DBD; they are relatively conserved in the putative ligand binding domain and less so in the N terminus (31). The crystal structure of their ligand-binding domain clearly supported other evidence that these orphan receptors may not be activated by a classical ligand as for other nuclear receptors (32). Nur factors have been implicated at all levels of the HPA axis. NGFI-B mRNA is rapidly induced in the paraventricular nucleus by stress stimuli (33), which constitute important regulators of hypothalamic CRH. Nur factors are expressed in the anterior pituitary (34) and NGFI-B, Nurr1, and NOR-1 mRNAs are increased in the pituitary tumor cell line AtT-20 after treatment with CRH (28, 35). It should be pointed out, however, that the POMC promoter NurRE has a clear preference for dimers that contain at least one molecule of NGFI-B, highlighting the importance of NGFI-B relative to other Nur factors in the POMC response to CRH (28). NGFI-B and Nurr1 transcripts are also strongly induced by stress in the adrenal cortex (36), and NGFI-B is implicated in regulation of the steroidogenic enzyme steroid-21 {alpha}-hydroxylase (27).

In the pituitary, we have demonstrated that signals elicited by CRH act on the NurRE for activation of POMC transcription and that these signals, mediated through the protein kinase A and MAPK pathways, involve Nur factors dimerization and coactivator recruitment (37, 38). Gc antagonize the NGFI-B-dependent transcriptional activation of POMC transcription (22). We now show that the other Nur factors, Nurr1 and NOR-1, are also subject to antagonism by GR and that this transrepression appears to involve direct protein-protein interactions between the DBDs of GR and Nur factors. However, the GR/Nur interaction does not appear to involve the same GR DBD residues as those required for GR homodimerization. In agreement with a model of transrepression involving direct interactions between GR and NGFI-B, both factors are corecruited to a common protein complex upon stimulation by hormones.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Essential Role of NurRE for Gc Repression
We have previously shown transrepression between GR and NGFI-B (22). Also, we have shown the stimulatory action of CRH-elicited signaling on both NurRE (37) and TpitRE (38) target sequences within the POMC promoter. Because CRH-induced POMC transcription is repressed by Gc, we tested whether either or both CRH promoter targets is (are) sensitive to Gc repression. Various POMC promoter constructs were used to this end in transfection experiments in AtT-20 cells followed by assessment of CRH and/or Gc [dexamethasone (Dex)] responsiveness. These experiments (Fig. 1Go) showed that mutagenesis of the NurRE (construct 2 compared with construct 1) did not significantly alter responsiveness to CRH, but completely abolished repression by Dex (a slightly higher response to CRH+Dex compared with CRH was even observed reproducibly). Deletion of the distal promoter region containing the NurRE (construct 5) behaved very similarly to the NurRE mutant (construct 2). In contrast, mutagenesis of the NBRE (construct 3), which may have been a NGFI-B target sequence, did not affect either CRH and/or Dex responsiveness. Mutagenesis of the TpitRE (construct 4), previously shown to support CRH response (38), did not significantly affect CRH response when assessed in the context of the intact promoter, but this mutation considerably reduced Dex repression. To test the intrinsic capacity of NurRE/Nur factors and TpitRE/Tpit targets to support hormone response independently of other promoter elements/factors, we used reporters containing either three copies of NurRE (construct 6) or TpitRE (construct 7). The activity of both reporters was enhanced by CRH as previously reported (37, 38) but only the NurRE reporters (but not the TpitRE) was repressed by Dex.



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Fig. 1. The NurRE, but Not the TpitRE, Supports Both CRH Responsiveness and Gc Repression

Different POMC promoter constructs fused to the luciferase reporter gene were assessed for activity by transfection into POMC-expressing AtT-20 cells. Reporter activity is shown with/without stimulation by 10–7 M CRH and/or 10–7 M Dex. Data are means ± SEM of at least three experiments each performed in duplicate.

 
These data clearly indicate that Nur factors are targeted by Gc and GR for repression whereas Tpit does not appear to be. It is noteworthy, however, that within the intact promoter context, both elements affect the ability to respond to Gc; this reflects the tight interdependence of POMC promoter-regulatory elements (39) and is supported by preliminary data suggesting the presence in AtT-20 cells of a large protein complex that contains many POMC transcription factors (data not shown).

