Binding of Estrogen Receptor ß to Estrogen Response Element in Situ Is Independent of Estradiol and Impaired by Its Amino Terminus

Jing Huang, Xiaodong Li, Casey A. Maguire, Russell Hilf, Robert A. Bambara and Mesut Muyan

University of Rochester School of Medicine and Dentistry, Department of Biochemistry and Biophysics (J.H., X.L., R.H., R.A.B., M.M.) and Department of Microbiology and Immunology (C.A.M.), Rochester, New York 14642

Address all correspondence and requests for reprints to: Mesut Muyan, University of Rochester School of Medicine and Dentistry, Department of Biochemistry and Biophysics, Rochester, New York 14642. E-mail: mesut_muyan{at}urmc.rochester.edu.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The functions of 17ß-estradiol (E2) are mediated by estrogen receptor (ER) {alpha} and ß. ERs display similar DNA- and ligand-binding properties in vitro. However, ERß shows lower transcriptional activity than ER{alpha} from the estrogen response element (ERE)-dependent signaling. We predicted that distinct amino termini contribute to differences in transcription efficacies of ERs by affecting in situ ER-ERE interactions. We used chromatin immunoprecipitation and a novel in situ ERE competition assay, which is based on the ability of ER to compete for ERE binding with a designer activator that constitutively induces transcription from an ERE-driven reporter construct. Interference of activator-mediated transcription by unliganded or liganded ERs was taken as an indication of ER-ERE interaction. Results revealed that ERs interacted with ERE similarly in the absence of E2. However, E2 enhanced the ERE binding of ER{alpha} but not that of ERß. The removal of the amino terminus increased the ERß-ERE interaction independent of E2. The ERß amino terminus also prevented E2-mediated enhancement of the chimeric ER{alpha}-ERE interaction. Thus, the amino terminus of ERß impairs the binding of ERß to ERE. The abrogation of ligand-dependent activation function 2 of the amino-terminally truncated ERß resulted in the manifestation of E2 effect on ERß-ERE interaction. This implies that E2-mediated enhancement of ERß-ERE interaction is masked by the activation function 2, whereas the intact amino terminus is a dominant region that decreases the binding of ERß to ERE. Thus, ERß-ERE interaction is independent of E2 and is impaired by its amino terminus. These findings provide an additional explanation for differences between ER{alpha} and ERß functions that could differentially affect the physiology and pathophysiology of E2 signaling.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
ESTROGENS, MAINLY 17ß-estradiol (E2), play critical roles in physiology and pathophysiology of many tissues (1, 2). E2 signaling is primarily conveyed by estrogen receptor (ER) {alpha} and ß (1, 2). ERs are members of a nuclear receptor superfamily and are encoded by two distinct genes. ERs consist of six functional domains. The amino-terminal A/B domain contains a ligand-independent transactivation function 1 (AF-1) and shows 17% amino-acid sequence identity between ERs. The highly conserved central C region, with 96% amino acid homology, is the DNA-binding domain (DBD). The flexible hinge, or D, domain contains a nuclear localization signal and links the C domain to the multifunctional carboxyl-terminal E/F domain. The E/F domain is involved in ligand binding, dimerization, and ligand-dependent transactivation function (AF-2). The E/F domains of ERs display 53% amino acid identity.

Although the sequence of events that lead to the conversion of inactive ER to a transcriptionally active form is unclear, the current model suggests that E2 binding to the nuclear ER allows the receptor to dissociate from heat shock proteins and to dimerize (1, 2). The ER dimer then interacts with permutations of a palindromic DNA sequence with three central nonspecific nucleotides, 5'-GGTCAnnnTGACC-3', the so-called consensus estrogen-responsive element (ERE) (3). The E2-ER-ERE complex subsequently recruits coactivators/regulators to promote local chromatin remodeling and to bridge with general transcription factors for transcription (4). Constituting a critical genomic effect of E2, this pathway is called ERE-dependent E2-ER signaling.

Despite similar in vitro ERE (5, 6) and E2 binding (7, 8) properties, ERß has a substantially lower transcriptional activity than ER{alpha} from the ERE-dependent E2-ER signaling pathway (9, 10, 11, 12, 13). The underlying mechanisms for differences in transcription efficacies of ER subtypes are unclear. The absence of a strong amino-terminal AF-1 (9, 10, 11, 12, 13) and of a functional interaction between the AF-1 and the carboxyl terminus AF-2 of ERß (13) appear to be responsible for the low transcription activity of the receptor in situ. It is also proposed that ERß contains a repression domain within its amino terminus that, when removed, increases the efficiency of the receptor to induce transcription (11, 12).

Because ER-ERE interaction is a critical step in ER-mediated transcription from the ERE-dependent E2-ER signaling, differences in the abilities of ERs to interact in situ with EREs could contribute to the differences in the activity of ERs in response to E2. We predicted here that the ER subtype-specific amino termini differentially regulate ER-ERE interactions in situ, hence contributing to subtype-specific transcriptional responses from the ERE-dependent E2-ER signaling pathway.

To examine ER-ERE interactions, we established a sensitive in situ competition assay and used a chromatin immunoprecipitation (ChIP) assay. We discovered that the amino terminus of ERß impairs the ability of the receptor to interact with ERE both in situ and in vitro. We also found that E2 enhances the in situ ERE binding of ER{alpha} but not of ERß. We observed that E2-mediated enhancement of ERß-ERE interaction is masked by a functional AF-2 function. In summary, we show here that the in situ ERE binding of ERß is regulated by its amino-terminal A/B domain but not by E2.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Studies have indicated that the distinct amino termini of ERs are critical in defining ER subtype-specific transcriptional responses from the ERE-dependent E2-signaling pathway (9, 10, 11, 12, 13). Because ER-ERE interaction has a pivotal importance in ER-mediated gene transcription, differential regulation of ER-ERE interaction in situ by ER subtype-specific amino termini could also contribute to the differences in the capacities of ERs to induce transcription. To test this prediction, we developed a sensitive in situ ERE competition assay that functionally assesses the ability of ER to interact with ERE. We also used a ChIP assay to independently verify our findings. Because of the absence of an ERß-specific antibody suitable for ChIP, we used a Flag antibody directed to the Flag epitope juxtaposed to the amino termini of ERs (Flag-ER{alpha} or Flag-ERß) to comparatively assess ER-ERE interaction in situ. The amino-terminal Flag epitope does not affect in vitro or in situ ER functions (Refs.8 and 13 and supplemental Fig. 1, published as supplemental data on The Endocrine Society’s Journals Online web site at http://mend.endojournals.org).

Development of an in Situ Competition Assay
ERs are modular such that distinct structural domains display a subset of functional properties of the intact protein (14, 15). We recently engineered a monomeric ERE-binding module, CDC, by genetically joining two DBDs (C domains) of ER{alpha} with the hinge domain (D domain) (16). The monomer CDC bound to ERE in a manner similar to the dimer ER{alpha}. Moreover, CDC effectively competed with ER{alpha} for binding to ERE. Because ERs share 96% amino acid identity in their DBDs, as reflected in their abilities to bind various ERE sequences with similar specificity and affinity (5, 6) by interacting with the same nucleosides (6), CDC also represents an ER subtype-independent ERE binder. Integration of strong activation domains from other transcription factors into this CDC module generated ERE-binding transactivators (16). These designer proteins specifically targeted and potently regulated ERE-driven gene transcription independent of dimerization, ER subtype, ER ligand, promoter, and cell type. One of the ERE-binding transactivators designated as PPVV (Fig. 1AGo) contains two tandem activation domains of the p65 subunit of the nuclear factor-{kappa}B, protein (residues 416–550) (17), and of the viral protein 16 (VP16) (residues 403–490) (18), genetically fused to the amino and carboxyl termini of CDC, respectively.



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Fig. 1. ERE Binding Activator and in Situ Competition Assay

A, Schematics of ER{alpha}, ERß, and PPVV, all of which contain an amino terminus Flag epitope. Two C domains of ER{alpha} were cojoined by the D domain to generate ERE binding module, CDC. PPVV was generated by genetically fusing two tandem activation domains of p65 and VP16 to the amino and carboxyl termini of CDC, respectively. B, PPVV potently induces luciferase activity independent of E2. CHO or HeLa cells were cotransfected with 300 ng expression plasmid bearing none (Vector, V), ER{alpha}, ERß, or PPVV cDNA and 125 ng of the TATA box promoter with one ERE, pERE-TATA, which drives the expression of the firefly luciferase cDNA as a reporter enzyme. A plasmid bearing CMV promoter driving the expression of the Renilla luciferase cDNA was used as an internal control (0.5 ng). Cells were then grown in the medium supplemented without or with 10–9 M E2 for 24 h. The cell extracts were assayed for luciferase activity and the normalized firefly/Renilla luciferase activities are presented as fold change compared with the control (V) without E2, a value that was set to 1. The mean ± SEM of three independent experiments performed in duplicate are shown. C, Schematic of the in situ competition assay to assess the E2-independent and E2-mediated ERE binding of ERs.

