High-Mobility Group (HMG) Protein HMG-1 and TATA-Binding Protein-Associated Factor TAFII30 Affect Estrogen Receptor-Mediated Transcriptional Activation

Carmel S. Verrier, Nady Roodi, Cindy J. Yee, L. Renee Bailey, Roy A. Jensen, Michael Bustin and Fritz F. Parl

Department of Pathology (C.S.V., N.R., C.Y., L.R.B., R.A.J., F.F.P.) Vanderbilt University Nashville, Tennessee 37232
Laboratory of Molecular Carcinogenesis (M.B.) National Cancer Institute Bethesda, Maryland 20892


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The estrogen receptor (ER) belongs to a family of ligand-inducible nuclear receptors that exert their effects by binding to cis-acting DNA elements in the regulatory region of target genes. The detailed mechanisms by which ER interacts with the estrogen response element (ERE) and affects transcription still remain to be elucidated. To study the ER-ERE interaction and transcription initiation, we employed purified recombinant ER expressed in both the baculovirus-Sf9 and his-tagged bacterial systems. The effect of high-mobility group (HMG) protein HMG-1 and purified recombinant TATA-binding protein-associated factor TAFII30 on ER-ERE binding and transcription initiation were assessed by electrophoretic mobility shift assay and in vitro transcription from an ERE-containing template (pERE2LovTATA), respectively. We find that purified, recombinant ER fails to bind to ERE in spite of high ligand-binding activity and electrophoretic and immunological properties identical to ER in MCF-7 breast cancer cells. HMG-1 interacts with ER and promotes ER-ERE binding in a concentration- and time-dependent manner. The effectiveness of HMG-1 to stimulate ER-ERE binding in the electrophoretic mobility shift assay depends on the sequence flanking the ERE consensus as well as the position of the latter in the oligonucleotide. We find that TAFII30 has no effect on ER-ERE binding either alone or in combination with ER and HMG-1. Although HMG-1 promotes ER-ERE binding, it fails to stimulate transcription initiation either in the presence or absence of hormone. In contrast, TAFII30, while not affecting ER-ERE binding, stimulates transcription initiation 20-fold in the presence of HMG-1. These results indicate that HMG-1 and TAFII30 act in sequence, the former acting to promote ER-ERE binding followed by the latter to stimulate transcription initiation.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The estrogen receptor (ER) belongs to a family of ligand-inducible nuclear receptors that exert their effects by binding to cis-acting DNA elements (hormone response elements) in the regulatory region of target genes (1, 2). The detailed mechanisms by which ER interacts with the estrogen response element (ERE) and affects transcription remains to be elucidated. The ER is commonly divided into regions A-F, with each region carrying out a specific function. The N-terminal regions A and B comprise a ligand-independent transactivation function, AF-1 (3). Region C is the cysteine-rich DNA-binding domain whereas region D harbors nuclear localization signals. The C-terminal region E contains the hormone-binding domain as well as a ligand-dependent transcription activation domain, AF-2 (4, 5). Functionally, it is believed that the estrogen-dependent activation of gene transcription occurs in a series of steps. In the first step, estrogen binds to the hormone-binding domain and induces the formation of stable ER homodimers (6, 7). In the second step, the hormone-activated ER dimer interacts with the ERE, characterized as a 13-bp palindrome with 5-bp stems separated by a 3-bp spacer and a consensus sequence of GGTCAnnnTGACC (8, 9, 10). In the final step, it is assumed, in analogy with the progesterone receptor, that the ER-ERE complex promotes the recruitment of general transcription factors and/or stabilizes their interaction with the promoter of estrogen-responsive genes such that high levels of transcription can ensue (11, 12, 13).

There is increasing evidence that the ER-ERE interaction is influenced by other proteins. For example, recombinant human ER purified from either HeLa or yeast cells fails to bind ERE (14, 15). Addition of yeast extract to purified ER restored formation of the ER-ERE complex. Mukherjee and Chambon (14) identified the yeast factor and characterized it as a 45-kDa single-stranded DNA-binding protein, termed ER DNA-binding stimulatory factor. Since then, various other proteins of sizes ranging from 30 to 160 kDa have been reported to associate with ER (16, 17, 18, 19, 20, 21, 22, 23). Some of these interacting proteins were identified by expressing the hormone-binding domain of ER fused to glutathione-S-transferase (GST-AF2). In the presence of 10-8 M 17ß-estradiol, several proteins from ZR-75 human breast cancer cells with molecular masses of approximately 160, 100, and 50 kDa were retained by GST-AF2 preloaded on glutathione-coupled beads (16). Using similar techniques, another group identified 160- and 140-kDa proteins (17). More recently, the cloned human TAFII30, which complexes with TATA-binding protein (TBP), has also been shown to interact directly with ER (18). Neither estradiol nor estrogen antagonists influenced this binding.

