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Molecular Endocrinology, doi:10.1210/me.2005-0257
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Molecular Endocrinology 20 (6): 1218-1230
Copyright © 2006 by The Endocrine Society

Activation Function 1 of Glucocorticoid Receptor Binds TATA-Binding Protein in Vitro and in Vivo

Alicja J. Copik, M. Scott Webb, Aaron L. Miller, Yongxin Wang, Raj Kumar and E. Brad Thompson

Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, Texas 77555-1068

Address all correspondence and requests for reprints to: E. Brad Thompson, Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, 301 University Boulevard, Galveston, Texas 77555-1068. E-mail: bthompso{at}utmb.edu.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The mechanism through which the glucocorticoid receptor (GR) stimulates transcription is still unclear, although it is clear that the GR affects assembly of the transcriptional machinery. The binding of the TATA-binding protein (TBP) to the TATA-box is accepted as essential in this process. It is known that the GR can interact in vitro with TBP, but the direct interaction of TBP with GR has not been previously characterized quantitatively and has not been appreciated as an important step in assembling the transcriptional complex. Herein, we demonstrate that the TBP-GR interaction is functionally significant by characterizing the association of TBP and GR in vitro by a combination of techniques and confirming the role of this interaction in vivo. Combined analysis, using native gel electrophoresis, sedimentation equilibrium, and isothermal microcalorimetry titrations, characterize the stoichiometry, affinity, and thermodynamics of the TBP-GR interaction. TBP binds recombinant GR activation function 1 (AF1) with a 1:2 stoichiometry and a dissociation constant in the nanomolar range. In vivo fluorescence resonance energy transfer experiments, using fluorescently labeled TBP and various GR constructs, transiently transfected into CV-1 cells, show GR-TBP interactions, dependent on AF1. AF1-deletion variants showed fluorescence resonance energy transfer efficiencies on the level of coexpressed cyan fluorescent protein and yellow fluorescent protein, indicating that the interaction is dependent on AF1 domain. To demonstrate the functional role of the in vivo GR-TBP interaction, increased amounts of TBP expressed in vivo stimulated expression of GR-driven reporters and endogenous genes, and the effect was also specifically dependent on AF1.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
THE GLUCOCORTICOID RECEPTOR (GR) is a ligand-activated transcription factor with the domain arrangement typical for steroid hormone receptors: an N-terminal domain that contains the powerful transcription-activating region activation function (AF)1 ({tau}1, enh2), a centrally located DNA-binding domain, and a C-terminal domain that contains nuclear localization signal sequences as well as the ligand-binding domain. Within the latter is a second important transcriptional activation function region (AF2). Removal of the ligand-binding domain including AF2, results in a GR that constitutively induces genes at 60–80% of the levels achieved by the steroid-treated, holo-GR (1) due to the unmasked function of AF1. In the human GR, AF1 maps to amino acids 77–262 (2). The core subdomain (AF1C, amino acids 187–244) accounts for 60–70% of AF1’s activity (3). AF1 and AF2 are critical for total GR gene induction capacity (4, 5, 6). Although relevant structural properties of AF2 have been well characterized, the mechanism of AF1 function is nebulous. Presumably, the AF1 region recruits other coregulatory proteins and heterologous transcription factors by creating binding surfaces for these proteins.

The interaction of AF1 with other transcription factors and proteins of the basal transcriptional machinery has not been well characterized, and understanding of such interactions from a structural perspective has been difficult due to the lack of a defined structure for AF1. Unlike AF2, which is a well-folded substituent within the globular ligand-binding domain, independently expressed recombinant GR AF1 (rAF1) is intrinsically disordered (7, 8, 9). Other AF1-like regions of several steroid receptors and of many important transcription regulatory proteins, including c-Myc, VP16, p53, and nuclear factor (NF)-{kappa}B p65 are similarly found to have disordered structures (reviewed in Refs. 10 and 11). Such domains share a commonality of having a high content of charged amino acids and low mean hydrophobicity, which manifests in random-coil characteristics and larger hydrodynamic dimensions than globular proteins with comparable molecular weight. Interestingly, many of these unstructured acidic transcription activation domains have been shown to bind TATA-binding protein (TBP) in vitro and even to acquire structure upon the interaction.

Although there have been several reports demonstrating that GR AF1 can bind to TBP in vitro (8, 9, 12, 13), the stoichiometry, affinity, and thermodynamics of this interaction have not been studied; therefore, any assumptions about the likelihood of this interaction occurring in vivo have been limited. To our knowledge, TBP-GR AF1 interaction has not been demonstrated under in vivo conditions nor has the physiological relevance of this interaction been addressed. Therefore, we have quantitatively determined the stoichiometry, affinity, and thermodynamics for binding of AF1 to TBP using native PAGE, sedimentation equilibrium, and isothermal titration microcalorimetry. AF1 binds TBP tightly with a dissociation constant in the high nanomolar range, suggesting that this interaction is likely to be relevant under physiological conditions. Furthermore, we demonstrate the occurrence of AF1-dependent GR-TBP interaction in cultured cells under conditions relevant for GR function in vivo using fluorescence resonance energy transfer (FRET). To test the functional significance of this interaction, two glucocorticoid response element (GRE)-driven promoter-reporter constructs were used to show that AF1-specific transcriptional activity correlates with in vivo TBP concentration. The induction of an endogenous gene by dexamethasone (Dex) also was enhanced by transfection of additional TBP. Taken together, our data have, for the first time, demonstrated that the GR AF1 makes a physical and functional interaction with TBP. Unlike the classical mechanism of the GR action, which does not consider direct interaction between GR and TBP, these results highlight the importance of direct interaction of GR AF1 and TBP as part of the GR-mediated transcriptional activation mechanism.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
In Vitro Characterization of the GR-TBP Interaction
Previous studies using pull-down assays indicated that AF1 and TBP can interact in vitro (13). These studies also defined the AF1C and the C-terminal core domain of TBP (TBPC), amino acids 168–339, as sufficient for the interaction, and thus all the in vitro experiments were performed using recombinant AF1C (rAF1C) and TBPC.

