Endocrine-Related Cancer (2009) 16 1073–1089 REVIEW Genomic actions of estrogen receptor a: what are the targets and how are they regulated? Willem-Jan Welboren, Fred C G J Sweep1, Paul N Span1,2 and Hendrik G Stunnenberg Department of Molecular Biology, Faculty of Science, Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen, PO Box 9101, HB Nijmegen, The Netherlands Departments of 1Chemical Endocrinology and 2Radiation Oncology, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands (Correspondence should be addressed to H G Stunnenberg; Email: [email protected]) Abstract The estrogen receptor a (ERa) is a ligand-dependent transcription factor that regulates a large number of genes in many different target tissues and is important in the development and progression of breast cancer. ERa-mediated transcription is a complex process regulated at many different levels. The interplay between ligand, receptor, DNA sequence, cofactors, chromatin context, and post-translational modifications culminates in transcriptional regulation by ERa. Recent technological advances have allowed the identification of ERa target genes on a genome- wide scale. In this review, we provide an overview of the progress made in our understanding of the different levels of regulation mediated by ERa. We discuss the recent advances in the identification of the ERa-binding sites and target gene network and their clinical applications. Endocrine-Related Cancer (2009) 16 1073–1089 Introduction thereby making the link between cancer and estrogens that was described almost a century before (Jensen Estrogens regulate many cellular processes in a wide et al. 1971, McGuire 1973). The subsequent cloning of variety of target tissues during growth, development, the ERa gene and the identification of specific domains and differentiation. Estrogens are mainly involved in demonstrated that ERa functions as a ligand-dependent the regulation and development of the female transcription factor (Green et al.1986, Greene et al. reproductive tract but also play a role in the central 1986, Kumar et al. 1987). The structures of ERa and its nervous system, cardiovascular systems, and in bone nongenomic regulation have been reviewed exten- metabolism (Katzenellenbogen 1996). In addition to sively (Ruff et al. 2000, Warner & Gustafsson 2006). their role in physiology, estrogens are also associated This review will focus on the regulation of ERa- with the development and progression of breast cancer mediated transcription and on the advances made in the (Anderson 2002). In 1896, Beatson discovered that elucidation of its target gene network. Furthermore, the removal of the ovaries resulted in breast cancer clinical significance and implications of ERa remission, connecting for the first time hormones expression and genome-wide chromatin immuno- with breast cancer, decades prior to the discovery of precipitation (ChIP) profiling is discussed. estrogens or estrogen receptors (ERs). Seventy years later, O’Malley observed changes in hybridizable RNA upon estrogen stimulation of the chick oviduct, Estrogen receptor indicating that estrogens regulate transcription The ERa (NR3A1) is a member of the super family of (O’Malley et al. 1968). Again several years later a nuclear receptors, which are ligand-dependent tran- specific estrogen-binding protein was discovered that scription factors. In addition to ERa, the family was present in breast tumors and its expression level includes other steroid hormone receptors such as the could predict the response to endocrine disruption, androgen receptor (AR, NR3C4), the glucocorticoid Endocrine-Related Cancer (2009) 16 1073–1089 Downloaded from Bioscientifica.comDOI: 10.1677/ERC-09-0086 at 09/27/2021 07:17:52AM 1351–0088/09/016–001073 q 2009 Society for Endocrinology Printed in Great Britain Online version via http://www.endocrinology-journals.orgvia free access W-J Welboren et al.: Genomic actions of estrogen receptor a receptor (GR, NR3C1), and other nuclear receptors to kB elements and regulates the expression of target such as the retinoic acid receptor (RAR, NR1B), genes. ERa has been shown to inhibit NF-kBinan retinoid X receptor (RXR, NR2B), and peroxisome E2-dependent manner. The ERa can directly inhibit the proliferators-activated receptor (PPAR, NR1C). Estro- binding of NF-kB transcription factors to the DNA, but gens are small lipophilic molecules that traverse the the mechanism is poorly understood. cell membrane and bind to cytoplasmic ERa associated with chaperone proteins such as hsp90. Upon binding a cascade of events occur; the receptor dissociates from Target gene network the chaperone proteins, dimerizes and associates with Single gene analysis chromatin. ERa can either bind directly to DNA To fully elucidate ERa function, the identification of (classical pathway) or indirectly via protein–protein its target gene network is essential. Many strategies interactions (nonclassical pathway). In the classical have been employed to gain insights into the target pathway, ERa homodimers bind to a specific DNA gene network governed by the