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Molecular Endocrinology, doi:10.1210/me.2006-0514
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Molecular Endocrinology 21 (8): 1847-1860
Copyright © 2007 by The Endocrine Society

Estrogen Induces Expression of BCAS3, a Novel Estrogen Receptor-{alpha} Coactivator, through Proline-, Glutamic Acid-, and Leucine-Rich Protein-1 (PELP1)

Anupama E. Gururaj, Shaohua Peng, Ratna K. Vadlamudi and Rakesh Kumar

Molecular and Cellular Oncology (A.E.G., S.P., R.K.), The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030; Obstetrics and Gynecology (R.K.V.), University of Texas Health Science Center at San Antonio, San Antonio, Texas 78229; and Molecular and Cellular Biology (R.K.), Baylor College of Medicine, Houston, Texas 77030

Address all correspondence and requests for reprints to: Rakesh Kumar, Molecular and Cellular Oncology, Unit 108, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, Texas 77030. E-mail: rkumar{at}mdanderson.org.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
We recently reported that the breast carcinoma amplified sequence-3 (BCAS3) gene is regulated by estrogen receptor (ER) {alpha}. However, the role of ER{alpha} coactivators in the regulation of BCAS3 expression remains unknown, and information regarding the function of the BCAS3 protein is lacking. Here, we define the contribution of ER{alpha} coactivators to BCAS3 regulation and identify BCAS3 itself as an ER{alpha} coactivator in breast cancer cells. We found that PELP1 (proline-, glutamic acid-, and leucine-rich protein-1), a newly described ER{alpha} coregulator, is recruited to BCAS3 chromatin and activates its expression. Analysis of the BCAS3 sequence for functional motifs and evidence from biochemical fractionation suggested that BCAS3 acts as a transcriptional coactivator. Results from chromatin immunoprecipitation, reporter assays, and expression studies further validated the coactivator function of BCAS3 for ER{alpha}. BCAS3 physically associated with histone H3 and histone acetyltransferase complex protein P/CAF (p300/CBP-associated factor) and possessed histone acetyltransferase activity. Unexpectedly, BCAS3 required PELP1 to function as a coactivator in ER{alpha} transactivation activity. In brief, these results highlight a mechanism whereby ER{alpha} activation triggers a positive feedback loop leading to signal amplification in the cell.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
THE ESTROGEN 17ß-ESTRADIOL (E2) plays a critical role in many biological processes, including regulation of growth and development and cell type-specific gene expression in the reproductive tract, central nervous system, skeleton, and immune system. Alterations in the response to estrogen are associated with a variety of hormone-dependent diseases, such as breast cancer, endometrial cancer, cardiovascular disease, and osteoporosis (1, 2). The biological actions of estrogen are mediated through the association of two distinct estrogen receptor (ER) proteins, ER{alpha} and ERß, with the estrogen-responsive elements (EREs) in the promoters of responsive genes. ERs can also regulate gene expression by associating with other promoter-bound transcription factors, such as activator protein-1 and SP-1. Estrogen-mediated activation of genes including cyclin D1, c-myc, c-fos, EBAG9, pS2, and cathepsin D is induced through the coordinated recruitment of ERs and specific coactivators to the genes’ promoters. The recruitment of coactivators, including steroid receptor coactivator (SRC)-1, AIB1/SRC-3, glucocorticoid receptor-interacting protein 1 (GRIP1), p300/cAMP response element binding protein-binding protein (CBP), p300/CBP-associated factor (P/CAF), and PELP1/MNAR (proline-, glutamic acid-, and leucine-rich protein-1/modulator of nongenomic activity of ER), to ER-bound promoters increases estrogen-regulated transcriptional activation (3, 4).

Biochemical and functional data suggest that PELP1/MNAR is a unique coactivator that participates in both genomic and nongenomic responses to ER signaling (4, 5). The action of PELP1 as an ER coactivator seems to be specific because an independent study confirmed that PELP1 coactivates ER but corepresses glucocorticoid receptors and non-nuclear receptor sequence-specific transcription factors, including activator protein-1, nuclear factor {kappa}B, and ternary complex factor/serum response factor (6). It was also recently reported that PELP1, which is up-regulated by retinoic acid acts as a coactivator of the retinoid X receptor pathway (7). PELP1 interacts with transcriptional activators such as CBP and p300 in vivo, suggesting that PELP1 functions as an ER coactivator by enhancing the recruitment of histone acetyltransferases (HATs) to increase ligand-mediated gene expression (8). Furthermore, PELP1 has been shown to induce chromatin remodeling by displacing histone H1 and has functional interactions with retinoblastoma protein that explains in part its role in ER genomic functions (9). PELP1 has also been shown to participate in estrogen receptor nongenomic signaling by activation of Src kinase (10).

Breast carcinoma amplified sequence-3 (BCAS3) is a gene of unknown function that is localized to 17q23, a chromosomal region amplified in approximately 20% of primary breast tumors, as assessed by comparative genomic hybridization (11). Amplification of 17q23 appears to be more common in high-grade tumors and is associated with poor prognosis, suggesting that genes affected by this amplification have a crucial role in breast cancer progression (11). Copy number gains at 17q23 have also been reported in tumors of the brain, lung, bladder, testis, and liver (12). The involvement of BCAS3 in breast cancer progression is further suggested by reports that BCAS3 is amplified in breast cancer cell lines (mainly ER+ cell lines) and in approximately 10% of primary breast tumors analyzed (13). Furthermore, overexpression of BCAS3 in primary breast tumors is associated with tumor grade and proliferation (14). In addition, the last two exons of BCAS3 can be translocated to 20q13, another commonly amplified region in breast cancers, resulting in a fusion product that is highly overexpressed in MCF-7 cells (13).

Recently, we showed that BCAS3 is an estrogen-inducible gene because ER{alpha} is recruited to a regulatory region of BCAS3 via a half ERE (1/2 ERE) (14). However, the role of ER{alpha} coactivators in the regulation of BCAS3 expression is not known, and information regarding the function of the BCAS3 protein is also lacking. In this study, we identify PELP1, an ER{alpha} coactivator, as a regulator of BCAS3 expression in response to estrogen. Unexpectedly, we also report a coactivator role of BCAS3 for ER{alpha} in breast cancer cells.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Estrogen Regulation of BCAS3 Expression in Vivo
To confirm that BCAS3 is estrogen inducible in vivo, we examined BCAS3 expression by Western blot analysis in the mammary glands of ovariectomized female nude mice that were either implanted with estrogen pellets or left untreated (controls). BCAS3 expression was barely detectable in the mammary glands of control mice (Fig. 1AGo, lanes 1–3), whereas its expression was clearly induced in mammary glands of estrogen pellet-implanted mice (Fig. 1AGo, lanes 4–7). These results were corroborated by in situ hybridization experiments for expression of BCAS3 transcript in both control and estrogen pellet-implanted mouse mammary gland tissues (Fig. 1BGo). More BCAS3 transcripts were detected in mammary gland epithelial cells with estrogen treatment (Fig. 1BGo, middle panel) than without estrogen treatment (Fig. 1BGo, upper panel). The sense probe was also hybridized as a negative control.


