AF-2-Dependent Potentiation of CCAAT Enhancer Binding Protein ß-Mediated Transcriptional Activation by Glucocorticoid Receptor

Marcin Boruk1, Joanne G. A. Savory1 and Robert J. G. Haché

Departments of Medicine (R.J.G.H.) and Biochemistry (M.B., J.G.A.S, R.J.G.H.) University of Ottawa Ottawa Civic Hospital Loeb Research Institute Ottawa, Ontario, Canada K1Y 4E9


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
We report glucocorticoid-dependent induction of transcription from the herpes simplex virus thymidine kinase gene promoter proximal regulatory region in the absence of glucocorticoid response elements and independent of the ability of glucocorticoid receptor (GR) to bind DNA. Examination of the thymidine kinase promoter localized glucocorticoid responsiveness to a binding site for CCAAT enhancer-binding proteins (C/EBPs). Further analysis indicated that GR specifically potentiated the induction of transcription by C/EBPß, but not C/EBP{alpha} or {delta}, and that full induction could be obtained by the ligand-binding domain (LBD) of GR alone. C/EBPß, but not C/EBP{alpha} or {delta}, reciprocally potentiated transcriptional activation by DNA-bound GR LBD. However, C/EBPß was unable to increase activation by a GR LBD with a short C-terminal truncation, indicating that the functional interaction between the two factors was dependent upon the GR AF-2. Surprisingly, despite the specificity in functional effects, all three C/EBPs bound indistinguishably to GR in GST pull-down and immunoprecipitation assays. Indeed, several nuclear receptors, including the estrogen (ER{alpha}), progesterone, retinoic acid (RAR), and androgen receptors, displayed a similar potential to bind C/EBPs. Previous reports have demonstrated that ER{alpha} and RARs repress transcriptional activation by C/EBPß in ways that were dependent on their related AF-2 functions. Therefore, the GR AF-2 may encode functional features that distinguish the transcriptional regulatory potential of GR from that of ER and RAR. Finally, C/EBP binding mapped to the GR DNA-binding domain, which was not required for functional interaction with C/EBPß. Thus, the potentiation of C/EBPß-mediated transcription by GR would appear to require the presence of an intermediary factor.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Transcription of the herpes simplex virus thymidine kinase (HSV-tk) promoter (-109/+51) is regulated by the synergistic interaction of CCAAT enhancer binding proteins (C/EBPs) and Sp1 (1, 2, 3, 4). Sp1 is a ubiquitous transcription factor with a zinc-fingered DNA-binding region that activates transcription of many mammalian genes but is of particular importance for the transcription of constitutively expressed structural genes lacking a TATA box (5).

By contrast, the C/EBPs are a subfamily of the tissue-restricted bZip transcription factors, which regulate transcription through CCAAT DNA sequence motifs (6, 7, 8, 9). There are several C/EBPs genes and many different isoforms of the C/EBP proteins (1, 10, 11, 12, 13). The bZip domains of most of the C/EBPs are highly conserved (8). However, the remainder of the proteins vary considerably between individual family members. C/EBPs have been shown to play determining roles in the differentiation (14) and function of hepatocytes (15, 16), adipogenesis (17, 18, 19, 20, 21, 22, 23, 24), and the functional regulation and homeostatic control of lymphoid (19, 22) and hematopoietic cells (19). Interestingly, C/EBPß (also known as NF-IL6, Il-6DBP, LAP, AGP/EBP, CRP2, and NF-M), but not C/EBP{alpha}, has been shown to specifically interact with Sp1 in a manner that allows it to regulate transcription from the rat CYP2D5 P450 gene (25).

HSV replication occurs more efficiently in cells treated with the synthetic glucocorticoid dexamethasone (dex) (26). However, glucocorticoids have not previously been reported to directly induce or otherwise influence transcription of the viral tk gene. Glucocorticoids mediate transcriptional regulation through an intracellular nuclear hormone receptor that binds as a homodimer with high affinity to specific glucocorticoid-responsive DNA sequences [glucocorticoid-responsive elements (GREs) (27, 28)]. The promoter-proximal regulatory region of the tk gene does not contain a sequence resembling GRE (4).

GR, like all nuclear receptors, is a modular protein with a central DNA- binding domain flanked by carboxy- and amino-terminal transcriptional regulatory functions (29, 30, 31, 32, 33). In the absence of hormone, glucocorticoid receptor (GR) normally occurs in the cytoplasm in a high molecular weight complex with heat shock proteins and immunophilins (34). Steroid binding induces a conformational change in the receptor ligand-binding domain (LBD), which promotes dissociation of the GR-heat shock protein complex and allows translocation of the free receptor to the nucleus (34).

The activation of transcription by nuclear receptors is accomplished through interactions with transcriptional coregulatory proteins that promote the modification of chromatin structure and that interact with the basal transcriptional machinery (35, 36, 37). While the N-terminal activation functions appear to be unique to each receptor, the AF-2 activation functions at the C terminus of GR and other nuclear receptors (38, 39, 40, 41, 42, 43) interact in an overlapping manner with a series of transcriptional coactivator protein complexes that include proteins such as SRC-1 (44), CBP (45, 46, 47), and GRIP-1 (48, 49) and have histone acetylase activity (50, 51). Interestingly, many other transcription factors also appear to interact with the same coactivator complexes. This creates the potential for competition by nuclear receptors and other transcription factors for a limited pool of coactivator molecules. For example, it is now clear that GR competes with other nuclear hormone receptors and transcription factors such as CREB and AP-1 for CBP-containing coactivator complexes (45, 52, 53, 54). However, the differential interaction of transcription factors with common coactivators also may explain elements of the transcriptional synergism observed between nuclear receptors and other sequence-specific transcription factors on complex promoters.

Not all effects of GR on transcription result from the direct binding of receptor homodimers to canonical GREs. A number of transcriptional effects resulting from direct protein-protein interaction of GR with other sequence-specific transcription factors have been described. For example, direct interaction between GR and AP-1 has been demonstrated to be required to direct transcription from composite response elements that bind both factors together (55). Transcription is enhanced or repressed by this complex, depending on the specific c-fos family member in the jun/fos AP-1 heterodimer (55, 56, 57, 58). Recently, it also has been demonstrated that GR can act essentially as a coactivator to potentiate the activation of transcription from PRL-responsive promoters in the absence of a GRE by binding to DNA-bound Stat5 (59).

In the present study we have determined that glucocorticoids activate transcription from the HSV-tk proximal promoter despite the absence of a GRE. The results of our analysis suggest that this effect is mediated through a functional interaction between the AF-2 of GR and C/EBPß. These results contrast with the recent demonstrations that the AF-2 activities of retinoic acid receptor and estrogen receptor-{alpha} (ER{alpha}) can act to repress C/EBPß-mediated transcription (17, 60). Therefore, our results indicate one way in which the GR AF-2 may be functionally distinct from the AF-2s of other steroid/retinoid receptors.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The HSV Promoter Is Activated by Glucocorticoids in the Absence of the Binding of GR to DNA
During the course of experiments examining the mechanism of transcriptional regulation by GR in Cos7 cells, we observed that the -109/+51 sequence from the HSV-tk promoter was strongly inducible by dex in the presence of coexpressed GR (data not shown). This was unexpected because, although this region of the tk promoter contains two Sp1 DNA-binding sites and one C/EBP-binding site, it does not contain a discernible GRE (Fig. 1AGo) (3, 4).



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Figure 1. Activation of HSV-tk Transcription by Glucocorticoids

A, Schematic of the HSV-tk promoter proximal (-109 to +51 bp) regulatory region linked to a CAT reporter gene. This portion of HSV-tk contains two GC-rich Sp1 DNA binding sites at -105 and -56 and a CCAAT DNA response element at position -87. B, Cos 7 cells were cotransfected with the HSV-tk CAT reporter construct and GR or AR expression plasmids as indicated. Ligand treatments were 0.2 µM dex, 1.0 µM RU 38486, or 0.05 µM DHT in ethanol as indicated. Relative CAT activity is expressed as fold induction over cells treated with ethanol alone and is corrected for variations in transfection efficiency. SD values were calculated from three independent experiments each performed in duplicate.

