Regulation of Estrogen Receptor-mediated Long Range Transcription via Evolutionarily Conserved Distal Response Elements
2008; Elsevier BV; Volume: 283; Issue: 47 Linguagem: Inglês
10.1074/jbc.m802024200
ISSN1083-351X
AutoresYou Fu Pan, K. D. Senali Abayratna Wansa, Mei Hui Liu, Bing Zhao, Shu Zhen Hong, Peck Yean Tan, Kar Sian Lim, Guillaume Bourque, Edison T. Liu, Edwin Cheung,
Tópico(s)Estrogen and related hormone effects
ResumoNuclear signaling by estrogens rapidly induces the global recruitment of estrogen receptors (ERs) to thousands of highly specific locations in the genome. Here, we have examined whether ER binding sites that are located distal from the transcription start sites of estrogen target genes are functionally relevant. Similar to ER binding sites near the proximal promoter region, ER binding sites located at distal locations are occupied by ERs after estrogen stimulation. And, like proximal bound ERs, ERs occupied at distal sites can recruit coactivators and the RNA polymerase transcription machinery and mediate specific structural changes to chromatin. Furthermore, ERs occupied at the distal sites are capable of communicating with ERs bound at the promoter region, possibly via long range chromosome looping. In functional analysis, disruption of the response elements in the distal ER binding sites abrogated ER binding and significantly reduced transcriptional response. Finally, sequence comparison of the response elements at the distal sites suggests a high level of conservation across different species. Together, our data indicate that distal ER binding sites are bona fide transcriptional enhancers that are involved in long range chromosomal interaction, transcription complex formation, and distinct structural modifications of chromatin across large genomic spans. Nuclear signaling by estrogens rapidly induces the global recruitment of estrogen receptors (ERs) to thousands of highly specific locations in the genome. Here, we have examined whether ER binding sites that are located distal from the transcription start sites of estrogen target genes are functionally relevant. Similar to ER binding sites near the proximal promoter region, ER binding sites located at distal locations are occupied by ERs after estrogen stimulation. And, like proximal bound ERs, ERs occupied at distal sites can recruit coactivators and the RNA polymerase transcription machinery and mediate specific structural changes to chromatin. Furthermore, ERs occupied at the distal sites are capable of communicating with ERs bound at the promoter region, possibly via long range chromosome looping. In functional analysis, disruption of the response elements in the distal ER binding sites abrogated ER binding and significantly reduced transcriptional response. Finally, sequence comparison of the response elements at the distal sites suggests a high level of conservation across different species. Together, our data indicate that distal ER binding sites are bona fide transcriptional enhancers that are involved in long range chromosomal interaction, transcription complex formation, and distinct structural modifications of chromatin across large genomic spans. Estrogens, such as 17β-estradiol (E2), 3The abbreviations used are: E2, estradiol; ER, estrogen receptor; SRC, steroid receptor co-activator; ERE, estrogen response element; ChIP, chromatin immunoprecipitation; pol, polymerase; 3C, chromosome conformation capture; siRNA, small interference RNA; CBP, CREB-binding protein; TSS, transcription start site; TPBM, theophylline, 8-[(benzylthio)methyl]-(7CI,8CI). 