Regulation of GREB1 Transcription by Estrogen Receptor α through a Multipartite Enhancer Spread Over 20 kb of Upstream Flanking Sequences

Estrogen receptors activate transcription in part through direct interactions with specific DNA motifs, called estrogen response elements (EREs). Here we show that the strong and sustained induction of the gene regulated in breast cancer 1 (GREB1), a gene of unknown function that has been previously suggested to play a role in the effects of estradiol on breast cancer cell proliferation (Rae, J. M., Johnson, M. D., Scheys, J. O., Cordero, K. E., Larios, J. M., and Lippman, M. E. (2005) Breast Cancer Res. Treat 92, 141–149), is mediated by binding of estrogen receptor α (ERα) to three consensus EREs spread over ∼20 kb of upstream flanking sequences. In addition to ERα, coactivator SRC-3, acetylated histones and phosphorylated RNA polymerase II (P-polII) were detected on all three EREs in the presence of estrogen, while basal recruitment of ERα and P-polII was observed only on the proximal element. Chromatin loops were formed between each ERE and the GREB1 transcriptional start site in the presence of estrogen but not of a total antiestrogen. Furthermore, estradiol induced physical association between EREs, suggesting that these elements function as a potent multipartite enhancer to regulate GREB1 transcription. Estrogen receptors activate transcription in part through direct interactions with specific DNA motifs, called estrogen response elements (EREs). Here we show that the strong and sustained induction of the gene regulated in breast cancer 1 (GREB1), a gene of unknown function that has been previously suggested to play a role in the effects of estradiol on breast cancer cell proliferation (Rae, J. M., Johnson, M. D., Scheys, J. O., Cordero, K. E., Larios, J. M., and Lippman, M. E. (2005) Breast Cancer Res. Treat 92, 141–149), is mediated by binding of estrogen receptor α (ERα) to three consensus EREs spread over ∼20 kb of upstream flanking sequences. In addition to ERα, coactivator SRC-3, acetylated histones and phosphorylated RNA polymerase II (P-polII) were detected on all three EREs in the presence of estrogen, while basal recruitment of ERα and P-polII was observed only on the proximal element. Chromatin loops were formed between each ERE and the GREB1 transcriptional start site in the presence of estrogen but not of a total antiestrogen. Furthermore, estradiol induced physical association between EREs, suggesting that these elements function as a potent multipartite enhancer to regulate GREB1 transcription. Estrogens are steroid hormones that have pleiotropic physiological actions in numerous target tissues including the reproductive system (1Couse J.F. Korach K.S. Endocr. Rev. 1999; 20: 358-417Crossref PubMed Scopus (0) Google Scholar, 2McEwen B.S. Alves S.E. Endocr. Rev. 1999; 20: 279-307Crossref PubMed Scopus (1338) Google Scholar, 3Jordan V.C. J. Natl. Cancer Inst. 2001; 93: 2-4Crossref PubMed Scopus (21) Google Scholar) but also play an important role in cancers, in particular breast cancer (4Pike M.C. Spicer D.V. Dahmoush L. Press M.F. Epidemiol. Rev. 1993; 15: 17-35Crossref PubMed Scopus (904) Google Scholar, 5Simpson E.R. J. Steroid Biochem. Mol. Biol. 2003; 86: 225-230Crossref PubMed Scopus (685) Google Scholar). Estrogens act through two receptors, ERα 6The abbreviations used are: ER, estrogen receptor; ERE, estrogen response element; TSS, transcriptional start site; FBS, fetal bovine serum; DMEM, Dulbecco's modified Eagle's medium; siRNA, small interfering RNA; ChIP, chromatin immunoprecipitation; E2, estradiol; P-polII, phosphorylated RNA polymerase II; 3C, chromatine conformation capture. and ERβ, members of the nuclear receptor superfamily of ligand-inducible transcription factors (6Mangelsdorf D.J. Thummel C. Beato M. Herrlich P. Schutz G. Umesono K. Blumberg B. Kastner P. Mark M. Chambon P. Evans R.M. 