 
It has been demonstrated previously that N6 methylation of adenine residues as a result of dam methylation of plasmids grown in dam + strains of Escherichia coli can lead to the artefactual creation of cryptic GREs (61). Therefore, to determine whether dam methylation had resulted in the creation of a cryptic GRE on our HSV-tk reporter plasmid, we repeated the transfections with a plasmid prepared in the dam- dcm- Rb404 E. coli strain (Fig. 1BGo). Dex treatment of cells cotransfected with the tkCAT reporter and a rat GR expression plasmid resulted in a 4-fold induction of chloramphenicol acetyltransferase (CAT) activity (lane 1). Thus, the glucocorticoid responsiveness of the tk promoter was not due to dam methylation. The activation of transcription was dependent upon GR and hormone agonist, as no induction was detected in the absence of cotransfected receptor expression plasmid (lane 4) or when GR-expressing cells were treated with the glucocorticoid antagonist RU486 (lane 2). Further, androgen receptor (AR) was unable to substitute for GR (lane 3). Indeed, dihydrotestosterone (DHT) treatment of cells expressing AR reproducibly led to a 2-fold repression of CAT activity. Finally, to confirm that the observed effect was not due to treatment of the cells with high levels of dex, we repeated the experiment at 33 nM dex, the optimal concentration for the use of this glucocorticoid in the Cos7 parental line CV1 (62), with similar results (data not shown).

To confirm that this effect was mediated in the absence of GR binding to DNA and to begin to localize the determinants on GR required for this effect, we examined the transcriptional response of the tk promoter to three additional GR constructs (Fig. 2Go). First, expression of full-length GR with an L501P mutation in the DNA-binding domain (DBD) that abrogates sequence-specific DNA binding (63) actually led to a slightly stronger response, with the induction of CAT activity from the tk promoter increased from 4- to 6-fold (lanes 1 and 2). However, expression of the N-terminal 525 amino acids of GR had no effect on reporter gene activity (lane 3). By contrast, N525 constitutively activates transcription from a GRE (64). Finally, expression of a GR fragment N-terminally truncated at amino acid 547 at the border of the LBD was as efficient in activating tk transcription as WT GR (lane 4). Thus, the ligand-binding domain of GR appeared to be sufficient for full induction of tk expression.



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Figure 2. The Ligand Binding Domain of GR Is Sufficient for the Activation HSV- tk Transcription by dex

Cos 7 cells were cotransfected with HSV-tk CAT and expression constructs for the mutant GRs whose structures and properties are summarized at the top of the figure. Below, CAT activity is presented as fold induction of activity in GR expressing cells over cells transfected with the tk reporter gene alone. For lanes 1, 2, and 4, cells were treated with 0.2 µM dex. Error bars represent the SD obtained from three independent experiments performed in duplicate. For lanes 1 and 2, similar effects were obtained with 33 nM dex (data not shown).

 
GR Activates Transcription from the tk CCAAT Element
The tk promoter used in these experiments contains binding sites for both Sp1 and C/EBP (3). To determine whether the potentiation of tk transcription by GR was mediated through one of these sequences, we prepared two CAT constructs with a minimal adenovirus E1B minimal promoter and four copies of an Sp1-binding site or the C/EBP response element (Fig. 3Go, top). As a control, a similar construct was prepared with four copies of an octamer motif, which does not occur in the tk promoter. In addition, we also recloned the -109/-29 HSV-tk promoter-proximal regulatory region in front of the E1B promoter to determine whether similar effects could be detected with a heterologous promoter. Transfections were then performed to determine the response of these constructs to the activation of GRL501P by dex (Fig. 3Go, bottom). In GRL501P-transfected cells, dex treatment failed to activate transcription appreciably from the SP1-responsive promoter or control construct with four octamer motifs inserted adjacent to the E1B sequence (lanes 1 and 2). By contrast, the CCAAT/E1B construct was hormone responsive, with dex treatment inducing CAT activity 4-fold (lane 3). This suggested that GR specifically targeted factors acting through the C/EBP-binding site in the tk promoter.



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Figure 3. GR Potentiates Transcription through a CCAAT Response Element

Cos7 cells were transfected with a vector expressing GRL501P and one of four E1B CAT reporter constructs or a construct with the tk promoter from -109 to +51 with an inactivating mutation in the C/EBP binding site. The structure of these constructs is summarized at the top. The fold induction of CAT activity in response to treatment of the transfected cells with 0.2 µM dex is shown at the bottom. Similar effects were observed at 33 nM dex.

 
Interestingly, when placed adjacent to the E1B promoter, the promoter-proximal tk-regulatory region was only weakly responsive to GRL501P and dex, with CAT activity being induced just under 2-fold (lane 4). One possibility suggested by this result was that the minimal tk promoter was somehow also making an important contribution to the GR responsiveness. However, recloning the tk sequences into the E1B promoter also resulted in an increase in spacing of 16 bp between the DNA response elements in the tk region and the TATA box element. Thus it is also possible that the decreased response was due to this change in relative positioning of the tk response elements and the TATA box, which is equivalent to 1.5 turns of the DNA helix.

Finally, to determine whether the entire effect of GR on tk transcription was mediated through the C/EBP-binding site in the tk promoter, we determined the response of a -109 to +51 tk reporter gene in which site-directed mutagenesis had been used to convert the C/EBP-binding site to a nonfunctional sequence that has previously been described (65). Dex treatment of cells cotransfected with GRL501P and the HSV-tk C/EBPmut reporter plasmid was completely unable to induce reporter gene activity. Therefore, the C/EBP binding site in the tk promoter was both required for and sufficient for the induction of transcription by GR in Cos7 cells.

GR Potentiates the Activation of Transcription by C/EBPß, but Not by C/EBP{alpha} or {delta}
The results obtained in the experiment shown in Fig. 3Go suggested that GR had the ability to potentiate the activation of transcription by one or more isoforms of C/EBP. To evaluate the selectivity of dex induction of transcription through the CCAAT element, we examined the effect of coexpressing GRL501P and three C/EBP proteins, C/EBP{alpha}, ß, and {delta}, on the induction of transcription of the CCAAT/E1B CAT reporter gene (Fig. 4Go). In the absence of dex, expression of C/EBP{alpha}, ß, and {delta} each resulted in a 5- to 8-fold induction of CAT activity. The same result was obtained in the absence of cotransfected GR, and no induction was observed on the parent E1B reporter construct lacking the CCAAT response elements (data not shown). Treatment of cells cotransfected to express C/EBP{alpha} or {delta} and GRL501P with dex had no significant additional effect on transcription (Fig. 4AGo, lanes 2 and 4). However, when C/EBPß was coexpressed with GRL501P, dex treatment led to a strikingly further induction of transcription (lane 3). Reexpression of the data as fold induction by dex (Fig. 4BGo) highlights that GRL501P induced CAT activity 4-fold above the level induced by C/EBPß, but had only a minimal effect on the transcription induced by C/EBP{alpha} and C/EBP{delta}.



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Figure 4. Enhancement of Transcription by GR through a CCAAT DNA Response Element Is Specific for C/EBPß

Cos 7 cells were cotransfected with p6RGRL501P, p4C/EBPG5E1BCAT, and pMSVC/EBP {alpha}, ß, and {delta} as indicated. Cells were treated with 0.2 µM dex in ethanol. CAT activities were corrected for ß-galactosidase. CAT activities relative to untreated cells transfected with p6RGRL501P are shown in panel A while the fold induction of CAT activity in response to dex treatment in the presence of C/EBP{alpha}, ß, and {delta} are displayed in panel B.

 
Functional Interaction between GR and C/EBPß Requires the C-Terminal AF-2 Activity of GR
Our results indicated that the LBD of GR contained a hormone-dependent ability to potentiate the activation of transcription by C/EBPß, in the absence of GR binding to DNA. One question raised by these results was whether C/EBPß could reciprocally potentiate the activation of transcription of DNA-bound GR in the absence of CCAAT response elements. To address this question, we tested the ability of the C/EBPs to potentiate the activation of transcription by a GR LBD construct fused to the yeast GAL4 DBD. This experiment was performed using a reporter gene with 5 GAL4-binding sites driving transcription from the E1B promoter (Fig. 5Go).



