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Cancer Research 67, 5017, May 15, 2007. doi: 10.1158/0008-5472.CAN-06-3696
© 2007 American Association for Cancer Research
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Endocrinology

Novel Estrogen Receptor-{alpha} Binding Sites and Estradiol Target Genes Identified by Chromatin Immunoprecipitation Cloning in Breast Cancer

Zhihong Lin1, Scott Reierstad1, Chiang-Ching Huang2 and Serdar E. Bulun1

Departments of 1 Obstetrics and Gynecology and 2 Preventive Medicine, Feinberg School of Medicine, Northwestern University, Chicago, Illinois

Requests for reprints: Serdar E. Bulun, Department of Obstetrics and Gynecology, Feinberg School of Medicine, Northwestern University, Chicago, IL 60611. Phone: 312-503-1600; Fax: 312-503-0095; E-mail: s-bulun{at}northwestern.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Estrogen receptor-{alpha} (ER{alpha}) and its ligand estradiol play critical roles in breast cancer growth and are important therapeutic targets for this disease. Using chromatin immunoprecipitation (ChIP)-on-chip, ligand-bound ER{alpha} was recently found to function as a master transcriptional regulator via binding to many cis-acting sites genome-wide. Here, we used an alternative technology (ChIP cloning) and identified 94 ER{alpha} target loci in breast cancer cells. The ER{alpha}-binding sites contained both classic estrogen response elements and nonclassic binding sequences, showed specific transcriptional activity in reporter gene assay, and interacted with the key transcriptional regulators, including RNA polymerase II and nuclear receptor coactivator-3. The great majority of the binding sites were located in either introns or far distant to coding regions of genes. Forty-three percent of the genes that lie within 50 kb to an ER{alpha}-binding site were regulated by estradiol. Most of these genes are novel estradiol targets encoding receptors, signaling messengers, and ion binders/transporters. mRNA profiling in estradiol-treated breast cancer cell lines and tissues revealed that these genes are highly ER{alpha} responsive both in vitro and in vivo. Among estradiol-induced genes, Wnt11 was found to increase cell survival by significantly reducing apoptosis in breast cancer cells. Taken together, we showed novel genomic binding sites of ER{alpha} that regulate a novel set of genes in response to estradiol in breast cancer. Our findings suggest that at least a subset of these genes, including Wnt11, may play important in vivo and in vitro biological roles in breast cancer. [Cancer Res 2007;67(10):5017–8]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Estrogen receptor-{alpha} (ER{alpha}; ref. 1) is a ligand-activated nuclear receptor that regulates transcription of estrogen-responsive genes important for cell growth, differentiation, and malignant transformation in various target cells (2). ER{alpha} and its ligand estradiol [17ß-estradiol (E2)] play critical roles in the growth of breast cancer tissue and are important therapeutic targets (3). Significant progress has been made in understanding the role of ER{alpha} as a transcription factor that regulates the expression of target genes by directly binding to an estrogen response element (ERE; ref. 4) or by association with other transcription factors on promoter targets (1, 4, 5). Until recently, however, little had been known about the distribution of ER{alpha}-binding sites within the genome and the identity of genes regulated by these cis-acting elements.

Two recent pioneering publications by Carroll et al. (6, 7) revolutionized our understanding of ER{alpha} action. Using chromatin immunoprecipitation (ChIP)-on-chip, this group mapped a large number of ER{alpha}-binding sites on a chromosome and genome-wide scale, identifying novel cis-regulatory sites and target genes in MCF-7 breast cancer cells (6, 7). The majority of these binding sites were distant from the transcription start sites of regulated genes (6, 7).

Identification of novel genomic targets and a deeper understanding of their transcriptional regulation by ER{alpha} and their physiologic function may lead to the development of more specific and effective treatments for breast cancer. In the current study, we used a technique alternative to ChIP-on-chip (i.e., ChIP-linked target site cloning) for unbiased and potentially genome-wide identification of regulatory targets of estradiol/ER{alpha} in breast cancer cells. Our aims were to determine the nature of ER{alpha} binding relative to the structure of a gene and increase our understanding of ER{alpha} action both in normal tissue and in the malignant state. We characterized these binding sites and regulation of proximal genes by estradiol/ER{alpha} both in vitro and in vivo. The majority of the 38 estrogen-regulated genes turned out to be previously unknown ER{alpha} targets. We showed in detail previously unknown biological roles of one of these estrogen/ER{alpha}–regulated genes, Wnt11, in breast cancer.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell lines and tissues. MCF-7, T47D, and MDA-MB-231 cells (American Type Culture Collection) were maintained in MEM (Invitrogen) containing 25 units/mL penicillin, 25 units/mL streptomycin, and 10% fetal bovine serum (FBS) at 37°C and 5% CO2. Snap-frozen ER+ (n = 25) and ER– (n = 20) breast cancer tissues were obtained from the Northwestern Breast Specialized Programs in Research Excellence Tissue Core Facility. These tissues were collected after obtaining written informed consent approved by the Institutional Review Board of Northwestern University.

