Mechanistic Aspects of Estrogen Receptor Activation Probed with Constitutively Active Estrogen Receptors: Correlations with DNA and Coregulator Interactions and Receptor Conformational Changes

Gwendal Lazennec, Tracy R. Ediger, Larry N. Petz, Ann M. Nardulli and Benita S. Katzenellenbogen

Department of Molecular and Integrative Physiology (G.L., L.N.P., A.M.N., B.S.K.) and Department of Cell and Structural Biology (T.R.E., B.S.K.) University of Illinois Urbana, Illinois 61801


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The estrogen receptor (ER) belongs to a large family of nuclear receptors, many of whose members function as ligand-dependent transcriptional activators. The mechanism by which the receptor is converted from an inactive into an activated state is not yet completely understood. To investigate the kind of changes in receptor conformation and interactions that are involved in this activation, we have used the wild type ER and a set of constitutively active ER point mutants that show from 20% to nearly 100% activity in the absence of estrogen. These mutants are of particular interest as they could mimic, in the absence of ligand, the activated state of the wild type receptor. We have analyzed several transcriptional steps that could be involved in the activation: the ability of these receptors 1) to interact with several coactivators (steroid receptor coactivator-1, SRC-1; transcription intermediary factor-1, TIF-1; and estrogen receptor-associated protein 140, ERAP 140) and with members of the preinitiation complex [TATA box-binding protein (TBP), transcription factor IIB (TFIIB)]; 2) to exhibit conformational changes revealed by proteolytic digest patterns similar to those observed for the wild type hormone-occupied ER; and 3) to bend estrogen response element-containing DNA, which is thought to be one of the important phenomena triggering transcriptional activation. Our results demonstrate that the interaction of these mutant receptors with coactivators is likely to be one of the features of the activated step, as the mutant receptors interacted with some coactivators in a ligand-independent manner in proportion to their extent of constitutive activity. However, the different degrees of ligand-independent interaction of the mutant ERs with the three coactivators suggest that SRC-1, TIF-1, and ERAP 140 may play different roles in receptor activity. Limited proteolytic digest experiments reveal that the activated state of the receptor corresponds to a particular conformation of the receptor, which is fully observed with the mutant ER showing the highest activity in the absence of estrogen. Finally, it appears that in inactive or active states, the receptor exhibits distinctly different DNA-bending abilities. Addition of estradiol is able to modify the bending ability of only the wild type receptor, whereas estradiol has no influence on the constitutive receptors, which exhibited the same bending ability as that observed for the ligand-occupied wild type receptor. These data document that the ER undergoes major changes in its conformation and also in its functional properties when it is turned from an inactive into an active state and that mutational changes in the ER protein that result in constitutive, hormone-independent activation mimic many of the changes in ER properties that are normally under hormone regulation.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The estrogen receptor (ER) is a ligand-dependent transactivator that belongs to a large superfamily of nuclear receptors. Some members are active only in the presence of ligand, as is the case for the ER, but a large number have no identified ligand at the present time and could be, in some cases, ligand-independent factors (for review, see Ref. 1). All these receptors share a common structure of five domains named A/B, C, D, E and F (2), and some key functions have been assigned to each domain. The N-terminal A/B domain contains the ligand-independent transcription activation function 1 (AF-1) (3, 4). The C domain has a characteristic helix-loop-helix structure stabilized by two zinc atoms and is responsible for the binding to estrogen response elements (EREs) (5, 6). The D domain appears to be a hinge region that can modulate the DNA-binding ability of the receptor (7). The E and F domains are involved in the ligand-binding function and exhibit also a strong ligand-dependent activation function (AF-2) (5, 8).

Numerous studies have focused on different aspects of the process by which the receptor is transformed from an inactive state in the absence of ligand to an activated state upon ligand exposure. However, this process is still not fully understood. In its inactive form, the receptor is associated with a number of other proteins (including at least hsp90, hsp70, and p23), forming a multiprotein complex with a sedimentation constant of 8–9 S (9, 10, 11). In the unliganded state, receptors for thyroid hormone (TR) or for retinoids [retinoic acid receptor (RAR) and retinoid X receptor (RXR)] can have an inhibitory effect on transcription and are associated with corepressors (12, 13, 14, 15, 16, 17). Upon hormone exposure, most of the associated proteins are released, and the receptor appears as a 4–5 S sedimenting complex (18, 19). In this state, the receptor is able to dimerize and to associate with coactivators (20, 21, 22, 23, 24, 25, 26, 27). The receptor is also known to undergo changes in its phosphorylation state (28, 29) and conformation (30, 31).

The aim of this study is to understand what kind of changes in function and conformation the receptor undergoes when it is converted to the active state. To address these questions, we have used the wild type ER and a set of three constitutive ER mutants that correspond to amino acid substitutions at residue 380 or residue 537 (32, 33). These mutants show constitutive activity in the absence of estradiol (E2) ranging from 20 to nearly 100% of the activity of wild type receptor in the presence of E2. By comparing the properties of wild type and these mutant receptors in the absence and in the presence of estrogens or antiestrogens, we have analyzed their ability to interact with several known coactivators and members of the preinitiation complex (PIC). Moreover, the conformation of these receptors was studied by limited proteolytic digest experiments. Finally, we analyzed the ability of these receptors to bend ERE-containing DNA, which is thought to reflect in part the transcriptional ability of transcription factors (34, 35, 36). Altogether, these results demonstrate that, upon hormone exposure, the wild type receptor undergoes major changes in its conformation and in its properties, whereas no effect or only limited effects of hormone are observed with the constitutively active receptors, as these appear to have already undergone those changes that render them ligand-independent transcriptional activators.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Certain ER Mutants Show Constitutive Activity
To understand the mechanisms underlying the activated state of a normally hormone-activated nuclear receptor, we have analyzed the properties of the wild type ER as well as those of constitutively active mutant ERs. Several steps presumed to be involved in receptor activation upon ligand treatment were analyzed, i.e. the interaction of the wild type and mutant ERs with coactivators and members of the PIC, the conformational state of these receptors before and after treatment with estrogen, and their ability to bend ERE-containing DNA fragments.

