Heterodimerization between the Glucocorticoid Receptor and the Unrelated DNA-Binding Protein, Xenopus Glucocorticoid Receptor Accessory Factor

Brian Morin, Glenna R. Woodcock, LaNita A. Nichols and Lené J. Holland

Department of Physiology University of Missouri-Columbia School of Medicine Columbia, Missouri 65212


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The adrenal steroid hormones, glucocorticoids, control many physiological responses to trauma, including elevated synthesis of fibrinogen, a major blood-clotting protein. Glucocorticoid regulation of the {gamma}-fibrinogen subunit gene in Xenopus laevis is mediated by a binding site for Xenopus glucocorticoid receptor accessory factor (XGRAF) and a contiguous glucocorticoid response element (GRE) half-site. Here, we characterize the protein:DNA complex formed by a cooperative interaction between XGRAF, GR, and the DNA. We demonstrate that the complex contains XGRAF by competition in a gel shift assay. The presence of GR is established by two criteria: 1) size dependence of the XGRAF:GR:DNA complex on the size of the GR component and 2) interference with complex formation by GR antibody. Cooperative binding of XGRAF and GR to the DNA was quantitated, showing that GR favors binding to XGRAF:DNA compared with free DNA by a factor of 30. The cooperative interaction between XGRAF and GR can occur on nicked DNA but is disrupted when 1 bp is inserted between the XGRAF binding site and half-GRE. Significantly, this loss of physical association in vitro correlates with loss of XGRAF amplification of GR activity in transiently transfected primary Xenopus hepatocytes. The simplest explanation for cooperativity between XGRAF and GR is formation of a DNA-bound heterodimer of these two proteins. This mechanism represents a new mode of transcriptional regulation in which GR and a nonreceptor protein form a heterodimer, with both partners contacting their specific DNA sites simultaneously.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Steroid hormones exert their myriad physiological effects by binding to specific intracellular proteins that selectively regulate gene transcription in target tissues (1). These hormone receptors belong to the large nuclear receptor superfamily, which is divided into two general classes based on DNA binding specificity (2, 3, 4). The first includes the receptors for glucocorticoid, mineralocorticoid, androgen, and progestin steroids (recognizing 5'-TGTTCT-3'). The second class encompasses the receptors for estrogenic steroids and nonsteroids such as retinoids, as well as many orphan receptors with no known ligand (recognizing 5'-TGACCT- 3'). In the classical mechanism of action, the steroid receptors bind as homodimers to inverted repeats of their recognition sequences, such as the consensus glucocorticoid response element (GRE), 5'-GGTACAnnnTGTTCT -3' (see Table 1GoA) (5).


View this table:
[in this window]
[in a new window]
 
Table 1. Nucleotide Sequences of DNA Fragments from the Upstream Region of the Xenopus {gamma}-Fibrinogen Gene Used in Gel Shift and Transfection Assays

 
Consistent with their large number and diversity of functions, the nuclear receptors operate by a variety of mechanisms in addition to homodimerization. For instance, members in the second class of receptors frequently heterodimerize on paired DNA sites (2, 3), whereas this mechanism has been described in only a few instances between members of the first class (6, 7, 8). Also, an association between receptors of the two different classes has been observed (9). Finally, interaction of the nuclear receptors with transcription factors in completely different protein families occurs (10). For example, the estrogen receptor can enhance binding of the promoter-specific transcription factor Sp1 to DNA (11) and GR can be tethered to the DNA by signal transducer and activator of transcription-5 (Stat5) (12) but, in both of these cases, a specific DNA binding site for the steroid receptor is not required.

A different steroid receptor mechanism controls transcription of the gene coding for the {gamma}-subunit of fibrinogen in the liver of the frog Xenopus laevis (13, 14, 15). The transcriptional regulatory region of the {gamma}-fibrinogen subunit gene has a binding site for a monomer of GR (TGTTCC) at positions –168 to –163 upstream of the transcription start site. The immediately adjacent sequence GAGTTAA at –175 to –169 (see Table 1GoA) binds Xenopus glucocorticoid receptor accessory factor (XGRAF) (13, 14, 15). The XGRAF binding site occupies the position of an upstream half-GRE in a full consensus GRE (5), but the DNA-binding domain of GR does not bind to this sequence (13). The recognition sequence for XGRAF does not correspond to the binding sites of nuclear receptors or other transcription factors, suggesting that XGRAF is a novel protein (13, 14). The segment of DNA containing the GR and XGRAF binding sites is sufficient to confer glucocorticoid induction of transcription (15). XGRAF alone does not mediate hormone responsiveness, but it increases fold induction by GR (15). The ability of XGRAF to bind to DNA and amplify hormonal induction defines it as an accessory factor (16). Here, we describe a new mechanism of GR action in which GR and XGRAF form a heterodimer on the DNA.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Demonstration of the Presence of XGRAF and GR in the XGRAF:GR:DNA Complex
We have shown previously in gel mobility shift assays that GR and XGRAF bind individually to their respective recognition sites on a fragment of the {gamma}-gene upstream DNA encompassing positions –189 to –157 (13, 14, 15). When both proteins are present, a protein:DNA complex forms that is larger than either GR:DNA or XGRAF:DNA (15). The formation of this complex implies that XGRAF and GR bind to the {gamma}-gene DNA simultaneously and that both proteins make contact with the DNA (15). Here we use gel shift assays that incorporate competition, size variation, and antibody displacement to confirm the identity of the protein components in the XGRAF:GR:DNA complex.