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Figure 5. Interaction of the GR LBD with C/EBPß in a Mammalian Two-Hybrid Assay Is Dependent on AF-2

Cos 7 cells were cotransfected with a G5E1BCAT reporter plasmid, GALGRLBD, and GALGRLBD781 fusion protein expression plasmids and C/EBP expression plasmids as indicated to assess the ability of C/EBPs to potentiate the activation of transcription by the LBD of GR. Data are expressed relative to expression of the GAL4 DBD alone (A) or as fold induction in the presence of GAL-LBD fusion protein vs. the GAL4 DBD (B). All experiments were performed in the presence of 0.2 µM dex, which is well above the concentration necessary to saturate GAL-LBD781. In panel C, the expression of levels of GAL-LBD and GAL-LBD781 were compared by Western analysis of whole-cell extracts probed with GAL4 antibody (Santa Cruz).

 
Cotransfection of the C/EBP expression plasmids with pGalO, a plasmid expressing GAL4 DBD alone, had no effect on E1B expression (Fig. 5AGo, lanes 1 and 4–6). Expression of the GAL-LBD construct in the presence of dex induced CAT activity approximately 8-fold above the level obtained with GalO (lane 2 Fig. 5Go B, lane 1). Coexpression of C/EBPß with GAL-LBD increased the induction of transcription in response to dex treatment a further 4-fold (Fig. 5BGo, lane 3). By contrast, coexpression of C/EBP{alpha} or {delta} resulted in no significant additional transcriptional activation above the level induced by GAL-LBD alone (lanes 2 and 4). Therefore, while the potentiation of transcription again appeared to be a specific property of the GR LBD and C/EBPß, which partner was tethered to DNA appeared to be unimportant.

The inability of RU486-treated GR to potentiate the activation of tk transcription (Fig. 1Go) suggested that the GR-C/EBPß interaction could be linked to AF-2 function of GR, which is unresponsive to RU486. Deletion of 14 amino acids from the C-terminal end of GR inactivates the AF-2 function, with a decrease in ligand-binding affinity that can be compensated for by treatment with pharmacological concentrations of hormone (66). To determine whether the AF-2 function of the GR LBD was required for C/EBPß to potentiate GAL-LBD-mediated E1B transcription, we repeated our experiment with GAL-LBD781 (Fig. 5Go). As expected, GAL-LBD781 was ineffective in activating E1B transcription (Fig. 5AGo, lane 3), and no additional activity was observed upon coexpression of C/EBP{alpha}, or {delta} (Fig. 5BGo, lanes 6–8). However, in this instance, coexpression of C/EBPß also failed to increase reporter gene transcription. Western blotting, shown in Fig. 5CGo, demonstrated that GAL-LBD and GAL-LBD781 were expressed at similar levels. Thus, our results indicate that functional interaction between the GR LBD and C/EBPß was dependent on the integrity of the GR AF-2.

The Potentiation of C/EBPß-Mediated Transcriptional Activation Occurs Independent of Binding to GR
To investigate whether the functional interactions observed between the GR LBD and C/EBPß might correlate with protein-protein interactions between the two factors, we tested the ability of in vitro translated GR peptides to bind to C/EBP{alpha} and C/EBPß expressed as GST fusion proteins. The results of this experiment are displayed in Fig. 6Go. Contrary to expectations, full-length, dex-treated GR bound strongly to both C/EBPs, not just C/EBPß (lanes 1 and 8). Deletion of the N terminus of GR up to the DBD (X795) had no effect on binding (lanes 2 and 9), nor did deletion of AF-2 (X781, lanes 3 and 10). Similarly, both C/EBPs were bound by a GR peptide containing amino acids 1–523 (lanes 6 and 13), a fragment of GR that was unable to potentiate C/EBP activity in transfection experiments (Fig. 2Go). By contrast, the LBD of GR (547C), which was sufficient for the potentiation of C/EBPß activity in transfection experiments, failed to bind either C/EBP (lanes 5 and 12). Finally, a GR peptide containing only the DBD (X616) retained full C/EBP-binding activity in this assay (lanes 4 and 11).



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Figure 6. The DBD, but not the LBD, of GR Binds Specifically to GST-C/EBP{alpha} and GST-C/EBPß

In vitro translated GR fragments or firefly luciferase were incubated with GST-C/EBP{alpha} (A), GST-C/EBPß (B), or GST alone (C). Specifically bound proteins were resolved on 10% SDS-PAGE gels. In panel D, 10% of the in vitro translated GRs were added to the binding assay. All GRs except X616 and N523 were activated by preincubation with 0.2 µM dex before binding. The GR peptides contained the following amino acid sequences of GR: GR, 1–795; X795, 407–795; X781, 407–781; X616, 407–616; 547C, 547–795; N523, 1–523.

 
The binding of GR to C/EBPß and {delta} was investigated further by incubating in vitro translated C/EBPs with GR immunoprecipitated from fibroblasts expressing a stably transfected WT GR with an N-terminal C-myc antibody tag (Fig. 7Go). To investigate the hormonal requirements for binding, GRs were prepared by salt extraction from cells treated with dex or RU486 and from untreated cells. In this experiment, both in vitro translated C/EBPß (Fig. 7Go A) and C/EBP{delta} (Fig. 7Go B) were coimmunoprecipitated to approximately the same extent from all three extracts prepared from GR-positive cells (lanes 5–7), while little binding was detected in extracts prepared from control cells lacking GR (lanes 2–4). In further contrast to the transcription results, C/EBP binding was the same for RU486-treated GRs and GRs transformed by heat and salt, as it was for dex-treated receptors.



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Figure 7. GR Binds Specifically to C/EBPß and C/EBP{delta} in an Immunoprecipitation Assay

A and B, Whole-cell extracts prepared from Sf7 cells with (lanes 5–7) or without (lanes 2–4) stably transfected myc-tagged GR treated for 15 min with 0.2 µM dex (lanes 2 and 5), RU486 (lanes 3 and 6) or 0.4 M KCl (lanes 4 and 7) to transform GR in the absence of ligand were incubated with in vitro translated C/EBPß (A) or C/EBP{delta} (B). Binding was detected on 15% SDS-PAGE gels. C, Western blot illustrating the loading of the GRs used in panel A.

 
C/EBP Binding Is a Conserved Property of Steroid/Retinoid Receptors
Two other nuclear hormone receptors in addition to GR, estrogen receptor-{alpha} (ER{alpha}) and retinoic acid receptor-{gamma} (RAR{gamma}), have been reported to interact functionally with C/EBPß in an AF-2-dependent manner (17, 60). However, in contrast to the inductive effects of GR, both ER{alpha} and RAR were observed to repress C/EBPß-mediated transcription. Further, peptides including the DBD of ER{alpha} have previously been shown to bind to C/EBPß in vitro (60). As GR bound C/EBPß in an apparently similar manner, we wondered whether C/EBP binding was a conserved property of steroid/retinoid receptors. Our results, displayed in Fig. 8Go, indicate that ER{alpha}, AR, and retinoic acid receptor ß (RARß) also bound to both C/EBP{alpha} and ß in a GST pull-down assay. The same result was also obtained with RAR{gamma} (data not shown). By contrast, an unrelated transcription factor, nuclear factor 1, and firefly luciferase did not interact with the C/EBPs. Thus, it appears that the ability to bind C/EBPs is a property of several nuclear hormone receptors and may be important for the repression of C/EBPß-activated transcription by ER{alpha}, RAR, and potentially AR, but is dispensable for the potentiation of C/EBPß-activated transcription by GR. However, the functional significance of this potential for direct binding between steroid/retinoid receptors and the three C/EBP isoforms remains to be completely elucidated.