Transcriptional activity of the wild type or mutant ERs was monitored in the presence and absence of hormone with estrogen-responsive CAT reporter genes containing a minimal (TATA) or a complex (pS2) promoter. As shown in Fig. 1Go, in the absence of ligand, the Y537S ER exhibited constitutive activity 65–90% that of the estrogen-occupied ER, the level of activity being dependent on the promoter used, while the Y537A and E380Q mutant ERs exhibited constitutive activity in the absence of ligand about 15% to 30% that of wild type activity in the presence of E2. Moreover, all three receptors were as potent as wild type ER in activating transcription in the presence of E2. The magnitude of constitutive activity of the ERs shown in Fig. 1Go is consistent with our earlier findings (32, 33). Referring to the structure of the related nuclear receptors hRAR{gamma} and rTR{alpha}1, for which crystallographic information is available (37, 38), the Y537 residue would be in helix 12, containing the hormone-dependent activation function (AF-2) of nuclear receptors, and the E380 residue would be at the end of helix 4 of the ligand-binding domain. [Note: The helix numbering is from the RAR{gamma} structure (37).]



View larger version (73K):
[in this window]
[in a new window]
 
Figure 1. Specific Mutations of the Tyrosine 537 and Glutamic Acid 380 Residues Create Constitutively Active ERs

Transcriptional activity of wild type or mutant (Y537S, Y537A, E380Q) ERs were monitored in the absence or presence of E2. A, CHO cells were cotransfected with wild type or mutant (Y537S, Y537A, E380Q) ER expression vectors, reporter gene construct 2ERE-TATA-CAT, and a pCH110 ß-galactosidase internal reporter. Transfected cells were treated for 24 h with no hormone or E2 (10-8 M). Values are the means and SDs of three experiments after standardization with ß-galactosidase activity and are expressed as the percentage of the CAT activity observed with wild type ER in the presence of E2. B, MDA-MB-231 cells were transfected in the same conditions as for CHO cells, except that the CAT reporter construct used was 2ERE-pS2-CAT.

 
Mutant and Wild Type ERs Interact in a Different Manner with Receptor-Associated Proteins
As numerous reports have emphasized the requirement of coactivator factors to promote full activity of nuclear receptors in the presence of their ligand, it was of interest to determine the extent to which wild type ER and the constitutively active mutants interacted with these factors. Pull-down experiments were performed utilizing glutathione-S-transferase (GST) fusion proteins with the hormone-binding domain of the different ERs. These fusions proteins were expressed in bacteria and adsorbed onto GSH-Sepharose columns. The interaction of coactivators with these ERs was then analyzed using in vitro translated coactivators, with equal inputs of coactivator in each experimental sample. Interactions were monitored in the absence of ligand and in the presence of estradiol or the antiestrogen trans-hydroxy-tamoxifen (TOT) (Fig. 2Go).



View larger version (33K):
[in this window]
[in a new window]
 
Figure 2. Interaction of ERAP140, TIF-1, and SRC-1 Coactivators with Wild Type ER and with Constitutively Active ERs

GST fusion proteins with the wild type (WT) or Y537S, Y537A, E380Q ERs were incubated in the presence of the same amount of in vitro translated [35S]methionine-labeled ERAP140, TIF-1, and SRC-1 coactivators. Incubations were performed in the absence of hormone (control 0.1% ethanol vehicle, C) or in the presence of E2 (E: 1 µM) or TOT (T: 1 µM). After incubation and extensive washings of the glutathione Sepharose, the beads were boiled in Laemmli buffer, and samples were analyzed by SDS-PAGE followed by autoradiography.

 
For the wild type ER in the absence of ligand (control vehicle only, lane labeled C), no interaction was observed with SRC-1, TIF-1, or ERAP-140, as expected (23, 26, 39). The addition of E2 promoted a good interaction with these factors, whereas TOT showed no ability to promote interaction between the wild type ER and these factors. When GST protein alone was used, no interaction was observed with any of the coactivators, either in the absence or in the presence of ligands (data not shown). Since equal inputs of radiolabeled coactivators were used in all samples, it is of note that in the presence of E2, ERAP-140 and TIF-1 showed a much lower extent of interaction with wild type ER than SRC-1, which could mean that these cofactors are somewhat less specific for ER than SRC-1.

The Y537S receptor showed a distinct interaction with each of these three coactivators. In the absence of E2, this mutant interacted with SRC-1 as strongly as did wild type receptor in the presence of E2; this interaction in the absence of E2 was not significantly enhanced by the addition of E2, but was completely suppressed by TOT. The Y537S ER showed a moderate but readily detected interaction with TIF-1 in the absence of E2, and again this interaction was suppressed by TOT. However, as opposed to the interaction of Y537S ER with SRC-1, which is already maximal in the absence of E2, the binding of TIF-1 to the Y537S ER was markedly enhanced by addition of E2. Y537S ER shows yet a third pattern of interaction with the coactivator ERAP-140. There is a small interaction in the absence of E2, which was easily seen with longer times of autoradiography (data not shown), whereas for the same exposure times, no interaction was observed with either the unliganded wild type ER or any of the other constitutively active receptors (Fig. 2Go). Moreover, as with TIF-1, the binding of ERAP-140 to the Y537S ER was greatly increased by addition of E2.

Therefore, the fully constitutively active mutant ER, Y537S, showed a different extent of ligand-independent interaction with the three coactivators: full interaction with SRC-1, some interaction with TIF-1, and little interaction with ERAP-140. Occupancy with E2 was needed to achieve maximal interaction with TIF-1 and ERAP-140.

Concerning Y537A and E380Q mutant ERs, both receptors showed the same type of interaction with SRC-1, namely a weak interaction in the absence of E2, which was greatly enhanced by addition of E2. The interaction in the absence of ligand (control vehicle, C) was abolished by incubation in the presence of TOT. Both the Y537A and E380Q receptors required E2 for interaction with TIF-1 or ERAP-140, but even with E2 treatment, the interaction of the E380Q mutant with ERAP-140 was much lower than that observed for E2-occupied wild type receptor.

Mutant and Wild Type ERs Interact in a Ligand-Independent Manner with the PIC
The interaction of the ER with the PIC could be another step of the transcriptional process involved in the activation. Transcription factor II B (TFIIB) and TATA-box binding protein (TBP) are two members of the PIC that have been shown to be the target of numerous transcription factors. We therefore expressed these factors by in vitro translation and used them in pull-down experiments to test their interactions with our ER proteins. The wild type ER and the three mutants showed a much stronger interaction with TBP compared with TFIIB, but for both the mutant ERs and the wild type ER, interaction with TFIIB and with TBP occurred in the absence or presence of E2 (Fig. 3Go). Among the three mutant receptors, the Y537S ER displayed the greatest ability to interact with TBP, being comparable to that of wild type ER. There was less difference in the degree of interaction of all four receptors with TFIIB.