Figure 1GoA shows the migration position of the {gamma} (–189 to –157) DNA bound to XGRAF (lane 1). This complex was eliminated by competition with nonradioactive DNA containing a binding site for XGRAF but not for GR (lane 2). Consistent with our previous work (13), binding of native GR in the nuclear extract to {gamma}-gene DNA was not seen. Because endogenous GR in a crude nuclear extract is generally not readily detectable in gel shift assays, experiments are usually carried out with GR synthesized from cDNA (17, 18, 19). Here, we used a truncated form of rat GR synthesized in Escherichia coli, designated GR(L) (see Materials and Methods). GR(L) bound to {gamma} (–189 to –157) DNA primarily as a monomer (lane 3, lower band), with a small amount of dimer present (lane 3, upper band). The upper band in lane 3 was deduced to be GR dimer by comparison to GR bound to a probe with a strong consensus GRE (lane 4), where binding as a dimer is expected to be the predominant form (13, 20). When XGRAF and GR(L) were both present, a new larger band, XGRAF:GR(L):DNA, also formed (lane 5). The complex was distinctly different from GR homodimer (compare lane 5 to lanes 3 and 4). Excess competitor DNA containing the XGRAF binding site completely eliminated both XGRAF:DNA and XGRAF:GR(L):DNA, without interfering with GR binding to the DNA (lane 6). Thus, XGRAF is required for formation of the XGRAF:GR(L):DNA complex. This result, together with our previous demonstration that XGRAF:GR(L):DNA does not form on a probe with a mutated XGRAF binding site (15), confirms that XGRAF is an integral part of the complex.



View larger version (39K):
[in this window]
[in a new window]
 
Figure 1. Characterization of the XGRAF:GR:DNA Complex Using Gel Mobility Shift Assays

The reactions were carried out as described in Materials and Methods with either wild-type (WT) DNA or consensus GRE (C) probe (see Table 1GoA) and, as indicated, 5–6 µg Xenopus liver nuclear extract and the nonradioactive competitor 1 DNA (Table 1GoA) at 100-fold molar excess over probe. A, WT DNA and C DNA were at 0.2 ng and GR(L) was at 20 ng. B, WT DNA was at 0.2 ng and C DNA at 0.01 ng. GR(S) was used at 5 ng (lane 4) or 10 ng (lanes 3, 5, and 6).

 
The presence of GR in the XGRAF:GR:DNA complex was established using the same experimental scheme with a smaller form of the rat GR, GR(S) (see Materials and Methods). When both XGRAF and GR(S) were present, XGRAF:GR(S):DNA formed (Fig. 1GoB, lane 5). This complex was shown to contain XGRAF by competition (lane 6). XGRAF:GR(S):DNA migrated slightly above XGRAF:DNA and, therefore, was in a distinctly different position from XGRAF:GR(L):DNA (compare Fig. 1BGo, lane 5, to Fig. 1GoA, lane 5). The size difference of the complexes is directly attributable to the two different sizes of GR used.

GR was confirmed to be required for assembly of XGRAF:GR(S):DNA by including a GR-specific antibody in the gel shift reactions. The antibody primarily blocked formation of GR(S):DNA (Fig. 2Go, compare lane 4 to lane 3), although a small amount of material was detectable as a large supershifted band on a darker image (lane 4'). When the DNA was incubated with both XGRAF and GR(S), the GR-specific antibody caused the GR(S):DNA and the XGRAF:GR(S):DNA bands to disappear, but did not interfere with XGRAF:DNA formation (compare lane 6 to lane 5). The largest band in lanes 6 and 6' is identified as GR(S):antibody:DNA since it comigrated with the supershifted GR(S):DNA in lane 4'. The nuclear extract apparently stabilized the supershifted complex. GR-specific antibody interference with XGRAF:GR(S):DNA assembly provides additional support for the presence of GR in the complex.