ERa. Classically, target sequence motif, the estrogen response element (ERE). genes have been identified using ‘single gene’ The ERE is a 15 bp palindrome consisting of two experiments. The egg-white proteins in the chicken PuGGTCA half sites separated by a 3 bp spacer. ERa is oviduct and the Xenopus laevis vitellogenin gene are also able to bind to imperfect EREs. Recent genome- among the first ERa target genes to be identified wide studies show that the ERE is the most (Hayward et al. 1982, Lai et al. 1983, Chambon et al. predominant motif in ERa-binding sites (Carroll 1984, Jost et al.1984). Later, by comparing cDNA et al. 2006, Lin et al. 2007, Welboren et al. 2009). libraries of nontreated and E -treated MCF-7 human In the nonclassical pathway, the ERa binds indirectly 2 breast cancer cells several other ERa-responsive genes to the DNA via tethering to other transcription factors were identified such as the classical and intensively such as specificity protein 1 (Sp1), activating protein 1 studied ERa target gene pS2/TFF1 (Brown et al. 1984, (AP-1), or nuclear factor kappa b (NF-kB), and Jakowlew et al. 1984). ERa-regulated genes identified regulates transcription in an ERE-independent manner. using differential cloning are e.g. the cell cycle The Sp1 family of transcriptional factors plays an regulators c-myc and cyclin D1, connecting prolifera- important role in proliferation, differentiation, survival, tion with E signaling (Dubik et al. 1987, Altucci and angiogenesis (Kaczynski et al. 2003, Safe & 2 et al. 1996). Abdelrahim 2005). Sp1 can bind to GC-rich regions, which are present in many estradiol (E2) responsive promoters. For example, mutational analysis revealed Estrogen response elements that the GC-rich region in the promoter of the The first ERE was identified in 1986 in the promoter of transforming growth factor a (TGFa) gene is required the Xenopus vitellogenin gene. Transfection experi- for E2-mediated gene activation (Vyhlidal et al. 2000). ments showed that this element also functions in Several other genes have been identified that are human cells, and that the core ERE could be defined activated by the ER/Sp1 pathway including e.g. the as a 13 bp palindrome (GGTCANNNTGACC; c-myc and progesterone receptor genes (Miller et al. Klein-Hitpass et al. 1986). The availability of the 1996, Petz & Nardulli 2000). The transcription factor entire human genome sequence allowed the compu- AP-1 is a complex containing fos, jun, and other family tational search for EREs and ERa target genes. This members. Several E2-regulated genes are dependent on approach led to the interesting observation that AP-1, such as insulin-like growth factor I (IGF-I), w70 000 EREs are present in the human genome, i.e. ovalbumin, progesterone receptor, and pS2/TFF1 one in every 43 kb of DNA (Bourdeau et al. 2004). By (Gaub et al. 1990, Savouret et al. 1994, Umayahara comparing these with the merely 9944 EREs identified et al. 1994, Barkhem et al.2002). The AP-1 complex in the mouse genome, a total of 660 evolutionary binds to promoters of genes involved in growth, conserved elements were found. The large number of differentiation, and development. Many ER/AP-1- EREs present in the genome and limited conservation responsive genes have been identified using microarray between human and mouse indicates that a large studies, indicating that E2-mediated regulation via the fraction of the EREs may not be ‘true’ ERa-binding nonclassical pathway occurs frequently (DeNardo sites. These in silico studies suggested that actual et al. 2005, Glidewell-Kenney et al.2005). The binding by ERa is dependent on more factors than just NF-kB family of transcriptional factors are involved the DNA sequence. The hypothesis was put forward in the immune and skeletal systems and inflammatory that the chromatin structure and hence the accessibility response (Kalaitzidis & Gilmore 2005). NF-kB binds of binding sites plays a major role. Furthermore, the Downloaded from Bioscientifica.com at 09/27/2021 07:17:52AM via free access 1074 www.endocrinology-journals.org Endocrine-Related Cancer (2009) 16 1073–1089 notion that sequence is not the only or decisive in karyotype, hormone receptor content, and growth determinant in ERa binding is underscored by the rate have been observed although morphologically the observation that ERa can bind indirectly via tethering cells look(ed) identical (Osborne et al. 1987, Bahia to other transcription factors and subsequently regulate et al. 2002, Burdall et al. 2003). target genes in an ERE-independent manner. It is important to note that a disadvantage of expression profiling is that indirect effects that affect the mRNA level are also measured which could be a Expression profiling major source of the observed differences. Lin et al. Microarrays, either cDNA or oligonucleotide, and used the translation inhibitor cyclohexamide combined serial analysis of gene expression are powerful tools with the ERa antagonist ICI 182,780 to show that only that can be used to assess global changes in gene a relatively small number (23%) of E2-responsive expression.