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Endocrinology Vol. 142, No. 5 2039-2049
Copyright © 2001 by The Endocrine Society

ARTICLES

A Splice Variant of Estrogen Receptor ß Missing Exon 3 Displays Altered Subnuclear Localization and Capacity for Transcriptional Activation1

Richard H. Price, Jr.1, Cheryl A. Butler, Paul Webb, Rosalie Uht2, Peter Kushner and Robert J. Handa

Department of Cell Biology, Neurobiology, and Anatomy, Loyola University Chicago (R.H.P.), Maywood, Illinois 60153; Department of Anatomy and Neurobiology, Colorado State University (R.H.P., C.A.B., R.J.H.), Fort Collins, Colorado 80523; and Metabolic Research Unit, University of California (P.W., R.U., P.K.), San Francisco, California 94143

Address all correspondence and requests for reprints to: Dr. Robert J. Handa, Department of Anatomy and Neurobiology, Colorado State University, Fort Collins, Colorado 80523. E-mail: rhanda{at}cvmbs.colostate.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
There are two separate estrogen receptors (ERs), ER{alpha} and ERß. The ERß gene is variably spliced, and in some cases variant expression is high. Besides the full-length ERß (equivalent to ERß1), splice variants can encode proteins bearing an insert within the ligand-binding domain (ß2), a deletion of exon 3 (ERß1{delta}3) disrupting the DNA-binding domain, or both (ERß2{delta}3). Here we examine the intracellular localization and transcriptional properties of each of the ERß splice variants heterologously expressed in cultured cells. In accordance with ER{alpha}, ERß1 and ERß2 are both distributed in a reticular pattern within the nucleus after exposure to ligand. In contrast, ERß1{delta}3 and ERß2{delta}3 localize to discrete spots within the nucleus in the presence of ER agonists. In the presence of ER antagonists, the {delta}3 variants are distributed diffusely within the nucleus. We also show that the spots are stable nuclear structures to which the {delta}3 variants localize in a ligand-dependent manner. Coactivator proteins of ER colocalize with {delta}3 variants in the spots in the presence of agonists. The {delta}3 variants of ERß can activate luciferase reporter constructs containing an activator protein complex-1 site, but not an estrogen response element (ERE). These data suggest that without an intact DNA-binding domain, ERß is functionally altered, allowing localization to discrete nuclear spots and activation from activator protein-1-containing reporter genes.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE ESTROGEN receptor (ER) is a ligand-activated transcription factor and a member of the nuclear receptor superfamily (1). The biological effects of estrogen are mediated by two forms of ER that are encoded by separate genes, ER{alpha} and ERß (2). Multiple splice variants of both ER{alpha} and ERß exist (3, 4). Splice variants of ER{alpha} messenger RNA (mRNA) are expressed at low levels in only the pituitary and some tumors (3). In contrast, we (5, 6) and others (4) have shown that splice variants of ERß mRNA are expressed in multiple tissues and in some cases at levels equivalent to or exceeding those of the full-length mRNA. The high expression level of some of the ERß mRNA splice variants suggests that if corresponding proteins are expressed, they too would be abundant. A recent report (7) demonstrates that multiple ERß variants can be seen with ERß-specific antisera and Western blot analysis of proteins derived from ovary, a tissue known to express ERß at high levels (8, 9). Therefore, the splice variants of ERß must be considered when assessing ERß function.

There are at least four variants of ERß mRNA, including the originally described wild-type form (ERß1). Transcripts designated ERß2 possess an in-frame insertion of 54 nucleotides between exons 5 and 6 that encode an additional 18 amino acids (aa) in the ligand-binding domain. The ERß2 variants bind hormone with a lower affinity as would be suggested by alteration of the ligand-binding domain (LBD) (4, 10). A deletion of exon 3 (designated by a {delta}3 after ß1 or ß2) corresponds to an in-frame loss of 117 nucleotides that encode 39 aa in the carboxyl-terminal half of the DNA-binding domain (DBD), including the second zinc finger. Consistent with this, transcriptional activation at estrogen response elements (ERE) requires a 100- to 1000-fold greater concentration of estrogen for full activation (4, 10). The {delta}3 variants do not bind to the classical ERE, and as a consequence, these forms are not active at an ERE. This region has been shown to be important for normal intranuclear localization of other nuclear receptors (11).

The ability of ERß to enhance transcription from promoters containing an ERE is similar to that of ER{alpha} (2). However, both ERs also enhance transcription by modulating the activity of the activator protein complex-1 (AP-1) (12). An important difference exists between ER{alpha} and ERß concerning activation through AP-1 sites. ER{alpha} is able to activate transcription from activator protein-1 (AP-1)-containing promoters in the presence of agonists, such as estradiol (E2) or diethylstilbestrol (DES), and the partial agonist/antagonist tamoxifen. In contrast, ERß is only able to activate transcription from AP-1 sites in the presence of antagonists (13).

The topic of the intracellular localization of ER has long been controversial. However, knowledge of the intracellular localization of nuclear receptors in living cells has been advanced recently by the use of chimeric proteins that have fused the Aquorea green fluorescent protein (GFP) to either the NH3- or COOH-terminus of a nuclear receptor. Such a strategy has been used to examine the intracellular distribution of glucocorticoid receptor (GR) (14), mineralocorticoid receptor (MR) (15), progesterone receptor (16), androgen receptor (17), thyroid hormone receptor (18), vitamin D receptor (19), and ER{alpha} (20). The use of GFP to monitor intracellular localization of nuclear receptors has allowed confirmation and extension of many previous studies that have painstakingly tracked their intracellular localization. For instance, androgen receptors, GR, and MR are found in the cytoplasm in the absence of hormone, whereas ER{alpha} has been localized largely to the nucleus regardless of whether hormone is present.