View larger version (38K):
[in this window]
[in a new window]
 
Figure 3. Patterns of Interaction of Wild Type and Mutant ERs with Two Members of the PIC, TBP and TFIIB

Pull-down experiments were performed as described in Fig. 2Go except that in vitro translated proteins were TBP and TFIIB in the absence or presence of E2 (1 µM). In vitro translated TFIIB appeared as two major products, which both interacted with the wild type ER. The autoradiograms show the interactions of GST-WT, GST-Y537S, GST-Y537A, and GST-E380Q ERs with TBP and TFIIB.

 
The Y537S Mutant Is in an Active Conformation in the Absence of Hormone: Proteolytic Digest Patterns
Previous studies have shown that differences in the conformation of unoccupied and hormone-occupied steroid receptors can be detected by differential sensitivity to protease digestion (30, 40). We therefore tested whether differences in proteolytic digestion patterns might provide a means to discriminate between constitutively active and inactive states among our mutant ERs. The proteolytic digestion patterns of [35S]methionine-labeled wild type or mutant ERs were analyzed by denaturing gel electrophoresis. The results of these analyses are shown in Fig. 4Go.



View larger version (76K):
[in this window]
[in a new window]
 
Figure 4. Proteolytic Digest Patterns of Radiolabeled Wild Type or Constitutively Active ERs after Incubation with Increasing Concentrations of Trypsin

A, Unliganded (0.1% ethanol control vehicle, lanes 1–4) or E2-occupied (lanes 5–8) wild type ER incubated for 10 min at 22 C with 0, 5, 15, or 25 µg/ml trypsin. B, Unliganded or E2-occupied wild type or Y537S ERs incubated for 10 min at 22 C with 0, 1.5, 5, or 15 µg/ml trypsin. C, Unliganded or E2-occupied Y537A, wild type, or E380Q ERs incubated with trypsin as described for panel B. After trypsin exposure, samples were analyzed by SDS-PAGE. The radiolabeled products were visualized by autoradiography.

 
In the absence of ligand, ER is highly sensitive to trypsin and gives a proteolytic digestion pattern in which the fragment sizes decrease rapidly with increasing concentrations of trypsin, until two bands of approximately equal intensity appear and remain relatively stable (panel A, lanes 1–4). One of these is approximately 28 kDa (arrow B), while the other is approximately 25 kDa (arrow A). When the receptor is occupied with E2, only the upper band is strongly stabilized (lanes 5–8, arrow B), suggesting that receptor in an active conformation is protected from further cleavage by trypsin. A transiently stabilized fragment at about 35 kDa appears with E2 treatment when receptor is digested with 5 µg/ml trypsin (lane 6, arrow C), although this fragment is further digested when the trypsin concentration is increased.

The Y537S ER mutant, which shows nearly full constitutive activity in the absence of ligand, showed a proteolytic digestion pattern in both its unliganded or liganded state (compare lanes 9–12 with 13–16), which closely resembled the pattern seen for wild type ER treated with E2. Also, the transiently stabilized band at 35 kDa (arrow C), which in wild type ER is stabilized only in the presence of E2 (lane 7), is strongly stabilized in Y537S ER without or with E2 (lanes 11 and 15). There is, however, a small amount of protein present in the lower/inactive form (arrow A) in the absence of hormone (lanes 11 and 12), which shifts to the upper/active form (arrow B) when E2 is added. Thus, the proteolytic digest pattern for the Y537S receptor reflects its constitutively active state.

The unoccupied E380Q and Y537A ERs, which possess partial constitutive activity, showed a slight enhancement of the transiently stabilized, high molecular mass bands, including the approximately 40-kDa band D and the 35-kDa band C, relative to the unoccupied wild type ER, especially noticeable at a trypsin concentration of 5 µg/ml (compare lanes 11 and 19 vs. 3). Perhaps these subtle differences are indicative that the unliganded E380Q and Y537A receptors have taken on a partially active conformation. However, E2 treatment resulted in a predominance of the B species after increasing trypsin treatment, as observed with the wild type E2-occupied receptor.

Transcriptionally Active Wild Type and Mutant ERs Induce Similar DNA-Bending Angles
Wild type and mutant ERs were expressed in a reticulocyte lysate system in the presence or absence of estrogen and used in DNA-phasing analysis experiments to determine the orientation and magnitude of ER-induced DNA bending (Fig. 5Go). The unoccupied and estrogen-occupied in vitro translated ERs were incubated with 32P-labeled DNA fragments, each of which contained an intrinsic DNA bend separated from a consensus ERE by either 26, 28, 30, 32, 34, or 36 bp (41). Thus, when the ER bound to the ERE and induced DNA bending, the intrinsic and ER-induced DNA bends would either be in phase and form a larger DNA bend or be out of phase and have the effect of straightening the DNA fragment. When wild type and mutant ERs were incubated with DNA fragments containing 26 or 36 bp between the intrinsic and ER-induced DNA bends, there was an increase in the mobilities of the receptor-DNA complexes through the acrylamide gel, indicating that the ER-induced and intrinsic DNA bends were in phase. When the wild type and mutant ERs were incubated with DNA fragments containing 32 bp between the intrinsic and ER-induced DNA bends, there was an increase in the mobilities of the receptor-DNA complexes indicating that the ER induced and intrinsic DNA bends were out of phase. Because the intrinsic and ER-induced DNA bends were on the same side of the DNA helix when they were out of phase (assuming 10.5 bp/helical turn), these findings demonstrate that the ER-induced DNA bend opposes the intrinsic DNA bend, which is directed toward the minor groove of the DNA helix (42). Therefore, the unoccupied and estrogen-occupied wild type and mutant ERs induced DNA bends that were directed toward the major groove of the DNA helix. The magnitudes of the ER-induced DNA bends were determined from replicate phasing analysis experiments (see Materials and Methods) and are summarized in Table 1Go. The unoccupied wild type ER induced a DNA bend of 15.6°, the largest bending angle measured. In contrast, the estrogen-occupied wild type ER induced a much smaller bend of 7.3°. Interestingly, the three mutant receptors, Y537S, Y537A, and E380Q, which are active in the presence and in the absence of hormone, had statistically similar directed DNA-bending angles of approximately 7.5°-9°, in the absence or presence of hormone. Thus, the mutant receptors induced directed bending angles that were similar in magnitude to the bend induced by the estrogen-occupied ER. These findings are in good agreement with previous studies carried out with estrogen-occupied wild type and mutant ERs that had been expressed in COS cells (43) and support the idea that transcriptionally active wild type and mutant ERs induce directed bending angles of similar magnitude.



