Published OnlineFirst May 25, 2010; DOI: 10.1158/0008-5472.CAN-09-4407 Published OnlineFirst on May 25, 2010 as 10.1158/0008-5472.CAN-09-4407 Tumor and Stem Cell Biology Cancer Research Genome-Wide Mapping of Estrogen Receptor-β–Binding Regions Reveals Extensive Cross-Talk with Transcription Factor Activator Protein-1 Chunyan Zhao1, Hui Gao1, Yawen Liu2, Zoi Papoutsi1, Sadaf Jaffrey1, Jan-Åke Gustafsson1,3, and Karin Dahlman-Wright1 Abstract Estrogen signaling can occur through a nonclassical pathway involving the interaction of estrogen receptors (ER) with other transcription factors such as activator protein-1 (AP-1) and SP-1. However, there is little mechanistic understanding about this pathway, with conflicting results from in vitro investigations. In this study, we applied the ChIP-on-chip approach to identify ERβ-binding sites on a genome-wide scale, identifying 1,457 high-confidence binding sites in ERβ-overexpressing MCF7 breast cancer cells. Genes containing ERβ-binding sites can be regulated by E2. Notably, ∼60% of the genomic regions bound by ERβ contained AP-1–like binding regions and estrogen response element–like sites, suggesting a functional association between AP-1 and ERβ signaling. Chromatin im- munoprecipitation (ChIP) analysis confirmed the association of AP-1, which is composed of the oncogenic tran- scription factors c-Fos and c-Jun, to ERβ-bound DNA regions. Using a re-ChIP assay, we showed co-occupancy of ERβ and AP-1 on chromatin. Short interfering RNA–mediated knockdown of c-Fos or c-Jun expression decreased ERβ recruitment to chromatin, consistent with the role of AP-1 in mediating estrogen signaling in breast cancer cells. Additionally, ERα and ERβ recruitment to AP-1/ERβ target regions exhibited gene-dependent differences in response to antiestrogens. Together, our results broaden insights into ERβ DNA-binding at the genomic level by revealing crosstalk with the AP-1 transcription factor. Cancer Res; 70(12); OF1–10. ©2010 AACR. Introduction tor complexes including Fos/Jun [activator protein-1 (AP-1)– responsive elements] or SP-1 (4, 5). However, there is little Estrogens bind to and activate two estrogen receptors mechanistic understanding about this pathway, with (ERα and ERβ), and exert their effects through a complex conflicting results from in vitro investigations (6, 7). A small array of signaling pathways that mediate genomic and non- number of genes containing an AP-1 site in their promoters genomic events (1, 2). ERs regulate gene expression through have been shown to be regulated by ERα, such as collagenase distinct DNA response elements. The classical mechanism of (8), human insulin-like growth factor I (9), and the human estrogen signaling is through an estrogen response element choline acetyltransferase gene (10). (ERE). In this process, ER dimerizes and interacts with EREs The AP-1 transcription factor is a key component of many associated with target genes, followed by the recruitment of a signal transduction pathways. AP-1 was first known as a 12-O- variety of coregulators to alter chromatin structure and facil- tetradecanoylphorbyl-13-acetate (TPA)–inducible transcrip- itate recruitment of the RNA polymerase II (PolII) transcrip- tion factor because the TPA response element was identified tional machinery (2, 3). Estrogen signaling could also occur as a binding site for AP-1 in many cellular and viral genes (11). through a nonclassical pathway in which liganded ERs are The AP-1 transcription factor is a dimeric complex consisting tethered to DNA via association with other transcription fac- of homodimers of Jun family members or heterodimers of Jun and Fos family members. AP-1 dimers regulate the expression of AP-1 target genes by binding to consensus DNA-regulatory elements (12). For example, c-Jun homodimers and c-Jun/c- Authors' Affiliations: 1Department of Biosciences and Nutrition, Novum, Fos heterodimers bind to the consensus TPA response ele- Karolinska Institutet, Huddinge, Sweden; 2Department of Epidemiology ment sequence TGAC/GTCA (13). and Biostatistics, School of Public Health, Jilin University, Changchun, In reporter assays, in the context of an isolated AP-1 ele- China; and 3Center for Nuclear Receptors and Cell Signaling, Department α β of Biology and Biochemistry, University of Houston, Houston, Texas ment, ER and ER were shown to signal in opposite ways when complexed with estrogen: with ERα, estrogen-activated Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). transcription, whereas with ERβ, estrogen-inhibited tran- Corresponding Author: Chunyan Zhao, Department of Biosciences and scription (7). However, it remains unclear whether AP-1 is Nutrition, Novum, Karolinska Institutet, S-141 57 Huddinge, Sweden. more generally recruited to promoter regions of ER target Phone: 46-8608-9273; Fax: 46-8774-5538; E-mail: [email protected]. genes, particularly in the context of intact chromatin and doi: 10.1158/0008-5472.CAN-09-4407 what is the potential molecular interplay between these