Fluorescent ligands can also be used for ER visualization. Coumestrol, a phytoestrogen, has been shown to emit a bright fluorescent signal after near UV excitation when bound to overexpressed ER{alpha} (21). When not bound to an ER, the fluorescent emission from coumestrol is nearly undetectable. Of particular significance to the present study, coumestrol has been recently shown to have a higher affinity for ERß than ER{alpha} (4, 22).

In this study we determined the intracellular localization of ERß variants derived from naturally occurring splice variants. We identify a novel trafficking pattern of ERß. In the presence of ER agonists, both ERß1{delta}3 and ERß2{delta}3 localize to discrete nuclear spots. This distribution is rapidly reversed by ER antagonists. We have also discovered that {delta}3 variants of ERß exhibit estrogen-dependent activation at AP-1-responsive reporters.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Vectors
Expression vectors bearing ERß1 (485 aa form) and splice variants (ERß2, ERß1{delta}3, and ERß2{delta}3) were obtained from Dr. T. Brown (Pfizer, Inc., Groton, CT). pEGFP C1 (CLONTECH Laboratories, Inc., Palo Alto, CA) was used as a GFP expression vector. The ERß splice variant coding sequences were inserted into the HindIII and ApaI sites downstream of the EGFP-coding sequence. Subsequent insertion of a PCR product, generated from rat complementary DNA (cDNA) using an upstream primer that overlapped the recently described 530-aa start site [containing a BglII site at the 5'-end and a downstream primer within the originally published A/B domain (13); nucleotides 756–776], served the dual purpose of maintaining the reading frame from EGFP to ERß and lengthening the expressed ERß from the originally described (2) 485 aa to the revised length of 530 aa. pEGFPc2 containing GR-interacting protein (GRIP1) was a gift from Dr. Steve Nordeen (University of Colorado Health Science Center, Denver, CO).

Cell culture and transfection
Cell lines. Chinese hamster ovary (CHO), African Green monkey kidney (COS-7; both gifts from Dr. S. Nelson, Colorado State University), human embryonic kidney (HEK-293), human uterine epithelial adenocarcinoma (HeLa; both gifts from Dr. K. Stroffokova, Colorado State University), immortalized embryonic muscle cells (BWEM; a gift from Dr. G. Engelman, Loyola University Chicago, Maywood, IL), and a neuroendocrine cell line (GT1–1; a gift from Dr. R. Weiner, University of California, San Francisco, CA) were grown in DMEM (Life Technologies, Inc., Gaithersburg, MD) without phenol red and supplemented with FBS (2 x charcoal stripped where noted; Life Technologies, Inc.), L-proline (100 mM; Sigma, St. Louis, MO), and gentamicin (25 mg/ml; Life Technologies, Inc.). All cells were transfected with expression vectors (1 µg/35-mm dish) using Lipofectamine (5 µl/35-mm dish; Life Technologies, Inc.). Twelve hours after transfection, cells were trypsinized and replated to poly-L-lysine (0.25 mg/ml; Sigma)-coated glass coverslips for fluorescence microscopy.

Steroid treatments. Coumestrol (Sigma), E2 (Sigma), DES (Sigma), a nonsteroidal antagonist CN-55 (23), genistein (GEN; Sigma/RBI), and testosterone (Steraloids, Newport, RI) were diluted into DMEM from 1-mM ethanol stocks. Transfected CHO cells maintained on coverslips in culture dishes were treated with steroids or other ligands according to the particular experiment. An association curve revealed that coumestrol fluorescence is first visible at 1 x 10-10 M with ERß1 and at 1 x 10-9 M with both ERß2 and ER{alpha}. However, for routine labeling purposes coumestrol concentrations were 1 x 10-6 M for 30 min before fixation.

Immunocytochemistry
Antisera specific for ERß (PA1–310), GFP, and GRIP-1 were obtained from Affinity BioReagents, Inc. (Golden, CO). Those specific for CREB-binding protein (CBP) and promyelocytic leukemia (PML) were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). All immunocytochemistry on cultured cells grown on glass coverslips was performed in six-well dishes. Briefly, cells were fixed with 10% buffered formalin for 5 min at room temperature. After extensive rinses in PBS, cells were permeabilized in 0.5% Triton X-100 in PBS for 5 min, then incubated in blocking solution (2% BSA/0.05% Tween 20 in PBS) for 30 min. After blocking, coverslips were incubated for 2 h in primary antiserum (1:500 for anti-ERß, anti-CBP, and anti-PML). Cells were again rinsed extensively in PBS and then incubated for 1 h in either antirabbit or antimouse IgG both conjugated with Texas Red. After extensive washing, coverslips were mounted onto glass slides for fluorescence microscopy using an aqueous mounting medium (50% glycerol in PBS).

Fluorescence microscopy
Fixed cells. Coverslips were fixed and inverted onto glass slides with an aqueous mounting medium (50% glycerol in PBS), and viewed using the differential interference contrast optics and epifluorescence (GFP using fluorescein isothiocyanate filters, coumestrol using near UV excitation, and broad pass emission) on a Carl Zeiss Axiophot microscope (New York, NY).

Quantification of intranuclear distribution of fluorescence. CHO cells were grown on glass coverslips and then transiently transfected with one of each of the four GFP-ERß constructs. Twenty-four hours after transfection, cells were exposed to ligands at the indicated concentrations (either 1 or 100 nM for E2, 10 nM or 1 µM for tamoxifen). One hundred cells showing fluorescence from each coverslip were examined, and the nuclear distribution of GFP fluorescence was rated as either punctate or diffuse (examples are shown in Fig. 4Go).