 
ER{alpha} and ERß have minimal effects on transcription from a single ERE placed upstream of a simple TATA box promoter in response to various concentrations of E2 (see supplemental Fig. 2, published as supplemental data on The Endocrine Society’s Journals Online web site at http://mend.endojournals.org), shown in Fig. 1BGo is 10–9 M E2, a physiological concentration. Both receptors require tandem ERE sequences to significantly induce transcription, the extent of which depends on ER subtype and cell context (13, 19, 20). PPVV, on the other hand, dramatically increased luciferase activity compared with E2-ER from the TATA box promoter bearing one (Fig. 1BGo) or tandem EREs in transiently transfected ER-negative Chinese hamster ovary (CHO) or HeLa cells independent of E2 (16).

Because PPVV constitutively and potently induces, whereas E2-ER has minimal effects on, transcription from the one ERE bearing TATA box promoter, we envisioned that a sensitive in situ competition assay could be established to examine the effects of E2 on ER-ERE interaction in situ (Fig. 1CGo). Moreover, the use of the TATA box promoter precludes the potential interaction between ER and gene-specific regulatory factors bound to complex promoter regions, hence allowing the assessment of the intrinsic abilities of ERs to interact in situ with ERE. We therefore reasoned that interference in the extent of PPVV-mediated gene transcription by ERs could be taken as an indication of ER-ERE interaction. If ER interacts with ERE in the absence of E2, the unliganded ERE-bound ER should decrease the luciferase activity compared with the activity induced by PPVV alone. Moreover, if E2 could enhance the ERE binding of ER, the E2-ER is expected to compete with PPVV more effectively than unliganded ER. Therefore, a further decrease of the luciferase activity should be observed.

E2 Differentially Regulates the Binding of ERs to ERE in Situ
We assessed the in situ ERE binding abilities of ER{alpha} and ERß in the absence and presence of E2. The reporter TATA box plasmid bearing none (TATA) or one ERE (ERE) was cotransfected with an expression vector encoding the PPVV cDNA into CHO or HeLa cells in the absence or presence of varying amounts of an expression vector bearing an ER cDNA. Cells were then treated with 10–9 M E2 for 24 h. Because E2 does not affect the luciferase activity induced by PPVV (Fig. 1BGo), the normalized luciferase activity mediated by PPVV alone in the absence of E2 was set to 100% (control). Alterations in the reporter enzyme activity as a result of a cotransfected Flag-ER cDNA without or with E2 are depicted as percentage (%) change compared with the activity induced by PPVV alone. None of the expression vectors had an effect on luciferase activity from the reporter plasmid bearing only the TATA box promoter (data not shown). On the other hand, increasing concentration of the expression vector bearing Flag-ER{alpha} cDNA gradually decreased the luciferase activity induced by PPVV such that, at the maximal amount of expression vector used (300 ng), the enzyme activity was decreased by 30% in the absence of E2 (Fig. 2AGo). The presence of 10–9 M E2 reduced the enzyme activity by 80%. This result implies that unliganded ER{alpha} interacts with ERE in situ, and E2 increases ER{alpha}-ERE interaction. In contrast, ERß decreased the enzyme activity by 20% whether or not 10–9 M E2 was present. Similarly, ER{alpha}, but not ERß, decreased the luciferase activity induced by PPVV in response to E2 in HeLa cells wherein the extent of ER-mediated suppression of luciferase activity was more pronounced than in CHO cells in the absence or presence of E2 (Fig. 2BGo).



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Fig. 2. The Differential Effect of E2 on the in Situ ERE Binding of ER{alpha} and ERß

CHO (A) or HeLa (B) cells were transfected with 125 ng reporter TATA box promoter bearing one ERE and 300 ng expression plasmid for PPVV, together with 0 (control), 100, 200, or 300 ng expression vector bearing ER{alpha} or ERß cDNA. After transfection, cells were grown in medium without (–E2) or with (+E2) 10–9 M E2 for 24 h. The luciferase activity in cell lysate was assayed, and the value for the relative luciferase activity was presented as percentage (%) change compared with control (PPVV alone in the absence of E2), which was set to 100%. Shown are the mean ± SEM of three independent experiments performed in duplicate. C and D, The abrogation of the ligand-dependent activation function in the carboxyl termini of ERs (AF-2) impairs transactivation (panel C) but does not affect receptor-subtype interaction with ERE as assessed by the in situ competition assay (panel D). CHO cells transfected with an expression vector bearing the AF-2 mutant ER{alpha} (ER{alpha}AF2) or ERß (ERßAF2) were processed as described in panel A. E, The binding of ERs to ERE is required for the decrease in the luciferase activity induced by PPVV. The in situ competition assay was performed as described in panel A, using the expression plasmid bearing DNA binding-defective ER{alpha} (ER{alpha}dbd) or ERß (ERßdbd) cDNA. F, ChIP assay. Expression vector with cDNA of ER{alpha} or ERß was cotransfected with the TATA box promoter without (TATA) or with one ERE (ERE) into CHO or HeLa cells. Cells were treated with 10–7 M E2 for 1 h before fixation with 1% paraformaldehyde. Cells were lysed and subjected to ChIP using Flag antibody-conjugated agarose beads. Shown are the PCR reactions subjected to 2% agarose gel electrophoresis from a representative experiment performed three times. Input indicates 1% of the diluted cell supernatant before ChIP. Sizes of the DNA fragments in base pairs are indicated.

 
We wanted to ensure that differences in the transcription efficiency of ERs to induce reporter enzyme activity in response to E2 from the ERE-containing TATA box promoter construct were not responsible for the differences in the extent of decrease in luciferase activity induced by PPVV. To examine this point, we used mutant ERs bearing three amino acid replacements (D538A, E542A, and D545A in ER{alpha} and D489A, E493A, and N496A in ERß) that abolish AF-2 activity (11, 13, 21, 22). The abrogation of AF-2 function completely prevented ER-induced transcription from one ERE bearing TATA box construct in CHO (Fig. 2CGo) or HeLa cells (data not shown), but had little effect on ER subtype-specific interaction with ERE in the absence or presence of E2 (Fig. 2DGo). These results also suggest that E2-mediated ERE interaction and transactivation of ERs are distinct events.

To further confirm that the decrease in luciferase activity results from the competition of ER with PPVV for binding to ERE, we used ERE binding-defective ERs: ER{alpha}dbd and ERßdbd. These mutants contain cysteine to histidine substitutions at residues 202 and 205 in ER{alpha} and at positions 166 and 169 in ERß in the first zinc finger of the DNA binding domain that prevent the binding of the receptors to ERE (8, 16). Results showed that ER{alpha}dbd and ERßdbd had no effect on PPVV-mediated luciferase activity whether or not cells were treated with E2 (Fig. 2EGo). This indicates that ER-mediated decreases in the reporter enzyme activity induced by PPVV in the absence or presence of E2 are due to the interaction of ER with the ERE sequence.

Collectively, these findings indicate that although unliganded-ERs interact with ERE similarly, E2 differentially regulates the in situ binding of the ER subtypes to ERE independently of cell type.

To corroborate our results, we employed a ChIP assay (Fig. 2FGo). The expression vector bearing none, Flag-ER{alpha}, or Flag-ERß cDNA was cotransfected with the reporter TATA box promoter vector bearing none (TATA) or one ERE (ERE) into CHO or HeLa cells. Cells were treated without or with 10–7 M E2 for 1 h and processed for ChIP. We observed no PCR products with the parent vector (data not shown). On the other hand, E2 further enhanced the in situ binding of ER{alpha}, but not of ERß, to ERE.

