Molecular Systems Biology 4; Article number 188; doi:10.1038/msb.2008.25 Citation: Molecular Systems Biology 4:188 & 2008 EMBO and Nature Publishing Group All rights reserved 1744-4292/08 www.molecularsystemsbiology.com Genomic analysis of estrogen cascade reveals histone variant H2A.Z associated with breast cancer progression Sujun Hua1,2,3,9, Caleb B Kallen4,9, Ruby Dhar1,2, Maria T Baquero5, Christopher E Mason6, Beth A Russell1,2,6, Parantu K Shah1,2, Jiang Liu1,2, Andrey Khramtsov7, Maria S Tretiakova8, Thomas N Krausz8, Olufunmilayo I Olopade7, David L Rimm5 and Kevin P White1,2,* 1 Joint Institute for Genomics and Systems Biology, The University of Chicago and Argonne National Laboratory, Chicago, IL, USA, 2 Department of Human Genetics, The University of Chicago, Chicago, IL, USA, 3 Interdepartmental Program in Computational Biology and Bioinformatics, Yale University, New Haven, CT, USA, 4 Department of Gynecology and Obstetrics, Emory University, Atlanta, GA, USA, 5 Department of Pathology, Yale University, New Haven, CT, USA, 6 Department of Genetics, Yale University, New Haven, CT, USA, 7 Center for Clinical Cancer Genetics, University of Chicago Medical Center, Chicago, IL, USA and 8 Department of Pathology, The University of Chicago Hospitals, Chicago, IL, USA 9 These authors contributed equally to this work * Corresponding author. Department of Human Genetics, The University of Chicago, 1639 Pierce Drive, WMB 4211, Chicago, IL 60637, USA. Tel.: þ 1 773 834 8259; Fax: þ 1 773 834 0505; E-mail: [email protected] Received 7.9.07; accepted 13.3.08 We demonstrate an integrated approach to the study of a transcriptional regulatory cascade involved in the progression of breast cancer and we identify a protein associated with disease progression. Using chromatin immunoprecipitation and genome tiling arrays, whole genome mapping of transcription factor-binding sites was combined with gene expression profiling to identify genes involved in the proliferative response to estrogen (E2). Using RNA interference, selected ERa and c-MYC gene targets were knocked down to identify mediators of E2-stimulated cell proliferation. Tissue microarray screening revealed that high expression of an epigenetic factor, the E2-inducible histone variant H2A.Z, is significantly associated with lymph node metastasis and decreased breast cancer survival. Detection of H2A.Z levels independently increased the prognostic power of biomarkers currently in clinical use. This integrated approach has accelerated the identification of a molecule linked to breast cancer progression, has implications for diagnostic and therapeutic interventions, and can be applied to a wide range of cancers. Molecular Systems Biology 15 April 2008; doi:10.1038/msb.2008.25 Subject Categories: functional genomics; chromatin and transcription Keywords: breast; cancer; chromatin; estrogen; histone variant This is an open-access article distributed under the terms of the Creative Commons Attribution Licence, which permits distribution and reproduction in any medium, provided the original author and source are credited. This licence does not permit commercial exploitation or the creation of derivative works without specific permission. Introduction indication that, while capable of predicting patient survival, the lists are only weakly informative with regard to mechan- Cancer is caused by the accumulation of genetic mutations isms of oncogenesis. Many genes with statistical significance combined with changes in the epigenetic state of affected cells. in a gene expression signature will not serve as viable These changes result in the initiation and progression of candidates for the development of prognostic assays or tumors, which can be described, in part, by the gene anticancer treatments (Ein-Dor et al, 2005; Fan et al, 2006). expression profiles of the cancer cells in question, a unique Use of genomic screening techniques to explore cancer cell gene expression ‘signature,’ reflecting the activation or physiology must therefore be followed by hypothesis testing to suppression of biological pathways important in generating validate the genes that are important for accurate prognosis the cancer phenotype (Perou et al, 1999; Sorlie et al, 2001; van and to determine which are involved in the initiation and/or de Vijver et al, 2002; Sotiriou et al, 2003). Importantly, gene progression of disease. expression profiling of tumors produces gene lists that do not Estrogens (E2) are steroid hormones that play critical roles necessarily contribute to a measured clinical distinction, such in the initiation, development, and metastasis of breast and as the presence or absence of distant metastases. In fact, the uterine cancers (Yager and Davidson, 2006). The E2 response lack of overlap