View larger version (115K):
[in this window]
[in a new window]
 
Figure 5. Phasing Analysis of Wild Type and Mutant ERs with ERE-Containing DNA Fragments

Translation of receptors was performed in the absence or in the presence of 1 µM E2. Reticulocyte lysate-expressed receptors were incubated with 32P-labeled DNA fragments containing an intrinsic bend separated from a consensus ERE by 26, 28, 30, 32, 34, or 36 nucleotides. The ER-DNA complexes were fractionated on a 8% polyacrylamide gel, dried, and visualized by autoradiography.

 

View this table:
[in this window]
[in a new window]
 
Table 1. Directed DNA Bending Angles Induced by Wild Type and Mutant ERs

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Our findings show that the active state of the ER is characterized by several features including functional and conformational/structural properties that are distinct from the ones observed when the receptor is in an inactive state. Interestingly, some of these features are common for the liganded wild type receptor and for constitutively active receptors in the absence or presence of hormone, suggesting that the mechanisms underlying their biological activity are similar.

Figure 6Go presents a model summarizing our findings regarding the interaction of several coregulators and basal transcription factors with the wild type and constitutively active Y537S ER and the effects of E2 on these interactions and on DNA bending by these receptors. The figure emphasizes that occupancy of the wild type ER by E2 elicits a reduction in the extent of DNA bending and an increase in the association with coactivators. The Y537S ER associates strongly with some, but not all, coactivators in the ligand-unoccupied state, and it exhibits the same DNA bend angle in its ligand-free or E2-occupied state as does the E2-occupied wild type receptor.



View larger version (25K):
[in this window]
[in a new window]
 
Figure 6. Model Summarizing Our Findings Regarding the Interaction of Several Coregulators and Basal Transcription Factors with the Wild Type and Constitutively Active Y537S ER and the Effects of E2 on These Interactions and on DNA Bending by These Receptors

The figure emphasizes that occupancy of the wild type ER by E2 elicits a reduction in the extent of DNA bending and an increase in the association with coactivators. The Y537S ER associates strongly with some but not all coactivators in the ligand-unoccupied state, and it exhibits the same DNA bend angle in its ligand-free or E2-occupied state as does the E2-occupied wild type receptor.

 
The most constitutively active ER (Y537S) had E2-occupied ER-like character in all assays but two, and in those (association with TIF-1 and ERAP-140) it showed some wild type-liganded ER character (Table 2Go). The partially constitutively active receptors showed liganded ER character in some assays but not others. Of the three ER coregulators evaluated, SRC-1 interaction correlated best with the degree of transcriptional activity displayed by the mutant ERs.


View this table:
[in this window]
[in a new window]
 
Table 2. Parameters Assessing the Transcriptionally Active State of Wild Type and Constitutively Active Mutant ERs

 
Of the various endpoints assessed, DNA bending was the most sensitive to the constitutively active character of the receptor (see Table 2Go). The receptor proteolytic digestion pattern and receptor coregulator association profiles only appear to clearly indicate the "active" character of the most constitutively active mutant ER Y537S; the other two receptors showed subtle differences in these assays consistent with some change toward the active state observed with the E2-liganded wild type ER, but overall the E380Q and Y537A receptors resembled wild type ER. Although DNA bending did not distinguish between partially and fully constitutively active receptors, it appears to be very sensitive to the propensity of the receptor to be in the active conformation. By contrast, the protease digestion pattern differences may require that receptor be very strongly in the active conformation, as the hormone-occupied wild type-like ER pattern was only observed with the most constitutively active ER, Y537S. The fact that both partially active and fully constitutively active receptors gave the same directed DNA-bending angle suggests that the DNA may form a scaffold for the accumulation of other protein factors and coregulators important in determining receptor transcriptional activity, with DNA bending being the first of several important steps leading to full receptor transcriptional effectiveness. Interaction of the ER with basal factors did not require ligand, and it is therefore not surprising that interaction with TFIIB and TBP does not discriminate wild type from constitutively active ERs.

The wild type ER was able to interact with the three coactivators tested (ERAP140, TIF-1, and SRC-1) in the presence of E2, but not in the presence of antiestrogens such as TOT. The strongest interaction was observed with SRC-1 and TIF-1 and to a lesser extent with ERAP140. In the presence of E2, the three constitutive mutants displayed the same ability to interact with these coactivators as the wild type ER. However, in the the absence of E2, these mutants exhibited different abilities to interact with the coactivators. Indeed, the Y537S mutant was able to interact with SRC-1 in the absence of E2 to the same extent as the wild type receptor in the presence of E2. Moreover, this interaction was not enhanced by addition of E2. With the two other coactivators, the interaction with Y537S ER was still markedly enhanced by (TIF-1), or dependent on, ligand (ERAP 140). Concerning E380Q and Y537A mutants, they showed basically a common pattern of interaction with the coactivators: no interaction in the absence of E2 with ERAP140 or TIF-1 and only a weak interaction with SRC-1 in the absence of ligand. Our observations suggest that SRC-1, TIF-1, and ERAP-140 play somewhat different roles in ER activity and may be involved to different degrees in the process of receptor-regulated transcription. That the activity of the mutants correlated well with binding to SRC-1 implies that SRC-1 is likely a functional mediator of ER transcriptional activity. The interaction of the three mutant receptors with coactivator in the absence of ligand was abolished by addition of trans-hydroxytamoxifen, suggesting that antiestrogen induces a different conformation of the receptor that is not compatible with the interaction with coactivator. Our data are in agreement with the model proposed by Chen and Evans (13), i.e. that the receptor bound to DNA could be in three states: in the absence of hormone, the receptor is inactive (or might be repressive as in the case of TR and RAR, which interact with corepressors). Once the ligand is added, the receptor is in an intermediary state, and basal transcription can take place. And finally, the liganded receptor is able to interact with coactivators, which would be a link with the PIC, and to activate strongly the transcription process.