Analysis of the Stoichiometry of the rAF1C-TBPC Interaction by Native PAGE.
To estimate the stoichiometry of the TBPC-rAF1C interaction, complexes of TBPC and rAF1C were examined by native, nondenaturing PAGE (Fig. 1Go). Under native, nondenaturing conditions [on Tris-HCl (pH 8.8) gel] only proteins that have an isoelectric point (pI) less than 7 migrate into the gel (toward the cathode), whereas proteins with pI greater than 7 are dispersed into the buffer. AF1C is fairly acidic, having a pI of approximately 4.3, and migrates toward the cathode (Fig. 1Go, lane 8), whereas TBPC is basic (pI ~ 10.3) and does not run into the gel (lane 7). The calculated pI values for AF1C-TBPC and (AF1C)2-TBPC are 8 and 6, respectively, suggesting that only the 2:1 complex could be detected on the basic gel. Thus, native PAGE is a perfect technique to rapidly assess the state of the AF1C-TBPC complex formation due to the significant change in overall charge of the complex. When TBPC and AF1C were combined at several different ratios, a band of free AF1C and a new, slower migrating band could be observed. Denaturing gel electrophoresis of the same samples confirmed that the ratios of the proteins in the mixtures were as calculated (data not shown). Therefore, we conclude that the slower migrating band comes from the (AF1C)2-TBPC complex. The band of the complex traveled on the gel toward the cathode, and its intensity was increased with increasing TBPC (Fig. 1BGo). At the same time, the intensity of the band of free AF1C was decreased, and at 2:1 AF1C-TBPC, it was undetectable. This would indicate that the observed complex may contain two AF1C molecules per molecule of TBPC because TBPC-AF1C would fail to enter the gel. Thus, crude analysis of the complex using electrophoresis in native condition confirms formation of the complex in vitro and indicates that it may exist in (AF1C)2-TBPC form.


Figure 1
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  		Fig. 1. Native PAGE Analysis of the rAF1C-rTBPC Complex

A, rTBPC and rAF1C mixed together at different ratios by varying TBPC while holding rAF1C constant (lanes 1–6). Lanes 7 and 8, AF1C and rTBPC controls, respectively. B, Plot of the band density vs. amount of rTBPC in the sample.

 
Analysis of the Stoichiometry of the AF1C-TBPC Interaction by Sedimentation Equilibrium.
To explore the behavior of the complex in solution, we performed a series of analytical centrifugation experiments. TBPC and rAF1C were mixed at 1:2 molar ratio, and sedimentation equilibrium data were collected at 25,000 rpm to determine the mass of the complex. Figure 2Go shows representative data from one of two experiments fitted to the self-association model. This model correlated well with the experimental data; the residuals are shown on the top panel. From these data an average molecular mass of 37.6 ± 1.0 kDa was obtained from the two experiments. This result corresponds closely to the theoretical molecular mass of 37.7 kDa of a 1:2, TBPC-(rAF1C)2 complex and is far from the 30.0 kDa expected for a 1:1, TBPC-rAF1C complex. Thus, the analytical centrifugation experiments are consistent with the results obtained from native, nondenaturing PAGE.


Figure 2
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  		Fig. 2. Analysis of Equilibrium Ultracentrifugation Data for rAF1C-TBPC Complex

Data were collected for a sample containing 2 equivalents of rAF1C GR and 1 equivalent of TBPC in 20 mM HEPES (pH 7.0), 200 mM NaCl, 10% glycerol, centrifuged at 25,000 rpm for 24 h at 5 C, and analyzed using self-association model from Beckman Coulter Software. Results from one of two experiments are shown. Upper panel, Residuals; lower panel, absorbance data.

 
Thermodynamics of TBPC-AF1C Binding Determined by Isothermal Titration Microcalorimetry (ITC).
To gain insight into the mechanism of binding of AF1 to TBP and to discern the effect of disorder on the thermodynamics of this interaction, we conducted a series of ITC titration experiments. At each of four different temperatures studied, rAF1C was titrated into the ITC cell containing a constant amount of TBPC. A representative titration recorded at 25 C is shown in Fig. 3Go. Raw data showing the heat evolved (negative peaks) upon successive injection of 9-µl aliquots of 600 µM rAF1C into a 25 µM solution of TBPC are depicted in Fig. 3AGo. After subtraction of the heats of dilution, the heat evolved was integrated to obtain {Delta}H, which was plotted against the molar ratio of rAF1C to TBPC (Fig. 3BGo). The resulting binding curve was analyzed for best fit, using single-site, sequential, or independent multiple-sites models. The sequential two-site binding model provided the best fit to the experimental data (Fig. 3BGo) yielding a binding stoichiometry of rAF1C-TBPC of 2:1. The fit of the ITC data to this model provides the association constant (Ka), the entropy of binding ({Delta}S), and the Gibbs free energy ({Delta}G) for the two sites (Table 1Go). The same measurements and calculations were performed at 15, 20, and 22.5 C to obtain the temperature dependence of the {Delta}H and {Delta}S for the two binding events. The heat capacity change ({Delta}Cp) associated with the binding was obtained from the slope of {Delta}H vs. T. The process of binding of rAF1C to TBPC is enthalpy driven, with favorable overall change of entropy at each temperature studied. The overall binding free energy was little affected by the temperature changes because of the enthalpy-entropy compensation, typical of bimolecular systems in water. The observed positive entropy is a result of the large contribution from the hydrophobic effect and solvent release outweighing the negative contribution from the loss of rotational and translational freedom. In our previous work, we have shown by various spectroscopic techniques that binding of AF1 to TBP is accompanied by a folding of AF1. Thus, the observed entropy of binding also has a negative component from the reduction of the conformational entropy. Consistent with this, formation of the TBPC-(rAF1C)2 complex is associated with a large negative heat capacity change, indicative of loss of hydration due to binding and/or folding. Based on available structural and thermodynamic data for a large group of well-characterized macromolecular complexes, Spolar and Record (14) developed an empirical method to correlate experimentally determined {Delta}Cp and {Delta}S with the number of residues participating in folding upon binding to a partner. Applied to our system, this calculation estimates that the first binding event induces folding in approximately 11 residues of rAF1C (R = 11, listed in Table 1Go), whereas the second event accounts for folding involving about nine amino acids of rAF1C (R = 9).