View larger version (69K):
[in this window]
[in a new window]
 
Figure 2. Supershift or Immunodepletion of Protein-DNA Complexes Containing GR

The gel shift reactions were carried out as described in Materials and Methods with WT DNA probe at 0.2 ng, GR antibody (Ab) at 1 µg, and GR(S) at 20 ng. Lanes 4'-6' are a darker image of lanes 4–6.

 
Quantitation of Cooperative Binding of XGRAF and GR to {gamma}-Fibrinogen Gene DNA
The existence of the XGRAF:GR:DNA complex raised the possibility that GR and XGRAF bind cooperatively to the {gamma}-gene DNA. To test this hypothesis, we adapted an approach described by La Baer and Yamamoto (21) to calculate a relative value for the equilibrium association constants for formation of XGRAF:GR:DNA and GR:DNA. The equilibrium association constants are derived from the following reaction scheme:

The equations for the equilibrium association constants KD and KB are:

Equation 1a: KD=[XGRAF:GR:DNA]/[XGRAF:DNA][GR]

Equation 1b: KB=[GR:DNA]/[DNA][GR]

The ability of GR to bind to the XGRAF:DNA complex as compared with free DNA is expressed as the ratio of KD to KB:

A value of 1.0 for KD/KB signifies that GR has equal affinity for XGRAF:DNA and free DNA, indicating that binding is not cooperative. Values greater than 1.0 denote that GR binds preferentially to XGRAF:DNA. Using the KD/KB ratio avoids uncertainties with regard to absolute concentrations of the reactants since a relative number is calculated (21). Thus, our modification of this powerful, yet simple, approach allows quantitative analysis of heterodimer formation using crude nuclear extract. The system can also be thought of in terms of the preference of XGRAF to bind GR:DNA compared with free DNA, and it is worthy of note that, upon rearrangement of terms, the ratio KC/KA is identical to the ratio KD/KB.

To determine KD/KB, the amount of radioactive wild-type {gamma} DNA in XGRAF:GR:DNA, XGRAF:DNA, GR:DNA, and free DNA in each lane of a gel shift assay was quantitated by phosphorimaging and substituted into Equation 2. Using GR(L), KD/KB was determined in six independent experiments (with 4–6 individual lanes per experiment) to be 30 ± 4 (mean ± SEM) (e.g. Fig. 3GoA, lanes 2–5). Using GR(S), a similar value of 29 ± 3 in eight experiments (with 3–8 individual lanes per experiment) was obtained (B. Morin and L. J. Holland, unpublished data). These values are substantially greater than 1.0, indicating that GR and XGRAF bind cooperatively to their adjacent binding sites on the DNA. In contrast, binding of GR(L) alone to {gamma} (–189 to –157) DNA yielded (when the above reaction scheme was rewritten for binding of two molecules of GR) a KD/KB value of 1.9 ± 0.3 (Fig. 3GoB), demonstrating minimal GR:GR cooperativity as predicted by the lack of a full GRE.



View larger version (40K):
[in this window]
[in a new window]
 
Figure 3. Gel Shift Assay for Quantitation of Cooperative Binding of XGRAF and GR to DNA

A, The gel shift reactions contained 4 ng GR(L) and 0.2 ng C DNA (lane 1) or 20 ng GR(L) and 0.2 ng WT DNA (lanes 2–5). Xenopus liver nuclear extract was used at 8, 7, 6, and 5 µg (lanes 2–5). B, WT DNA was at 0.2 ng and GR(L) was at 100 ng (lanes 1–3).

 
Demonstration of Cooperativity by Competition between Labeled GR:DNA and Unlabeled Free DNA
We have used a competition gel shift assay previously to quantitate the preference of XGRAF to bind wild-type {gamma}-gene DNA compared with mutated competitors (14). Here this assay was adapted to examine the preference of XGRAF to bind radioactively labeled GR:DNA instead of unlabeled competitor free DNA. In this assay, a large amount of GR was used to form a substantial amount of the GR:DNA complex (Fig. 4Go). Under these conditions XGRAF:DNA was not visible because the XGRAF:GR:DNA complex was formed preferentially. A nonradioactive competitor DNA, which contains the XGRAF binding site but no GR binding site, was added to the reactions. Since this competitor can only form the XGRAF:DNA complex, this method computes the affinity of XGRAF for the radioactive GR:DNA complex compared with its affinity for nonradioactive free DNA. The amount of radioactivity in the XGRAF:GR:DNA complex over a range of competitor concentrations was quantitated by phosphorimaging (lanes 2–9). A Scatchard analysis of the data, described in Materials and Methods, revealed that a 25-fold excess of competitor over GR:DNA was required to reduce the concentration of XGRAF:GR:DNA by 50%. The 25-fold preference of XGRAF to bind GR:DNA compared with free DNA determined by this approach is in excellent agreement with the 30-fold value calculated by the more direct method in the previous section.



