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Molecular Endocrinology, doi:10.1210/me.2005-0040
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Molecular Endocrinology 19 (6): 1584-1592
Copyright © 2005 by The Endocrine Society

Functional Genomics Identifies a Mechanism for Estrogen Activation of the Retinoic Acid Receptor {alpha}1 Gene in Breast Cancer Cells

Josée Laganière, Geneviève Deblois and Vincent Giguère

Molecular Oncology Group (J.L., G.D., V.G.), McGill University Health Center, and Departments of Biochemistry (J.L., V.G.), Medicine (V.G.), and Oncology (V.G.), McGill University, Montréal, Québec, Canada H3A 1A1

Address all correspondence and requests for reprints to: Dr. Vincent Giguère, Molecular Oncology Group, McGill University Health Centre, Room H5–21, 687 Pine Avenue West, Montréal, Québec, Canada H3A 1A1. E-mail: vincent.giguere{at}mcgill.ca.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The identification of estrogen receptor (ER{alpha}) target genes is crucial to our understanding of its predominant role in breast cancer. In this study, we used a chromatin immunoprecipitation (ChIP)-cloning strategy to identify ER{alpha}-regulatory modules and associated target genes in the human breast cancer cell line MCF-7. We isolated 12 transcriptionally active genomic modules that recruit ER{alpha} and the coactivator steroid receptor coactivator (SRC)-3 to different intensities in vivo. One of the ER{alpha}-regulatory modules identified is located 3.7 kb downstream of the first transcriptional start site of the RARA locus, which encodes retinoic acid receptor {alpha}1 (RAR{alpha}1). This module, which includes an estrogen response element (ERE), is conserved between the human and mouse genomes. Direct binding of ER{alpha} to the ERE was shown using EMSAs, and transient transfections in MCF-7 cells demonstrated that endogenous ER{alpha} can induce estrogen-dependent transcriptional activation from the module or the ERE linked to a heterologous promoter. Furthermore, ChIP assays showed that the coregulators SRC-1, SRC-3, and receptor-interacting protein 140 are recruited to this intronic module in an estrogen-dependent manner. As expected from previous studies, the transcription factor Sp1 can be detected at the RARA {alpha}1 promoter by ChIP. However, treatment with estradiol did not influence Sp1 recruitment nor help recruit ER{alpha} to the promoter. Finally, ablation of the intronic ERE was sufficient to abrogate the up-regulation of RARA {alpha}1 promoter activity by estradiol. Thus, this study uncovered a mechanism by which ER{alpha} significantly activates RAR{alpha}1 expression in breast cancer cells and exemplifies the utility of functional genomics strategies in identifying long-distance regulatory modules for nuclear receptors.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
NUCLEAR RECEPTORS CONSTITUTE a superfamily of transcription factors that control reproduction, embryonic development, homeostasis, and play important roles in the initiation, progression, and treatment of numerous diseases, including cancer. The cloning of the glucocorticoid and estrogen receptors 20 yr ago (1, 2), together with the development of a rapid assay for receptor activity in transfected cells (3), set the stage for investigating the molecular mechanisms of small lipophilic ligand-regulated transcription. Whereas early work focused on the receptors themselves, defining their functional domains (3, 4) and their interaction with DNA (5), it is now well understood that nuclear receptor-regulated gene expression requires the recruitment, in a multistep fashion, of diverse sets of coregulatory proteins. To ensure modulated hormonal control of gene expression, these regulatory complexes can induce changes in chromatin structure, control the basal transcription machinery activity, and regulate the degradation of the receptors and associated proteins (reviewed in Refs. 6, 7, 8). The ability of nuclear receptors to modulate transcription of target genes is achieved through recognition by the receptors of short sequences referred to as hormone response elements located in the promoters and enhancers of these genes (9). In addition, molecular cross-talk allows nuclear receptors to regulate the expression of genes via association, either on DNA or in solution, with other transcription factors such as Sp1 and activator protein 1 (reviewed in Refs. 10, 11, 12). However, whereas nuclear receptors are expected to control a large number of genes, given their expansive roles in development and physiology, relatively few genomic targets have been identified to date.

