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Molecular Endocrinology, doi:10.1210/me.2005-0288
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Molecular Endocrinology 20 (2): 291-301
Copyright © 2006 by The Endocrine Society

Altered Target Gene Regulation Controlled by Estrogen Receptor-{alpha} Concentration

Amy M. Fowler, Natalia M. Solodin, Christopher C. Valley and Elaine T. Alarid

Department of Physiology, University of Wisconsin-Madison, Madison, Wisconsin 53706

Address all correspondence and requests for reprints to: Elaine T. Alarid, Ph.D., Department of Physiology, University of Wisconsin-Madison, 120 Service Memorial Institute, 1300 University Avenue, Madison, Wisconsin 53706. E-mail: alarid{at}physiology.wisc.edu.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Estrogen receptor-{alpha} (ER{alpha}) is a transcriptional activator whose concentration is tightly regulated by the cellular environment. In breast tumors of postmenopausal women, elevated receptor concentrations can be associated with negative clinical outcomes, yet it remains poorly understood how such high levels impact ER{alpha} function. We previously demonstrated that high nuclear concentrations of ER{alpha} in breast cancer cells bypass the requirement for ligand and are sufficient to activate transcription and accelerate proliferation. Here, we extended those studies and asked whether the transcriptional targets and activation mechanism are similar or different from that of estrogen-stimulated ER{alpha}. We found that at elevated levels, ER{alpha} activated, but could not repress, known estrogen-responsive genes. Moreover, the set of activated genes was expanded to include the uterine-restricted target gene, complement component 3. The activation mechanism of ER{alpha} under these conditions depends both on activation function-1 and residues in the proximal region of the ligand-binding domain. Mutations of aspartate 351 and leucine 372 can inhibit ER{alpha} transcriptional activity gained at high concentrations and discriminate concentration-inducible ER{alpha} function from that induced by estrogen. Moreover, we demonstrate that at high levels, ER{alpha} stimulates transcription without recruiting steroid receptor coactivator-3 and without interference by a Gal4-receptor interaction domain box fusion protein containing LxxLL motifs, further distinguishing this mode of regulation from known activation mechanisms. Together these results demonstrate that the concentration of receptor in breast cancer cells can influence the pattern of target gene expression through a noncanonical activation mechanism.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
ESTROGEN RECEPTOR-{alpha} (ER{alpha}) serves dual functions in the cell as both a regulator of gene transcription and an environmental sensor. A member of the nuclear receptor superfamily, ER{alpha} controls transcription through the recruitment of macromolecular transcriptional complexes to estrogen-responsive elements (EREs) in target genes (1). ER{alpha} binds to DNA through a centrally located DNA-binding domain (DBD) and activates transcription through two activation domains, activation function (AF)-1 and AF-2, located in the N and C termini of the receptor, respectively (2, 3, 4). ER{alpha} also sensitizes the cells to estrogens and thus, its intracellular concentration is strictly regulated. Estrogen decreases ER{alpha} levels through multiple mechanisms, including direct transcriptional repression of ER{alpha} gene expression (5), posttranscriptional destabilization of ER{alpha} mRNA (5), and destruction of ER{alpha} protein through the ubiquitin-proteasome pathway (6, 7, 8). Conversely, removal of 17ß-estradiol (E2) results in an up-regulation of cellular ER{alpha} concentration.

The up-regulation of ER{alpha} expression is a hallmark of the majority of breast cancers in postmenopausal women. In these cases, ER{alpha} is expressed in the majority of cells (9, 10), and there is also a striking increase in the intracellular amount of ER{alpha} protein (11). The highest levels of ER{alpha} measured in invasive breast carcinoma cells by either computer-aided image analysis of immunohistochemical data or flow cytometry are approximately 11- to 13-fold greater than those found in the commonly used ER{alpha}+ MCF-7 human breast cancer cell model system (12, 13). Tumors expressing the highest amounts of ER{alpha} have been associated with decreased recurrence-free survival (14, 15, 16) and reduced response to tamoxifen treatment (17). The reasons underlying poor clinical outcomes in tumors with high ER{alpha} concentrations are unknown, yet these observations raise the possibility that ER{alpha} functions aberrantly when expressed at high levels.

To begin to understand how high concentrations affect ER{alpha} function, we created a tetracycline (Tet)-inducible MCF-7 model system in which treatment with doxycycline (Dox) induces the overexpression of a hemagglutinin (HA)-tagged ER{alpha} to levels approximating the high range in human breast cancer samples. We demonstrated that elevated ER{alpha} levels lead to induction of endogenous target gene expression, accelerated cell proliferation, and anchorage-independent tumor growth in the absence of estrogen (Ref.18 and unpublished observations, A. Fowler). We refer to this activity gained at high concentrations of receptor as "concentration-inducible" ER{alpha} function. One possible mechanism that could explain concentration-inducible receptor activity is that at elevated levels, ER{alpha} may adopt an active conformation similar to that stabilized by estrogen. If so, it would be predicted that the concentration- and estrogen-inducible activation pathways would converge at events downstream of ligand binding. We tested this possibility through examination of endogenous target gene regulation and dissection of the molecular mechanisms governing concentration- vs. estrogen-inducible receptor function. We found that concentration-inducible ER{alpha} activity can be distinguished from estrogen- and growth factor-stimulated pathways by a lack of dependence on p160 coactivators and through differences in required structural elements. The activity was mapped to AF-1 and the proximal region of the C-terminal ligand-binding domain (LBD). Importantly, specific point mutants in the LBD were identified that abolish concentration-inducible activity without altering estrogen-inducible activity. Thus, under conditions that simulate high concentrations of receptor in breast cancer cells, ER{alpha} activates transcription through a distinct mechanism that results in alterations in the pattern of ER{alpha} target gene expression.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Elevated Concentrations of ER{alpha} Lead to Increased Target Promoter Occupancy but Result in Different Transcriptional Outputs
ER{alpha}HA cells are a Tet-inducible cell line that respond to Dox treatment by an 8-fold overexpression of an HA-tagged ER{alpha} (18). Previous studies had demonstrated by Northern analysis that treatment with 1 µg Dox for 24 h induced expression of the endogenous pS2 gene in the absence of E2 (18). The induction of ER{alpha}HA and pS2 by Dox was confirmed by real-time quantitative RT-PCR shown in Fig. 1Go, A and B. Initial experiments were undertaken to determine whether concentration-inducible and ligand-inducible ER{alpha} activity stimulated the expression of an overlapping set of genes. Quantitative RT-PCR was performed on RNA isolated from samples treated with Dox or 10 nM E2. Ethanol treatment served as a vehicle control. The genes examined were chosen based on whether they were activated or repressed by ER{alpha} in the presence of estrogen. Activated genes included progesterone receptor (PR) and cathepsin D (CATD), whereas repressed genes included a p160 coactivator (steroid receptor coactivator; SRC-3) and ER{alpha}. In the case of activated genes, quantitative RT-PCR analysis showed that PR and CATD are up-regulated by both high concentrations of ER{alpha} as well as E2 treatment relative to control samples (Fig. 1Go, C and D). However, the magnitude of gene induction by these two modes of ER{alpha} activation varied. PR expression was more highly stimulated by concentration-inducible activity than E2, whereas the opposite was the case for pS2. CATD, which is only weakly activated, was similarly induced by both stimuli. Examination of repressed target genes showed that E2 treatment diminished the expression of SRC-3 and ER{alpha} (Fig. 1Go, E and G), as expected (19, 20). Lactoferrin expression was also inhibited by E2 in these cells (Fig. 1FGo), which contrasts with its stimulatory effect observed in endometrium (21). However, the expression of these repressed genes was unaffected by high concentrations of ER{alpha} (Fig. 1Go, E–G). Thus, the pattern of gene regulation revealed both similarities and differences between ER{alpha} functions induced by high receptor concentrations and by E2.