Chromatin immunoprecipitation. After MCF-7 cells were grown to 75% to 80% confluence in MEM supplemented with 10% FBS, the cells were serum starved in DMEM/F-12 without phenol red (Invitrogen) and FBS for 24 h. After 3 h of treatment with 10–9 mol/L E2, cells were washed twice with cold PBS and cross-linked with 1% formaldehyde at room temperature for 10 min. The cross-linking reaction was stopped by adding glycine containing a cocktail of protease inhibitors (Sigma) to a final concentration of 125 nmol/L for 5 min at room temperature. Cells were rinsed twice with cold PBS, harvested, and stored at –80°C before use. Cell pellets were lysed and sonicated to shear the DNA into 0.6- to 3.0-kb fragments. Insoluble material was removed by centrifugation, and the extract was precleared by incubation with blocked protein A-agarose/Salmon Sperm DNA (Upstate) for at least 1 h at 4°C to reduce nonspecific interactions. After centrifugation, the supernatant (50 µL) was collected as input, and the remainder was diluted in buffer [1% Triton X-100, 2 mmol/L EDTA, 50 mmol/L Tris-HCl (pH 8.1)] and subjected to immunoprecipitation overnight at 4°C with a monoclonal antibody against ER{alpha} (Upstate).

After immunoprecipitation, 60 µL protein A-agarose/Salmon Sperm DNA beads were added and the incubation was continued for another 1 h. To decrease nonspecific binding, DNAs/protein complexes were washed under high-stringency wash conditions. Precipitates were washed sequentially for 10 min, two times in buffer I [0.1% SDS, 1% Triton X-100, 2 mmol/L EDTA, 20 mmol/L Tris-HCl (pH 8.0), 150 mmol/L NaCl], six times in buffer II [0.1% SDS, 1% Triton X-100, 2 mmol/L EDTA, 20 mmol/L Tris-HCl (pH 8.0), 500 mmol/L NaCl], two times in buffer III [0.25 mol/L LiCl, 1% NP40, 1% deoxycholate, 1 mmol/L EDTA, 10 mmol/L Tris-HCl (pH 8.0)], and twice with 1 mmol/L EDTA and 10 mmol/L Tris-HCl (pH 8.0). Precipitated chromatin complexes were removed from the beads through a 15-min incubation with 50 µL of 1% SDS and 0.1 mol/L NaHCO3 and vortexing at room temperature. This step was done twice. Eluates were pooled and heated at 65°C overnight to reverse the formaldehyde cross-linking. DNA fragments were purified with a MinElute Reaction Cleanup kit (Qiagen). The immunoprecipitated and input DNA samples were assayed for binding to the MYC promoter region as a positive control before ChIP cloning. For PCR, 1 µL of purified DNA extraction and 40 cycles of amplification were used.

Cloning, sequencing, and analysis of ER{alpha}-binding fragments. ChIP-derived DNA was subjected to AvaII restriction digestion and PCR linker ligation followed by PCR amplification (40 cycles) generating sufficient material for cloning into the pGEM-T Easy Vector System (Promega). Clones were sequenced and every retrieved ChIP fragment was mapped to the human genome using the BLAT search function of University of California Santa Cruz genome browser or ENSEMBL search. The presence of EREs was analyzed by ERE Finder (8), and activator protein-1 (AP-1) sites were identified using the Transcription Element Search System.3

Microarray-based mRNA profiling. MCF-7 cells at 70% to 80% confluence maintained in MEM as described above were treated with vehicle or E2 at a concentration of 10–9 mol/L for 3 and 6 h. Total RNA was extracted as described below. Gene expression profiles of the MCF-7 cells were analyzed on Human Genome U133 Plus 2.0 microarray chips (Affymetrix), which contained 54,613 probe sets. Sample labeling and subsequent hybridization to the array were carried out according to the manufacturer's instructions in the Microarray Core Facility within the Center for Genetic Medicine, Northwestern University. Expression data normalization was done as described previously (9).

Validation of the in vivo ER{alpha}-binding sites by real-time PCR. To examine the enrichment of specific ER{alpha}-binding fragments in an independent ChIP assay, primers were generated corresponding to the regions examined within each ChIP-derived genomic fragment. Primers were synthesized by Integrated DNA Technologies. For each PCR assay, DNA after ChIP of ER{alpha} and IgG as well as input DNA was quantified by PicoGreen dsDNA dye (Invitrogen) from E2-treated or vehicle (ethanol) MCF-7 cells. Real-time PCR was done using Applied Biosystems SYBR Green Master kit following manufacturer's instructions. The estrogen-mediated fold enrichment of ER{alpha}-binding regions relative to IgG was compared with its vehicle (ethanol) control. For every fragment analyzed, enrichment was measured after three independent immunoprecipitations. To further validate the in vivo binding of ER{alpha} to its putative binding regions, ChIP of nuclear receptor coactivator-3 (NCOA3; also known as SRC-1), RNA polymerase II (PolII; Santa Cruz Biotechnologies), and IgG were done.