Transrepression and Direct Interactions between GR and Nur Factors
We had previously demonstrated transrepression between Gc/GR and NGFI-B (22). The other Nur factors are quite homologous (Fig. 2AGo), and in many systems, more than one Nur factor is induced by the same signals (28, 40, 41, 42, 43, 44). To determine whether all three Nur factors can be antagonized by Gc, we used a NurRE reporter in cotransfection experiments to test the ability of Gc and GR to inhibit the transcriptional activity of each Nur factor (Fig. 2BGo). In the presence of Dex, GR antagonized the activity of all three Nur factors, albeit with slightly different potencies. We had previously shown that GR cannot bind the NurRE (22). We therefore assessed the hypothesis of a direct protein-protein interaction between GR and NGFI-B. Having previously shown that the GR DBD is involved in NGFI-B antagonism (22), we used a resin-bound glutathione-S-transferase (GST)-GR(DBD) fusion protein (45) in an in vitro pull-down assay to test for interaction with in vitro translated Nurr1, NOR-1, and NGFI-B (Fig. 2CGo). All three Nur factors, but not luciferase, interacted with GST-GR(DBD) but not with GST itself. These results indicate that, at least in vitro, Nur factors interact with GR DBD by direct protein-protein interactions.



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Fig. 2. Transcriptional Antagonism between Nur Factors and GR Involves Direct Protein-Protein Interaction

A, Schematic representation of the three known mouse Nur factors and their degrees of homology in the N-terminal, DBD, and putative ligand-binding domains (LBDs). Comparisons are relative to mouse Nur77. NGFI-B, the rat homolog of Nur77, is 96.3% identical to Nur77. B, Transcriptional antagonism of all three Nur factors by GR. CV-1 cells were cotransfected with expression vectors (50 ng) for Nur factors, GR expression vector (200 ng), and NurRE reporter (750 ng). RSV-GH (100 ng) was used as internal control. C, Direct protein-protein interaction between Nur factors and GR DBD. GST/GR(DBD) specifically retains NGFI-B, Nurr1, and NOR-1 in pull-down assays. GST retained neither Nur factors nor luciferase (Luc) in the assay.

 
Nur Factor Domains Required for GR Antagonism and Interaction
Previous analysis of NGFI-B (rat), Nur77 (mouse), and Nurr1 using monomer-dependent NBRE reporter genes had indicated that the N-terminal activation function 1 (AF-1) domain of these two orthologs was primarily responsible for transcriptional activity (37, 46, 47, 48). Because the POMC NurRE shows marked preference for Nur factor dimers that contain at least one NGFI-B moiety (28), indicating that this Nur factor plays a preferential role in POMC NurRE activity, we first analyzed the structure-function activity of NGFI-B mutants. Using a POMC NurRE-dependent reporter that is bound by Nur factor homo- or heterodimers (22, 28), we also found that the NGFI-B N-terminal domain accounts for most of its transcriptional activity (Ref.37 and Fig. 3AGo). Expression levels of all mutants were found to be similar as assessed by gel retardation using nuclear extracts of Cos-1 cells overexpressing each mutant protein (Fig. 3DGo). Gel retardation was used to assess activity and, indirectly, expression levels of different mutants because Western blots could not be used for all mutants (the available antisera recognizing either N- or C termini but not both) and mostly because the DNA binding activity of Nur factors is modulated by phosphorylation within the DBD (36, 37, 49). Gel retardation reveals expression of DNA binding-proficient Nur factors. Next, we mapped the domain of NGFI-B required for repression of GR action by testing NGFI-B mutants (Fig. 3BGo) for their capacity to repress GR-induced transcription of GRE reporter (Fig. 3CGo). Neither deletion of the N- or C termini was sufficient to prevent suppression of GR-dependent transcription, although the N-terminal deletion mutants appeared to be slightly less active even at higher concentrations of the expression vector (data not shown) than wild-type NGFI-B. The N terminus of NGFI-B may therefore contribute slightly to the repressor activity which otherwise appears to reside primarily with the DBD. This interpretation is supported by an internal deletion mutant devoid of the DBD. This mutant no longer repressed GR-dependent transcription (Fig. 3CGo). Because it was difficult to assess precisely the level of expression of this deletion mutant, we confirmed this interpretation with a protein that only contains the NGFI-B DBD. A protein flag was added to the DBD to verify its expression and to compare it to the level of full-length flag-NGFI-B expression (Fig. 3EGo). The flagged-DBD was sufficient to repress GR-dependent transcription (Fig. 3CGo). Similar Nurr1 mutants were constructed and assessed for their capacity to activate the NurRE reporter and to repress GR and GRE-dependent transcription (Fig. 4Go). As for NGFI-B, deletion of either C- or N termini did not impair repression of GR-dependent transcription, and a flagged Nurr1 DBD was sufficient for antagonism of GR activity (Fig. 4CGo). Expression levels of Nurr1 mutant proteins were assessed in gel shift experiments (Fig. 4DGo). Next, we wanted to ascertain whether the same NGFI-B and Nurr1 domains are implicated in direct protein-protein interaction with GR as in transcriptional antagonism. We used the in vitro pull-down assay (Fig. 2Go) and in vitro translated NGFI-B and Nurr1 mutant proteins. All NGFI-B (Fig. 5AGo) and Nurr1 (Fig. 5BGo) mutants bound the GST-GR(DBD) column except for the NGFI-B {Delta}DBD deletion mutant, in agreement with the model of an interaction mediated through the Nur DBD. Further, the N-terminal deletion mutants bind less efficiently than the intact factors to GR DBD. This difference in in vitro interaction may account for the lesser activity of the N-terminal deletion mutants in repression of GR activity. Thus, the N-terminal domain may facilitate the direct interaction between GR and Nur DBDs. Taken together, these findings suggest that the DBD of NGFI-B or Nurr1 is essential for transcriptional antagonism and for direct protein-protein interaction between Nur factors and GR.