Figure 1
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  		Fig. 1. Target Gene Regulation by ER{alpha}

Tet-inducible ER{alpha}HA cells were treated with ethanol (EtOH), 1 µg/ml Dox, or 10 nM E2 for 24 h. Total RNA was isolated and quantitative RT-PCR was performed using primers for ER{alpha}HA (A), pS2 (B), PR (C), CATD (D), SRC-3 (E), lactoferrin (F), and ER{alpha} (G). mRNA levels were calculated relative to ethanol-treated cells. Data are presented as mean ± SE of three independent experiments. LTF, Lactoferrin.

 
To determine whether promoter occupancy by ER{alpha} could explain differences in regulation between activated and repressed targets, chromatin immunoprecipitation (ChIP) analysis was performed using pS2 and ER{alpha} as models for activated and repressed targets, respectively. Promoter binding by ER{alpha} was measured using antibodies directed against ER{alpha} (Fig. 2AGo) and the HA tag (data not shown). Concentration-inducible conditions resulted in increased ER{alpha} occupancy of the ERE-containing region of the pS2 promoter relative to ethanol and IgG controls (Fig. 2AGo). The level of promoter occupancy was approximately 3-fold greater than that induced by E2. This result is consistent with an increase in total ER{alpha} favoring greater promoter occupancy. However, increased ER{alpha} at the promoter did not translate into greater transcriptional activation because E2 was more potent than Dox in stimulating pS2 gene expression (Fig. 1BGo). With respect to genes repressed by ER{alpha}, both high receptor concentrations and E2 stimulated receptor occupancy of the ER{alpha} 5'-untranslated region, yet only E2-inducible ER{alpha} activity resulted in down-regulation of ER{alpha} gene (Figs. 1GGo and 2BGo). Examination of a distal region upstream of the pS2 gene indicated that concentration-inducible conditions do not generally lead to ER{alpha} occupancy at nonspecific sites on DNA (Fig. 2AGo).


Figure 2
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  		Fig. 2. ER{alpha} Occupancy of Activated and Repressed Target Genes

Tet-inducible ER{alpha}HA cells were treated with ethanol (–), 1 µg/ml Dox (+ Dox), or 10 nM E2 (+ E2) for 24 h. ChIP was performed using normal rabbit IgG or an ER{alpha}-specific antibody. Quantitative PCR was performed on input and immunoprecipitated DNA with primers (A) near the ERE (–353 to –31) and approximately 3 kb upstream of the transcription start site (–3376 to –3229) of the pS2 gene and (B) within the 5'-untranslated region (+60 to +167) of the ER{alpha} gene. The amount of immunoprecipitated DNA was calculated relative to ethanol-treated controls. Data are presented as mean ± SE of three independent experiments.

 
Recent studies in yeast identified a class of transcription factors, termed "condition-expanded" activators, which bind to an increased number of promoter elements when their nuclear concentrations increase in response to nutrient starvation (22). We therefore asked whether, at high concentrations, ER{alpha} may expand its target genes beyond those typically supported by ER{alpha} in the breast. Our previous studies suggested that concentration-inducible ER{alpha} activity was dependent on AF-1 function. Thus, as a potential expanded target, we identified the AF-1-dependent gene, complement component 3 (C3). C3 is a known ER{alpha} target gene the expression of which is regulated by estrogen in the uterus (23). Quantitative RT-PCR analysis confirmed that E2 treatment failed to activate endogenous C3 expression in ER{alpha}HA cells (Fig. 3AGo). However, when its expression was analyzed under concentration-inducible conditions, C3 expression was induced 6-fold over vehicle controls. ChIP analysis showed that C3 activation correlated with an increase in C3 promoter occupancy by ER{alpha} in cells expressing high concentrations of receptor (Fig. 3BGo). Thus, similar to the findings in yeast, conditions that favor high nuclear concentrations facilitate receptor occupancy at an additional regulatory element not typically bound by liganded ER{alpha} in these cells.


Figure 3
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  		Fig. 3. Regulation of C3 Gene by ER{alpha}

Tet-inducible ER{alpha}HA cells were treated with ethanol (EtOH), 1 µg/ml Dox, or 10 nM E2 for 24 h. A, Total RNA was isolated and quantitative RT-PCR was performed using primers for C3. B, ChIP was performed using normal rabbit IgG or an ER{alpha}-specific antibody. Quantitative PCR was performed on input and immunoprecipitated DNA with primers specific for the C3 promoter (–300 to –151 relative to the transcription start site). The amount of immunoprecipitated DNA was calculated relative to ethanol-treated controls. Data are presented as mean ± SE of three independent experiments.

 
Concentration-Inducible ER{alpha} Transactivation Functions Independently of the p160 Coactivator Family
We next asked whether events within the estrogen-signaling pathway, downstream of DNA binding, were necessary for concentration-dependent transactivation. The E2-induced activation mechanism, as well as that stimulated by growth factor-signaling pathways, involves coactivators of the p160 family (24, 25). Initial studies were undertaken to examine the recruitment of SRC-3 to C3 and pS2 promoters. SRC-3 represents the major p160 coactivator required for the mitogenic activity of ER{alpha} in breast cancer cells (26). Comparison of SRC-3 recruitment induced by E2 vs. Dox treatment showed that SRC-3 was recruited to the pS2 promoter, but not the C3 promoter, in the presence of E2 (Fig. 4AGo). In contrast, SRC-3 was not recruited to either promoter under concentration-dependent conditions, despite the presence of ER{alpha} and activation of gene transcription (Figs. 3Go and 1Go, respectively). ChIP studies using a second antibody that recognizes a different epitope of SRC-3 yielded similar results (data not shown).