View larger version (82K):
[in this window]
[in a new window]
 
Figure 4. Demonstration of Cooperative Binding of XGRAF and GR to DNA by Competition

The gel shift reactions contained 7 µg Xenopus liver nuclear extract, 50 ng GR(L), 0.05 ng WT probe, and competitor 2 (see Table 1GoA) at the indicated fold excess over GR:DNA.

 
Interaction between XGRAF and GR on a Nicked DNA Probe
Cooperativity could be due to a protein-induced conformational change in the DNA that increases the binding affinity of the second protein (22). To examine this possibility, in the gel shift assay we used a DNA probe with a nick in one strand between the GR and XGRAF binding sites. The nick should disrupt transmission of an alteration in the DNA structure. For these experiments, we used a probe of 42 nucleotides from positions –189 to –148. Figure 5Go shows that this probe has similar binding characteristics for GR(L) (lane 1), XGRAF (lane 2), and the XGRAF:GR(L):DNA complex (lane 3) as the 33 mer {gamma} (–189 to –157) DNA (see Figs. 1GoA, 3A, and 4). When this probe had a nick in the sense strand between the XGRAF and GR binding sites, binding of GR(L) (lane 5), XGRAF (lane 6), and XGRAF:GR(L) (lane 7) also occurred. For both probes, the presence of XGRAF in the XGRAF:DNA and XGRAF:GR(L):DNA bands was confirmed by including an unlabeled competitor DNA that has a binding site for XGRAF but not for GR (lanes 4 and 8, respectively). XGRAF bound equally well to the intact or nicked probes. However, GR binding was substantially impaired by the nick, lowering the absolute amount of XGRAF:GR(L):DNA that formed on the nicked probe relative to the intact probe. Nonetheless, the KD/KB ratios, calculated from three separate reactions, showed that cooperativity was similar for the intact and nicked 42-mer probes. These values were comparable to that calculated for the 33-mer probe. Since a nick in the DNA failed to reduce cooperativity, it is unlikely that a conformational change in the DNA accounts for the preferential simultaneous binding of XGRAF and GR to the DNA.



View larger version (70K):
[in this window]
[in a new window]
 
Figure 5. Analysis of the Effect of a Nick in the DNA between the XGRAF and GR Binding Sites on Cooperative Binding

The gel shift reactions contained, as indicated, 6 µg Xenopus liver nuclear extract and either 2 ng (lanes 1, 3, and 4) or 20 ng (lanes 5, 7, and 8) GR(L). The 42-mer intact probe (lanes 1–4) or 42-mer nicked probe (lanes 5–8) was present at 0.2 ng and competitor 1 (see Table 1GoA) at 20 ng. See Materials and Methods for a description of the DNA probes.

 
Disruption of Cooperativity by Separation of the XGRAF and GR Binding Sites
A second possibility to account for the cooperativity is a direct protein-protein interaction that stabilizes XGRAF:GR binding to the DNA (22). To determine the importance of protein-protein contacts, the contiguity of the XGRAF and GR binding sites was disrupted. Gel shift experiments were carried out with a DNA probe containing 1 bp inserted between the XGRAF binding site and GRE half-site (Table 1GoA). This DNA was able to bind GR or XGRAF singly (Fig. 6Go, lanes 1 and 2, respectively) in a manner similar to the wild-type probe (lanes 4 and 5, respectively). However, the XGRAF:GR:DNA band observed using wild-type DNA (lane 6) was undetectable with the DNA containing the 1-bp insertion (lane 3). While XGRAF and GR may still bind to this DNA simultaneously, without the cooperative interaction the amount of the trimeric complex would be reduced by 30-fold and would be indiscernible. Since the immediate adjacency of the sites is required for cooperativity, we conclude that a direct interaction between XGRAF and GR occurs.



View larger version (67K):
[in this window]
[in a new window]
 
Figure 6. Analysis of the Effect of Separation of the XGRAF and GR Binding Sites on Cooperative Binding to DNA

Gel shift reactions contained, as indicated, 6 µg Xenopus liver nuclear extract, 20 ng GR(S), and 0.2 ng either WT or +1 bp probe (see Table 1GoA).