Figure 1
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  		Fig. 1. Estrogen Stimulation of BCAS3 in Vivo

A, Stimulation of BCAS3 expression in ovariectomized mice implanted with E2 pellets (upper panel). Lower panel shows actin staining as a loading control. MG, Mammary gland; WB, Western blot. B, Expression of BCAS3 in mouse mammary gland by in situ hybridization. Top panel, gland from ovariectomized mice, middle panel, gland from ovariectomized mice implanted with E2 pellets, and lower panel, sense probe. Bar, 20 µm. C, Activity of BCAS3 enhancer-luciferase reporter in MCF-7 cells incubated with known ER{alpha} coactivators. RLU, Relative light units. D, Luciferase activity in various cell lines transfected with BCAS3-luciferase reporter and T7-PELP1 or pcDNA. HSG and HSY are epithelial cell lines from submandibular (HSG) and parotid (HSY) salivary glands. Top, Western blot to show efficient expression of the transfected protein. Values in C and D are normalized to ß-galactosidase activity (n = 3), and error bars indicate SEM.

 
Having established a role for ER{alpha} in regulating BCAS3 expression (14), we sought to identify the nature of coactivators of ER{alpha} that are involved in this regulation. We first conducted a preliminary screen by cotransfecting MCF-7 cells with a luciferase vector containing the BCAS3 regulatory region to which ER{alpha} binds and expression vectors for various known coactivators of ER{alpha}. The activity of the reporter gene increased in response to PELP1 expression (Fig. 1CGo) but not expression of any of the other coactivators, indicating that PELP1 could function as a coactivator of BCAS3.

The enhancer function of PELP1 was confirmed in another breast cancer cell line, ZR-75, in which overexpression of PELP1 resulted in increased estrogen-induced reporter gene activity (Fig. 1DGo). Similar results were obtained in two other estrogen-responsive endometrial cell lines, Hec1A and Ishikawa (Fig. 1DGo). In contrast, PELP1 did not significantly activate reporter gene expression in response to estrogen in two ER{alpha}-negative salivary gland cell lines HSG and HSY (15) (Fig. 1DGo), suggesting that PELP1-induced activation of BCAS3 via the BCAS3 enhancer sequence seems to be tissue specific.

Role of PELP1 in BCAS3 Expression
To confirm the finding that PELP1 modulates the expression of BCAS3 via the BCAS3 regulatory region, we examined the recruitment of PELP1 to the BCAS3 region in the second intron encompassing about 600 bp (17427070–17427655) (14). Immunoprecipitation of T7 epitope-tagged PELP1 from MCF cells stably expressing PELP1 (9), followed by PCR amplification of the BCAS3 enhancer sequence, indicated that PELP1 is strongly recruited to the BCAS3 regulatory region upon estrogen induction (Fig. 2AGo).


Figure 2
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  		Fig. 2. Regulation of BCAS3 Expression by ER{alpha} Coactivator PELP1

A, ChIP assay showing recruitment of T7-PELP1 onto BCAS3 regulatory region in response to E2 stimulation in MCF-7/PELP1. B, ChIP assay for T7-PELP1 in PELP1-Teton cells treated with E2, ICI-182780, or both. DNA from whole-cell lysates was used as the input control. IP, Immunoprecipitation. C, ChIP assay showing recruitment of endogenous PELP1 onto BCAS3 regulatory region in response to E2 stimulation in MCF-7 cells. D, Occupancy of BCAS3 regulatory sequence by T7-PELP1 and ER{alpha} at different times during E2 treatment in PELP1-Teton cells, as measured by ChIP (left panel). Right panel shows Western blot analysis for induction of T7-PELP1 upon doxycycline (Dox) treatment. WB, Western blot. E, Occupancy of BCAS3 regulatory sequence by endogenous PELP1 at different times during E2 treatment in MCF-7 cells, as measured by ChIP. F, BCAS3-luciferase reporter activity in PELP1-Teton cells treated with E2 or left untreated. Values are normalized to ß-galactosidase activity (n = 3) and error bars indicate SEM. RLU, Relative light units. G, RT-PCR analysis of BCAS3 expression in PELP1-Teton cells stimulated with E2 (upper panel). RT-PCR for actin was carried out in the same samples as a control (lower panel). H, Northern blot analysis of BCAS3 mRNA in MCF-7/pcDNA and MCF-7/PELP1 cells treated with E2 or left untreated. Actin was used as a loading control. I, Effect of PELP1 depletion on BCAS3 expression in MCF-7 cells treated with or without E2.

 
We also examined recruitment in PELP1-Teton cells, which express T7-PELP1 under the control of a tetracycline-regulated promoter (9) (Fig. 2DGo, right panel). T7-PELP1 was recruited upon doxycycline treatment onto the BCAS3 regulatory region as shown in Fig. 2BGo. Doxycycline-mediated induction of PELP1 in these cells resulted in persistent occupancy of the enhancer sequence by PELP1 (Fig. 2BGo, compare lanes 1 and 5). Treatment of the cells with the antiestrogen ICI-182780 decreased the recruitment of PELP1, indicating that PELP1 occupancy on the BCAS3 enhancer sequence depends on ER{alpha} (Fig. 2BGo, compare lanes 2 and 4 and lanes 6 and 8). To validate our results with exogenously expressed PELP1 protein, we performed chromatin immunoprecipitation (ChIP) experiments with estrogen treatment using the endogenous protein. Endogenous PELP1 protein was strongly recruited to the BCAS3 regulatory region upon estrogen treatment (Fig. 2CGo, lane 2). This recruitment was abrogated by pretreatment with ICI-182780 as observed in the case of T7-PELP1, indicating the dependence of PELP1 recruitment on ER{alpha} (Fig. 2CGo, lane 4).

A time course of estrogen treatment in PELP1-teton cells showed that T7-PELP1 was strongly recruited to the BCAS3 regulatory region within 15 min of stimulation and remained associated with the sequence for up to 1 h without derecruitment (Fig. 2DGo, left panel), indicating that PELP1 might be continuously needed for BCAS3 gene expression. We performed ChIP analysis using ER{alpha} in the same system to validate that ER{alpha} pathway was active in these cells (Fig. 2DGo, left panel). We next performed ChIP analysis using antibodies raised against the endogenous PELP1 protein after treatment of estrogen for various time points. Results showed that PELP1 was recruited by 15 min with a maximal recruitment by 30 min (Fig. 2EGo, please compare lanes 1, 2, and 3). As observed with the transfected protein, PELP1 remained associated with the regulatory region of BCAS3 until 90 min of estrogen treatment (Fig. 2EGo, lane 6).