Figure 4
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  		Fig. 4. Role of p160 Coactivators in ER{alpha}-Mediated Gene Regulation

A, Tet-inducible ER{alpha}HA cells were treated with ethanol (–), 1 µg/ml Dox (+ Dox), 100 nM 4-hydroxytamoxifen (+ OHT), or 10 nM E2 (+ E2) for 24 h. ChIP assay was performed using an anti-SRC-3 antibody. Input and immunoprecipitated DNA was amplified using primers specific for the C3 and pS2 promoters. The amount of DNA immunoprecipitated with SRC-3 was quantified by quantitative PCR and calculated relative to ethanol-treated controls. B, Diagram of the competitive p160 coactivator fragment encompassing the RID, which contains three LxxLL motifs conserved among SRC-1, -2, and -3. C4–12 cells were transfected with Gal4 DBD RID or its vector control (Gal4 DBD), LHL-CA (–) or LHL-CA-ER{alpha} (+) expression vectors, and the ERE-tk-Luc reporter plasmid. After a 24-h treatment with (C) 10 nM E2 or (D) ethanol (EtOH), ER{alpha} transcriptional activity was assessed by measuring luciferase activity. Data represent the mean ± SE of three independent experiments.

 
To test the possible involvement of other coactivators that possess the conserved LxxLL motifs [receptor-interaction domain (RID)], concentration-inducible transcriptional activity was assessed in the presence and absence of a Gal4 DBD-RID box fusion protein. The Gal4-RID chimera, containing three LxxLL motifs conserved among SRC-1, -2, and -3 (Fig. 4BGo), has been previously shown to competitively inhibit coactivator function (27). As expected, E2-inducible activation of a reporter gene was diminished in the presence of the Gal4-RID fusion (Fig. 4CGo). Concentration-dependent ER{alpha} transcriptional activity, however, was augmented rather than repressed (Fig. 4DGo). This result is supported by our previous finding that tamoxifen is unable to inhibit concentration-inducible activity in ER{alpha}HA cells (18). Together these data suggest that coactivators that bind to the ER{alpha} LBD through an LxxLL motif are not essential for the transcriptional function of ER{alpha} acquired at high receptor concentrations.

The potential role of protein-protein interactions was further analyzed in competition experiments using a fusion construct consisting of the Gal4 DBD coupled to the ER{alpha} A/B domains (A/B-Gal4 DBD). Compared with vector controls, expression of the A/B fusion protein resulted in an 80% inhibition of concentration-dependent ER{alpha} transcriptional activity (Fig. 5Go). The ability of an isolated A/B domain to squelch the activity of a full-length ER{alpha} confirms a critical role of AF-1 in mediating the transcriptional activity at high receptor concentrations (30). Importantly, despite the lack of dependence on the LxxLL-containing coactivators, a Gal4-fusion expressing the C-terminal D/E/F domains was also capable of squelching concentration-inducible ER{alpha} transcriptional activity (Fig. 5Go). These data indicate that both the N and C termini of ER{alpha} contribute to activity driven by high concentrations of receptor.


Figure 5
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  		Fig. 5. Transcriptional Interference by the Isolated A/B and LBDs

Tet-inducible ER{alpha}HA cells were treated with (+) or without (–) 1 µg/ml Dox and transfected with the A/B-Gal4 DBD or Gal4 DBD-D/E/F fusion constructs, or its vector control (Gal4 DBD) and the ERE-tk-Luc reporter plasmid. Transcriptional activity of the receptor chimeras was assessed 2 d later by measuring luciferase activity. Data represent the mean ± SE of three independent experiments.

 
Specific LBD Residues Discriminate Concentration-Inducible from E2-Inducible ER{alpha} Transcriptional Activity
In an attempt to map the N-terminal and C-terminal elements involved in concentration-inducible ER{alpha} transcriptional function, we generated a series of point mutants within the B and E domains. Mutations in the B domain targeted potential sites of posttranslational modification [S104/106A, S118A, S118E, Phospho-null, K48R, K171R, and K180R, K-null (Fig. 6AGo)]. LBD point mutants included those previously recognized for their function in either estrogen-activated [transcription activation function 1–3X (TAF1–3X), L540Q] or tamoxifen-activated (D351G, L372R) ER{alpha} transcriptional activity (28, 29, 30, 31, 32). Tamoxifen-sensitive residues (D351 and L372) were considered because results in Fig. 4Go suggested that, like tamoxifen, concentration-inducible activity does not involve interactions with LxxLL-containing proteins. These ER{alpha} mutant constructs were assayed for their concentration-inducible activity in ER{alpha}-negative MDA-MB-231 cells to avoid potential complications arising from the presence of endogenous wild-type (WT) ER{alpha}. Figure 6BGo shows that transient overexpression of ER{alpha} in both MCF-7 and MDA-MB-231 cells resulted in ligand-independent transcriptional activity that is dependent on sequences in the B domain, similar to results obtained in ER{alpha}HA cells. Examination of transcriptional activity of the ER{alpha} point mutants in the B domain revealed no differences from the wild-type receptor. However, point mutants that disrupt either the DNA binding domain (C202/205H and HE82G) or the LBD (D351G, L372R, TAF1–3X, and L540Q) resulted in a complete loss of concentration-inducible activity (Fig. 6AGo). Significantly, mutation of D351 and L372 selectively impaired concentration-inducible ER{alpha} activity without effecting E2-stimulated transcription. In contrast, mutations at D538/E542/D545 (TAF1–3X), and L540 diminished both concentration-inducible and E2-inducible transcriptional activity. Western blot analysis was performed to ensure that the expression levels of the mutant and wild-type receptors were similar (data not shown).


Figure 6
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  		Fig. 6. Identification of Residues that Discriminate Concentration-Inducible from Ligand-Inducible ER{alpha} Transcriptional Activity

A, Receptor transcriptional activity was measured in MDA-MB-231 cells transfected with the indicated mutant ER{alpha} expression vectors and treated for 24 h with ethanol (EtOH) or 10 nM E2. Gene activation measured in cells transfected with wild-type (WT) ER{alpha} was set to 100% for each treatment. The activities of the WT receptor in the absence or presence of E2 were 8.4 (±0.6)- or 37 (±8)-fold, respectively, compared with the vector control. B, MCF-7 and MDA-MB-231 were transfected with WT, {Delta}B, or LHL-CA (vector) expression vectors and the ERE-tk-Luc reporter plasmid. Receptor transcriptional activity was assessed 2 d later by measuring luciferase activity. C, Tet-inducible ER{alpha}HA cells treated with 1 µg/ml Dox were transfected with WT, mutant, or LHL-CA expression vectors and ERE-tk-Luc. Receptor transcriptional activity was assessed 2 d later by measuring luciferase activity. Gene activation measured in Dox-treated cells transfected with LHL-CA vector was set to 100%. Dox stimulated a 5.1 (±0.7)-fold increase in gene activation compared with untreated control cells. Data represent the mean ± SE of at least three independent experiments.