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  		Figure 4. ER ligands alter the localization of GFP-ERß1{delta}3 and GFP-ERß2{delta}3 within the nucleus of transfected cells. A, CHO cells overexpressing either GFP-ERß1{delta}3 (left plots) or ERß2{delta}3 (right plots) were exposed to either E2 (upper plots) or tamoxifen (lower plots) for the indicated times, and nuclear fluorescence was scored as punctate or diffuse. B, Plot showing the number of nuclei (per 100) with either punctate or diffuse fluorescence within the nuclei of CHO cells transfected with GFP-ERß1{delta}3 after 30-min exposure to the ER agonists DES (100 nM) and GEN (100 nM) or the ER antagonist CN-55 (100 nM). All values are reported as the number of cells that expressed punctate nuclear fluorescence per 100 counted (described as percentage of punctate in text) and are representative of results obtained from at least two separate experiments.

 
Visualization of GFP in live cells. Cells were grown as described above, except that glass coverslips were affixed to 35-mm culture dishes that and had the center of their plastic growth surface drilled out (here termed viewing plates). This provided a growth surface that was thin enough and adequate for high power inverted epifluorescence microscopy. Live imaging was accomplished by using a Nikon Diaphot inverted microscope (Melville, NY) with a heated and motorized stage controller. Illumination through fluorescein isothiocyanate filters was via a mercury vapor lamp. Sixteen-bit images (x2 photomultiplied) were captured with a photometrics cooled chip camera with a x100 objective (Carl Zeiss). Both illumination and image capturing were controlled using Metamorph software (version 3.1, Universal Imaging Corp., Downington, PA). Twenty-four to 48 h after transfection (described above) the medium in the viewing plates was changed to 2 ml phenol-red free medium including 10 mM HEPES, which enabled better room air buffering than bicarbonate alone (regular DMEM). Plates were removed from the incubator just before imaging and transferred immediately to the heated stage. Once a transfected cell was located with the eyepieces, light was diverted to the Photometrics camera, and exposure could be controlled with Metamorph. Exposures were typically 1 sec each. All digital images were captured and converted to output tagged image format files with Metamorph. Time series were acquired with Metamorph to preset (typically 15-sec) intervals. Pharmacological manipulation of cells was accomplished by applying 100-fold hormone solutions (1 mM hormone in 95% ethanol stocks were diluted accordingly in phenol red-free DMEM without serum).

Binding studies
Preparation of extracts. CHO cells were grown in phenol red-free medium supplemented with 2 x charcoal-stripped FBS on 100-mm culture plates until 60–80% confluence was reached. The cells were then transfected with a control vector (pEGFPc1), the non-GFP-tagged ERß expression vectors (pcDNA-ERß1, pcDNA-ERß2, pcDNA-ERß1{delta}3, pcDNA-ERß2{delta}3), or the GFP-tagged ERß-containing expression vectors (GFP-ERß1, GFP-ERß2, GFP-ERß1{delta}3, GFP-ERß2{delta}3). Cells were harvested by trypsinization and pelleted at 0.5 x g for 5 min. The pelleted cells were resuspended in ice-cold TEGMD (10 mM Tris-Cl, 1.5 mM EDTA, 10% glycerol, 25 mM molybdate, and 1 mM dithiothreitol, pH 7.4) buffer (250 µl) and homogenized with glass homogenizers (Dounce Co., Vineland, NJ). Homogenates were then centrifuged at 40,000 rpm (100,000 x g) in an ultracentrifuge (Beckman Coulter, Inc., Palo Alto, CA) with a fixed angle rotor (Sorvall TI 60, Dupont-Sorvall, Wilmington, DE) for 15 min at 4 C to separate the extract from the nuclear and membrane fractions.

Saturation isotherms. One hundred-microliter aliquots of supernatant (extract) were incubated for 4 h at room temperature with increasing (0.01–50 nM) concentrations of [3H]E2. Nonspecific binding was assessed using a 200-fold excess of the ER agonist, DES, in parallel tubes. After 4 h, bound and unbound [3H]E2 was separated by passing the incubation reaction through a 1-ml lipophilic Sephadex LH-20 column. Columns were constructed by packing a disposable pipette tip (1 ml; Labcraft, Curtin Matheson Scientific, Inc., Houston, TX) with TEGM-saturated Sephadex according to a previously published protocol (24). For chromatography, the columns were reequilibrated with TEGMD (100 µl), and the incubation reactions were added individually to each column and allowed to incubate for 30 min at 4 C. After the 30-min incubation, 600 µl TEGMD were added to each column, flow-through was collected, 4 ml scintillation fluid were added, and samples were counted (5 min each) in an LS 7000 scintillation counter (Beckman Coulter, Inc.).

Competition studies. For competition studies, extracts were incubated with 0.5 nM [3H]E2 in the presence of increasing concentrations of E2 (0.1 nM to 10 nM) and tamoxifen (1–100 nM) or high concentrations (10 nM) of testosterone or progesterone.

Transcription assays
Vectors used. Reporter vectors have been described previously (25). Expression vectors containing ERß splice variants both with and without GFP were as described above.