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Figure 8. Nuclear Receptors, but Not NF-1, Bind Specifically to GST-C/EBP{alpha} and GST-C/EBPß

In vitro translated, 35S-labeled proteins were incubated with GST-C/EBP{alpha} (A), GST-C/EBPß (B), or GST alone (C). Specifically bound proteins were resolved by 10% SDS-PAGE gels. Panel D shows 10% of the proteins added to the binding assay. All gels were exposed equally. Before binding, the nuclear receptors were pretreated with specific ligands as described in Materials and Methods.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Glucocorticoids and C/EBPß converge in the regulation of a large variety of cellular processes, including responses to inflammation and stress, and in tissue differentiation, e.g. differentiation of preadipocytes to mature adipocytes. It is well established that glucocorticoids and C/EBPß interact cooperatively in the regulation of the transcription of many of the genes whose induction contributes to these processes (67, 68, 69). The results presented in this work suggest that cooperative interactions between C/EBPß and GR in the activation of specific gene transcription need not be dependent upon the close proximity of DNA-binding sites for each factor or upon direct protein-protein interactions between the two factors. Rather, they suggest that GR can potentiate the transcriptional regulatory potential of C/EBPß indirectly in a manner that is independent of DNA binding by GR. Indeed, this effect appeared to be dependent solely upon the receptor LBD. Moreover, the ability of C/EBPß to potentiate AF-2 dependent transcriptional activation by the DNA-bound GR LBD suggests that this process may be effective from both GREs and CCAAT-response elements. Interestingly, however, the transcriptional effects were specific for C/EBPß, as neither C/EBP{alpha} nor C/EBP{delta} interacted productively with GR in our experiments.

To date, three main mechanisms for the regulation of gene transcription by glucocorticoids have been established: direct activation through GREs; direct repression through negative GREs; and transcriptional interference resulting from the direct interaction of GR with other sequence-specific transcription factors (29, 70, 71). Our results suggest a fourth mechanism, transcriptional cooperativity mediated indirectly through an interaction between GR and the transcriptional machinery downstream from the binding of C/EBPß to DNA.

The activation of transcription by GR and C/EBPß, like most transcription factors, is mediated through interactions with transcriptional coactivator molecules, proteins with histone acetyltransferase activity that do not bind DNA themselves, but function as bridging molecules between sequence-specific transcription factors and the basal transcriptional machinery (72, 73). Recently, it has become apparent that many of these coactivator molecules exist in larger coregulatory complexes that include several different coactivator molecules (74). For example, p300/CBP occurs in complexes with SRC-1, p/CAF, GRIP-1, and potentially other factors (75).

The activation functions of many sequence-specific transcription factors bind directly to a variety of sites on individual coactivators (76). Recently, it has been demonstrated that nuclear receptors and other transcription factors exhibit different requirements for coactivators within a coactivator complex, and it has been suggested that coactivator complexes exist in multiple alternative configurations (74). Thus, liganded nuclear receptors, including GR, RAR, and ER{alpha}, interact with p/CAF and SRC-1, while C/EBPß and CREB interact with p300/CBP (45, 49, 52, 74, 77, 78).

Two schemes to explain how the functional interaction between GR and C/EBPß observed in our experiments might take place in the absence of DNA binding by GR are presented in Fig. 9Go. As the reciprocal potentiation of transcriptional activation of GR and C/EBPß in our experiments was mediated indirectly, and both factors interact differently with coactivators that occur in the same complex, it is plausible that the functional interaction between GR and C/EBPß occurred at the level of the coactivator complex (Fig. 9Go, panel 1). In this scenario, in response to dex treatment, liganded GR interacting at a second site on the C/EBPß-coactivator complex would enhance the ability of the coactivators to activate transcription. The effect on transcriptional activation could be mediated allosterically or by inducing changes in the composition of the complex. Certainly, the feasibility for the formation of such a complex has been established (74, 79). It should also be noted that this model also suggests a mechanism for transcriptional synergism when both GR and C/EBPß are bound to DNA. In this instance, the binding of GR and C/EBPß to DNA might be expected to further stabilize the recruitment of the larger regulatory complex.



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Figure 9. Schematic Presentation of Possible Ways in Which the GR AF-2 Function Could Stimulate the Activation of Transcription by C/EBPß

C/EBPß is known to interact with p300/CBP, transcriptional coactivator molecules that occur within larger transcriptional coactivator complexes that include molecules such as SRC-1, p/CAF, and GRIP-1. SRC-1 and potentially other molecules in the complex are also direct targets of the AF-2 domain of GR. In the first scenario (panel 1), upon ligand binding, GR enters into the coactivator complex that is associated with C/EBPß and acts to stimulate the activity of this complex. Possible mechanisms for this effect are discussed in the text. In a second scenario (panel 2), the GR AF-2 domain interacts with a factor (X) still unknown, but that functions in the context of the coactivator complex, or downstream from the complex, to decrease the efficiency of C/EBPß-mediated transcriptional activation.

 
A second possibility (Fig. 9Go, panel 2) would be that GR interacts with a molecule [X] that acts negatively downstream from the C/EBPß-coactivator complex to decrease transcriptional activation. In this instance, GR would remove or titrate a block on the communication of the C/EBPß-coactivator complex with basal transcription factors. This possibility seems less likely as there are presently no potential candidates for this activity. Interestingly, one potential compromise between these two possibilities is that the binding of GR to the coactivator complex could relieve a repressive activity that occurs directly within the coactivator complex.

Our results clearly dissociated the binding of GR to C/EBPß from the potentiation of C/EBPß-mediated transcription. Indeed, the minimum GR fragment required for the potentiation of transcription activated by C/EBPß was the only GR fragment tested in binding assays that failed to bind C/EBPß. Further GR also bound to C/EBP{alpha} and C/EBP{delta}, but had no effect on the activation of transcription by these factors under our experimental conditions. These results clearly sever the previously proposed linkage between GR-C/EBPß binding and the potentiation of C/EBPß-mediated transcription. However, it remains possible that GR-C/EBP binding will prove to be biologically relevant in other cell types or in response to additional signaling molecules not included in the present study. Alternatively, it is also possible that the binding observed here for C/EBP{alpha}, ß, and {delta}, and reported previously for C/EBPß, does not reflect a productive association between these factors in the cell.

In the present study, we observed that GR-C/EBPß binding in vitro requires the GR DBD, while a previous study demonstrated that binding required the bZIP DBD of C/EBPß (80). As this is the conserved region of the C/EBPs, it would seem probable that the binding of GR to C/EBP{alpha} and {delta} would also be to the bZIP domain. Thus, a third possibility would be that the binding of the C/EBPs to DNA could interfere with the protein-protein interaction with GR. The DBDs of some factors, including GR, can simultaneously accommodate protein-protein and protein-DNA interactions (59, 80, 81, 82, 83, 84, 85). By contrast, we have recently demonstrated that a direct interaction between the GR DBD and the POU DNA binding domain of transcription factors Oct-1 and Oct-2 was dissociated by the binding of GR to a GRE (86). For GR and the octamer factors, the protein-protein interaction is nonetheless productive, as it serves to promote the binding of the octamer factors to response elements adjacent to DNA-binding sites for GR. Thus determining how GR-C/EBP binding responds to the presentation of GREs and CCAAT elements may suggest how this interaction might occur productively in the cell.

While GR potentiates the ability of C/EBPß to activate transcription, there are reports that ER{alpha} and RAR act to repress the activation of transcription by C/EBPß (17, 60). In our study, we also observed that AR repressed the activation of transcription by C/EBPß and that RARß, ER{alpha}, and AR bound C/EBPß similarly to GR. For ER{alpha} and RAR, repression also required AF-2 (17, 60), which would appear to suggest a difference in the function of the AF-2 of GR and that of ER{alpha} and RAR. For example, it is possible that the differences in effect reflect differences in the association of GR and ER{alpha}-RAR with a common coactivator complex.

For ER{alpha}, however, the repression of transcription induced by C/EBPß also was dependent upon the receptor DBD (60). Indeed, we note that, upon deletion of the DBD, ER{alpha} reverted from a repressor of C/EBPß-induced transcription to an activator similar to GR. Thus a second possibility is that the difference in the effect of ER{alpha} and GR on C/EBPß may be explained by differences in the way ER and GR bind to C/EBPß. A resolution of the molecular basis for the differences in the interaction of GR, ER{alpha}, and RAR with C/EBPß will require a direct comparative study of their individual effects.