 
To further test the requirement of residues D351 and L372 in concentration-inducible ER{alpha} activity, mutant constructs were transiently transfected into the Tet-inducible ER{alpha}HA treated with Dox. Introduction of a wild-type ER{alpha} construct in the presence of Dox did not further augment concentration-inducible activity (5-fold over untreated controls), indicating that the increase in concentration-dependent transcriptional activity achieved by Dox treatment was maximal. The reporter gene activity measured in the presence of Dox was therefore considered as 100% activation. Mutants lacking the A/B domain ({Delta}A/B) inhibited the transcriptional activity by approximately 50%, consistent with the requirement for the AF-1 domain. Because the {Delta}A/B ER{alpha} mutant completely lacks activity in the absence of wild-type ER{alpha}, it is likely that this decrease represents the maximal inhibition that can be achieved under these experimental conditions. Like the {Delta}A/B ER{alpha} mutant, introduction of point mutants, D351G and L372R mutants were able to dominantly inhibit the concentration-inducible activity stimulated by Dox (Fig. 6CGo). The inhibitory activity of D351G and L372R is specific because the overexpression of the P-null ER{alpha} mutant (S104/106/118/167A) was without effect. Expression levels of the transiently transfected receptors were equivalent (data not shown). Collectively, these results indicate that elements in both the N-terminal AF-1 and residues D351 and L372 mediate concentration-inducible ER{alpha} transcriptional activity.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Based on the observation that ER{alpha} protein is dramatically up-regulated in preneoplastic and malignant lesions of the breast, Fabris et al. (11) first presented the theory that increased ER{alpha} expression could enhance receptor-mediated transcriptional pathways and growth potential leading to their selective expansion, presumably through hormone-dependent mechanisms. This notion is supported by the work of Webb et al. (33), which demonstrated a correlation between the amount of ER{alpha} protein and the magnitude of estrogen-stimulated activation of reporter genes in Chinese hamster ovary (CHO) cells. Our previous work supports the idea that increased expression of ER{alpha} may provide a selective advantage through both increased transcriptional activation and proliferation; however, this benefit is gained only under conditions of hormone deprivation in vitro (18). In trying to understand the basis for this advantage, we tested whether increased expression of receptor allowed unliganded ER{alpha} to adopt the properties of an E2-activated receptor.

We observed a promoter-specific pattern of endogenous target gene regulation at elevated ER{alpha} concentrations and a regulatory set of genes distinct from that of an estrogen-bound receptor. Whereas genes that classically represent estrogen-activated targets, PR and pS2, were induced by high receptor concentrations, gene targets repressed by estrogen treatment (lactoferrin, SRC-3, and ER{alpha}) are unaffected and remain elevated. In the case of the ER{alpha} gene, this represents a loss of negative feedback control and thus a potential perpetuation of receptor signaling. In addition, high concentrations of ER{alpha} resulted in the activation of C3 gene, a target that was not subject to regulation by the ligand-activated receptor. The C3 gene is constitutively expressed in the liver and is up-regulated by estrogen and tamoxifen in the endometrium. Previous studies have reported estrogen- and tamoxifen-inducible activation of a transiently introduced reporter gene driven by the C3 regulatory region in breast and other cell lines (34). However, here we examined the regulation of endogenous C3, the expression of which is restricted by the native chromatin environment. The "expansion" of ER{alpha} target genes to include C3 in a breast cancer cell is unlikely to result from a global increase in ER{alpha} target gene expression because another tissue-restricted gene, prolactin, remained silent under the same conditions (data not shown). Rather, the up-regulation of C3 is consistent with our previous findings that concentration-dependent ER{alpha} transactivation occurs through AF-1. The induction of C3 in cells with elevated levels of receptor brings forward the intriguing possibility that such conditions may allow ER{alpha} to escape the boundaries that maintain both ligand-dependent and tissue-specific regulation of select ER{alpha} target genes.

The condition-expansion hypothesis proposed in yeast predicts that expansion would occur at sites of lower affinity because mass action would strengthen low affinity receptor-DNA interactions (22). Increased transcription factor binding to lower affinity binding sites at high activator concentrations is also used to explain how morphogens such as Bicoid and Dorsal in Drosophila establish position-specific gene expression in the early embryo (35, 36). We therefore examined the pS2 and C3 regulatory regions amplified in our ChIP analysis using a computer-based algorithm, NUBIScan, to quantify the degree of sequence similarity to a canonical ER{alpha} binding site of AGGTCAnnnTGACCT (37). Based on the NUBIScan program, the Xenopus vitellogenin A2 ERE has a raw score of 1.0 (P < 0.0001). The score for pS2 is 0.76 (P < 0.0001), but the C3 region amplified by ChIP has three ERE-containing sites with raw scores of 0.53, 0.53, and 0.51 with higher P values (<0.05). This analysis suggests that the C3 promoter contains highly divergent EREs, which is consistent with the findings of Fan et al. (38). Thus, high concentrations of receptor may allow expansion of ER{alpha} binding sites to include those that contain less stringent ERE sequences and that may have decreased affinity for the receptor. A genome-wide approach is underway to identify additional putative "condition-expanded" target genes and to determine whether differences in promoter structure correlate with gene induction at high ER{alpha} concentrations.