Cell culture and transfection. HeLa, CHO, and MCF-7 cells were used for transcriptional activation studies. Each cell type was grown in phenol-red free DMEM supplemented with 10% FBS (2 x charcoal stripped). Cells were initially grown on 100-mm plates. Just before reaching confluence, cells were trypsinized, pelleted by centrifugation (1000 x g), resuspended in 1 ml/100-mm plate and counted. Two million cells were used per transfection. For transfection by electroporation, cells were again pelleted, resuspended in electroporation buffer (PBS supplemented with 5% dextrose) to a concentration of 2 million cells/0.5 ml, and transferred to electroporation cuvettes (Bio-Rad Laboratories, Inc.). Vectors were then added depending on the experiment, and cuvettes were subjected to 0.24 kV each. After a brief recovery, cells were plated to either 6- or 12-well plates (2 million cells divided equally across the 6 or 12 wells) and exposed to either steroid or vehicle. For transfection by liposome, CHO cells were plated onto 60-mm dishes 24 h before transfection with Lipofectamine (12 µl/ml; Life Technologies, Inc.). DNA was combined with the Plus reagent and then with Lipofectamine before exposure to the cells. Incubation with Lipofectamine-DNA complexes was carried out for 5 h in serum- and antibiotic-free medium. After the transfection, cells were allowed to recover for 4 h in fresh medium supplemented with 10% FBS (2 x stripped) and 25 µg/ml gentamicin. The cells were then trypsinized and replated to 12- or 6-well plates where they were treated with hormone or vehicle.

Twenty-four to 48 h after plating, cells were washed twice with cold PBS and disrupted with a passive lysis buffer (Tris-Cl, 0.5% Triton X-100, and 2.5 mM dithiothreitol; 0.2 ml/well for a 6-well plate; 0.1 ml/well for a 12-well plate). The cell lysates were then stored at 4 C until assayed for luciferase and ß-galactosidase expression. Luciferase expression levels were assayed from a 20-µl aliquot of the lysate in a luminometer (Promega Corp.) following combination with the assay reagent (50 µl) from a firefly luciferase kit (Promega Corp.). Transfection efficiency was assessed by assaying ß-galactosidase activity. Luciferase expression levels were expressed as relative light units after correction based on transfection efficiency (the ß-galactosidase activity of the condition with the greatest transfection efficiency was set at 1 and the others were converted to fractions thereof).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
ERß variants show unique intranuclear trafficking patterns in cultured cells
ERs are known to be nuclear even in the absence of ligand (20). Recently, we and others have found immunocytochemical evidence for localization of ERß in the cytoplasm of neuronal cells (5, 26). Therefore, we sought to determine the intracellular localization of ERß splice variants. We used fluorescence microscopy to monitor CHO cells that were transiently transfected with ERß splice variants fused to GFP. Our initial observations indicated that the precise nuclear localization pattern could be altered depending on whether the maintenance medium was stripped of steroids (see Table 1Go). Therefore, subsequent experiments were conducted using medium that was stripped of steroids and only then supplemented with specific hormones.


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  		Table 1. Intranuclear localization patterns of transiently expressed GFP-ERß splice variants in CHO cells grown in whole or stripped serum

 
Cells that express GFP alone exhibit a uniform distribution of green fluorescence throughout the cytoplasm and nucleus (Fig. 1Go, A and B). The presence of 100 nM E2 did not alter the localization of GFP (not shown). However, in the absence of steroids, ER{alpha}, ERß1, and ERß2 fused to GFP (Fig. 1Go, C, E, G, respectively) show exclusively diffuse nuclear localization. When exposed to 100 nM E2 for 20 min, each of these ER isoforms relocalizes in a reticular or hyperspeckled distribution within the nucleus (Fig. 1Go, D, F, and H).



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  		Figure 1. GFP-ERß splice variants localize differentially within the nucleus of transiently transfected cells (CHO cells are used in all figures unless otherwise noted). CHO cells were transfected with GFP alone (A and B), ER{alpha} (C and D), and the indicated splice variants of ERß (E-L). Images in C–H were subjected to digital deconvolution to enhance visualization of intranuclear distribution. In the left panels (C, E, G, I, and K), cells were maintained in medium containing 2 x charcoal-stripped FBS after transfection. The right panels (D, F, H, J, and L) show localization after exposure to 100 nM E2 for 20 min. The scale bar in A = 10 µm and applies to each panel. Schematic diagrams at the left show the protein-encoding regions (boxes; deletions indicated by single line; insertions are shaded gray) of each splice variant used in this study.

 
Curiously, the distribution of GFP-ERß1{delta}3 and GFP-ERß2{delta}3 differed considerably. In the absence of steroids, localization of GFP-ERß1{delta}3 exists in one of two states. In approximately 25% of the cells, the distribution of fluorescence was in discrete spots that were fewer in number and larger than the hyperspeckles seen above for ER{alpha} and ERß1 or ERß2. The rest of the nuclei expressing GFP-ERß1{delta}3 showed diffuse localization in the absence of hormone. In the presence of 100 nM E2, nearly all nuclei displayed the discrete spotted pattern. ERß2{delta}3 differed slightly, as all transfected nuclei showed diffuse fluorescence in the absence of steroids (Fig. 1KGo). This isoform similarly shows the dramatic hormone-dependent localization to spots (Fig 1LGo). The same localization pattern can be seen in many different cell lines (HeLa, COS-7, HEK-293, GT1–1, and BWEM; not shown). In summary, ERß1 and ERß2 isoforms are nuclear and, like ER{alpha}, relocalize in a hyperspeckled distribution after exposure to hormones. The variants missing exon 3 localize to larger, discrete spots within the nucleus in a ligand-dependent manner.

The GFP tag does not alter [3H]E2 binding characteristics of ERß splice variants
To ensure that the transiently transfected GFP-ERß variants are able to specifically bind estradiol, we conducted [3H]E2 binding assays. First, extracts from transfected CHO cells were isolated, and estrogen binding affinity was determined using a Scatchard analysis. Specific Kd values are shown in Fig. 2AGo. No specific binding was seen when CHO extract alone was incubated with [3H]E2. These results are equivalent to those previously reported for the non-GFP-tagged variants (4). Notably, the ß2 variants bind estrogen with lower affinity, and {delta}3 does not affect ligand binding. Similarly, the specificity of binding is not altered by the GFP tag. [3H]E2 binding to GFP-ERß variants is competed by concentrations of E2 and tamoxifen ranging from 0.5–10 nM (E2) and 1–100 nM (tamoxifen). No competition is seen when other gonadal steroids, testosterone and progesterone (10 nM), are coincubated with receptor and [3H]E2 (Fig. 2BGo). These results confirm that ERß splice variants can bind hormone normally.