3The abbreviations used are: E2, estradiol; ER, estrogen receptor; SRC, steroid receptor co-activator; ERE, estrogen response element; ChIP, chromatin immunoprecipitation; pol, polymerase; 3C, chromosome conformation capture; siRNA, small interference RNA; CBP, CREB-binding protein; TSS, transcription start site; TPBM, theophylline, 8-[(benzylthio)methyl]-(7CI,8CI). are pleiotropic hormones whose effects are responsible for many physiological processes, including normal growth, development, and the precise and coordinated regulation of gene expression in tissues of the reproductive tract, central nervous system, and bone (1Nilsson S. Makela S. Treuter E. Tujague M. Thomsen J. Andersson G. Enmark E. Pettersson K. Warner M. Gustafsson J.A. Physiol. Rev. 2001; 81: 1535-1565Crossref PubMed Scopus (1574) Google Scholar, 2Heldring N. Pike A. Andersson S. Matthews J. Cheng G. Hartman J. Tujague M. Strom A. Treuter E. Warner M. Gustafsson J.A. Physiol. Rev. 2007; 87: 905-931Crossref PubMed Scopus (1306) Google Scholar). Estrogens also have important functions in hormone-dependent diseases, such as breast cancer and osteoporosis (1Nilsson S. Makela S. Treuter E. Tujague M. Thomsen J. Andersson G. Enmark E. Pettersson K. Warner M. Gustafsson J.A. Physiol. Rev. 2001; 81: 1535-1565Crossref PubMed Scopus (1574) Google Scholar, 2Heldring N. Pike A. Andersson S. Matthews J. Cheng G. Hartman J. Tujague M. Strom A. Treuter E. Warner M. Gustafsson J.A. Physiol. Rev. 2007; 87: 905-931Crossref PubMed Scopus (1306) Google Scholar). Selective estrogen receptor modulators, therapeutic agents that act as agonists or antagonists depending on the target tissue, are currently used in the treatment and prevention of these and other hormone-related disorders (1Nilsson S. Makela S. Treuter E. Tujague M. Thomsen J. Andersson G. Enmark E. Pettersson K. Warner M. Gustafsson J.A. Physiol. Rev. 2001; 81: 1535-1565Crossref PubMed Scopus (1574) Google Scholar, 2Heldring N. Pike A. Andersson S. Matthews J. Cheng G. Hartman J. Tujague M. Strom A. Treuter E. Warner M. Gustafsson J.A. Physiol. Rev. 2007; 87: 905-931Crossref PubMed Scopus (1306) Google Scholar, 3Green K.A. Carroll J.S. Nat. Rev. Cancer. 2007; 7: 713-722Crossref PubMed Scopus (173) Google Scholar). Estrogens and selective estrogen receptor modulators exert their effects through two estrogen receptors (ERs), ER alpha (ERα/ESR1/NR3A1) and ER beta (ERβ/ESR2/NR3A2), which belong to a large superfamily of nuclear hormone receptor proteins (2Heldring N. Pike A. Andersson S. Matthews J. Cheng G. Hartman J. Tujague M. Strom A. Treuter E. Warner M. Gustafsson J.A. Physiol. Rev. 2007; 87: 905-931Crossref PubMed Scopus (1306) Google Scholar, 3Green K.A. Carroll J.S. Nat. Rev. Cancer. 2007; 7: 713-722Crossref PubMed Scopus (173) Google Scholar). ERs share a conserved structural and functional organization with other members of the nuclear hormone receptor superfamily, including domains responsible for ligand binding, dimerization, DNA binding, and transcriptional activation (2Heldring N. Pike A. Andersson S. Matthews J. Cheng G. Hartman J. Tujague M. Strom A. Treuter E. Warner M. Gustafsson J.A. Physiol. Rev. 2007; 87: 905-931Crossref PubMed Scopus (1306) Google Scholar, 3Green K.A. Carroll J.S. Nat. Rev. Cancer. 2007; 7: 713-722Crossref PubMed Scopus (173) Google Scholar).As their domain structures imply, ERs behave as ligand-inducible, DNA binding transcription factors (2Heldring N. Pike A. Andersson S. Matthews J. Cheng G. Hartman J. Tujague M. Strom A. Treuter E. Warner M. Gustafsson J.A. Physiol. Rev. 2007; 87: 905-931Crossref PubMed Scopus (1306) Google Scholar, 3Green K.A. Carroll J.S. Nat. Rev. Cancer. 