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Geistlinger T.R. Fox E.A. Silver P.A. Brown M. Cell. 2005; 122: 33-43Abstract Full Text Full Text PDF PubMed Scopus (1090) Google Scholar, 16Vega V.B. Lin C.Y. Lai K.S. Kong S.L. Xie M. Su X. Teh H.F. Thomsen J.S. Yeo A.L. Sung W.K. Bourque G. Liu E.T. Genome Biol. 2006; 7: R82Crossref PubMed Scopus (44) Google Scholar, 17Carroll J.S. Meyer C.A. Song J. Li W. Geistlinger T.R. Eeckhoute J. Brodsky A.S. Keeton E.K. Fertuck K.C. Hall G.F. Wang Q. Bekiranov S. Sementchenko V. Fox E.A. Silver P.A. Gingeras T.R. Liu X.S. Brown M. Nat. Genet. 2006; 38: 1289-1297Crossref PubMed Scopus (1105) Google Scholar). Some estrogen target genes, such as those encoding cyclin D1 (CCND1), c-myc (MYC), and E2F transcription factors have been shown to mediate the proliferative effects of estrogen in breast cancer cell lines (18D'Cruz C.M. Gunther E.J. Boxer R.B. Hartman J.L. Sintasath L. Moody S.E. Cox J.D. Ha S.I. Belka G.K. Golant A. Cardiff R.D. Chodosh L.A. Nat. Med. 2001; 7: 235-239Crossref PubMed Scopus (346) Google Scholar, 19Hui R. Finney G.L. Carroll J.S. Lee C.S. Musgrove E.A. Sutherland R.L. Cancer Res. 2002; 62: 6916-6923PubMed Google Scholar, 20Butt A.J. McNeil C.M. Musgrove E.A. Sutherland R.L. Endocr. Relat. Cancer. 2005; 12: S47-S59Crossref PubMed Scopus (206) Google Scholar, 21Mukherjee S. Conrad S.E. J. Biol. Chem. 2005; 280: 17617-17625Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). It has also been recently suggested that the gene regulated in breast cancer 1, GREB1 (22Ghosh M.G. Thompson D.A. Weigel R.J. Cancer Res. 2000; 60: 6367-6375PubMed Google Scholar), whose function remains unknown, contributes to the growth-promoting effects of estrogens in MCF-7 cells (23Rae J.M. Johnson M.D. Scheys J.O. Cordero K.E. Larios J.M. Lippman M.E. Breast Cancer Res. Treat. 2005; 92: 141-149Crossref PubMed Scopus (188) Google Scholar). ERs can bind to specific DNA motifs, called estrogen response elements (EREs) through a central conserved DNA binding domain (24Mader S. Chambon P. White J.H. Nucleic Acids Res. 1993; 21: 1125-1132Crossref PubMed Scopus (102) Google Scholar). Consensus EREs, which were defined by compiling natural response elements in estrogen-responsive genes and correspond to the highest affinity binding site in vitro, are 15-bp palindromes composed of two PuGGTCA motifs spaced by 3 bp (25Klinge C.M. Nucleic Acids Res. 2001; 29: 2905-2919Crossref PubMed Scopus (820) Google Scholar, 26Sanchez R. Nguyen D. Rocha W. White J.H. Mader S. Bioessays. 2002; 24: 244-254Crossref PubMed Scopus (174) Google Scholar). Although most high affinity EREs were initially characterized in the proximal regulatory sequences of target genes, recent studies have indicated that elements binding ERα can be found at large distances from transcriptional start sites (TSS). Our genome-wide mapping of high affinity EREs combined with characterization of elements by chromatin immunoprecipitation identified functional ERα binding sites up to 10 kb from TSS (27Bourdeau V. Deschenes J. Metivier R. Nagai Y. Nguyen D. Bretschneider N. Gannon F. White J.H. Mader S. Mol. Endocrinol. 2004; 18: 1411-1427Crossref PubMed Scopus (277) Google Scholar). In addition, genome-wide mapping of chromatin fragments bound by ERα revealed sites located much further from adjacent genes, and use of chromatin conformation capture (3C) assays showed that some of these elements can form chromatin loops with promoters located at distances of up to 100 kb (15Carroll J.S. Liu X.S. Brodsky A.S. Li W. Meyer C.A. Szary A.J. Eeckhoute J. Shao W. Hestermann E.V. Geistlinger T.R. Fox E.A. Silver P.A. Brown M. Cell. 2005; 122: 33-43Abstract Full Text Full Text PDF PubMed Scopus (1090) Google Scholar, 17Carroll J.S. Meyer C.A. Song J. Li W. Geistlinger T.R. Eeckhoute J. Brodsky A.S. Keeton E.K. Fertuck K.C. Hall G.F. Wang Q. Bekiranov S. Sementchenko V. Fox E.A. Silver P.A. Gingeras T.R. Liu X.S. Brown M. Nat. Genet. 