GR is required for viability, as mice lacking a GR gene die shortly after birth from a defect that results in the lack of production of surfactant proteins in the lung (87). The recent demonstration, that mutant GRs compromised for DNA-binding and DNA-dependent dimerization are viable (88), highlights that many important functional activities of GR are mediated in the absence of direct contact of the receptor with DNA. The most intensively investigated DNA-independent effects of GR have been in the interference with the activities of NF{kappa}B and AP1. Our results suggest that potentiation of the transactivation potential of C/EBPß may be another important way in which GR may exert physiological effects in the absence of DNA binding. Functional interaction between GR and C/EBPß is most obvious in their effects on inflammation and in the differentiation of preadipocytes. It will be interesting to determine to what degree the effects of glucocorticoids on these processes are dependent on the interaction between GR and C/EBPß reported here.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Plasmids
The rat GR eukaryotic expression constructs, p6RGR (89), p6RGRN525 (64), and p547C (90), have been previously described. Other constructs were derived from these vectors as described below. p6RGRL501P was created by site- directed mutagenesis of a p6RGR DNA fragment. pGaLLBD and pGaLLBD781 were created by PCR amplification of GR LBD fragments encoding amino acids 540–795 and 540–781, respectively, into the SalI-XbaI sites of pGalO (91). C/EBP expression vectors pMSV C/EBP{alpha}, ß, and {delta} have been previously described (9). The rat AR was expressed from pSV40 AR (92). The HSV-tk CAT reporter vector was essentially that previously described containing HSV-tk sequences -109/+51 (93). Adenovirus E1B reporter constructs were prepared by cloning into the XbaI site at -45 adjacent to the minimal E1B promoter of pG5E1BCAT (94). Four copies of C/EBP (5'-CTA GGA GTG TCA TTG GCG AGG-3') binding sites, octamer motifs (5'-AGGAGC TTG CTT ATG CAA ATA AGG TG-3'), and Sp1 (5'-CTA GCG ACC CCG CCC AGC GTG-3') binding sites were cloned into pG5E1BCAT to generate p4Sp1G5E1BCAT, p4C/EBPG5E1BCAT, and p4OctG5E1BCAT reporter plasmids. PCR amplification was used to clone the -109/-29 promoter-proximal regulatory region of HSV-tk adjacent to the E1B promoter at -45 in pG5E1BCAT to generate pG5tkE1BCAT. Mutagenesis of the C/EBPß response element in the tk promoter was performed by changing the sequence of the CCAAT element at -88 to -90 -GAGTCGGACA-80 (65), by performing PCR amplification with a mutated oligo and recloning the amplified product into the BamHI/RsrII sites of original HSV-tkCAT reporter gene. All constructs were verified by DNA sequencing. In all experiments pRSV ß-gal was cotransfected to monitor transfection efficiency. C/EBPß and C/EBP{delta} plasmids for in vitro translation were created by isolating EcoRI/BamHI ß and {delta} fragments from pMSVC/EBP ß and {delta} and recloning into pGEM-7Z (Promega, Madison, WI).

The plasmids used for in vitro translations, GRWT (pRDN93) (95), X795, X781, X616, and 547C (66), have all been described previously. N523 was generated by digesting T7N556 (66) with PstI. The AR (92), ER (96), RARß (97), and nuclear factor 1 (98) vectors have been described previously. pSP6Luciferase was from Promega. The pTL-MTG GR expression vector has been described previously (86).

In initial experiments, and for all plasmids not used as reporters in transient transfections, DNA was prepared from E. coli-DH5{alpha}. Reporter plasmids were prepared from E. coli Rb404 (strain) to preclude the presence of cryptic GRE resulting from bacterial methylation (61).

Transient Transfection Analyses
Cos 7 cells were maintained in DMEM containing 10% FBS at 37 C. Sixteen hours before transfection, 2 x 105 cells were seeded onto 35-mm plates. Transfections were performed using Lipofectamine (Life Technologies, Gaithersburg, MD; 5 µl per 35-mm plate). Each transfection was performed using 0.3 µg CAT reporter plasmid, 0.3 µg ß-galactosidase reporter, and, as indicated, 0.6 µg steroid receptor expression plasmids and 0.3 µg C/EBP expression plasmids. Sixteen hours posttransfection, the medium was replaced with DMEM-10% FBS supplemented with steroidal ligands or ethanol alone as described in individual experiments. Dex (Steraloids, Wilton, NH) was added to 0.2 µM, RU38486 (RU486) was added to 1.0 µM, and DHT (Steraloids) was added to 0.05 µM. In selected transfections the lower concentration of 33 nM dex was used with similar results. Cells were then allowed to grow for an additional 48 h.

ß-Galactosidase (used to normalize results for variations in transfection efficiency) and CAT assays were performed essentially as previously described (86). Conversion of acetylated chloramphenicol was quantified using phosphorimager analysis (Bio-Rad, Richmond, CA). CAT activity was corrected for ß-activity. Each data point represents the average of a minimum of three independent experiments each performed in duplicate. All error bars represent the SEM.

GR-C/EBP Binding Assays
GST fusion proteins were prepared and purified on glutathione Sepharose essentially as described (23). 35S-labeled proteins were produced using the Coupled Transcription-Translation TNT Reticulocyte Lysate System (Promega). Steroid binding to in vitro translated receptors was done by adding 1 µM all-trans-retinoic acid to RAR, DHT to AR, diethlystilbestrol (DES) to ER, and dex to GR for 2 h at 4 C. For GST-binding assays, 35S-labeled proteins were incubated with 0.5 µg immobilized GST-fusion protein in 200 µl binding buffer [(15 mM HEPES, pH 7.9, 60 mM KCl, 12% glycerol, 1 mM EDTA, 1 mM dithiothreitol, 0.1 mM phenylmethylsulfonyl fluoride] for 90 min at 4 C and washed three times. The proteins retained on the affinity matrix were eluted in SDS sample buffer, resolved by SDS-PAGE, and visualized by autoradiography. A fraction representing 10% of the in vitro translated proteins added to the binding assay was loaded on identical gels and exposed together with the gels containing the bound fractions.

For immunoprecipitation assays, myc-tagged GR was immunoprecipitated from cellular extracts of Sf-7 murine fibroblasts stably transfected with a vector expressing WT GR with an N-terminal myc tag (86). The protein A-Sepharose beads complex was preblocked in 150 µl binding buffer and 5 µl rabbit reticulocyte lysate at 4 C for 2 min. Samples were then centrifuged at 4000 rpm (4 C), after which the precipitate was resuspended in another 150 µl binding buffer. Equivalent amounts (as determined by phosphoimage analysis) of the desired in vitro translated C/EBP isoform were added and allowed to bind to the affinity-purified GR for 2 h at 4 C. Samples were washed three times with 500 µl binding buffer. After the washes, samples were resuspended in 20 µl SDS sample buffer, boiled for 5 min, and run on SDS-PAGE. The gel was then dried and bands were quantified by phosphorimager analysis. Binding to immunoprecipitates from MTG GR-containing extracts was compared with that of the parental Sf-7 cells, which do not express MTG GR. MTG GR loading in each experiment was confirmed by Western immunoblotting.

Western blotting for GR was done as previously described (99). After SDS-PAGE, protein samples were electroblotted from the SDS-PAGE gel to a polyvinylidene fluoride membrane. The primary antibody used was anti-myc antibody, 9E10 (1:2000 dilution). Detection of 9E10 signal was done by enhanced chemiluminescence (ECL, Amersham, Arlington Heights, IL) using horseradish peroxidase-conjugated sheep antimouse antibody (1:50,000 dilution) (Amersham), as the secondary antibody. Expression levels for the pGALO constructs were verified by Western blotting with the an anti-GAL4 antibody (Santa Cruz Biotechnology, Santa Cruz, CA).


    ACKNOWLEDGMENTS
 
We would like to thank Drs. K. Yamamoto, S. Liao, G. Tomaselli, and W. H. Lee for generously providing us with plasmid DNAs. The pMSVC/EBP constructs were graciously provided by Tularik (San Francisco, CA). We are also grateful to our colleagues, Y. Lefebvre, G. Préfontaine, and C. Schild-Poulter, for their critical commentary on the manuscript.