In addition to observing differences in target gene regulation, our results indicate that high concentrations of receptor activate transcription through a mechanism that is distinct from that signaled by estrogen and growth factors. First, the presence of the LBD is sufficient for estrogen-induced receptor activity but is insufficient for concentration-inducible ER{alpha} transactivation. Second, estrogen-, but not concentration-inducible, ER{alpha} activity could be inhibited by either competition with a RID box-containing fragment or tamoxifen, both of which impair p160 coactivator interactions with receptor. Furthermore, SRC-3 was not recruited to the promoters of either of the genes up-regulated by concentration-inducible receptor activity (pS2 and C3). Concentration-inducible ER{alpha} transactivation was also insensitive to knockdown of SRC-3 expression by short interfering RNA and to overexpression of SRC-3 (data not shown). These results are in agreement with previous studies by Lee et al. (39), which showed that p160 coactivators do not increase receptor transcriptional activity at high receptor concentrations in CV-1 cells. Experiments using Gal4-ER{alpha} fusions do suggest, however, that protein-protein interactions are critical for concentration-inducible ER{alpha} transactivation. Third, concentration-inducible activity requires AF-1, but not posttranslational modification, within the B domain. In addition, it depends on sequences in the C terminus of the receptor, specifically residues D351 and L372. ER{alpha} point mutants, D351G and L372R, inhibited, in a dominant fashion, the concentration-dependent activity of ER{alpha} in the breast cancer cells used here. In contrast, these mutants supported estrogen-induced receptor activity. Taken together, these results argue against the possibility that at high concentrations, ER{alpha} activates transcription through the assumption of an active conformation that mimics the ligand-bound receptor or through increased sensitivity to E2. Instead, the evidence supports a model in which high concentrations of ER{alpha} induce active transcription through a unique mechanism that is distinct from the classical mode of activation.

We show that unliganded ER{alpha} increases receptor association with the pS2 promoter, most likely by increasing the probability of receptor-response element interaction. The greater promoter occupancy was not matched by higher transcriptional activity. The relative ratios of promoter occupancy to the magnitude of transcriptional output for the unliganded receptor and the E2-bound receptor are approximately 9 and 1.3, respectively. This observation implies a difference in transcriptional efficiency between these two activating situations. Modulation of receptor transcriptional efficiency can be attributed to coactivators that are recruited in response to estrogen, but these were not found on target promoters under concentration-dependent conditions. The diminished role of p160 coactivators might explain the weaker transcriptional output achieved in the absence of ligand.

Based on data presented here and information gained by others regarding unliganded ER{alpha} receptor function, we can speculate into potential mechanisms by which high concentrations of ER{alpha} activate transcription. It is likely that high concentrations of receptor strengthen weak interactions based on the principles of mass action. We show that elevated expression of unliganded ER{alpha} increases receptor occupancy at target promoters, either through increased probability or rate of interaction. It is also likely that high concentrations of receptor favor interactions with low-affinity coregulatory proteins. It has previously been demonstrated in ChIP experiments that components of the basal transcriptional machinery, including TATA-binding protein (TBP), transcription factor IIA (TFIIA), and TBP-associated factor p130 (TAFp130), can bind dynamically with ER{alpha} at the pS2 promoter in the absence of hormone (40). Direct hormone-independent interactions of ER{alpha} with TBP, TAFII30, and transcription factor IIB have also been demonstrated in vitro (41, 42, 43). In particular, TBP binds to ER{alpha} AF-1 with micromolar affinity and promotes a more ordered structure of the amino terminus (44). We hypothesize that overexpression of unliganded ER{alpha} may stabilize interactions with the basal transcriptional machinery, which at normal receptor levels may be too weak to support effective transcription.

The implication of this study explains, in part, the necessity for multiple levels of control of ER{alpha} levels in cells. As mentioned previously, ER{alpha} levels are strictly limited by transcriptional, posttranscriptional, and posttranslational mechanisms. A reduction in negative feedback by ligand or alterations in other pathways controlling ER{alpha} expression can lead to an increase in the steady-state level of ER{alpha}. Currently, it is unclear why ER{alpha}+ tumors expressing the highest levels of ER{alpha} are associated with poor prognosis and decreased response to tamoxifen (14, 16, 17, 45). Our demonstration that heightened expression of ER{alpha} causes estrogen-independent, tamoxifen-insensitive receptor transactivation and proliferation provides one possible explanation for the altered phenotype of breast cancers with high levels of ER{alpha}. Furthermore, the expansion in target genes regulated by ER{alpha} concentration offers a potential reason for why the correlation between ER{alpha} expression and the expression of ER{alpha}-regulated genes identified in human breast tumors by microarray analysis deteriorates at high receptor concentrations (46). Thus, understanding the mechanism of receptor transactivation and target gene regulation arising from up-regulation of ER{alpha} levels could have important diagnostic and therapeutic implications.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Plasmids
Mutants of ER{alpha} including K48R, {Delta}B, S118E, S167A, Phospho-null (S104/106/118/167A), K171R, K180R, K-null (K48/171/180R), HE82G (E203G/G204S/A207V), D351G, L372R, and TAF1–3X (D538N, E542Q, D545N) were generated by QuikChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA) using wild-type ER{alpha} containing a C-terminal hemagglutinin (HA) tag in pBluescript cloning vector (Stratagene) as a template. Primer sequences are available upon request. The mutant receptors were verified by sequencing at the University of Wisconsin Biotechnology Center. S118A (HE457), {Delta}A/B (HE19), and C202/205H (HE27) ER{alpha} mutants were generously provided by Dr. Pierre Chambon. pCMV S104/106A ER{alpha}, pCMV S118A ER{alpha}, and pCMV L540Q ER{alpha} was kindly sent to us from Dr. Benita Katzenellenbogen. Valine 400 mutations were corrected to glycine, where necessary, and HA tags were fused to the C terminus. The cDNA for these receptors were subcloned into the LHL-CA vector, which drives their expression under the control of a cytomegalovirus (CMV)-ß-actin fusion promoter (47). Expression constructs for ER{alpha}-Gal4 DBD fusions were kindly provided by Dr. Zafar Nawaz and are described elsewhere (48). The plasmid encoding the receptor interaction domain of SRC-1 (amino acids 621–765) fused to the DBD of the Gal4 transcription factor, pM-SRC-1-NR (27), was generously given to us by Dr. J. Wesley Pike. The reporter plasmid ERE-thymidine kinase (tk)-luciferase was used to assess ER transcriptional activity and consists of three copies of estrogen response element (ERE)-containing sequences from the chicken vitellogenin gene upstream of herpes simplex virus tk promoter and luciferase cDNA. To control for transfection efficiency, a CMV-ß-galactosidase plasmid was used.

Cell Culture
Tissue culture reagents were obtained from Mediatech, Inc. (Herndon, VA) unless indicated otherwise. All media were supplemented with 100 U/ml penicillin G and 100 µg/ml streptomycin (Life Technologies, Inc., Carlsbad, CA). MDA-MB-231 and MCF-7 cell lines were maintained at 37 C and 10% CO2 in high-glucose DMEM with phenol red and L-glutamine supplemented with 10% fetal bovine serum (FBS, Hyclone Laboratories, Inc., Logan, UT). Tet-inducible ER{alpha}HA cells (18) were maintained under these conditions in the presence of 0.5 µg/ml puromycin (Sigma Chemical Co., St. Louis, MO) and 200 µg/ml G418 (Life Technologies) to maintain clonal selection. C4–12 cells are an ER{alpha}-negative MCF-7 cell derivative (49) and were grown in phenol red-free {alpha} minimal essential medium with L-glutamine, ribonucleosides, and deoxyribonucleosides (Life Technologies) supplemented with 5% steroid-depleted FBS. Steroid-depleted serum was prepared by undergoing the dextran-coated charcoal stripping procedure outlined by Reddel et al. (50) six times.