RNA preparation and validation of ChIP-derived target gene expression by real-time PCR. Total RNA was extracted from the MCF-7, T47D, and MDA-MB-231 breast cancer cell lines and breast cancer tissue samples (n = 45; wet weight {approx} 100 mg) using Tri-Reagent (Sigma) according to the manufacturer's instruction. Total RNA samples were treated with DNase I (Ambion) for 20 min at 37°C according to the product manual. One microgram of total RNA was then reverse transcribed in a final volume of 20 µL using Reverse Transcriptase III (Invitrogen). Real-time PCR was done for measurement of gene expression. A dissociation curve was analyzed for each sample to ensure that a single amplification product was obtained. For real-time PCR, 10 µL of 2x SYBR Green PCR Master Mix (Applied Biosystems), 5 to 10 µmol/L forward and reverse primers of each gene, and 1 µL cDNA template were added in a 20 µL reaction in triplicate. Forty cycles of PCR amplification (95°C for 30 s and 60°C for 1 min) were done on an Applied Biosystems Prism 7000 or 7900 HT Sequence Detection System. Data are reported as the mean fold change ± SD for experiments done in triplicate.

Plasmids, transfections, and luciferase assays. ER{alpha}-binding regions identified from ChIP cloning for Wnt11, Adora1, and SAPS2 were amplified by PCR. To investigate the effect of orientation of ER{alpha}-binding fragments on transcriptional activities, ER{alpha}-binding fragments were cloned in both the sense and the antisense orientations in reporter gene expression vectors. For orientation cloning, primers were used to introduce KpnI and XhoI sites into amplified fragments of Wnt11 and Adora1 and KpnI and SacI sites into the fragment of SAPS2. The primers for the sense fragments were the following: Wnt11, 5'-GGTACCAGTTGTTAGGAATGGCTATGTTCC-3' (forward) and 5'-CTCGAGTAGGCTACCAAGTCTATGGTATTCTG-3' (reverse); Adora1, 5'-GGTACCTCTGTCTATGCACACCATGCA-3' (forward) and 5'-CTCGAGCCAGTCTGAAAACTGGTAATAATACCT-3' (reverse); and SAPS2, 5'-GGTACCGTCCAACTGGTTTCAACT-3' (forward) and 5'-GAGCTCCGTAACCGGAGTCCATGTTT-3' (reverse). The primers for the antisense fragments were the following: Wnt11, 5'-CTCGAGAGTTGTTAGGAATGGCTATGTTCC-3' (forward) and 5'-GGTACCTAGGCTACCAAGTCTATGGTATTCTG-3' (reverse); Adora1, 5'-CTCGAGTCTGTCTATGCACACCATGCA-3' (forward) and 5'-GGTACCAGTCTGAAAACTGGTAATAATACCT-3' (reverse); and SAPS2, 5'-GAGCTCGTCCAACTGGTTTCAACT-3' (forward) and 5'-GGTACCGTAACCGGAGTCCATGTTT-3' (reverse).

The PCR profile was 3 min at 94°C, followed by 35 cycles of 30 s at 94°C, 30 s at 57°C, and 30 s at 72°C, and a final extension of 10 min at 72°C. The amplified fragments were analyzed on a 1% agarose gel. The PCR fragments were directly cloned into the pGEM-T Easy Vector System as described in the manufacturer's protocol and sequenced to check their fidelity. The inserts were then released from the vector by appropriate restriction enzymes indicated above and subcloned into a pGL4-SV40 vector in sense and antisense orientations. Briefly, SV40 promoter was cloned in the location between a synthetic poly(A) signal/transcriptional pause site and the luc2 gene of the pGL4.10[luc2] vector (Promega). All constructs were reconfirmed by sequencing.

Hormone-depleted MCF-7 cells were transfected with each of the ER{alpha}-binding domain vectors with Fugene 6 transfection reagent (Roche Applied Science) according to the manufacturer's protocol. Reporter plasmid (0.2 µg) and pCMVßGal internal control (0.1 µg) were transfected per well. Twenty-four hours after transfection, DMEM/F-12 was added containing ethanol (vehicle) or E2 (10–7 mol/L), and total protein lysate was collected and assayed for luciferase and ß-galactosidase activities after 20- to 24-h treatment.

