Oncogene (2011) 30, 1608–1614 & 2011 Macmillan Publishers Limited All rights reserved 0950-9232/11 www.nature.com/onc SHORT COMMUNICATION Differential recruitment of coregulators in ligand-dependent transcriptional repression by -a

KW Merrell, JD Crofts, RL Smith, JH Sin, KE Kmetzsch, A Merrell, RO Miguel, NR Candelaria and C-Y Lin1

Department of Microbiology and Molecular Biology, Brigham Young University, Provo, UT, USA

Estrogen receptors (ERs) are normally expressed in breast endocrine-related diseases, including breast cancers tissues and mediate hormonal functions during develop- (Gruber et al., 2002; Yager and Davidson, 2006). Two ment and in female reproductive physiology. In the estrogen receptor (ER) subtypes, ERa and ERb,have majority of breast cancers, ERs are involved in regulating been identified and exhibit differential target tissue tumor cell proliferation and serve as prognostic markers distribution and functions. In the majority of breast and therapeutic targets in the management of hormone- cancers, ERa is expressed in tumor cells and is involved dependent tumors. At the molecular level, ERs function as in promoting tumor cell proliferation and invasiveness ligand-dependent transcription factors and activate target- (Ali and Coombes, 2000). The role of ERb in breast expression following hormone stimulation. Recent cancer biology remains to be determined, although some transcriptomic and whole-genome-binding studies suggest, findings suggest that it may attenuate ERa functions however, that ligand-activated ERs can also repress the (Lindberg et al., 2003; Cappelletti et al., 2004; Lin et al., expression of a significant subset of target . To 2007a). ER status is an important prognostic marker characterize the molecular mechanisms of transcriptional in breast cancer management and a determinant for repression by ERs, we examined recruitment of nuclear treatment with selective estrogen receptor modulators. receptor coregulators, histone modifications and RNA Treatment with selective estrogen receptor modulators polymerase II docking at ER-binding sites and cis- has also been shown to be effective in reducing the risk regulatory regions adjacent to repressed target genes. of breast cancer (Fisher et al., 1998, 2005; Vogel et al., Moreover, we utilized data from patient 2006). However, inhibiting estrogen or ER functions samples to determine potential roles of repressed target elicits menopausal symptoms in premenopausal women genes in breast cancer biology. Results from these studies and negates other beneficial effects, such as bone indicate that nuclear receptor recruitment is a maintenance and cardiovascular protection. Long-term key feature of ligand-dependent transcriptional repression application of endocrine therapy may result in drug by ERs, and some repressed target genes are associated resistance and hormone-independence in tumor cells with disease progression and response to endocrine (Bookman, 2005). Overcoming these challenges will therapy. These findings provide preliminary insights into necessitate a more complete understanding of the a novel aspect of the molecular mechanisms of ER molecular mechanisms of ER function. functions and their potential roles in hormonal carcino- ER is a nuclear receptor and affects target-gene genesis and breast cancer biology. expression directly as a ligand-dependent transcription Oncogene (2011) 30, 1608–1614; doi:10.1038/onc.2010.528; factor (Nilsson and Gustafsson, 2002). Upon activation, published online 22 November 2010 ER upregulates target-gene expression by binding cis-regulatory estrogen response elements (EREs) and Keywords: estrogen receptor; transcriptional regula- nucleating coregulator complexes that modify histones tion; hormonal carcinogenesis; coregulators and render the DNA accessible to the transcriptional machinery (Shang et al., 2000; Metivier et al., 2003). ER coregulators include those which enhance transcrip- tional activity by affecting nucleosome spatial orienta- Estrogens and their cellular receptors play key roles tioin or by modifying histones through acetylation during development and in reproductive physiology and (SRC1, CBP/p300, p/CAF and p/CIP/AIB1) and methylation (CARM1 and PRMT1) (Halachmi et al., 1994; Onate et al., 1995; Lin et al., 1996; Ogryzko et al., Correspondence: Dr C-Y Lin, Department of Microbiology and Molecular Biology, Brigham Young University, 753 WIDB, Provo, 1996; Wang et al., 1996, 2001; Anzick et al., 1997; Chen UT 84602, USA. et al., 1999; Phelan et al., 1999; Sterner and Berger, E-mail: [email protected] 2000). Coregulators, such as NCoR, SMRT, NRIP1, 1Current address: Center for Nuclear Receptors and Cell Signaling, LCoR and REA, function as nuclear receptor corepres- Department of Biology and Biochemistry, University of Houston, Houston, TX 77204, USA. sors, and have been shown to bind ER following Received 7 January 2010; revised 5 October 2010; accepted 6 October selective estrogen receptor modulator treatments, but 2010; published online 22 November 2010 their roles in normal ER transcriptional activity are not Differential recruitment of nuclear receptor coregulators KW Merrell et al 1609 known (Chen and Evans, 1995; Treuter et al., 1998; Hu involve potential interactions with AP-1 factors (Carroll and Lazar, 1999; Montano et al., 1999; Webb et al., et al., 2006). They also demonstrated that the corepres- 2000; Fernandes et al., 2003).Research into ER tran- sor NRIP1 is involved in the late repression of target scriptional regulatory mechanisms has focused mainly genes adjacent to AP-1-binding site motif containing on genes, such as vitellogenin and pS2/TFF1, whose ER-binding sites. They did not address experimentally, expression levels are upregulated following estrogen however, the observed early repression events and stimulation. Consequently, most of the current models mechanisms, the role of AP-1 factors, the general effects of transcriptional regulation by ER are based on of NRIP1 in transcriptional repression by ER and the molecular and biochemical studies of the EREs adjacent involvement of other . A report by Stossi to these upregulated target genes. Microarray analyses et al. showed that the nuclear receptor corepressor of transcriptomic alterations following estrogen NCoR is recruited to the promoter region of the cyclin treatments suggest, however, that a large number of G2 gene, previously shown to be repressed by ER responsive genes are transcriptionally repressed (Frasor (Frasor et al., 2003; Lin et al., 2004), at two hours et al., 2003; Lin et al., 2004). Whole-genome cartogra- following estrogen treatment, a much earlier time point phy of ER-binding sites by chromatin immunoprecipita- than the repression events examined by Carroll et al. tion (ChIP) coupled with either microarray (ChIP–chip) (Stossi et al., 2006), and they also demonstrated the or high-throughput sequencing (ChIP-seq) technologies involvement of p300 and CtBP1 in a recent study (Stossi has mapped ER to cis-regulatory regions of both et al., 2009). Much of the mechanisms of transcriptional activated and repressed target genes (Carroll et al., repression by ER still remain to be elucidated. 2005, 2006; Lin et al., 2007b). Mechanisms of ligand- Based on known general and ER-specific transcrip- dependent transcriptional repression of ER direct target tional regulatory mechanisms, we hypothesize that, in genes have not been studied in detail. Carroll et al. cases of transcriptional repression, ligand-activated ER propose that transcriptional repression by ER occurs at interacts with specific factors and binding site sequence late time points following estrogen exposure and may motifs adjacent to repressed target genes, and these