We wanted to ensure that ER subtype-specific ERE binding in response to E2 is not due to promoter context. We assessed the binding of ER to the ERE sequence of the pS2 promoter by ChIP in transiently transfected CHO cells (Fig. 3AGo) or to the ERE of the endogenous pS2 gene promoter in adenovirus-infected MDA-MB-231 cells (Fig. 3Go, B–E). The pS2 gene contains a nonconsensus ERE sequence that mediates the E2-ER responsiveness of the gene in breast cancer cells (23) and in infected MDA-MB-231 cells (24). We observed that E2 enhances the binding of ER{alpha}, but not that of ERß, to the ERE of the pS2 promoter in transfected CHO cells (Fig 3AGo). We also found that ERs were synthesized at similar amounts (Fig. 3BGo) to functional proteins (Fig. 3DGo) and localized to the nuclei (Fig. 3BGo) of infected MDA-MB-231 cells. As observed in transfected cells, E2 augmented the interaction of ER{alpha} with the endogenous pS2 promoter without affecting the binding of ERß to the pS2-ERE in infected MDA-MB-231 cells. These results collectively indicate that E2 does not affect the ability of ERß to interact with EREs in situ, while enhancing that of ER{alpha}.



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Fig. 3. Binding of ERs to ERE in Situ

A, ChIP assay of CHO cells transiently transfected with an expression vector bearing cDNA of ER{alpha} or ERß together with a reporter vector bearing the TATA box promoter (TATA), or the pS2 promoter (pS2). Cells were treated with 10–7 M E2 for 1 h before fixing the cells with 1% paraformaldehyde. Cells were lysed and extracts were subjected to ChIP assay using Flag antibody-conjugated agarose beads. Shown are the PCR results resolved on a 2% agarose gel from a representative experiment performed three independent times. Input indicates 1% of the diluted cell supernatant before ChIP. Sizes of the DNA fragments in base pairs are indicated. Immunocytochemical (ICC) (panel B), Western blot (WB) (panel C), and EMSA (panel D) detection of ER proteins in MDA-MB-231 cells infected with a recombinant adenovirus bearing none (Ad5), Flag-ER{alpha}, or Flag-ERß cDNA. For ICC, receptor proteins were probed with ER{alpha} (HC-20) or ERß (PA1–313) specific antibody and visualized by fluorescein-conjugated secondary antibody [fluorescein isothiocyanate (FITC)]. 4',6-Diamidino-2-phenylindole (DAPI) staining indicates the nucleus. A Flag antibody (Flag) was used in WB and EMSA. Shown are cells treated with 10–9 M E2 for 24 h. For WB, molecular mass in kilodaltons is indicated. E, ChIP assay of the endogenous pS2 gene in MDA-MB-231 cells infected with indicated recombinant adenoviruses. Cells were treated 24 h after infection with 10–7 M E2 for 1 h before fixation with 1% paraformaldehyde. ChIP was achieved with Flag antibody-conjugated agarose beads. Shown are the PCR results resolved on 2% agarose gel from a representative experiment performed three independent times. Input indicates 1% of the diluted supernatant before ChIP. Non-PS2 indicates PCR product using primers about 3000 bp away from the ERE sequence of the promoter of the pS2 gene (pS2). P-ERE denotes receptor protein bound to ERE. Free indicates unbound ERE.

 
The Effect of E2 Binding and ER Dimer Status on the Differences in ER-Specific Interaction with ERE in Situ
Although studies have clearly established that in vitro or in situ synthesized ER{alpha} and ERß bind similarly to E2 in vitro (7, 8, 13), an inability of ERß to bind in situ to E2 could explain differences in E2-mediated ER-ERE interactions. To address this point, we employed an in situ E2 binding assay. CHO cells were transfected with expression vector bearing none, or a Flag-ER, cDNA and incubated with various concentrations (0–10–7 M) of 3H-labeled E2 ([3H]E2) in the absence or presence of 10–6 M 4-hydroxytamoxifen, an antiestrogenic compound (25). ER{alpha} and ERß were synthesized at comparable levels in the absence or presence of E2 (shown is 10–9 M) as assessed by Western blotting (Fig. 4AGo). Cells transfected with ER{alpha} and ERß cDNA, but not with the parent vector, retained similar amounts of [3H]E2 (Fig. 4BGo). That 4-hydroxytamoxifen effectively decreased the amount of [3H]E2 detected indicates that [3H]E2 is specifically retained in cells by ERs. Thus, differences in E2-mediated in situ ER-ERE interactions are not due to variations in the amount of ERs in the absence or presence of E2 or in the abilities of ERs to bind to E2. It should be noted that the maximal retention of [3H]E2 occurred at 10–8 M in cells synthesizing either ER, whereas both receptors maximally induced reporter enzyme activity in response to 10–9 M E2 independent of promoter- and cell-type (supplemental Fig. 2). Although the reason for the concentration difference is not clear, one explanation could be that only a portion of total E2-bound ER is sufficient, or available, to induce transcription.



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Fig. 4. Effects of E2 Binding and Dimer Status of ER{alpha} and ERß on the Abilities of ERs to Interact with ERE in Situ

A, Western blot (WB) analysis of transiently transfected CHO cells with the expression vector bearing none (V), ER{alpha}, or ERß in the absence (–) or presence (+) of 10–9 M E2. Equal amounts of whole cell extracts were subjected to SDS 10%-PAGE. Proteins were transferred onto nitrocellulose membrane and probed with a Flag antibody. Molecular mass in kilodaltons is indicated. B, ER{alpha} and ERß bind to E2 similarly. CHO cells were transiently transfected with the expression vector without (V) or with ER{alpha} or ERß cDNA. Cells were incubated 24 h after transfection with fresh medium in the absence or presence of varying concentrations (10–12 to 10–7 M) of [3H]E2 without or with 10–6 M 4-hydroxytamoxifen (4-OHT) for 1 h. Cells were washed, trypsinized, and collected. Radioactivity in cells was quantified with a scintillation counter. C, The in situ competition assays for WT (ER{alpha} and ERß), dimerization defective (ER{alpha}3L{Delta}E and ERß3L{Delta}E), and the single-chain ({alpha}-{alpha} and ß-ß) ERs were accomplished in transiently transfected CHO cells as described in the legend of Fig. 2Go. Results are the mean ± SEM of three independent experiments performed in duplicate.

 
It is well established that ER{alpha} interacts with EREs as a dimer, which is primarily mediated by a surface located within the carboxyl termini of each monomer (26, 27). It is possible that differential effects of E2 on the dimer state of ERs could contribute to E2-mediated ER-ERE interaction in situ. To address this point, we used dimerization-defective ER mutants and single-chain ERs (Fig. 4CGo). We previously showed that changing three Leu residues at positions 504, 508, and 511 to Glu in ER{alpha} (ER{alpha}3L{Delta}E) and at positions 455, 459, and 462 in ERß (ER3L{Delta}E) prevents dimerization and subsequent interaction of the mutant ERs with ERE (8). Single-chain {alpha}-{alpha} and ß-ß proteins were generated by genetically conjugating two ER cDNAs in tandem (8, 28). Circumventing the pivotal dimerization step in receptor action, the monomeric single-chain receptors display biochemical and functional properties that mimic those of the ER dimers both in vitro and in situ (8, 28). In transiently transfected CHO cells, the dimerization-defective ERs had little effect on luciferase activity mediated by PPVV in the absence or presence of 10–9 M E2. The single-chain ERs, on the other hand, simulated the effects of the parent ER dimers on the reporter enzyme activity in the absence or presence of E2. We observed that the treatment of cells with 10–9 M E2 further suppressed the luciferase activity decreased by {alpha}-{alpha} but not by ß-ß.

Consistent with previous observations that ERs form dimers independent of E2 (29), these results collectively indicate that ERs decrease the PPVV-mediated enzyme activity as dimers and that the differences in the dimer state of ERs in the absence or presence of E2 do not contribute to differences in the receptor subtype-specific interaction with EREs.

The A/B Domain of ERß Impairs the Ability of the Receptor to Interact with ERE
The amino-terminal A/B domains of ER{alpha} and ERß share a low amino acid identity (17%) and are critical in defining the ER subtype-specific functional features. To address whether the receptor-specific A/B domains contribute also to differences in the binding of ERs to ERE in situ, we used truncated ER{alpha} ({alpha}CDEF) and ERß (ßCDEF) variants that contain C, D, and E/F domains only (13). ER{alpha} and {alpha}CDEF suppressed the luciferase activity mediated by PPVV to a similar extent in the absence or presence of E2 (Fig. 5AGo). On the other hand, the amino-terminally truncated ERß, i.e. ßCDEF, decreased the luciferase activity more effectively than the parent ERß in CHO cells (Fig. 5BGo). However, the in situ ERE binding of ßCDEF remained independent from E2 binding. Similar results were also observed in HeLa cells (Fig. 5CGo). To ensure that the robust suppression of PPVV-induced luciferase activity by ßCDEF did not mask the effect of E2 on ßCDEF-ERE interaction, we also transfected cells with the ßCDEF expression vector at lower concentrations. We found that ßCDEF suppressed luciferase activity independent of E2 in CHO (Fig. 5BGo, inset) or HeLa (data not shown) cells.