Transfection of small interfering RNA. RNA interference was carried out by using SMARTpool small interfering RNA (siRNA) designed against Wnt11 and siCONTROL nontargeting siRNA as a negative control (Dharmacon). After 3 days of culture in MEM containing 10% charcoal-stripped calf serum, siRNA against Wnt11 or control siRNA at a final concentration of 100 nmol/L was transiently transfected into the MCF-7 cells for 48 h. The cells were then stimulated with E2 (10–7 mol/L) or vehicle for 20 to 24 h and harvested for analysis. Total RNA and protein were prepared from harvested cells using Tri-Reagent. Knockdown efficiency of target genes was examined by real-time PCR and Western blot.

Cell viability and apoptosis assay. Cell viability was determined by trypan blue exclusion. After various treatments, cells were harvested, washed, and treated with trypan blue at a concentration of 0.4% (w/v). After 10 min, trypan blue uptake (indicating dead cells) was determined by counting on a hemocytometer. Apoptosis in cells was evaluated by a poly(ADP-ribose) polymerase (PARP) cleavage Western blot assay.

Cell cycle distribution analysis. MCF-7 cells were transiently transfected with control siRNA or Wnt11 siRNA (100 nmol/L) for 48 h; the cells were then stimulated for 20 to 24 h with E2 (10–7 mol/L) or vehicle and harvested for cell cycle distribution analysis using propidium iodide (PI) staining and flow cytometry as in Keeton and Brown (10) with slight modification. Briefly, 1 x 106 cells were harvested and washed with PBS and then fixed in cold 70% ethanol at –20°C for 2 h. Fixed cells were treated with 1 mL PI solution (50 µg/mL PI, 0.2 mg/mL RNase A, and 0.1% Triton X-100) for 20 min at 37°C and analyzed for DNA content by flow cytometry by a core facility.

Western blotting. Western blot was done for cleaved PARP analysis and for detection of Wnt11 protein level knockdown. Aliquots of 20 µg of total protein were electrophoresed on an 8% (for PARP cleavage) or 10% (for Wnt11 western) SDS-polyacrylamide gel and transferred to a nitrocellulose membrane. The membrane was blocked overnight at 4°C with 5% milk in TBS followed by hybridization with a rabbit anti-human cleaved PARP antibody at a dilution of 1:1,000 (Cell Signaling) or with rabbit anti-human Wnt11 antibody at a dilution of 1:1,000 (kindly provided by Len Eisenberg, Medical University of South Carolina, Charleston, SC; ref. 11) for confirmation of specificity of Wnt11. The hybridization with antibodies was done for 3 h at room temperature. After washing, the membrane was then incubated for 1 h at room temperature with horseradish peroxidase–conjugated secondary antibody (Sigma) at a dilution of 1:3,000. Immunoreactive bands were stained by a chemiluminescent procedure (Pierce) and visualized by autoradiography.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Identification of genomic ER{alpha}-binding sites in human breast cancer cells. To identify the ER{alpha}-binding sites, we developed a technique to clone sequences from DNA immunoprecipitated by an anti-ER{alpha} antibody. The ChIP-PCR procedure was optimized to achieve amplification using an ER{alpha} antibody to detect proteins bound to the promoter of a prototypical ER{alpha} target gene, Myc, in the absence of any nonspecific IgG binding after 40 cycles of PCR (Supplementary Fig. S1). Once these conditions were reproducibly achieved in five consecutive experiments, DNA fragments immunoprecipitated by the ER{alpha} antibody were extracted, cloned, and sequenced. A total of 130 cloned fragments with insert sizes ranging from ~100 to 1,000 bp were then identified by BLAT or ENSEMBL searches for human genome matches.

Ninety-four cloned fragments were mapped to the genome (Supplementary Table S1). The remaining 36 could not be mapped because either the fragment could not be sequenced due to a high GC-content or the cloned fragments were mapped to repetitive sequences across the genome. Of these, as shown in Table 1 , ~46% were localized within an intron of the open reading frame (ORF) of a gene. Forty percent were located in the 5'-region of a gene (upstream of the transcription start site), and 14% were located in the 3'-region of a gene (downstream of the 3'-untranslated region). Further analysis showed that 23% of the ER{alpha}-ChIP fragments were located within the 50 kb 5'-flanking region of a gene, whereas the remaining 17% were located >50 kb 5' upstream of a gene. Approximately 12% of ER{alpha}-ChIP fragments were located within 50 kb 3'-flanking region of a gene.