 
Correlation between Loss of Cooperativity in Vitro and Loss of Function in Vivo
The effect of separation of the binding sites on function in vivo was analyzed by transient transfection. DNA vectors containing the {gamma}-gene regulatory region were introduced into Xenopus primary hepatocytes (15). Hormonal induction of transcription by the XGRAF and GR binding sites was shown using the GRU({gamma}-104) construct (Fig. 7Go). Consistent with our previous demonstrations that XGRAF enhances GR function (13, 14, 15), mutation of the XGRAF binding site in the construct GRUmutX({gamma}-104) reduced glucocorticoid responsiveness. A 1-bp separation of the two binding sites in the GRU+1bp({gamma}-104) construct also decreased hormonal activation in comparison to the GRU({gamma}-104) control. The level of induction was equivalent whether the XGRAF binding site was completely inactivated by mutation or was moved away from the GR binding site. Thus, when the binding sites are noncontiguous, the loss of cooperative binding in the gel shift assay correlates with the reduced glucocorticoid responsiveness in intact cells. This observation strongly supports the hypothesis that interaction between XGRAF and GR, and not simply binding of the two proteins to the DNA, is crucial for XGRAF to exert its stimulatory effect on GR.



View larger version (10K):
[in this window]
[in a new window]
 
Figure 7. Analysis of the Effect of Separation of the XGRAF and GR Binding Sites on Hormonal Activation of Transcription

The diagrams show the presence of the XGRAF binding site (white box), the GRE half-site (gray box), and a 9-bp linker (black box) in the constructs containing Xenopus {gamma}-fibrinogen gene regulatory DNA (see Table 1GoB). The vectors were transiently transfected into Xenopus primary hepatocytes as described in Materials and Methods. Hormonal induction for each construct is expressed as the percentage ± SEM of that observed for the GRU({gamma}-104) control in four separate experiments. The fold induction for GRU({gamma}-104) was 2.5. The responses of the two mutated constructs were significantly reduced compared with the control, but were not different from each other (P < 0.01).

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
We have shown here that the preferential simultaneous binding of GR and XGRAF to their respective sites on the DNA involves a cooperative interaction between the two proteins. Quantitation of the cooperativity established that GR favored binding to XGRAF:DNA compared with free DNA by a factor of 30, as determined by direct comparison of equilibrium binding constants. This value was corroborated by a competition binding analysis. One explanation for cooperative binding is that protein-induced changes in DNA conformation increase individual binding affinity (Fig. 8Go). This interpretation is unlikely since a nick in the DNA between the binding sites, which is expected to disrupt transmission of changes in DNA structure (22), did not interfere with cooperative binding (Fig. 5Go). An alternative explanation for cooperativity is that XGRAF and GR physically contact one another when bound to the DNA (Fig. 8Go). Support for this model was obtained when separation of the two binding sites, which would reposition potential interaction surfaces of the proteins, eliminated cooperativity (Fig. 6Go).



View larger version (12K):
[in this window]
[in a new window]
 
Figure 8. Models of Formation and Disruption of the XGRAF:GR Heterodimer

XGRAF is represented by the white triangle, GR by the gray oval, and DNA by the black bars. The 1-bp insertion is denoted by the white box.

 
While GR classically binds to DNA as a homodimer, monomeric GR has been shown to function with other transcription factors (23, 24). Therefore, the XGRAF:GR:DNA complex could contain either one or two molecules of GR. We have shown previously that XGRAF binds to DNA in the major groove (14), occluding the position where a second molecule of GR normally binds to DNA as a homodimer (25). Thus, if the complex contains two molecules of GR, one GR monomer must be tethered rather than contacting the DNA directly (13). To distinguish between one or two molecules of GR, we examined the size of the XGRAF:GR:DNA complex. Although the native gels of the gel shift assay cannot be used for absolute mass determination, XGRAF behaves as a protein somewhat larger than GR(L), since XGRAF:DNA migrated more slowly than GR(L):DNA (Fig. 1GoA, compare lane 1 to lane 3). Furthermore, the XGRAF:GR:DNA complex migrated slightly above the GR(L) dimer (Fig. 1GoA, compare lane 5 with lanes 3 and 4), indicating that the complex most likely contains one molecule each of XGRAF and GR(L). If the complex contained two molecules of GR in addition to XGRAF, its apparent size would be larger than three molecules of GR(L) bound to the DNA. However, we have determined that GR(L):GR(L):GR(L):DNA migrates much more slowly than XGRAF:GR(L):DNA (L. J. Holland, unpublished data).

Taken together, the in vitro binding results are most consistent with a model of heterodimerization between GR and XGRAF. This mechanism, in which GR and an unrelated partner protein both contact the DNA simultaneously, has not been described previously. The region of GR that interacts with XGRAF was delineated using truncated forms of GR. Both GR(L) and GR(S), which have only amino acids 407–525 in common, interact cooperatively with XGRAF. Thus, the surface of GR responsible for heterodimerization with XGRAF must be contained within these amino acids. This region also contains domains for DNA binding (25), GR homodimerization (25), and physical or functional interaction with other transcription factors (26, 27). Furthermore, this region is conserved across the nuclear receptor superfamily (2), suggesting that XGRAF could heterodimerize with other members of the family.