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Fig. 5. The Effect of Amino Termini of ERs on the in Situ ERE Binding

A–C, The in situ competition assay for {alpha}CDEF and ER{alpha} in CHO cells (panel A), ßCDEF and ERß in CHO cells (panel B), or HeLa cells (panel C) cells. The assay was performed as described in the legend of Fig. 2Go. The mean ± SEM indicates three independent experiments performed in duplicate. The inset in Fig. 4BGo indicates the in situ competition assay conducted with the ßCDEF expression vector at 25-, 50-, and 75-ng concentrations. D, Comparison of the in situ ERE binding of ßCDEF and ERß using ChIP assay in CHO cells. The expression vector encoding ßCDEF or ERß was cotransfected with the reporter TATA box promoter plasmid bearing none (data not shown) or a single ERE into CHO cells. Cells were treated and processed for ChIP assay as described in the legend of Fig. 2EGo. Shown are the results of PCR products resolved on a 2% agarose gel from a representative experiment performed at least three times. Sizes of the DNA fragments in base pairs are indicated.

 
ChIP assays using the TATA box reporter plasmid bearing one ERE in transiently transfected CHO cells confirmed that ßCDEF indeed interacts with ERE more effectively compared with ERß whether or not cells were treated with E2 (Fig. 5DGo). These results imply that ßCDEF interacts in situ with ERE more readily than ERß.

To further assess the effect of the amino terminus of ERß in ERE binding, we used EMSAs. An expression vector bearing a cDNA that encodes ERß or ßCDEF was transiently transfected into CHO cells in the absence or presence of 10–9 M E2. Whole-cell extracts (WCEs) were prepared and subjected to Western blotting (Fig. 6AGo). Images were scanned, and bands corresponding to receptor proteins were quantified by ImageQuant. Adjusted amount of WCEs containing similar amounts of receptor proteins were then subjected to EMSA (Fig. 6BGo). At comparable levels, ßCDEF interacted with more EREs (~10-fold) than ERß in the absence or presence (data not shown) of E2.



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Fig. 6. Assessments of in Vitro ERE Binding of in Situ or in Vitro Synthesized ERß or ßCDEF

A, An expression vector encoding ERß or ßCDEF was transiently transfected into CHO cells. WCEs (10 µg) were subjected to SDS 10%-PAGE followed by Western blotting as described in Fig. 3AGo. B, 1, 2, 4, 8 or 16 µg (lanes 1–5, respectively) WCEs containing ERß or 0.125, 0.25, 0.5, 1, 2, 4, 8, or 16 µg (lanes 1–8, respectively) WCEs containing ßCDEF were incubated with 50 pM labeled ERE for 1 h. A Flag antibody (Flag) was added in lane 6 of ERß and in lane 9 of ßCDEF that contained 16 µg protein to confirm the specificity of receptor proteins. Reactions were subjected to nondenaturing 5% PAGE. C and D, Assessments of ERE binding of in vitro synthesized ERß and ßCDEF. C, Proteins were synthesized in vitro with rabbit reticulocyte lysate in the presence of radiolabeled [3H]methionine. Reaction mixtures (5 µl) were subjected to SDS 10%-PAGE followed by fluorography. D, Reaction mixtures containing none (vector, V) or equimolar concentrations of ERß and ßCDEF were incubated with labeled ERE for 1 h. A Flag (Flag) or an ERß-specific antibody, N19 (directed to the A/B domain of ERß), was used to indicate the specificity of receptor-ERE interactions. Reactions were resolved on nondenaturing 5% PAGE and visualized with a PhosphorImager.

 
This is also consistent with the ERE binding pattern of the proteins synthesized in vitro. We used radiolabeled methionine ([3H]Met) to readily quantify the amount of receptor proteins (16, 28). Reaction mixtures containing the same amount of ERß and ßCDEF as assessed by fluorographs (Fig. 6CGo) were then subjected to EMSA (Fig. 6DGo). As observed with the in situ synthesized protein, at an equimolar concentration, ßCDEF retarded more radiolabeled ERE (~10-fold) than ERß. Similar results were obtained in the presence of E2 (data not shown).

Collectively, these results indicate that the A/B domain of ERß impairs the ability of the receptor to interact in situ with EREs whether or not E2 is present.

The Inhibitory Function of the ERß A/B Domain Is Not Specific for the Receptor Subtype
To address whether the inhibitory function of the ERß A/B domain is autonomous, we used the chimeric ßAB{alpha}CDEF receptor (13). ßAB{alpha}CDEF contains the amino-terminal A/B domain of ERß and the C, D, and E/F domains of ER{alpha}. We tested the ERE binding ability of ßAB{alpha}CDEF in comparison with those of ER{alpha} and {alpha}CDEF by the in situ competition assay (Fig. 7AGo). ßAB{alpha}CDEF decreased the reporter enzyme activity less efficiently than {alpha}CDEF and ER{alpha} in response to E2. This suggests that the A/B domain of ERß also decreases the ERE binding of {alpha}CDEF.



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Fig. 7. The Amino Terminus of ERß Contains an Autonomous Inhibitory Domain for ERE Binding

A, The in situ competition assay for ERß, ßCDEF, and the chimeric ßAB{alpha}CDEF that contains the amino-terminal A/B domain of ERß and the CDEF domain of ER{alpha} in CHO cells. Transfection and processing of cells was accomplished as described in the legend of Fig. 2Go. Results are the mean ± SEM of three independent experiments performed in duplicate. B, WCEs (10 µg) were subjected to Western blotting as described in Fig. 3AGo. C, WCEs (1, 2, 4, 8 or 16 µg; lanes 1, 2, 3, 4, 5, respectively) were incubated with 50 pM end-labeled ERE for 1 h. An antibody specific to the carboxyl terminus of ER{alpha} (H-222) in the amount of 1 µl was added in lane 6, which contains 16 µg protein, to confirm the specificity of receptor proteins. Reactions were subjected to nondenaturing 5% PAGE. D and E, The assessments of ERE binding of in vitro synthesized ERE binding module CDC, and CDC bearing A/B domain of ER{alpha} ({alpha}A/B-CDC) or ERß (ßA/B-CDC) genetically fused to the amino terminus of CDC. D, Proteins were synthesized by an in vitro transcription/translation reaction in the presence of radiolabeled [3H]methionine. Reaction mixtures (10 µl) were subjected to SDS 15%-PAGE followed by fluorography. Molecular mass in kilodaltons is indicated. E, The reaction mixture containing equimolar concentrations (4, 8, 16, 28, and 36 pM for lanes 1, 2, 3, 4 and 5, respectively. Lane 6 indicates free DNA) of receptors were incubated with 50 pM end-labeled ERE for 1 h. Reactions were resolved with nondenaturing 5% PAGE. A representative experiment performed in three independent times is presented. V, Vector; Ab, antibody; P-ERE indicates receptor proteins bound to ERE.

 
To further assess the effect of the amino terminus of ERß on ERE binding, we used EMSA. An expression vector bearing a cDNA that encodes ER{alpha}, {alpha}CDEF, or ßAB{alpha}CDEF was transiently transfected into CHO cells in the absence or presence of 10–9 M E2. WCEs were prepared for Western blotting (Fig. 7BGo). Images were quantified, and adjusted amounts of WCEs containing similar amounts of receptor species were then subjected to EMSA (Fig. 7CGo). {alpha}CDEF and ER{alpha} retarded the 32P-end-labeled ERE comparably. ER{alpha} synthesized in vitro or in situ forms three distinct ERE-bound complexes in EMSA due to a truncation in the amino-terminus (8). The slowest migrating complex (complex 3) represents the wild-type ER{alpha} homodimer. The fastest migrating band, the least prominent complex, corresponds to the amino-terminally truncated ER{alpha} homodimer (complex 1). The intermediate ER-ERE complex is the heterodimer of the wild-type and the truncated ER{alpha} species (complex 2). Thus, three major ER{alpha}-ERE complexes are observed in EMSA. The extent of interaction of ßAB{alpha}CDEF with ERE was significantly lower than those observed with ER{alpha} and {alpha}CDEF. In contrast to the in situ result, we observed that the ERE binding of ER{alpha} is minimally affected by E2 treatment. Although recapitulating ER-ERE interaction in situ in the absence of E2, these results also indicate that EMSA is not a suitable approach to examine the effect of a ligand on ER-ERE interaction.