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  		Figure 2. GFP-ERß splice variants expressed after transient transfection are able to bind [3H]E2 normally. A, Saturation isotherms (left) and Scatchard analyses (right) indicate that GFP-ERß1 and GFP-ERß1{delta}3 both bind estradiol. B, Competition studies show that GFP-ERß1 binding is specific. Both unlabeled E2 ({square}) and unlabeled tamoxifen () compete with [3H]E2 for GFP-ERß1, whereas testosterone () and progesterone ({blacktriangleup}) do not.

 
Intranuclear trafficking of ERß splice variants can be visualized with coumestrol
The surprising distribution of GFP-tagged ERß variants missing exon 3 prompted us to ask whether non-GFP-tagged ERß splice variants localize in a similar fashion. CHO cells were transfected with expression vectors encoding ERß variants without fused GFP and were incubated for 1 h with the ER agonist coumestrol (1 x 10-8 M), which emits bright blue fluorescence under near UV excitation when bound to an ER (21). No coumestrol fluorescence was observed when cells were mock transfected (not shown). However, bright blue fluorescence was visible from coumestrol-exposed ER{alpha}, ERß1, and ERß1{delta}3 (Fig. 3Go, A–C; respectively). The distribution of each is similar to that seen when their GFP-tagged counterparts are exposed to agonist.



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  		Figure 3. Specific coumestrol binding reveals the intracellular localization of non-GFP-tagged ERß splice variants. Photomicrographs of fluorescence from coumestrol in CHO cell nuclei expressing ER{alpha} (A), ERß1 (B), and ERß1{delta}3 (C). The scale bar in A = 10 µm and applies to A–F. The specificity of coumestrol binding is shown in D–F: coumestrol fluorescence from a CHO cell expressing ERß1 exposed to coumestrol alone (10-9 M; D), coumestrol (10-9 M) plus E2 (10 -8 M; E), or coumestrol (10-9 M) plus testosterone (10-6 M; F). G–I, GFP, coumestrol, and red immunofluorescence, respectively, from two adjacent CHO cells expressing GFP-ERß1. The scale bar in G = 10 µm and applies to G–I.

 
To determine whether coumestrol fluorescence results specifically from binding to an ER, we conducted an experiment with E2 and testosterone as nonfluorescent competitors. Normal coumestrol fluorescence (Fig. 3DGo) can be attenuated by E2 (Fig. 3EGo), but not testosterone (Fig. 3FGo). The specificity of coumestrol fluorescence as a marker of ERß-expressing cells was further confirmed by immunostaining GFP-ERß1-transfected cells with a specific ERß antiserum. The distribution of GFP and coumestrol fluorescence (Fig. 3Go, G and H) from GFP-ERß1 matched the red fluorescence from the immunostaining (Fig. 3IGo). These results show that GFP does not alter the localization of the ERß splice variants, and they support the use of GFP fusions as a useful marker of the ERß splice variants within transfected cells.

ER agonists enhance the punctate nuclear distribution of {delta}3 variants of ERß
Our initial experiments suggested that the intranuclear distribution of {delta}3 variants of ERß is dependent on whether agonist or antagonist was present. Further, the presence or absence of the ß2 insertion within the LBD (ERß1{delta}3 vs. ERß2{delta}3) offered us a chance to evaluate the effect of ligand on intranuclear distribution of the {delta}3 variants because of their differing abilities to bind ligand (see Fig. 2AGo). Therefore, we incubated transfected cells with ERß agonists and antagonists and recorded the percentage of cells that showed punctate fluorescence within the nucleus. We first determined the time course of {delta}3 isoform redistribution within the nucleus. Transfected cells were exposed to low and high concentrations of agonist (E2) or antagonist (tamoxifen) for 0, 5, 20, or 60 min, then formalin-fixed and evaluated with fluorescence microscopy. Addition of E2 to the medium caused a change in the overall percent punctate value for both GFP-ERß1{delta}3 and GFP-ERß2{delta}3. As shown in Fig. 4AGo, redistribution occurs in a time- and concentration-dependent manner. The low concentration of E2 (1 nM) causes accumulation of GFP-ERß1{delta}3 to nuclear spots in a majority of cells within 60 min, whereas only one third of GFP-ERß2{delta}3-expressing cells have punctate fluorescence within 60 min. The high concentration of E2 (100 nM) allows accumulation of GFP-ERß1{delta}3 to spots within 20 min and GFP-ERß2{delta}3 to spots within 60 min. Therefore, it appears that the altered LBD (ß2) does change the kinetics of ERß redistribution in the presence of estrogen.

Figure 4AGo also shows that some cells expressing GFP-ERß1{delta}3 in the absence of hormone have fluorescence in nuclear spots. This may be a result of low levels of residual E2 or stimulation of other ER activation pathways. Differences between ß1 and ß2 forms suggest the former. The presence of {delta}3 in spots in the absence of added ligand allowed us to evaluate the effect of tamoxifen on intranuclear distribution. The high (1 µM) concentration of tamoxifen reduced the percentage of transfected cells with fluorescence in nuclear punctae to 7% within 5 min, whereas the lower concentration of tamoxifen (10 nM) required up to 20 min to reduce the number of cells with punctate nuclear fluorescence to an equivalently low level (4%).