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  		Table 1. Genome-wide location of ER{alpha}-binding sites relative to a gene

 
We next identified the genes that contained an ER{alpha}-binding intron or that contained an ER{alpha}-binding site within 50 kb of its 5'- or 3'-flanking regions. First, we determined the mRNA expression levels of the most proximal gene after treatment with E2 (10–9 mol/L) at 3 or 6 h. Expression levels of the second most proximal gene were also determined after E2 treatment at similar time points, provided that the coding region of the second gene was closer than 50 kb to the ER{alpha}-binding site. If more than two genes were found within 50 kb, we evaluated only the two most proximal genes. Following this strategy, we identified 88 E2-regulated genes located proximal to the 94 binding sites identified by ChIP cloning. Expression of 38 of these 88 (43%) genes was significantly regulated after 3 or 6 h of E2 treatment by ≥1.5-fold or ≤0.67-fold compared with baseline (P < 0.05; Table 2 ). Importantly, 82% (31 of 38) of genes identified were novel E2 targets, 24% of which were uncharacterized previously. Interestingly, 54% of genes with intronic sequences that bind ER{alpha} were regulated by E2.


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  		Table 2. ER{alpha} target genes regulated by estradiol

 
Confirmation of ER{alpha} binding to E2-regulated genes. Conventional ChIP experiments showed binding of ER{alpha}, RNA PolII, NCOA3 (SRC-1), and IgG to 11 randomly selected putative ER{alpha}-binding sites (each labeled by the closest gene in Fig. 1 ). We showed that treatment with E2 (versus ethanol) significantly enhanced binding of these three transcription factors to these sites. These results further validated these sites as functional ER{alpha}-binding sequences (Fig. 1). ER{alpha}, RNA PolII, and NCOA3 associations were observed in all 11 binding sites in an estrogen-dependent manner; the range of E2-dependent fold enrichments for ChIP-derived DNA were 2.6 to 7.2 (ER{alpha}), 2.9 to 11.0 (RNA poIII), and 2.6 to 6.5 (NCOA3) compared with the vehicle (Supplementary Figs. S2–S4). On the other hand, there were no E2-dependent fold enrichment of DNA fragments after ChIP with nonspecific IgG (range of fold change, 0.9–1.1; Supplementary Fig. S5). This independent demonstration of enrichment of these binding sites in response to E2 suggests that the fragments identified by ChIP cloning were bona fide ER{alpha}-binding sites in MCF-7 cells.


Figure 1
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  		Figure 1. Validation of the binding of a transcription complex to ER{alpha}-bound DNA fragments cloned after ChIP. Left, conventional ChIP of ER{alpha} and standard PCR of sites adjacent to randomly selected 11 genes; right, ChIP of ER{alpha}, RNA PolII, NCOA3, or IgG (control) and real-time PCR of above ER{alpha}-binding regions. Data represent estrogen-mediated fold enrichment compared with vehicle (ethanol) control. DNA samples after ChIP of ER{alpha}, RNA poIII, NCOA3, and IgG, as well as input DNA (Inp) from samples treated with or without E2 were quantified by PicoGreen dsDNA dye. Negative control (N) was PCR without DNA. The color intensity reflects the fold change as described in the legend. The detailed enrichment graphs of ChIP with ER{alpha}, RNA PolII, NCOA3, and IgG are available as Supplementary Figs. S2 to S5. Data are the average of three replicates ± SD.

 
Genes identified by ChIP cloning are highly regulated by E2. Using the Affymetrix U133 Plus 2.0 chip, which contains probe sets representing a majority of the human genes, we identified E2-regulated genes in MCF-7 cells. We found that 3% of the probe sets (1,618/54,613) showed differential expression (≥1.5-fold or ≤0.67-fold regulation; P < 0.05, t test) after 3 or 6 h of E2 treatment. We compared the E2-regulated genes identified by the microarray with the 88 genes that contained proximal ER{alpha} targets identified by genome-wide ChIP cloning. We found that genes proximal to ER{alpha} target sequences have a much higher chance (43%) of being regulated by E2 compared with E2-regulated probe sets (3%) determined by a genome-wide microarray experiment (P < 0.0001, two sample proportion test).

Characterization of ER{alpha} target genes in breast cancer cell lines. As described above, we confirmed that 38 of the genes containing ER{alpha}-binding sites identified by ChIP cloning were significantly up-regulated or down-regulated by E2 (P < 0.05). These genes encoded novel E2 targets with various functions, such as proliferation (Myc and SPOCK), apoptosis (Adora1), cell motility (SPOCK), Wnt signaling (Wnt11), transcription factors or coregulators (Myc, ZBTB9, HSF1, MYOG, PLXDC1, and GRIP1), ion transport (SLC9A1, AP1B1, and CACBN1), and signal transduction (OR7A17, Wnt11, GPR173, STK32B, and PPM1K; Table 2). Of note, that several genes known previously to be regulated by E2, including Myc (12–14), CTSD (13, 15), ADORA1 (16), and GRIP1 (17), were identified in our experiments validates the ChIP cloning technique.