Transfection of BCAS3-luciferase reporter into PELP1-overexpressing MCF-7 cells induced reporter gene expression upon estrogen stimulation (Fig. 2FGo). RT-PCR analysis of BCAS3 mRNA in PELP1-overexpressing teton clones indicated that BCAS3 mRNA is up-regulated upon stimulation with doxycycline (Fig. 2GGo). These results were corroborated by Northern blot analysis in stable MCF-7 clones constitutively overexpressing PELP1 (Fig. 2HGo), showing that the level of PELP1 is positively associated with BCAS3 expression. To rule out the possibility that a factor required for BCAS3 expression might be squelched by overexpression of PELP1 in MCF7 cells, we knocked down PELP1 in MCF7 cells and looked for BCAS3 expression by Western blotting analysis. Use of scrambled RNA interference (RNAi) as a control demonstrated that BCAS3 expression was stimulated by estrogen treatment (Fig. 2IGo, lanes 1 and 2) as previously reported (14). Under PELP1 knockdown conditions, BCAS3 levels were decreased both in basal as well as estrogen stimulated conditions (Fig. 2IGo, lanes 3 and 4) indicating that PELP1 is required for BCAS3 expression in MCF7 cells. Collectively, these results demonstrate that PELP1 is a coactivator with a prominent role in the regulation of BCAS3 gene expression in an ER{alpha}-dependent manner.

Coactivator Function of BCAS3
To determine the possible physiological consequences of PELP1 and ER{alpha} regulation of BCAS3, we sought to elucidate the role of BCAS3 in breast cancer cells. Analysis of the BCAS3 protein sequence for functional motifs using the GenomeNet MOTIF search (http://motif.genome.jp) revealed the presence of a bromodomain, an atypical zinc-finger motif, three WD-40 domains, four potential DNA-binding domains, and two phosphorylated kinase-inducible activation domains (Fig. 3AGo), motifs that are typically found in a variety of transcriptional coactivators (16).


Figure 3
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  		Fig. 3. ER{alpha} Coactivator Function of BCAS3

A, Functional motifs in BCAS3 protein. TRAF2, TNF receptor-associated factor 2. B, Western blotting for endogenous BCAS3, ER{alpha}, and CBP in fractionated, E2-treated MCF-7 cells. Paxillin, poly(ADP-ribose) polymerase (PARP), and lamin B1 were used as positive controls for cytosol, chromatin, and nuclear matrix, respectively. WB, Western blot. C, E2 signaling promotes BCAS3 interaction with the endogenous ER{alpha} pathway. ZR-75 cells were transfected with T7-BCAS3 and treated with E2 or left untreated. ChIP analysis was done using anti-T7 antibodies followed by PCR analysis of the pS2 (304 bp) promoter fragment (left panel). ChIP analysis using ER{alpha} in the same cell lysate was performed as a positive control (left panel). Western blot analysis was performed simultaneously to check for expression of the transfected protein (right panel). IP, Immunoprecipitation. D, Occupancy of estrogen-responsive promoter elements (pS2 promoter and c-myc promoter) by endogenous BCAS3 at upon E2 treatment in ZR-75 cells, as measured by ChIP. BCAS3 recruitment onto the actin promoter was also determined as a negative control for the experiment. ChIP analysis using ER{alpha} in the same cell lysate was performed as a positive control (left panel). Ab, Antibody. E, Dose-dependent stimulation of pS2-luciferase reporter activity by BCAS3 in MCF-7 cells. Top, Western blot to show efficient expression of the transfected protein. RLU, Relative light units. F, pS2-luciferase reporter activity in MCF-7 cells transfected with T7-BCAS3 or vector and treated with E2. Top, Western blot to show efficient expression of the transfected protein. G, pS2-luciferase reporter activity, in the presence or absence of E2, in MCF-7 cells depleted of BCAS3 by RNAi. Knockdown of BCAS3 was confirmed by RT-PCR, and GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was used as a loading control. Values in E–G are normalized to ß-galactosidase activity (n = 3), and error bars indicate SEM. H, Northern blot analysis of pS2 gene expression after BCAS3 knockdown in MCF-7 cells (upper panel). The same blot was probed for BCAS3 expression to confirm efficient knockdown of BCAS3 (middle panel) and for actin as a loading control (lower panel).

 
Based on the presence of these motifs and our preliminary data on the role of BCAS3 in ER{alpha} transactivation responses (data not shown), we formulated the working hypothesis that BCAS3 is a transcriptional coactivator. To explore this idea, we first fractionated MCF-7 cells to yield purified cellular components and analyzed BCAS3 expression in the different compartments by Western blotting. Substantial amounts of BCAS3 protein were extracted with Nonidet P-40 buffer, suggesting that some BCAS3 was present in the soluble fraction (Fig. 3BGo, lane 2). In addition, a substantial amount of BCAS3 was present in the chromatin fraction (Fig. 3BGo, lane 4). ER{alpha} was present in the cytoplasm/nucleoplasm, chromatin, and nuclear matrix fractions, but most of the ER{alpha} was found in the chromatin fraction (Fig. 3BGo). These results suggest that BCAS3 is localized in both the cytoplasmic and nuclear compartments and is associated with chromatin.

Because BCAS3 was strongly associated with the chromatin fraction, we next determined whether BCAS3 could be recruited onto a classic ER{alpha}-induced promoter, such as that of pS2, in MCF-7 cells. MCF-7 cells were transfected with a T7-BCAS3 expression vector and stimulated with estrogen. The cells were then subjected to ChIP analysis using a T7-specific antibody and PCR amplification of the pS2 promoter using primers against the ER{alpha}-binding region (17). BCAS3 was found to occupy the promoter region under basal conditions, and estrogen stimulation increased its occupancy on the promoter, as is typically seen with ER{alpha} coactivators (Fig. 3CGo, left panel). We also performed ChIP analysis with ER{alpha}-specific antibody under the same conditions as a positive control for our experiment (Fig. 3CGo, left panel). An aliquot of the cells were used for Western blotting analysis to verify expression of T7-BCAS3 in the transfected cells (Fig. 3CGo, right panel). Next, we sought to validate the recruitment of endogenous BCAS3 protein to the proximal regions of estrogen response elements of E2-responsive genes in vivo using ChIP assay. ZR-75 cells were treated with E2 for 45 min, and BCAS3-bound chromatin was immunoprecipitated, eluted, and PCR amplified using primers that are specific to the ERE gene promoters such as trefoil factor 1 precursor (pS2), and c-myc. Results showed increased recruitment of PELP1 to pS2, and c-myc promoters in response to estrogen treatment (Fig. 3DGo). In addition, we used ER{alpha} ChIP on pS2 promoter as a positive control and ChIP for BCAS3 on actin promoter as a negative control (Fig. 3DGo).