Other ER ligands were screened for their ability to induce subnuclear relocalization of the {delta}3 variants. Saturating concentrations (100 nM) of DES, GEN, and a compound related to tamoxifen, CN-55, each showed distinct abilities to induce punctate {delta}3 localization when exposed to GFP-ERß1{delta}3-transfected cells for 30 min. The pure agonist DES induced a punctate nuclear distribution in 79%, GEN in 68%, and CN-55 in less than 5% of cells after 30 min (Fig. 4BGo). These results suggest that accumulation of GFP-ERß1{delta}3 and GFP-ERß2{delta}3 into nuclear spots is enhanced after exposure to ER agonists and reduced by ER antagonists, and further that the redistribution occurs in a time- and concentration-dependent manner. The difference between ß1 and ß2 forms of the {delta}3 variants further suggest that the relative ability to bind ligand is crucial to the mechanism governing intranuclear localization.

Trafficking of {delta}3 variants to nuclear spots is dynamically regulated by ER agonists and antagonists
We next monitored redistribution dynamics by imaging the same cell over time to determine whether the redistribution process is reversible. We also wanted to determine whether the spots are stable structures within the nucleus to which the agonist-bound {delta}3 variants localize before and after disruption by antagonist. To observe {delta}3 isoforms of ERß being redistributed in the nucleus in real time, we used live cell fluorescence imaging (see Materials and Methods). We used GFP-ERß1{delta}3-transfected cells and a high concentration of ligands to achieve rapid relocalization. Figure 5AGo shows that E2 (100 nM) can induce a punctate nuclear localization of {delta}3 within 10 min. To observe tamoxifen’s effect on the {delta}3 spots, we chose cells that express {delta}3 in spots even in the absence of any hormone (about one quarter of the transfected cells, as described in Table 1Go and Figs. 1IGo and 4BGo). Figure 5BGo shows that 1 µM tamoxifen is able to disperse GFP-ERß1{delta}3 from nuclear spots within 5 min.



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  		Figure 5. Intranuclear distribution of ERß1{delta}3 is dynamically regulated by estradiol and tamoxifen. A and B, Successive fluorescence images taken at 5-min intervals after exposure to E2 (100 nM; A) or tamoxifen (1 µM; B). Before hormone exposure, transfected cells with diffuse (A) and punctate (B and C) localization patterns were selected for real-time imaging. For tamoxifen-induced changes in localization (B and C) cells were chosen that showed fluorescence that was already organized into spots. Successive fluorescence images of an HEK-293 cell nucleus (results were identical with those for CHO cell; not shown) expressing GFP-ERß1{delta}3 taken at 5-min intervals after exposure to tamoxifen (1 µM at 0 min) and then estrogen (100 nM at 10 min). The scale bar in C = 10 µm and applies to all panels.

 
We next addressed whether the {delta}3 nuclear spots, dispersed by tamoxifen, could be reassembled into the same nuclear spots when exposed to E2. Figure 5CGo shows that GFP-ERß1{delta}3 in nuclear spots can be disrupted by tamoxifen and subsequently reassembled to the same spots after exposure to E2. These findings show that {delta}3 variants of ERß are rapidly and reversibly distributed into stable nuclear compartments in a ligand-dependent manner.

ERß variants colocalize with the nuclear receptor coactivator proteins, GRIP1 and CBP
Previous studies have shown that coactivator proteins involved in ER trans-activation also localize to nuclear spots. To determine whether ER coactivators localize to the same nuclear spots as ERß1{delta}3, we performed multilabeling studies. Two examples of such coactivators are GRIP-1 and CBP (27, 28). We found that there is a good correspondence between the localization of ERß1 and GFP-GRIP-1 in the presence of coumestrol (Fig. 6Go, A and B). We also found that in all cases, when expressed in the same cell, GFP-GRIP1 and ERß1{delta}3 (as visualized with coumestrol fluorescence) colocalized to the same punctate nuclear structures (Fig. 6Go, C and D). In addition, when ERß1{delta}3 was expressed in CHO cells and CBP was identified with immunocytochemistry, there was excellent correspondence between the subnuclear distribution of each (Fig. 6Go, E and F). We are also able to detect ERß1 and CBP distributed in a similar manner within the nucleus of transfected cells (data not shown). Finally, we used an antiserum to PML protein, a suspected transcription factor that defines nuclear domains called PODs (PML oncogenic domains) (28). When ERß1{delta}3 was visualized with PML in the same cell there was not complete colocalization (62 ± 8% of the PML spots were also ERß1-{delta}3 positive; see Fig. 6Go, G and H). Thus, in the presence of an ER agonist (coumestrol) both ERß1 and ERß1{delta}3 can colocalize with the coactivators GRIP1 and CBP.



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  		Figure 6. ERß variants colocalize with the coactivators GRIP1 and CBP. ERß variants are shown in the right panels (B, D, F, and H) and are visualized with coumestrol fluorescence. A and B, Micrographs of a CHO cell coexpressing GFP-GRIP1 and ERß1. C and D, CHO cell coexpressing GFP-GRIP1 and ERß1{delta}3. E and F, CHO cell expressing ERß1{delta}3 and CBP (visualized by immunofluorescence using specific primary antibody and a Texas Red-coupled secondary antibody). G and H, A HeLa cell expressing ERß1{delta}3 (coumestrol) and endogenous PML (visualized with immunofluorescence). The scale bar in A = 10 µm and applies to A–D. Bars in E apply to E and F, and those in G apply to G and H; both = 10 µm.