sig- ©2010 American Association for Cancer Research. naling pathways at the level of chromatin. www.aacrjournals.org OF1 Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 2010 American Association for Cancer Research. Published OnlineFirst May 25, 2010; DOI: 10.1158/0008-5472.CAN-09-4407 Zhao et al. Recently, the combined technique of chromatin immuno- the University of California Santa Cruz genome browser. precipitation (ChIP) with genomic DNA microarrays (ChIP- Clover (21) was used to screen the sequences against a on-chip; ref. 14) or with DNA sequencing (ChIP-PET and precompiled library of motifs, JASPAR, to find the statisti- ChIP-Seq; refs. 15, 16) has been used for the identification cally overrepresented motifs as described in ref. (22). The of ERα-binding sites at the whole genome level. In this study, similarity of enriched motifs was analyzed using the web using ChIP-on-chip, we mapped ERβ-binding DNA regions tool STAMP at default settings (23). on a global scale and explored mechanisms of cross-talk between ERβ and AP-1. Quantitative real-time PCR Total RNA was extracted using the RNeasy kit (Qiagen). Materials and Methods Real-time PCR was performed as previously described (18). Generation of a stable MCF7 tet-off ERβ clone RNA interference The generation of a stable MCF7 tet-off ERβ clone has pre- Short interfering RNA (siRNA) targeting c-Fos (si_c-Fos) or viously been described by Papoutsi and colleagues (17). c-Jun (si_c-Jun) and nonspecific siRNA (si_Control) were pur- chased from Santa Cruz Biotechnology. siRNA transfections Western blot analysis were carried out using a final concentration of 50 nmol/L oligo Western blotting was carried out as previously described using the Interferin transfection reagent (Polyplus) according (18) using the following antibodies: anti-Flag (M5; Sigma), to the recommendations of the manufacturer. anti–c-Fos (H-125; Santa Cruz Biotechnology), and anti–c- Jun (sc-45; Santa Cruz Biotechnology). Luciferase assay ERβ-binding regions identified from ChIP-on-chip for an ChIP/re-immunoprecipitation association with the EREG,FOXA1,MYC,VAV3,CAPN2, ChIP analysis was performed as previously described (18). and LTBP1 genes were amplified from genomic DNA using The antibodies used were as follows: anti-ERβ rabbit poly- PCR amplification and cloned into the pGL3 promoter clonal antibody LBD (19), anti-ERα (HC-20; Santa Cruz Bio- luciferase vector (Promega) at MluIandXhoIsites.The technology), anti–c-Fos (H-125; Santa Cruz Biotechnology), primer pairs used to amplify these regions are shown in anti–c-Jun (sc-45; Santa Cruz Biotechnology), anti-Flag (M5; Supplementary Table S1. Hormone-depleted HeLa cells were Sigma), and normal rabbit IgG (Santa Cruz Biotechnology). transfected using LipofectAMINE 2000 (Invitrogen Life Re-ChIP was carried out as described (17). The immunopre- Technologies). Luciferase activity was measured using the cipitated DNA was amplified by real-time PCR using Plati- Dual-Luciferase Reporter Assay (Promega). To control for num SYBR green quantitative PCR supermix uracil DNA transfection efficiency, luciferase activity was normalized to glycosylase (Invitrogen). Renilla luciferase activity. Probe labeling and microarray hybridization Fluorescent-activated cell sorting ChIP DNA samples (three E2-treated ERβ ChIP samples and MCF7 tet-off ERβ cells were seeded in phenol red–free three input DNA samples) were amplified and labeled as de- DMEM supplemented with 5% charcoal dextran–stripped scribed (19). Six micrograms of labeled products were hybrid- fetal bovine serum for 24 hours and were transfected with ized to Affymetrix human tiling 2.0R arrays (Affymetrix). control siRNA or c-Fos siRNA for 48 hours; the cells were then stimulated with 10 nmol/L of E2 or vehicle for 72 hours. Affymetrix data analysis Cells were collected and stained with propidium iodide. To obtain high-confidence ERβ-binding sites, the scanned Staining was measured using flow cytometry by the fluores- output files were analyzed by two independent methods: cence intensity (FL-1, 530 nm) of 10,000 cells. Tiling Analysis Software version 1.1 (Affymetrix) using a threshold of P ≤ 0.0005, and Model-based Analysis of Results Tiling arrays (MAT; ref. 20) using a MAT score cutoff of 120. A total of 1,710 and 1,878 ERβ DNA-binding regions Global mapping of ERβ-binding regions in human were identified using either the Tiling Analysis Software breast cancer cells or the MAT analysis method. The predicted sites had a high A stable cell line, MCF7 ERβ, that expresses a tetracycline- degree of concordance, and a total of 1,457 ERβ-binding re- regulated version of ERβ, was used as a model system gions were identified by both analysis strategies. The same for mapping ERβ-binding regions on a genome-wide data set was also analyzed only by MAT using a MAT score scale. Figure 1A shows that high levels of ERβ protein cutoff of 50 to obtain a more extensive set of ERβ-binding were expressed in the absence of tetracycline, but not in 4,766 sites.