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a Instituto de Investigaciones Biomédicas, CSIC, Madrid, Spain.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSREFERENCES |
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Key Words: growth hormone gene • transcriptional regulation • nuclear receptors
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSREFERENCES |
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The pituitary-specific transcription factor GHF-1/Pit-1, a member of the homeobox POU family of DNA-binding proteins, plays an important role in growth hormone (GH) gene expression (8, 9). GHF-1/Pit-1 binds to two sequences (-65/-95 and -107/-137 base pairs) in the rat GH promoter, and expression of GHF-1 in nonpituitary cells stimulates the activity of the GH promoter in transient transfections showing that this pituitary factor is limiting for GH gene expression. Evidence strongly suggests that GHF-1 activates cell-specific gene transcription by cooperating with other transcription factors or coactivator proteins. Thus, the thyroid hormone receptor has been reported to act synergistically with GHF-1 to activate this promoter (10, 11), and a similar synergism with the RA receptors has been observed (12). This interaction may be significant in vivo since it has been reported that the two GHF-1 binding sites are necessary, but not sufficient, for efficient transcriptional activation of the rat GH gene promoter in transgenic mice. The inclusion of additional sequences that contain the T3/RA response element results in much higher levels of transgene expression suggesting the existence of cooperation between GHF-1 and this element (13). In addition to the direct effect on the GH promoter, we have very recently shown that these receptors can indirectly influence rat GH gene transcription by modulating GHF-1 gene expression (14, 15).
In this report we demonstrate that, as analyzed by gel retardation assays and in vitro binding experiments with GST-fusion proteins, the functional cooperation between the nuclear receptors and GHF-1 involves a direct physical association. Although cooperative binding was not detected, the protein-protein interactions facilitate the formation of complexes containing GHF-1 and receptor heterodimers with the rat GH promoter that contains binding sites for both types of transcription factors.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSREFERENCES |
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(17), the human RAR (18), and the human RXR (19) cloned into the expression vector pSG-5 have been described previously.
Cell culture and transfections
GH4C1 cells cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal calf serum were transfected by electroporation as previously described (14, 15). Ten micrograms of the reporter plasmids were mixed with 20 to 30 million cells and exposed to a high-voltage pulse (200–250 V, 960 µF) by using a Bio-Rad (Hercules, Calif.) electroporator with a capacitor extender. The cells from each electroporation were split into different culture plates in DMEM containing 10% AG1x8 resin-charcoal stripped newborn calf serum. Treatments with RA and T3 were administered in this hormone-depleted medium. In cotransfection experiments the reporter plasmids were transfected with the expression vectors for GHF-1 and the nuclear receptors, and the total amount of DNA was kept constant by adding empty noncoding expression vectors (RSV-0 and pSG5-0). Each treatment with the ligands was performed at least in duplicate cultures that normally exhibited less than 10% variation in CAT activity. CAT activity was determined by incubation of the cell extracts with [14C]chloramphenicol. The unreacted and acetylated [14C]chloramphenicol were separated by thin-layer chromatography, identified by autoradiography, and quantitated. The data are expressed as the percentage of acetylated forms after each treatment.
Protein preparations
The coding region of GHF-1 cloned in Bluescript SK-, and the vectors for TR, RAR, and RXR cloned in pSG5 were used for in vitro transcription and translation. For this purpose 1 µg of the different vectors was transcribed and translated by using the TNT kit (Promega, Madison, Wisc.) following the manufacturer recommendations. All reactions were split into two aliquots: one was translated in the presence of 40 µCi of [35S]methionine (Amersham) and the other in the presence of the same amount of the unlabeled aminoacid. Three µl of the reaction product were resolved in 12% sodium dodecyl sulfate-polyacrylamide (SDS-PAGE) gel electrophoresis. The gel was dried and autoradiographed overnight. Recombinant GHF-1 expressed as described (16) in the bacterial strain BL21(DE3) was also used. Highly purified preparations of TR, RAR and RXR (>80% purity) (20) were a kind gift from T. Iglesias and D. Barettino.