Thus, our in vitro results confirm the in situ observation that the A/B domain of ERß impairs the ability of the receptor to interact with ERE.

To more directly assess this conclusion, we genetically fused the A/B domain of ER{alpha} or ERß to the amino terminus of our ERE-binding module CDC. [3H]Met-labeled proteins were synthesized in vitro (Fig. 7DGo), quantified, and subjected to EMSA (Fig. 7EGo). At equimolar concentrations the amino terminus of ERß, but not that of ER{alpha}, indeed decreased the binding of CDC to ERE.

Collectively, these results show that the amino terminus of ERß impairs the ability of receptor to interact with ERE independent of E2.

Delineation of an Inhibitory Subdomain in the Amino Terminus of ERß
Because the amino terminus of ERß impairs the ERE binding of the receptor, we sought a subregion(s) responsible for this inhibitory function. We generated a series of truncated ERß variants at the amino terminus (Fig. 8AGo). CHO cells were transiently transfected and treated without or with 10–9 M E2 for 24 h. Western blot analysis was performed with a Flag antibody (Fig. 8BGo). Images were scanned, quantified, and adjusted amounts of WCEs containing comparable amounts of receptor proteins were then subjected to EMSA (Fig. 8CGo). We observed that ERß{Delta}1–53, ERß{Delta}1–100, and ERß{Delta}1–148, i.e. ßCDEF, retarded more radiolabeled ERE than ERß, for which relative binding was plotted as the ratio of bound vs. total ERE in Fig. 8DGo. Although gradual truncation of the amino terminus of ERß led to an increase in ERE binding of the receptor variants, ERß{Delta}1–100 and ERß{Delta}1–148 bound to ERE similarly in the absence (data not shown) or presence of E2. This suggests that an inhibitory subdomain is present within the first 100 amino acids of the amino terminus. Interestingly, the deletion of amino acids from 101–148 also dramatically decreased the ability of receptor to bind to ERE. This implies that this subdomain is required for ERß to interact with EREs. Similar results were also obtained with in vitro synthesized proteins (data not shown).



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Fig. 8. Delineation of a Structural Region in the Amino Terminus of ERß Involved in the Impairment of Receptor-ERE Interaction

A, Schematics of amino-terminal truncations of ERß. A Flag epitope is present at the amino terminus of each protein. B, Western blot analysis of the ERß variants. WCEs (10 µg) from transiently transfected CHO cells were subjected to Western blotting as described in Fig. 3AGo. C, The assessment of in vitro ERE binding of ERß variants with EMSA. WCEs of transfected cells treated without (data not shown) or 10–9 M E2 containing the similar amount of truncated ERß variants were incubated with 50 pM radiolabeled ERE, and the reaction was resolved on nondenaturing 5% PAGE. The intensities of free (Free) and bound ERE (P-ERE) were analyzed by a PhosphorImager. D, EMSA results are presented as the relative percent (%) ERE binding that describes the ratio of bound ERE vs. the total ERE. E, The evaluation of the in situ ERE binding of ERß variants with the in situ competition assay, which was carried out as described above. Relative repression, as percent (%) decrease in luciferase activity induced by PPVV, for each truncated receptor obtained with 300 ng expression vector is shown. F, The transactivation capacities of ERß variants in comparison with ER{alpha} and {alpha}CDEF in CHO cells. An expression vector bearing none (Vector, V) or an ER variant cDNA was cotransfected with the TATA box promoter bearing three consensus EREs in tandem (3x ERE-TATA) or a reporter vector bearing the E2-responsive promoter from the pS2 gene. Cells were processed as described in the legend of Fig. 2Go. Results are the mean ± SEM of three independent experiments performed in duplicate. P-ERE indicates receptor proteins bound to ERE.

 
We also assessed the in situ ERE binding of truncated ERß variants with the in situ ERE competition assay in transiently transfected CHO cells (Fig. 8EGo). The assay, plotted as relative repression of the luciferase activity induced by PPVV in response to E2, confirmed that the inhibitory domain is localized to the first 100 amino acids of the amino-terminal domain, whereas a positive regulatory domain resides in a region that encompasses amino acids from 101–148.

In contrast, the removal of the amino terminus of ER{alpha}, {alpha}CDEF, had little effect on ER{alpha}-specific interaction with ERE in the absence (data not shown) or presence of E2 (Fig. 8EGo).

The Role of the Amino Terminus of ERß in Transactivation
Previous studies showed that the removal of the A/B domain of ERß resulted in higher transactivation capacity in transiently transfected cells (11, 12). Because the ER-ERE interaction is the pivotal step in ERE-mediated transactivation, we wanted to address whether the increased binding of an ERß variant to ERE would correlate with an increased transcription efficacy of the receptor. Expression vector bearing none or a cDNA for an ERß variant was cotransfected into CHO or HeLa cells with a reporter plasmid bearing three consensus EREs placed upstream of the TATA box promoter (3x ERE-TATA) or a reporter vector bearing the estrogen-responsive pS2 gene promoter (pS2). Both promoter constructs drive the expression of the firefly luciferase cDNA as the reporter enzyme. Cells were treated without or with 10–9 M E2 for 24 h (Fig. 8FGo). We found that an increase in binding to ERE indeed correlates with an enhanced transcription efficacy of an ERß variant from both the 3x ERE-TATA and the pS2 promoter in CHO cells. That the pattern of reporter enzyme activation was independent of the amount of expression vector used (supplemental Fig. 2B) suggests transactivation capacity is specific to a receptor species.

However, ERß54–100, which showed an intermediary ERE binding in vitro and in situ compared with ERß and ERß1–148, was the most potent ERß variant to induce luciferase activity from the pS2 promoter. This effect appears to be specific to the promoter, because ERß54–100 showed an intermediary transcriptional activity from the ERE bearing estrogen-responsive complement 3 gene promoter in both CHO and HeLa cells (supplemental Fig. 3 published as supplemental data on The Endocrine Society’s Journals Online web site at http://mend.endojournals.org). Thus, these results suggest that the extent of ERß-ERE interaction is critical for activity of the receptor.

{alpha}CDEF had a significantly reduced activity compared with that observed with ER{alpha} (Fig. 8FGo). This is consistent with the requirement for the AF-1 function for the high transcription activity of the receptor (13, 21, 30, 31).

The Role of the AF-2 Function in the Ability of ERs to Interact with ERE in Situ
In search of the structural basis for the unresponsiveness of ERß to E2 to bind to ERE in situ, we found that the AF-2 mutation in ßCDEFCDEF{Delta}AF2) resulted in the manifestation of an E2 effect on the in situ ßCDEF-ERE interaction. In transfected CHO or HeLa (data not shown) cells, ßCDEF{Delta}AF2 showed a lesser effect in suppressing the luciferase activity induced by PPVV in the absence of E2 compared with that observed with the presence of E2. This contrasted to ßCDEF that interacted with ERE independent of E2 (Fig. 9AGo). The AF-2 mutant of ERß (ßCDEF{Delta}AF2), as ERß, on the other hand, had minimal effect on PPVV-mediated luciferase activity whether or not cells were treated with E2 (Fig. 2Go). The abrogation of AF-2 function did not alter the ability of the amino-terminally truncated {alpha}CDEF ({alpha}CDEF{Delta}AF2) (Fig. 9BGo) or ER{alpha}{Delta}AF2 to interact with ERE in response to E2 (Fig. 2Go). Because E2 does not alter the intracellular levels of ERs as assessed by Western blotting (Fig. 9CGo), the responsiveness of ßCDEF to E2 is apparently specific to the disruption of the AF-2 function. ChIP assay of transiently transfected CHO cells using the TATA box reporter construct bearing one ERE further confirmed that ßCDEF{Delta}AF2 interacts more readily with ERE in the presence of E2 than in the absence of E2 (Fig. 9DGo). Thus, our results suggest that the manifestation of E2 effect on the ability of ßCDEF to interact with ERE is suppressed by a functional AF-2 function, whereas the intact amino terminus in ERß is a dominant region that impairs the ability of the receptor to interact with ERE.



