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 –8–7–6–5–4–3–2 +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].