Although it has been known for decades that estrogens are potent stimulators of estrogen receptor-positive breast cancer cell proliferation (13), the exact mechanisms underlying their growth-stimulating effects are still unknown. Delineation of estrogen action in these cells has been hindered by the paucity of bona fide ER{alpha}-regulated genes identified discovered thus far (14). Recently, gene expression profiling experiments have identified genes with altered expression upon estrogen treatment of human breast cancer cells, but very few have been confirmed as ER{alpha} primary targets (15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25). In an attempt to identify direct genomic targets of ER{alpha}, we recently developed a chromatin immunoprecipitation (ChIP)-derived approach to isolate genomic fragments bound to the receptor in MCF-7 cells (26). Interestingly, among the cloned targets, endogenous ER{alpha} was found to bind in vivo to an estrogen response element (ERE) located in the first intron of the RARA locus, the gene encoding retinoic acid receptor {alpha} (RAR{alpha}). In this report, we investigated the role played by this regulatory region in the control of RARA in response to estradiol. We found that, in addition to recruiting coactivators, this intronic ERE provides the major estrogen response of the RARA gene in MCF-7 cells. This study highlights the predominant role that functional genomics will play in the near future in defining gene networks directly regulated by nuclear receptors and how, mechanistically, members of this superfamily of transcription factors exert this control.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Identification of ER{alpha} Genomic Targets in Human Breast Cancer Cells
To identify regulatory modules directly bound by ER{alpha} in human breast cancer cells, we performed ChIP using a specific antibody against ER{alpha} in MCF-7 cells followed by cloning and sequencing of the fragments obtained, as previously described (26). The location of each fragment in the human genome was determined using the University of California Santa Cruz (UCSC) human genome database (http://genome.ucsc.edu). Twelve fragments containing either EREs or multiple half-sites were selected for further analysis (see Table 1Go). First, the binding of ER{alpha} to these modules was reevaluated using standard ChIPs and quantified by real-time PCR using primers specific for each fragment isolated. As shown in Fig. 1Go, we found that the genomic sequences examined were significantly bound by ER{alpha} in vivo, being enriched at least 2-fold over the control (no antibody) when retested by standard ChIP PCR with specific primers. This series of fragments included a genomic region located 220 bp upstream of the TFF1 (pS2) start site (clone ER4282), a promoter region known to contain a well-defined ERE and to be estrogen responsive (27). Quantitative PCR quantification of the enrichment obtained by an independent ER{alpha} standard ChIP assay using primers specific for the TFF1 promoter showed an enrichment of 108-fold over the control when cells were treated with estradiol for 45 min before chromatin preparation (Fig. 1Go). Other fragments located near or at promoter regions were isolated and recognized in vivo by ER{alpha}: the promoters of FLJ10618, which encodes a predicted protein, NAP1L4 (nucleosome assembly protein 1-like 4), as well as a fragment located 2.2 kb upstream of RNF14, which encodes a coactivator of the androgen receptor also known as ARA54 (28). Interestingly, in addition to binding to promoters, ER{alpha} also recognized modules distal from known transcriptional start sites. We found ER{alpha}-regulatory modules located from 4–103 kb from the DDEF2 (development and differentiation enhancing factor 2), GPR81 (G protein-coupled receptor 81), EDF1 (endothelial differentiation-related factor 1), EDG1 (endothelial differentiation, sphingolipid G protein-coupled receptor 1), FLJ16032, BCR (breakpoint cluster region), FLJ41849, and RARA genes. All modules except DER005 (Table 1Go) were well conserved between the mouse and human genomes (data not shown).


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  		Table 1. ERE and Nuclear Receptor Half-Site Binding-Related Sequences in the MCF-7 Genomic Fragments Isolated by ChIP Cloning Using an ER{alpha} Antibody

 


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  		Fig. 1. Identification of Direct ER{alpha} Target-Regulatory Modules in MCF-7 Cells

A, In vivo binding of ER{alpha} and SRC-3 to novel regulatory modules identified by ChIP cloning. Standard ER{alpha} and SRC-3 ChIP assays in MCF-7 cells, followed by quantitative PCR using primers specific for the region cloned. The graph shows enrichment in fold over the control (no antibody) by quantitative PCR. The results shown here are representative of three independent ChIP experiments. Clone, Clone number of the fragments obtained by ChIP cloning; gene, closest annotated gene from the UCSC human genome database; locus link, locus link number of the gene; distance, distance from the closest annotated gene. B, Modulation of target gene expression by estradiol. MCF-7 cells were treated with estradiol for various time points. The total RNA was extracted and used for cDNA production followed by quantitative RT-PCR using specific primers. C, Control; E2, estradiol.