16 Input ChIP 14 EtOH E2 EtOH E2 12 10 αER PSCA 8 αHA 6 4 αER SLC35A1 2 α HA Change E2+/E2- 0 ER Recruitment Fold αER -2 GREB1 -4 α 45m 2h 12h 24h 45m 2h 12h 24h 45m 2h 12h 24h HA -6 PSCA SLC35A1 GREB1 Figure 1 Binding of ER to select repression-associated ER-binding sites is validated and characterized by ChIP. (a) Before hormone treatment, MCF-7 cells were grown in phenol red-free Dulbecco’s modified Eagle’s medium (Invitrogen, Carlsbad, CA, USA) supplemented with 5% charcoal–dextran-stripped fetal bovine serum (HyClone, Logan, UT, USA) for 72 h. Following estrogen deprivation, cells were either incubated with 10 nM estradiol (E2; Sigma-Aldrich, St Louis, MO, USA) or mock treated with the ethanol carrier for E2. Treatments ranged from 45 min and 2, 12 and 24 h. Chromatin was cross-linked with 1.5% formaldehyde in phosphate buffered saline solution for 15 min at room temperature, followed by a series of nucleus/chromatin preparation (NCP) buffer washings. Cells were washed sequentially with 1 ml of phosphate buffered saline, NCP I (10 mM EDTA, 0.5 mM EGTA, 10 mM HEPES, pH ¼ 6.5, 0.25% Triton X-100) and NCP II (1 mM EDTA, 0.5 mM EGTA, 10 mM HEPES, 200 mM NaCl). Following NCP buffer washings, isolated nuclei were resuspended in lysis buffer (10 mM EDTA, 20 mM Tris–HCl, pH ¼ 8.1, 0.5% Empigen BB (Sigma-Aldrich, St Louis, MO, USA), 1% SDS) and incubated at room temperature for 10 min. Nuclear extracts were then sonicated to solubilize –DNA complexes and clarified by centrifugation. Aliquots of extracts were retained as input controls for subsequent analysis. Immunoprecipitation buffer (2 mM EDTA, 150 mM NaCl, 20 mM Tris–HCl, pH ¼ 8.1, 1% Triton X-100, protease inhibitors) and anti-ERa antibodies (HC-20; Santa Cruz Biotechnology, Santa Cruz, CA, USA) were added to the nuclear extracts and incubated overnight at 4 1C. Following incubation, 50% Protein A-sepharose bead (Invitrogen) slurry in beads buffer (10 mM Tris–HCl pH ¼ 8.1, 1mM EDTA) was added and immunoprecipitation continued for 3 additional h. Beads were then washed sequentially with wash buffer (WB) I (2 mM EDTA, 20 mM Tris–HCl, pH ¼ 8.1, 0.1% SDS, 1% Triton X-100, 150 mM NaCl), WB II (same as WB I except 500 mM NaCl), WB III (10 mM Tris–HCl, pH ¼ 8.1, 1 mM EDTA, 250 mM LiCl, 1% Deoxycholate, 1% NP-40) and WB IV (10 mM Tris–HCl, pH ¼ 8.1, 1 mM EDTA) at 4 1C. Protein–DNA complexes were extracted three times with extraction buffer (1% SDS, 0.1 M NaHCO3) and de-cross-linked at 65 1C overnight. ChIP and input control DNA were isolated using the QIAquick PCR purification kit (Qiagen, Valencia, CA, USA). Specific PCR primers targeting the sites adjacent to two repressed target genes (PSCA and SLC35A1) were used to determine binding of ER at 45 min following estrogen treatment (PSCA ERE, forward primer, 50-CACAGGGATGTGTGTTCCAG- 30, reverse primer, 50-TCCCTGGAGATCCTGTCTTC-30; SLC35A1 ERE, forward primer, 50-TTTGCAACCACAAAGGTGAC-30, reverse primer, 50-GCCTCCCTCCCTATTCTGTC-30). The previously characterized GREB1 ERE (Lin et al., 2004) was included as a positive control for ER binding (GREB1 ERE, forward primer, 50-AGCAGTGAAAAAAGTGTGGCAACTGGG-30, reverse primer, 50-CGACCCACAGAAATGAAAAGGCAGCAAACT-30). Control ChIPs using anti-hemagglutinin (Santa Cruz Biotechnology) antibodies showed no non-specific binding of the sites following hormone treatment. (b) Quantitative PCR was performed on a Roche LightCycler 480 system using the SYBR Green master mix (Roche, Indianapolis, IN, USA) to determine the extent of ER recruitment following 45 min and 2, 12 and 24 h of estrogen treatment. Results were normalized to input DNA data and presented as relative average fold-change between treated and untreated samples for three biological replicates. Error bars represent s.e.m. fold changes.