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Fig. 3. The DBD of NGFI-B Is Sufficient to Repress GR-Dependent Transcription

A, Transcriptional activity of NGFI-B mutants using the NurRE-reporter. CV-1 cells were transfected with expression vectors (10 ng) and NurRE-reporter (750 ng). CMV-ßGal (50 ng) was used as internal control (ctrl), and the total amount of DNA was increased to 2.5 µg with pSP64. Data are presented as relative activity with Nur77 wild-type activity set at 100%. B, Schematic representation of NGFI-B mutants, including position of salient amino acid residues for rat NGFI-B. C, Antagonism of GR-dependent activity by NGFI-B and its mutants. Transfections were in the same conditions as above, using GRE reporter (750 ng), GR expression vector (75 ng), and NGFI-B expression vectors (500 ng). Data are presented as relative activity, and GR transcriptional activity in the presence of Dex is set at 100%. D, The NGFI-B constructs are expressed at similar levels in nuclear extracts of transfected Cos-1 cells. Levels of wild-type and mutant Nur factors were assessed by gel retardation using NBRE probe. E, Both flag constructs are expressed at the same level in nuclear extracts of CV-1 cells. Antiflag antibody was used to detect expression of flag-NGFI-B and flag-NGFI-B/DBD by Western blot. LBD, Ligand-binding domain.

 


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Fig. 4. The Nurr1 DBD Is Sufficient to Repress GR Transcriptional Activity

A, Transcriptional activity of Nurr1 mutants using the NurRE-reporter. Data are presented as relative activity with Nurr1 wild-type activity set at 100%. B, Schematic representation of Nurr1 mutants, including position of salient amino acid residues for rat Nurr1. C, Nurr1 transcriptional antagonism of GR-dependent activity. Data are presented as relative activity, and GR activity in the presence of Dex is set at 100%. D, The Nurr1 constructs are expressed at similar levels in nuclear extracts of Cos-1 transfected cells. Levels of wild-type and mutant Nur factors were assessed by gel retardation using NBRE and NurRE consensus probes. LBD, Ligand-binding domain; conc, consensus; ctrl, control.

 


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Fig. 5. Nur77 and Nurr1 Domains Required for Direct Interaction with GR(DBD)

A, Pull-down assay with NGFI-B and deletion mutants. The first panel shows protein input (10%) used for pull-down assays. The second panel shows all NGFI-B mutants retained specifically by GST-GR(DBD) but not GST (third panel) except for the DBD-deleted mutant ({Delta}DBD). B, Similar pull-down assay with Nurr1 constructs. C-terminal or N-terminal deletion mutants of Nurr1 interact with GR DBD.

 
NGFI-B/GR Transcriptional Antagonism Is Independent of GR Homodimerization Interface
Because GR can form homodimers that involve an interface within the DBD, we investigated whether this interface could also be involved in the interaction with NGFI-B. This interface was revealed by crystallography (50, 51) and defined functionally with mutations that prevent dimerization (52). Two mutants were used: a single amino acid change of alanine to threonine GRA458T or together with changes of three of five positions in the D-loop GR(D4X) (Fig. 6AGo). The GRA458T mutation was used in gene knock-in experiments to show the importance of GR dimerization and DNA binding for different GR functions (20). In transrepression experiments using NGFI-B and the NurRE reporter, both mutants were able to transrepress as efficiently as wild-type GR (Fig. 6BGo). These findings suggest that the interaction/antagonism between NGFI-B and GR does not take place via the same protein interface as GR homodimerization.