Protein-protein interactions
pGST-TR, which expresses a fusion protein between glutathione S-transferase (GST) and wild-type cTR, and pGST-RXR, which contains the entire mouse RXR cDNA, were a kind gift of H. Samuels. pGST-RAR was constructed by fusing the human RAR cDNA to the pGEX vector. GST and GST fusion proteins were expressed and purified by using standard techniques. For the pull-down assays, 5 µl of in vitro translated L-[35S]methionine labeled GHF-1 was incubated with 100 ng of GST fusion proteins immobilized in glutathione-agarose beads as described elsewhere (21). Where indicated, 1 µM T3 or 1 µM 9-cis-RA were included in the incubation mixture. The bound proteins were analyzed by SDS-PAGE and autoradiography.
Mobility shift assays
Gel retardation analysis were performed with recombinant proteins, or with in vitro translated proteins. As probes we used oligonucleotides corresponding to the proximal GHF-1/Pit-1 binding site of the rat GH promoter (5'-CCAGCCATGAATAAATGTATAAGGG-3'), and to the perfect palindromic T3/RA responsive element TREPAL (5'-AGCTCTAGGTCATGACCTGA-3') derived from the natural element in the rat GH gene. For the binding assay, the proteins were incubated on ice for 15 min in a buffer (20 mM Tris HCl [pH7.5], 75 mM KCl, 1mM dithiothreitol, 13% glycerol) containing 3 µg Poly(dI-dC) and then for 15–20 min at room temperature with approximately 50,000 cpm double-stranded oligonucleotide end-labeled with [32P]ATP, using T4-polynucleotide kinase. In addition, a labeled fragment of the rat GH promoter (from -68 to -210) was obtained by PCR using the sequences 5'-TGGGCAAAGGCGGC-3' and 3'-TTTCCCTATACATTTATTC-5'. Each binding reaction contained the same amount of extract or proteins, which was obtained by adding the appropriate amount of mock lysate or bacterial proteins. For band-shift with purified recombinant proteins, binding buffer was supplemented with 5 µg of bovine serum albumin. For competition experiments, an excess of unlabeled double-stranded oligonucleotides or PCR fragment was added to the binding reaction mixture. DNA-protein complexes were resolved on 7.5 or 5% polyacrylamide gels in 0.5% TBE buffer. The gels were then dried and autoradiographed at -70°C.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSREFERENCES |
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Figure 1
compares the effect of a 48 h incubation with T3, RA and the combination of both in cells transfected with a reporter plasmid containing 530 bp of the 5'-flanking region of the rat GH gene (-530GH-CAT). In agreement with previous observations (6, 12, 22), T3 and RA stimulate the activity of this construct in GH4C1 cells, which contain endogenous receptors for both ligands, and expression of GHF-1 further increases this response (data not shown). Since T3 and RA induce GH gene expression through the same responsive element, it was of interest to analyze the influence of the expression of T3 and RA receptors on the response to the ligands as well as on the response to GHF-1. For this purpose the reporter plasmid was co-transfected with TR and RAR vectors in the presence or absence of a GHF-1 expression vector. In TR-overexpressing cells, T3 increased CAT activity and a further stimulation was found in cells transfected with GHF-1, supporting the existence of functional cooperation between the TR and GHF-1 ( Fig. 1A). However, the transcriptional response to RA was strongly blocked in TR-transfected cells, and a synergistic activation in the presence of RA and GHF-1 was not observed. Similarly, in cells transfected with RAR, there was a response to RA, but T3-stimulation was blocked ( Fig. 1B). GHF-1 significantly increased the response to RA, but the cooperation between T3 and GHF-1 was repressed in these cells. As can be observed in both panels in Fig. 1, the combination of T3 plus RA has a similar effect to the corresponding ligand alone.
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The influence of GHF-1 was also examined in a construct that contains a shorter fragment of the rat GH promoter (-145GH-CAT). In this fragment the T3/RA responsive element is deleted, although both the distal and proximal binding sites for GHF-1 are present. GHF-1 increased about fivefold the activity of -145GH-CAT. On this shorter promoter T3 had a strong negative effect and very significantly decreased CAT activity even in the presence of GHF-1. The negative effect was not shared by RA, which did not alter CAT levels with respect to those found in control cells (data not shown).