2007; 7: 713-722Crossref PubMed Scopus (173) Google Scholar). Their transcriptional activities require the recruitment of a variety of co-regulatory proteins by the receptors to estrogen-regulated promoters through either direct or indirect interactions (2Heldring N. Pike A. Andersson S. Matthews J. Cheng G. Hartman J. Tujague M. Strom A. Treuter E. Warner M. Gustafsson J.A. Physiol. Rev. 2007; 87: 905-931Crossref PubMed Scopus (1306) Google Scholar, 3Green K.A. Carroll J.S. Nat. Rev. Cancer. 2007; 7: 713-722Crossref PubMed Scopus (173) Google Scholar). A group of factors, including the p160/steroid receptor co-activator (SRC) family of proteins and the Mediator-like complexes (e.g. TRAP, DRIP, and ARC), have been shown to interact with and stimulate the transcriptional activities of ERs by interacting directly with the ligand binding domain in a ligand and activation function-2-dependent manner (2Heldring N. Pike A. Andersson S. Matthews J. Cheng G. Hartman J. Tujague M. Strom A. Treuter E. Warner M. Gustafsson J.A. Physiol. Rev. 2007; 87: 905-931Crossref PubMed Scopus (1306) Google Scholar, 3Green K.A. Carroll J.S. Nat. Rev. Cancer. 2007; 7: 713-722Crossref PubMed Scopus (173) Google Scholar). Other factors that contain enzymatic activities, such as the histone acetyltransferase p300/CBP and the histone methyltransferase CARM-1, are recruited indirectly by ERs mainly via interactions with the SRC proteins (2Heldring N. Pike A. Andersson S. Matthews J. Cheng G. Hartman J. Tujague M. Strom A. Treuter E. Warner M. Gustafsson J.A. Physiol. Rev. 2007; 87: 905-931Crossref PubMed Scopus (1306) Google Scholar, 3Green K.A. Carroll J.S. Nat. Rev. Cancer. 2007; 7: 713-722Crossref PubMed Scopus (173) Google Scholar). A smaller subset of ER-interacting factors has been shown to bind primarily to the N-terminal A/B region of the receptors, including the RNA-binding protein p68/p72 and SRA (2Heldring N. Pike A. Andersson S. Matthews J. Cheng G. Hartman J. Tujague M. Strom A. Treuter E. Warner M. Gustafsson J.A. Physiol. Rev. 2007; 87: 905-931Crossref PubMed Scopus (1306) Google Scholar, 3Green K.A. Carroll J.S. Nat. Rev. Cancer. 2007; 7: 713-722Crossref PubMed Scopus (173) Google Scholar). Together, these co-regulatory proteins are recruited by ERs in a precise temporal and coordinated manner in response to estrogen to promote local changes in histone modifications, chromatin structure, and the recruitment of RNA polymerase II to the promoters of target genes.Numerous estrogen target genes have been identified through expression microarray studies (reviewed in Ref. 4Kininis M. Kraus W.L. Nucl. Recept. Signal. 2008; 6: e005Crossref PubMed Scopus (91) Google Scholar); however, it is unclear what fraction of these genes are directly regulated by ERs. Direct regulation by estrogen is largely due to the recruitment of ERs to genomic regions containing sequence specific cis-regulatory motifs (2Heldring N. Pike A. Andersson S. Matthews J. Cheng G. Hartman J. Tujague M. Strom A. Treuter E. Warner M. Gustafsson J.A. Physiol. Rev. 2007; 87: 905-931Crossref PubMed Scopus (1306) Google Scholar, 3Green K.A. Carroll J.S. Nat. Rev. Cancer. 2007; 7: 713-722Crossref PubMed Scopus (173) Google Scholar). These sequences mostly contain a 13-bp palindromic motif (GGTCAnnnTGACC) called estrogen response elements (EREs) (2Heldring N. Pike A. Andersson S. Matthews J. Cheng G. Hartman J. Tujague M. Strom A. Treuter E. Warner M. Gustafsson J.A. Physiol. Rev. 2007; 87: 905-931Crossref PubMed Scopus (1306) Google Scholar, 3Green K.A. Carroll J.S. Nat. Rev. Cancer. 