We next investigated whether the expression pattern of E2-regulated genes identified in MCF-7 cells was similar to that in the T47D cell line, which is also ER{alpha}+. We determined the mRNA levels of seven randomly selected E2-regulated ER{alpha} target genes in T47D cells. As shown in Supplementary Table S2, the results confirmed a very high degree of concordance between MCF-7 and T47D cells with respect to E2 regulation of novel ER{alpha} target genes. On the other hand, none of these seven genes were regulated in the ER{alpha}– cell line MDA-MB-231, in which expression of two genes (MYOG and Wnt11) were undetectable at both time points tested. Moreover, the ER antagonist ICI 182780 blocked E2-dependent regulation of these genes (data not shown). These observations strongly support the notion that ER{alpha} is required for E2 regulation of the target genes identified by the ChIP cloning method.

Association between ER{alpha} target gene expression and ER{alpha} status of breast cancer tissues. ER{alpha} status (the presence or absence of ER{alpha} protein determined by immunohistochemistry in a tumor sample) is a prognostic factor in breast cancer and the single most important predictor for response to hormonal treatment (3). To investigate whether expression of ER{alpha} target genes identified in E2-treated MCF-7 cells are associated with the ER{alpha} status in breast cancer tissues, the mRNA levels of three novel ER{alpha} target genes were measured in 25 ER{alpha}+ and 20 ER{alpha}– breast cancer tissues.

We selected three prototypical E2-regulated genes, each of which represented a distinct ER{alpha}-binding pattern: (a) 40 kb 5' (Wnt11), (b) intron 2 (Adora1), and (c) 2 kb 3' (SAPS2; Fig. 1; Table 2). We investigated in vivo regulation of these three genes by ER{alpha} using regression analyses. The mRNA levels of Wnt11, Adora1, and SAPS2 were plotted against ER{alpha} mRNA levels. Regression analysis showed statistically significant correlations between ER{alpha} mRNA and Wnt11 mRNA (r = 0.567), ER{alpha} mRNA and Adora1 mRNA (r = 0.609), and ER{alpha} mRNA and SAPS2 mRNA (r = 0.792; P < 0.00005 for each r value; Fig. 2 ). These findings indicate that E2-responsive genes that lie proximal to ER{alpha}-binding sites identified by ChIP cloning in breast cancer cell lines are also regulated by ER{alpha} in vivo.


Figure 2
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  		Figure 2. Correlation between mRNA levels of ER{alpha} and Wnt11, Adora1, and SAPS2 in 45 breast tumor tissues. Total RNA was isolated from immunohistochemically determined 25 ER{alpha}+ and 20 ER{alpha}– breast cancer tissues. mRNA levels of ER{alpha}, Wnt11, Adora1, and SAPS2 were measured by real-time PCR and normalized by glyceraldehyde-3-phosphate dehydrogenase. The correlation between mRNA levels of ER{alpha} and the three ER{alpha} target genes were carried out by regression analysis.

 
ER{alpha}-binding sites exert regulatory activities. To determine whether the ER{alpha}-binding sites cloned by ChIP contained sequences with enhanced transcriptional activity, we cloned the ER{alpha}-binding fragments proximal to the Wnt11, Adora1, and SAPS2 genes in both the sense and the antisense orientations into an SV40 promoter/luciferase reporter construct. As described above, the ER{alpha}-binding site for each gene represents one of three binding patterns: 5' proximal for Wnt11, intronic for Adora1, and 3' proximal for SAPS2. The ER{alpha}-binding site located 40 kb 5' of Wnt11 was not proximal to another gene. On the other hand, the ER{alpha}-binding site within intron 2 of Adora1 is also 13 kb 5' upstream of the Myogenin gene, which is regulated by E2 in MCF-7 cells. The ER{alpha} target site located 2 kb 3' downstream of the SAPS2 gene is also 1 kb 3' downstream of the SBF1 gene and is also regulated by E2.

Individual ER{alpha}-binding site/luciferase reporter constructs were transfected into ER{alpha}+ MCF-7 cells, which were then treated with vehicle or E2 for 20 h and assayed for luciferase reporter gene activity. The ER{alpha}-binding site 40 kb 5' of Wnt11 enhanced transcriptional activity by 3.7-fold in the sense direction and 5.0-fold in the antisense direction on treatment with E2 (10–7 mol/L; Fig. 3A ). Similar results were obtained with transfection of luciferase reporter constructs containing ER{alpha}-binding sites within intron 2 Adora1 and 2 kb 3' of SAPS2 genes. E2 treatment enhanced transcription by 4.1-fold and 3.7-fold in cells transfected with ER{alpha}-Adora1 sense and antisense constructs, respectively. Cells transfected with ER{alpha}-SAPS2 sense construct increased transcription by 6.5-fold on E2 treatment, whereas transcriptional activity was augmented by 16-fold on E2 treatment of cells transfected with ER{alpha}-SAPS2 antisense construct.