To determine whether BCAS3 occupancy of the promoter results in transcriptional activation of the pS2 gene, a pS2 promoter-luciferase reporter was transfected into MCF-7 cells along with various concentrations of either pcDNA (control) or the T7-BCAS3 vector. Transfection of BCAS3 increased pS2 promoter activity by up to fivefold compared with transfection of pcDNA (Fig. 3EGo). Next we determined the effect of BCAS3 on the regulatory region of pS2-luciferase reporter vector in response to estrogen signaling. Cotransfection of BCAS3 expression plasmid with pS2-luciferase reporter vector resulted in increased reporter activity. Interestingly, estrogen stimulation did not augment the activity of the reporter gene (Fig. 3FGo) indicating that although BCAS3 has a specific role in regulating pS2 gene, overexpression of BCAS3 appeared to saturate pS2 reporter expression in estrogen-stimulated situation. However, depletion of BCAS3 from MCF-7 cells using BCAS3-specific small interfering RNA (siRNA) resulted in down-regulation of reporter gene activity under both basal and estrogen-induced conditions (Fig. 3GGo), suggesting that BCAS3 is an important coactivator of ER{alpha} in breast cells. Efficient knockdown of BCAS3 was confirmed by RT-PCR (Fig. 3GGo, inset). The effect of BCAS3 on estrogen-induced pS2 expression was confirmed by the finding that pS2 mRNA expression decreased in response to estrogen in the presence of BCAS3 siRNA but not in the presence of control scrambled siRNA (Fig. 3HGo).

BCAS3 as an ER{alpha}-Interacting Partner
The above findings demonstrated that both BCAS3 and ER{alpha} reside in the chromatin fraction and that BCAS3 induces the expression of classic ER{alpha}-responsive genes in response to estrogen in breast cancer cells. We therefore looked for a physical association between BCAS3 and ER{alpha}. In a glutathione S-transferase (GST)-pull-down assay, recombinant GST-BCAS3 protein coprecipitated 35S-labeled ER{alpha} efficiently, whereas the control GST did not (Fig. 4AGo, top panel). Ponceau staining of the blot used in the above experiment indicate loading of the GST-tagged ER{alpha} and GST control proteins (Fig. 4AGo, bottom panel).


Figure 4
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  		Fig. 4. Interaction between BCAS3 and ER{alpha} in Vitro and in Vivo

A, GST-pull-down assay for 35S-labeled ER{alpha} translated in vitro and incubated with GST-BCAS3 (upper panel). Ponceau staining was used for normalization (lower panel). Asterisks denote the GST-tagged bands of interest. B, Immunoprecipitation (IP) of endogenous ER{alpha} and Western blotting (WB) for BCAS3 in cell lysates from MCF-7 cells treated with ICI-182780 or left untreated (upper panel). Western blotting for ER{alpha} indicated immunoprecipitation of ER{alpha} in coimmunoprecipitation experiments (middle panel). C, Pull-down assay using GST-tagged ER{alpha} deletion constructs with 35S-labeled BCAS3 to identify the region of ER{alpha} that binds to BCAS3. Upper panel shows the autoradiogram, and the lower panel shows Ponceau staining of the same membrane. Asterisks denote the GST-tagged bands of interest. D, Immunoprecipitation (IP) of endogenous BCAS3 and Western blotting (WB) for ER{alpha} in cell lysates from MCF-7 cells treated with ICI-182780 or left untreated (upper panel). Western blotting for BCAS3 indicated immunoprecipitation of BCAS3 in coimmunoprecipitation experiments (middle panel). E, Pull-down assay using GST-tagged BCAS3 deletion constructs with 35S-labeled ER{alpha} to identify the region of BCAS3 that binds to ER{alpha}. Upper panel shows the autoradiogram, and the middle panel shows Ponceau staining of the same membrane. The lower panel depicts the domains found in the BCAS3 deletion constructs. Asterisks denote the GST-tagged bands of interest.

 
To validate the observed in vitro interaction of BCAS3 with ER{alpha} in a physiological setting, we subjected cellular extracts to coimmunoprecipitation and Western blot analysis using ER{alpha}-specific and control IgG antibodies. When ER{alpha} was immunoprecipitated from MCF-7 cells, BCAS3 coprecipitated with ER{alpha} as determined by Western blotting (Fig. 4BGo, top panel, lane 3). This association was disrupted by treatment with ICI-182780, indicating that the in vivo interaction between BCAS3 and ER{alpha} was very specific (Fig. 4BGo, top panel, lane 4). Western blotting for ER{alpha} on the same blot confirmed the efficient immunoprecipitation and expected decrease in ER{alpha} expression after ICI-182780 treatment (Fig. 4BGo, bottom panel).

To identify the BCAS3-binding regions of ER{alpha}, a series of GST-tagged deletion constructs of ER{alpha} were used in a GST-pull-down assay with in vitro-translated BCAS3. BCAS3 did not bind to any of the ER{alpha} deletion constructs but efficiently interacted with the full-length protein, suggesting that the fully folded ER{alpha} is necessary for interaction with BCAS3 (Fig. 4CGo, top panel). Ponceau staining of the blot used in the above experiment indicates equal loading of the proteins (Fig. 4CGo, bottom panel).

The in vivo interaction between ER{alpha} and BCAS3 was further substantiated using coimmunoprecipitation studies. Immunoprecipitation of BCAS3 and Western blotting for ER{alpha} showed that the endogenous proteins interact with one another in a physiological setting (Fig. 4DGo, lane 3). This interaction was specific because immunoprecipitation of BCAS3 in the presence of ICI-182780 did not result in coimmunoprecipitation of ER{alpha} protein (Fig. 4DGo, lane 4). Because the BCAS3 amino acid sequence does not have the typical NR boxes (LXXLL motifs), we next mapped the ER{alpha}-binding site in BCAS3. The full-length BCAS3-GST fusion protein, but not GST, efficiently interacted with the 35S-labeled full-length ER{alpha} protein. GST-tagged BCAS3 deletion mutants were then used in a GST-pull-down assay with in vitro-translated ER{alpha}. We mapped the ER{alpha}-binding domain of BCAS3 to the construct C of the protein because the deletion construct encompassing this region of BCAS3 was sufficient for strong binding to ER{alpha}, whereas removal of this region abolished binding (Fig. 4EGo, left panel, construct C). The deletion construct, construct C, includes the bromodomain motif (Fig. 4EGo, right panel) and thus, our finding implicates the bromodomain in the role of BCAS3 as a coactivator of ER{alpha}.