Oncogene Differential recruitment of nuclear receptor coregulators KW Merrell et al 1610 molecular interactions confer a repressive receptor and selected ER-binding sites adjacent to two repressed conformation. The repressive conformation then target genes (PSCA and SLC35A1) for validation and promotes recruitment of corepressors, expulsion of characterization. ER binding to these sites following co-activators, alteration of histone modifications, and estrogen treatment was first validated by conventional disruption of RNA polymerase II (Pol II) docking. ChIP. PCR of the ChIP samples confirmed ER binding Furthermore, we posit that genes involved in the at 45 min following hormone treatment as compared mechanisms of transcriptional repression by ER and with the mock-treated controls (Figure 1a). ER binding repressed target genes constitute a significant and little to the previously characterized ERE adjacent to the understood aspect of ER biology, and are likely GREB1 gene was included as a positive control. There is associated with disease parameters and outcomes in no binding of these sites in the immunoprecipitation breast cancer patients. controls using the irrelevant anti-hemagglutinin anti- To determine the recruitment of the co-activators and bodies, and there were either negligible or no estrogen- corepressors to repression-associated ER-binding sites, dependent binding in control exonic regions of the we examined the overlap between our previously repressed target genes (data not shown). ER binding to published mapping results (Lin et al., 2007b) and these sites was then further characterized by ChIP and available microarray data (Finlin et al., 2001; Soulez quantitative PCR at 45 min and 2, 12 and 24 h following and Parker, 2001; Frasor et al., 2003; Lin et al., 2004), hormone treatment (Figure 1b). ER recruitment to these