2006; 38: 1289-1297Crossref PubMed Scopus (1105) Google Scholar). Often, ERα binds several chromatin regions within this range of distances from the TSS of estrogen-regulated genes, raising the possibility of cooperativity between widely separated enhancer units for transcriptional regulation of target genes. This may result in the formation of multiple chromatin loops with the TSS of these target genes and possibly between enhancers themselves. GREB1 stands out as an estrogen target gene because of the presence in its flanking region of three consensus EREs spread over 20 kb of upstream sequences. We have previously observed in vivo recruitment of ERα to the EREs present at –1.5 and –9.5 kb (27Bourdeau V. Deschenes J. Metivier R. Nagai Y. Nguyen D. Bretschneider N. Gannon F. White J.H. Mader S. Mol. Endocrinol. 2004; 18: 1411-1427Crossref PubMed Scopus (277) Google Scholar). Here we address whether the three GREB1 consensus EREs are functional enhancers that cooperate for transcriptional induction of the GREB1 gene by ERα and present evidence for a complex chromatin loop structure that implicates all three EREs and the TSS. Cell Culture—MCF-7 breast carcinoma cells and Ishikawa cells were maintained in α-minimal Eagle's medium (Wisent, St-Bruno, Quebec, Canada) supplemented with 10% fetal bovine serum (FBS) (Sigma-Aldrich, Oakville, Ontario, Canada). ZR75 and T47D were maintained in RPMI-1640 (Wisent) supplemented with 10% FBS. MDA-MB-231::ERα cells were maintained in Dulbecco's modified Eagle's medium (DMEM) (Wisent) supplemented with 5% FBS with 0.15 mg/ml of hygromycin (Sigma). Three days before experiments, cells were switched to phenol red-free DMEM (Wisent) containing 10% charcoal-treated FBS (FBS-T), 1% sodium pyruvate (Wisent), 1% penicillin/streptomycin (Wisent), and 1% l-glutamine (Wisent). The day before hormonal stimulation, the medium was changed to phenol red-free DMEM supplemented with 0.5% charcoal-treated FBS. Cells were then treated with 17-β-estradiol (E2, 25 nm, Sigma), ICI 182,780 (ICI, 100 nm, Tocris, Ellisville, MO) or vehicle (0.1% ethanol) for variable periods of time as indicated in the figure legends. RNA Extraction—Cells were seeded in 10-cm plates (MCF-7, T47D, ZR75, MDA-MB-231::ERα) or 6-cm plates (Ishikawa) at a density such that near confluence was obtained at the end of the treatment. Cells were treated with 25 nm E2, 100 nm ICI 182,780, or vehicle (ethanol) for different times, as indicated in the figure legends. For pretreatments with actinomycin D (2 μg/ml) or cycloheximide (10 μg/ml), incubation was initiated 1 h before hormonal treatment. siGenome siRNAs were transfected according to the instructions of the manufacturer (Dharmacon, Chicago, IL), medium was changed 24 h after transfection and hormonal stimulations were initiated after another 24 h. At the end of the treatments, medium was removed, cells were collected in 1 ml of TRI Reagent (Sigma), and total RNA was extracted as recommended by the manufacturer. Reverse Transcription and Real-time PCR—Total RNA (2 μg) was reverse transcribed using the RevertAid H first minus strand cDNA synthesis kit (MBI Fermentas, Burlington, Ontario, Canada) as recommended by the manufacturer. The reverse transcription product was diluted 10 times prior to real-time PCR. Each real-time PCR amplification reaction contained the reverse transcription dilution (2 μl), forward and reverse primers (150–300 nm), MgCl2 (3–4.5 mm according to primer pairs), dNTP (0.2 mm), SYBR Green (0.33×, Invitrogen, Burlington, Ontario, Canada), buffer for Jump Start Taq and Jump Start Taq (0.5 unit, Sigma) in a final volume of 20 μl. After denaturation at 95 °C for 7 min, samples went through a one-degree annealing temperature touchdown of 7 cycles starting from 60 °C (30 s at 95 °C, 30 s at annealing temperature, 30 s at 72 °C) followed by 40 cycles of amplification (30 s at 95 °C, 30 s at 58 °C and 30 s at 72 °C). A dissociation protocol followed the amplification program to characterize the amplified product(s). PCR was performed