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Fig. 6. GR Dimerization Is Not Required for Antagonism of NGFI-B-Dependent Activity

A, Schematic representation of human GR second zinc finger showing mutated residues within the D loop for mutants GRD4X and GRA458T. These mutant proteins are unable to form GR homodimers. B, CV-1 cells were cotransfected with NurRE-reporter and NGFI-B (100 ng) and/or GR, GRD4X, or GRA458T (800 ng) expression plasmids. RSV-GH was used as internal control. The two GR mutants repressed NGFI-B activity as well as wild-type GR.

 
NGFI-B/GR Transcriptional Antagonism Is Independent of GR Residue K461
The GR mutant K461A (Fig. 7AGo) was shown to modify the indirect protein-protein interaction-dependent transrepressive effect of GR on both AP-1 and NF{kappa}B into an activation function (53, 54, 55). This mutant still exerted repressive activity by DNA binding of the osteocalcin promoter (53). When we assessed its activity on NurRE/NGFI-B-dependent transcription (Fig. 7BGo), GR K461A was even more effective than GR itself for repression of NGFI-B-dependent transcription. To compare these data with the ability of GR K461A to interference with NF{kappa}B or AP-1 activity, we tested GR K461A in our conditions using NF{kappa}B (Fig. 7CGo) and AP-1 (Fig. 7DGo) reporters. We found that the mutant GR did not repress p65-dependent activity, but our experimental conditions did not exhibit the GR K461A-dependent activation of transcription observed in other conditions/cells (54). In contrast to published data (53, 55), we found that GR K461A repressed tetradecanoyl phorbol acetate-dependent activity at the Col73 reporter just as well as wild-type GR (Fig. 7DGo); this discrepancy could be due to the difference in cell lines or promoter context. Because GR does not bind the NurRE (22) and thus account for repressor activity of K461A (53), we conclude that protein-protein interactions between GR and NGFI-B are responsible for repression and that these interactions must be different by comparison to those of GR/NF{kappa}B; the GR/AP-1 interactions may resemble those of GR/NGFI-B.



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Fig. 7. In Contrast to NF{kappa}B-Dependent Transcription, GR Mutant K461A Is as Effective as GR for Repression of NGFI-B-Dependent Activity

A, Schematic representation of rat GR zinc fingers showing the mutation K461A. B, CV-1 cells were cotransfected with NGFI-B (100 ng) and GR or GR K461A expression plasmids (800 ng). NurRE-reporter (750 ng) was used together with CMV-ßGal (25 ng) as internal control. GR K461A repressed NGFI-B-dependent activity as well as wild-type GR. Similar experiments were performed with a NF{kappa}B reporter containing two copies of NF{kappa}B-RE from the Ig{kappa} promoter placed upstream of the c-fos promoter (C) and with the collagenase gene promoter (Col73) that is AP-1dependent (D). NF{kappa}B activity was enhanced by expression of its p65 subunit, whereas AP-1 activity was stimulated with tetradecanoyl phorbol acetate (TPA).

 
Interaction between NGFI-B/Nur77 and GR in Vivo
To verify that NGFI-B and GR interact in vivo, we assessed their interaction by coimmunoprecipitation using nuclear extracts of AtT-20 cells pretreated or not with Dex and CRH. Nuclear extracts immunoprecipitated with a Nur77 antibody were analyzed by Western blotting with a GR antibody (Fig. 8AGo). This experiment showed that GR was immunoprecipitated together with Nur77 from AtT-20 nuclear extracts after treatment with Dex and CRH, but not from extracts of untreated cells. We further supported these data by immunoprecipitation of treated/untreated AtT-20 cells with a GR antibody and analysis of immunoprecipitates with a Nurr1 antibody (Fig. 8BGo). This showed recruitment of endogenous Nurr1 in treated, but not from untreated, cells; unfortunately, suitable NGFI-B antibodies were not available to probe these immunoprecipitates. To test directly the ability of GR and NGFI-B to interact in vivo, we set up a mammalian two-hybrid system. In this system, the activity of a Gal4/DBD-NGFI-B fusion protein was enhanced in the presence of VP16-GR1–513 (Fig. 8CGo). GR1–513 is deleted from the LBD, and therefore its activity is not ligand dependent. These results suggest that Nur77 and GR could interact together in vivo and that the signals elicited by CRH and Dex converge on Nur77.



