Review TRENDS in Endocrinology and Metabolism Vol.15 No.2 March 2004

Anatomy of the estrogen response element

Christian J. Gruber, Doris M. Gruber, Isabel M.L. Gruber, Fritz Wieser and Johannes C. Huber

Division of Gynecologic Endocrinology and Reproductive Medicine, Department of Obstetrics and Gynecology, University of Vienna Medical School, Wa¨ hringer Gu¨ rtel 18–20, A-1090, Vienna, Austria

Estrogens exert their regulatory potential on gene therefore yield different transcriptional effects at the same expression through different nuclear and non-nuclear site [4]. On activation of the ER by agonist ligand binding, mechanisms. A direct nuclear approach is the inter- conformational changes are induced and intracytoplasmic action of estrogen with specific target sequences of chaperones, such as heat-shock proteins 70 and 90, DNA, estrogen response elements (ERE) or units. EREs dissociate from the receptor molecule [5]. The receptor can be grouped into perfect and imperfect palindromic then interacts with DNA and the transcriptional response sequences with the imperfect sequences differing from is modulated by the recruitment of co-regulatory proteins the consensus sequence in one or more nucleotides and [6]. A site of attachment for nuclear co-activators within being less responsive to the activated estrogen–estro- the ligand-binding domain (LBD) of the ERa is formed by gen receptor (ER) complex. Differences in the ERE helix 12 when the receptor is occupied by an agonist ligand sequence and the ER subtype involved can substantially [7]. Anti-estrogens displace part of the receptor, which alter ER–ERE interaction. In addition, cross-talk between then occludes the site and blocks co-activator access [8]. ERs and other nuclear transcription factors profoundly ERs are members of a large family of nuclear receptors influences gene expression. Here, we focus on the that probably arose from a common ancestral receptor recent advances in the understanding of the structure molecule [9]. This hypothesis is supported by the similar of EREs and how ERs are recruited to these. Identifying modes of action of nuclear steroid receptors and a rather known target genes for estrogen action could help us conserved DNA-binding domain (DBD). The LBDs of to understand the potential risks and benefits of the nuclear steroid receptors differ markedly in structure, administration of this steroid to humans. even between the receptor subtypes ERa and ERb, reflecting different binding affinities for physiological Estrogens are now known to influence the expression of a ligands [10]. Glucocorticoid, mineralocorticoid, androgen wide range of genes by different mechanisms in the and progesterone receptors bind to derivatives of a reproductive tract and other areas [1]. A direct genomic common response element [11] (Figure 1) but the ERE is interaction occurs between the estrogen receptor (ER) different to this response element. However, when the ligand complex and specific sequences of DNA known as DBD of ERa is switched experimentally with the DBD of estrogen response elements (ERE). Alternatively, ERs can the glucocorticoid receptor, the chimeric receptor binds to be activated independently of a hormonal ligand [2]. However, the different anatomy of EREs, the two subtypes of ERs involved (ERa and ERb), the variety of interacting 0 Ð8Ð7Ð6Ð5Ð4Ð3Ð2 +2+3+4+5+6+7+8 nuclear co-regulatory proteins and the substantial cross- talk between nuclear transcription factors can yield 5′-C A G G T C A nnn T G A C C T G-3′ various responses to estrogen stimulation. By focusing ERE ′ ′ on the structure of natural EREs and on how ERs are 3 -G T C C A G T nnn A C T G G A C-5 recruited to these, a better understanding of the complex- ity of the action of this steroid when administered to humans can be achieved. ′ ′ 5 -G G T A C A nnn T G T T C T-3 GRE