2007; 7: 713-722Crossref PubMed Scopus (173) Google Scholar). Through classic molecular analyses, estrogen-regulated genes were typically found to be associated with a single ER binding site, containing either a full ERE, half ERE, or other binding elements, located near the TSS at the proximal promoter region (5Klinge C.M. Nucleic Acids Res. 2001; 29: 2905-2919Crossref PubMed Scopus (790) Google Scholar). Well known examples of genes directly regulated by ER whose binding site is at the proximal promoter include CATD, EBAG9, and pS2/TFF1 (5Klinge C.M. Nucleic Acids Res. 2001; 29: 2905-2919Crossref PubMed Scopus (790) Google Scholar).To obtain a better understanding of how ER binding directly regulates gene expression under estrogen signaling, we recently mapped the genomic landscape of ERα binding sites in breast cancer cells using a ChIP-and-clone approach called ChIP-PET (Chromatin immunoprecipitation-Paired End diTags) (6Lin C.Y. Vega V.B. Thomsen J.S. Zhang T. Kong S.L. Xie M. Chiu K.P. Lipovich L. Barnett D.H. Stossi F. Yeo A. George J. Kuznetsov V.A. Lee Y.K. Charn T.H. Palanisamy N. Miller L.D. Cheung E. Katzenellenbogen B.S. Ruan Y. Bourque G. Wei C.L. Liu E.T. PLoS Genet. 2007; 3: e87Crossref PubMed Scopus (368) Google Scholar). From this study, 1234 ERα binding sites were identified in estradiol-stimulated MCF-7 cells. The majority of these ER binding sites contained full EREs (71%), whereas the rest harbored either half EREs (25%) or had no identifiable ERE motif (4%). Surprisingly, most of the ER binding sites were not concentrated at the proximal promoter region of genes as anticipated but distributed throughout the genome. A large proportion of the binding sites were found at distal regions, 5-100 kb from the 5′- and 3′-ends of the adjacent transcripts. Furthermore, these ER binding sites often occurred as groups of two or more distributed far apart from each other. Whether ERs recruited to these distal binding sites are functional and how they are able to regulate the transcription of target genes from large genomic distances is unclear. Herein, we have examined in detail the functional consequences of ERα recruitment at distal binding sites on coactivator recruitment, histone modification, and ER-dependent transcription. Our results suggest that multiple ERs function cooperatively with each other by communicating through long distance interaction to modify chromatin structure and formation of a stable and active transcription machinery to directly regulate the expression of ER target genes.EXPERIMENTAL PROCEDURESReagents and Antibodies—17β-estradiol (E2), 4-hydroxytamoxifen, and raloxifene were purchased from Sigma. TPBM (NSC 95910) was obtained from the Drug Synthesis and Chemistry Branch, Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis, NCI, National Institutes of Health. Commercially available antibodies raised against the following proteins and histone modifications were purchased from Abcam, Santa Cruz Biotechnology, Upstate, or Labvision and used in chromatin immunoprecipitation: ERα (ab-10; MS-315), ERα (HC-20; sc-543), NCOA3/AIB1 (c-20; sc-7216), GCN5 (N-18; sc-6303), CBP (c-20; sc-583), CARM1 (07-080), p68 (05-850), SRA (H-190; sc-20660), SRC-1 (05-522), unphosphorylated RNA pol II (8WG16; MMS-126R), RNA pol II phospho ser 5 (ab5131), H3K4me1 (ab8895), H3K4me2 (07-030), H3K4me3 (07-473), H3K9me1 (ab9045), H3K9me2 (07-441), H3K9me3 (ab8898), acH4 (06-598), acH3 (06-599), acH3K9 (06-942), H3Arg17me2 (07-214), bulk histone H3 (ab1791), normal goat IgG (sc-2028), normal rabbit IgG (sc-2027), and normal mouse IgG (sc-2025).Cell Culture and Transient Transfection Reporter Assays—Human mammary cancer MCF-7 cells, obtained from the American Type Culture Collection (ATCC, Manassas, VA), were routinely maintained in Dulbecco's modified Eagle's medium supplemented with 5% fetal bovine serum, penicillin, streptomycin, and gentamycin in a 37 °C incubator at 5% CO2. Two days prior to transfection, cells were transferred and maintained in phenol red-free Dulbecco's modified Eagle's medium/F-12 containing 