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Fig. 9. The Role of the AF-2 Function in the E2-Mediated ER-ERE Interaction in Situ

A and B, The assessment of in situ ERE binding of ßCDEF and ßCDEF{Delta}AF2 (panel A), and {alpha}CDEF and {alpha}CDEF{Delta}AF2 (panel B) in transiently transfected CHO cells in the absence (–E2) or presence (+E2) of 10–9 M E2. C, Western blot analysis of WCEs from CHO cells transiently transfected with the indicated constructs with a Flag antibody in the absence (–E2) and presence (+E2) of 10–9 M E2. D, ChIP assay. CHO cells were cotransfected with the expression vector bearing ßCDEF{Delta}AF2 cDNA and the TATA box reporter vector bearing no (TATA, data not shown) or one ERE (ERE) were subjected to ChIP assay as described in legend of Fig. 2EGo. Sizes of the DNA fragments in base pairs are indicated. Shown are the results of PCR products resolved on a 2% agarose gel.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
We developed a novel in situ competition assay to address the role of the amino terminus of ERß in receptor-ERE interactions in situ. Providing a simple, rapid and sensitive approach, the competition assay together with a ChIP assay revealed that the in situ ERE binding of ERß is independent of E2 and is impaired by its amino-terminal domain.

Previous studies using a promoter interference assay (32), ChIP (33, 34), and chromatin modeling (35) approaches showed that the unliganded ER{alpha} interacts with ERE and that E2 increases the ability of the receptor to bind to ERE. Our results are consistent with these observations. We also recently reported using the interference assay (32) that unliganded or E2-bound ER{alpha} and ERß interact similarly with EREs in situ (13). The interference assay is based on the ability of ER to bind to two consensus EREs in tandem placed between the strong cytomegalovirus (CMV) promoter and the transcription initiation site of a reporter enzyme. In this assay, a decrease in the reporter enzyme activity as a result of interference of the CMV promoter-driven transcription by ER is taken as an indication of ER-ERE interactions. Herein, high sensitivities of the in situ competition and ChIP assays allowed us to decipher and demonstrate that ERs interact with EREs in situ differently: E2 enhances ER{alpha}-ERE interactions in contrast to the ERE binding of ERß that is independent of E2. This finding confirms a previous conclusion that ERß interacts with EREs independent of E2, which was based on the observation that the transcription capacity of a constitutively active chimeric ERß is not altered by E2, whereas E2 further enhances the activity of the chimeric ER{alpha} (11).

Our results also show that the in situ ERE binding of ERß is impaired by its amino terminus. Studies have indicated that the differential activities of the ER{alpha} and ERß primarily arise from differences in the amino termini of ERs. The amino terminus of ER{alpha} contains an independent AF-1 that operates in a promoter- and cell type-dependent manner (30). A functional interaction between AF-1 and the hormone-dependent AF-2 of ER{alpha} provides the receptor with high transcription activity for the ERE-dependent E2 signaling pathway (13, 30, 31). The amino terminus of ERß, on the other hand, lacks significant transcriptional activity and capability of functionally interacting with the AF-2 of the receptor (9, 10, 11, 12, 13). It was also suggested that the amino terminus of ERß contains a repressor domain that decreases the transcription activity of the receptor (11, 12). Our results indicate that, in contrast to the amino terminus of ER{alpha} that does not affect the interaction of the receptor with ERE, the ERß amino terminus acts as a dominant region that impairs the ability of the receptor to bind to ERE independent of E2. This consequently affects the transcription activity of the receptor in response to E2.

We observed that at similar protein concentrations ßCDEF more readily interacted with ERE than ERß both in vitro and in situ, such that about 10-fold more ERß was required to achieve similar levels of ERE binding compared with that observed with ßCDEF. It is possible that an intermolecular interaction of the amino terminus with a factor could decrease the ability of ERß to bind to ERE. Conversely, differences in abilities of ERs to interact with a protein that augments ER-ERE interaction could contribute to the subtype-specific ER-ERE interaction. Studies have indicated that high-mobility group proteins facilitate ER{alpha}-ERE interactions (36, 37, 38) through the DBD of the receptor (37). Similarly, template-activating factor 1ß (39), and pp32 (40), were shown to increase the binding of ER{alpha} to ERE through regions also encompassing the DBD. Despite the fact that ERs share a high amino acid identity in their DBDs, the folding of the ERß amino terminus could allosterically affect the folding of the DBD. This could impair the ability of the receptor not only to bind to ERE but also to interact with coregulatory proteins that augment the extent of ERß-ERE interactions. It is also possible that differential posttranslational modifications of the amino terminus of ERß could lower the ERE binding affinity of the receptor.

Studies clearly established that in vitro or in situ synthesized as well as highly purified recombinant ER{alpha} and ERß bind to EREs in vitro with similar affinities (3, 5, 6, 41, 42). This raises another possibility that the amino terminus of ERß could impair the ERE binding by decreasing the fraction of ERß molecules capable of interacting with EREs. Studies have shown that the DNA binding of p53 is negatively regulated by its carboxyl terminus, which interacts with the DBD, thereby blocking its DNA binding (43, 44). An intramolecular interaction between the amino terminus and the DBD could therefore influence the ability of the receptor to interact with EREs. It is also likely that interaction of a factor with the ERß amino terminus sterically masks the DBD, thereby decreasing the extent of ERß-ERE interactions. A reduction in the amount of ERß capable of ERE binding implies that the primary role of the ERß amino terminus is to keep the receptor sequestered in a population that is incapable of interacting with EREs.

A subdomain in the amino terminus of progesterone receptor (45) and mineralocorticoid receptor (46) was shown to decrease the ability of receptors to induce transcription. Although it is not yet explored whether the reduced transcription activity is associated with a decrease in receptor-DNA interaction, a small ubiquitin-like modifier 1 consensus binding motif, IKXE, appears to be responsible for the autoinhibition of progesterone receptor (47) and mineralocorticoid receptor (46). The amino -terminus of ERß lacks such a motif. Because the removal of the amino terminus enhanced ERß-ERE interactions and the magnitude of transactivation, an increase in binding to ERE apparently correlates with enhanced transcription efficiency. Studies showed that the ER{alpha}/ß heterodimer induces transcription in a manner similar to that of the ER{alpha} homodimer (8, 48). Heterodimerization could serve as a mechanism to overcome the inhibitory function imposed by the amino terminus of ERß. Studies have indicated that phosphorylation of the amino terminus of murine ERß affects the transcription efficacy of the receptor (49). It is therefore possible that posttranslational modifications of ERß could directly, or indirectly through recruitment of a factor, modify the ability of receptor to interact with ERE and subsequent transcription efficiency of the receptor. This also implies that other signaling pathways that cross-talk with ERß, rather than, or in addition to, E2, could be critical for ERß-mediated ERE-dependent signaling.

It appears that the structural basis for the differential E2 effect on the ability of ERs to bind to ERE in situ resides in the carboxyl termini of the receptors. We observed that although ßCDEF with an intact AF-2 function binds to ERE independently of E2 in situ, ßCDEF with point mutations that block the AF-2 function requires E2 for an efficient ERE binding. In contrast, the E2-mediated increase in the in situ ERE binding of ßCDEF is independent of AF-2. These observations indicate that, in addition to the amino termini, the carboxyl termini of ERs are also distinct, and suggest that the presence of a functional AF-2 masks the effect of E2 on the ability of ERß to bind EREs. Because the manifestation of E2-mediated ßCDEF-ERE interaction requires the abrogation of AF-2 function that specifically abolishes the interaction of cofactors, the binding of a factor(s) likely induces a conformation in ERß that resembles that mediated by the E2 binding. The transcriptional regulation of thyroid hormone-responsive genes involves the recruitment of cofactors to regulatory regions by the DNA-bound thyroid hormone receptor (T3R) (50, 51). Several coregulatory proteins that include corepressor proteins (silencing mediator of retinoid and thyroid responsive transcription/nuclear receptor corepressor) interact with T3R in the absence of ligand (50, 51). The binding of hormone leads to the dissociation of these corepressors and subsequent recruitment of coactivator proteins (50, 51). This hormone-dependent coregulatory protein exchange is critical for T3R-mediated transactivation. Studies indicate that ER{alpha} through the amino terminus domain interacts, albeit weakly, with corepressor proteins in the absence of E2 (52, 53). The binding of E2 to ER{alpha} leads to release of corepressor proteins from the E2-ER{alpha} complex (52, 53), likely through an intramolecular interaction between the amino and carboxyl termini (Yi, P., and M. Muyan, unpublished observation). This E2-mediated dissociation of corepressor proteins from the amino terminus with or without recruitment of AF-2- dependent coactivator proteins could lead to an increase in the affinity and/or the capability of the E2-ER{alpha} complex to interact with EREs. The unliganded ERß also interacts with silencing mediator of retinoid and thyroid responsive transcription/nuclear receptor corepressor through a region in the carboxyl terminus of the receptor that encompasses the AF-2 domain (Ref.54 , and Yi, P., and M. Muyan, unpublished observation). However, in contrast to ER{alpha}, the E2 binding does not promote the dissociation of these corepressors from ERß (Ref.54 Yi, P., and M. Muyan, unpublished observation). This could mask the E2-mediated increase in ERß-ERE interactions. Because E2 is necessary for transcription induction, E2 binding to ERß must act as a switch in converting the inactive ERE-bound ERß to a transcriptionally active state through a coregulator exchange or through concurrent recruitment of coactivators mediated by a distinct cofactor-interacting surface(s).