ERa and ERb TRENDS in Endocrinology & Metabolism Activated ERs are transcription factors that bind in dimeric form to specific sequences of DNA in the Figure 1. Sequence of the ERE and GRE. (a) A consensus ERE has been derived regulatory region of target genes, the EREs. Generally, from several highly estrogen-responsive sequences from the African clawed frog Xenopus laevis genes encoding vitellogenin A1, A2, B1, B2 and the chicken apo- ERa and ERb can form both homo- and heterodimers VLDL II gene. It is a 13 bp perfect palindromic inverted repeat with a 3 bp spacing before attaching to DNA [3]. Both receptor subtypes have of variable bases (red). (b) The sequence of the consensus GRE [11]. As indicated, replacement of the adenine base at position þ4 by thymine results in the gener- different affinities for different response elements and can ation of a GRE. Positions þ2, þ3 and þ6 are conserved in both the ERE and GRE. Abbreviations: ERE, estrogen response element; GRE, glucocorticoid response Corresponding author: C.J. Gruber ([email protected]). element. www.sciencedirect.com 1043-2760/$ - see front matter q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.tem.2004.01.008 74 Review TRENDS in Endocrinology and Metabolism Vol.15 No.2 March 2004 the glucocorticoid response element (GRE) but is activated within the ERE are contacted with highest affinity by the by 17b-estradiol [12]. This indicates the high specificity of P-Box amino acids because different laboratory techniques the DBD of steroid receptors. give different results [23,24]. However, recent studies Highly conserved regions in the DBD of ERa and ERb indicate that both ERa and ERb contact the same are the two cysteine–cysteine zinc fingers which allow nucleotides in the consensus ERE [25]. It is therefore contact between the major groove of DNA and the sugar– assumed at this time that the two ER subtypes interact phosphate backbone. The resulting ER–ERE complex is with EREs in a similar mode. stabilized by ligand binding [13] and the high mobility As mentioned previously, ER conformation differs when group proteins 1 and 2 [14], which are architectural occupied by different ligands. Also, the structure of the proteins that facilitate chromatin function. ERE alters the conformation of the receptor. As seen in crystallographic studies, the ERa reacts to a specific single The ERE nucleotide alteration within the ERE by changing its DBD Highly estrogen-responsive and perfectly palindromic conformation by means of a side-chain rearrangement sequences have been found in the African clawed frog [26]. With these modifications, a rearrangment in the local Xenopus laevis genes encoding vitellogenin A1, A2, B1 and hydrogen bond network between DNA bases and receptor B2 [15]. From these natural EREs and similar sequences amino acids is achieved and alternative base contacts [16] a minimal consensus sequence for EREs has been enabled. derived (Figure 1). At position þ2, the ER attaches to Thus, ER conformation is dependent on two factors: the first thymine base of a half site by interacting with the 0 (i) the ligand and (ii) the specific ERE sequence. As 5 -methyl group. The thymine base at this position is also indicated by protease sensivity assays, which rely on the conserved in thyroid hormone and glucocorticoid/pro- ability of a protease to cleave the receptor protein at þ gesterone response elements (GRE/PRE). At position 3, accessible amino acids, the ERa obtained a different the guanine base is also conserved throughout all three conformation when complexed with the different EREs þ hormone reponse elements but the adenine at position 4 from the Xenopus laevis vitellogenin A2 and B1 genes and of the half site is crucial for discrimination between the the genes encoding human pS2 and human oxytocin [27]. three [17]. Replacement of the adenine base by thymine is This can yield a differential recruitment of co-regulatory incompatible with ER binding, resulting in the generation proteins to the ER–ERE complex and might therefore of a GRE. An ERE with a cytosine or guanine base at constitute a mechanism for modulation of gene transcrip- position þ3 is still functional although it is less tran- tion at different EREs. The recruitment of activation scriptionally active. In all EREs position þ5 is occupied by function 2 (AF-2) dependent cofactor to ERa and ERb, for a cytosine in at least one-half-palindrome. Position þ6is instance,isaffectedbyboth theligandandtheEREsequence occupied by a cytosine in both ERE and GRE [17]. [25]. When the ERs are liganded with 17b-estradiol (E ) However, in the human genome, most estrogen target 2 the recruitment is primarily dependent on the ERE genes do not contain an ERE palindrome in their promoter but have non-palindromic EREs through which estrogen sequence. By contrast, when the ERs are occupied by regulation is mediated [18]. Sequence requirements for anti-estrogens, the ERE sequence loses its influence on imperfect EREs have been determined in vitro [19].Ifan AF-2-dependent cofactor recruitment [25]. ERa homodimer attempts to bind to an ERE differing in a single base pair from the consensus sequence, binding is ER affinity and transcriptional activation from different abolished unless rescued by appropriate flanking. A EREs purine base immediately flanking the element on the 50 Few natural EREs have been examined with respect to their side at position 27 of each strand is required in an ERE exact ER affinity and strength of transcriptional activation. with one base mutation. Two, but not three mutations can These points are especially difficult to summarize because of be compensated for by appropriate 27 and 28 flanking. the multiple detection systems that have been used. Mutations in both halves of the element also abolished Generally, mutant variants of the consensus sequence receptor binding in this study [19]. In accordance, natural functionwithlesstranscriptionalpotencyandtheirabilityto EREs in vivo lose affinity for the ER with increasing bind the ER is significantly weaker [28]. For the few genes numbers of nucleotide changes from the consensus that have been examinedthat containnatural EREs, a direct sequence, particularly if these alterations affect both correlation between ER–ERE binding affinity and tran- halves of the ERE palindrome [20]. The flanking sequence scriptional activation has been observed for both ER immediately adjacent to the ERE was also shown in vivo to subtypes [18]. In a further study, it was confirmed that impact the transcriptional potency of the element. An both ERa and ERb bind to the same EREs but that ERa does enhancement of the transcriptional potency was particu- so with an approximately twofold higher affinity [29]. larly noticeable with a AT-rich flanking DNA [21]. When the Xenopus laevis vitellogenin A2 and B1 ERE were compared with the human pS2 and oxytocin ERE by The ER–ERE interaction means of transient co-transfection and chloramphenicol Three specific amino acids within the proximal box (P-Box) acetyltransferase assay in HeLa cells, the most potent of the first zinc finger of the ER bind to the ERE in a transcriptional activation (10.5-fold) was observed with sequence-specific manner [22]. The second zinc finger is vitellogenin A2, followed by a 9.5-fold induction with the involved in receptor molecule dimerization and ERE half- oxytocin ERE, a 2.7-fold induction with the pS2 ERE and site spacing recognition. It is unclear which nucleotides 1.6-fold induction with the vitellogenin B1 ERE [27]. www.sciencedirect.com Review TRENDS in Endocrinology and Metabolism Vol.15 No.2 March 2004 75