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Fig. 8. Direct Interaction between Nur77/NGFI-B and GR in Vivo

A, Coimmunoprecipitation of Nur77 and GR from nuclear extracts of AtT-20 cells treated with Dex and CRH (T) but not from untreated cells (NT). An anti-Nur77 antibody (N19, Santa Cruz Biotechnology, Inc.) was used for immunoprecipitation and anti-GR (P20, Santa Cruz) was used for analysis by Western blotting. B, Coimmunoprecipitation of Nurr1 with GR from nuclear extracts of similarly treated (T) cells. An anti-GR antibody (M20, Santa Cruz) was used for Immunoprecipitation, and the BD Bioscience (Toronto, Ontario, Canada) Nurr1 antibody (N83220) was used for Western blot analysis. An asterisk identifies the specific Nurr1 band present in treated cells (T) but not in nontreated (NT) cells nor in IgG control precipitates. C, Two-hybrid assays were performed in CV-1 cells using expression vectors for fusion proteins Gal4-DBD (Gal4), Gal4-DBD/NGFI-B, and VP16/GR1–513 as indicated. Cells were cotransfected with a UAS-containing luc reporter plasmid. D, ChIP analysis of GR recruitment to the POMC promoter in AtT-20 cells treated with/without 10–7 M CRH and/or 10–7 M Dex. Promoter recruitment is shown for POMC promoter (–450 to –319 bp) relative to POMC exon 3 (+5036 to +5253 bp). The asterisk indicates statistical significance of P ≤ 0.05 (n = 6).

 
The chromatin immunoprecipitation (ChIP) technique was used to show recruitment of GR to the POMC promoter upon treatment of AtT-20 cells with Dex (Fig. 8DGo). Recruitment of GR to the promoter was slightly higher in cells treated with both Dex and CRH compared with Dex alone. This may reflect NGFI-B-dependent recruitment of GR to NurRE. Taken together, these observations are consistent with models of transrepression involving promoter-bound protein complexes that contain both GR and NGFI-B.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The repressor activity of GR is an essential feature of its antiinflammatory activity. It was shown that GR exerts its repressor activity through antagonism of positively acting transcription factors such as NF{kappa}B and AP-1 (2, 3). We have shown recently that GR antagonizes transcription elicited by immediate early response genes of the nuclear receptor family. Indeed, transcription elicited by NGFI-B (Nur77) is antagonized by GR. The mechanism of this antagonism appears to be quite similar to transrepression observed between GR and NF{kappa}B or AP-1 (22). In the present work, we have further defined the mechanism of transrepression between GR and the orphan nuclear receptors related to NGFI-B (Nur77) by showing that the two other related orphan nuclear receptors, Nurr1 and NOR-1, are also targets of GR antagonism (Figs. 2–4GoGoGo). Further, we have dissected mechanistic requirements for this antagonism by identifying the Nur factor DBD as the primary target for direct protein-protein interactions between Nur factors and GR (Figs. 3–5GoGoGo). Direct interaction between these proteins was also supported in vivo using a mammalian two-hybrid system (Fig. 8CGo) and, most importantly, in vivo corecruitment of endogenous Nur77 or Nurr1 and GR was shown to occur upon hormone treatment of AtT-20 cells. This was shown both by coimmunoprecipitation of endogenous NGFI-B and GR from nuclear extracts (Fig. 8AGo) and of GR and Nurr1 (Fig. 8BGo). Further, POMC promoter recruitment of GR (Fig. 8DGo) was observed after Dex treatment. Taken together, these data present a reciprocal picture for GR and Nur factors in the sense that antagonism between these proteins appears to be primarily dependent on their DBDs. Such a model is consistent with prior work on the antagonistic activity of GR with AP-1 (4, 5, 6), NF{kappa}B (7, 8, 9), or Nur factors (22).

We also show that GR mutant K461A is capable of repressing NGFI-B-dependent transcription as well as wild-type GR (Fig. 7BGo), whereas this mutant has lost the capacity to repress NF{kappa}B-dependent (Fig. 7CGo), but not AP-1-dependent (Fig. 7DGo), transcription. Because these three targets of GR transrepression depend on protein-protein interactions rather than direct GR DNA binding, the nature of these interactions appear different as they are not dependent on residue K461 for NGFI-B and AP-1, but they are for NF{kappa}B. Thus, antagonism between two nuclear receptors (GR and Nur factors) appears to have particular requirements by comparison with antagonism between GR and other structural classes of DNA binding proteins.