The complex and dynamic regulation of E2 functions by either or both ERs through integrated effects on nuclear and nonnuclear events is critical for the physiology of estrogen-responsive tissues. Despite the comparable ligand- and DNA-binding properties in vitro, emerging evidence clearly indicates that different structural features of ERs also give rise to distinct functions in mediating E2 signaling in situ (1, 2). Our findings here that E2 is important for both the increased binding to and transactivation from EREs of ER{alpha}, as opposed to ERß, for which E2 appears to be important for only transactivation, provide an additional mechanism for functional differences between ERs. One speculation is that ER{alpha} is the primary target for fluctuating levels of circulating E2, whereas ERß acts as a permissive modulator in the temporal regulation of estrogen-responsive genes through the ERE-dependent E2 signaling pathway. A prediction is therefore that the primary regulatory route for ERß involves other ERE-independent nuclear (55, 56) and/or cytoplasmic/perimembrane (57) E2 signaling pathways. Disruption of these regulatory balances in E2 signaling resulting from aberrant decrease/loss or gained/amplified expression of either or both ER subtypes could lead to malignancy in estrogen target tissues. Another complexity in E2 signaling stems from the development of mutant receptors (58). ER{alpha} and ERß variants, derived largely from alternative splicing, appear in both normal and neoplastic estrogen target tissues and exhibit altered functional features that may include modified ERE binding abilities in situ and subsequent transcription efficiencies. These variant ERs, as homodimers or heterodimers with wild-type ERs, could adversely affect E2 signaling pathways contributing to tumor development and/or progression.

In addition to estrogens, ER also binds compounds that act as estrogen competitors that display agonist and/or antagonist properties depending upon ER subtypes, promoter, and cell context (25, 59). These compounds have been valuable not only in the treatment of ER-positive breast cancers but also in the understanding of the mechanism of action of ERs. Whether these estrogen competitors affect the abilities of ER subtypes to interact in situ with ERE remains to be explored. Nevertheless, development of ER subtype-specific ligands that also modify the in situ ER-ERE interactions could be useful pharmacological agents for experimental biology and medicine.

In conclusion, we found, using a novel in situ competition assay, that the amino terminus of ERß impairs the ERE binding independent of E2 and the transactivation ability of the receptor in response to E2. These findings provide an additional mechanism for the functional difference between ER{alpha} and ERß. Our in situ competition assay could be extended to other members of the steroid/thyroid hormone receptors to study receptor-DNA interaction as an additional/alternative approach.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Plasmids
The human ER{alpha}, ERß, mutant, truncated, and chimeric ER cDNAs without or with the Flag epitope were described previously (13, 16, 28). The engineering of the cDNA for the ERE-binding protein, PPVV, was detailed previously (16). The DNA binding-defective mutant (26) of ER{alpha} or ERß contains cysteine to histidine substitutions at positions 202 and 205 in ER{alpha} or 166 and 169 in ERß. {alpha}CDEF and ßCDEF consist of residues 181–595 for ER{alpha} and 149–530 for ERß. The ßAB{alpha}CDEF contains the A/B domain (residues 1–148) of ERß and C, D, and E/F domains (residues 181–595) of ER{alpha}. The dimerization defective mutants were described previously (8). All cDNAs were inserted into the expression vector pM2 as previously described (28). In engineering of {alpha}A/B-CDC and ßA/B-CDC, the PCR products of the A/B domain encoding residues 1–180 for ER{alpha} and 1–148 of ERß were inserted in frame into the amino terminus of the ERE-binding module CDC cDNA. Resulting cDNAs were sequenced.

Reporter pGL3 (Promega Corp., Madison, WI) plasmids bearing the TATA box promoter without or with a single ERE or multiple EREs were described previously (13, 22, 28). The distance between the center nucleotide in the 13-base core of the first ERE and the TATA box promoter is 106 nucleotides (10 helical turns). Tandem EREs contain 38-bp center-to-center spacing. We also described pGL3 reporter vector bearing the promoter of the pS2 or the complement 3 gene (13). All promoters drive the expression of the firefly luciferase cDNA as the reporter enzyme. A reporter vector driving the expression of the Renilla luciferase cDNA (Promega Corp.) was also described (13, 16, 28). A Flag antibody (M2), ER{alpha}-specific HC-20, and ERß-specific PA1–313 were purchased from Sigma-Aldrich, (St. Louis, MO), Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), and Affinity BioReagents, Inc., (Golden, CO), respectively. The ER{alpha}-specific antibody H222 was kindly provided by Dr. G. Greene (University of Chicago, Chicago, IL).

In Vitro Synthesis of Receptor Proteins
In vitro transcription/translation.
The pBluescript II-KS plasmid (Stratagene, La Jolla, CA) bearing the cDNA for an ER was used for in vitro transcription/translation reactions performed with a rabbit reticulocyte lysate system (Promega Corp.). The proteins were labeled with L-[methyl-3H]methionine (72 Ci/mmol) (NEN Life Sciences, Boston MA) as described previously (28). Reaction mixture (5 µl) was subjected to electrophoresis on SDS 10%-PAGE under reducing condition. The gel was immersed in 1 M sodium salicylate for 30 min, dried, and visualized by an X-OMAT film (Eastman Kodak, Rochester, NY). Bands corresponding to each protein were excised from the gel and counted using a scintillation counter (Beckman Coulter, Fullerton, CA). The relative amount of each protein was determined by the number of methionines specific to each protein.

EMSA.
Oligomers were annealed and end labeled with [32P] as described (6, 28). End-labeled DNA (1 fmol in 20 µl binding buffer) was incubated for 30 min with rabbit reticulocyte lysate that contained equimolar concentrations of receptor proteins (6, 16, 28). One microliter of Flag antibody (M2, Sigma-Aldrich) or ERß-specific antibody, N19 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA.), directed to the amino terminus of the receptor was added into the reaction and incubated for an additional 30 min. Reactions were then subjected to nondenaturing 5% PAGE. The dried gel was visualized and quantified by a PhosphorImager (Molecular Dynamics, Inc., Sunnyvale, CA). Image densities were determined by using ImageQuant version 1.2 software (Molecular Dynamics).

In Situ Synthesis of Receptor Proteins
Western Blot.
CHO and HeLa cells, grown as described previously (13, 16, 28) in six-well tissue culture plates, were transiently transfected with TransIT-CHO and TransIT-HeLa MONSTER transfection reagents, respectively, using 2.4 µg expression vector (Mirus Corp., Madison, WI). Cells were incubated 3 h after transfection with fresh medium supplemented without or with 10–9 M E2 for 24 h. At the termination of an experiment, cells were lysed in a buffer [20 mM Tris-HCl (pH 7.5), 400 mM KCl, 2 mM dithiothreitol, 1 mM phenylmethylsulfonylfluoride, 1:100 (vol/vol) protease inhibitor cocktail (Sigma-Aldrich), 20% glycerol] that contained none or 10–9 M E2 and subjected to three cycles of freeze/thaw at –80 C and 4 C. Total protein (whole cell extract, WCE, 10 µg) was subjected to SDS 10%-PAGE. Western blotting was performed using the FLAG Western detection kit (Stratagene, La Jolla, CA).

EMSA.
Western blot images were scanned. Bands corresponding to receptor proteins were quantified by ImageQuant version 1.2 software (Molecular Dynamics). The amount of cell extracts was adjusted to contain similar amounts of receptor proteins. Extracts were then subjected to EMSA as described above.