10 SRC1 10 p/CIP

8 8 6 6 4 4 2 0 2 * -2 * 0 -4 * -2 -6 * * * -8 -4 p/CIP Recruitment Fold Change E2+/E2- SRC1 Recruitment Fold Change E2+/E2- * -10 45 minutes 2 hours -6 45 minutess 2 hours

PSCA SLC35A1 pS2

15 NCoR 15 NRIP1 15 SMRT

10 10 10 * * * 5 * * 5 5 * * * 0 0 0

* -5 -5 -5

-10 -10 -10 SMRT Recruitment Fold Change E2+/E2- NRIP1 Recruitment Fold Change E2+/E2- NCoR Recruitment Fold Change E2+/E2- -15 -15 -15 45 minutes 2 hours 45 minutes 2 hours 45 minutes 2 hours

PSCA SLC35A1 pS2 Figure 2 Repression-associated ER-binding sites exhibit differential recruitment of nuclear receptor coregulators. (a) Recruitment of nuclear receptor coregulators was determined by sequential ChIP steps or by the so-called ‘re-ChIP’ method (Metivier et al., 2003). Following the initial anti-ERa ChIP, up to the final wash step, ER–chromatin complexes were extracted by the addition of 10 mM dithiothreitol (DTT) and incubated for 20 min at 37 1C. Supernatants from the extraction procedure were pooled together and diluted with re-ChIP buffer (20 mM Tris–HCl, pH ¼ 8.1, 2 mM EDTA, 150 mM NaCl, 1% Triton X-100), and additional IP buffer and 1 mgof antibodies against the selected coregulator were added, and re-ChIP mixtures were incubated overnight at 4 1C. Isolation of specific coregulator complexes and purification of associated DNA was carried out as described for standard ChIP experiments. Coregulator binding was determined by quantitative PCR as previously described. Recruitment to the extensively characterized pS2 ERE (Shang et al., 2000; Metivier et al., 2003) was included as a control (pS2 ERE, forward primer, 50-CCATGTTGGCCAGGCTAGTC-30, reverse primer, 50-ACAACAGTGGCTCACGGGCT-30). Control PCR experiments were also carried out using primers designed against exonic regions of repressed target genes (PSCA exon 3, forward primer, 50-TGGCCCAGCATTCTCCACCCTT-30, reverse primer, 50-GGGACACCACGGACCAGACCT-30; SLC35A1 exon 4, forward primer, 50-GACCTACCAGTTGAAGATTCCG-30, reverse primer, 50-AAGCGTAACTCCAGCACACA-30), and detected either no binding or no differences between mock- and E2- treated ChIP and re-ChIP samples (data not shown). Co-activator recruitment re-ChIP studies were performed using antibodies against SRC1 (Santa Cruz Biotechnology) and p/CIP (Santa Cruz Biotechnology). (b) Specific antibodies against NCoR (Santa Cruz Biotechnology), SMRT (Santa Cruz Biotechnology) and NRIP1 (Abcam, Cambridge, MA, USA) were used for the corepressor studies. Statistically significant (Po0.05; two-tailed Student’s t-test) differences between repressed the target gene sites and the activated control pS2 ERE are denoted by asterisks for co-activator and corepressor studies.