In view of the direct interaction between the DBDs of GR and NGFI-B (Fig. 5Go), it was formally possible that GR and NGFI-B might antagonize each other’s activity by formation of heterodimers such as those that form between many different nuclear receptors like GR and AR (56, 57), Nurr1 and retinoid X receptor (48), or NGFI-B and chicken ovalbumin upstream promoter transcription factor (58). We have shown that specific D-loop residues of GR required in its homodimerization (52) are not involved in the interaction with NGFI-B because their mutation did not impair GR repression of NGFI-B activity (Fig. 6BGo), ruling out the involvement of this well-documented dimerization interface. This observation is consistent with prior demonstration that in vitro interaction between these two proteins impairs their respective DNA binding activities (22). Although the Nur factor DBD appears to be the primary determinant for antagonism of GR activity, other Nur factor domains may also contribute to antagonism. Indeed, we showed in cotransfection assays that Nurr1 and NGFI-B DBD are sufficient to antagonize GR-dependent activity, but with less efficiency than full-length Nur factors (Figs. 3CGo and 4CGo). The N-terminal domain of NGFI-B may have an auxiliary role in interaction and transcriptional antagonism as revealed with N-terminal deletion mutants (Fig. 3CGo). Consistent with this model, the N-terminal truncated receptors did not interact as efficiently with GR DBD as full-length NGFI-B (Fig. 5AGo). Similar observations were made for Nurr1 (Fig. 5BGo). The C-terminal domain of nuclear receptors such as GR has been implicated in dimerization but, again highlighting the unique features of their antagonistic interactions, the NGFI-B or Nurr1 C-terminal domains are not involved in GR antagonism as revealed with deletion mutants (Figs. 3Go and 4Go). Similarly, the NGFI-B C terminus was not involved in formation of heterodimers with chicken ovalbumin upstream promoter transcription factor (58). Previous studies have demonstrated that GR DBD is essential for interaction with AP-1 (4) and that this interaction is stabilized by the GR C-terminal domain. In contrast, the GR DBD is sufficient for interaction with Nur factors as shown in pull-down assays (Fig. 2BGo). In summary, the DBDs of GR and Nur factors appear to be the primary determinants for interaction and antagonism of transcription.

The mechanism of repression through protein-protein interactions remains poorly understood (3). Originally, it was proposed that antagonistic factors prevented each other’s DNA binding ability through complex formation (4). Later, this model was revised to take into account the maintenance of promoter occupancy (i.e. DNA binding), even in the presence of the antagonistic factors, and the tethering model was proposed (59, 60). It is only very recently that further insight into this model was provided through investigation of in vivo recruitment of factors and cofactors on the IL-8 and intercellular adhesion molecule 1 promoters upon hormone treatment (54). This study revealed a unique pattern of serine-2 phosphorylation of the RNA polII C-terminal repeat on the promoters of genes subjected to antagonism between NF{kappa}B and GR. GR transrepression may also involve recruitment of histone deacetylase 2 to the NF{kappa}B/CBP complex (61).

Antagonism between GR and Nur factors has been implicated in two essential roles of maintenance and homeostasis. First, during maturation of T cells, Nur factors responsive to the T cell receptor (TCR) signaling promote apoptosis, and this response is antagonized by Gc (40, 41, 42). We have shown previously that Nur factor/GR antagonism operates in TCR-induced T cell lines on NurRE reporters that are targeted by Nur factor dimers (22). Also, Nur factors mediating the action of hypothalamic CRH activate transcription of the pituitary POMC gene (23, 37), and this action is antagonized by GR (22). This feedback interaction may be altered in patients with Cushing’s disease or in patients and animals under chronic stress.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Plasmids
The NurRE trimer reporter was constructed in pXP1-luc (62) containing the minimal (–34 to +63) POMC promoter as described previously (28). The GRE dimer was previously described (19). NGFI-B, Nurr1, and NOR-1 plasmids were described previously (28). {Delta}N1 NGFI-B mutant is an internal deletion between amino acid (aa) D3 and P214, {Delta}N2 is a deletion between L3 and L174, the {Delta}C mutant ends at aa 380, and {Delta}DBD/NGFI-B is an internal deletion within two Sma1 restriction sites. {Delta}C Nurr1 mutant is a C-terminal deletion at aa 352, and {Delta}N1 is an internal deletion between aa 72 and aa 162. An oligonucleotide flag (5'-gactacaaggacgacgatgacaag-3') was synthesized for insertion at the N-terminal region of NGFI-B and Nurr1. The flag-NGFI-B/DBD starts at T216 and ends at K362, and the flag-Nurr1/DBD starts at Q247 and ends at G397. In both cases, the flag is inserted in the N terminus. In vitro translated GR protein was produced using plasmid rBal117 (63). GST-GR(DBD) was graciously provided by M. Michalak (45). GRD4X and GRA458T mutants were described (52) and provided by A. Cato. GR K461A was described (55) and provided by K. Yamamoto. The NF{kappa}B reporter (64) was provided by J. Hiscott (Montréal, Quebec, Canada), and the Col73 reporter was from Michael Karin (University of California, San Diego). Vectors for the two-hybrid system were described previously (44, 65) except for the VP16/GR1–513, which was cloned at the EcoRV site of pCMX-VP16.