5% charcoal-stripped fetal bovine serum, penicillin, streptomycin, and gentamycin. A day before transfection cells were plated in 24-well plates at a density of ∼60%. Cells were transfected with FuGENE™ 6 as recommended by the manufacturer (Roche Diagnostics). Firefly luciferase reporter constructs were transfected together with the pRenilla luciferase-TK plasmid as the internal control at a ratio of 50:1 of firefly to Renilla luciferase. Following an overnight incubation with the transfection mixture, the cells were treated with ethanol or 10 nm E2. After 48 h cells were harvested and firefly and Renilla luciferase assays were performed using the Dual-Luciferase Reporter Assay System (Promega, Madison, WI). The relative reporter gene activity was obtained after normalization of the firefly luciferase activity with Renilla luciferase activity. Each experiment was repeated at least three times to ensure reproducibility.Small Interfering RNA Studies—MCF-7 cells were seeded in Dulbecco's modified Eagle's medium/F-12 containing 5% charcoal-stripped fetal bovine serum 1 day prior to transfection. 100 nm siGENOME Non-Targeting siRNA Pool #1 or ERα ON-TARGETplus SMARTpool siRNA (Dharmacon) was then transfected into MCF-7 cells using Lipofectamine 2000 (Invitrogen) according to manufacturer's protocol. 48 h following siRNA transfection, the cells were treated with either E2 or vehicle for 45 min (for Western blot analysis, 3C, and ChIP assays) or 8 h (for mRNA analysis). Total cellular RNA was isolated with TRI® reagent (Sigma) and chloroform, ethanol precipitated, and purified using Qiagen RNeasy. The RNA was then reverse transcribed in the presence of oligo(dT)15 primer (Promega), dNTP Mix, and M-MLV RT (Promega). Quantitative PCR of the cDNA was carried out using SYBR Green PCR Master Mix (Applied Biosystems) on an ABI 7900 real-time PCR machine.Gel Mobility Shift Assays—Recombinant FLAG-tagged hERα-(1-595) was expressed and purified from Sf9 cells as described previously (7Cheung E. Schwabish M.A. Kraus W.L. EMBO J. 2003; 22: 600-611Crossref PubMed Scopus (40) Google Scholar). Gel mobility shift assays were performed as described previously (8Kraus W.L. Montano M.M. Katzenellenbogen B.S. Mol. Endocrinol. 1994; 8: 952-969Crossref PubMed Scopus (176) Google Scholar). Briefly, 32P-radiolabeled probes containing sequences for either TFF1 EREs or the Xenopus vitellogenin ERE were incubated with or without 20 nm of ERα on ice for 15 min in the presence of 100 nm E2. The samples were separated on a 4.8% non-denaturing polyacrylamide gel and exposed on x-ray film.ChIP, 3C, and ChIP-3C/ChIP-Loop Assays—ChIP experiments were performed as essentially described (6Lin C.Y. Vega V.B. Thomsen J.S. Zhang T. Kong S.L. Xie M. Chiu K.P. Lipovich L. Barnett D.H. Stossi F. Yeo A. George J. Kuznetsov V.A. Lee Y.K. Charn T.H. Palanisamy N. Miller L.D. Cheung E. Katzenellenbogen B.S. Ruan Y. Bourque G. Wei C.L. Liu E.T. PLoS Genet. 2007; 3: e87Crossref PubMed Scopus (368) Google Scholar). Briefly, after 45 min of drug treatment cells were cross-linked with 1% (v/v) formaldehyde (Sigma) for 10 min at room temperature and stopped with 125 mm glycine for 5 min. Crossed-linked cells were washed with phosphate-buffered saline, resuspended in lysis buffer, and sonicated for 8-10 min in a Biorupter (Diagenode) to generate DNA fragments with an average size of 500 bp. Chromatin extracts were diluted 5-fold with dilution buffer, pre-cleared with Protein-A- and/or -G-Sepharose beads, and immunoprecipitated with specific antibody on Protein-A- and/or -G-Sepharose beads. After washing, elution and de-cross-linking, the ChIP DNA was detected by either traditional PCR (25-35 cycles) or by quantitative real-time PCR analyses with SYBR green master mix on the ABI Prism 7900.3C was performed as described previously (9Hagege H. Klous P. Braem C. Splinter E. Dekker J. Cathala G. de Laat W. Forne T. Nat. Protoc. 