Although the number of ER-associated proteins is growing, their interaction and precise role in DNA binding or transcriptional activation remains to be defined. Recently, it was reported that binding of purified progesterone receptor to its response element is enhanced by HMG-1 (24, 25). HMG-1 is a 28-kDa-member of the high-mobility group (HMG) family of nonhistone chromosomal proteins that is involved in diverse aspects of eukaryotic gene expression, including determination of nucleosome structure and stability, as well as transcription and/or replication (26, 27). Higher eukaryotes contain three families of HMG proteins, the HMG-1/-2 family, the HMG-14/-17 family, and the HMG-I/-Y family, each of which contains distinct sequence motifs (28). HMG-1 is an abundant, highly conserved protein present in all vertebrate nuclei that has been shown to nonspecifically bind and bend different DNA structures as well as facilitate the binding of transcription factors to template DNA (28, 29). Two groups conclusively proved the ability of HMG-1 to induce curvature in double-stranded DNA by testing its effect on ligase-dependent cyclization of short linear DNA fragments. Covalently closed circles were formed only in the presence of HMG-1, indicating that HMG-1 is capable of introducing bends into the linear duplex (30, 31). Because the two groups used different DNA fragments, their results also indicate that the effect of HMG-1 is independent of DNA sequence.

In addition, HMG-1 and the related HMG-2 have been shown to facilitate the DNA-binding of general and specific transcription factors, such as TFIID-TFIIA, MLTF, HOX, and octamer transcription factor 2 (Oct2) (32, 33, 34, 35). Thus, HMG-1 should be considered as an "architectural element" (29, 32), which bends DNA and facilitates binding of DNA-binding proteins to their target. Based on this interaction, it has been proposed that HMG-1 may function as a general class II transcription factor by stimulating the formation of transcription initiation complexes of RNA polymerase II and III (36, 37, 38). Conversely, HMG-1 has been proposed to inhibit formation of the preinitiation complex by interacting with TBP, leading to an inhibition of RNA polymerase II transcription (39).

In light of these findings, we decided to determine whether HMG-1 and TAFII30 could facilitate ER to ERE binding and whether that promotion of binding is associated with any changes in the level of estrogen-dependent transcription. We find that recombinant, purified human ER binds to its cognate response element only in the presence of HMG-1. HMG-1 and TAFII30 act in sequence, the former acting to promote ER-ERE binding followed by the latter to stimulate transcription initiation.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
ER-ERE Binding Requires Additional Factors
To assess binding requirements of ER to its specific DNA element, we overexpressed ER in baculovirus. Western blot analysis of the baculovirus-generated ER demonstrated that the overexpressed protein had electrophoretic and immunological properties identical to those of ER expressed in the ER-positive breast cancer cell line, MCF-7 (Fig. 1AGo). Moreover, the ER overexpression increased with time, reaching a peak 48 h post infection. When tested for ERE binding by electrophoretic mobility shift assay (EMSA), the overexpressed ER failed to show binding with ERE (Fig. 1BGo), even though hormone-binding assays revealed high estradiol binding (Fig. 1CGo). ER overexpressed in bacteria (Fig. 1Go, D and E) similarly failed to bind with ERE (results not shown). This failure of recombinant ER to bind ERE, therefore, suggested that additional proteins may be necessary to promote formation of the complex.



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Figure 1. Recombinant ER Does Not Bind ERE

A, ER in MCF-7 (lane 1) and Sf9-hER (lanes 2–4) cells was analyzed by Western blot using anti-ER monoclonal antibody, D547; B, EMSA; and C, hormone-binding assay with results expressed in femtomoles per mg. Sf9-hER cells, analyzed at 24, 48, and 72 h post infection, contain increasing amounts of immunochemically detectable ER with some degradation products at the later time points. An ER-ERE complex formed with MCF-7 whole-cell extract is indicated by the arrow. While the hormone binding increases in parallel with the overexpressed ER, no corresponding ER-ERE complexes are observed with Sf9-hER cells at all three time points. Identical results were obtained with recombinant ER expressed in bacteria as His-tagged protein. D, Purified His-tagged ER (lane 2) was run on an SDS-polyacrylamide gel and stained with Coomassie blue (lane 1, mol wt markers) as well as analyzed by Western blot (E).

 
HMG-1 Promotes ER-ERE Binding and Interacts with ER
To determine whether HMG-1 can promote ER-ERE binding, EMSAs were carried out. Under the conditions of our assays, when purified HMG-1 was incubated alone with ERE, no detectable complex was formed. However, in the presence of HMG-1, a strong ER-ERE complex was formed, which comigrated with the MCF-7 control (Fig. 2AGo). In competition experiments, addition of increasing concentrations of cold ERE led to a progressive decrease in the intensity of the ER-ERE complex (Fig. 2BGo). To determine whether the complex contained ER, we carried out an antibody supershift analysis. Incubation with the monoclonal anti-ER antibody, H222, further retarded band migration, indicating the presence of ER in the complex (Fig. 2CGo). On the other hand, using a rabbit antiserum to HMG-1, we were not able to demonstrate the presence of HMG-1 in the ER-ERE complex. EMSAs carried out with histone H1 failed to show a promoting effect on ER-ERE binding, ruling out the possibility of a nonspecific effect of nuclear proteins on ER-ERE complex formation.