Physical Interaction of BCAS3 with PELP1
We next asked whether PELP1, a known coactivator of ER{alpha}, interacts with BCAS3, a newly identified ER{alpha} coactivator (this study). We next examined the in vivo protein-protein interaction between BCAS3 and PELP1 using coimmunoprecipitation studies. We first used anti-T7 antibody and T7-PELP1-overexpressing stable MCF7 clones for the coimmunoprecitation/Western blotting studies (9). Immunoprecipitation of T7-PELP1 and Western blotting for BCAS3 in MCF-7/PELP1 cells showed that the proteins interact with one another in a physiological setting (Fig. 5AGo, top panel). As a control, we used MCF-7/pcDNA clones simultaneously and observed no interaction, indicating that the interaction seen in MCF-7/PELP1 cells was specific. The same blot was reprobed with the anti-T7 antibody to demonstrate efficient immunoprecipitation of T7-PELP1 (Fig. 5AGo, lower panel). To establish the physiological nature of the BCAS3-PELP1 interaction, we showed that immunoprecipitation of endogenous PELP1 resulted in coimmunoprecipitation of BCAS3 in ZR-75 cells (Fig. 5BGo). Likewise, immunoprecipitation of endogenous BCAS3 resulted in coimmunoprecipitation of PELP1 (Fig. 5CGo), indicating that both BCAS3 and PELP1 interact with each other in vivo.


Figure 5
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  		Fig. 5. Interaction between BCAS3 and PELP1 in Vivo and in Vitro

A, Immunoprecipitation (IP) of T7-PELP1 and Western blotting (WB) for BCAS3 in lysates from MCF-7/PELP1 cells (upper panel). Western blotting for PELP1 indicated immunoprecipitation of PELP1 in coimmunoprecipitation experiments (lower panel). B, Immunoprecipitation of endogenous PELP1 and Western blotting for BCAS3 in cell lysates from MCF-7 cells (upper panel). Western blotting for PELP1 indicated immunoprecipitation of PELP1 in coimmunoprecipitation experiments (middle panel). C, Immunoprecipitation of endogenous BCAS3 and Western blotting for PELP1 in cell lysates from MCF-7 cells (upper panel). Western blotting for BCAS3 indicated immunoprecipitation of BCAS3 in coimmunoprecipitation experiments (middle panel). D, Pull-down assay using GST-tagged PELP1 deletion constructs with 35S-labeled BCAS3 to identify the region of PELP1 that binds to BCAS3. Upper panel shows the autoradiogram, and the lower panel shows a schematic diagram of PELP1 and its deletion constructs. Positions of NR-box motifs in PELP1 are indicated by black bands. Asterisks denote the GST-tagged bands of interest. E, Pull-down assay using GST-tagged BCAS3 deletion constructs with T7-PELP1 from lysate of MCF7 cells overexpressing T7-PELP1 to identify the region of BCAS3 that binds to PELP1. Upper panel shows the Western blot, and the middle panel shows Ponceau staining of the same membrane. Lower panel depicts the domains found in the BCAS3 deletion constructs. Asterisks denote the GST-tagged bands of interest.

 
Results from the GST pull-down assays using GST-tagged PELP1 deletion constructs and 35S-labeled BCAS3 indicated that the N-terminal 400 amino acids of PELP1, a region that contains seven of the nine NR boxes in PELP1 (7), has the highest binding affinity to BCAS3 (Fig. 5DGo, left panel). A comparatively lower binding affinity was also shown by the PELP1 construct encompassing amino acids 600–866, which contains the remaining two NR boxes in PELP1. No binding was observed for the deletion constructs encompassing the rest of the protein.

In vitro interaction of BCAS3 and PELP1 was also established by a GST-pull-down assay, in which T7-PELP1derived from PELP1 stable clone coprecipitated with GST-BCAS3 (Fig. 5EGo, left panel, lane A). To map the region of BCAS3 that interacts with PELP1, we used GST-tagged deletion constructs of BCAS3 in a GST-pull-down assay with T7-PELP1. Among the deletion constructs, only the one encompassing the bromodomain efficiently interacted with PELP1 (Fig. 5EGo, left panel, lane C). Together, these results suggest that PELP1 and BCAS3 physically interact with each other under physiological conditions.

Mechanism of Coactivator Function of BCAS3
Having established that BCAS3 acts as a coactivator of ER{alpha} by physically interacting with ER{alpha} and its coactivator PELP1, we sought to dissect the mechanism of the coactivator functions of BCAS3. Because bromodomains in protein have been shown to be important for binding to acetylated histone tails (16), we used far-Western analysis with 35S-labeled BCAS3 and histones H1 and H3 immobilized on a membrane to determine whether BCAS3 interacts directly with the histones. There was robust association between BCAS3 and histone H3 (Fig. 6AGo, upper panel). The blot was stained using Ponceau-S to affirm equal loading of proteins (Fig. 6AGo, lower panel).


Figure 6
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  		Fig. 6. BCAS3 Is an ER{alpha} Coactivator with Associated HAT Activity

A, Far-Western analysis showing interaction of BCAS3 with histones. Membrane-immobilized total histones and purified H1 and H3 were probed with 35S-labeled BCAS3. Ponceau staining indicates the amounts of histones used. Asterisks denote the bands of interest. B, GST-pull-down assay of 35S-BCAS3 using GST-tagged histones H1 and H3. Ponceau staining shows equal loading. Asterisks denote the GST-tagged bands of interest. C, Immunoprecipitation (IP) of endogenous histone H3 and Western blotting (WB) for BCAS3 in nuclear extracts from MCF-7 cells treated with E2 (upper and middle panel). Upper panel, shorter exposure showing the band in the lane from the immunoprecipitated sample. Middle panel, longer exposure showing the band in the input lane. Asterisks denote the band of interest. Western blotting for histone H3 was used as a control for immunoprecipitation (lower panel). D, Association of BCAS3 with HAT activity (n = 2). Error bars indicate SEM. E, Immunoprecipitation of T7-tagged protein and Western blotting for P/CAF or T7 in total lysates from cells transfected with pcDNA or T7-BCAS3.

 
To validate the observed interaction between histone H3 and BCAS3, we performed a GST-pull-down assay and coimmunoprecipitation followed by Western blotting. In vitro-translated BCAS3 coprecipitated with GST-histone H3 in the GST-pull-down assay (Fig. 6BGo, upper panel). The blot was stained using Ponceau-S to confirm equal loading of proteins (Fig. 6BGo, lower panel). Immunoprecipitation of histone H3 and Western blotting for BCAS3 in MCF-7 cells substantiated this association, showing that the endogenous proteins interacted with one another in a physiological setting (Fig. 6CGo). This interaction was dependent on estrogen because immunoprecipitation in the absence of estrogen stimulation did not pull down any BCAS3 protein.