The mechanism by which the nuclear receptors activate transcription remains unclear, but it is proposed that the receptors could stimulate PIC formation either by recruiting the different members of the PIC or by positioning a preformed PIC on the DNA. The PIC consists of at least seven basal transcription factors, namely TFIIA, TFIIB, TFIID [comprised of the TBP interacting with TBP-associated factors, TFIIE, TFIIF, TFIIH, and TFIIJ (for review, see Ref. 44)]. A number of transcription factors, including nuclear receptors and, in particular, the ER, have been shown to interact in vitro with TFIID (45, 46, 47, 48) or TFIIB (49, 50, 51, 52, 53). We have now investigated the interaction of the wild type and mutant ERs with members of the PIC. Basically, all the ERs tested, whether wild type or constitutively active, interacted with TBP and TFIIB in the absence or presence of hormone. However, of note was the fact that TBP interaction, which was substantial in the absence of estrogen but was enhanced by E2 in the case of wild type receptor, was not increased by E2 for mutant receptors. These data suggest that the ability of a receptor to interact with the PIC is not a specific feature of activated receptors. However, it is possible that the interaction of inactive receptors (i.e. unoccupied wild type ER) with the PIC could be nonproductive. The transformation of the receptor into an active state would then not lead to an increase of the interactions but rather to a modification of the nature of these interactions.

To further investigate the conformational changes that could arise from treatment with estrogen, we performed limited proteolytic digestion experiments. Using the wild type receptor, we present evidence that the conformation of the receptor in the absence and in the presence of E2 are different, which is in agreement with previous work (30, 54). Indeed, in the absence of ligand, the receptor was highly sensitive to trypsin and gives a proteolytic digestion pattern in which, for high concentrations of trypsin, two stable bands (~25 and 28 kDa) of approximately equal intensity appeared. Treatment with E2 stabilized strongly the upper, 28-kDa band. The transformation of the receptor into a distinct, active conformation upon hormone exposure has been reported not only for ER but also for progesterone receptor and RAR/RXR (30, 31), suggesting that this is a phenomenon common to the ligand-activatable nuclear receptors. Of note, the fully constitutively active Y537S ER exhibited a proteolytic digestion pattern in the absence of E2, which was very similar to the pattern of the active wild type estrogen-occupied ER, suggesting that this mutant receptor was in the conformationally active state.

If the receptor can undergo conformational changes after activation, it is possible that these changes could modify the nature of its interactions with DNA. It has been reported by several groups that the binding of transcription factors to DNA can induce bending of DNA (34, 35, 36, 41, 43, 55, 56, 57). A change in the bending state of the DNA could reflect a difference in the ability of the receptor to activate transcription. Our results show that the unliganded wild type receptor induced a larger bend (~16°) and that the E2-occupied wild type receptor induced a dramatically smaller DNA bend (~ 7°). Of particular interest is the fact that the three consitutively active unoccupied and estrogen-occupied ER mutants induced DNA bends that were similar to the bending angle induced by the E2-occupied wild type receptor. This smaller DNA bend would be a characteristic of the active state. We propose that the distinct conformations induced by active and inactive receptors could be interpreted as activating and silencing signals. We should emphasize that the difference in DNA bending observed for the active and inactive wild type ER could be obtained using a totally cell-free system, suggesting that cell context is not a factor in the effect of receptor on DNA bending.

Altogether, the data from studies with these constitutively active ERs provide evidence that the transformation of receptor into an active state involves a complex set of events that include conformational changes as well as distinct alterations in the ability of the receptor to interact with coregulator proteins and with DNA. Collectively, these changes in conformation and interactions "mark" the receptor as being transcriptionally productive.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Chemicals
E2 was from Sigma (St. Louis, MO), TOT and ICI 164,384 were kindly provided by Dr. Alan Wakeling (Zeneca Pharmaceuticals, Macclesfield, U.K.). [35S]Methionine was from ICN (Costa Mesa, CA).

Plasmid Construction
The plasmid encoding SRC-1 (26) was kindly provided by Drs. Ming Tsai and Bert O’Malley. The plasmid encoding TIF-1 (amino acids 434–750) (39) was a gift from Dr. Pierre Chambon. The plasmid pGEX-2TK-ER, which contains the human ER spanning amino acids 282–595, and the plasmid encoding ERAP140 (23) were kindly provided by Dr. Myles Brown. The two expression vectors encoding TBP and TFIIB were kindly provided by Dr. Danny Reinberg. The GST-ER mutant plasmids for Y537A and Y537S were previously described (33). PCR was used to generate the E380Q ER fragment from amino acids 282 to 595 bearing BamHI and EcoRI sites and cloned into PGEX-2TK.

Production of GST Fusion Proteins
Bacteria expressing GST fusion proteins were grown at 37 C in 500 ml of LB (Luria Bertani) broth until the absorbance (600 nm) reached 0.8. Then the induction was performed for 3 h at 30 C with 1 mM isopropyl ß-D-thiogalactopyranoside (IPTG). Cells were collected by centrifugation at 4 C at 3000 x g for 15 min. The supernatant was discarded, and the pellet was rapidly frozen in liquid nitrogen and then kept for 1 h at -80 C. Frozen pellets were then thawed on ice and resuspended in 0.01 volume of NET buffer (20 mM Tris, pH 8.0/100 mM NaCl/1 mM EDTA) and sonicated twice for 30 sec at maximum level. The suspension was centrifuged for 10 min at 12,000 x g, and the supernatant was then transferred to other tubes and centrifuged at 105,000 x g (30 min, 4 C). Protein concentration was estimated by the Bradford method. The levels of expressed fusion proteins were determined by in vitro binding assays followed by Western analysis with H222 monoclonal antibody.

In Vitro Translation of Receptor-Associated Proteins and Human ER Proteins
In vitro translation was performed using the TNT Promega kit (Promega, Madison, WI). Briefly, 1 µg of expression vector was mixed with 25 µl TNT rabbit reticulocyte lysate, 2 µl TNT buffer, 1 µl of mix containing all amino acids except methionine, 1 µl RNAsin (50 U/µl), 1 µl T3 RNA polymerase (20 U/µl), and 4 µl of [35S]methionine (15 µCi/µl). The final reaction volume was 50 µl. The reaction was performed for 1.5 h at 30 C. The translation efficiency was checked by loading 1 µl of lysate on an SDS-PAGE gel.