 
It is now well established that ER{alpha} recruits coactivators in response to estradiol to modulate gene expression (6, 29), and that recruitment of coactivators is a good indicator of the transcriptional activity of a transcription factor-binding site. We thus investigated whether estradiol treatment would lead to SRC-3 recruitment to these modules. As shown in Fig. 1Go, steroid receptor coactivator 3 (SRC-3) was indeed recruited to the ER{alpha}-bound targets. We next investigated the estrogen responsiveness of a number of genes located near the ER{alpha}-bound modules. We performed quantitative RT-PCR for genes that were located close to an ER{alpha} target region, because regions located at very far distances are likely to control closer transcripts not yet annotated or annotated but not reviewed. As shown in Fig. 2Go, we observed, as expected, that TFF1 and RARA mRNA amounts were increased after estradiol treatment. In addition, we observed that FLJ10618and RNF14 were also up-regulated after estradiol treatment, following distinct response profiles to the hormone. In contrast, GPR81 was down-regulated by 2-fold whereas BCR was not significantly modulated by estradiol.



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  		Fig. 2. An ERE Is Located in the First Intron of RARA Gene

A, The RARA transcripts are produced from two distinct promoters, {alpha}1 and {alpha}2. The ERE isolated by ChIP cloning is located 3.7 kb downstream of the first RARA {alpha}1 promoter and 8.6 kb upstream of the second RARA {alpha}2 promoter (UCSC annotation). B, Complete sequence of the DER001 genomic fragment. The ERERARA is boxed. The fragment obtained by ER{alpha} ChIP cloning is well conserved among the human, mouse, and rat genomes. Asterisk represents the bases that are conserved. Overall, 70 bp of the 130 bp are conserved in the three genomes shown. C, The ERERARA is conserved among these genomes. The bases corresponding to the ERE consensus sequence are shown in capital letters.

 
Our laboratory was particularly interested in the finding that enrichment of ER{alpha} by ChIP corresponded to a region, according to the UCSC genome database, located 8.36 kb upstream of the RARA gene that encodes a receptor for retinoic acid, RAR{alpha}. In fact, it has been determined that transcription of the RARA gene is achieved through two distinct promoters, generating two different mRNAs encoding the RAR{alpha}1 and RAR{alpha}2 isoforms (30). Close examination of the RARA gene shows that the ERE-containing module isolated by ChIP cloning is indeed located within the first intron of RARA, 3.7 kb downstream of its first transcriptional start site (RAR{alpha}1) and 8.36 kb upstream of the second (RAR{alpha}2) promoter (Fig. 2AGo). In addition, recent studies have shown that RAR{alpha} expression in MCF-7 cells depends solely on the RARA {alpha}1 promoter because the RARA {alpha}2 promoter is inactivated by methylation in these cells (31). Therefore, we decided to focus our investigation on the significance of this ERE (herein referred to as ERERARA) in the control of the expression of the RAR{alpha}1 isoform in MCF-7 cells.

The ERERARA Is Functional in Vitro and in Vivo
We were next interested in establishing the functionality of the novel ERE found in the first intron of the RARA gene. The ERERARA differs by only one base from the established ERE consensus (Fig. 2CGo), but an ERE with this sequence had not been previously reported to be functional in vivo. As mentioned above, both the ERERARA itself and the genomic fragment obtained by ChIP cloning (130 bp in length) are well conserved from the mouse to the human genome (Fig. 2Go, B and C). We first tested the ability of ER{alpha} to bind the ERE in vitro. As expected from the ChIP-cloning experiment and shown in Fig. 3AGo, an EMSA demonstrates that in vitro translated ER{alpha} recognized the ERE probe directly. In addition, endogenous ER{alpha} contained in total MCF-7 cell extract also affected the migration of the ERE probe in the gel. The presence of ER{alpha} in the retarded complex was confirmed by a supershift of the complex using an anti-ER{alpha} antibody (Fig. 3BGo). To determine whether ER{alpha} could modulate gene transcription using this ERE, we performed transient transfections in MCF-7 cells of a TK-Luc reporter containing one copy of either ERERARA or the whole 130-bp fragment (DER001) isolated by ChIP cloning. Our results show that treatment of MCF-7 cells with estradiol leads to a large induction of luciferase activity in cells transfected with either reporter plasmid, indicating that endogenous ER{alpha} can utilize the ERERARA to activate gene transcription (Fig. 3CGo).