2007; 2: 1722-1733Crossref PubMed Scopus (495) Google Scholar) with modifications. Briefly, MCF-7 cells were treated as mentioned in the ChIP protocol up to the cross-linking step with 1% formaldehyde. Nuclei were resuspended in 500 μl of 1.2× restriction enzyme buffer at 37 °C for 1 h. 7.5 μl of 20% SDS was added, the mixture was incubated for 1 h, followed by addition of 50 μl of 20% Triton X-100, and then incubation for an additional 1 h. Samples were then incubated with 400 units of selected restriction enzyme at 37 °C overnight. After digestion, 40 μl of 20% SDS was added to the digested nuclei, and the mixture was incubated at 65 °C for 10 min. 6.125 ml of 1.15× ligation buffer and 375 μl of 20% Triton X-100 was added, the mixture was incubated at 37 °C for 1 h, and then 2000 units of T4 DNA ligase was added at 16 °C for a 4-h incubation.Samples were then de-cross-linked at 65 °C overnight followed by phenol-chloroform extraction and ethanol precipitation. All primers had to be within a region of ±150 bp from the restriction enzyme digestion site. PCR products were amplified with AccuPrime Tag High Fidelity DNA Polymerase (Invitrogen) for 40 cycles. PCR products were run on a 2% agarose gel. Each validation experiment was repeated at least twice.ChIP-3C/ChIP-Loop assays were performed as essentially described previously with slight modifications (10Carroll 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 (1068) Google Scholar, 11Wang Q. Carroll J.S. Brown M. Mol. Cell. 2005; 19: 631-642Abstract Full Text Full Text PDF PubMed Scopus (362) Google Scholar). Briefly, antibody-specific immunoprecipitated chromatin was obtained as described above for ChIP assays. Chromatin still bound to the antibody-Protein-A-Sepharose beads was digested with restriction enzyme, ligated with T4 DNA ligase, eluted, and de-cross-linked. After purification, the ChIP-3C material was detected for long range interaction with primers from the distal and promoter ER binding regions. Primer sequences used for ChIP, 3C, and ChIP-3C assays are available upon request.Evolutionary Conservation Analysis—The proximal promoter sequence of TFF1 (from 1 kb upstream to 200 bp downstream of the TSS) in human (hg17), chimp (panTro1), rhesus (rheMac2), mouse (mm7), rat (rn4), dog (canFam2), and cow (bosTau2) were retrieved and aligned using ClustalW. A similar procedure was used for the region upstream of TFF1 and intronic to TMPRSS3 in which the distal EREs are located. Here, a 2-kb window (chr21: 42,668,352-42,670,351) was used to include the full intron and the two flanking exons.RESULTSEstrogen Up-regulated Genes Are Often Associated with Multiple ER Binding Sites—Our previous genome-wide ERα binding site study in MCF-7 breast cancer cells showed that estrogen up-regulated genes in general were more significantly associated with ER binding compared with down-regulated genes (6Lin C.Y. Vega V.B. Thomsen J.S. Zhang T. Kong S.L. Xie M. Chiu K.P. Lipovich L. Barnett D.H. Stossi F. Yeo A. George J. Kuznetsov V.A. Lee Y.K. Charn T.H. Palanisamy N. Miller L.D. Cheung E. Katzenellenbogen B.S. Ruan Y. Bourque G. Wei C.L. Liu E.T. PLoS Genet. 