    FOOTNOTES
 
Address requests for reprints to: Robert J. G. Haché, Ottawa Civic Hospital Loeb Research Institute, 1053 Carling Avenue, Ottawa, Ontario, Canada K1Y 4E9. E-mail: rhache{at}lri.ca

This work was supported by operating funds from the Medical Research Council of Canada (to R.J.G.H.). R.J.G.H. is a Scholar of the Medical Research Council of Canada and the Cancer Research Society, Inc.

1 M. Boruk and J. Savory contributed equally to this work and should be considered co-first authors. Back

Received for publication December 31, 1997. Revision received July 1, 1998. Accepted for publication July 23, 1998.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Osada S, Yamamoto H, Nishihara T, Imagawa M 1996 DNA binding specificity of the CCAAT/enhancer-binding protein transcription factor family. J Biol Chem 271:3891–3896[Abstract/Free Full Text]
  2. Seo SJ, Kim HT, Cho G, Rho HM, Jung G 1996 Sp1 and C/EBP-related factor regulate the transcription of human Cu/Zn SOD gene. Gene 178:177–185[CrossRef][Medline]
  3. Graves BJ, Johnson PF, McKnight SL 1986 Homologous recognition of a promoter domain common to the MSV LTR and the HSV tk. Cell 44:565–576[Medline]
  4. McKnight SL, Kingsbury R 1982 Transcriptional control signals of eukaryotic protein-coding gene. Science 217:316–324[Medline]
  5. Azizkhan JC, Jensen DE, Pierce AJ, Wade M 1993 Transcription from TATA-less promoters: dihydrofolate reductase as a model. Crit Rev Eukaryot Gene Expr 3:229–254[Medline]
  6. Piccolo S, Marigo V, Girotto D, Volpin D, Bressan GM 1995 Identification of a recognition element for CAAT-enhancer binding proteins (C/EBPs) in the elastin promoter. Biochim Biophys Acta 1264:40–44[Medline]
  7. Landschulz WH, Johnson PF, Adashi EY, Graves BJ, McKnight SL 1988 Isolation of a recombinant copy of the gene encoding C/EBP. Genes Dev 2:786–800[Abstract]
  8. Landschulz WH, Johnson PF, McKnight SL 1988 The leucine zipper: a hypothetical structure common to a new class of DNA binding proteins. Science 240:1759–1764[Medline]
  9. Cao Z, Umek RM, McKnight SL 1991 Regulated expression of three C/EBP isoforms during adipose conversion of 3T3–L1 cells. Genes Dev 5:1538–1551[Abstract]
  10. Akira S, Isshiki H, Sugita T, Tanabe O, Kinoshita S, Hirano T, Kishimoto T 1990 A nuclear factor for Il-6 expression (NF-IL6) is a member of a C/EBP family. EMBO J 9:1897–1906[Abstract]
  11. Chumakov AM, Grillier I, Chumakova E, Chih D, Slater J, Koeffler HP 1997 Cloning of the novel human myeloid-cell-specific C/EBP{epsilon} transcription factor. Mol Cell Biol 17:1375–1386[Abstract]
  12. Descombes P, Chojkier M, Lichtseiner S, Falvey E, Schibler U 1990 LAP, a novel member of the C/EBP gene family, encodes a liver-enriched transcriptional activator protein. Genes Dev 4:1541–1551[Abstract]
  13. Muller C, Kowenz-Leutz E, Grieser-Ade S, Graf T, Leutz A 1995 NF-M (chicken C/EBPß) induces eosinophilic differentiation and apoptosis in a hematopoietic progenitor cell line. EMBO J 14:6127–6135[Abstract]
  14. Diehl AM, Johns DC, Yang S, Lin H, Yin M, Matelis LA, Lawrence JH 1996 Adenovirus-mediated transfer of CCAAT/enhancer-binding protein-{alpha} identifies a dominant antiproliferative role for this isoform in hepatocytes. J Biol Chem 271:7343–7350[Abstract/Free Full Text]
  15. Trautwein C, Rakemann T, Pietrangelo A, Plumpe J, Montosi G, Manns MP 1996 C/EBP-ß/LAP controls down-regulation of albumin gene transcription during liver regeneration. J Biol Chem 271:22262–22270[Abstract/Free Full Text]
  16. Soriano HE, Bilyeu TA, Juan TS, Zhao W, Darlington GJ 1995 DNA binding by C/EBP proteins correlates with hepatocyte proliferation. In Vitro Cell Dev Biol 31:703–709
  17. Schwarz EJ, Reginato MJ, Shao D, Krakow SL, Lazar MA 1997 Retinoic acid blocks adipogenesis by inhibiting C/EBPß-mediated transcription. Mol Cell Biol 17:1552–1561[Abstract]
  18. Zhang DE, Hetherington CJ, Meyers S, Rhoades KL, Larson CJ, Chen HM, Hiebert SW, Tenen DG 1996 CCAAT enhancer-binding protein (C/EBP) and AML1 (CBF {alpha}2) synergistically activate the macrophage colony-stimulating factor receptor promoter. Mol Cell Biol 16:1231–1240[Abstract]
  19. Screpanti I, Romani L, Musiani P, Modesti A, Fattori E, Lazzaro D, Sellitto C, Scarpa S, Bellavia D, Lattanzio G, Bistoni F, Frati L, Cortese R, Gulino A, Ciliberto G, Constantini F, Poli V 1995 Lymphoproliferative disorder and imbalanced T-helper response in C/EBP ß-deficient mice. EMBO J 14:1932–1941[Abstract]
  20. Mandrup S, Lane MD 1997 Regulating adipogenesis. J Biol Chem 272:5367–5370[Free Full Text]
  21. Lin FT, Lane MD 1994 CCAAT/enhancer binding protein alpha is sufficient to initiate the 3T3–L1 adipocyte differentiation program. Proc Natl Acad Sci USA 91:8757–8761[Abstract]
  22. Chen X, Liu W, Ambrosino C, Ruocco MR, Poli V, Romani L, Quinto I, Barbieri S, Holmes KL, Venuta S, Scala G 1997 Impaired generation of bone marrow B lymphocytes in mice deficient in C/EBPß. Blood 90:156–164[Abstract/Free Full Text]
  23. Chen PL, Riley DJ, Chen Y, Lee WH 1996 Retinoblastoma protein positively regulates terminal adipocyte differentiation through direct interaction with C/EBPs. Genes Dev 10:2794–2804[Abstract]
  24. Brun RP, Kim JB, Hu E, Altiok S, Spiegelman BM 1996 Adipocyte differentiation: a transcriptional regulatory cascade. Curr Opin Cell Biol 8:826–832[CrossRef][Medline]
  25. Lee Y-H, Williams SC, Baer M, Sterneck E, Gonzalez FJ, Johnson PF 1997 The ability of C/EBPß but not C/EBP{alpha} to synergize with an Sp1 protein is specified by the leucine zipper and activation domain. Mol Cell Biol 12:2038–2047
  26. Sawiris GP, Sydiskis RJ, Bashirelahi N 1994 Hormonal modulation of herpes simplex virus replication in a mouse neuroblastoma cell line. J Clin Lab Anal 8:135–139[Medline]
  27. Beato M 1989 Gene regulation by steroid hormones. Cell 56:335–344[Medline]
  28. Strahle U, Klock G, Schütz G 1987 A DNA sequence of 15 base pairs is sufficient to mediate both glucocorticoid and progesterone induction of gene expression. Proc Natl Acad Sci USA 84:7871–7875[Abstract]
  29. Beato M, Herrlich P, Schutz G 1995 Steroid receptors: many actors in search of a plot. Cell 83:851–857[Medline]
  30. Laudet V, Hanni C, Coll J, Catzeflis F, Stehelin D 1992 Evolution the nuclear receptor gene superfamily. EMBO J 11:1003–1013[Abstract]
  31. Mangelsdorf DJ, Evans RM 1995b The RXR heterodimers and orphan receptors. Cell 83:841–850
  32. Mangelsdorf DJ, Thummel C, Beato M, Herrlich P, Schütz G, Umesono K, Blumberg B, Kastner P, Mark M, Chambon P, Evans RM 1995a The nuclear receptor superfamily: the second decade. Cell 83:835–839
  33. Thummel CS 1995 From embryogenesis to metamorphosis: the regulation and function of Drosophila nuclear receptor superfamily members. Cell 83:871–877[Medline]