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  		Fig. 3. The Novel ERERARA Isolated in the RARA First Intron Is Functional

A, EMSA using in vitro translated ER{alpha} and the ERERARA as a probe. Comp, Vitallogenin ERE competitor probe. B, EMSA using endogenous ER{alpha} from MCF-7 total cell extracts. Ab, ER{alpha} antibody supershift. C, Transient transfection in MCF-7 cells. TK-Luc reporters containing either the whole DER001 fragment obtained by ChIP cloning or ERERARA were transfected in MCF-7 cells, in the presence of estradiol (E2) or vehicle (C).

 
The Intronic ERERARA Is Transcriptionally Active in MCF-7 Cells
The recruitment of coregulators to the intronic ERERARA in the presence of estradiol would suggest that the ERE is actively engaged in transcriptional regulation of RARA. In addition, because it was previously suggested that ER{alpha} controls the expression of the RAR{alpha}1 isoform through an ER{alpha}-Sp1 interaction occurring at the promoter (32, 33), we also evaluated the binding of specific transcriptional modulators at the first RARA promoter. We thus performed ChIP experiments using primers specific for the RARA {alpha}1 promoter, the intronic ERERARA, and a generic control region located 4 kb upstream of the TFF1 promoter. As previously shown in Fig. 1Go, ER{alpha} recognized the intronic region containing the ERERARA in the absence of estradiol, and its binding was significantly enhanced in the presence of the hormone (Fig. 4Go). Data presented in Fig. 4Go also demonstrate that both SRC-1 and SRC-3 are recruited to the ERERARA region in the presence of estradiol, further supporting the in vivo activity of the ER{alpha}-bound ERERARA. Interestingly, the coregulator RIP140 (receptor-interacting protein 140) was also strongly (>40-fold enrichment) recruited to the region containing the ERERARA (Fig. 4Go). RIP140 recruitment was also observed at the TFF1 promoter and other regulatory modules isolated (data not shown). Modest binding of Sp1 (6-fold enrichment) was also found at the ERERARA region. In sharp contrast, none of the coactivators tested or ER{alpha} was significantly detected at the RARA (RAR{alpha}1) promoter region. Similar results were also obtained after estradiol treatment at various time points taken between 5 min and 5 h (data not shown). In addition, strengthening of protein-protein interactions with the cross-linking agent, dimethyl 3,3'-dithiobispropionimidate 2·HCl, before formaldehyde treatment of the prepared chromatin did not improve the detection of ER{alpha} at the promoter (data not shown). However, a significant presence of Sp1 could be detected by ChIP at the promoter, but addition of estradiol had no significant effect on the recruitment of the protein.



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  		Fig. 4. The ERERARA Is Active in Vivo

Sp1, ER{alpha}, SRC-1, SRC-3, and RIP140 ChIP assays were performed using MCF-7 cells in the presence of estradiol (E2) or vehicle (C), followed by PCR using primers specific for the RARA a1 promoter, the intronic ERE (3.7 kb downstream of the promoter) and a control region located 4 kb upstream of the TFF1 promoter (control). No ab, No antibody control.

 
The Intronic ERERARA Controls RAR{alpha}1 Response to Estrogens
Finally, we sought to determine whether the newly discovered ERERARA plays a direct role in the control of the RAR{alpha}1 gene expression. To this end, we cloned a 5-kb genomic DNA fragment containing the RARA {alpha}1 promoter and a segment of the first intron that includes the ERERARA. The same construct with a deleted ERERARA was also made (Fig. 5AGo). When the intact construct was transfected in MCF-7 cells, we observed a strong induction of luciferase activity after estradiol treatment, whereas less than 2-fold activation was obtained when the ERERARA-deleted construct was assayed in the same cells (Fig. 5BGo). Our data thus suggest that the main estrogenic response of the RARA gene is driven by the intronic ERERARA in MCF-7 cells. To confirm that ER{alpha} is responsible for this activity, we performed transient transfections of the same constructs in MDA-MB-231, an ER-negative breast cancer cell line, in the presence or absence of exogenous ER{alpha}. Figure 5CGo shows that these cells depend on both ER{alpha} and the ERERARA to provide estradiol-induced activation of the RARA locus, because the activation by estradiol is observed only in the presence of the receptor and the intronic ERERARA. We also wanted to determine whether the induction of RAR{alpha}1 through this ERE could be observed in other ER-positive cell lines. In Fig. 5DGo, we show that endogenous ER{alpha} from BT-474 breast cancer cells activates the reporter in a similar fashion.