2007; 3: e87Crossref PubMed Scopus (368) Google Scholar). When we examined the location of ER binding with respect to the up-regulated genes, we observed that many genes, including previously known estrogen direct targets such as TFF1, CYP1B1, SIAH2, CTSD, and GREB1, were associated with multiple ER binding sites that are distributed across large genomic distances of up to 100 kb apart (Fig. 1). From these observations, it appears that transcriptional regulation of a large subset of estrogen up-regulated genes may require the actions of not one but multiple ERs functioning collectively through a mechanism involving long distance chromatin interaction.To understand the molecular basis of how ERs function together across large genomic distances, we examined the recruitment of ERα to the regulatory region of a well characterized E2-responsive gene, TFF1. From our ERα ChIP-PET analysis, ERα appears to be recruited at two main locations, 1) at the proximal promoter (∼-400 bp) which has been previously characterized and 2) a novel region ∼10 kb upstream of the TSS (Fig. 1). The ERα binding site at the proximal promoter has been shown to harbor a functional ERE motif in previous studies (12Berry M. Nunez A.M. Chambon P. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 1218-1222Crossref PubMed Scopus (295) Google Scholar, 13Nunez A.M. Berry M. Imler J.L. Chambon P. EMBO J. 1989; 8: 823-829Crossref PubMed Scopus (250) Google Scholar). Sequence analysis of the distal ER binding site shows there are two potential ERE motifs (Fig. 2A). To determine whether ERα can bind directly to these 2 EREs, we performed gel mobility shift assays with purified recombinant FLAG-tagged ERα (Fig. 2B). As expected, ERα bound efficiently to radiolabeled double stranded probes containing sequences of the promoter ERE (ERE I) and the positive control, vitellogenin ERE. ERα also bound to probes containing sequences of the two EREs (ERE II and III) found in the distal ER binding site (Fig. 2B). Mutating the ERE sites in the gel mobility shift probes completely abrogated the binding of ERα to all 3 ERE sites (data not shown). In addition to gel shift assays, ChIP analyses were performed in MCF-7 cells to assess the in vivo binding of ERα at the corresponding ERE locations. ERα was recruited in a ligand-dependent manner to the promoter region, which encompasses ERE I, and to the distal ER binding site, which contains ERE II and III (Fig. 2, C and D). Taken together, these results indicate that ERα occupies multiple EREs at the proximal and distal regulatory regions of TFF1.FIGURE 2Multiple ERs are recruited to the TFF1 locus. A, distal ER binding sites of TFF1 identified by ChIP-PET contain potential EREs. DNA sequence and location of the previously identified ERE (ERE I) at the proximal ER binding site and two potential EREs (ERE II and III) at the distal binding sites of TFF1. B, gel mobility shift assay was performed using purified recombinant ERα protein and radiolabeled, double-stranded probes for TFF1 ERE I, ERE II, ERE III, and Xenopus vitellogenin A2 ERE as control. The samples were analyzed by native polyacrylamide gel electrophoresis with subsequent exposure to x-ray film. C, ChIP assays using anti-ERα and IgG were performed with MCF-7 cells treated with or without E2 for 45 min. Total input and ChIP material were detected by traditional PCR with primer pairs located at ERE I, II, and III of TFF1. A schematic diagram of the TFF1 locus with the three ERE sites is shown below. D, ChIP-quantitative PCR was performed on ChIP material from C. The result represents the average of three independent experiments ± S.E.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Estrogen Stimulates the Accumulation of ERα, Coactivators, and RNA pol II to the Proximal and Distal ER Binding Sites of TFF1—Estrogen-dependent transcription involves a large number of coactivators that function with the liganded ER to modify histones, alter chromatin structure and recruit RNA polymerase II (1Nilsson S. Makela S. Treuter E. Tujague M. Thomsen J. Andersson G. Enmark E. Pettersson K. Warner M. Gustafsson J.A. Physiol. Rev. 2001; 81: 1535-1565Crossref PubMed Scopus (1574) Google Scholar, 2Heldring N. Pike A. Andersson S. Matthews J. Cheng G. Hartman J. Tujague M. Strom A. Treuter E. Warner M. Gustafsson J.A. Physiol. Rev. 2007; 87: 905-931Crossref PubMed Scopus (1306) Google Scholar, 3Green K.A. Carroll J.S. Nat. Rev. Cancer. 