Oncogene Differential recruitment of nuclear receptor coregulators KW Merrell et al 1611 sites started to diminish at 2 h following hormone three corepressors, perhaps components of the same treatment, and ER binding at 12 and 24 h were corepressor complex, are recruited to the ER bound to comparable with the mock-treated controls. Subsequent both of the repressed target gene sites following 45 min analyses of coregulator recruitment and changes in the and 2 h of estrogen treatment, with the exception of chromatin structure were thus limited to the two earlier NRIP1 recruitment to the PSCA ER-binding sites at 2 h time points. after treatment (Figure 2b). Increases in NCoR and Following the validation and characterization of the SMRT recruitment to the repressed target gene sites, as selected ER-binding sites, we performed ‘re-ChIP’ compared with the pS2 ERE, are statistically significant experiments to determine the binding of the co-activator (Po0.05; Student’s t-test) at both time points, whereas to the ER complex bound to the sites. Briefly, differences in NRIP1 binding are significant or nearly ChIP was carried out using anti-ER antibodies first, significant (Po0.1) at 2 h, although the repressed target followed by a second ChIP with antibodies against gene sites appeared to recruit NRIP1 at 45 min. In all either SRC1 or p/CIP. MCF-7 cells were treated with instances, the corepressors are present at the pS2 ERE in estrogen for 45 min and 2 h before the assays. The ERE the absence of estrogen and are expelled following in the promoter region of the previously characterized hormone treatment, suggesting a mechanism for suppres- pS2 gene was included as a positive control. Both SRC-1 sing basal transcriptional activity for this upregulated ER and p/CIP are recruited to the pS2 ERE following target gene. These findings suggest that recruitment of the hormone treatment (Figure 2a). This is consistent with nuclear receptor corepressors is a common mechanism previously published results (Shang et al., 2000; Metivier for transcriptional repression by ER. et al., 2003). For the repressed target gene sites, SRC-1 is Nuclear receptor corepressors NCoR, NRIP1 and expelled from the ER complex at both sites examined SMRT are known to interact with histone deacetylases after 2 h of hormone treatment (Figure 2a), whereas, and block transcriptional activity by altering interac- in contrast, p/CIP is expelled from one of the two tions between DNA and histones (Wei et al., 2000; repression-associated sites following estrogen treatment Guenther et al., 2001). As these corepressors are (Figure 2a). Differences in the co-activator recruitment recruited to the ER complex adjacent to repressed between the repressed target gene sites and the pS2 ERE target genes, we performed ChIP experiments to were statistically significant (Po0.05; Student’s t-test), determine the effect of their recruitment on the histone with the exception of the PSCA site at 45 min. There- acetylation status in the promoter regions of these genes fore, our theory regarding the expulsion of the co- and the impact on Pol II docking. MCF-7 cells were activators appears partially correct, and the actual treated with estrogen for 45 min or 2 h, and ChIPs were model may differ depending on the binding site and carried out using specific antibodies against acetylated co-activator examined. histones H3 (K14) and H4 (K16), both associated with To test whether the corepressors are recruited to the transcriptionally active promoters (Hebbes et al., 1988), repression-associated ER-binding sites, we performed or Pol II. re-ChIP experiments as described for the co-activators. For both of the repressed ER target gene promoters Specifically, we examined the recruitment of NCoR, tested, histones H3 and H4 acetylation decreased at both NRIP1 and SMRT (Figure 2b). As we had posited, all 45 min and 2 h following hormone treatment (Figure 3).