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Figure 2. EMSA of ER-ERE Interactions in the Presence of HMG-1

A, ER complexes were incubated for 30 min at 4 C with 32P-labeled ERE in the presence or absence of purified HMG-1 protein. Human ER-ERE complexes formed (denoted by the arrow) were resolved on a 4.5% polyacrylamide gel. MCF-7 whole-cell extracts were used as a positive control. HMG-1 appears to promote ER-ERE binding. This binding can be competed off with cold ERE. B, Competition experiment demonstrating that the ER-ERE complex promoted by HMG-1 can be suppressed by addition of cold ERE. C, The human ER monoclonal antibody, H222, was first incubated with ER, and the complex was further incubated for 30 min at 4 C with [32P]ERE in the presence of HMG-1. The supershifted band demonstrates the presence of ER in the complex formed.

 
To test whether HMG-1 can interact with ER, we performed coimmunoprecipitation experiments. When recombinant ER was mixed with purified HMG-1, it could be coimmunoprecipitated with affinity-purified antibody specific for HMG-1 coupled to protein A-Sepharose (Fig. 3Go). This coprecipitation was dependent on the presence of both the HMG-1 protein and the antibody against HMG-1, confirming the specificity of the precipitation reaction. When recombinant TAFII30 or BSA as a control were substituted for ER, no coimmunoprecipitation occurred. These results suggest that the HMG1-ER interaction is a property of the native proteins.



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Figure 3. Coimmunoprecipitation Assays

Using an affinity-purified antibody to HMG-1, the latter was precipitated in the presence of either purified ER or purified TAFII30. Unbound proteins were collected in the supernatant. Bound proteins were washed three times, then eluted off the Protein A-Sepharose by boiling in SDS-sample buffer. Proteins were transferred onto nitrocellulose membrane, which was probed with: A, the ER antibody, H222; or B, the TAFII30 antibody, 2F4; or C, the affinity-purified antibody to HMG-1. Whereas ER can be coprecipitated with HMG-1, TAFII30 cannot.

 
HMG-1-Induced ER-ERE Binding Is Dependent Upon Oligonucleotide Length and Position of the ERE within the Oligonucleotide
Having identified HMG-1 as a factor capable of promoting ER-ERE binding, we next sought to elucidate the mechanism by which HMG-1 promotes formation of the complex. To accomplish that task, we took into consideration that HMG-1 is a DNA-bending protein. We reasoned that the time at which HMG-1 is added to the reaction mixture might determine whether or not an ER-ERE complex is formed. Thus, HMG-1 was incubated with ERE before or after addition of the overexpressed receptor. As can be seen in Fig. 4Go, to promote ER-ERE binding, HMG-1 must be incubated with the DNA probe before addition of ER. When the order is reversed, HMG-1 no longer facilitates ER-ERE complex formation. In addition, the promotion of ER-ERE binding brought about by HMG-1 is concentration-dependent (Fig. 4Go). Moreover, if DNA bending by HMG-1 is a critical factor for ER-ERE complex formation, the length of the oligonucleotide as well as the position of the palindromic ERE within the synthetic oligo deserve consideration. Two 25-mers containing the ERE consensus sequence, either at the end or in the middle of the oligo, were employed instead of the usual 35-mer. The binding of ER to these two 25-mers differed significantly when tested in the presence of HMG-1. Figure 5AGo, lane 1, shows binding obtained with the usual 35-mer used in previous EMSAs. With the 25-mers, when the ERE consensus is placed at the end of the oligo, a significant reduction in ER-ERE interaction was observed. In contrast, when the ERE is centrally placed within the oligo, ER-ERE binding is restored to levels higher than that observed with the asymmetric 25-mer but still lower than that obtained with the 35-mer. The effect of HMG-1 was also dependent on the sequence flanking the ERE consensus. When the ERE consensus was flanked symmetrically by GCs to create another symmetric 25-mer, ER-ERE binding mediated by HMG-1 was reduced as compared with the symmetric 25-mer flanked by mostly ATs. Yet, the symmetry of this GC-rich 25-mer ERE allowed it to bind ER at a much higher level than did the asymmetric 25-mer (Fig. 5AGo, compare lane 2 vs. 4). Competition experiments showed that increasing amounts of cold 25-mer suppressed ER-ERE complex formation in a dose-dependent manner, whereas equimolar concentrations of cold 15-mer containing the ERE without flanking sequences reduced the ER-ERE formation much less (Fig. 5BGo). Thus, the effectiveness of HMG-1 to stimulate ER-ERE binding in the EMSA depends on oligonucleotide length and position of the ERE within the nucleotide.



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Figure 4. EMSA of ER-ERE in the Presence of HMG-1: Promotion of ER to ERE Binding by HMG-1 is Dependent Upon Time of Addition and Concentration of HMG-1

Lane 1, MCF-7 whole-cell extract was incubated with [32P]ERE for 30 min at 4 C. ER-ERE complex (arrow) was resolved on a 4.5% nondenaturing polyacrylamide gel. Lanes 2–10 represent the incubation of [32P]ERE with increasing concentrations (10, 50, 200 ng, respectively) of HMG-1 in the absence (lanes 2–4) or presence (lanes 5–10) of ER extracted from Sf9-hER. To promote ER-ERE binding, HMG-1 must be incubated with the probe before the addition of ER (lanes 8–10). When ER is incubated with HMG-1 before the addition of labeled probe, ER-ERE binding is no longer facilitated by HMG-1, regardless of concentration (compare lanes 5–7 vs. lanes 8–10).