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  		Fig. 5. The Intronic ERERARA Controls RARA {alpha}1 Response to Estradiol

A, Schematic representation of the region of the RARA locus inserted in the pRL-Null vector. RAR{alpha}1ERE and RAR{alpha}1{Delta}ERE represent the locus with and without ERERARA, respectively. B, Transient transfections in MCF-7 cells in the presence of estradiol (E2) or vehicle (C). C, Transient cotransfections of the two reporter constructs in MDA-MB-231 cells together with ER{alpha} or empty vector (CMX), in the presence of estradiol or vehicle. D, Transient transfections of the reporters described in panel A, in an ER-positive breast cancer cell line BT-474. RLU, Relative light units.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
In this report, we described the identification of direct genomic targets of endogenous ER{alpha} in the MCF-7 breast cancer cell line using a ChIP assay-derived approach. We found novel direct regulatory sequences directly bound in vivo by ER{alpha}, which were located upstream, downstream, or in annotated promoter regions. Among the 12 ER{alpha} binding modules described in this study, two were associated with genes (TFF1 and RARA), previously known to be regulated by estradiol in human breast cancer cells. Although less high-throughput than promoter arrays (34), this functional approach has the advantage of identifying binding sites located outside of promoter regions. This is exemplified by the focus of our investigation on the ERERARA located in the first intron of the RARA gene, which was strongly bound by ER{alpha} in vitro and in vivo. In addition, the presence of the ERERARA was found to be essential for significant activation by estradiol of the RARA {alpha}1 promoter in transfected MCF-7 cells. Most importantly, we also showed that, in addition to ER{alpha}, the SRC-1 and SRC-3 coactivators were recruited to the ERERARA in an estrogen-dependent manner, demonstrating that this intronic site becomes transcriptionally operational in MCF-7 cells after estradiol treatment. Recent evidence indeed states the important regulatory role of the first intron in the regulation of gene expression. Based on chromosome 21 and 22 gene location analyses of Sp1 and other transcription factors, a great proportion of all functional transcription factor-binding sites are located a few hundred or thousand bases downstream of annotated promoters, strongly predicting an important regulatory role of the first intron for gene transcription in general (35). Our study supports the view that, similar to other transcription factors such as c-Myc and Sp1, ER{alpha} action is not restricted to promoters in vivo. Therefore, whole-genome strategies will be essential to reveal distal but functional EREs, indeed providing a much improved picture of ER{alpha} action in breast cancer cells and at other sites of estradiol action.

As mentioned above, RARA is a well known estrogen-regulated gene with potential as a target for cancer prevention and therapy because treatment with retinoids leads to cell growth arrest and apoptosis in ER{alpha}-positive breast cancer cells (36, 37). RARA can produce two different transcripts, encoding the RAR{alpha}1 and RAR{alpha}2 isoforms, originating from two distinct promoters (30, 38). In MCF-7 cells, the RAR{alpha}1 isoform is predominant because the RARA {alpha}2 promoter is methylated in these cells (31). It has also been demonstrated that RAR{alpha}1 is the only estrogen-regulated isoform in breast cancer cells (39). Up-regulation of RAR{alpha}1 expression by estrogens was previously attributed to an ER{alpha}-Sp1 interaction occurring at a GC-rich sequence in the first RARA promoter (32). Another laboratory had previously found an imperfect ERE half-site and Sp1 motif in the promoter presumably responsible for the estrogen induction of RAR{alpha}1 (40). However, no direct binding of ER{alpha} was detected at these sites. In addition, the studies were performed with overexpressed exogenous proteins and restricted to proximal sections of the promoter. To have a more physiological representation of RARA {alpha}1 regulation, we transiently transfected a luciferase reporter construct of the genomic region containing the RARA {alpha}1 promoter and a portion of the first intron including the ERERARA, or its ERE-deleted mutant. Interestingly, the treatment of cells with estradiol led to a strong increase in luciferase activity, indicating that endogenous ER{alpha} was sufficient to promote RARA {alpha}1 up-regulation by the hormone. Moreover, in the ERERARA deletion mutant, the luciferase activity was nearly unchanged after estradiol treatment; hence, the ERERARA confers the estrogenic response of RAR{alpha}1 in MCF-7 cells.