Cell Culture and Transfection
African green monkey kidney fibroblast-like CV-1 cells were grown in DMEM supplemented with 10% of bovine fetal serum and maintained at 37 C in an atmosphere of 5% CO2. CV-1 cells were plated in 12-well plates at a density of 50,000 cells per well the evening before transfection and then transfected by the calcium phosphate coprecipitation method. The next day cells were rinsed with PBS and fed with DMEM-10% FBS containing either vehicle or dexamethasone 10–7M for 24 h. Results show a representative experiment that was repeated three to five times, except for Figs. 2CGo and 3CGo where results are presented as the means ± SEM, n = 6. Plasmids cytomegalovirus (CMV)-ßGal or rous sarcoma virus (RSV)-GH were used as internal control for transfection efficiency.

Recombinant Protein Production and Pull-Down Assays
The GR/DBD-glutathione-S transferase (GST) and the GST fusion proteins were produced as described elsewhere (45). The GR(DBD) construct contains human GR sequences from aa 420–506, whereas the DBD has been described from residues 421–486. 35S-labeled in vitro synthesized NGFI-B, Nurr1, NOR-1, and luciferase, as well as NGFI-B and Nurr1 mutants, were obtained by using the TNT coupled reticulocyte lysate system (Promega Corp., Madison, WI). Protein-protein interaction assays (28, 66) were performed with 500 ng of fusion protein coupled to glutathione beads (Pharmacia Biotech, Piscataway, NJ) and about 50 ng of 35S-labeled protein in the binding buffer (150 mM NaCl; 10 mM Tris, pH 8; 0.3% 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate; 1 mM dithiothreitol; 10 µM ZnCl2; and 1 mM NaN3) for 4 h at 4 C.

AtT-20 Nuclear Extracts and Coimmunoprecipitation
AtT-20 D16V cells were grown under the same condition as CV-1 cells. AtT-20 cells were plated in 150-mm dishes at a density of 6,000,000 cells per dish 36 h before treatment with vehicle or 10–7 M dexamethasone and 10–7 M CRH for 3 h. Cells were washed twice with cold PBS and harvested in PBS containing 1 mM EDTA. The cells were then centrifuged and resuspended in 800 µl of buffer A (10 mM Tris, pH 7.9; 10 mM KCl; 0.1 mM EDTA; 0.1 mM EGTA; 0.1 mM phenylmethylsulfonylfluoride; 1 µg/ml aprotinin; 1 µg/ml leupeptin; and 1 µg/ml pepstatin). Cells were allowed to swell on ice for 15 min before addition of 50 µl Nonidet P-40 (10%) followed by vigorous vortexing. After centrifugation, the nuclear pellet was resuspended in 50 µl of buffer C (20 mM Tris, pH 7.9; 400 mM NaCl; 1 mM EDTA; 1 mM EGTA; 10–7 M Dex; and the same protease inhibitor cocktail that was in buffer A) and shaken vigorously at 4 C for 1 h. The protein concentration of the nuclear extract was estimated by the Bradford assay. Coimmunoprecipitation was performed using 100 µg AtT-20 nuclear extract, which was precleared with 4 µg of purified rabbit IgG (Sigma Chemical Co., St. Louis, MO). Nur77 antibody, 100 ng (N19; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) was used for immunoprecipitation. GR was revealed by Western blotting with a GR antibody (GR P-20; Santa Cruz Biotechnology) and an antirabbit antibody-horseradish peroxidase conjugate (Sigma). Revelation was performed by chemiluminescence as described by the manufacturer (ECL+plus; Amersham Pharmacia, Arlington Heights, IL).