 


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Figure 5. EMSA of ER-ERE in the Presence of HMG-1: Length of Oligonucleotide, Position of ERE, and Bases Flanking the ERE Affect HMG-1-Mediated ER-ERE Binding

ER complexes were incubated for 30 min at 4 C with equimolar amounts of each of the respective labeled probes indicated above in the presence of HMG-1. HMG-1-mediated ER-ERE complexes were resolved on 4.5% polyacrylamide gel. A, When a 35-mer ERE is used, strong ER-ERE complexes are formed when HMG-1 is present (lane 1). Reducing the length of the ERE to a 25-mer causes a significant reduction in ER-ERE complex formation (lanes 2–4). This reduction in complex formation is further reduced when the ERE is either positioned at the end of the oligo (lane 2) or flanked by GCs (lane 4). B, When competition experiments are carried out with 50- or 100-fold excess of either the symmetrical, AT-rich 25- or the 15-mer ERE, it can be seen that the 25-mer is a much better competitor than the 15-mer ERE.

 
HMG-1 and TAFII30 Are Important Components in ER-Mediated Transcription Initiation
To determine whether the ER-ERE binding promoted by HMG-1 has any functional significance, we carried out in vitro transcription assays using purified ER, HMG-1, and TAFII30. TAFII30 was bacterially expressed and purified by means of a nickel-nitrilotriacetic acid (Ni-NTA) column. In contrast to HMG-1, TAFII30 appears to have no effect on ER-ERE interaction (Fig. 6Go). By itself, TAFII30 neither promotes ER-ERE binding nor does it further induce the HMG-1-mediated ER-ERE binding. In light of these findings, we assessed the functional role, if any, that HMG-1 and TAFII30 might have on transcription initiation. The results of the in vitro transcription are presented in Fig. 7Go. When the plasmid pLovTATA, which is devoid of any ERE, is used, only basal level or no transcription can be obtained with ER alone or in the presence of HMG-1 or TAFII30 or both (lanes 1–3). Similarly, when the ERE-containing plasmid, pERE2LovTATA, was used, transcription remained basal in the presence of ER alone or in combination with HMG-1 or TAFII30 (lanes 4, 5, and 7). However, transcription was enhanced 20-fold by addition of TAFII30 to ER and HMG-1 (lane 6). Addition of estradiol further increased transcription (lanes 9–11). The hormone induction was approximately 5-fold for ER alone, ER plus HMG-1, and ER plus HMG-1 and TAFII30.



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Figure 6. EMSA of ER-ERE Binding in the Presence of TAFII30

TAFII30 was expressed in E. coli as a histidine-tagged protein for one-step purification onto a Ni-NTA column. By EMSA, ER-ERE complexes formed were resolved on a 4.5% nondenaturing polyacrylamide gel. Lane 1 is the ER-ERE complex formed using MCF-7 cell extracts, a positive control. TAFII30 alone neither complexes with ERE (lane 2) nor promotes ER-ERE binding (lane 3). The HMG-1-mediated ER-ERE complex formation is not further induced by the presence of TAFII30 (compare lane 4 vs.5).

 


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Figure 7. In Vitro Transcription Assay

The control template, pLovTATA, and test template, pERE2LovTATA, were incubated with either ER, ER and HMG-1, ER and TAFII30, or ER and HMG-1 and TAFII30. The transcription reactions were initiated by addition of 5 U of Drosophila embryo nuclear extract and incubated at 30 C for 1 h. Transcripts were recovered by ethanol precipitation and analyzed by electrophoresis on a 6% acrylamide, 8 M urea sequencing gel. As an internal control, the plasmid pMLcas190, which contains the AdML promoter linked to a G-free cassette of 180 bp, was used. With the control template, pLovTATA, only basal or undetectable levels of transcription are obtained (lanes 1–3). With the test template, pERE2LovTATA, HMG-1 has no effect on transcription in the absence or presence of hormone (lanes 4–5 vs. 8–9). However, when TAFII30 is added to ER and HMG-1, a 20-fold increase in transcription is seen (lane 6), and this increase is further enhanced by addition of hormone (lane 11). Interestingly, while TAFII30 appears to be needed in ER-dependent transcription initiation, it failed to initiate ER-dependent transcription in the absence of HMG-1 (lane 7). Ethanol as a vehicle has no significant effect on transcription (lane 5 vs. 10).

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The experiments show that overexpressed ER, although it displays high hormone binding and is electrophoretically and immunologically identical to MCF-7 ER, does not by itself bind ERE. This observation suggested that other factors may be necessary to promote ER-ERE binding. We find that the HMG chromatin protein, HMG-1, enhances binding of ER to ERE in a dose-dependent fashion in mobility shift assays. In addition, coimmunoprecipitation experiments show direct interaction of HMG-1 with ER in the absence of DNA. To study in greater detail the mechanism by which HMG-1 promotes ER-ERE binding, we felt it was necessary to obtain purer ER fractions. Although the baculovirus expression system allows high level expression of recombinant ER, we as well as others (40) encountered several problems when an attempt was made to purify the receptor. First, high levels of degradation products are obtained post infection from the baculovirus system; second, additional breakdown products are generated when conventional column chromatography techniques are carried out. Therefore, we opted for expression in the bacterial system that not only offers better control over expression via alterations in growth conditions but also allows ER to be obtained by one-step purification onto the Ni-NTA column (see Materials and Methods). Even though expression of ER in the bacterial system yields much lower levels of full-length ER as had been reported by others (41), we have successfully expressed the receptor in bacteria with less degradation products being generated. EMSAs in which the bacterially expressed ER was used further support the findings obtained with baculovirus ER.