Figure 3
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  		Fig. 3. ITC of rAF1C Binding to TBPC at 25 C

A, Raw data showing the heat released during binding of increasing amounts of rAF1C to TBPC. B, Plot of integrated heat of binding vs. molar ratio of the two proteins fitted to a two-site sequential model. NDH, Normalized heat change.

 

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  		Table 1. Thermodynamic Parameters of the Binding of rAF1cto TBPc

 
Taken together, our data clearly indicate that AF1 and TBP physically interact with each other in vitro. The stoichiometry of the complex reveals two rAF1C binding one TBPC. The thermodynamic parameters of this binding predict feasibility of this interaction taking place under in vivo conditions and that this interaction may lead to induced folding, consistent with our previous studies (9).

GR-TBP Interaction in Vivo
TBP and GR500 Interact in Vivo, FRET Microscopy.
The current standard models assume that the GR interacts indirectly with the TBP-based basal transcription multiprotein complex. However, biophysical in vitro data show that GR AF1 can bind to TBP directly and with fairly high affinity. To probe for interaction of GR AF1 and TBP in vivo, we used FRET microscopy, which can provide measurements of spatial relationship between the fluorophores at the Angstrom scale.

Plasmids expressing cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) were obtained, and from that we generated constructs that express the fluorophores linked to GR500 (CFP-GR500) and TBP (YFP-TBP). The GR500 construct is constitutively active as a transcription factor while avoiding the possibility of any contribution from AF2. The constructs were cotransfected into GR-deficient CV-1 cells with cotransfection of a promoter-reporter construct containing GRE sites [GRE-SEAP (secreted alkaline phosphatase)] or lacking them (pTAL-SEAP). Several control experiments were included. Independent CFP and YFP-expressing constructs were cotransfected with GRE-SEAP and tested for FRET as negative control. As a positive control, a CFP-YFP construct that linked CFP-YFP by eight amino acids was coexpressed with GRE-SEAP. Figure 4AGo shows examples of results from such controls. All three constructs show a pan-fluorescence, with greater intensity over the nucleus (somewhat distorted due to lipofection). Here and on images B and C, the area tested by photobleaching (PB) is shown within the white box, expanded to show the detail in the Fig. 4AGo insets. FRET is obviously much greater in the positive control. Figure 4BGo shows two nuclei from cells expressing CFP-GR500 and YFP-TBP, cotransfected with the GRE-SEAP plasmids. Again the before and after PB images show evidence of FRET. In these transfections, there was little fluorescence in the cytoplasm, which has been excluded for the most part from this figure: the entire white-boxed area shown was photobleached for this particular cell. Further controls are shown in Fig. 4CGo. From left to right they are: coexpression of YFP-TBP and a GR500 lacking all of AF1 ({Delta}AF1_GR500) cotransfected with the promoter-reporter containing GREs (GRE-SEAP); and the same arrangement except for a GR500 lacking only the core of AF1({Delta}AF1C_GR500); cotransfection of GR500 and TBP with the promoter-reporter construct lacking a GRE (pTAL). FRET efficiency in seven to 12 cells for each condition was determined, and the average FRETs ± SD are shown in Fig. 4DGo. The numbers under the bars correspond to the conditions used in the correspondingly numbered sections of Fig. 4Go.


Figure 4
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  		Fig. 4. GR500 AF1 Interacts Directly with TBP in the Nuclei of CV-1 Cells Transfected with a Promoter Containing GRE

CV1 cells were transfected with plasmids expressing CFP, YFP, a CFP-YFP fusion protein (CFP-YFP), and GR500 lacking the AF1 region ({Delta}AF1_GR500) or lacking only the AF1C ({Delta}AF1C_GR500). All cells but those in panel A were cotransfected with an expression plasmid conveying YFP-TBP. The cells were also cotransfected with a promoter-reporter construct, GRE-SEAP, except these in panel C [no.(6 )], which received pTAL, which lacks GRE but is otherwise the same as GRE-SEAP. Data are from one of three such experiments and show examples of same-cell images in the donor channel before and after PB; the areas within the white boxes were photobleached. Microscopy was with a 63x1.4 objective; slight differences in apparent magnification are due to electronic manipulations that occurred while arranging the figure. A, Controls receiving fluorescent proteins without TBP or GR, to establish basal (no interaction) and maximal FRETs. B, GRE-dependent FRET between GR500 and TBP (entire white box was photobleached). Panel C [nos. (4 ) and (5 )] show lack of FRET when GR lacks AF1 or AF1C and no. (6 ) shows lack of FRET in the absence of a GRE. Panel D displays calculated average FRET efficiencies ± 1 SD of n cells for each of the six conditions. The abscissa numbers correspond to the numbered conditions [nos.(1 )–(6 )].

 
These FRET analyses indicate that in these cells interaction of GR AF1 with TBP in the cellular environment appears to occur when GR is bound to its GRE site. Lack of any significant FRET between GR500 devoid of AF1 and AF1C suggests that these protein-protein interactions are specific to AF1 and TBP. Furthermore, the lack of significant FRET between GR500 and TBP in cells transfected with a promoter lacking any GRE site indicate that these interaction may be promoter specific, suggesting a functional significance of this direct AF1-TBP interaction in vivo.