using a RotorGene 3000 (Corbett, Australia) and analyzed using expression levels of the p36b4 gene for normalization. For each set of primers, non-template control reactions were performed as a negative control. Each sample was assayed in triplicate and each experiment was reproduced at least two times. A typical experiment is shown. All primer pairs were designed using the Primer3 software and chosen to span an intron. The primer sequences and individual conditions used for polymerase chain reaction amplification are available upon request. Western Blot Analysis—Whole cell extracts and blot analysis were performed as described previously (28Rocha W. Sanchez R. Deschenes J. Auger A. Hebert E. White J.H. Mader S. Mol. Pharmacol. 2005; 68: 1852-1862Crossref PubMed Scopus (14) Google Scholar) using anti-ERα mouse monoclonal (B10, kind gift from Prof. P. Chambon) and anti-β-actin mouse monoclonal antibody (AC-15, Sigma Diagnostics). Chromatin Immunoprecipitation—For chromatin immunoprecipitation (ChIP) assays, cells in phenol red-free DMEM containing 0.5% FBS-T were treated with vehicle or ICI for Chromatin was by cells with for 10 at temperature and by as previously (27Bourdeau V. Deschenes J. Metivier R. Nagai Y. Nguyen D. Bretschneider N. Gannon F. White J.H. Mader S. Mol. Endocrinol. 2004; 18: 1411-1427Crossref PubMed Scopus (277) Google Scholar), fragments of ERα or were from and phosphorylated polymerase II or acetylated were from The sequences of the primers used in assays are available upon request. Chromatin immunoprecipitation were performed at least two times with A set of is shown. assays were performed as described E. F. W. 2004; PubMed Scopus Google Scholar), with only In 3 days prior to the MCF-7 cells were switched to phenol red-free DMEM containing 10% FBS-T and for h at cells The serum was to 0.5% 24 h before treatment with nm), ICI nm), or vehicle (ethanol) for medium was removed, and cells were with for 10 at Cells were then with collected in of buffer mm 10 mm and with and in buffer (10 mm 10 mm and were in ml of buffer (MBI supplemented with was to the The DNA was with units of or (MBI and then with another 100 units for h at The was by addition of and incubation at DNA were in for between The reactions were diluted with buffer mm 10 mm 10 mm 1 mm and 25 bovine serum supplemented with final and for 1 h at The DNA was using at was and samples were at °C to reverse the The samples were for h at °C with and the DNA was by and Chromatin loop formation was by PCR amplification out using conditions as for amplification but SYBR Green and with two primer pairs for each PCR were on and by sequences are available upon request. characterize the of regulation of the GREB1 gene by estrogen, we first the of estradiol treatment on its expression in different cell lines (MCF-7, T47D, and induction was detected in all cells with treatment the ICI 182,780 levels with these a strong between GREB1 levels and ERα expression in breast tumors can be observed in studies by the of ERα in transcriptional regulation by estrogen, we the of of ERα on GREB1 expression levels in MCF-7 shown in 1, levels of ERα were by the specific in a of basal and levels of GREB1 which with the of ICI treatment that ERα is important for basal and GREB1 further that GREB1 is regulated at the transcriptional by E2, MCF-7 cells were by the transcription actinomycin D. This treatment induction of GREB1 that GREB1 transcription the the synthesis cycloheximide on the induction by in MCF-7 cells as (22Ghosh M.G. Thompson D.A. Weigel R.J. Cancer Res. 2000; 60: 6367-6375PubMed Google Scholar), to was observed with the target gene encoding the receptor while induction of the gene was by cycloheximide and that of was as previously G. J. P.A. J.D. PubMed Scopus Google Scholar) of induction of GREB1 by in MCF-7 cells that of the target at and sustained over a h the induction of or was but while that of was and that of in time these that GREB1 is a ERα target induced in an and sustained by ERα in MCF-7 The sequences of GREB1 three consensus EREs and we have previously shown that two of the three GREB1 EREs bound ERα in in MCF-7 cells and in cells transfected with ERα (27Bourdeau V. Deschenes J. Metivier R. Nagai Y. Nguyen D. Bretschneider N. Gannon F. White J.H. Mader S. Mol. Endocrinol. 