HMG-1 has previously been shown to enhance the sequence-specific DNA binding of the progesterone receptor (24, 25). However, the binding of the retinoic acid receptor {alpha} to its cognate responsive element is not enhanced by HMG-1 (34), indicating that the effect of HMG-1 on DNA binding is not generalized for all nuclear receptors. HMG-1 also enhances the sequence-specific DNA binding of HOX proteins, developmentally active transcription factors (34). Addition of HMG-1 to the HOX-DNA binding reaction did not result in the formation of slower migrating complexes in EMSA, indicating that a DNA-HMG1-HOX ternary complex was not formed or dissociated very rapidly, similar to the lack of a ternary ERE-HMG1-ER complex in the present study. In any case, coimmunoprecipitation experiments demonstrated that HMG-1 formed protein-protein contacts with HOX proteins in the absence of DNA (34), again similar to the HMG1-ER interaction in this study. HMG-1 and the closely related HMG-2 were also shown to increase the sequence-specific DNA binding of Oct proteins (35). Interestingly, HMG-2 protein, although not present in the Oct-DNA complex detected by EMSA, forms protein-protein contacts with Oct, as demonstrated by coimmunoprecipitation. Thus, HMG1/2 is capable of enhancing the sequence-specific DNA binding of several unrelated transcriptional activators and of establishing protein-protein contacts with these same activators in the absence of DNA.

It has been suggested that HMG1-like proteins may exert a ‘DNA chaperone’ action by binding transiently to DNA, bending it into a thermodynamically unfavorable conformation and then exchanging with the protein that must eventually form a stable complex with its DNA target (42). This scenario is indeed attractive also in the context of ER-mediated transcription. HMG-1 enhanced binding of ER to ERE in a time-dependent fashion, i.e. the promotion of binding was apparent only when HMG-1 was incubated with the probe before the addition of ER. However, the ‘DNA chaperone’ model does not necessarily predict any form of direct protein-protein interaction as demonstrated for HOX, Oct, and ER. To account for the protein-protein interaction, Zappavigna et al. (34) favor an alternative interpretation, although not mutually exclusive with the one described above. They propose that the physical contact between HMG-1 and its protein partner directs both to adjacent or overlapping DNA segments, generating a complex that is endowed with both geometric and sequence specificity.

Using purified ER, we sought to determine how the length as well as the position of the ERE within the oligonucleotide might affect the HMG-1-mediated ER-ERE binding. We found that when the ERE was positioned at the very end of the oligonucleotide, a significant reduction in binding was noted. Flanking sequences around the ERE were also important factors contributing to the ER-ERE complex formation induced by HMG-1. ERE flanked by ATs bound ER at a higher yield than did the ERE flanked by GCs. While HMG-1 has no consensus DNA-binding site, it shows a preference for binding AT-rich sequences (38). In terms of length, we have also noted a significant reduction in binding when a 25-mer ERE was used in place of the usual 35-mer. We believe that this reduction in binding is a result of decreasing the DNA site HMG-1 needs for efficient interaction with sequences flanking the ERE. The footprint size of HMG-1 was determined to be 14 [plusm] 3 bp (28). Therefore, it is conceivable that as the number of bases flanking the ERE is reduced, HMG-1-mediated ER-ERE binding becomes affected as well. In the case of PR, the effect of HMG-1 on DNA binding was assessed in relation to the position of the progesterone response element within a 142-bp oligonucleotide (25). In EMSA, Prendergast et al. (25) found a subtle difference in band migration that was dependent upon the position of the progesterone response element and reflected the degree of DNA flexure induced by HMG-1. In this study, no difference in migration was noted between the 35-mer and 25-mer, because both are much smaller that the 142-bp PRE.