Effect of TBP on AF1-Driven Transcription.
We examined the functional interaction of TBP and GR using two GR-responsive promoters, in transient transfection-based reporter assays in GR-deficient CV-1 cells. The promoter-reporter plasmid (GRE-SEAP) contains three GREs upstream from a TATA-box and a reporter gene that encodes alkaline phosphatase secreted into the medium. To test the effect of TBP on transcription driven by full-length GR, we cotransfected CV-1 cells with a constant amount of GR expression vector and increasing amounts of the plasmid pcDNA3.1_TBP. After a day for recovery, the cells were treated with 1 µM synthetic glucocorticoid Dex and incubated for an additional 24 h; then the medium bathing the cells was tested for SEAP activity. Increasing the input of the plasmid expressing the TBP gene enhanced the GR induction of the GRE-SEAP reporter in a dose-dependent fashion (Fig. 5Go, A and C). Expression of the reporter was increased more than 7-fold at the highest concentration of TBP tested within the working range for optimal transfection efficiency. This value does not correspond to the maximum amount of stimulation, because a saturation level of TBP input was not reached (Fig. 5CGo). To examine whether the stimulation of transcription by TBP is mediated through AF1, AF2, or both, we compared the TBP effect on holo-GR and GR with AF1 deleted ({Delta}AF1 GR, Fig. 5DGo). The results indicate that the TBP enhancement of transcription depends mainly on the AF1 domain (Fig. 5CGo). A small enhancement of activity also appears to be mediated through AF2 (Fig. 5CGo, solid squares). To further test whether AF1 alone is sufficient to cause TBP-mediated transactivation activity of GR, we determined stimulation by TBP of a form of GR with ligand-binding domain deleted (GR500). The GR500 is transcriptionally active and can induce apoptosis in cells, to nearly the same extent as steroid-bound holo-GR. Our data indicate that GR500 is still strongly stimulated by added TBP (Fig. 5Go, B and C, closed circles). When either AF1 or AF1C is deleted (Fig. 5CGo; closed triangles, open circles), the stimulation by TBP is significantly diminished, suggesting that the effect of TBP is, in fact, transduced primarily through AF1 and, in particular, through AF1C. These results strongly suggest that the enhancement of GR-induced transcription by TBP is achieved predominantly through the AF1 region.


Figure 5
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  		Fig. 5. TBP Increases AF1-Dependent GR-Mediated Transcription Activation of an Artificial Promoter Containing a GRE

A, AF1 in holo-GR. CV-1 cells were cotransfected with 133 ng DNA of the pGRE_SEAP plasmid plus 133 ng of pRS_GR (black bar) or pRS_{Delta}AF1 GR (gray bar), in the presence of increasing amounts (0, 133, 266, 333, 399, and 467 ng) of pcDNA3.1_TBP, indicated by the TBP ramp. The growth medium was made 1 µM in Dex 24 h later. After an additional 24 h, the medium was tested for the presence of SEAP (see Materials and Methods). B, Test of GR lacking an ligand-binding domain; CV-1 cells were cotransfected with 133 ng of pECFP_GR500 (light gray bar), pECFP_ {Delta}AF1C GR500 (medium gray bar) or pECFP_ {Delta}AF1 GR500 (dark gray bar) and increasing amounts (0–467 ng) of pcDNA3.1_TBP. Control (black bar) cells received no GR construct, only pECFP and pcDNA3.1_TBP. The medium was assayed for SEAP activity 24 h (GR500) or 48 h (deletions) later. C, Data from panels A and B plotted as a fold increase in SEAP activity over control (no exogenous TBP). In panels A, B, and C, results are shown as averages of data from three independent transfections ± 1 SD. D, Schematic representation of the GR constructs used in the transient transfection experiments. DBD, DNA-binding domain; LBD, ligand-binding domain; RLU, relative light units.

 
Effect of TBP on the Induction of a Transfected Natural Promoter.
To test the response of a more natural promoter to added TBP, we transfected CV-1 cells with a construct driven by a 223-bp fragment of the glucocorticoid-responsive mouse mammary tumor virus (MMTV) promoter. This region contains GREs, an NF-1 site, and two Oct-binding sites preceding a TATA box. It is known that the action of the NF-1 and Oct factors is involved in the GR-mediated induction from this site (15). As with the simple GRE-driven gene, induction from this MMTV-driven reporter stimulated by GR500 was enhanced by increasing amounts of TBP (Fig. 6Go).


Figure 6
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  		Fig. 6. TBP Increases AF-1-Dependent GR500-Stimulated Transcription Activation on a Natural Promoter

CV-1 cells were cotransfected with 133 ng DNA of the pHHluc plasmid plus 133 ng of phCMV2-GR500, in the presence of no (first gray bar) or increasing amounts (133, 266, 333, 399, and 467 ng) of pcDNA3.1_TBP, indicated by the TBP ramp, abscissa. Control cells received no phCMV2-GR500 (black bar). After 24 h, the cells were assayed for luciferase activity (Materials and Methods). Error bars indicate range of duplicates. RLU, Relative light units.

 
Effect of TBP on the Induction of Endogenous GR-Regulated Genes.
We tested whether TBP has the same stimulatory effect on the induction of endogenous GR-regulated genes. Based on gene microarray data performed in the Dex-sensitive leukemic CEM-C1–6 cell line (16), we screened four induced genes. One of those (GILZ), very strongly induced by Dex, seemed to show no TBP effect. The others all appeared to respond to added TBP. Based on several preliminary PCR experiments, we selected for a more detailed experiment RTP801, which in the CEM cells is 3- to 4-fold induced 2–4 h after Dex addition (our unpublished results). The CEM-C1–6 cells were transfected with 5, 10, or 15 µg of empty pEYFP or pEYFP-TBP and allowed to recover for 7 h, and then the level of expression of the fluorophore was assessed by fluorescence microscopy. It was clear that the increasing fluorescent protein was being expressed with increasing amounts of transfected DNA (data not shown). To look for the early effect of the postulated TBP recruitment, cells were subsequently treated with 1 µM Dex or vehicle for 1 h and total RNA was extracted. The level of RTP801 mRNA was than assayed by semiquantitative RT-PCR (Fig. 7Go). In the control samples, transfected with the empty vector, the RTP801 was induced approximately 1.5-fold (Fig. 7Go, solid lines). This level was independent of the amount of empty vector transfected into the cells. In the Dex-treated cells transfected with TBP, the RTP801 mRNA was induced to higher levels dependent on the amount of transfected and expressed TBP (Fig. 7Go, broken line).