The cooperative binding between XGRAF and the small GR fragments in vitro parallels XGRAF enhancement of native GR function in vivo. Therefore, it is likely that the transcriptional activation of the {gamma}-fibrinogen gene by full-length GR in intact cells involves a cooperative interaction with XGRAF. Since the in vitro binding studies were carried out with mammalian GR, heterodimerization with XGRAF is not restricted to the amphibian receptor. In addition, we have evidence for a protein with similar DNA-binding specificity to XGRAF in a human liver-derived cell line (K. D. Fohey and L. J. Holland, unpublished data). Thus, this mechanism is potentially applicable to activation of other glucocorticoid-regulated genes in a wide variety of animals, including humans.

We have shown that glucocorticoid-induced gene transcription can be mediated through a heterodimer of GR and an unrelated DNA-binding protein, in which both proteins bind to their specific sites on the DNA. Heterodimerization between some related nuclear receptors is common (2, 3). However, this is the first demonstration, to our knowledge, of any member of the large nuclear receptor superfamily forming a DNA-bound heterodimer (as opposed to a tethered ternary complex) with a protein that has a completely different DNA-binding specificity. This mechanism provides a new explanation for how the DNA can dictate the assembly of specific transcriptional regulatory complexes on different genes, to achieve diverse patterns of gene activation in response to a hormone signal.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Gel Mobility Shift Assays and Phosphorimager Analysis
The 33-mer oligonucleotide probes used in Figs. 1Go, 2Go, 3Go, 4Go, and 6Go are described in Table 1GoA. Oligonucleotide probes were purified and radioactively labeled at the 5'- end as described (13). The 42-mer probes used in Fig. 5Go had the following sequence on the sense strand: -189CTCCAGACAGAAAAGAGTTAATGTTCCCTCTTATacTaACTA-148. The XGRAF binding site is in bold and the GRE half-site is underlined. The lower case letters indicate mutations that inactivate a second GR binding site at positions –156 to –151 (13, 15). The nicked 42-mer probe contained a single-strand break between nucleotides –169 and –168. This probe was assembled by annealing two independently synthesized (Genosys, The Woodlands, TX) 21-mer sense strands, –189 to –169 and –168 to –148, to an intact antisense 42-mer from –148 to –189. A 42-mer instead of 33-mer probe was used to ensure a stable double-stranded structure. Both the intact and nicked 42-mer probes were radioactively labeled at the 5'-end as described above. We confirmed by denaturing gel electrophoresis (28) that the nicked probe consisted of labeled 21-mer and 42-mer strands and that the nick did not become ligated during incubation for the gel shift assay (L. J. Holland, unpublished data).

Bacterially synthesized, truncated forms of rat GR were kindly provided by the laboratory of Dr. Keith Yamamoto. T7EX525, here designated GR(L), comprises amino acids 106–318 adjoined to amino acids 407–525, which includes the DNA-binding domain (29). T7X556, here designated GR(S), consists of amino acids 407–556 (17, 30). It is necessary to use truncated forms of GR because full-length GR is too large for optimal electrophoretic separation and quantitation of all the protein-DNA complexes. The GR antibody BuGR 2 (Affinity BioReagents, Inc., Golden, CO) recognizes amino acids 410–416 (31). Xenopus liver nuclear extract served as the source of XGRAF (15). The binding reactions and gel electrophoresis conditions were as described previously (15).

Quantitation of products in the gel shift assays was performed with a phosphorimager and ImageQuant 3.3 software (Molecular Dynamics, Inc., Sunnyvale, CA). In each lane the amount of radioactivity in XGRAF:GR:DNA, XGRAF:DNA, GR:DNA, and free DNA was determined. An individual background for each lane was determined and subtracted from the values for the protein-DNA complexes. As an example, normalized data for Fig. 3AGo are shown in Table 2Go.


View this table:
[in this window]
[in a new window]
 
Table 2. Example of Normalized Data to Calculate Cooperativity

 
Quantitation of Preferential Binding by Competition Assay
The competition gel shift assays (14) were carried out under conditions where XGRAF was visible only in XGRAF:GR:DNA. Unlabeled competitor DNA containing an XGRAF binding site but no GR binding site was added in increasing amounts. Radioactivity was quantitated by phosphorimaging, and the data were analyzed with a Scatchard plot (14) generated by the following equation:

The amount of XGRAF bound to the competitor DNA, designated [DNA·XGRAF], was computed as the fraction lost from XGRAF:GR:DNA. [DNA]t, representing total competitor DNA, was expressed as fold excess over GR:DNA, since the ability of XGRAF to bind either GR:DNA or the competitor was being compared. The quantity of GR:DNA in each reaction was calculated as the fraction of radioactivity in the GR:DNA complex compared with radioactivity in the entire lane. The term C50 represents the fold excess of competitor required to displace 50% of XGRAF from the XGRAF:GR:DNA. The y-intercept was used to obtain a value for C50. This value was normalized to the C50 value obtained in a parallel experiment for competition of XGRAF from wild-type DNA in the absence of GR.