20 H3 K 8 H4 K 20 RNA Pol II 14 16 15 6 15

4 10 10

2 5 5 0 0 0 -2 * -5 -5 -4 * * H3 Acetylation Fold Change E2+/E2-

-10 H4 Acetylation Fold Change E2+/E2- -10

-6 Pol II Recruitment Fold Change E2+/E2-

-15 45 minutes 2 hours -8 45 minutes 2 hours -15 45 minutes 2 hours

PSCA SLC35A1 pS2 Figure 3 Transcriptional repression by ER is associated with histone deacetylation and reduced RNA Pol II docking in the promoter regions of target genes. Histone acetylation and Pol II binding in the promoter regions of PSCA and SLC35A1 genes were determined by ChIP assays using specific antibodies against acetylated histones H3 and H4 (Upstate Biotech/Millipore, Billerica, MA, USA) and Pol II (Santa Cruz Biotechnology), and quantitative PCR as described for the ER binding studies (PSCA promoter, forward primer, 50-AGGGAGAGGGAGGTGCAGGT-30, reverse primer, 50-CAAATGGGGAGAGGCTGGAC-30; SLC35A1 promoter, forward primer, 50-ATCCGCGTTCTCCCCATTTT-30, reverse primer, 50-ACCGCCCCTTGTGGTCAT-30; pS2 promoter, primers same as pS2 ERE) was carried out. Statistically significant (Po0.05; two-tailed Student’s t-test) differences between the repressed target gene promoters and the activated control pS2 promoter are denoted by asterisks.

Oncogene Differential recruitment of nuclear receptor coregulators KW Merrell et al 1612 In contrast, histone acetylation increased in the promoter whereas recruitments at 2 h to the SLC35A1 promoter region of the pS2 gene, consistent with an increase in its were nearly significant and clearly showed similar trends. expression following estrogen treatment. Decreases in the In these studies, we examined potential mechanisms histone H4 acetylation were statistically significant of transcriptional repression by ER involving the (Po0.05; Student’s t-test) at 2 h for both repressed target nuclear receptor co-activators and corepressors. The genes as compared with pS2, and differences at 45 min most consistent trend is the recruitment of the cor- and also in histone H3 acetylation were nearly significant epressors to the repressed target gene ER-binding sites, (Po0.1). In line with the observed changes in histone although NRIP1, which is reported to have both the acetylation status, Pol II is recruited to the pS2 promoter corepressor and co-activator activity (Cavailles et al., but not the promoters of the two repressed target genes 1995; Treuter et al., 1998), appeared to be present in the (Figure 3) where it’s binding was actually decreased complex at the earlier time point and absent in the latter following estrogen treatment. Pol II recruitment to the for the PSCA ER-binding site. We did not measure PSCA promoter was significantly different at 45 min, NRIP1 recruitment to these sites at 12 h following

SRC1 p/CIP 10 10 8 8 6 6 4 4 2 2 0 * -2 0 -4 * -2 -6 * -4 Fold Change (+E2/-E2) Fold Change (+E2/-E2) -6 MME -8 45 minutes 2 hours 45 minutes 2 hours COBL CYP1A1 DICER1 NCoR NRIP1 SMRT 10 15 15 5 * 10 10 1.0 * Cluster 1 0 5 5 * xx x * x Cluster 2 -5 0 0 xx x 0.8 x Cluster 3 x -10 -5 * -5 x -15 -10 -10 xxxx x Fold Change (+E2/-E2) 0.6 xxxxxx x Fold Change (+E2/-E2) x Fold Change (+E2/-E2) -20 45 minutes 2 hours -15 45 minutes 2 hours -15 45 minutes 2 hours xxxx