Figure 7
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  		Fig. 7. Effect of TBP on an Endogenous Gene (RTP801) Induction by Dex in CEM-C1–6 Cells

Cells were transfected with TBP-YFP or YFP; 7 h later Dex was added to the cultures, and 1 h after that, RNA was extracted. Upper panel, Results of RT-PCRs on mRNA from cells transfected with vectors expressing TBP-YFP (TBP) or YFP only. Lower panel, Data expressed quantitatively, after normalization to time-matched ß-actin controls. Veh, Vehicle.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Regulation of tissue-specific gene transcription lies at the heart of eukaryotic differentiation. This regulation is driven by transcription factors at their specific target genes. In a currently popular model, the initiation of transcription driven by GR is believed to require independent recognition and binding of the GR and TBP to their respective response elements (17, 18). Each protein then is thought to provide the basis for recruitment of other factors, which modify chromatin and somehow form a bridge to bring in the basal transcription machinery to the promoter. The present data in no way contradict this model but suggest that, in addition, there is direct interaction between the GR and TBP. This interaction depends on the GR AF1 domain. The functional significance of this interaction, to our knowledge, has not been presented in the context of transcription initiation by GR in vivo.

The prevailing models of transcription activation by GR do not accommodate the direct interaction between GR AF1 and TBP. Even though several studies have indicated such an interaction, it has been thought to be relatively weak, and its functional relevance has been doubted (12, 19). This conclusion was based upon work in which the interaction between AF1 and TBP could not be detected in vitro by affinity chromatography (19). However, when AF1 is caused to fold by several means, it binds TBP well, and we have shown that bound TBP can cause rAF1 to fold (9). Herein, we performed a set of ITC titrations, starting with unfolded rAF1C, to quantitatively characterize the protein-protein interaction and determine its thermodynamics. The Kd values determined for the rAF1C:TBPC binding are in the hundred nanomolar range (~200 nM). These values correlate well with reported Kd values for binding of TBP to the AF1-like domains of other transcription factors such as estrogen receptor (ER){alpha} (1–10 µM), VP-16 (0.02–0.2 µM), and c-Myc (~0.4 µM) (20, 21, 22, 23). These values were determined using surface plasmon resonance and/or fluorescent titration and were fitted assuming 1:1 stoichiometry. The rAF1C:TBPC experimental binding curves determined by ITC could be rationalized only by 2:1 stoichiometry. This model was corroborated by results of analytical centrifugation experiments and native PAGE analysis. Because GR forms a dimer when bound to the GRE (24, 25, 26), it is reasonable that two AF1 domains would participate in binding one TBP. The TBPC has internal 2-fold symmetry, reflected both in amino acid sequence (TBP consists of two 80-amino acid imperfect direct repeats) and in secondary and tertiary structural elements (27). Thus, we hypothesize that the two AF1s interact with the similar structural motifs located on opposite sites of the saddle-like TBPC molecule. To further support this hypothesis, the thermodynamic parameters determined for the two sites are of the same order of magnitude, indicating similarities in the two sites. How TBP interacts with holo-GR is not settled by the data available. Although our ITC data with simplified AF1 TBP constructs give two binding sites, results based on pull-down assays show that GR’s AF2 can also weakly associate with TBP (13). Additionally, our transient transfection assays suggest that TBP has a slightly greater effect on the stimulation of transcription by full-length GR as compared with the GR500 fragment (Fig. 5CGo). Thus, one can speculate that both AF domains synergize to bind one TBP molecule giving 1:1 GR:TBP stoichiometry. Whether holo-GR binds TBP 2:1 or 1:1, and which molecular interactions (AF1 only or AF2), remains to be seen. Furthermore, our data do not rule out kinetic behavior that gives preference to one site.

We have recently shown, using nuclear magnetic resonance and Fourier transform infrared spectroscopies, that AF1 assumes a three-dimensional fold with significant helical content upon interaction with TBP (9). The estimated increase in helical content of from 27% in free AF1 to 42% in the complex with TBP (based on calculation from second-derivative Fourier transform infrared spectra) corresponds to approximately 28 residues participating in folding in the AF1 domain. Consistent with this, the heat capacity associated with complex formation and the temperature dependence of entropy yielded a rough estimate of nine and 11 residues folding within the AF1C domain upon interaction with TBPC at the two binding sites. Thus, a large part of the folding takes place in the AF1C region. Phenotypic screening of mutants with decreased or increased transcriptional activity suggested that formation of two helices in AF1C is important for AF1 functions, with the first helix being the more crucial for transcription activation (28). Moreover, the activity of these variants was later shown to correlate to their ability to bind TBP (as estimated by pull-down assays followed by SDS-PAGE analysis) (13). The screening also identified tryptophan 213 of the putative loop region as absolutely crucial for TBP binding and important for transcriptional activity. We have later shown by nuclear magnetic resonance and near-UV circular dichroism that the tryptophan 213 is affected by the binding of TBP, and the environment/conformation around this residue is changed upon interaction with TBP (9). The cumulative evidence suggests that a functional fold in AF1 is induced when TBP interacts with two AF1 GR molecules. This induced fold most likely spans the minimum core between residues 189 and 244.

In vivo interactions between AF1 and TBP in cell nuclei were demonstrated by FRET analysis. As might be expected, the FRET efficiency for AF1:TBP was lower than that for the CFP-YFP positive control. Several factors could account for the lower FRET in the experimental set up, such as suboptimal orientation between CFP and YFP in the CFP-GR500:TBP-YFP complex, or an imperfect ratio of the transfected CFP-GR500 and YFP-TBP inside the nuclei of the tested cells. Also, among the transfected cells, some could have an insufficient amount of the promoter-reporter construct (which cannot be checked by fluorometry) reducing the mean FRET efficiency. Nevertheless, the calculated FRET is clearly significant. Pointing to the importance of AF1 in the interaction, the deletion variants, which were connected to CFP in the same manner as the CFP-GR500 (so that the orientation between CFP and YFP in the complex should be similar), did not show any detectable FRET. Moreover, the presence of DNA-containing GRE sites seems to be required to observe FRET, indicating localization at the promoter. Because the GRE in our constructs is close to 100 bp upstream of the TATA element, this raises the question of how these two proteins can interact directly. A recent report, which used GAL4-VP16 cross-linked as a dimer at its cognate site to a nonchromatin DNA at various distances from the TATA element, has shown that the GAL4-VP16 can physically interact with TBP-TFIIA-TFIIB complex assembled on a distant TATA element (29). The efficiency of the transcription in the cell-free extracts was dependent on the precise location of the GAL4-binding site on the face of the helix relative to TATA rather than the distance, implying that bending and looping of the promoter DNA may permit this interaction. Similar bending of DNA could occur in our system. Observations have been made also for the acidic activation domain of c-Rel and v-Rel (of the transcription factor NF-{kappa}B) and TBP, which associate in vitro and probably interact in vivo on a DNA template containing {kappa}B and TATA sites (30, 31). TBP capable of binding to a TATA site is required for synergistic transactivation by c-Rel.