Transfection of Primary Hepatocytes and Assay of Hormonal Induction
The transfection vectors containing Xenopus {gamma}-fibrinogen gene regulatory DNA were constructed with either wild-type or mutated {gamma} (–187 to –157) (see Table 1GoB) linked to {gamma} (–104 to +41) (15). The vectors were transiently transfected into Xenopus primary hepatocytes following published methods (15) except cells were plated at 4 x 105 per well in 24-well plates with 1.6 ml of medium, with a final composition as described (32). Experiments were conducted in accordance with the NIH Guide for the Care and Use of Laboratory Animals. As described in detail previously (15), hormonal induction is expressed as a percentage of the control using the following equation: Fold Induction (% of control) = [(Fold induction of test construct – 1)/(Fold induction of control construct – 1)] x 100. The value 1 was subtracted from each fold induction to account for the baseline, representing no hormone response. The induction for each construct is expressed as the percentage ± SEM of that observed for the GRU({gamma}-104) control in four separate experiments. Statistical analyses of raw data for fold hormonal induction in each independent transfection were done with the Student- Newman-Keuls test (33).


    ACKNOWLEDGMENTS
 
We thank the laboratory of K. Yamamoto for generously providing the purified GR fragments and M. Hannink, M. Martin, A. McClellan, and R. Woodward for helpful comments on the manuscript.


    FOOTNOTES
 
Address requests for reprints to: Lené J. Holland, Department of Physiology, University of Missouri-Columbia School of Medicine, Columbia, Missouri 65212.

This work was supported by the American Heart Association (Grants-in-Aid 9708034A and 0051320Z).