Influence of GHF-1 and the receptors on each other binding to DNA
The cooperative response of the GH promoter to T3 or RA receptors and GHF-1 could involve protein-DNA and/or protein-protein interactions. Figure 2A
shows the effect of GHF-1 on binding of TR/RXR to an isolated thyroid hormone response element (TREPAL). In vitro translated TR binds predominantly in monomeric form to this element (lanes 3 and 8), although some homodimer formation can be detected at longer exposures or when higher amounts of protein are used. RXR, that by itself does not significantly bind this element (not shown), produces the appearance of TR/RXR heterodimers that bind strongly to DNA (lanes 4 and 10). The heterodimers are supershifted in the presence of an anti-RXR antibody (lane 11), which did not alter binding of TR (lane 9). GHF-1 did not produce retardation with the TRE (lane 5). However, incubation with GHF-1 decreases the intensity of the TR/RXR heterodimeric complex (lane 7), which could be compatible with the existence of a protein-to-protein association between the pituitary factor and the receptors. As shown in lane 6, GHF-1 did not significantly alter the abundance of receptor monomers. Figure 2B shows binding of RA receptors to this element. Binding of RAR alone either as a monomer or as a homodimer was not observed (not illustrated), but the heterodimer RAR/RXR caused the appearance of a retarded band (lanes 4), which was displaced in the presence of GHF-1 (lane 5). Again the RAR/RXR heterodimeric band (lane 7) was supershifted by the anti-RXR antibody (lane 8).
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To analyze whether the nuclear receptors had a reciprocal effect on GHF-1 binding, we conducted similar experiments in which binding of GHF-1 to the proximal site in the GH gene (GHF-1P) was performed in the absence and presence of receptors. Lanes 2 and 7 in 
Figure 2C show binding of GHF-1 to this sequence which was super-retarded in the presence of an anti-GHF-1 antibody (lane 8). TR and RAR neither as homodimers nor as heterodimers with RXR bind to this element (data not shown). However, TR, RAR, and RXR (lanes 3 to 5) produced a significant decrease in the abundance of the retarded band produced by GHF-1 (lane 2).
The finding that GHF-1 and the receptors inhibit each other binding to their corresponding DNA sites is apparently paradoxical with the functional results showing that GHF-1 cooperates with the nuclear receptors increasing GH promoter activity. However, the inhibition observed with the individual cognate elements does not occur on the GH promoter, which contains binding sites for both the receptors and GHF-1. Figure 3
shows formation of DNA-protein complexes with purified recombinant receptors, GHF-1 and a DNA fragment which encompasses the sequences -68 to -210 of the rat GH promoter. This promoter fragment contains the binding sites for GHF-1 ( Figure 3A, lanes 7 and 8), and the T3/RA response element which binds TR/RXR (lanes 9 and 10) and RAR/RXR (lanes 11 and 12) heterodimers. TR, RAR or RXR alone did not bind with high affinity to this fragment neither in the absence or presence of GHF-1 (not illustrated). Figure 3A also shows that limiting concentrations of TR/RXR and RAR/RXR, which by themselves did not cause retardation (lanes 3, and 5), in the presence of GHF-1 caused the appearance of retarded bands with the expected mobilities of the heterodimers (lanes 4 and 6). Therefore, in contrast with the results shown in Fig. 2with an isolated TRE, GHF-1 did not decrease but rather increased binding of TR/RXR and RAR/RXR to the natural GH promoter. The increase in binding was mutual and GHF-1 bound more strongly in the presence than in the absence of receptors. Figure 3B shows that GHF-1 did not alter TR/RXR or RAR/RXR binding when higher amounts of receptors than those shown in panel A were used. Under these conditions the heterodimers produced a retardation (lanes 3 and 5) that was not affected by GHF-1 (lanes 4–6). The identity of the retarded complexes was again demonstrated by supershift with anti-GHF-1 and anti-RXR antibodies (lanes 8–11).