Because the growth-suppressive effect of retinoids is limited to ER-positive cells, it has been suggested that the action of retinoids is mainly antiestrogenic (41, 42, 43, 44). A recent study has shown that induction by retinoic acid of the coregulator RIP140, a known corepressor of ER activity (45, 46, 47), could mediate the antiestrogenic effects of retinoic acid in ER-positive human breast cancer cells (48). Interestingly, we showed that endogenous RIP140 was recruited to the ERERARA, an event that we also observed to occur at the estrogen-inducible TFF1 promoter. This is, to our knowledge, the first demonstration that endogenous RIP140 is recruited to ER{alpha} target-regulatory modules and promoters, a finding that further supports the view that RIP140 plays a central role in the estrogenic response of breast cancer cells. In a manner analogous to the switch between coactivators and the corepressor nuclear receptor corepressor in apo-ER{alpha}-bound promoters recently shown by Métivier et al. (49, 50), our finding suggests that a dynamic exchange may occur between the coactivators SRC-1 and SRC-3 and the corepressor RIP140 to provide subtle modulation of gene control in response to estradiol and retinoic acid in breast cancer cells.

In conclusion, this and other recent studies (34, 51) clearly demonstrate the importance that functional genomics will play in the near future in identifying, in a genome-wide and unbiased manner, hormone response elements and associated genes directly controlled by all members of the nuclear receptor superfamily. After 20 yr of work dedicated to the receptors themselves and their interactions with DNA and other regulatory proteins, the tools are finally available to reveal the vast networks of genes regulated by nuclear receptors in a cell-specific manner.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Plasmids
To construct TK-ERERARALuc, we used the synthetic oligonucleotides 5'-GATCCAGCGAGTGGGTCACGGTGACACTGCCTGCG-3' and 5'-TCGACGCAGGCAGTGTCACCGTGACCCACTCGCTG-3' that were annealed and subsequently ligated in TK-Luc, using SalI and BamHI restriction sites. For the pRL-RAR{alpha}1 construct (Fig. 5Go), the RARA promoter/intron 1 locus was amplified from total MCF-7 genomic DNA with the following PCR primers: 5'-GTCTCTCAGATGGAGGGTGATTCAGATCC-3' and 5'-CGACGCGTCAGGAAGT-GACAGCCACGTGACAGGAAGAC-3'. The PCR product was digested with HindIII and MluI and ligated in corresponding pRL-Null vector (Promega Corp., Madison, WI) restriction sites. The pRL-RAR{alpha}1{Delta}ERE construct was made using PCR products from two oligonucleotide sets, one designed for a PCR product upstream of ERERARA and the other for the region downstream of ERERARA. PCRs were performed using Expand Long Template PCR System (Roche Applied Science, Mannheim, Germany). For the region upstream, the oligonucleotides used were 5'-GTCTCTCAGATGGAGGGTGATTCAGATCC-3' and 5'-GACTAGTGCCTGCGGGGTACAGTGACACATGGAGAC-3' followed by digestion with HindIII and SpeI. The region downstream of the ERE was amplified using oligos 5'-GACTAGTGTACACACCTACCTTGGAGTGGCTTTATCC-3' and 5'CGACGCGTCAGGAAGTGACAGCCACGTGACAGGAAGAC-3'. The digested PCR products were ligated into pRL-Null vector.