For gel mobility shift assays, the translation was performed in the presence of control vehicle (0.1% ethanol) or 1 µM E2 as above, except that labeled methionine was replaced with unlabeled methionine.

In Vitro Binding Assays with Glutathione Sepharose
Glutathione Sepharose (Pharmacia, Piscataway, NJ) was equilibrated with IP binding buffer (25 mM Tris-HCl (pH 7.9), 10% vol/vol glycerol, 0.1% NP-40, 0.5 mM dithiothreitol, 100 mM KCl). The in vitro translated products were first precleared for 2 h by incubation with 100 µl of beads and 300 µg of GST (which does not contain any insert). Crude bacterial extract (500 µg) containing GST fusion proteins was incubated at 4 C with 25 µl of beads for 2.5 h in the presence of vehicle (0.1% ethanol) or hormone (E2 or TOT, at 1 µM concentration). After three washes, the beads were incubated with 5 µl of in vitro translated product for 2.5 h in the presence of vehicle or hormone at 4 C. The beads were washed three times with 1 ml of IP buffer and two times with 1 ml of IP buffer containing 300 mM KCl. After washing, beads were boiled in SDS sample buffer, and a quarter of the proteins were run on SDS-PAGE. The gel was fixed, dried, and submitted to autoradiography.

Protease Digestion Assays
Bluescript vector (Stratagene, La Jolla, Ca) was used for insertion of cDNA sequences of wild type ER, E380Q, Y537A, and Y537S ER mutants. Aliquots of in vitro translated, [35S]-labeled proteins (25 µl) were treated with control (0.1% EtOH) vehicle or ligand at a final concentration of 9 x 10-6 M for 20 min at room temperature. Aliquots (5 µl) of the ligand-treated receptor were incubated without trypsin or with trypsin to a final concentration of 1.5, 5, 15, or 25 µg/ml (Worthington Biochemicals, Freehold, NJ) After a 10-min incubation at room temperature, the digestion was stopped with 20 µl of Laemmli buffer, and the samples were boiled for 5 min and then separated on a 12% SDS-PAGE gel. The radiolabeled products were visualized by autoradiography.

DNA-Bending Gel Mobility Shift Assays
The ERE-containing DNA-phasing vectors, ERE 26, ERE 28, ERE 30, ERE 32, ERE 34, and ERE 36 (41), were digested with EcoRI and HindIII, isolated on an acrylamide gel, and electroeluted. The 281- to 291-bp DNA fragments containing the intrinsic DNA bend and the ERE were filled in with Klenow in the presence of [32P]dATP and [32P]dGTP and then purified using a G-25 Sephadex Quick Spin column (Boehringer Mannheim, Indianapolis, IN). Gel mobility shift assays were carried out as previously described (55) with minor modifications. Briefly, 10,000 cpm of the 32P-labeled DNA phasing fragment was combined with 4 µl (188 µg total protein) reticulocyte lysate-expressed wild type or mutant ER and 1 µg poly(deoxyinosinic-deoxycytidylic)acid (Sigma) in a buffer containing 10% glycerol, 50 mM KCl, 15 mM Tris, pH 7.9, 0.2 mM EDTA, and 0.4 mM dithiothreitol (20 µl final volume) for 15 min at room temperature. Low ionic strength gels and buffers were prepared as described (58). Twenty-centimeter gels were prerun for 1 h at 300 V. Samples were fractionated for 3 h on an 8% (75:1 acrylamide to bis-acrylamide ratio) polyacrylamide gel. Water recirculation was used to maintain the gels at 4 C. Radioactive bands were visualized by autoradiography. The relative mobilities of the ER-DNA complexes and free probes were quantitated with a Molecular Dynamics PhosphorImager and Imagequant software (Molecular Dynamics, Sunnyvale, CA). The magnitudes of the receptor-induced directed DNA bending angles ({alpha}B) were determined for the wild type and mutant ERs using the empirical formula (59):

where {alpha}C is the intrinsic DNA bending angle, APH is the phasing amplitude, and k is a coefficient used to adjust for electrophoretic conditions. A value of k = 0.991 was determined by comparing the relative mobility of five sets of DNA-bending standards with their known bending angles (60).


    ACKNOWLEDGMENTS
 
We thank Myles Brown, Danny Reinberg, Pierre Chambon, Ming-Jer Tsai, and Bert O’Malley for providing plasmids.


    FOOTNOTES
 
Address requests for reprints to: Dr. Benita S. Katzenellenbogen, Department of Molecular and Integrative Physiology, University of Illinois at Urbana-Champaign, 524 Burrill Hall, 407 South Goodwin Avenue, Urbana, Illinois 61801-3704.

This research was supported by NIH Grant R37 CA-18119 (to B.S.K.), a Susan G. Komen Foundation Postdoctoral Fellowship (to G.L.), and NIH Grant R29 HD-31299 (to A.M.N.).