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In the experiments illustrated in Figure 3in which the receptors and GHF-1 were added together in the presence of an excess of the DNA fragment, we find no indication for the formation of a ternary complex containing GHF-1 and the receptor heterodimer on the same promoter fragment. However, as shown in Figure 4,
this complex was clearly observed in band shift experiments that used longer gels and limited amounts of the GH promoter fragment. Under these conditions ( Fig. 4A), when GHF-1 and TR/RXR were added together (lane 8), the retarded complexes with the mobility of either component (lanes 2 and 5, respectively) disappeared, and instead new complexes migrating slower than TR/RXR or GHF-1 were detected. The new retarded bands appear to contain both GHF-1 and the receptor heterodimer as demonstrated by competition with specific binding sites. As shown in lanes 3 and 4, binding of GHF-1 was competed by an excess of the proximal GHF-1 binding site in the GH gene but was not affected by the TREPAL oligonucleotide. Similarly, the retardation caused by TR/RXR was competed by the receptor binding site (lane 6), but not by the GHF-1 binding site (lane 7). Moreover, the ternary complex was disrupted in the presence of excess TREPAL (lane 9), and under these conditions binding of GHF-1 with the same mobility as in lane 2 was observed. In addition, an excess of the GHF-1P oligonucleotide produced the reappearance of the original receptor bands, whereas GHF-1 binding was no longer detected (lane 10). Simultaneous binding of RAR/RXR and GHF-1 to the same DNA molecule is illustrated in Figure 4B. As was found with TR/RXR (lane 4), combination of RAR/RXR and GHF-1 (lane 6) using limiting amounts of promoter fragment led to the formation of complexes of slower mobility than RAR/RXR (lane 5) or GHF-1 (lane 2) alone. Lanes 7 and 8 demonstrate that these complexes were super-shifted in the presence of an anti-RXR antibody. Figure 4C demonstrates the presence of GHF-1 in the ternary complexes, since the anti-GHF-1 antibody produced the supershift not only of the retarded band formed by GHF-1 alone (lane 3), but also of the bands formed with the combination of GHF-1 with TR/RXR (lane 7) or RAR/RXR (lane 12). Figure 4C also illustrates the results obtained with specific antibodies against TR and RAR. These antibodies caused the supershift of the retarded bands containing TR/RXR (lane 5) and RAR/RXR (lane 10), as well of the ternary complexes (lanes 8 and 13). Together, these results demonstrate that these complexes contain both GHF-1 and receptor heterodimers.
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The affinity of the different protein-DNA complexes was assessed by off-rate experiments challenging the performed complexes with an excess of the unlabeled GH promoter fragment and applying aliquots at different times after addition of the competitor onto a running gel. As shown in Fig. 5,
binding of GHF-1 was stable on challenge (lanes 1 to 4), whereas binding of TR/RXR gradually disappeared (lanes 5 to 8). This result agrees with the idea that the natural TRE present in the GH promoter is a weak response element. Lanes 9 to 12 show the results obtained with the combination of TR/RXR and GHF-1. There was the possibility that the association with GHF-1 could stabilize binding of the receptor heterodimer to the promoter fragment; however, the ternary complex was inhibited with the same kinetics as TR/RXR alone, showing that the interaction does not significantly enhance the apparent affinity of receptor binding to the promoter.
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GHF-1 interacts with T3 and RA receptors
The results illustrated in Figures 2–5 are compatible with the existence of a direct association between the receptors and the pituitary-specific transcription factor. These interactions were studied by assessing the binding of 35S-labeled GHF-1 to GST-TR, GST-RAR and GST-RXR immobilized on glutathione-agarose beads. The results obtained are shown in Fig. 6.
GST protein alone did not interact with GHF-1 (6A, lane 2). However, both GST-TR and GST-RAR interacted with GHF-1. GST-RAR associated with GHF-1 more strongly than TR. Figure 6B shows that GST-RXR also interacts significantly with GHF-1. Quantification of the corresponding bands showed that GST-RXR binds GHF-1 at least fourfold more efficiently than GST-TR. Approximately 10–20% of the 35S-labeled GHF-1 bound to GST-RXR in different, independent experiments. Binding of GST-TR and GST-RXR to the pituitary factor was not enhanced by incubation of both receptors together. Furthermore, the interaction of the T3 or retinoid receptors with GHF-1 was constitutive and did not increase in the presence of the corresponding ligand.
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DISCUSSION
In GH4C1 pituitary cells T3 and RA caused a significant increase in GH gene expression and overexpression of GHF-1 enhanced the response to both ligands. Our studies therefore corroborate the synergism between the nuclear receptors and GHF-1 (10–12), and agree with data obtained in transgenic mice (13) that suggest that while GHF-1 is necessary for GH promoter activity, other factors are required for maximal expression. Since the receptor heterodimers TR/RXR and RAR/RXR bind to the same DNA element in the promoter, an excess of TRs blocks the stimulation by RA even in the presence of high amounts of GHF-1 and, similarly, an excess of RARs over TRs blocks synergistic activation by T3 and GHF-1. Since both receptors share the same heterodimerization partner (RXR) (5) and the same coactivators (5, 23, 24) it is also possible that competition by these factors could be involved in this effect.