ERE versus estrogen response unit enhance the transcriptional activity of AP-1. In this path- Often more than one ERE-like sequence is seen in the way, the ER is part of the co-activator complex for Jun/Fos regulatory region of estrogen target-genes, which lead to without binding to the AP-1 response element [39].It the concept of the estrogen response unit (ERU) [30].An thereby confers estrogen responsiveness to genes encoding ERU comprises imperfect palindromic elements or even human ovalbumin, c-fos, collagenase, and insulin-like half elements, sometimes separated by hundreds of base growth factor [40]. Interestingly, estrogenic activity at AP-1 pairs. Estrogenic inducibility of gene transcription is then sites depends on both the ER subtype and the ligand, and can accomplished by transcriptional synergism of these separ- either yield induction or inhibition of transcription [41]. ate elements. Transcriptional synergism has been Similarily to AP-1, the binding of ERa and ERb to the reported for ER binding to natural ERUs, such as the nuclear transcription factor Sp-1 and the transcriptional Xenopus laevis vitellogenin B1 ERU [31], and also to effect of this interaction is cell type-, ligand- and promoter- synthetic ERUs [32]. The exact mechanism of transcrip- dependent. The ER–Sp-1 complex binds to a consensual tional synergism remains to be elucidated, but might GC-rich promoter sequence of a gene in which only the include cooperative recruitment of co-activators, direct Sp-1 protein attaches to DNA [42]. Physical interaction interaction between ER dimers, and allosteric modu- between ERs and AP-1 or Sp-1 proteins is mediated by the lations between the DNA–ER complexes. Sometimes, ER domains that harbour the transactivation function EREs overlap with other hormone response-elements, (AF) 1 and 2. Binding of Sp-1 to its recognition site confers creating elements that mediate multihormonal sensitivity estrogen responsiveness to the human creatine kinase B, [33]. Additionally, ERa was shown to bind to direct repeats c-myc, retinoic acid receptor a, heat shock protein 27, of ERE half-sites and activate transcription, presumably cathepsin D and uteroglobin genes [43]. with highest affinity when the direct repeats are separated Another pathway of estrogen action involves the by 15–20 nucleotides [34]. ER-related receptors (ERR), nuclear orphan receptors Because rules for sequence requirements of ERUs with significant homology to ERs, which do not bind await further clarification, putative estrogen responsive estrogen and have unknown physiological ligands. ERRs sequences must be verified experimentally to determine are known to bind to the steroidogenic factor 1 response their biological importance [35]. Multiple systems have been element (SFRE), an ERE half-site preceeded by a used to demonstrate the functionality of EREs or ERUs. thymine–cytosine–adenine trinucleotide [44], but they Band-shift assays and electrophoretic gel mobility-shift also bind to classic EREs, in which they exert a con- assays test the binding of the ER protein to a putative ERE stitutive transcriptional activity. In turn, ERa but not the sequence in vitro and the specificity of the reaction can be ERb homodimers can activate transcription through enhanced by ER-reactive antibodies. However, these assays binding to SFREs. This alternative mode of action has do not demonstrate whether a putative ERE is biologically been demonstrated for the murine osteopontin gene [45]. functional or not. Functionality can be verified in transient Similar cross-talk between ERs and ERRs has been transfection experiments by fusing the putative ERE observed in the transcriptional regulation of the human sequence with a reporter gene (chloramphenicol acetyl- breast cancer marker gene pS2, the promoter of which is transferase assay, luciferase assay) and measuring the not only activated by ERs but also by ERRs, particularly resulting enzyme activity. Subsequent deletion or mutagen- ERRa. Consistent with the well-characterized ERE, a esisofthetestedsequenceinthetransienttransfectionassay functional SFRE is present in the pS2 promoter at position further increases the specificity of the result. 2269 to 2260. Both the ERE and the SFRE are required However, multiple genes known to be influenced by the for full response to ER and ERR pathways [46]. action of the ER and its ligand lack classic EREs or ERUs. Here, we summarize examples of natural EREs and Here, we summarize alternative pathways of ER action. ERUs in human DNA. It has become evident in recent years that substantial cross-talk between the classic ERE- Alternative pathways of ER action mediated pathway and alternative pathways can occur. Some genes are regulated by an estrogenic pathway referred to as tethering, in which the ER does not neces- EREs differing by one to three nucleotides from the sarily bind to DNA but interacts with another DNA-bound consensus sequence transcription factor. Well-characterized examples of these Examples of EREs differing by one to three nucleotides include the transcription factors activating protein (AP)-1 from the consensus sequence are described below and and specificity protein 1 (Sp-1). No direct receptor binding, listed in Table 1. As indicated above, base mutations from but E2-dependent loading of the upstream stimulatory the consensus sequence decrease the binding affinity factors (USF)-1 and -2 to the promoter is required for between DNA and ERs. activation of the gene encoding cathepsin D, which is also (i) Oxytocin: An early characterized ERE differing in one known to harbour a functional ERE [36]. nucleotide is the human oxytocin ERE. Optimal function of The AP-1 transcription factor is the heterodimer from this sequence is independent of other regulatory elements in the Jun/Fos gene products that binds to the phorbol diester the promoter in transient transfection experiments [47]. (TPA) response element as well as to the AP-1 binding site (ii) pS2: Another example of this category is the gene of DNA. Activated ERs directly interact with the nuclear co- encoding human pS2, in which expression is controlled by activators CREB-binding protein (CBP) [37] and gluco- estrogens in human breast cancer cells via mediation of an corticoid receptor interacting protein (GRIP) [38] recruited imperfect ERE (2405 to 2394) [48]. In transfected HeLa by Jun/Fos, which completes the co-activator complex to cells a 4,5-fold increase in the estrogen induction of a www.sciencedirect.com 76 Review TRENDS in Endocrinology and Metabolism Vol.15 No.2 March 2004