Received for publication April 2, 1997. Accepted for publication May 21, 1997.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Evans RM 1988 The steroid and thyroid hormone receptor superfamily. Science 240:889–895[Medline]
  2. Krust A, Green S, Argos P, Kumar V, Walter P, Bornert JM, Chambon P 1986 The chicken oestrogen receptor sequence: homology with V-erbA and the human oestrogen receptor and glucocorticoid receptors. EMBO J 5:891–897[Abstract]
  3. Metzger D, Ali S, Bornert JM, Chambon P 1995 Characterization of the amino-terminal transcriptional activation function of the human estrogen receptor in animal and yeast cell. J Biol Chem 270:9535–9542[Abstract/Free Full Text]
  4. Tora L, Gronemeyer H, Turcotte B, Gaub M-P, Chambon P 1988 The N-terminal region of the chicken progesterone receptor specifies target gene activation. Nature 333:185–188[CrossRef][Medline]
  5. Danielian PS, White R, Lees JA, Parker MG 1992 Identification of a conserved region required for hormone dependent transcriptional activation by steroid hormone receptors. EMBO J 11:1025–1033[Abstract]
  6. Härd T, Kellenbach E, Boelens R, Maler BA, Dahlman K, Freedman LP, Carlstedt-Duke J, Yamamoto KR, Gustaffson JA, Kaptein R 1990 Solution structure of the glucocorticoid receptor DNA-binding domain. Science 2:157–160
  7. Mader S, Chambon P, White JH 1993 Defining a minimal estrogen receptor DNA binding domain. Nucleic Acids Res 21:1125–1132[Abstract]
  8. Kumar V, Green S, Staub A, Chambon P 1986 Localization of the oestradiol-binding and putative DNA binding domains of the human oestrogen receptor. EMBO J 5:2231–2236[Abstract]
  9. Bresnick EH, Dalman FC, Sanchez ER, Pratt WB 1989 Evidence that the 90-kDa heat shock protein is necessary for the steroid binding conformation of the L cell glucocorticoid receptor. J Biol Chem 264:4992–4997[Abstract/Free Full Text]
  10. Miller MA, Mullick A, Greene GL, Katzenellenbogen BS 1985 Characterization of the subunit nature of nuclear estrogen receptors by chemical crosslinking and dense amino acid labeling. Endocrinology 117:515–522[Abstract]
  11. Smith DF, Faber LE, Toft DO 1990 Purification of inactivated progesterone receptor and identification of novel receptor associated proteins. J Biol Chem 265:39996–40003
  12. Burris TP, Nawaz Z, Tsai M-J, O’Malley BW 1995 A nuclear hormone receptor-associated protein that inhibits transactivation by the thyroid hormone and retinoic acid receptors. Proc Natl Acad Sci USA 92:9525–9529[Abstract]
  13. Chen JD, Evans RM 1995 A transcriptional co-repressor that interacts with nuclear hormone receptors. Nature 377:454–457[CrossRef][Medline]
  14. Chen JD, Umesono K, Evans RM 1996 SMRT isoforms mediate repression and anti-repression of nuclear receptor heterodimers. Proc Natl Acad Sci USA 93:7567–7571[Abstract/Free Full Text]
  15. Hörlein AJ, Näär AM, Heinzel T, Torchia J, Glass B, Kurokawa R, Ryan A, Kamel Y, Söderström M, Glass CK, Rosenfeld MG 1995 Ligand-independent repression by the thyroid hormone receptor mediated by a nuclear receptor co-repressor. Nature 377:397–404[CrossRef][Medline]
  16. Kurokawa R, Söderström M, Hörlein A, Halachmi S, Brown M, Rosenfeld MG, Glass CK 1995 Polarity-specific activities of retinoic acid receptors determined by a co-repressor. Nature 377:451–454[CrossRef][Medline]
  17. Yen PM, Wilcox EC, Hayashi Y, Refetoff S, Chin WW 1995 Studies on the repression of basal transcription (silencing) by artificial and natural human thyroid hormone receptor-ß-mutants. Endocrinology 136:2845–2851[Abstract]
  18. Nemoto T, Ohara-Nemoto Y, Denis M, Gustafsson JA 1990 The transformed glucocorticoid receptor has a lower steroid-binding affinity than the non-transformed receptor. Biochemistry 29:1880–1886[Medline]
  19. Sanchez ER, Faver LE, Henzel WJ, Pratt WB 1990 The 56–59-kilodalton protein identified in untransformed receptor complexes is a unique protein that exists in cytosol in a complex with both the 70- and 90-kilodalton heat shock proteins. Biochemistry 29:5145–5152[Medline]
  20. Baur EV, Zechel C, Heery D, Heine M, Garnier JM, Vivat V, Ledouarin B, Gronemeyer H, Chambon P, Losson R 1996 Differential ligand-dependent interactions between the AF-2 activating domain of nuclear receptors and the putative transcriptional intermediary factors msug1 and TIF1. EMBO J 15:110–124[Abstract]
  21. Cavaillés V, Dauvois S, Danielian PS, Parker MG 1994 Interaction of proteins with transcriptionally active estrogen receptors. Proc Natl Acad Sci USA 91:10,009–10,013
  22. Cavaillés V, Dauvois S, L’Horset F, Lopez G, Hoare S, Kushner PJ, Parker MG 1995 Nuclear factor RIP140 modulates transcriptional activation by the estrogen Receptor. EMBO J 14:3741–3751[Abstract]
  23. Halachmi S, Marden E, Martin G, MacKay H, Abbondanza C, Brown M 1994 Estrogen receptor-associated proteins: possible mediators of hormone-induced transcription. Science 264:1455–1458[Medline]
  24. Hong H, Kohli K, Trivedi A, Johnson DL, Stallcup MR 1996 GRIP1, a novel mouse protein that serves as a transcriptional coactivator in yeast for the hormone binding domains of steroids receptors. Proc Natl Acad Sci USA 93:4948–4952[Abstract/Free Full Text]
  25. Kamei Y, Xu L, Heinzel T, Torchia J, Kurokawa R, Gloss B, Lin SC, AHR, Rose DW, Glass CK, Rosenfeld MG 1996 A CBP integrator complex mediates transcriptional activation and AP-1 inhibition by nuclear receptors. Cell 85:403–414[Medline]
  26. Onate SA, Tsai SY, Tsai MJ, O’Malley BW 1995 Sequence and characterization of a coactivator for the steroid hormone receptor superfamily. Science 270:1354–1357[Abstract]
  27. Voegel JJ, Heine M, Zechel C, Chambon P, Gronemeyer H 1996 TIF2, a 160 kDa transcriptional mediator for the ligand-dependent activation function AF-2 of nuclear receptors. EMBO J 15:3667–3675[Abstract]
  28. Ali S, Metzger D, Bornert JM, Chambon P 1993 Modulation of transcriptional activation by ligand-dependent phosphorylation of the human oestrogen receptor A/B region. EMBO J 12:1153–1160[Abstract]
  29. Orti E, Bodwell JE, Munck A 1992 Phosphorylation of steroid hormone receptors. Endocr Rev 13:105–128[Abstract]