Protein Overexpression and EMSAs
CV-1 cells were plated at 1,000,000 cells per 100-mm dish 16 h before transfection and then transfected the next day by the calcium phosphate coprecipitation method using 10 µg expression vector; the total amount of DNA was completed to 20 µg with pSP64. The next day, dishes were rinsed twice with PBS and harvested for nuclear extract preparation as for AtT-20 cells nuclear extract. Flag proteins were detected by Western blot (antibody M5, Sigma) and an antimice horseradish conjugate (Sigma). Revelation was performed by chemiluminescence as above. Cos-1 cells were grown under the same conditions as CV-1 and AtT-20 cells. Cells were plated at a density of 1,000,000 cells per 100-mm dish 16 h before transfection by the calcium phosphate coprecipitation method with 10 µg of expression vector, and we increased the total amount of DNA to 20 µg with pSP64. Cells were washed twice with cold PBS and harvested in PBS containing 1 mM EDTA. The cells were then centrifuged and resuspended in 400 µl of buffer A (10 mM Tris, pH 7.9; 10 mM KCl; 0.1 mM EDTA; 0.1 mM EGTA; 0.1 mM phenylmethylsulfonylfluoride; 1 µg/ml aprotinin; 1 µg/ml leupeptin; and 1 µg/ml pepstatin). Cells were allowed to swell on ice for 15 min before addition of 50 µl of Nonidet P-40 (10%) followed by vigorous vortexing. After centrifugation, the nuclear pellet was resuspended in 50 µl buffer C (20 mM Tris, pH 7.9; 400 mM NaCl; 1 mM EDTA; 1 mM EGTA; and the same protease inhibitor cocktail as in buffer A) and shaken vigorously at 4 C for 1 h. The protein concentration of the nuclear extract was estimated by the Bradford assay. Nuclear extract (5 µg) was used for gel shift experiments as previously described (28).

ChIP Assays
AtT-20 cells were prepared for ChIP as described elsewhere (67). Sonicated chromatin corresponding to 107 cells was subjected to immunoprecipitation at 4 C with 3 µg of GR antibody-matched nonimmune IgG (Sigma) as negative control. Immunoprecipitates were collected with protein A/G agarose beads saturated with tRNA. Beads were washed as described by Upstate Biotechnology, Inc. (Lake Placid, NY). Quantitative real-time PCR (Stratagene MX-4000; Stratagene, La Jolla, CA) was performed with the SYBR Green kit (QIAGEN, Chatsworth, CA). For the POMC promoter, PCR amplification of sequences between –450 to –319 bp was performed using primers 5'-TGGTTTCACAAGATATCACACTTTCCC-3' and 5'-TCGGAGTGGAATTACCTATGTGCG-3'. For exon 3, a 124-bp fragment was amplified using primer 5'-AAGTACGTCATGGGTCACTTCCG-3' and 5'-TCGGCTCT- GGACTGCCAT-3'.


    ACKNOWLEDGMENTS
 
We thank V. Giguère (NGFI-B), O. Conneely (Nurr1), N. Ohkura (NOR-1), T. Perlmann (Gal4-Nurr1, Gal4-NGFI-B), K. Yamamoto (6RGR, rBal117, GR K461A), M. Michalak [GST-GR(DBD)] and A. Cato (GRD4X, GRA458T) for generously providing plasmids. We thank K. R. Yamamoto and Inez Rogatsky for the GR antibody used in ChIP experiments and L. Laroche for excellent secretarial assistance.


    FOOTNOTES
 
This work was supported by the Canadian Institutes of Health Research (CIHR). M.M. is recipient of a CIHR studentship, S.B. is the recipient of studentships from le Fonds pour la Formation de Chercheurs et l’Aide a la Recherche-Fonds de la Recherche en Santé du Québec and Canadian Institutes of Health Research.

First Published Online December 9, 2004

Abbreviations: aa, Amino acid; AP-1, activator protein 1; CBP, cAMP response element-binding protein (CREB)-binding protein; ChIP, chromatin immunoprecipitation; CMV, cytomegalovirus; DBD, DNA-binding domain; Dex, dexamethasone; Gc, glucocorticoids; GR, glucocorticoid receptor; GRE, glucocorticoid response element; GST, glutathione-S-transferase; HPA, hypothalamo-pituitary-adrenal; NBRE, NGFI-B response element; NF{kappa}B, nuclear factor {kappa}B; nGRE, negative GRE; NurRE, Nur response element; POMC, proopiomelanocortin; RSV, rous sarcoma virus.

Received for publication August 24, 2004. Accepted for publication December 1, 2004.


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