The cooperation of the nuclear receptors and GHF-1 could involve a physical association between them. We have found that, as examined by gel retardation analysis, GHF-1 inhibits binding of receptor heterodimers to a strong hormone response element, and that the receptors inhibit GHF-1 binding to its cognate element. The simpler explanation for these observations is the formation of complexes between the receptors and GHF-1, which are no longer able to bind the corresponding DNA element. The existence of direct protein-to-protein interactions between the receptors and GHF-1 was unequivocally demonstrated by in vitro binding experiments with GST-fusion proteins. GHF-1 bound very strongly RXR and RAR, and more weakly TR. The stronger interaction of GHF-1 with RXR agrees with the finding that binding of TR/RXR heterodimers to the TRE was easily inhibited in the presence of GHF-1, whereas binding of TR monomers was not affected by the pituitary factor. In contrast to the association with nuclear coactivators, which requires ligand binding, the interaction of the receptors with GHF-1 was also observed in the absence of ligand. A constitutive association of receptors with other transcription factors such as c-Jun (25), general transcription factors such as TFIIB and TBP (21, 26–28), or viral proteins (29) has been previously described and has been implicated in both positive and negative transcriptional regulation by the receptors.
We have previously demonstrated that the activity of a GHF-1 promoter fragment, which contains an autoregulatory GHF-1 binding site but does not bind the thyroid hormone receptor, is repressed by T3 (14). This finding is compatible with sequestration of GHF-1 by the interaction with the receptor. By contrast, the mutual inhibition of binding between the receptors and GHF-1 is apparently paradoxical with the activation of the GH promoter, since these proteins have synergistic effects in its activation. However, the inhibition is obtained with isolated DNA binding elements for the receptors and the transcription factor, rather than with the natural GH promoter, which contains binding sites for both. Our results clearly demonstrate that GHF-1 does not inhibit binding of TR/RXR or RAR/RXR to this promoter.
Two models have been proposed to explain synergism that are relevant to the regulation of the GH promoter by the receptors and GHF-1. First, binding of transcription factors to DNA response elements can be cooperative. In this model binding of one of the factors facilitates the binding of the second resulting in greater occupancy of the cis-acting elements, thus promoting transcription. This type of synergism would be oberved only if the transcription factors are in limited concentration (30). In fact we have found that, when present in limiting amounts, the receptors and GHF-1 increase each other binding to a fragment of the rat GH promoter that contains binding sites for the receptors and GHF-1. It is likely that protein-protein interactions between them facilitate this cooperation. However, our results also show that, only under conditions of low DNA concentrations, the formation of a ternary complex composed of receptor heterodimers and GHF-1 with the promoter is favored. Furthermore, the presence of GHF-1 did not stabilize receptor binding to DNA, so little cooperative binding between the receptors and GHF-1 is observed, making it unlikely that this mechanism accounts for the total level of synergism observed. A second mechanism proposes that interaction of receptors and transcription factors with multiple target sites (some of them presumably basal transcription factors as well as coactivator proteins) may play a role in synergism. In this respect it has been shown that different nuclear receptors, including T3 and RA receptors, as well as GHF-1 can associate with different components of the basal transcriptional machinery (26–28, 31). The simultaneous binding of the receptors and GHF-1 to their relatively close cognate sites in the GH promoter, as well as the direct interactions between them and with other nuclear factors could facilitate promoter occupancy and are most likely involved in the functional cooperation observed.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Abbreviations: CAT, chloramphenicol acetyltransferase; DMEM, Dulbecco's modified Eagle's medium; GH, growth hormone; GST, glutathion-S-transferase; HRE, hormone response element; RA, retinoic acid; RAR, retinoic acid receptor; RSV, Rous sarcoma virus; RXR, retinoid X receptor; SDS-PAGE, sodium dodecyl sulphate-polyacrylamide gel electrophoresis; T3, 3-5-3' triiodo-L-thyronine; TR, thyroid hormone receptor; TREPAL, palindromic thyroid hormone response element.
Received for publication December 1, 1997. Accepted for publication April 1, 1998.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSREFERENCES |
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