Figure 3
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  		Figure 3. Transcriptional activities of ER{alpha}-binding sites proximal to E2-induced genes. ER{alpha}-binding fragments for Wnt11, Adora1, and SAPS2 were cloned in the sense or antisense orientation into a pGL4-SV40 plasmid. Expression of the empty vector (Empty) was used as a reference standard. Reporter plasmids (0.2 µg) and pCMVßGal (0.1 µg) were cotransfected into ER{alpha}+ MCF-7 cells (A) and ER{alpha}– MDA-MB-231 cells (B) in 24-well plates. After 20 to 24 h, cells were washed with PBS and treated with 10–7 mol/L E2 or ethanol for an additional 20 h. Cells were then lysed and assayed for luciferase and ß-galactosidase activities. Columns, mean of a representative experiment done from at least three experiments; bars, SE. Constructs oriented in the sense direction for the Wnt11, Adora1, and SAPS2 genes are denoted Wnt11-S, Adora1-S, and SAPS2-S, respectively; whereas constructs that contain the antisense ER{alpha}-binding fragments are denoted Wnt11-AS, Adora1-AS, and SAPS2-AS. *, P < 0.05, t test, statistically significant differences compared with empty vector.

 
To show that E2 regulated the transcriptional activity of these constructs in an ER{alpha}-dependent fashion, we transfected them into the ER{alpha}– MDA-MB-231 cell line. The absence of induction by E2 supported our conclusion that these sequences confer responsiveness to E2 in an ER{alpha}-dependent manner (Fig. 3B). Overall, these observations confirmed that ER{alpha}-binding regions identified for Wnt11, Adora1, and SAPS2 contain E2-responsive functional cis-acting elements.

Knockdown of E2-induced Wnt11 is associated with increased breast cancer cell death. We showed that Wnt11 is located proximally to an ER{alpha}-binding site and its expression is induced by E2 (Supplementary Table S2). Because Wnt11 belongs to an oncogene family (18), we hypothesized that E2-induced Wnt11 expression could be involved in breast cancer pathobiology. MCF-7 cells were cultured in steroid-deprived medium for 3 days, and siRNA against Wnt11 or control siRNA was transiently transfected into the MCF-7 cells for 48 h followed by 20- to 24-h treatment with E2 or vehicle. To show the efficiency and specificity of depletion of Wnt11, both real-time PCR and Western blot analysis were done (Fig. 4D ).


Figure 4
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  		Figure 4. siRNA-mediated inhibition of Wnt11 in MCF-7 leads to increased cell death. MCF-7 cells were cultured in hormone-depleted medium for 3 days, and siRNA against Wnt11 or control siRNA was transiently transfected into the MCF-7 cells for 48 h. The cells were then stimulated with E2 (10–7 mol/L) or vehicle for 20 to 24 h and harvested for analysis. MCF-7 cells were harvested for determination of percentage of nonviable cells by trypan blue exclusion (A) and for PARP cleavage with rabbit anti-human cleaved PARP antibody (B). Lanes 1 and 2, control siRNA or Wnt11 siRNA-transfected MCF-7 cells treated with vehicle; lanes 3 and 4, control siRNA or Wnt11 siRNA-transfected MCF-7 cells treated with E2 (10–7 mol/L). Blots were reprobed with a ß-actin antibody to control for loading. Analysis of cell cycle progression after silencing of Wnt11 (C). Knockdown efficiency and specificity of the Wnt11 gene were examined by both real-time PCR and Western blotting using a Wnt11 peptide antibody (D). Columns, mean of three independent experiments; bars, SE. *, P < 0.05, t test, statistically significant differences.

 
As shown in Fig. 4, knockdown of Wnt11 in MCF-7 cells treated with or without E2 resulted in a significantly decreased cell survival (12.2% and 13.9%, respectively) compared with cells transfected with control siRNA (5.3% and 6.7%, respectively; P < 0.05 for each case; Fig. 4A). We next determined whether this change in cell survival was due to changes in apoptosis and/or proliferation. Knockdown of Wnt11 increased apoptosis as shown specifically by a striking increase in PARP cleavage (Fig. 4B). The highest level of PARP cleavage was observed in Wnt11-depleted cells incubated with vehicle followed by Wnt11-depleted cells treated with E2. The results suggested that Wnt11 depletion increased apoptosis, and E2 blunted this effect. On the other hand, depletion of Wnt11 by siRNA did not result in significant changes in the fractions of MCF-7 cells in S phases compared with control samples (Fig. 4C). However, significant changes in cell populations in S phases were observed in an E2-dependent manner, suggesting that Wnt11 does not have a major role in cell cycle progression in MCF-7 cells. Thus, the observed changes in cell viability seem to be due to an effect of Wnt11 on apoptosis but not on proliferation.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Using a ChIP-linked target site cloning strategy, we identified 94 ER{alpha}-binding sites within the human genome, many of which represent novel E2 targets in MCF-7 breast cancer cells. We showed both in vivo and in vitro that a representative portion of these genes are highly regulated by E2/ER{alpha} in breast cancer cells and tissues. We also showed a novel biological role of the E2-regulated gene Wnt11 in breast cancer.