in gene lists from different studies is an in breast cancer cells is predominantly mediated by the & 2008 EMBO and Nature Publishing Group Molecular Systems Biology 2008 1 Genomic analysis of estrogen response in cancer S Hua et al estrogen receptor-a (ERa), a ligand-activated transcription ERα ChIP sites factor. ERa regulates transcription of target genes through 1% direct binding to its cognate recognition sites, known as 24% estrogen response elements (EREs), or by modulating the 21% activity of other DNA-bound transcription factors at alternative DNA sequences (Bjornstrom and Sjoberg, 2005). The proto- 56% oncogene c-myc is upregulated by ERa in response to E2 25% (Dubik and Shiu, 1992) and encodes a transcription factor, 5Ј UTR 10% c-MYC (henceforth MYC), which regulates a cascade of gene 16% CDS 1st intron targets whose products mediate cellular transformation 2%0.4% (Ponzielli et al, 2005). Synergy between MYC and ERa at a other introns 3Ј UTR subset of E2-responsive promoters has been reported, suggest- MYC ChIP sites intergenic regions ing a cooperative role between ERa and MYC in breast cancer 2% 24% 0–10 kb progression (Cheng et al, 2006). 12% 10–50 kb Endocrine therapy, often effective for ERa-positive breast >50 kb tumors, impairs the hormone–receptor complex or inhibits E2 production. Unfortunately, a significant fraction (B20–50%) 37% of ERa-positive breast tumors fails to respond (EBCTC, 1998), 14% 11% or eventually develops resistance, to antiestrogen treatments 28% (Schiff et al, 2005). The lack of integrated and comprehensive approaches to study pathways regulated by E2 has delayed our 5% 4% understanding of the progression to hormone resistance in Figure 1 Genomic distribution of ERa- and MYC-binding sites. The cancer. distributions of 1615 ERa-(A) and 311 MYC- (B) binding sites in E2-stimulated Using an E2-dependent breast cancer cell model, MCF7 MCF7 cells relative to known genes. Where multiple genomic probes indicated a cells, we employed an integrated genomic approach that transcription factor-bound region, the center of each ERa- or MYC-binding region was designated as the bound position. Within annotated genes, binding sites combined E2-stimulated gene expression profiling and gen- 0 0 ome-wide transcription factor-binding site detection for two were classified as follows: within 5 untranslated regions (5 UTR), within coding sequences (CDS), within 50-most intron or the first intron, within other introns, and essential transducers of the E2 response, ERa and MYC. Recent within 30 untranslated regions (30 UTR). ERa or MYC binding in intergenic reports have similarly identified ERa-binding sites and E2- regions was further classified based on the distance to the nearest annotated activated gene expression in this cell model (Frasor et al, 2003; gene (0–10, 10–50, and 450 kb). Carroll et al, 2005, 2006; Laganiere et al, 2005). However, integration of genomic screening data must be followed by functional and clinical studies to develop useful tools for cells were stimulated to proliferate with E2 before chromatin diagnosing and treating cancer. Therefore, validation of direct immunoprecipitation (ChIP). transcription factor-regulated gene targets, using gene-specific We identified a total of 1615 ERa-bound regions (Po1eÀ5) knockdown in cell proliferation assays, was followed by throughout the human genome (Supplementary Table 1). The clinical correlation using primary cancer specimens with distribution of ERa-binding regions ranged from proximal associated data regarding patient outcomes. We demonstrate (o1 kb) to the nearest transcription start site (TSS) of a gene to that hormone-stimulated MYC enhances production of an over 500 kb from the closest TSS (Figure 1A). Similarly, we epigenetic factor, histone variant H2A.Z, the expression of detected 311 MYC-bound regions across the human genome in which correlates with increased probability of metastasis and MCF7 cells (Supplementary Table 2). The distribution of MYC- decreased patient survival. This integrated systems approach bound loci in relation to annotated TSSs is depicted in is potentially applicable to the study of other oncogenic Figure 1B. A total of 62.4% of ERa-bound regions detected in pathways. our study were confirmed in an independently derived data set using similar methods (Carroll et al, 2006) (common sites are indicated in Supplementary Table 1). Results We examined ERa- and MYC-binding regions for the presence of predicted transcription factor-binding motifs and Genome-wide identification of ERa- and detected statistically significant enrichment of canonical EREs MYC-binding sites in MCF7 cells (AGGTCAnnnTGACCT) in ERa-bound regions and E-boxes Recent studies have performed location analysis for ERa in (CACGTG) in MYC-bound