2007; 7: 713-722Crossref PubMed Scopus (173) Google Scholar). To examine the repertoire of coactivators recruited to ER binding sites of TFF1, we performed ChIP assays targeting the proximal and distal binding sites with antibodies against various proteins that are known coactivators of ERα. As expected, stimulation of MCF-7 cells with E2 resulted in enhanced recruitment of activation function-2-dependent coactivators, including SRCs (SRC-1 and AIB1), CBP, GCN5, CARM1, and the AF-1-dependent coactivators, p68 and SRA, to ERE I in the ER binding site near the proximal promoter (Fig. 3). These same coactivators also bound to both ERE II and ERE III in the distal ER binding site in a similar manner. Interestingly, in addition to coactivators, RNA pol II (both the phosphorylated and unphosphorylated forms) was also recruited to all three ERE sites, indicating that the distal EREs may have similar functional properties as the proximal ERE.FIGURE 3The distal ER binding site of TFF1 functions by recruiting coactivators and the basal transcription machinery. ChIP assays using antibodies against ERα, coactivators, and RNA pol II were performed with MCF-7 cells treated with or without E2 as described in Fig. 2.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The recruitment of ER, coactivators, and the RNA polymerase machinery to the distal ER binding site of TFF1 suggests that regulatory elements located far away may play an important role in estrogen-dependent transcription. Because our binding experiments were limited to only the ERE sites, it is possible that we may have overlooked important information regarding the recruitment of ER, coactivators, and RNA polymerase to other regulatory regions of TFF1. We therefore expanded our analysis by scanning across a 19-kb region of the TFF1 locus by ChIP quantitative PCR, which included not only the proximal and distal ER binding sites, but also surrounding upstream and downstream regions such as the coding and noncoding region of the TFF1 gene. As shown in Fig. 4, estrogen induced the recruitment of ERα at three major locations along the 19-kb TFF1 locus with one sharp distinct peak at the promoter corresponding to ERE I, and another two (one small and one large) peaks at 9 and 10 kb corresponding to ERE II and ERE III, respectively. The recruitment of coactivators AIB1 and CBP also generated similar binding profiles as ERα. In contrast, estrogen induced a very different pattern for RNA pol II recruitment compared with ERα and the coactivators. Both the phosphorylated and un-phosphorylated RNA pol II bound to three main regions with very broad occupancy spanning 4-5 kb in width. Two of these regions co-localized with the proximal and distal ER binding sites, whereas a third RNA pol II peak appeared after the TFF1 gene. Collectively, our ChIP analyses suggest that the distal EREs can recruit the same set of factors as the ERE at the proximal promoter of TFF1.FIGURE 4Estrogen induces the recruitment of ERα and coactivators to specific sites while inducing RNA pol II spreading across large genomic regions. ChIP scanning of the TFF1 locus. ChIP assay was performed with MCF-7 cells treated with or without E2 as described in Fig. 2 using antibodies against ERα, SRC3, CBP, phosphorylated (pol II Ser 5), and un-phosphorylated (pol II) RNA pol II and IgG. ChIP material
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