  34. Pratt WB, Toft DO 1997 Steroid receptor interactions with heat shock protein and immunophilin chaperones. Endocr Rev 18:306–360[Abstract/Free Full Text]
  35. Abraham SE, Lobo S, Yaciuk P, Wang HG, Moran E 1993 p300, and p300-associated proteins, are components of TATA-binding protein (TBP) complexes. Oncogene 8:1639–1647[Medline]
  36. Dallas PB, Yaciuk P, Moran E 1997 Characterization of monoclonal antibodies raised against p300: both p300 and CBP are present in intracellular TBP complexes. J Virol 71:1726–1731[Abstract]
  37. McEwan IJ, Wright AP, Dahlman-Wright K, Carlstedt-Duke J, Gustafsson JA 1993 Direct interaction of the tau 1 transactivation domain of the human glucocorticoid receptor with the basal transcriptional machinery. Mol Cell Biol 13:399–407[Abstract]
  38. Baniahmad A, Leng X, Burris TP, Tsai SY, Tsai MJ, O’Malley BW 1995 The tau 4 activation domain of the thyroid hormone receptor is required for release of a putative corepressor(s) necessary for transcriptional silencing. Mol Cell Biol 15:76–86[Abstract]
  39. Baniahmad A, Thormeyer D, Renkawitz R 1997 Tau4/tau c/AF-2 of the thyroid hormone receptor relieves silencing of the retinoic acid receptor silencer core independent of both tau4 activation function and full dissociation of corepressors. Mol Cell Biol 17:4259–4271[Abstract]
  40. Barettino D, Vivanco Ruiz MM, Stunnenberg HG 1994 Characterization of the ligand-dependent transactivation domain of thyroid hormone receptor. EMBO J 13:3039–3049[Abstract]
  41. Durand B, Saunders M, Gaudon C, Roy B, Losson R, Chambon P 1994 Activation function 2 (AF-2) of retinoic acid receptor and 9-cis retinoic acid receptor: presence of a conserved autonomous constitutive activating domain and influence of the nature of the response element on AF-2 activity. EMBO J 13:5370–5382[Abstract]
  42. Leng X, Blanco J, Tsai SY, Ozato K, O’Malley BW, Tsai MJ 1995 Mouse retinoid X receptor contains a separable ligand-binding domain and transactivation domain in its E region. Mol Cell Biol 15:255–263[Abstract]
  43. Schulman IG, Juguilon H, Evans RM 1996 Activation and repression by nuclear hormone receptors: hormone modulates an equilibrium between active and repressive states. Mol Cell Biol 16:3807–3813[Abstract]
  44. Onate SA, Tsai SY, Tsai MJ, O’Malley BW 1995 Sequence and characterization of a coactivator for the steroid hormone receptor superfamily. Science 270:1354–1357[Abstract]
  45. Kamei Y, Xu L, Heinzel T, Torchia J, Kurokawa R, Gloss B, Lin SC, Heyman RA, Rose DW, Glass CK, Rosenfeld MG 1996 A CBP integrator complex mediates transcriptional activation and AP-1 inhibition by nuclear receptors. Cell 85:403–414[Medline]
  46. Nordheim A 1994 CREB takes CBP to tango. Nature 370:177–178[CrossRef][Medline]
  47. Smith CL, Onate SA, Tsai MJ, O’Malley BW 1996 CREB binding protein acts synergistically with steroid receptor coactivator-1 to enhance steroid receptor-dependent transcription. Proc Natl Acad Sci USA 93:8884–8888[Abstract/Free Full Text]
  48. Hong H, Kohli K, Trivedi A, Johnson DL, Stallcup MR 1996 GRIP1, a novel mouse protein that serves as a transcriptional coactivator in yeast for the hormone binding domains of steroid receptors. Proc Natl Acad Sci USA 93:4948–4952[Abstract/Free Full Text]
  49. Hong H, Kohli K, Garabedian MJ, Stallcup MR 1997 GRIP1, a transcriptional coactivator for the AF-2 transactivation domain of steroid, thyroid, retinoid, and vitamin D receptor. Mol Cell Biol 17:2735–2744[Abstract]
  50. Ogryzko VV, Schiltz RL, Russanova V, Howard BH, Nakatani Y 1996 The transcriptional coactivators p300 and CBP are histone acetyltransferases. Cell 87:953–959[Medline]
  51. Spencer TE, Jenster G, Burcin MM, Allis CD, Zhou J, Mizzen CA, McKenna NJ, Onate SA, Tsai SY, Tsai MJ, O’Malley BW 1997 Steroid receptor coactivator-1 is a histone acetyltransferase. Nature 389:194–198[CrossRef][Medline]
  52. Chakravarti D, LaMorte VJ, Nelson MC, Nakajima T, Schulman IG, Juguilon H, Montminy M, Evans RM 1996 Role of CBP/P300 in nuclear receptor signalling. Nature 383:99–103[CrossRef][Medline]
  53. Chrivia JC, Kwok RP, Lamb N, Hagiwara M, Goodman MR, Montimy RH 1993 Phosphorylated CREB binds specifically to the nuclear protein CBP. Nature 365:855–859[CrossRef][Medline]
  54. Kwok RPS, Lundblad JR, Chrivia JC, Richards JP, Bachinger HP, Brennan R, Roberts SGE, Goodman MRGaRH 1994 Nuclear protein CBP is a coactivator for the transcription factor CREB. Nature 370:223–226[CrossRef][Medline]
  55. Diamond MI, Miner JN, Yoshinaga SK, Yamamoto KR 1990 c-Jun and c-Fos levels specify positive or negative glucocorticoid regulation from a composite GRE. Science 249:1266–1272[Medline]
  56. Heck S, Kulmann M, Gast A, Ponta H, Rahmsdorf HJ, Herrlich P, Cato AC 1994 A distinct modulating domain in glucocorticoid receptor monomers in the repression of activity of the transcription factor AP-1. EMBO J 13:4087–4095[Abstract]
  57. Schüle R, Rangarajan P, Klieweer S, Ransone LJ, Bolado J, Yang N, Verma IM, Evans RM 1990 Functional antagonism between oncoprotein c-jun and the glucocorticoid receptor. Cell 62:1217–1226[Medline]
  58. Yang-Yen H-F, Chambard J-C, Sun Y-L, Smeal T, Schmidt TJ, Drouin J, Karin M 1990 Transcriptional interference between c-Jun and the glucocorticoid receptor: mutual inhibition of DNA binding due to direct protein-protein interaction. Cell 62:1205–1215[Medline]
  59. Stocklin E, Wissler M, Gouilleux F, Groner B 1996 Functional interaction between Stat5 and the glucocorticoid receptor. Nature 383:726–728[CrossRef][Medline]
  60. Stein B, Yang MX 1995 Repression of the interleukin-6 promoter by estrogen receptor is mediated by NF-kappa B and C/EBP ß. Mol Cell Biol 15:4971–4979[Abstract]
  61. Truss M, Bartsch J, Chalepakis G, Beato M 1992 Artificial steroid hormone response element generated by dam-methylation. Nucleic Acids Res 20:1483–1486[Abstract]
  62. Lim-Tio S, Keightley M-A, Fuller PJ 1997 Determinants of specificity of transactivation by the mineralocorticoid of glucocorticoid receptor. Endocrinology 138:2537–2543[Abstract/Free Full Text]
  63. Schena M, Freedman LP, Yamamoto KR 1989 Mutations in the glucocorticoid receptor zinc finger region that distinguish interdigitated DNA binding and transcriptional enhancement activities. Genes Dev 3:1590–1601[Abstract]
  64. Godowski PJ, Rusconi S, Miesfeld R, Yamamoto KR 1987 Glucocorticoid receptor mutants that are constitutive activators of transcriptional enhancement. Nature 325:365–368[CrossRef][Medline]
  65. Strahle U, Schmid W, Schutz G 1988 Synergistic action of the glucocorticoid receptor with transcription factors. EMBO J 7:3389–3395[Abstract]
  66. Rusconi S, Yamamoto KR 1987 Functional dissection of the hormone and DNA binding activities of the glucocorticoid receptor. EMBO J 6:1309–1315[Abstract]