0.4 H3K H4K RNA Pol II 20 4 8

Survival Probability x xxx x 14 16 6 15 2 0.2 10 4 Disease-Free Survival 0 2 5 p=0.00417 -2 0 0.0 0 * -2 -4 024681012-5 * -4 Fold Change (+E2/-E2) Fold Change (+E2/-E2) Time (years) Fold Change (+E2/-E2) -10 45 minutes 2 hours -6 45 minutes 2 hours -6 45 minutes 2 hours MME pS2 Figure 4 Expression profiles of some repressed ER target genes are correlated with disease recurrence in breast cancer patients. (a) The original patient material consisted of freshly frozen breast tumors that came from a population-based cohort of 315 women and that represented 65% of all the breast tumors resected in Uppsala County, Sweden, from 1 January 1987 to 31 December 1989 (Sjogren et al., 1996). The follow-up RNA expression profiling study was approved by the ethical committee at the Karolinska Institute, Stockholm, Sweden. Affymetrix (Santa Clara, CA, USA) oligonucleotide microarrays (U133A&B) were used to determine gene expression profiles in breast tumor samples. After exclusions based on RNA integrity and array quality control, expression profiles of 260 tumors were deemed suitable for further analysis. To assess the role of repressed ER target genes in breast tumor biology, we selected the 69 ER-positive tumors from patients who underwent adjuvant tamoxifen therapy. Clinicopathological characteristics were derived from the patient records and routine clinical measurements at the time of diagnosis. Histopathological reexamination and grading according to Elston–Ellis were performed by an experienced breast cancer pathologist without previous knowledge of the selected therapies and outcomes as described previously (Miller et al., 2005). Gene expression data are presented as mean-centered log2-transformed normalized intensity for the probe set. Distances between expression profiles were calculated based on Pearson correlation, and the average-linkage method from the Eisen Cluster program (http://rana.lbl.gov/EisenSoftware.htm) was used to cluster patient samples. Expression profiles of four recurrence-associated ER-repressed target genes (MME, COPL, CYP1A1 and Dicer 1) were used to cluster patients. (b) Kaplan–Meier plot of 10-year-censored disease-free survival curves for patients clustered by expression profiles of ER-repressed target gene is shown with the associated P-value from likelihood-ratio analysis. (c) Nuclear receptor co-activator recruitment to the MME ER-binding site was determined by re-ChIP experiments as described previously (MME ERE, forward primer, 50-GCCCCTTCCAGCTTCTCTAT-30, reverse primer, 50-GCAGGAAGGGAATGAGCA-30). Control PCR experiments were carried out using primers designed against exon 23 of the MME gene (MME exon 23, forward primer, 50-TCAGAAGCCTTTCACTGCCGCA-30, reverse primer, 50-GCAAGTCTAGCCAAGGGCTGCA-30) and detected either no binding or no differences between mock- and E2-treated ChIP and re-ChIP samples (data not shown). Statistically significant (Po0.05; two-tailed Student’s t-test) differences between the MME ER-binding site and the activated control pS2 ERE are denoted by asterisks. (d) Corepressor recruitment was similarly studied by re-ChIP. (e) Histone acetylation and RNA Pol II binding in the promoter region of the MME gene were examined by conventional ChIP and quantitative PCR (MME promoter, forward primer, 50-CCCCGAAGCTAAGCAATTC-30, reverse primer, 50-AAGGTTTCCGCTAGGTCTCC-30). Statistically significant (Po0.05; two- tailed Student’s t-test) differences between the MME promoter region and the activated control pS2 promoter are denoted by asterisks.