Although HMG-1 plays a clearly defined role in DNA binding, its function in transcriptional activation remains uncertain (36, 37, 38, 39). For this reason, we decided to determine its interaction with a bona fide ER-specific transcriptional activator, TAFII30 (18). By EMSA, we find that TAFII30 alone does not promote ER-ERE binding and does not have an effect on HMG-1-mediated ER-ERE binding. However, for transcription to be initiated from an ERE-containing template, TAFII30 must be present. Thus, although HMG-1 promotes ER-ERE binding, it fails to stimulate transcription initiation either in the presence or absence of hormone. This is consistent with the notion that although DNA bending may be involved in transcriptional regulatory mechanisms, it is not sufficient, by itself, for transcription (43, 44). Therefore, HMG-1 might fall into the growing class of transcription factors that act by bending DNA to facilitate assembly of higher order nucleoprotein complexes (45, 46, 47, 48, 49). In this context, HMG-1 may be providing the structural framework necessary for other transcription factors to interact and function. In fact, as seen by in vitro transcription assay, TAFII30, while not affecting ER-ERE binding, stimulates transcription initiation when in the presence of HMG-1. We observed a 20-fold induction of transcription initiation, even in the absence of hormone, when ER, TAFII30, and HMG-1 are incubated with the test template, pERE2LovTATA. In the presence of hormone, an additional effect on transcription initiation was apparent. We believe that the induction of transcription initiation in the absence of hormone is due to the lack of repressors in our in vitro system. A repressor protein, SSN6, that specifically interacts with the N-terminal AF1 of the ER was identified in yeast (50). Estradiol promotes dissociation of SSN6 allowing interaction of AF1 and AF2 with the transcription apparatus. However, a mammalian counterpart of yeast SSN6 has not been identified. Other repressors have been identified for the thyroid hormone and retinoic acid receptors as SMRT (silencing mediator for retinoid and thyroid receptors) and N-Cor (nuclear receptor corepressor) that have the ability to silence thyroid hormone receptor- and retinoic acid receptor-dependent gene expression (51, 52, 53). Upon addition of ligand, SMRT and N-Cor dissociate from the receptor, thereby permitting ligand-induced transcriptional activation.

In summary, HMG-1 enhances the binding of ER to ERE. The ER thus joins the progesterone receptor and the HOX and Oct proteins as a group of transcription factors whose sequence-specific DNA binding is promoted by HMG-1. These findings are in agreement with the proposed role of HMG-1 as an "architectural element" (29, 32) that bends DNA and facilitates the stable binding of DNA binding proteins to their target. Once formed, the stable ER-ERE complex enhances the recruitment of specific transcription factors, such as TAFII30, that can assume the role of a bridging protein between ER, TBP, and other components of the transcriptional machinery that are essential for ER-induced transcription initiation. In this process, HMG-1 and TAFII30 appear to act in sequence, the former acting to promote ER-ERE binding followed by the latter to stimulate transcription initiation. Extensive work will be required to define the role of additional proteins in this process and to gain a complete understanding of ER-mediated gene transcription.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Baculovirus Expression of ER
The insect cell line Spodoptera frugiperda (Sf9) was obtained from American Type Culture Collection (Rockville, MD) and grown at 27 C in EXCELL 400 (JR Scientific; Lenexa, KS) medium. Autographa californica nuclear polyhedrosis virus (AcNPV) was purchased from Invitrogen (San Diego, CA). Sf9 cells were infected with a multiplicity of infection of >=10 plaque-forming units/cell for protein expression studies and 0.1–1.0 plaque forming units/cell for virus stock production. The baculovirus transfer vector pVL1392 was purchased from Invitrogen. The plasmid pSG5 HEGO containing the human ER cDNA was a generous gift from Dr. Pierre Chambon (Illkirch, France). Insertion of the ER cDNA into pVL1392 was accomplished by digesting pSG5 HEGO with EcoRI. This fragment was then cloned into pVL1392, which had been similarly digested and purified by isolation on NA45 membranes (Schleicher & Schuell, Keene, NH). This fragment was oriented by digestion with SmaI and designated pVL1392-hER. Recombinant baculovirus was produced by cotransfecting 2 x 106 Sf9 cells with AcNPV DNA (1 µg) and pVL1392-hER (2 µg) using the calcium phosphate transfection. The resulting culture supernatants were harvested after 4 days and screened for homologous recombination by visual inspection of plaques, which were confirmed by dot-blot hybridization using the respective 32P-labeled, nick-translated cDNA probes. Purified recombinant baculovirus was obtained after three rounds of plaque purification and designated Ac-hER. Sf9 cells (9 x 106) infected with Ac-hER were harvested at 24, 48, and 72 h post infection by centrifugation, and lysed in 500 µl of buffer A (20 mM HEPES, pH 7.5, 0.1 mM EDTA, 40 mM KCl, 20% glycerol) containing 5 mM dithiothreitol (DTT), 1 mM phenylmethylsulfonyl fluoride, 2.5 µg/ml aprotinin, 2.5 µg/ml pepstatin, and 2.5 µg/ml leupeptin. The lysates were clarified by centrifugation at 14,000 x g for 10 min and stored frozen at -70 C.

Purification of ER from Baculovirus Extracts
Approximately 35 x 106 cells were homogenized in 3.2 ml of ice-cold extraction buffer (0.4 M KCl, 10 mM Tris, pH 7.9, 1 mM EDTA, 5 mM DTT, 10% glycerol) containing protease inhibitors as listed above. The resulting homogenate was centrifuged for 3 min at 4 C in an Eppendorf microfuge at 14,000 rpm. The salt concentration of the supernatant (3 ml) was reduced to 40 mM KCl with extraction buffer containing no salt. The sample was loaded onto a Heparin-Sepharose column that had been preequilibrated with extraction buffer containing 40 mM KCl. The column was washed with the same buffer used for equilibration and step-eluted with extraction buffer containing 200, 400, and 800 mM KCl, respectively. A 10-µl aliquot of the 400 and 800 mM fractions containing ER was used for protein determination by standard methods to adjust the protein concentration to 1.0 mg/ml using incubation buffer (10 mM Tris, pH 7.9, 1 mM EDTA, 10 mM monothioglycerol, 10% glycerol) containing protease inhibitors before carrying out EMSAs.