The in vivo TBP titrations show that TBP has a stimulatory effect on GR-driven transcription and that AF1 is required for this enhancement. Because in the presence of GR AF1, the effect of TBP on GR-driven transcription is much greater, one can speculate that the transcriptional enhancement is due to the direct interaction between AF1 and TBP. A stimulatory effect of TBP was also observed on induction of endogenous GR-regulated genes. The question arises as to what might be the role of this interaction in the mechanism for the transcriptional activation. One possibility is that the TBP-AF1 GR interaction serves to establish the directionality of the preinitiation complex, providing a vector for the bidirectional nature of the TBP binding to TATA box in the absence of cofactors. It has been demonstrated that the asymmetry provided by TBP at most TATA boxes is not sufficient to determine the directionality of preinitiation complex formation (32). Recent elegant work by Kays and Schepartz (33) provided evidence that the orientation of TBP, and thus the preinitiation complex, is likely dictated by the presence of additional basal and gene-specific factors. DNA affinity cleavage and a TBP-phenanthroline-copper conjugate were used to monitor the orientation of TBP. Their use revealed that Gal4-VP16 pushed the equilibrium toward the correct, productive orientation of TBP on TATA. The natively unstructured N-terminal activation domain of VP-16, previously shown to directly interact with TBP, was absolutely required to observe this shift in equilibrium. Similar enhancement, but to a lesser extent, has also been observed with transcription factor (TF)IIA, TFIIB, and activator Gal4AH (33). Therefore, AF1 GR-TBP interaction may be important for enhancement of the formation of correctly oriented preinitiation complex.

Another possible effect of TBP-GR AF1 interaction could involve recruitment of TBP to TATA by AF1 GR. Binding of TBP (as part of TFIID) to the promoter constitutes a critical rate-limiting step at which activators and/or chromatin-remodeling factors can control transcription (34, 35, 36, 37, 38, 39). Interaction of unstructured AF1-like domains with TBP, with subsequent folding, has been observed for VP-16, ER{alpha}, and c-Myc, supporting a common mechanism (20, 21, 23). For ER{alpha} and c-Myc, the kinetic parameters of the binding to TBP have been determined. In both cases the interaction proceeds in a two-step manner with initial very fast, low-affinity association, followed by a slow, folding event and tighter association. Based on these results, it has been proposed that the initial association occurs by electrostatic interactions between the acidic residues of these AF domains and the positively charged TBP. This unstable complex is thought to subsequently convert to a more stable form by the folding of the AF and the formation of specific contacts between the two proteins. Recently, this two-step mechanism of cofactor recruitment by unstructured acidic activators has been tested on broader groups of target proteins (including TBP and chromatin-remodeling complexes) (40), and the results suggest that target-induced folding may be a more general mechanism of cofactors recruitment by unstructured activation domains. Our data, obtained using alternative techniques, support this model.

Schoemaker et al. (41) proposed a very attractive fly-casting mechanism to rationalize the advantage of protein disorder in protein-protein interaction and target protein recruitment. According to this proposal the unstructured protein would have a greater capture radius than a well-folded, globular protein. In this hypothetical mechanism the unfolded polypeptide binds weakly at relatively long distance and folds as it reels in its target. As a consequence, the mechanism predicts an increased rate of binding, which would be of great importance when the cellular concentrations of regulatory proteins are low, as in the case of transcription regulation processes. Applying this mechanism to AF1 GR-activated transcription, we postulate that when GR binds to the GRE, the unstructured or partially structured AF1 interacts with TBP, and as AF1 folds, TBP is recruited to the TATA box. As noted earlier, this may be an example of a general mechanism by which transactivation domains activate transcription. It is believed that, in many systems, recruitment of TBP to the promoter is rate limiting for transcription (34, 35, 36, 37, 42, 43, 44); hence, AF domains may stimulate transcription of a particular gene by increasing the rate of this step. Early studies on GR-activated transcription carried out with the long terminal repeat of MMTV as a model inducible promoter, showed that binding of TBP to TATA promoter occurred only in the presence of hormone, suggesting a direct receptor-mediated event and role of GR in TBP recruitment (45). Thus, the available evidence seems to support our hypothesis that TBP enhances the GR-driven transcription through direct interaction with AF1_GR on the GRE site, with folding of AF1 induced by TBP.

In summary, our FRET results show that TBP and AF1 GR do interact in vivo, and this interaction is important, based on the observed enhancement of transcription of both transfected and endogenous genes in the presence of TBP. Based on the available data, together with the results presented herein, we hypothesize that one of the mechanisms by which GR activates transcription is by direct interaction/recruitment of TBP and, possibly, by orienting its function as well (Fig. 8Go). In this hypothetical model, ligand-stimulated GR samples the DNA until it finds a GRE site. Upon DNA binding domain-GRE binding, a conformational change occurs in the GR AF1, preorganizing it for enhanced interaction with TBP (46). The extended, charged arm of AF1 searches for TBP and forms an initial weak association followed by binding-induced folding, which brings TBP to the TATA-box while properly orienting it. TBP, bound to the TATA site and the more structured AF1, form the platform for further interactions and preinitiation complex formation (Fig. 8Go).