  30. Allan GF, Leng X, Tsai SY, Weigel NL, Edwards DP, Tsai MJ, O’Malley BW 1992 Hormone and anti-hormone induce distinct conformational changes which are central to steroid receptor activation. J Biol Chem 267:19513–19520[Abstract/Free Full Text]
  31. Valcarcel R, Holz H, Garcia Jeménez C, Barettino D, Stunnenberg HG 1994 Retinoid-dependent in vitro transcription mediated by the RXR/RAR heterodimer. Genes Dev 8:3068–3079[Abstract]
  32. Pakdel F, Reese JC, Katzenellenbogen BS 1993 Identification of charged residues in an N-terminal portion of the hormone binding domain of the human estrogen receptor important in transcriptional activity of the receptor. Mol Endocrinol 7:1408–1417[Abstract]
  33. Weis KE, Ekena K, Thomas JA, Lazennec G, Katzenellenbogen BS 1996 Constitutively active human estrogen receptors containing amino acid substitutions for tyrosine 537 in the receptor protein. Mol Endocrinol 10:1388–1398[Abstract]
  34. Frankel AD, Kim PS 1991 Modular structure of transcription factors: implication for gene regulation. Cell 65:717–719[Medline]
  35. Horikoshi M, Bertuccioli C, Takada R, Wang J, Yamamoto T, Roeder RG 1992 Transcription factor TFIID induces DNA bending upon binding to the TATA element. Proc Natl Acad Sci USA 89:1060–1064[Abstract]
  36. Nardulli AM, Greene GL, Shapiro DJ 1993 Human estrogen receptor bound to an estrogen response element bends DNA. Mol Endocrinol 7:331–340[Abstract]
  37. Renaud JP, Rochel N, Ruff M, Vivat V, Chambon P, Gronemeyer H, Moras D 1995 Crystal structure of the RAR-g ligand-binding domain bound to all-trans retinoic acid. Nature 378:681–689[CrossRef][Medline]
  38. Wagner RL, Apriletti JW, McGrath ME, West BL, Baxter JD, Fletterick RJ 1995 A structural role for hormone in the thyroid hormone receptor. Nature 378:690–697[CrossRef][Medline]
  39. Le Douarin B, Zechel C, Garnier JM, Lutz Y, Tora L, Pierrat B, Heery D, Gronemeyer H, Chambon P, Losson R 1995 The N-terminal part of TIF-1, a putative mediator of the ligand-dependent activation function (AF-2) of nuclear receptors, is fused to B-RAF in the oncogenic protein T18. EMBO J 14:2020–2033[Abstract]
  40. McDonnell DP 1995 Unraveling the human progesterone receptor signal transduction pathway. Trends Endocrinol Metab 6:133–138[CrossRef]
  41. Nardulli AM, Grobner C, Cotter D 1995 Estrogen receptor-induced DNA bending: orientation of the bend and replacement of an estrogen response element with an intrinsic DNA bending sequence. Mol Endocrinol 9:1064–1076[Abstract]
  42. Zinkel SS, Crothers DM 1987 DNA bend direction by phase sensitive detection. Nature 328:178–181[CrossRef][Medline]
  43. Potthoff SJ, Romine LR, Nardulli AM 1996 Effects of wild type and mutant estrogen receptors on DNA flexibility, DNA bending and transcription activation. Mol Endocrinol 10:1095–1106[Abstract]
  44. Roeder RG 1991 The complexities of eukaryotic transcription initiation: regulation of preinitiation complex assembly. Trends Biochem Sci 16:402–408[CrossRef][Medline]
  45. Sadovsky Y, Webb P, Lopez G, Baxter JD, Fitzpatrick PM, Gizang-Ginsberg E, Cavaillés V, Parker MG, Kushner PJ 1995 Transcriptional activators differ in their responses to overexpression of TATA-box-binding protein. Mol Cell Biol 15:1554–1563[Abstract]
  46. Brou C, Wu J, Ali S, Scheer E, Lang C, Davidson I, Chambon P, Tora L 1993 Different TBP-associated factors are required for mediating the stimulation of transcription in vitro by the acidic transactivator GAL-VP16 and the two nonacidic activation functions of the estrogen receptor. Nucleic Acids Res 21:5–12[Abstract]
  47. Ham J, Steger G, Yaniv M 1994 Cooperativity in vivo between the E2 transactivator and the TATA box binding protein depends on core promoter structure. EMBO J 13:147–157[Abstract]
  48. Schulman IG, Chakravarti D, Juguilon H, Romo A, Evans RM 1995 Interactions between the retinoid x receptor and a conserved region of the TATA-binding protein mediate hormone-dependent transactivation. Proc Natl Acad Sci USA 92:8288–8292[Abstract]
  49. Blanco JCG, Wang IM, Tsai SY, Tsai MJ, O’Malley BW, Jurutka PW, Haussler MR, Ozato K 1995 Transcription factor TFIIB and the vitamin D receptor cooperatively activate ligand-dependent transcription. Proc Natl Acad Sci USA 92:1535–1539[Abstract]
  50. Ing NH, Beekman JM, Tsai SY, O’Malley BW 1992 Members of the steroid receptor superfamily interact with TFIIB(S300-ll). J Biol Chem 267:17617–17623[Abstract/Free Full Text]
  51. Lin YS, Green MR 1991 Mechanism of action of an acidic transcriptional activator in vitro. Cell 64:971–981[Medline]
  52. Malik S, Karathanasis S 1995 Transcriptional activation by the orphan nuclear receptor ARP-1. Nucleic Acids Res 23:1536–1543[Abstract]
  53. McDonald PN, Sherman DR, Dowd DR, Jefcoat SC, Delisle RK 1995 The vitamin D receptor interacts with general transcription factor IIB. J Biol Chem 270:4748–4752[Abstract/Free Full Text]
  54. Beekman JM, Allan GF, Tsai S, Tsai M, O’Malley BW 1993 Transcriptional activation by the estrogen receptor requires a conformational change in the ligand binding domain. Mol Endocrinol 7:1266–1274[Abstract]
  55. Nardulli AM, Lew D, Erijman L, Shapiro DJ 1991 Purified estrogen receptor DNA binding domain expressed in Escherichia coli activates transcription of an estrogen-responsive promoter in cultured cells. J Biol Chem 266:24070–24076[Abstract/Free Full Text]
  56. Nardulli AM, Romine LE, Carpo C, Greene GL, Rainish B 1996 Estrogen receptor affinity and location of consensus and imperfect estrogen response elements influence transcription activation of simplified promoters. Mol Endocrinol 10:694–704[Abstract]
  57. Ptashne M 1986 Gene regulation by proteins acting nearby and at a distance. Nature 322:697–701[Medline]
  58. Chodosh LA 1989 Mobility Shift DNA-Binding Assay Using Gel Electrophoresis. Greene Publishing Associates and Wiley Interscience, New York, pp 12.2.1–12.2.10
  59. Kerppola TK, Curran T 1991 DNA bending by Fos and Jun: the flexible hinge model. Science 254:1210–1214[Medline]
  60. Thompson JF, Landy A 1988 Empirical estimation of protein-induced DNA bending angles: Applications to site-specific recombination complexes. Nucleic Acids Res 16:9687–9705[Abstract]