 
ERß splice variants activate transcription from artificial promoter-reporter constructs
Finally, we wanted to determine whether {delta}3 variants of ERß have any unique transcriptional properties. ERß is known to activate transcription from at least two classes of promoter response element, the classical ERE and the activator protein complex (AP-1) response element (13). Thus, splice variants of ERß were tested for their ability to activate transcription of the reporter gene firefly luciferase under the control of either EREs or AP-1 sites.

First, the ability of the ERß splice variants to activate transcription from an ERE-containing reporter gene was tested. As shown in Fig. 7AGo, in the absence of an ER, there was no E2 induction of the reporter. In contrast, transiently expressed ERß1 and ERß2 activated luciferase expression from the ERE-containing promoter in response to 10 nM estrogen (at least 4-fold over vehicle-treated cells). Neither ERß1{delta}3 nor ERß2{delta}3 activate transcription from an ERE-containing promoter (Fig. 7AGo), as might be deduced from their inability to bind to DNA. Importantly, the GFP-tagged ERß variants do not differ significantly from their non-GFP-tagged counterparts (Fig. 7AGo). Thus, the present results agree with previous studies of ERß splice variants at ERE-containing reporters (4).



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  		Figure 7. ERß splice variants activate transcription from estrogen-responsive promoters. A, Effect of either tamoxifen or E2 on luciferase expression in HeLa cells cotransfected with the ERE-luciferase reporter and either no ER or the indicated ERß splice variant. For all transfections, activation of luciferase was corrected for transfection efficiency with ß-galactosidase activity levels (Rous sarcoma virus-ß-galactosidase was cotransfected with each), and all data are represented accordingly as relative light units. GFP-tagged ERß1 and GFP-ERß1{delta}3 are shown at the right for comparison with their non-GFP-tagged counterparts. B, Effect of no hormone, tamoxifen, or E2 on luciferase expression in HeLa cells cotransfected with the AP-1 ({delta}col73-luciferase) reporter and either no ER or the indicated ERß splice variant. All results shown are representative of at least three separate transfection experiments.

 
ERß splice variants were also tested for their ability to activate an AP-1-luciferase reporter. ERß1 and ERß2 only activate the reporter in the presence of the ER antagonist tamoxifen (~4-fold over no hormone). In agreement with previous studies, the agonist E2 has no effect (see Fig. 7CGo). Interestingly, ERß1{delta}3 and ERß2{delta}3 both activate the AP1-LUC reporter in the presence of E2 (2- to 4-fold over no hormone), but not tamoxifen. This contrasts with their inability to activate transcription from an ERE as shown above. As with activation of the ERE-containing reporter, GFP-tagged variants do not differ significantly from their non-GFP-tagged counterparts. Thus, unlike full-length ERß, the {delta}3 variants of ERß exhibit the ability to enhance AP-1-responsive transcription in the presence of estrogens.


   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This report describes the intracellular localization and transcriptional activation of ERß splice variants when transiently expressed in cultured cells. For localization studies, we have primarily used N-terminally fused GFP which we and others (29) show does not have an effect on the function of the tagged protein. We find that ERß1 and ERß2 localize to the nucleus in a distribution that is highly reminiscent of ER{alpha} (20, 30). In the presence of ER agonists (coumestrol, E2) this distribution is reticular/hyperspeckled within the nucleus, distinguishing it from the diffuse distribution seen in the absence of any hormone. Although not previously reported for ERß, this reticular/hyperspeckled distribution of full-length ER has been reported for ER{alpha} (30). Surprisingly, our results show that protein products from the ERß splice variants lacking exon 3 ({delta}3) localize to discrete punctae (spots) in the nucleus in a ligand-dependent manner. Importantly, these discrete spots are larger and less numerous than the hyperspeckles seen when full-length ERß is bound by ligand. The assembly of {delta}3 variants to the discrete intranuclear spots is enhanced by ER agonists and disrupted by antagonists. Real-time imaging has revealed that this redistribution process is rapid and reversible. This discrete intranuclear distribution and its dynamic response to ligands are quite different from those seen with ER{alpha}, ERß1, and ERß2 and, importantly, have not been previously reported. We also show that unlike full-length ERß, the {delta}3 variants are able to activate transcription from AP-1 sites in the presence of natural ER agonists.

Previously, investigators have localized androgen receptor, MR, progesterone receptor (A and B forms), thyroid hormone receptor ({alpha} and ß forms), and vitamin D receptor in living cells by using GFP fusions (14, 15, 16, 17, 18, 19). The majority of these reports suggest that when localized to the nucleus, the distribution of nuclear receptors is reticular, conforming to previous studies using localization by immunofluorescence (31). Importantly, all of the previously described nuclear receptors are at least partially cytoplasmic in the absence of ligand. In distinction, ER{alpha} (20, 30) and ERß (herein) are both present in the nucleus even in the absence of ligand. Thus, our results support the idea that ER ({alpha} and ß forms) are nuclear and contribute to the growing literature describing discrete localization of nuclear receptors using fusions with GFP.

Our finding that the {delta}3 isoforms of ERß can be reversibly localized to discrete spots within the nucleus of transfected cells adds new information to what we know about intranuclear localization of ERs. This information is important in the context of a recent report (32) showing that an exon 3-deleted ER{alpha} (also eliminates second zinc finger of DBD) possesses normal intranuclear localization. Such a difference suggests that the intact DBD of ERß is an important domain guiding localization of this receptor while it appears to be dispensable for normal localization of ER{alpha} (32).