Because BCAS3 promoted transcription from the estrogen-inducible promoter and interacted effectively with ER{alpha} target gene chromatin (Fig. 3Go, C–H), we suspected that BCAS3 could influence the status of chromatin remodeling, presumably through HAT activity. We therefore examined whether such activity was intrinsic or associated with T7-BCAS3. Immunoprecipitation analysis revealed increased functional HAT activity associated with T7-BCAS3 in estrogen-induced MCF-7 cells (Fig. 6DGo).

We next examined whether BCAS3 interacts with HAT enzymes or other coactivators. To that end, T7-BCAS3 was transfected into MCF-7 cells, and immunoprecipitation was carried out 48 h later using T7-specific antibody followed by Western blotting for P/CAF. P/CAF coprecipitated with BCAS3; this interaction was very specific because immunoprecipitation from control vector-transfected cells did not pull-down P/CAF (Fig. 6EGo). These findings indicated that the ER{alpha}-coactivating function of BCAS3 after estrogen stimulation might occur through modulation of the chromatin structure via associated HAT activity.

Cooperative Interactions between BCAS3 and PELP1
The finding that BCAS3 is present in the ER{alpha} coactivator complex led us to ask whether BCAS3 depends on PELP1 for its function as an ER{alpha} coactivator. Stable MCF-7 clones expressing T7-PELP1 were transiently transfected with myc-tagged BCAS3. Forty-eight hours later, the cells were cross-linked with formaldehyde and subjected to ChIP analysis using anti-myc antibodies to immunoprecipitate myc-tagged BCAS3. Elutes from the first ChIP were reimmunoprecipitated with T7-specific antibody to immunoprecipitate any associated T7-PELP1 molecules. Results from this double-ChIP experiment indicated that BCAS3 and PELP1 were recruited to the pS2 promoter region together in E2-stimulated cells (Fig. 7AGo), suggesting in vivo existence of an active BCAS3/PELP1 complex.


Figure 7
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  		Fig. 7. Cooperative Interactions between BCAS3 and PELP1

A, Double-ChIP analysis for myc and T7 in MCF7/PELP1 cells transfected with myc-BCAS3 and treated with E2. Recruitment to the pS2 promoter was detected by PCR using specific primers. IP, Immunoprecipitation. B, pS2-luciferase activity in MCF-7 cells in the presence of PELP1, BCAS3, or both, with or without estrogen treatment. Top, Western blot (WB) to show efficient expression of the transfected proteins. C, Western blot analysis of BCAS3 and PELP1 in HeLa, MCF-7, and ZR-75 cells. D, Effect of BCAS3 overexpression on pS2-luciferase activity in the presence of ER{alpha} in HeLa cells, with or without estrogen treatment. Top, Western blot to show efficient expression of the transfected protein. RLU, Relative light units. E, pS2-luciferase activity with BCAS3 overexpression after depletion of PELP1 by PELP1 siRNA in HeLa cells, with or without estrogen treatment. Knockdown of PELP1 was confirmed by Western blotting, and Vinculin was used as a loading control. F, Double-ChIP analysis of myc and T7 in MCF7/PELP1-WT and MCF7/PELP1-H1 mutant cells transfected with myc-BCAS3 and treated with E2. Recruitment to the pS2 promoter was detected by PCR using specific primers. G, pS2 luciferase activity was measured in MCF-7 cells in the presence of either PELP1-WT, PELP1-H1-mutant and BCAS3 by themselves or together with or without estrogen treatment. Inset, Western blot to show efficient expression of the transfected proteins. Values in B, D, E, and G are normalized to ß-galactosidase activity (n = 3), and error bars indicate SEM.

 
To determine whether PELP1 and BCAS3 complement each other in activating pS2 gene expression, a pS2-luciferase reporter assay was carried out with transient overexpression of T7-PELP1, myc-BCAS3, or both in MCF-7 cells. We observed induction of pS2-luciferase activity by T7-PELP1 or myc-BCAS3 compared with the vector-cotransfected cells (Fig. 7BGo). Coexpression of T7-PELP1 and myc-BCAS3 further augmented transcriptional activity of the pS2 promoter (Fig. 7BGo).

Our next goal was to elucidate the role of PELP1 in the coactivator function of BCAS3. We first screened PELP1 and BCAS3 expression in HeLa, MCF-7, and ZR-75 cells. HeLa and MCF-7 cells expressed high amounts of both BCAS3 and PELP1, whereas ZR-75 expressed moderate amounts of BCAS3 and very low amounts of PELP1 protein (Fig. 7CGo). Because BCAS3 also exists as a fusion protein with BCAS4 in MCF-7 cells (13), we used HeLa cells as a model to implicate PELP1 in BCAS3 coactivator function. We verified whether BCAS3 could activate pS2 in HeLa cells with exogenous expression of ER{alpha}. As shown in Fig. 7DGo, overexpression of T7-BCAS3 in HeLa cells strongly induced pS2-luciferase reporter activity in the presence of ER{alpha}, as seen in ER{alpha}-positive cells. Knockdown of PELP1 expression by PELP1-specific siRNA (7) in HeLa cells transfected with ER{alpha} and pS2-luciferase reporter resulted in decreased activation of the reporter in both basal and estrogen-stimulated conditions when T7-BCAS3 was overexpressed (Fig. 7EGo). These results suggested that BCAS3 coactivator function is mediated, at least in part, through its interaction with PELP1. The inset shows efficiency of knockdown of PELP1 in these cells.