A significant volume of work has focused on identifying essential domains within the proximal promoters of E2-regulated genes (6, 12, 13, 19–24). However, many functionally relevant binding sites for transcription factor likely exist in regions outside of gene promoters, particularly in introns (25, 26). These sites would not have been identified using promoter microarray (21) or CpG island-enriched DNA array (20, 27), which identify binding sites only within the region proximal to the transcription start site. A recently published pioneering study showed by ChIP-on-chip that ER{alpha} binds to thousands of sites genome-wide and interact with transcription factors binding to specific cis-acting elements as a master regulator of many genes (7).

We compared the ER{alpha}-binding sites found in this study with those published by Carroll et al. (7). Approximately 95%, 86%, 57%, 39%, 17%, 11%, and 6% of the 94 binding sites that we cloned were located 500, 300, 100, 50, 20, 11, and 5 kb away from the closest binding sequences published by Carroll et al, respectively (7). The distribution of binding sequences across the chromosomes was fairly similar in both studies. We identified one ER{alpha}-binding site mapped to the Y chromosome, whereas no binding sites were mapped to this chromosome in the Carroll et al. study (7). We showed the proximity of the 11 sites, which bind ER{alpha}, RNA PolII, and NCOA3, from our study to the closest possible binding sites identified in the Carroll et al. study in Supplementary Table S3. The distance ranged from 1.24 to 304 kb. This possibly suggested that the identification of exact binding sites may vary with the technique used. In general, our findings agree with the conclusions published by Carroll et al. For example, both groups found that the majority of the ER{alpha}-binding sites fell outside of classically defined promoter regions.

In this study, there were only 18 (of 94) binding sites that did not reside within 50 kb of a gene. Thus, we arbitrarily chose this distance to limit the group of genes to be tested for regulation by estrogen treatment. It is quite possible that ER{alpha}-binding sites can also regulate genes that lie more than 50 kb away. In fact, recently published articles from Carroll et al. (7) showed the importance of these far distant sites as regulators of transcription.

The fact that 54% of the genes containing intronic ER{alpha}-binding sites were regulated by E2 suggests that this is an interesting phenomenon with functional significance. ER{alpha}-binding fragments located distal to genes or within introns might function through long-range interactions that involve looping of chromatin to bring the elements within proximity of gene promoters (26, 28). Indeed, the intronic ER{alpha}-binding sites identified in the present study indicate that ER{alpha} may regulate target gene transcription by altering local chromatin structure. Recent reports about intronic binding of other transcription factors, such as cAMP-responsive element binding protein and BARX2, provide further support that intronic binding of ER{alpha} may be an important mechanism of gene regulation by E2 (25, 29).

E2 has been shown to regulate transcription through either direct binding of EREs or indirectly by interacting with transcription factor complexes. Analysis of the ER{alpha}-binding sequences associated with the 38 identified E2-regulated genes revealed that 61% of these sequences contained at least one classic (palindromic) ERE. Eighty-seven percent of binding sites missing a palindromic ERE contained one or more AP-1 sites. The comparison of these results with previously published data suggests that there is a higher likelihood of the occurrence of canonical EREs in ER{alpha}-binding sites proximal to an E2-regulated gene (4, 6). Furthermore, in the absence of an ERE, AP-1 or other cis-acting elements may confer E2/ER{alpha} responsiveness possibly via tethering of ER{alpha} on AP-1–binding transcription factors (19).

It has been shown that Wnt11 signaling induces proliferation (30), transformation (31), and prostate cancer progression (32) through a noncanonical pathway (33–35). However, the role of Wnt11 in the progression of breast cancer remains unknown. Our studies provide an initial insight into the mechanism by which Wnt11 may promote tumor progression in the breast. Up-regulation of Wnt11 mRNA on E2 exposure may activate the Wnt11 signaling pathway and inhibit apoptosis, thus favoring tumor growth. Further investigation of the biological functions of Wnt11 and other identified ER{alpha}-regulated genes in breast cancer may lead to the development of specific, targeted treatments.


   Acknowledgments
 
Grant support: NIH grant RO1-CA67167.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   Footnotes
 
Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).

3 http://www.cbil.upenn.edu/cgi-bin/tess/tess Back

Received 10/ 6/06. Revised 1/31/07. Accepted 3/15/07.


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 Introduction
 Materials and Methods
 Results
 Discussion
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