Table 1. EREs differing from consensus ERE in 1, 2 or 3 nucleotides in humans

ERE consensus sequence: 50-GGTCAnnnTGACC-30 Peptide/protein Gene symbol No. of nucleotides differing from the consensus Sequence Refs Oxytocin OXT 1 GGTGA nnn TGACC [47] pS2 PS2 1 GGTCA nnn TGGCC [48] Angiotensinogen AGT 1 GGGCA nnn TGACC [49] Cathepsin D CTSD 2 GGCCG nnn TGACC [51] Glial fibrillary acidic protein GFAP 2 GGGTA nnn TGACC [53] Complement C3 C3 2 GGTGG nnn TGACC [54] Vascular endothelial growth factor VEGF 3 AATCA nnn TGACT [55] reporter gene was observed with the pS2–ERE [48]. was observed in cells co-transfected with ERb.Site- Additionally,a cooperative SFRE is required in the promoter directed mutagenesis of this sequence abolished induc- region for full response to ER and ERR pathways [46]. tion of gene expression by estradiol. (iii) Angiotensinogen: The imperfect palindromic sequence at positions 224 to 211 between the TATA box Half-palindromic elements that mediate estrogen and transcriptional initiation site of the gene encoding inducibility human angiotensinogen contains a cytosine/adenine poly- Although half-EREs only confer weak estrogen respon- morphism at 220. When adenine is present at 220, the siveness, genes are known for which transcription is sequence binds to the estrogen receptor and increases induced by two or more ERE half-sites. Examples are reporter expression [49]. described below and listed in Table 2. (iv) Cathepsin D: Estrogens induce the expression of (i) Corticotropin-releasing hormone (CRH): Four half the gene encoding cathepsin D, a lysosomal proteinase EREs sites occur in the promoter of human CRH gene [56] produced in excess in human breast cancer cell-lines [50]. that respond to estrogen administration. In co-transfected Within the proximal promoter sequence, three EREs have CV-1 cells by chloramphenicol acetyltransferase been found, among which one is functional (2270 to 2249) analysis, these five half-elements induced estrogen [51]. This sequence requires cooperation with other regu- responsiveness by ,10%–20% of the strength of the latory elements, a homologous sequence upstream, general consensus ERE [56]. regulatory sites downstream (e.g. Sp1) and a TATA box [36]. (ii) Platelet-activating factor receptor (PAFR): The gene (v) Glial fibrillary acidic protein (GFAP): The induction encoding the human PAFR has two different promoters, of transcription of the GFAP is consistent with the which yield two transcripts of the receptor DNA in a presence of a functionally imperfect ERE (2149 to tissue-specific manner [57]. In PAFR transcript 2, an ERU 2137) in the upstream region of the rat gene promoter occurs in the gene promoter, comprising two half ERE [52]. The identical sequence is present in the human GFAP sites separated by 153 bp (2257 to 293) [58].Ina promoter region (at 2150) and verified by site-directed chloramphenicol acetyltransferase assay of transfected mutagenesis experiments [53]. JR-St cells, estrogen induced transcriptional activity 2.5- (vi) Complement C3: The human complement C3 ERE fold [58]. is located at position 2226 to 2214. Another sequence in (iii) Prothymosin a: Prothymosin a gene expression is that promoter (þ33 to þ45) also exhibits similarity to the increased by estrogen. The 5 kb promoter region consensus ERE, but has been characterized as nonfunc- contains two half-palindromic EREs at position 2750 tional [54]. In transfected HeLa cells a fourfold activation and 21051 [59]. of promoter activity was observed [54]. (vii) Vascular endothelial growth factor (VEGF): A Combinations with half-palindromic elements mediating variant ERE (21560 to 21548), differing by three estrogen inducibility nucleotides from the consensus sequence, is located ERE half-sites can act in synergy with other regulatory 1.5 kb upstream from the transcriptional start site of the elements within the promoter region of a gene. Examples gene encoding human VEGF [55]. In cells co-trans- are described below and listed in Table 2. fected with ERa, estradiol induced 3.2-fold induction in (i) Lactoferrin: In the lactoferrin gene promoter, a VEGF-promoter luciferase activity. A 2.3-fold increase composite hormone-response element can be found that