Bacterial Expression and Purification of His-tagged ER and TAFII30
The ER cDNA was amplified using the sense primer, 5'-CGGGATCCATGACCATGACCCTCCACACCAAAGC-3' and the antisense primer, 5'-GGGGTACCCGTGTGGGAGCCAGGGAGCT-3'. TAFII30 cDNA was amplified from a cDNA stock of the normal mammary cell line, HBL-100. Primers for amplification of TAFII30 were the sense primer, 5'-CGGGATCCAGCTGCAGCGGCT CC-3' and the antisense primer, 5'-GGGGTACCTACATTTAGGTTGGGTGGCTCAG GTG-3'. Both sets of primers were designed to contain BamHI and KpnI sites, respectively, at the 5'-ends. The amplification reaction was carried out in 100 µl volume containing 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl2, 200 mM each of the four deoxyribonucleotides, native Pfu DNA Polymerase (2.5 U; Stratagene, La Jolla, CA), and each oligonucleotide at either 50 ng/ml or 150 ng/ml. Amplification conditions for ER cDNA consisted of a denaturing step at 97 C, annealing at 67 C, and extension at 72 C for a total of 20 cycles. For TAFII30, conditions consisted of a denaturing step at 95 C, annealing at 64 C, and extension at 72 C for a total of 30 cycles. The amplified cDNAs were purified using the QIAEX gel purification kit (QIAGEN, Chatsworth, CA), digested with BamHI and KpnI, and repurified by the same method. Ligation of each cDNA into the similarly digested vector pQE-30 (QIAGEN), which encodes an N-terminal hexahistidine tag, followed. Each ligated vector/insert was used to transform M15 Escherichia coli strain (QIAGEN) as described by the manufacturer. A picked colony harboring the correct size insert (as judged by restriction digest and DNA sequencing) was used to express the ER or TAFII30 protein. Expression conditions consisted of growing the cells in Luria-Broth (supplemented with kanamycin at 25 mg/ml and Ampicillin at 100 mg/ml) at 30 C until an A600 reading of 0.6 was reached, induction with 0.5 mM isopropyl-ß-D-thiogalactopyranoside, and collection of cells 1.5 h after induction. The cells were lysed by sonication and freeze/thaw cycles. Tagged ER or TAFII30 was purified using the Ni-NTA resin as specified by the manufacturer (QIAGEN).

HMG-1
Human HMG-1 was purified as previously reported (54, 55).

Histone H1
Histone H1 was purified by 5% perchloric acid extraction followed by ion exchange chromatography on Amberlite IRC-50 columns (ICN Pharmaceuticals, Costa Mesa, CA) (56).

ER Immunoblotting
Whole-cell lysates from either Sf9 or E. coli were separated by SDS-PAGE and transferred to Immobilon-P membranes (Millipore; Bedford, MA) in 25 mM Tris, 192 mM glycine, 0.025% SDS, and 15% methanol for 2 h at 200 mA. Nonspecific binding was blocked with nonfat dry milk, and the blots were incubated with rat anti-human ER monoclonal antibodies D547 or H222 (Abbott Laboratories, North Chicago, IL). The filters were washed four times with Tris-buffered saline/0.05% Tween-20 and bound antibody was detected with enhanced chemiluminescence (Amersham; Arlington Heights, IL).

Hormone-Binding Assay
The ER content of whole-cell extracts from the baculovirus or bacterial system was determined using dextran-coated charcoal to absorb free hormone in a six-point binding assay of 17ß-[3H]estradiol. Scatchard analysis was performed to calculate binding capacity and affinity.

Oligonucleotides
All double-stranded oligonucleotides used in EMSAs were purchased from Integrated DNA Technologies (Coralville, IA). EREs used were: 1) 35-mer ERE of the Xenopus vitellogenin A2 gene 5'-GTCCAAAGTCAGGTCACAGTGACCTGATCAAAGTT-3'; 2) two 25-mers from the vitellogenin A2 gene differing in the position of the ERE, 5'-AGGTCACAGTGACCTGATCAAAGTT-3' and 5'-AAGTCAGGTCACAGTGACCTGA TCA-3'; 3) a consensus ERE flanked by GCs, 5'-GGCCCCGGTCACAGTGACCGG CCCC-3'. The oligos were dissolved in TE buffer (10 mM Tris, 1 mM EDTA, pH 8.0), and equimolar amounts of each strand were annealed by heating to 95 C and cooling to room temperature over a period of 1.5 h. Double-stranded oligos were end-labeled using [{gamma}-32P]ATP and T4 polynucleotide kinase. Free isotope was removed by passing the labeled, double-stranded oligos through Chroma spin-10 columns (Clontech; Palo Alto, CA).