At least 3 d before experiments, cells maintained in phenol red-containing media were washed with PBS and grown at 5% CO2 in phenol red-free DMEM supplemented with 4 mM L-glutamine and 10% steroid-depleted FBS or phenol red-free DMEM/F12 media containing the supplements listed above. Chemical concentrations and incubation times for each experiment are indicated in the figure legends. The final concentration of ethanol vehicle control was 0.1% in all samples. E2 was purchased from Steraloids, Inc. (Newport, RI). 4-Hydroxytamoxifen (OHT) and Dox were purchased from Sigma Chemical Co.

Transient Transfections
Transfection conditions were optimized for each cell line. The day before transfection, cells were counted and equal numbers were dispersed into six-well culture plates. Cells were transfected by Lipofectamine 2000 (Invitrogen) or Superfect (QIAGEN, Valencia, CA) according to the manufacturers’ instructions. To control for transfection efficiency in all experiments, cells were cotransfected with CMV-ß-galactosidase. Hormone treatments indicated in the figure legends began the day after transfection and continued for 24 h, at which point the cells were harvested. Luciferase (Promega Corp., Madison, WI) and ß-galactosidase activity (Tropix, Inc., Bedford, MA) were assayed according to manufacturers’ instructions.

Quantitative RT-PCR
Total RNA from a confluent 10-cm plate was isolated using Trizol Reagent (Invitrogen) according to the manufacturer’s instructions. After quantification, 40 µg of RNA was treated with DNAse and purified using RNeasy Micro Kit (QIAGEN). DNAse-treated RNA (1 µg) was reverse-transcribed in a total volume of 20 µl (iScript cDNA Synthesis Kit; Bio-Rad Laboratories, Hercules, CA) according to the manufacturer’s protocol. Reactions lacking reverse transcriptase were also prepared to test for contaminating genomic DNA. The resulting cDNA was diluted to a total volume of 100 µl with nuclease-free water. Quantitative PCR (25 µl total volume) consisted of 1x iQ SYBR Green Supermix (Bio-Rad), 100 nM of forward and reverse primers, and 1 µl diluted cDNA. Primer sequences were designed using Beacon Designer software (Premier Biosoft International, Palo Alto, CA) and are listed in Supplemental Table 1, published on The Endocrine Society’s Journals Online web site at http://mend.endojournals.org. Thermocycling was performed for 40 cycles (95 C for 15 sec, annealing for 60 sec) after an initial 3-min incubation at 95 C using a Bio-Rad iCycler iQ real-time PCR detection system. Relative mRNA levels were calculated using the {Delta}{Delta}Ct method (51) with the ribosomal protein P0 mRNA as the internal control (52) and the vehicle-treated (EtOH) samples as the calibrator.

ChIP
ChIP assays were performed essentially as previously described (18). The antibodies used for immunoprecipitation are as follows: ER{alpha} (HC-20), HA (Y-11), and SRC-3 (M-397; Santa Cruz Biotechnology, Inc., Santa Cruz, CA). The protocol used for immunoprecipitating SRC-3 is described by Métivier et al. (53). Resulting DNA fragments were purified and eluted in 50 µl Buffer EB (QIAquick PCR purification kit, QIAGEN). Quantitative PCR was performed as described above with the following reaction modifications: 200 nM each of forward and reverse primers and 1 or 4 µl of input or immunoprecipitated DNA, respectively. Sequences of the primers used for amplifying the pS2 and C3 promoters are included in Supplemental Table 1. The relative amount of immunoprecipitated DNA was calculated using the {Delta}{Delta}Ct method (51) with input samples as the internal control and vehicle-treated (EtOH) or IgG immunoprecipitated samples as the calibrator.


   ACKNOWLEDGMENTS
 
We thank Drs. Pierre Chambon, Benita Katzenellenbogen, Zafar Nawaz, and J. Wesley Pike for gifts of reagents. We are also grateful to Drs. Michael Fritsch, Fern Murdoch, Tamara Greco, and Shigeki Miyamoto for insightful discussions and critical reading of the manuscript.


   FOOTNOTES
 
This work was supported by U.S. Department of Defense Breast Cancer Research Program Grant W81XWH-04-1-0327 (to A.M.F.) and National Institutes of Health Grant DK64034 (to E.T.A.).

First Published Online September 22, 2005

Abbreviations: AF-1, Activation function 1; C3, complement component 3; CATD, cathepsin D; ChIP, chromatin immunoprecipitation; CMV, cytomegalovirus; DBD, DNA-binding domain; Dox, doxycycline; E2, 17ß-estradiol; ER, estrogen receptor; ERE, estrogen response element; FBS, fetal bovine serum; HA, hemagglutinin; LBD, ligand-binding domain; PR, progesterone receptor; RID, receptor interaction domain; SRC, steroid receptor coactivator; TAF, TBP-associated factor; TBP, TATA-binding protein; Tet, tetracycline; tk, thymidine kinase.