Received for publication September 22, 2000. Revision received November 28, 2000. Accepted for publication November 29, 2000.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Tsai M-J, O’Malley BW 1994 Molecular mechanisms of action of steroid/thyroid receptor superfamily members. Annu Rev Biochem 63:451–486[CrossRef][Medline]
  2. Mangelsdorf DJ, Thummel C, Beato M, Herrlich P, Schütz G, Umesono K, Blumberg B, Kastner P, Mark M, Chambon P, Evans RM 1995 The nuclear receptor superfamily: the second decade. Cell 83:835–839[Medline]
  3. Enmark E, Gustafsson J-Å 1996 Orphan nuclear receptors– the first eight years. Mol Endocrinol 10:1293–1307[Medline]
  4. Nuclear Receptors Nomenclature Committee 1999 A unified nomenclature system for the nuclear receptor superfamily. Cell 97:161–163[Medline]
  5. Beato M, Chalepakis G, Schauer M, Slater EP 1989 DNA regulatory elements for steroid hormones. J Steroid Biochem 32:737–747[CrossRef][Medline]
  6. Trapp T, Rupprecht R, Castrén M, Reul JMHM, Holsboer F 1994 Heterodimerization between mineralocorticoid and glucocorticoid receptor: a new principle of glucocorticoid action in the CNS. Neuron 13:1457–1462[Medline]
  7. Liu W, Wang J, Sauter NK, Pearce D 1995 Steroid receptor heterodimerization demonstrated in vitro and in vivo. Proc Natl Acad Sci USA 92:12480–12484[Abstract]
  8. Chen S, Wang J, Yu G, Liu W, Pearce D 1997 Androgen and glucocorticoid receptor heterodimer formation. A possible mechanism for mutual inhibition of transcriptional activity. J Biol Chem 272:14087–14092[Abstract/Free Full Text]
  9. Lee Y-F, Shyr C-R, Thin TH, Lin W-J, Chang C 1999 Convergence of two repressors through heterodimer formation of androgen receptor and testicular orphan receptor-4: a unique signaling pathway in the steroid receptor superfamily. Proc Natl Acad Sci USA 96:14724–14729[Abstract/Free Full Text]
  10. Diamond MI, Miner JN, Yoshinaga SK, Yamamoto KR 1990 Transcription factor interactions: selectors of positive or negative regulation from a single DNA element. Science 249:1266–1272[Medline]
  11. Porter W, Saville B, Hoivik D, Safe S 1997 Functional synergy between the transcription factor Sp1 and the estrogen receptor. Mol Endocrinol 11:1569–1580[Abstract/Free Full Text]
  12. Cella N, Groner B, Hynes NE 1998 Characterization of Stat5a and Stat5b homodimers and heterodimers and their association with the glucocortiocoid receptor in mammary cells. Mol Cell Biol 18:1783–1792[Abstract/Free Full Text]
  13. Woodward RN, Li M, Holland LJ 1997 Novel accessory factor-binding site required for glucocorticoid regulation of the {gamma}-fibrinogen subunit gene from Xenopus laevis. Mol Endocrinol 11:563–576[Abstract/Free Full Text]
  14. Li M, Ye X, Woodward RN, Zhu C, Nichols LA, Holland LJ 1998 Analysis of the DNA-binding site for Xenopus glucocorticoid receptor accessory factor. Critical nucleotides for binding specificity in vitro and for amplification of steroid-induced fibrinogen gene transcription. J Biol Chem 273:9790–9796[Abstract/Free Full Text]
  15. Morin B, Zhu C, Woodcock GR, Li M, Woodward RN, Nichols LA, Holland LJ 2000 The binding site for Xenopus glucocorticoid receptor accessory factor and a single adjacent half-GRE form an independent glucocorticoid response unit. Biochemistry 39:12234–12242[CrossRef][Medline]
  16. Lucas PC, Granner DK 1992 Hormone response domains in gene transcription. Annu Rev Biochem 61:1131–1173[CrossRef][Medline]
  17. Freedman LP, Luisi BF, Korszun ZR, Basavappa R, Sigler PB, Yamamoto KR 1988 The function and structure of the metal coordination sites within the glucocorticoid receptor DNA binding domain. Nature 334:543–546[CrossRef][Medline]
  18. Srinivasan G, Thompson EB 1990 Overexpression of full-length human glucocorticoid receptor in Spodoptera frugiperda cells using the baculovirus expression vector system. Mol Endocrinol 4:209–216[Abstract]
  19. Aumais JP, Lee HS, DeGannes C, Horsford J, White HJ 1996 Function of directly repeated half-sites as response elements for steroid hormone receptors. J Biol Chem 271:12568–12577[Abstract/Free Full Text]
  20. Tsai SY, Carlstedt-Duke J, Weigel NL, Dahlman K, Gustafsson J-Å, Tsai M-J, O’Malley BW 1988 Molecular interactions of steroid hormone receptor with its enhancer element: evidence for receptor dimer formation. Cell 55:361–369[Medline]
  21. La Baer J, Yamamoto KR 1994 Analysis of the DNA-binding affinity, sequence specificity and context dependence of the glucocorticoid receptor zinc finger region. J Mol Biol 239:664–688[CrossRef][Medline]
  22. Dahlman-Wright K, Siltala-Roos H, Carlstedt-Duke J, Gustafsson J-Å 1990 Protein-protein interactions facilitate DNA binding by the glucocorticoid receptor DNA-binding domain. J Biol Chem 265:14030–14035[Abstract/Free Full Text]
  23. Heck S, Kullmann M, Gast A, Ponta H, Rahmsdorf HJ, Herrlich P, Cato ACB 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]
  24. Reichardt HM, Kaestner KH, Tuckermann J, Kretz O, Wessely O, Bock R, Gass P, Schmid W, Herrlich P, Angel P, Schütz G 1998 DNA binding of the glucocorticoid receptor is not essential for survival. Cell 93:531–541[Medline]
  25. Luisi BF, Xu WX, Otwinowski Z, Freedman LP, Yamamoto KR, Sigler PB 1991 Crystallographic analysis of the interaction of the glucocorticoid receptor with DNA. Nature 352:497–505[CrossRef][Medline]
  26. 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]
  27. Kerppola TK, Luk D, Curran T 1993 Fos is a preferential target of glucocorticoid receptor inhibition of AP-1 activity in vitro. Mol Cell Biol 13:3782–3791[Abstract]
  28. Maniatis T, Fritsch EF, Sambrook J 1982 Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
  29. Starr DB, Matsui W, Thomas JR, Yamamoto KR 1996 Intracellular receptors use a common mechanism to interpret signaling information at response elements. Genes Dev 10:1271–1283[Abstract]
  30. Freedman LP, Yoshinaga SK, Vanderbilt JN, Yamamoto KR 1989 In vitro transcription enhancement by purified derivatives of the glucocorticoid receptor. Science 245:298–301[Medline]
  31. Simons Jr SS 1994 Function/activity of specific amino acids in glucocorticoid receptors. Vitam Horm 49:49–130[Medline]
  32. Roberts LR, Nichols LA, Holland LJ 1993 Transcriptional regulation of the Xenopus laevis Bß fibrinogen subunit gene by glucocorticoids and hepatocyte nuclear factor 1: analysis by transfection into primary liver cells. Biochemistry 32:11627–11637[Medline]
  33. Glantz SA 1997 Primer of Biostatistics, ed. 4. McGraw-Hill, New York