EMSA
The assay was performed essentially as previously described (57). HMG-1 (1 ng) was first incubated with 0.3–0.5 ng of the 32P-labeled double-stranded ERE oligomer for 15 min at 4 C. Aliquots of partially purified ER (10 µg/ml) and 1.0 µg poly(deoxyinosinic)·poly(deoxycytidylic)acid were then added to the reaction mixture for a final volume of 20 µl. After a 15-min incubation at 4 C, the protein-ERE complexes were separated by electrophoresis through 4.5% acrylamide (38:2, acrylamide:bis) gels using 1x TBE buffer (10 mM Tris, 10 mM boric acid, 0.02 mM EDTA, pH 8.0). Gels were vacuum-dried and autoradiographed. The ER-containing human breast cancer cell line MCF-7 was used as a positive control for ERE binding. For antibody shift experiments, ER was preincubated with the monoclonal antibody H222 before the assay.

Coimmunoprecipitation Assays
For coimmunoprecipitation experiments, 25 µl preswollen protein A insolubilized on Sepharose CL-4B (Sigma, St. Louis, MO) were washed twice with 500 µl binding buffer (100 mM NaCl, 20 mM Tris, pH 7.0, 10% glycerol, 1% Triton-X). Between washes, the protein A-Sepharose was recovered by centrifugation for 2 min at 3500 rpm. To the washed beads, affinity-purified anti-HMG-1 (1 µg), purified HMG-1 (100 ng), and 100 ng purified bacterial ER or TAFII30 were added. The reaction volume was brought up to 400 µl using binding buffer containing DTT and the protease inhibitors phenylmethylsulfonylfluoride, aprotinin, and leupeptin. The reactions were incubated overnight at 4 C on a rotating wheel. Immunoprecipitates were collected by centrifugation, washed three times with 500 µl binding buffer, and recovered by boiling the precipitate for 5 min in SDS-sample buffer. The supernatant and pooled washes were each concentrated using Centricon-10 concentrators (Amicon, Beverly, MA). Supernatant, wash, and precipitate fractions were run on an SDS-PAGE gel. Proteins were transferred onto nitrocellulose membranes, and Western blot analysis was carried out with the ER-antibody, H222 (Abbott Laboratories) or TAFII30 antibody, 2F4 (kindly provided by Dr. Pierre Chambon). The same membranes were stripped in 100 mM 2-mercaptoethanol, 2% SDS, 62.5 mM Tris/HCl, pH 7.0, at 50 C and reprobed with the affinity-purified anti-HMG-1.

In Vitro Transcription Assays
Assays were performed using the test template, pERE2LovTATA, and the control template, pLovTATA, kindly provided by Drs. Ming-Jer Tsai and Bert O’Malley (Baylor University, Houston, TX). Both templates are ultimately derived from pML(C2AT)19, a plasmid containing a 377-bp ‘G-free cassette’ linked to the TATA box region of the adenovirus-2 major late promoter (58). Accurate initiation 30 bp downstream from the TATA box is expected to generate a 360-nucleotide transcript devoid of G residues. As an internal control, the plasmid pMLcas190, which contains the AdML promoter linked to a G-free cassette of 180 bp (58), was used. Typical reactions contained the following components in a 30 µl volume: 1) 7.5 mM HEPES, pH 7.6, 60 mM potassium glutamate, 3.75 mM MgCl2, 0.03 mM EDTA, 1.5 mM DTT, 3% glycerol, and 0.5 mM each ATP, CTP, GTP, 20 µM UTP, and 15 µCi of [{alpha}-32P]UTP; 2) 500–700 ng ERE-test or control template and internal control template; 3) ER-containing extract at 100 ng/ml and, when indicated, ER was preincubated with 10-8 M estradiol dissolved in ethanol; 4) 1 ng HMG-1; 5) 10 ng TAFII30; and 6) 10 U of ribonuclease T1. Reactions were initiated by adding 5 U of Drosophila embryo nuclear extract (Promega; Madison, WI). After a 60-min incubation at 30 C, the transcription reactions were terminated by treatment with 170 µl of stop solution [20 mM Tris-HCl, pH 7.5, 10 mM EDTA, 0.5% SDS] containing 200 µg/ml yeast tRNA, and 400 µg/ml proteinase K. After addition of 200 µl of 7 M urea (in 10 mM Tris-HCl, 1 mM EDTA, pH 8.0), transcripts were recovered by ethanol precipitation and analyzed by electrophoresis on 6% acrylamide (38:2, acrylamide-bis), 8 M urea sequencing gels.


    ACKNOWLEDGMENTS
 
We thank P. Chambon for HEGO ER cDNA and anti-TAFII30 antibody, M. J. Tsai and B. W. O’Malley for pLovTATA and PERE2LovTATA, and Abbott Laboratories for anti-ER antibodies. We also thank Martha Bass for excellent technical help.


    FOOTNOTES
 
Address requests for reprints to: Fritz F. Parl, M.D., Ph.D., 4918 The Vanderbilt Clinic, 22nd Ave South at Pierce, Nashville, Tennessee 37232.

This research was supported by Public Health Service Grant HD-07043 (to C.S.V.) and US Army Breast Cancer Training Grant DAMD-17-94-J4024 (to C.J.Y. and L.R.B.).

Received for publication August 15, 1996. Accepted for publication April 17, 1997.


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