To show that PELP1 is essential for BCAS3 coactivator function, we used MCF-7 cells stably expressing either wild-type (PELP1-WT) or a mutant of PELP1 that is unable to bind to histone H1 and lacks the ability to activate transcription (PELP1-H1) (9). We transfected myc-tagged BCAS3 into PELP1-WT and PELP1-H1 cells and performed double-ChIP experiments as described above. Myc-BCAS3 was recruited to the pS2 promoter along with PELP1-WT in response to estrogen stimulus, as demonstrated previously; in contrast, we found no recruitment of myc-BCAS3 and the PELP1-H1 mutant (Fig. 7FGo). This finding indicated that the ability of PELP1 to bind and displace histone H1 might be essential for the recruitment of BCAS3 onto pS2 chromatin. We corroborated this finding in a pS2-luciferase reporter assay in which cotransfection of BCAS3 and PELP1-H1 did not lead to enhanced activation of the reporter, whereas cotransfection of BCAS3 and PELP1-WT increased the activity of pS2-luciferase, as reported earlier (Fig. 7GGo). In conclusion, we demonstrate here that BCAS3 and PELP1 act cooperatively to enhance ER{alpha} genomic functions in the cell and that the H1-displacing activity of PELP1 might be important for BCAS3 coactivator function.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
In recent years, an increasing number of coregulators of nuclear receptors have been identified. These coregulators interact with DNA-bound transcription factors and play essential roles in the action of these transcription factors. Moreover, these coregulators can recruit multiprotein complexes that also modulate transcription. Transcriptional coregulators can be subdivided into coactivators, which mediate gene activation; corepressors, which mediate gene silencing; or bifunctional coregulators that can do both. In a previous investigation, we defined BCAS3 as an estrogen-inducible gene whose regulatory region binds ER{alpha} in response to estrogen stimulation (14). In the present study, we further define the ER{alpha} coactivator molecule(s) that regulate BCAS3 expression and identify the BCAS3 protein itself as an ER{alpha} coactivator. The fact that BCAS3 could be induced by estrogen and is an ER{alpha} coactivator suggests that this is a mechanism for signal amplification in the cell. Estrogen-inducible BCAS3 is itself a coactivator of ER{alpha} and is up-regulated and amplified in breast cancers. Our study underscores this autocrine regulation by ER{alpha}, which could be an important way of amplifying ER{alpha} signaling in breast cancer cells (Fig. 8Go).


Figure 8
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  		Fig. 8. Schematic Representations of the Activation and Action of ER{alpha} and BCAS3

Induction of BCAS3 expression by estrogen occurs via ER{alpha} in conjunction with PELP1 as its coactivator. Upon estrogen stimulation of ER{alpha}, BCAS3 associates with ER{alpha} as well as ER{alpha} coactivators such as PELP1 and HAT enzymes such as P/CAF. Such a complex or complexes are recruited to ER{alpha} target gene chromatin where it functions as an ER{alpha} coactivator to bring about estrogen-induced changes in the cell. CoA, Coactivator.

 
Our studies also define PELP1 as an important molecule involved in both the regulation and function of BCAS3. Unexpectedly, members of the family of p160 coactivators—the best-studied group of coactivators of ER{alpha} that we tested in our system—were incapable of activating the BCAS3 reporter system. There are two possible reasons: either these coactivators require additional binding regions on BCAS3 chromatin to activate BCAS3 expression, or the p160 family is not involved in ER{alpha} regulation of BCAS3. The latter seems to be a more likely explanation because coactivators have been reported to demonstrate preferential association with nuclear receptors depending on the genetic context that the nuclear receptor regulates (3, 18, 19, 20).

Functional characterization of BCAS3 allowed us to conclude that it is an ER{alpha} coactivator. Whether it is specific for ER{alpha} or has similar effects on other nuclear receptors remains to be determined. It is of interest that BCAS3 was ineffective as a coactivator for ER{alpha} in the context of synthetic promoters (3X ERE; data not shown) but was able to induce reporter gene expression from natural promoters such as pS2 and PELP1. This result emphasizes the physiological importance of BCAS3 as an ER{alpha} coactivator. Furthermore, as seen in Fig. 3FGo, overexpression of BCAS3 stimulated pS2 gene expression only under basal conditions and knockdown of BCAS3 resulted in down-regulation of reporter gene activity under both basal and estrogen-induced conditions (Fig. 3GGo). This suggests that BCAS3 is an important regulator of pS2 gene in breast cells. Because BCAS3 has been shown earlier to be overexpressed in breast tumors (14) and could lead to tamoxifen resistance (21), it implies that overexpression of BCAS3 in the physiological setting could lead to estrogen independence of the breast cells. However, this is a hypothesis that still need to be supported by an independent investigation. Also, because BCAS3 lacks the typical LXXLL motif found in most coregulators of nuclear receptors (17), we speculated that the interaction occurs through either a hitherto-unidentified motif or another protein. Because we were able to pull down BCAS3 along with ER{alpha} in an in vitro GST-pull-down assay, the presence of an intermediary linker protein seems unlikely. Our results demonstrated that the region encompassing the bromodomain in BCAS3 is the ER{alpha}-interacting region. This agrees well with previous studies in which bromodomains were found in classic coactivator molecules (16). There is no known nuclear localization signal in BCAS3, and the protein is distributed in both the cytoplasmic and nuclear compartments of the cell, suggesting that BCAS3 has unidentified functions outside of its role as a coactivator.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Cell Culture and Reagents
Human cancer cells were cultured in DMEM/F12 medium supplemented with 10% fetal bovine serum. For the estrogen-treatment experiments, the regular medium was replaced with medium containing 5% dextran-coated, charcoal-stripped medium.

Mouse Studies
For the in vivo estrogen stimulation studies, four 4-wk-old ovariectomized female athymic nude mice (Charles River, Wilmington, MA) were implanted with a 60-d-release estrogen pellet. The mice were killed 6 wk later, and their mammary glands were dissected and used for extraction of proteins. All animal procedures were performed in compliance with our Institutional Animal Care and Use Committee guidelines and the National Institutes of Health Policy on Humane Care and Use of Laboratory Animals.

RNA in Situ Hybridization
To prepare digoxigenin-labeled cRNA probes for in situ hybridization, cDNA fragments of the BCAS3 were subcloned into the vector pDP18 (Ambion, Austin, TX), and linearized with XbaI for antisense probe, and SacI for sense probe respectively. The probes were synthesized using in vitro transcription with a digoxigenin RNA labeling kit (Roche Diagnostics, Indianapolis, IN). Mammary gland tissues were fixed in 4% paraformaldehyde, sectioned, and processed for in situ hybridization using standard procedures (22).

Plasmid Construction and Generation of GST-Tagged BCAS3 Deletion Constructs
Full-length BCAS3 cDNA was purchased from OriGene Technologies (Rockville, MD) and subcloned into the EcoR1 and XbaI sites of pcDNA3.1 (Invitrogen, Carlsbad, CA) vector (forward primer, 5'-GCGAATTCTATGAATGAAGCTATGGCTACAG-3'; reverse primer, 5'-GCAGGTTGCTCTAGACTACGGGAAGCCAGTCGG-3'). Full-length BCAS3 was also subcloned into the EcoR1 and Not1 sites of the pGEX 5X3 vector (Amersham, Piscataway, NJ); forward primer, 5'-GCGAATTCTATGAATGAAGCTATGGCTACAG-3'; reverse primer, 5'-GCAGGTTGCGGCCGCCTACGGGAAGCCAGTCG-3') and into the EcoR1 and Not1 sites of the pCMV vector (Stratagene, La Jolla, CA) (forward primer, 5'-GCGAATTCGTATGAATGAAGCTATGGCTACAG-3'; reverse primer, 5'-GCAGGTTGCGGCCGCCTACGGGAAGCCAGTCGG-3').