Received for publication July 13, 2005. Accepted for publication September 14, 2005.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Kumar V, Green S, Stack G, Berry M, Jin J-R, Chambon P 1987 Functional domains of the human estrogen receptor. Cell 51:941–951[CrossRef][Medline]
  2. Webster NJG, Green S, Jin JR, Chambon P 1988 The hormone-binding domains of the estrogen and glucocorticoid receptors contain an inducible transcription activation function. Cell 54:199–207[CrossRef][Medline]
  3. Tora L, White J, Brou C, Tasset D, Webster N, Scheer E, Chambon P 1989 The human estrogen receptor has two independent nonacidic transcriptional activation functions. Cell 59:477–487[CrossRef][Medline]
  4. Lees JA, Fawell SE, Parker MG 1989 Identification of two transactivation domains in the mouse oestrogen receptor. Nucleic Acids Res 17:5477–5488[Abstract/Free Full Text]
  5. Saceda M, Lippman ME, Chambon P, Lindsey RL, Ponglikitmongkol M, Puente M, Martin MB 1988 Regulation of the estrogen receptor in MCF-7 cells by estradiol. Mol Endocrinol 2:1157–1162[Abstract/Free Full Text]
  6. Alarid ET, Bakopoulos N, Solodin N 1999 Proteasome-mediated proteolysis of estrogen receptor: a novel component in autologous down-regulation. Mol Endocrinol 13:1522–1534[Abstract/Free Full Text]
  7. El Khissiin A, Leclercq G 1999 Implication of proteasome in estrogen receptor degradation. FEBS Lett 448:160–166[CrossRef][Medline]
  8. Nawaz Z, Lonard DM, Dennis AP, Smith CL, O’Malley BW 1999 Proteasome-dependent degradation of the human estrogen receptor. Proc Natl Acad Sci USA 96:1858–1862[Abstract/Free Full Text]
  9. Clarke RB, Howell A, Potten CS, Anderson E 1997 Dissociation between steroid receptor expression and cell proliferation in the human breast. Cancer Res 57:4987–4991[Abstract/Free Full Text]
  10. Petersen OW, Høyer PE, van Deurs B 1987 Frequency and distribution of estrogen receptor-positive cells in normal, nonlactating human breast tissue. Cancer Res 47:5748–5751[Abstract/Free Full Text]
  11. Fabris G, Marchetti E, Marzola A, Bagni A, Querzoli P, Nenci I 1987 Pathophysiology of estrogen receptors in mammary tissue by monoclonal antibodies. J Steroid Biochem 27:171–176[CrossRef][Medline]
  12. Riera J, Simpson JF, Tamayo R, Battifora H 1999 Use of cultured cells as a control for quantitative immunocytochemical analysis of estrogen receptor in breast cancer. The Quicgel method. Am J Clin Pathol 111:329–335[Medline]
  13. Gritzapis AD, Baxevanis CN, Missitzis I, Katsanou ES, Alexis MN, Yotis J, Papamichail M 2003 Quantitative fluorescence cytometric measurement of estrogen and progesterone receptors: correlation with the hormone binding assay. Breast Cancer Res Treat 80:1–13[Medline]
  14. Thorpe SM, Christensen IJ, Rasmussen BB, Rose C 1993 Short recurrence-free survival associated with high oestrogen receptor levels in the natural history of postmenopausal, primary breast cancer. Eur J Cancer 29A:971–977
  15. Romain S, Chinot O, Guirou O, Soullière M, Martin P-M 1994 Biological heterogeneity of ER-positive breast cancers in the post-menopausal population. Int J Cancer 59:17–19[Medline]
  16. Sancho-Garnier H, Delarue J-C, Mouriesse H, Contesso G, May-Levin F, Gotteland M, May E 1995 Is the negative prognostic value of high oestrogen receptor (ER) levels in postmenopausal breast cancer patients due to a modified ER gene product? Eur J Cancer 31A:1851–1855
  17. Gaskell DJ, Sangsterl K, Hawkins RA, Chetty U, Forrest APM 1989 Relation between immunocytochemical estimation of oestrogen receptor in elderly patients with primary breast cancer and response to tamoxifen. Lancet 1:1044–1046[Medline]
  18. Fowler AM, Solodin N, Preisler-Mashek MT, Zhang P, Lee AV, Alarid ET 2004 Increases in estrogen receptor-{alpha} concentration in breast cancer cells promote serine 118/104/106-independent AF-1 transactivation and growth in the absence of estrogen. FASEB J 18:81–93[Abstract/Free Full Text]
  19. Lauritsen KJ, List H-J, Reiter R, Wellstein A, Riegel AT 2002 A role for TGF-ß in estrogen and retinoid mediated regulation of the nuclear receptor coactivator AIB1 in MCF-7 breast cancer cells. Oncogene 21:7147–7155[CrossRef][Medline]
  20. Read LD, Greene GL, Katzenellenbogen BS 1989 Regulation of estrogen receptor messenger ribonucleic acid and protein levels in human breast cancer cell lines by sex steroid hormones, their antagonists, and growth factors. Mol Endocrinol 3:295–304[Abstract/Free Full Text]
  21. Teng CT, Gladwell W, Beard C, Walmer D, Teng CS, Brenner R 2002 Lactoferrin gene expression is estrogen responsive in human and rhesus monkey endometrium. Mol Hum Reprod 8:58–67[Abstract/Free Full Text]
  22. Harbison CT, Gordon DB, Lee TI, Rinaldi NJ, Macisaac KD, Danford TW, Hannett NM, Tagne J-B, Reynolds DB, Yoo J, Jennings EG, Zeitlinger J, Pokholok DK, Kellis M, Rolfe PA, Takusagawa KT, Lander ES, Gifford DK, Fraenkel E, Young RA 2004 Transcriptional regulatory code of a eukaryotic genome. Nature 431:99–104[CrossRef][Medline]
  23. Sundstrom SA, Komm BS, Ponce-de-Leon H, Yi Z, Teuscher C, Lyttle CR 1989 Estrogen regulation of tissue-specific expression of complement C3. J Biol Chem 264:16941–16947[Abstract/Free Full Text]
  24. Dutertre M, Smith CL 2003 Ligand-independent interactions of p160/steroid receptor coactivators and CREB-binding protein (CBP) with estrogen receptor-{alpha}: regulation by phosphorylation sites in the A/B region depends on other receptor domains. Mol Endocrinol 17:1296–1314[Abstract/Free Full Text]
  25. Cavarretta ITR, Mukopadhyay R, Lonard DM, Cowsert LM, Bennett CF, O’Malley BW, Smith CL 2002 Reduction of coactivator expression by antisense oligodeoxynucleotides inhibits ER{alpha} transcriptional activity and MCF-7 proliferation. Mol Endocrinol 16:253–270[Abstract/Free Full Text]
  26. List H-J, Lauritsen KJ, Reiter R, Powers C, Wellstein A, Riegel AT 2001 Ribozyme targeting demonstrates that the nuclear receptor coactivator AIB1 is a rate-limiting factor for estrogen-dependent growth of human MCF-7 breast cancer cells. J Biol Chem 276:23763–23768[Abstract/Free Full Text]