Oncogene Differential recruitment of nuclear receptor coregulators KW Merrell et al 1613 estrogen treatment, as was reported by Carroll et al. for involvement in disease parameters and outcome, we the repressed target genes they examined, as in our analyzed the expression profiles of all of the putative studies, ER does not appear to be bound to our sites repressed target genes identified in mapping and after 12 h of hormone exposure (Figure 1b). These microarray studies (Finlin et al., 2001; Soulez and disparate findings suggest that a more detailed analysis, Parker, 2001; Frasor et al., 2003; Lin et al., 2004, 2007b) including sites from both studies, is warranted to resolve in a previously published microarray gene expression the apparent differences. In contrast to the corepressors, dataset of 69 ER-positive breast cancer patient samples the co-activator recruitment to the repression-associated (Lin et al., 2007a). All of the patients received selective ER-binding sites was much more varied. SRC1 and estrogen receptor modulator (tamoxifen) treatment p/CIP were recruited to some of the binding sites and following surgery. When patients were grouped into expelled from others. The recruitment and expulsion of those with recurrent disease and those without, we the co-activators appeared to be both site- and time identified four repressed target genes, which were dependent. Even when the co-activators were present in significantly (Po0.05; Wilcoxon rank-sum test) differ- the ER complex, however, the reduction in histone entially expressed between the two patient groups. acetylation in the promoter regions of repressed target Hierarchical clustering of patients using the expression genes suggests that the histone deacetylases associated profiles of these four genes revealed three major clusters with the corepressors were exerting a greater influence (Figure 4a). Kaplan–Meier analysis of disease-free on the chromatin structure and transcriptional activity. survival (Figure 4b) indicated a significant (likelihood What is unclear is how ER is able to recruit corepressors ratio test, P ¼ 0.00417) association of one of the clusters to the repression-associated cis-regulatory sites as with poor prognosis. We then confirmed ER binding to opposed to the recruitment of co-activators in the the site adjacent to one of these genes, membrane canonical model of transcriptional activation of the metallo-endopeptidase, by ChIP (data not shown) and pS2 gene following estrogen treatment. No differences in observed similar patterns of the co-activator expulsion, the ERE motifs in repression-associated ER-binding corepressor recruitment, histone deacetylation and sites were detected when compared with those associated blockage of Pol II docking in the promoter region with transcriptional activation by ER (data not shown). (Figures 4c–e) as the two repressed target genes (PSCA There is a lower frequency of EREs in repressed sites and SLC35A1) selected for the mechanistic studies. and an enrichment of ER-binding sites in the promoter These results suggest that some of the repressed target regions of activated target genes; ER-binding sites genes are associated with disease progression and adjacent to repressed target genes are more dispersed response to endocrine therapy, and these genes and the and does not appear to be localized to a specific region mechanisms of transcriptional repression by ER warrant relative to the target gene (Lin et al., 2007b). Recent further study and evaluation as potential prognostic analysis has also revealed the enrichment of a number of markers and therapeutic targets in the treatment of ER- transcription factor-binding site motifs within the ER- positive breast cancers. binding regions of the repressed target genes, and initial studies suggest the involvement of the transcription factor RUNX1 in the repression of SLC35A1 by ER Conflict of interest (unpublished data). Another import question regarding mechanisms of transcriptional repression by ER is how The authors declare no conflict of interest. these mechanisms are affected by different ER agonists and antagonists. Preliminary results show that tamox- ifen functions as an agonist on repressed target genes, whereas raloxifene exhibits antagonistic activity; some Acknowledgements phytoestrogens also function as agonists on repressed This work was supported by a Mentoring Environment Grant ER target genes (unpublished data). (CYL) from Brigham Young University, a research grant Given the involvement of estrogens and ER in (CYL) from the Women’s Research Institute, NIH award breast cancer, we further posit that these mechanisms number R03CA143981 (CYL) and summer research fellow- and repressed target genes likely play key roles in ships (KWM, JDC, RLS, KEK and NRC) from the BYU breast cancer biology. To determine their potential Cancer Research Center.

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