Figure 8
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  		Fig. 8. Proposed Mechanism of Transcription Stimulated by GR, Involving Direct Interaction of GR AF1 and TBP, Mediated by the Folding of the GR AF1 Domain

 

   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Plasmids
The pGRE_SEAP vector (BD Biosciences, Palo Alto, CA) contains three copies of a GRE consensus sequence in tandem, fused to a TATA-like promoter (PTAL) upstream from the reporter gene for secreted alkaline phosphatase (SEAP); pTAL_SEAP lacks the GREs but is otherwise identical. The pRS_hGR and pRS_{Delta}AF1 GR plasmids were generated as previously reported (4). GR500 encodes amino acids 1–500 of the hGR, plus a five-residue nonspecific extension (47). The AF1 ({Delta}77–262) and AF1C ({Delta}187–244) GR500 deletion variants were generated via PCR using phCMV2-GR500 as the starting template and inserting the PCR fragments into pECFP-C1 (BD Biosciences) using XhoI/ SmaI cloning sites. TBP was cloned into the pcDNA3.1(+) expression vector (Invitrogen, Carlsbad, CA) and into pEYFP-C1 (BD Biosciences). DNA sequencing was performed on all clones to confirm correct sequence. The CFP-YFP fusion protein was generated as described elsewhere (48). TBPC encoding 181 C-terminal residues of human TBP was expressed in pET-21d vector (27). pHHluc plasmid containing –223 to +105 bp of the MMTV promoter with luciferase as reporter was purchased from American Type Culture Collection (Manassas, VA).

Cell Culture, Transient Transfection
CV-1 monkey kidney epithelial cells (American Type Culture Collection) were grown at 37 C in MEM with Earle’s salts (Invitrogen) supplemented with 10% (vol/vol) fetal bovine serum (Atlanta Biologicals, Norcross, GA). Cells were subcultured every 2–3 d. CV-1 cells were plated on a 24-well plate (500 µl/well) 1 d before the transfection and transfected using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol. Transfected cells were maintained at 37 C in 5% CO2/95% air for the duration of the experiment (24–48 h).

CEM-C1–6 leukemic cells were grown at 37 C in RPMI medium with L-glutamine (Invitrogen) supplemented with 5% (vol/vol) heat-inactivated fetal bovine serum (Atlanta Biologicals). Cells were subcultured every 2–3 d to maintain log phase growth. Cells were transfected with the desired DNA using electroporation as follows: 400 µl of cells concentrated to 107 viable cells/ml in serum-free medium supplemented with 1.25% of dimethylsulfoxide were placed in a cuvette along with varying amount of plasmid DNA. The cells were electroporated at 975 microfarads, 270 V with time constant of 25–26 msec. Cells were diluted in 8 ml of serum containing medium supplemented with 1.25% dimethylsulfoxide and placed in a 37 C incubator for 6 h. The level of transfection was estimated to be 50% by use of fluorescence microscopy of the cells receiving CFP. Cells were then treated with Dex or vehicle for 1 h, after which they were collected and RNA was isolated.

Protein Expression and Purification
His-tagged rAF1C (residues 187–244) and C-terminal core of human TBP (residues 159–339) were expressed in Escherichia coli BL21(DE3), and purified on the NiNTA column (QIAGEN, Valencia, CA) using imidazole step-gradient. rAF1C was further purified on a Resource Q column (Amersham Biosciences, Piscataway, NJ). Final protein purity of both proteins was greater than 98% as verified by presence of a single band on SDS-PAGE.

Native Gel Analysis
Native PAGE samples were prepared by mixing 32 µg of AF1C and varying amounts of TBPC (4.5–45 µg) in 20 mM HEPES (pH 7.0), 200 mM NaCl, 10% glycerol (without sodium dodecyl sulfate and 2-mercaptoethanol). Samples were run in Tris-HCl (pH 8.8), 10–20% polyacrylamide gel, and stained with Coomassie blue.

Analytical Ultracentrifugation
Sedimentation equilibrium experiments were carried out with a Beckman XL-A analytical ultracentrifuge, using equilibrium six-channel Epon centerpiece (Beckman Coulter, Miami, FL). Purified rAF1C was combined with purified TBPC in 20 mM HEPES (pH 7.0), 200 mM NaCl, 10% glycerol in molar ratio 2:1 (100 µM:50 µM). The complex was centrifuged at 25,000 rpm at 5 C with absorbance scans taken at 280 nm. The samples were judged to be at equilibrium when successive scans showed no change in the distribution of protein. Data were analyzed using a self-association model (Beckman Software).

Isothermal Microcalorimetry Measurements
ITC experiments were performed on the VP-ITC instrument (MicroCal, Northampton, MA). Purified rAF1C (4–5.5 mg/ml) and TBPC (0.5–0.9 mg/ml) in a buffer containing 50 mM HEPES (pH 7.0), 200 mM NaCl, and 10% glycerol were filtered and degassed before use. For titrations, 1.4 ml of 0.025 mM TBPC was placed in the ITC cell; with stirring at 480 rpm to assure rapid mixing, 35–37 successive 9-µl injections of 0.6 mM rAF1C followed at 6-min intervals to allow for complete equilibration. The heats of dilution of TBPC into the ITC buffer were small as compared with the actual heat of complex formation and were subtracted from the experimental titration results. The data were analyzed using single-site and sequential or independent two-site models by the Windows-based Origin Software p (MicroCal). Best fit was given by sequential model. Titrations at 15, 20, 25, and 28 C allowed determination of the heat capacity changes ({Delta}Cp) upon complex formation.