2004; 18: 1411-1427Crossref PubMed Scopus (277) Google Scholar). of ERα to the three EREs was also observed in a recent genome-wide mapping of ERα binding sites in the presence of estrogen J.S. Meyer C.A. Song J. Li W. Geistlinger T.R. Eeckhoute J. Brodsky A.S. Keeton E.K. Fertuck K.C. Hall G.F. Wang Q. Bekiranov S. Sementchenko V. Fox E.A. Silver P.A. Gingeras T.R. Liu X.S. Brown M. Nat. Genet. 2006; 38: 1289-1297Crossref PubMed Scopus (1105) Google Scholar). Here we show that in MCF-7 cells all three EREs are bound with a after as observed previously for the gene Y. X. J. Brown M. Cell. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, R. G. M.R. G. H. M. Gannon F. Cell. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). that the basal levels of ERα to the ERE at –1.5 kb from the GREB1 TSS were those for binding of ERα to the GREB1 TSS was observed In addition, recruitment to the EREs of the ERα coactivator acetylated as as phosphorylated RNA polymerase II (P-polII) was also detected by that binding of P-polII was strong basal conditions on the these that the two EREs are functional enhancers and that the proximal ERE contributes to the of the recruitment of ERα and its coactivator SRC-3, of and binding of P-polII to sites that are to the GREB1 TSS to gene is located kb from this not that these EREs play a role in direct of whether all three EREs with the GREB1 TSS, we used a chromatin conformation capture (3C) J. K. M. N. 2002; PubMed Scopus Google Scholar), which physical between DNA sites by of DNA fragments that are maintained by that at least between consensus EREs and the GREB1 TSS, we observed PCR amplification of from the of each of the three EREs and the TSS of and the TSS was which the basal of receptor and P-polII with this ERE or the between the ERE and the TSS. the of chromatin loop formation in the presence of ICI formation of a chromatin loop in the of interactions between the two DNA chromatin loops were detected with several regions located between the EREs and the TSS and not or between the TSS and a at kb not that chromatin with the TSS is specific for fragments containing the that the PCR obtained were to product and that all primer for led to amplification of a and this genomic region not that the consensus EREs in the GREB1 gene as enhancers ERα, SRC-3, and P-polII, are with acetylated and with the TSS of GREB1 in the presence of The presence of three strong enhancers the of whether these sequences with the TSS in a and possibly or may in interactions as this we analyzed by whether chromatin loop formation between ERE enhancers PCR amplification from chromatin loop formation between of two consensus EREs be detected in the presence of but not in its or in the presence of In addition, interactions were detected with regions and not the of these interactions for the presence of that EREs can with each in a large complex with the TSS or in interactions in on the we that the region of a gene of as unknown function but that may play an important role in cell proliferation (23Rae J.M. Johnson M.D. Scheys J.O. Cordero K.E. Larios J.M. Lippman M.E. Breast Cancer Res. Treat. 2005; 92: 141-149Crossref PubMed Scopus (188) Google Scholar), a multipartite enhancer composed of three consensus EREs separated by to ∼20 kb of sequences. studies be to whether this multipartite enhancer also with ERα binding regions and with the TSS of target genes and whether transcriptional regulation by the association of several widely spread enhancer We Metivier and for on the and and for on the We are also to for and Rocha for with

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