Cell Culture and Transient Transfections
MCF-7, MDA-MB 231, and BT-474 cells were cultured in DMEM containing penicillin (25 U/ml), streptomycin (25 U/ml), and 10% fetal calf serum at 37 C with 5% CO2. At least 3 d before transfection, cells were cultured in phenol red-free DMEM supplemented with 10% charcoal-dextran-stripped fetal bovine serum. The cells were transfected with FuGENE 6 transfection reagent (Roche Applied Science), according to the protocol supplied by the manufacturer. Typically, 0.5 µg of reporter plasmid and 0.3 µg of pCMVßGal internal control were transfected per well. Twelve hours after transfection, fresh medium was added containing ethanol (vehicle) or estradiol (10–7 M). Cells were then harvested 24 h later and assayed for luciferase and ß-galactosidase. For pRL vector constructs, Renilla Luciferase was assayed using the Renilla Luciferase kit (Promega).

EMSA
ER{alpha} proteins were synthesized by in vitro transcription-translation using rabbit reticulocyte lysates (Promega). DNA-binding reactions were conducted as previously described (52) using 5 µl of programmed lysates in each binding reaction or 50 µg of protein obtained from a MCF-7 total cell extract.

RT-PCR
Total RNA was extracted from MCF-7 cells using RNeasy mini kit (QIAGEN, Chatsworth, CA). Reverse transcription reactions were performed using Superscript II (Invitrogen) according to the manufacturer’s recommendations. Quantitative RT-PCR was performed using Light Cycler (Roche Applied Science) and QuantiTech SYBR Green PCR kits (QIAGEN) according to the manufacturers’ recommendations. Fold increase of transcription by estradiol were calculated using ribosomal protein large P0 as an internal control. Primer sequences used for RT-PCR are available upon request.

ChIP and ChIP Cloning
The ChIP-cloning procedure has been described previously (26). Briefly, the fragments obtained after ER{alpha} ChIP by double immunoprecipitation using an ER{alpha} antibody (HC20, Santa Cruz Biotechnology, Inc., Santa Cruz, CA) were repaired using T4 DNA polymerase, cloned in Ready-to-go pUC19-Sma1 BAP+Ligase (Pharmacia Biotech, Piscataway, NJ) and sequenced. Quantitative PCR was performed using Light Cycler and SYBR Green Light cycler kit (Roche Diagnostics, Mannheim, Germany) according to the manufacturer’s recommendations. For standard ChIP, SRC-1, P/CPB (cAMP response element binding protein-binding protein) interacting protein, and RIP140 antibodies were purchased from Santa Cruz Biotechnology, and the Sp1 antibody was obtained from Upstate Biotechnology, Inc. (Lake Placid, NY). Primers used for standard ChIP at the RARA {alpha}1 promoter were 5'-TCTCCACCGAGCGCTATTTTCATTCTTTCC-3' and 5'-CTGACTGGTGATTGGTCGGTGGGCGGGCAG-3'; the ERERARA region, 5'-GAGGCTCAGGACAGGGCAAGAGTGGGGCAC-3' and 5'-GACAGAGGGAAGGA-GGGCTGAGGACCTGCG-3'; the control region, 5'-CTGGGCAATGCGAGGAGAGTGAAGACTG-3' and 5'-GGGGAGGGAGGAGTTTGGAGGAAGTGG-3'. All primers used for standard ChIP on novel regulatory modules are available upon request.


   ACKNOWLEDGMENTS
 
We thank Yoshi Kiriyama for primers, Céline Lefebvre for expert technical assistance, and Catherine Dufour for critical reading of the manuscript.


   FOOTNOTES
 
This work was supported by Genome Quebec/Canada and the Canadian Institutes for Health Research (CIHR); a CIHR senior scientist career award (to V.G.); and US Department of Defense Breast Cancer Research Program Predoctoral Traineeship Award W8IWXH-04-1-0399 (to J.L.).

First Published Online April 14, 2005

Abbreviations: ChIP, Chromatin immunoprecipitation; ER{alpha}, estrogen receptor {alpha}; ERE, estrogen response element; RAR{alpha}, retinoic acid receptor {alpha}; SRC, steroid receptor coactivator; RIP140, receptor-interacting protein 140; UCSC, University of California Santa Cruz.

Received for publication January 14, 2005. Accepted for publication April 5, 2005.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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NURSA Molecule Pages Link:

Nuclear Receptors:   RARα  |  ERα
Coregulators:   RIP140  |  SRC-1  |  AIB1
Ligands:   17β-Estradiol



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