  27. Chang C-y, Norris JD, Gron H, Paige LA, Hamilton PT, Kenan DJ, Fowlkes D, McDonnell DP 1999 Dissection of the LXXLL nuclear receptor-coactivator interaction motif using combinatorial peptide libraries: discovery of peptide antagonists of estrogen receptors {alpha} and ß. Mol Cell Biol 19:8226–8239[Abstract/Free Full Text]
  28. Anghel SI, Perly V, Melancon G, Barsalou A, Chagnon S, Rosenauer A, Miller Jr WH, Mader S 2000 Aspartate 351 of estrogen receptor {alpha} is not crucial for the antagonist activity of antiestrogens. J Biol Chem 275:20867–20872[Abstract/Free Full Text]
  29. Schafer JM, Liu H, Bentrem DJ, Zapf JW, Jordan VC 2000 Allosteric silencing of activating function 1 in the 4-hydroxytamoxifen estrogen receptor complex is induced by substituting glycine for aspartate at amino acid 351. Cancer Res 60:5097–5105[Abstract/Free Full Text]
  30. Huang H-J, Norris JD, McDonnell DP 2002 Identification of a negative regulatory surface within estrogen receptor {alpha} provides evidence in support of a role for corepressors in regulating cellular responses to agonists and antagonists. Mol Endocrinol 16:1778–1792[Abstract/Free Full Text]
  31. 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[Medline]
  32. Ince BA, Zhuang Y, Wrenn CK, Shapiro DJ, Katzenellenbogen BS 1993 Powerful dominant negative mutants of the human estrogen receptor. J Biol Chem 268:14026–14032[Abstract/Free Full Text]
  33. Webb P, Lopez GN, Greene GL, Baxter JD, Kushner PJ 1992 The limits of the cellular capacity to mediate an estrogen response. Mol Endocrinol 6:157–167[Abstract/Free Full Text]
  34. Tzukerman MT, Esty A, Santiso-Mere D, Danielian P, Parker MG, Stein RB, Pike JW, McDonnell DP 1994 Human estrogen receptor transactivational capacity is determined by both cellular and promoter context and mediated by two functionally distinct intramolecular regions. Mol Endocrinol 8:21–30[Abstract/Free Full Text]
  35. Struhl G, Struhl K, Macdonald PM 1989 The gradient morphogen bicoid is a concentration-dependent transcriptional activator. Cell 57:1259–1273[CrossRef][Medline]
  36. Jiang J, Kosman D, Ip YT, Levine M 1991 The dorsal morphogen gradient regulates the mesoderm determinant twist in early Drosophila embryos. Genes Dev 5:1881–1891[Abstract/Free Full Text]
  37. Podvinec M, Kaufmann MR, Handschin C, Meyer UA 2002 NUBIScan, an in silico approach for prediction of nuclear receptor response elements. Mol Endocrinol 16:1269–1279[Abstract/Free Full Text]
  38. Fan JD, Wagner BL, McDonnell DP 1996 Identification of the sequences within the human complement 3 promoter required for estrogen responsiveness provides insight into the mechanism of tamoxifen mixed agonist activity. Mol Endocrinol 10:1605–1616[Abstract/Free Full Text]
  39. Lee Y-H, Koh SS, Zhang X, Cheng X, Stallcup MR 2002 Synergy among nuclear receptor coactivators: selective requirement for protein methyltransferase and acetyltransferase activities. Mol Cell Biol 22:3621–3632[Abstract/Free Full Text]
  40. Métivier R, Penot G, Carmouche RP, Hübner MR, Reid G, Denger S, Manu D, Brand H, Kos M, Benes V, Gannon F 2004 Transcriptional complexes engaged by apo-estrogen receptor-{alpha} isoforms have divergent outcomes. EMBO J 23:3653–3666[CrossRef][Medline]
  41. Sadovsky Y, Webb P, Lopez G, Baxter JD, Fitzpatrick PM, Gizang-Ginsberg E, Cavailles 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]
  42. Sabbah M, Kang K-I, Tora L, Redeuilh G 1998 Oestrogen receptor facilitates the formation of preinitiation complex assembly: involvement of the general transcription factor TFIIB. Biochem J 336:639–646[Medline]
  43. Jacq X, Brou C, Lutz Y, Davidson I, Chambon P, Tora L 1994 Human TAFII30 is present in a distinct TFIID complex and is required for transcriptional activation by the estrogen receptor. Cell 79:107–117[CrossRef][Medline]
  44. Wärnmark A, Wikstrom A, Wright APH, Gustafsson J-Å, Hard T 2001 The N-terminal regions of estrogen receptor {alpha} and ß are unstructured in vitro and show different TBP binding properties. J Biol Chem 276:45939–45944[Abstract/Free Full Text]
  45. Hupperets PS, Volovics L, Schouten LJ, Jager JJ, Schouten HC, Hillen HF, Blijham GH 1997 The prognostic significance of steroid receptor activity in tumor tissues of patients with primary breast cancer. Am J Clin Oncol 20:546–551[CrossRef][Medline]
  46. Gruvberger-Saal SK, Edén P, Ringnér M, Baldetorp B, Chebil G, Borg A, Fernö M, Peterson C, Meltzer PS 2004 Predicting continuous values of prognostic markers in breast cancer from microarray gene expression profiles. Mol Cancer Ther 3:161–168[Abstract/Free Full Text]
  47. Van Antwerp DJ, Verma IM 1996 Signal-induced degradation of I{kappa}B{alpha}: association with NF-{kappa}B and the PEST sequence in I{kappa}B{alpha} are not required. Mol Cell Biol 16:6037–6045[Abstract]
  48. Castles CG, Oesterreich S, Hansen R, Fuqua SAW 1997 Auto-regulation of the estrogen receptor promoter. J Steroid Biochem Mol Biol 62:155–163[CrossRef][Medline]
  49. Oesterreich S, Zhang P, Guler RL, Sun X, Curran EM, Welshons WV, Osborne CK, Lee AV 2001 Re-expression of estrogen receptor {alpha} in estrogen receptor {alpha}-negative MCF-7 cells restores both estrogen and insulin-like growth factor-mediated signaling and growth. Cancer Res 61:5771–5777[Abstract/Free Full Text]
  50. Reddel RR, Murphy LC, Sutherland RL 1984 Factors affecting the sensitivity of T-47D human breast cancer cells to tamoxifen. Cancer Res 44:2398–2405[Abstract/Free Full Text]
  51. Livak KJ, Schmittgen TD 2001 Analysis of relative gene expression data using real-time quantitative PCR and the 2–{Delta}{Delta}CT method. Methods 25:402–408[CrossRef][Medline]
  52. Laborda J 1991 36B4 cDNA used as an estradiol-independent mRNA control is the cDNA for human acidic ribosomal phosphoprotein PO. Nucleic Acids Res 19:3998[Free Full Text]
  53. Métivier R, Penot G, Hübner MR, Reid G, Brand H, Kos M, Gannon F 2003 Estrogen receptor-{alpha} directs ordered, cyclical, and combinatorial recruitment of cofactors on a natural target promoter. Cell 115:751–763[CrossRef][Medline]

NURSA Molecule Pages Link:

Nuclear Receptors:   ERα  |  PR
Coregulators:   SRC-1  |  GRIP1  |  AIB1
Ligands:   17β-Estradiol



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