Deletion constructs of PELP1 (7) and ER{alpha} (8) have been previously described. BCAS3 deletion constructs were generated using PCR followed by cloning into pGEX 5X1. Deletion A used EcoR1 and Xho1 with primers 5'-CTTGTGGAATTCGATTCTGACAGTGATGGC-3' and 5'-GTTCAATCTCGAGCTCTCCAGCACTTTTCTGGAAACG-3'; deletion B used EcoR1 and Not1 with primers 5'-CAAGCAGCGAATTCGGGTCACCCTTGCATGGG-3' and 5'-CCCAGCGGCCGCAGGGGGAACCAGGCC-3'; and deletion C used EcoR1 and Xba1 with primers 5'-CAATGGAATGAATTCCAGCCACCGTTTAATGCAAAC-3' and 5'-GCAGGTTGCTCTAGACTACGGGAAGCCAGTCGG-3'.

Transient Transfections and Luciferase Assay
Transient transfection studies were done using FuGENE 6 transfection reagent following the recommended protocol (Roche Molecular Biochemicals). The luciferase assay was done using a Promega luciferase assay system (Promega, Madison, WI).

ChIP Assay
The ChIP assay was conducted as reported previously (23). Antibodies against myc (NeoMarkers, Fremont, CA), ER{alpha} (Chemicon International, Temecula, CA), T7 (Novagen, Darmstadt, Germany), BCAS3 (Bethyl Laboratories, Montgomery, TX) or PELP1 (Bethyl Laboratories) were used to immunoprecipitate protein-bound chromatin. The precipitated DNA was then amplified using specific primers for the BCAS3 enhancer (forward, 5'-CCTGAAGATGCCTCCTAGACA-3'; reverse, 5'-CCCTTCCCTCCCTCATTTATT-3'),the pS2 promoter (forward, 5'-GAATTAGCTTAGGCCTAGACGGAATG-3'; reverse, 5'-AGGATTTGCTGATAGGACAGAG-3'), the c-myc promoter (forward, 5'-CCGAAAACCGGCTTTTATAC-3'; reverse, 5'-CTGAGTCTCCTCCCCACCTT-3'), and the actin promoter (forward, 5'-AGTGCCCAAGAGATGTCCAC-3'; reverse, 5'-TCGAGCCATAAAAGGCAACT-3').

RT-PCR and Northern Blot Analysis
Total RNA from cells was extracted using TRIzol reagent (Invitrogen). After deoxyribonuclease treatment, RT-PCR was done using Access RT-PCR kit (Promega) with primers for BCAS3 (forward, 5'-GAAGAATGGCTTTCCCAGGT-3'; reverse, 5'-GTCACGCTCCTGTCAAAGG-3') and actin (forward, 5'-GGACTTCGAGCAAGAGATGG-3'; reverse, 5'-ACATCTGCTGGAAGGTGGAC-3'). Northern blot analysis was done as described previously (23).

Subcellular Protein Extraction
Cellular components were sequentially extracted using a widely adopted biochemical fractionation and sequential extraction procedure (9). The purity of the isolated fractions was established by Western blot analysis of marker proteins.

siRNA Transfection
siRNA transfections were performed using Oligofectamine (Invitrogen) according to the manufacturer’s protocol. BCAS3 siRNA was purchased from QIAGEN (Valencia, CA), and PELP1 siRNA and control-scrambled siRNA were purchased from Dharmacon (Lafayette, CO).

GST-Pull-Down Assay
In vitro transcription and translation of BCAS3, and ER{alpha} were done using a T7-TNT kit (Promega). The procedure used for the binding studies with GST-tagged proteins and 35S-labeled proteins was described previously (23). For GST-pull-down studies of PELP1 with deletion constructs of BCAS3, T7-PELP1 overexpressing cells were lysed in RIPA lysis buffer to obtain the cell lysate. The cell lysate was diluted with NP-40 lysis buffer and appropriate quantities of GST-tagged constructs of BCAS3 immobilized on Sepharose beads was added to equal amounts of the cell lysate. Subsequent to rotation for 3 h at 4 C, the beads were washed thoroughly and the bound proteins were resolved on SDS-polyacrylamide gel, transferred to a nitrocellulose membrane and subjected to Western blot analysis using T7 antibody (Bethyl Laboratories).

Cell Extracts, Immunoprecipitation, and Western Blot Analysis
For immunoprecipitation of ER{alpha}, cells were suspended in high-salt lysis buffer [0.5 M NaCl, 50 mM Tris-HCl (pH 8.0), and 25 µM EDTA]. Cell lysates were diluted with no-salt buffer and immunoprecipitation was performed for 3 h at 4 C using anti-ER{alpha} antibody (1 µg antibody per mg protein). Immunoprecipitated proteins were resolved on SDS-polyacrylamide gel, transferred to a nitrocellulose membrane, and subjected to Western blot analysis. For immunoprecipitation of T7-tagged BCAS3, endogenous BCAS3 or endogenous PELP1, radioimmunoprecipitation assay lysis buffer [10 mM Tris-HCl (pH 7.4), 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, and 0.1 M NaCl] was used to lyse cells, and immunoprecipitation and Western blotting were performed as described earlier (7, 23).

Far-Western Analysis
Far-Western analysis using 35S-labeled BCAS3 was performed as described previously (9, 24).

HAT Assay
T7-tagged BCAS3 was expressed in MCF-7 breast cancer cells. The HAT assay was performed using a HAT-Check activity assay kit (Pierce, Rockford, IL) as described earlier (9, 24).


   ACKNOWLEDGMENTS
 
We thank R. Rajesh Singh for Northern and immunoprecipitation analyses and Amjad Talukder for Far-Western analysis.


   FOOTNOTES
 
Disclosure Statement: The authors have nothing to disclose.

This study was supported in part by National Institutes of Health Grants CA098823 and CA90970 (to R.K.).

First Published Online May 15, 2007

Abbreviations: BCAS3, Breast carcinoma amplified sequence-3; ChIP, chromatin immunoprecipitation; E2, 17ß-estradiol; ER, estrogen receptor; ERE, estrogen-responsive element; GST, glutathione S-transferase; HAT, histone acetyltransferases; MNAR, modulator of nongenomic activity of ER; P/CAF, p300/CBP-associated factor; PELP1, proline-, glutamic acid-, and leucine-rich protein-1; RNAi, RNA interference; siRNA, small interfering RNA.

Received for publication December 1, 2006. Accepted for publication May 10, 2007.


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 DISCUSSION
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NURSA Molecule Pages Link:

Nuclear Receptors:   ERα
Coregulators:   P/CAF  |  CBP  |  SRC-1  |  PELP1  |  GRIP1  |  AIB1
Ligands:   17β-Estradiol  |  Fulvestrant




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