Fluorescence Microscopy and FRET Analysis
CV-1 cells grown on glass cover slips (Fisher Scientific, Houston, TX) 1 d before transfection were cotransfected with 1 µg of pGRE_SEAP reporter and 3 µg of pECFP-YFP (positive control), 1.5 µg of pECFP-C1, and/or 1.5 µg of pEYFP-C1 (negative control). To test the dependence of FRET on AF1 in the GR or on GRE in the gene promoter, cells were cotransfected with 1.5 µg of pEYFP_TBP and 1 µg of pGRE_SEAP or pTAL_SEAP (no GRE). Pairs of these pGRE_SEAP and pTAL_SEAP received 1.5 µg of either pECFP_GR500, pECFP_{Delta}AF1C GR500, or pECFP_{Delta}AF1 GR500. The cells were washed 24 h later twice with isotonic pH 7.4 PBS, fixed with 4% formaldehyde/PBS for 10 min and washed twice with PBS. Cells were visualized using a Zeiss LSM-510 META confocal microscope (Carl Zeiss, Thornwood, NY) with a Plan-Apochromat 63 x 1.4 oil-immersion objective and 6.1 Amp Argon laser. Pre- and postbleach images were collected at 12-bits resolution on two channels: 458 nm for CFP and 514 nm for YFP. Five images were taken, two before and three after the PB, with 15-sec intervals. To assure more than 90% PB, an arbitrarily selected region of interest, containing examples of both nuclear and cytoplasmic compartments, was irradiated with the 100% intensity laser line at 514 nm at 200-2000 iteration. Increased CFP (donor) fluorescence intensity upon YFP (acceptor) PB was indicative of positive FRET, and its efficiency was calculated by the equation:

Formula
Where, IDA is donor intensity after PB (extracted from image 2 of time series) corrected for background and fractional PB; IDB is donor intensity before PB background corrected (estimated from image 3 of the PB time series). Images that showed any focal plane drift were eliminated. In addition, we tested CFP, CFP-GR500, YFP, and YFP-TBP alone each time to account for any bleed-through and background FRET as recommended (data not shown) (49).

Reporter Gene Assays
We employed the SEAP reporter system due to its high signal-to-noise ratio and quantifiable transcriptional activity without the need for cell disruption. In the experiments with holo-GR, CV-1 cells were cotransfected as described above with 0.13 µg of pGRE_SEAP reporter vector, 0.13 µg of pRS_GR, or pRS_{Delta}AF1 GR and increasing amounts (0–0.5 µg) of pcDNA3.1_TBP. The total amount of DNA added was kept fixed at 0.8 µg by addition of empty pRS vector. Cells were allowed 24 h to recover and then were treated with 1 µM Dex. Medium (50 µl) was collected 24 h later and tested for the presence of SEAP (Great EscAPe SEAP Detection Kit; BD Biosciences) according to the manufacturer’s protocol. For experiments with the constitutively active GR500, CV-1 cells were cotransfected as described above with 0.13 µg of pGRE_SEAP reporter vector, 0.13 µg of pECFP_GR500, pECFP_{Delta}AF1C GR500, or pECFP_{Delta}AF1 GR500, and increasing amounts (0 to 0.5 µg) of pcDNA3.1_TBP. The total amount of DNA added was kept fixed at 0.8 µg by addition of pEGFP-C1 (BD Biosciences). After 36 h, 50 µl of medium was collected and tested for the presence of SEAP. Experiments were performed twice, in triplicate. Data from different experiments were normalized to GR500 activity.

The MMTV-driven pHHluc reporter was luciferase, which was assayed according to the protocol of the supplier (Promega Corp., Madison, WI). Triplicate lipofections and reporter assays were carried out. After the means and SDs were determined, values that deviated from the mean by more than 1 SD were removed and averages were recalculated. The same trend of increasing transcription with increasing TBP was observed by either method of calculation.

Semiquantitative RT-PCR Analysis
Total RNA was extracted from the CEM C1–6 cells using RNeasy Mini Kit with QIAshredder (QIAGEN) according to the manufacturer’s protocol. The RNA was quantified at this point. cDNA was prepared from 250 ng of RNA using ImProm-II Reverse Transcriptase System (Promega). After the inactivation of the enzyme by incubation at 75 C for 10 min, the cDNA was amplified by PCR with the primers designed for RTP801 and ß-actin (as a control), based on the nucleic acid sequences available from GenBank databases. Aliquot samples from the PCR products were run on a 2.5% agarose gel containing ethidium bromide. The spots were visualized using AlphaDigiDoc photoimager (Alpha Innotech Corp., San Leandro, CA) and quantified with Spot Densitometry Software supplied by the maker. To compare the relative mRNA levels, the ratios between the target mRNA and the corresponding ß-actin were calculated.


   ACKNOWLEDGMENTS
 
We thank Drs. Sean Juo (Yale University, New Haven, CT) and Vincent Giguere (McGill University Health Center, Montreal, Quebec, Canada) for providing plasmids, Drs. Christopher Chin (Sealy Center for Structural Biology Analytical Ultracentrifugation Core), Leoncio A. Vergara (Optical Imaging Laboratory), both at University of Texas Medical Branch (Galveston, TX), and Allan C. Ferreon (Scripps Research Institute, San Diego, CA) for technical assistance.


   FOOTNOTES
 
This work was supported by National Institutes of Health Grants DK 58829 (to R.K.) and CA41407 (to E.B.T.) and by Jeane B. Kempner Postdoctoral and Nowinsky Fellowships (to A.J.C.).

We have nothing to disclose.

First Published Online February 9, 2006

Abbreviations: AF1, Activation function 1; AF1c, core subdomain of AF1; CFP, cyan fluorescent protein; Dex, dexamethasone; ER, estrogen receptor; FRET, fluorescence resonance energy transfer; GR, glucocorticoid receptor; GRE, glucocorticoid response element; ITC, isothermal titration microcalorimetry; MMTV, mouse mammary tumor virus; NF, nuclear factor; PB, photobleaching; rAF1c, recombinant AF1c; SEAP, secreted alkaline phosphatase; TBP, TATA-binding protein; TBPc, terminal core domain of TBP; TFIIA, transcription factor IIA; YFP, yellow fluorescent protein.

Received for publication June 29, 2005. Accepted for publication January 31, 2006.


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 DISCUSSION
 MATERIALS AND METHODS
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NURSA Molecule Pages Link:

Nuclear Receptors:   GR
Coregulators:   TBP
Ligands:   Dexamethasone




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