We used ChIP-Seq to map ERa-binding sites and to profile changes in RNA polymerase II (RNAPII) occupancy in MCF-7 cells in response to estradiol (E2), tamoxifen or fulvestrant. We identify 10 205 high confidence ERa-binding sites in response to E2 of which 68% contain an estrogen response element (ERE) and only 7% contain a FOXA1 motif. Remarkably, 596 genes change significantly in RNAPII occupancy (59% up and 41% down) already after 1 h of E2 exposure. Although promoter proximal enrichment of RNAPII (PPEP) occurs frequently in MCF-7 cells (17%), it is only observed on a minority of E2-regulated genes (4%). Tamoxifen and fulvestrant partially reduce ERa DNA binding and prevent RNAPII loading on the promoter and coding body on E2-upregulated genes. Both ligands act differently on E2-downregulated genes: tamoxifen acts as an agonist thus downregulating these genes, whereas fulvestrant antagonizes E2-induced repression and often increases RNAPII occupancy. Furthermore, our data identify genes preferentially regulated by tamoxifen but not by E2 or fulvestrant. Thus (partial) antagonist loaded ERa acts mechanistically different on E2-activated and E2-repressed genes. IntroductionEstradiol (E2) is a key regulator in the growth and differentiation of many target tissues and is involved in the development and progression of breast cancer (Anderson, 2002). Its genomic activity is to a large extent mediated by the estrogen receptor a (ERa; NR3A1), a member of the nuclear receptor super family. ERa regulates expression of target genes classically by binding directly to its cognate sequence, the estrogen response element (ERE). ERa binds to its cognate-binding sites as homodimer, recruits co-factors and activates or represses transcription in response to E2 (Shang et al, 2000). Alternatively, nonclassical regulation involves protein-protein interactions with other DNA-binding proteins such as Sp1, AP-1 and NF-kB. Identification of the ERa target gene network regulated by agonist and/or antagonist treatment is essential to understand the role of ERa in normal physiological processes and in cancer.Several gene expression profiling studies in MCF-7 cells identified E2-responsive genes in the range of 100-1500 (Charpentier et al, 2000;Coser et al, 2003;Frasor et al, 2003;Rae et al, 2005;Carroll et al, 2006;Kininis et al, 2007;Kwon et al, 2007;Lin et al, 2007;Stender et al, 2007), whereas large scale ERa ChIP profiling showed that ERa interacts with many thousands genomic regions (Carroll et al, 2006;Kininis et al, 2007;Lin et al, 2007). This discordance is in part due to the fact that mRNA levels do not necessarily reflect gene activity because it is subject to degradation and regulation, and that likely not all ERa-binding sites are active under all conditions. Genome-wide profiling of RNA polymerase II (RNAPII) occupancy, however, does provide a much more direct readout and, thus, could yield insights beyond what is typically obtained by mRNA expression profiling.Recent studies have shown that the promoters of a large number...

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 genomewide 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.

The estrogen receptor (ER) is a ligand inducible transcription factor that regulates a large number of target genes. These targets are particularly relevant in breast cancer, where the sensitivity of the tumor to estrogens determines whether the patients can be treated with endocrine therapy such as tamoxifen. Identifying genomic ER targets is a daunting task. Quantifying expression levels of suspected target genes after estradiol stimulation or, more recently, using expression microarrays to this effect will reveal which genes are regulated by estradiol, however, without discriminating between direct and indirect tar- Published by Elsevier B.V. All rights reserved. IntroductionIn 1896 Beatson observed that removing the ovaries could lead to remission of breast cancer (Beatson, 1896). Although at that time hormones were not yet discovered, his experiments were the first to connect estrogens with breast cancer. More than 60 years later it was demonstrated that estrogens were retained in target tissues (Jensen and Jacobson, 1962), laying the foundation for the subsequent identification of steroid receptors. Indeed, in 1968 O'Malley described that changes in gene expression occurred after estrogen stimulation, indicating estrogen receptor (ER) functions as a transcription factor (O'Malley et al., 1968). Soon after, a protein that specifically bound estrogens was found in breast tumors, and its quantity could predict the response of these tumors to endocrine disruption (Jensen et al., 1971;McGuire, 1973). With the cloning of the ER in 1986 (Green et al., 1986;Greene et al., 1986) and subsequent identification of its functional domains the role for the ER as a ligand dependent transcription factor became apparent (Green et al., 1986;Greene et al., 1986;Kumar et al., 1987). The ER is a member of the superfamily of nuclear receptors, which are structurally related ligand-inducible transcription factors, including steroid receptors (SRs), thyroid/ retinoids receptors (TR, RARs and RXRs), vitamin D receptors (VDR), LXR, PPARs, and orphan receptors for which no ligand