  67. Savoldi G, Fenaroli A, Ferrari F, Rigaud G, Albertini A, Di Lorenzo D 1997 The glucocorticoid receptor regulates the binding of C/EPBbeta on the alpha-1-acid glycoprotein promoter in vivo. DNA Cell Biol 16:1467–1476[Medline]
  68. Renkawitz R, Kaltschmidt C, Leers J, Martin B, Muller M, Eggert M 1996 Enhancement of nuclear receptor transcriptional signalling. J Steroid Biochem Mol Biol 56:39–45[CrossRef][Medline]
  69. Ben-Or S, Okret S 1993 Involvement of a C/EBP-like protein in the acquisition of responsiveness to glucocorticoid hormones during chick neural retina development. Mol Cell Biol 13:331–340[Abstract]
  70. Karin M 1998 New twists in gene regulation by glucocorticoid receptor: is DNA binding dispensable? Cell 93:487–490[Medline]
  71. Lefstin JA, Yamamoto KR 1998 Allosteric effects of DNA on transcriptional regulators. Nature 392:885–888[CrossRef][Medline]
  72. Glass CK, Rose DW, Rosenfeld MG 1997 Nuclear receptor coactivators. Curr Opin Cell Biol 9:222–232[CrossRef][Medline]
  73. Imhof A, Yang XJ, Ogryzko VV, Nakatani Y, Wolffe AP, Ge H 1997 Acetylation of general transcription factors by histone acetyltransferases. Curr Biol 7:689–692[Medline]
  74. Korzus E, Torchia J, Rose DW, Xu L, Kurokawa R, McInerney EM, Mullen TM, Glass CK, Rosenfeld MG 1998 Transcription factor-specific requirements for coactivators and their acetyltransferase functions. Science 279:703–707[Abstract/Free Full Text]
  75. Yang X-J, Ogryzko V, Nishikawa J-i, Howard B, Nakatani Y 1996 A p300/CBP-associated factor that competes with the adenoviral oncoprotein E1A. Nature 382:319–324[CrossRef][Medline]
  76. Heery DM, Kalkhoven E, Hoare S, Parker MG 1997 A signature motif in transcriptional co-activators mediates binding to nuclear receptors. Nature 387:733–736[CrossRef][Medline]
  77. Mink S, Haenig B, Klempnauer KH 1997 Interaction and functional collaboration of p300 and C/EBPß. Mol Cell Biol 17:6609–6617[Abstract]
  78. Voegel JJ, Heine MJS, Zechel C, Chambon P, Gronemeyer H 1996 TIF2, a 160 kDA transcripitonal mediator for the ligand-dependent activation function AF-2 of nuclear receptors. EMBO J 15:3667–3675[Abstract]
  79. Chen H, Lin RJ, Schiltz RL, Chakravarti D, Nash A, Nagy L, Privalsky ML, Nakatani Y, Evans RM 1997 Nuclear receptor coactivator ACTR is a novel histone acetyltransferase and forms a multimeric activation complex with P/CAF and CBP/p300. Cell 90:569–580[Medline]
  80. Nishio Y, Isshiki H, Kishimoto T, Akira S 1993 A nuclear factor for interleukin-6 expression (NF-IL6) and the glucocorticoid receptor synergistically activate transcription of the rat alpha 1-acid glycoprotein gene via direct protein-protein interaction. Mol Cell Biol 13:1854–1862[Abstract]
  81. Treisman R, Marais R, Wynne J 1992 Spatial flexibility in ternary complexes between SRF and its accessory proteins. EMBO J 11:4631–4640[Abstract]
  82. Giffin W, Torrance H, Rodda DJ, Préfontaine GG, Pope L, Haché RJG 1996 Sequence-specific DNA binding by Ku autoantigen and its effects on transcription. Nature 380:265–268[CrossRef][Medline]
  83. Giffin W, Kwast-Welfeld J, Rodda DJ, Préfontaine GG, Traykova-Andonova M, Zhang Y, Weigel NL, Lefebvre YA, Haché RJ 1997 Sequence-specific DNA binding and transcription factor phosphorylation by Ku autoantigen/DNA-dependent protein kinase. Phosphorylation of Ser-527 of the rat glucocorticoid receptor. J Biol Chem 272:5647–5658[Abstract/Free Full Text]
  84. Chan SK, Jaffe L, Capovilla M, Botas J, Mann RS 1994 The DNA binding specificity of Ultrabithorax is modulated by cooperative interactions with extradenticle, another homeoprotein. Cell 78:603–615[Medline]
  85. Guichet A, Copeland JW, Erdelyi M, Hlousek D, Zavorszky P, Ho J, Brown S, Percival-Smith A, Krause HM, Ephrussi A 1997 The nuclear receptor homologue Ftz-F1 and the homeodomain protein Ftz are mutually dependent cofactors. Nature 385:548–552[CrossRef][Medline]
  86. Préfontaine GG, Lemieux ME, Giffin W, Schild-Poulter C, Pope L, LaCasse E, Walker P, Haché RJG 1998 Recruitment of octamer transcription factors to DNA by glucocorticoid receptor. Mol Cell Biol 18:3416–3430[Abstract/Free Full Text]
  87. Cole TJ, Blendy JA, Monaghan AP, Krieglstein K, Schmid W, Aguzzi A, Fantuzzi G, Hummler E, Unsicker K, Schutz G 1995 Targeted disruption of the glucocorticoid receptor gene blocks adrenergic chromaffin cell development and severely retards lung maturation. Genes Dev 9:1608–1621[Abstract]
  88. Reichardt HM, Kaestner KH, Tuckermann J, Kretz O, Wessely O, Bock R, Gass P, Schmid W, Herrlich P, Angel P, Schutz G 1998 DNA binding of the glucocorticoid receptor is not essential for survival. Cell 93:531–541[Medline]
  89. Pearce D, Yamamoto KR 1993 Mineralocorticoid and glucocorticoid receptor activities distinguished by nonreceptor factors at a composite response element. Science 259:1161–1165[Medline]
  90. Picard D, Yamamoto KR 1987 Two signals mediate hormone-dependent nuclear localization of the glucocorticoid receptor. EMBO J 6:3333–3340[Abstract]
  91. Sadowski I, Ma J, Trienzenberg S, Ptashne M 1988 GAL4-VP16 is an unusually potent transcriptional activator. Nature 335:563–564[CrossRef][Medline]
  92. Chang CS, Kokontis J, Chang CT, Liao SS 1987 Cloning and sequence analysis of the rat ventral prostate glucocorticoid receptor cDNA. Nucleic Acids Res 15:9603[Medline]
  93. Truss M, Chalepakis G, Slater EP, Mader S, Beato M 1991 Functional interaction of hybrid response elements with wild-type and mutant steroid hormone receptors. Mol Cell Biol 11:3247–3258[Medline]
  94. Lillie JW, Green MR 1989 Transcription activation by the adenovirus E1a protein. Nature 338:39–44[CrossRef][Medline]
  95. Miesfeld R, Rusconi S, Godowski PJ, Maler BA, Okret S, Wilkstrom A-C, Gustafsson J-A, Yamamoto KR 1986 Genetic complementation of a glucocorticoid receptor deficiency by expression of cloned receptor cDNA. Cell 46:389–399[Medline]
  96. Greene GL, Gilna P, Waterfield M, Baker A, Hort Y, Shine J 1986 Sequence and expression of human estrogen receptor complementary DNA. Science 231:1150–1154[Medline]
  97. Benbrook D, Lernhardt E, Pfahl M 1988 A new retinoic acid receptor identified from a hepatocellular carcinoma. Nature 333:669–672[CrossRef][Medline]
  98. Gander I, Foeckler R, Rogge L, Meisterernst M, Schneider R, Mertz R, Lottspeich F, Winnacker EL 1988 Purification methods for the sequence-specific DNA-binding protein nuclear factor I (NFI)–generation of protein sequence information. Biochim Biophys Acta 951:411–418[Medline]
  99. Towbin H, Staehelin T, Gordon J 1979 Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedures and some applications. Proc Natl Acad Sci USA 76:4350–4354[Abstract]