The reversibility of the localization pattern of {delta}3 isoforms of ERß suggests that there are distinct regions of the nucleus that strongly attract agonist-bound ERß (spots) and other regions that attract antagonist-bound ERß (diffuse localization). As {delta}3 isoforms of ERß do not specifically bind DNA (4), the forces of attraction are most likely determined by protein-protein interactions. Furthermore, the difference in movement of ERß1 in the presence of either agonist or antagonist suggests that the DBD is crucial for the localization pattern of ERß1. Importantly, we believe that the present results reveal that ERß activities that are not readily seen from the wild-type receptor. That is, when the DBD is intact, the ERß is localized according to interactions facilitated by this domain, possibly with the nuclear matrix or promoter DNA. However, when the DBD is truncated (exon 3 deletion), these localizing forces are diminished and thus other localizing forces predominate, allowing the spotted distribution in the presence of agonist and the diffuse distribution in the presence of antagonist.

The ligand dependence of the {delta}3 localization pattern is reminiscent of the ligand dependence of nuclear receptor interaction with coactivator proteins (33, 34). In the case of ER{alpha}, this interaction depends on the conformation of the LBD. Bound agonist induces a conformation of the LBD that enables coactivator binding, whereas bound antagonist changes the LBD conformation to one in which the coactivator binding surface is obstructed and unavailable for coactivator interaction (34, 35, 36).

In accordance with this, we show that {delta}3 variants colocalize completely with cotransfected GFP-GRIP1 and endogenous CBP. This lends strength to the ligand-dependent interaction between {delta}3 variants and coactivators being at least partially responsible for {delta}3 localization to spots. We also see ERß1 colocalized with GFP-GRIP1 and CBP in the presence of the agonist, coumestrol, suggesting that the nuclear structures to which activated ERß variants can bind (hyperspeckles in the case of ERß1/ERß2 and discrete spots in the case of the {delta}3 isoforms) may also have affinity for coactivators. This and other evidence showing CBP and RNA polymerase II localized to nuclear punctae (37, 38) suggest that intranuclear structures contain many of the proteins required for transcription, and that they may be called into action (i.e. redistributed) by ligand-activated transcription factors such as the ER (28).

Another potential intranuclear site that partially overlaps with {delta}3 isoforms of ERß is the PML oncogenic domain (POD). PODs are identified as sites of virus immediate-early protein localization (such as ICP0 of herpes simplex virus) after infection of eukaryotic cells (25). Little is known about the function of these domains, although the proteins that are found localized there may include some coactivators of nuclear receptors (28). In fact, a recent report by LeMorte et al. (38) suggests that the PODs are sites of active transcription based on the finding of nascent RNA polymerase II transcripts in these structures. It is possible that association of the {delta}3 variants of ERß with structures such as the POD may indicate that the spots identified in the present study are indeed sites of active transcription.

To our knowledge, no previous reports exist concerning any nuclear receptor with a reversibly modifiable distribution similar to that presently described for the {delta}3 variants of ERß. Although other nuclear receptors, such as MR and GR, have been found concentrated in foci within the nucleus in an agonist-dependent manner (14, 15), there remains high levels of receptor elsewhere in the nucleus, suggesting that these areas of concentration contain only one localizing influence on the liganded receptor.

We have also found that the {delta}3 variants of ERß possess unexpected transcriptional properties. These variants are capable of activating a reporter gene from a promoter containing an AP-1 response element. This response element has been shown to be regulated by the ER in a manner that does not require DNA binding (12, 39). Interestingly, the {delta}3 variants activate at AP-1 sites in the presence of ER agonists unlike the full-length ERß, which activates at AP-1 only in the presence of ER antagonists such as tamoxifen (13, 39).

Previous studies have suggested that ER action at AP-1 sites proceeds through two pathways. ER{alpha} acts through an activation function (AF)-dependent pathway, which does not require the DBD but relies on intact AF-1 or AF-2 and allows activation with agonists such as E2 (39). ERß activates AP-1-responsive transcription through an AF-independent pathway that requires an intact DBD and allows activation of AP-1 sites with ER antagonists. However, ER{alpha} can also act through the AF-independent pathway if its activation functions are mutated or in the presence of antiestrogens (39). The present results suggest another situation in which ERß can be active in the AF-dependent pathway at AP-1 sites. ERß lacking exon 3 (which deletes the C-terminal half of the DBD) is able to activate AP-1 reporters in the presence of estrogens. Ultimately the ability of ER{alpha} and ERß to act at AP-1 sites with both AF dependence and AF independence suggests a balance exists between the two pathways that may be shifted by particular mutations (AF-1 or 2 in ER{alpha}) or splice variations ({delta}3 in ERß).

The physiological relevance of {delta}3 forms of ERß is contingent on whether they are expressed as protein. In some tissues the {delta}3 forms represent a relatively high proportion of total ERß mRNA (6). ER{alpha} splice variants are not normally expressed in tissues other than the pituitary (3) and appear predominantly in neoplastic tissue when they are expressed (40). In fact, a recent study (7) showing ERß in the ovary by Western blot has detected multiple bands that might represent expressed isoforms such as those discussed here. Nonetheless, if not expressed normally, the {delta}3 isoforms still serve as useful model mutants of ERß function.

In summary, we found that the ERß splice variants have dramatically different localization patterns in living cells, and that this localization pattern can be altered by agonists and antagonists. The ligand dependence of this localization pattern correlates with the transcriptional response of {delta}3 isoforms of ERß at AP-1 sites. In addition, we have shown that the intranuclear structures to which the {delta}3 isoforms localize also contain coactivator proteins, GRIP1 and CBP. We believe that the present study underscores the importance of the intact DBD in ERß localization and also sheds light on the dynamic forces that act upon ERß in the nucleus.


   Footnotes
 
1 Present address: Metabolic Research Unit, University of California, San Francisco, California 94143. Back

2 Present address: Department of Pathology, University of Virginia, Charlottesville, Virginia 22908-0214. Back

Received August 18, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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