In situ E2 Binding.
Cells in 48-well tissue culture plates were maintained for 24 h in medium containing 5% charcoal-dextran-stripped fetal bovine serum (CD-FBS). Cells were then transiently transfected with 300 ng expression vector bearing none (control), or an ER cDNA. Cells were incubated 24 h after transfection with 250 µl fresh medium without or with various concentrations, 10–12 to 10–7 M of [2,4,6,7,16,17-3H]E2 (118 Ci/mmol, NEN Life Sciences, Boston, MA) in the absence or presence of 10–6 M 4-hydroxytamoxifen (Sigma-Aldrich) for 1 h. Cells were then washed extensively with PBS, trypsinized, and collected, and radioactivity was measured in a scintillation counter. Experiments were repeated three independent times in duplicate.

In Situ Competition and Transactivation Assays
Cells grown in 48-well tissue culture plates in medium supplemented with 5% CD-FBS for 24 h were transfected with TransIT LT1 transfection reagent (Mirus Corp.). For the in situ competition assay, cells were transfected with 125 ng reporter and 300 ng expression vector bearing the PPVV cDNA together with 0 (control), 100, 200, or 300 ng expression vector containing the cDNA for an ER. Appropriate amounts of the parent expression vector were added into a given reaction to equalize the total plasmid DNA concentration.

For the transactivation assay, 125 ng reporter and 300 ng expression vectors were cotransfected into CHO or HeLa cells. A plasmid bearing the Renilla luciferase cDNA was used as an internal control in the amount of 0.5 ng to normalize the transfection efficiency (13, 16, 28). Three hours after transfection, cells were maintained in fresh medium supplemented with 5% CD-FBS in the absence or presence of various concentrations (10–12 to 10–7 M) of E2 for 24 h.

The relative firefly/Renilla luciferase activity of the cell lysate was determined using a dual luciferase assay kit (Promega Corp.).

ChIP Assay.
ChIP assay was performed using a ChIP kit (Upstate Biotechnology, Inc., Lake Placid, NY). Cells grown in six-well tissue culture plates as described above were cotransfected with 2.4 µg expression vector and 1 µg reporter vector. After transfection, cells were maintained in medium containing 5% CD-FBS for 24 h. We then incubated cells in fresh medium without or with 10–7 M E2 for 1 h. Due to the short incubation period (1 h), 10–7 M E2, a saturating concentration, was necessary to observe a significant E2 effect in ChIP assays in transfected cells (data not shown). Cells were fixed with 1% paraformaldehyde at 37 C for 10 min and lysed with sodium dodecyl sulfate lysis buffer. Sonication was performed to shear DNA to 200-1000 bp. Cell debris was pelleted and supernatant was collected. The supernatant was diluted with ChIP dilution buffer. To preclear, diluted samples were incubated with salmon sperm DNA/protein A agarose slurry with rotation for 1 h at 4 C. The beads were then pelleted and the supernatant was collected. The Flag-M2 antibody-conjugated agarose beads (Sigma-Aldrich) were added into the supernatant and incubated overnight at 4 C with rotation to precipitate antibody-ER-ERE complexes. Immunoprecipitates were then sequentially washed once each with immune complex wash buffers containing low salt, high salt, and lithium chloride followed by two washes with Tris-EDTA buffer. ER-ERE complexes were eluted with fresh elution buffer (1% sodium dodecyl sulfate-0.1 M NaHCO3). The cross-linking was reversed by the addition of 1/25 volume of 5 M NaCl followed by incubation at 65 C for 6 h. The DNA was recovered with a QIAGEN PCR cleaning kit (QIAGEN, Valencia, CA) in 30 µl nuclease-free water. Eluate (1 µl in a 10 µl reaction) was subjected to PCR (two cycles of 95 C for 2 min; 50 C for 1 min; 72 C for 30 sec and 24 cycles of 95 C for 1 min; 50 C for 1 min; 72 C for 30 sec). The PCR products were resolved on 2% agarose gel. Primers used for PCRs for the TATA box reporter vector bearing none (TATA) or one ERE (ERE-TATA) were specific to the backbone of the reporter vector, pGEM, (5'-GATAGTACTAACATACGCTCTCCATC, forward primer) and the luciferase cDNA (5'-GCTCTCCAGCGGTTCCATCTTCCAG, backward primer). The generation of 324-bp and 366-bp PCR fragments indicates the specificity of PCRs for the TATA and ERE-TATA reporter vectors, respectively. Primers specific to the pS2 gene ChIP assay (5'-GAATTAGCTTAGGCCTAGACGGAATGGG, forward primer; and 5'-GCTACATGGAAG GATTTGCTGATAGACAG, backward primer) flank the ERE sequence and produce a 315-bp DNA fragment. For examining the ER-ERE interaction in transiently transfected cells with the reporter vector bearing the pS2 promoter, we used the pGEM-specific primer and the pS2 gene-specific primer to distinguish specific ER-ERE interaction on the reporter vector DNA from that on the endogenous pS2 gene. PCR reaction produces a 595-bp DNA fragment from the pGL3 reporter plasmid bearing the pS2 promoter. The nonspecific interaction of ERs with cellular DNA of ER-negative MDA-MB-231 cells derived from human breast adenocarcinoma was assessed with primers (5'-CCTGTGGCCCAGCCACTGCGTCTTTCAG, forward primer; and 5'-CCTATCTCCTTGGGAGAGCTGTGAG, backward primer) that are specific to sequences at 3 kb away from the forward primer specific to the pS2 gene. A 348-bp DNA fragment indicates the non-pS2 DNA amplification.

Adenoviral Gene Delivery.
We used the AdEasy XL system (Stratagene, La Jolla, CA) to effectively deliver the gene of interest into breast cancer cell lines to examine the ER-ERE interaction in native chromatin context. We cloned the cDNA for Flag-ER{alpha} or Flag-ERß into the shuttle vector, pJH-Shuttle-CMV, which contains a modified multiple cloning site. In this vector, the strong CMV promoter drives the expression of the inserted cDNA. The resultant vector was transformed into Escherichia coli strain BJ5183-pAD-1 pretransformed with the adenoviral backbone plasmid pAdEasy-1 for homologous recombination. Bacterial colonies containing the recombinant adenoviral plasmid were selected against kanamycin, propagated and purified by a DNA Midi prep kit (QIAGEN). The recombinant vector (Ad5) was then linearized and transfected into AD-293 cells using Liopofectamine-2000 transfection reagent (Invitrogen, San Diego, CA) for virus production. AD-293 cells are derived from human embryonic kidney cells, HEK 293, that were transformed by the sheared Ad5 DNA. Cells were propagated to produce lysate containing infectious recombinant virus. Recombinant virus was purified and used in the infection of MDA-MB-231 cells. We used the recombinant adenovirus bearing the ERß cDNA at 600 multiplicity of infection (MOI) determined with an Adeno-X Rapid Titer kit (BD Biosciences, Palo Alto, CA). At this MOI, ERß was synthesized at amounts comparable to those observed with ER{alpha} in cells infected by the recombinant adenovirus at 100 MOI together with the parent recombinant adenovirus bearing no cDNA at 500 MOI to equalize the total amount of adenovirus used in infections. Cells were incubated 24 h after infection in fresh medium supplemented without or with 10–7 M E2 for 1 h. Cells were collected and subjected to a ChIP assay as described above.

Immunocytochemistry of infected MDA-MB-231 cells was carried out using ER{alpha}-specific HC-20 and ERß-specific PA1–313 as described previously (13, 16, 28).


    ACKNOWLEDGMENTS
 
We thank Dr. Geoffrey Greene for kindly providing the ER{alpha}-specific antibody, H-222. We thank Drs. Gwendal Lazennec, Eun Jig Lee, and Larry Jameson for the advice regarding adenovirus infection into breast cancer cell lines. We thank Dr. Jay Reeder for allowing us to access the fluorescent microscopy facility. We also thank Drs. Mini Balakrishnan, Steve Dewhurst, Mark Sowden, and Melanie Baker for critical reading of the manuscript.


    FOOTNOTES
 
This work was supported by National Institutes of Health Grant HD 24459 (to R.H., R.A.B., and M.M.).

Current address for J.H.: The Wistar Institute, Philadelphia, Pennsylvania 19104.

First Published Online June 23, 2005

Abbreviations: AF-1, Activation function-1; CD-FBS, charcoal-dextran-stripped fetal bovine serum; ChIP, chromatin immunoprecipitation; CMV, Cytomegalovirus; DBD, DNA-binding domain; E2, 17ß-estradiol; ER, estrogen receptor; ERE, estrogen response element; MOI, multiplicity of infection; WCE, whole-cell extract.

Received for publication March 10, 2005. Accepted for publication June 15, 2005.


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