Table 2. Half-palindromic elements and combinations that mediate estrogen inducibility

ERE consensus sequence: 50-GGTCAnnnTGACC-30 Peptide/protein Gene Symbol Sequence Refs (a) Half-palindromic elements Corticotropin releasing hormone CRH Four half-palindromes [56] Platelet activating receptor transcript 2 PAFR2 Two half-palindromes [58] Prothymosin a PTMA Two half-palindromes [59] (b) Combinations with half-palindromic elements Lactoferrin HLF AGGTCA n AGGTCA [33] Heat shock protein 27 HSP27 GGGCGGG (Sp1) n(10) GGTCA [60] www.sciencedirect.com Review TRENDS in Endocrinology and Metabolism Vol.15 No.2 March 2004 77

Table 3. Other variations involving ERE-like sequences

ERE consensus sequence: 50-GGTCAnnnTGACC-30 Protein/peptide Gene symbol Sequence Refs c-fos CFOS CGGCA gcg TGACC overlapping with two sequences [61] homologous to the core sequence of AP-1 transcription binding sites Transforming growth factor a TGFA GGTCA nnnn TGCCC n(20) GGTGA nnn TAGCC [62]

Retinoic acid receptor a1 RXRA GGTGA and GGCGGG [63] Progesterone receptor PGR TGACC nnnn CCGCCC nnnn CCGCCC [64] comprises a retinoic acid response element that overlaps estrogenregulationoccurotherthanthosecurrentlyknown? with an ERE (2349 to 2337). The two AGGTCA motifs Second, more insight into the nuclear environments and the can be activated by the retinoic acid X receptor and one mechanisms leading to estrogen-induced gene stimulation AGGTCA half also responds to estrogen application [33]. or restriction is needed. From these points, a better under- (ii) Heat shock protein (HSP) 27: In the promoter of the standing of the tissue-selective effects of estrogenic com- gene encoding HSP 27, an ERE half-element (288) is pounds can be achieved and the knowledge more easily present, which acts synergistically with a GGGCGGG Sp1 transferred to clinical settings. Because estrogens have site 10 bp away [60]. direct access to the human genome, administration of this steroid in humans should therefore occur with care. Under- Other variations involving ERE- like sequences standing the molecular actions of estrogens and the variety Often ERE-related sequences display synergistic action of genes affected might help us to understand the potential with or overlap with other regulatory elements within the risks and benefits of hormone replacement therapy and of promoter region of a gene. Examples are described below the oral contraceptive pill. and listed in Table 3. (i) c-fos: Functional EREs have been identified in the References c-fos 50-flanking DNA, comprising one imperfect palin- 1 Gruber, C.J. et al. (2002) Production and action of estrogens. N. Engl. drome (21211 to 21199) overlapping with two sequences J. Med. 346, 340–352 2 El-Tanani, M.K. and Green, C.D. 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