Estrogen response element sequence impacts the conformation and transcriptional activity of estrogen receptor α1
Introduction
Estrogen receptor (ER) is a member of the steroid/thyroid superfamily of proteins that act as hormone-inducible transcription factors (Mangelsdorf et al., 1995). The products of two ER genes, i.e. ERα and ERβ, mediate the actions of estrogens in target tissues, thus regulating estrogenic effects on reproduction, bone homeostasis, and mammary gland structure and function (Katzenellenbogen and Korach, 1997). Estradiol (E2) and other ligands bind in the ligand-binding domain (LBD) of ERα and induce conformational changes leading to dimerization and high-affinity ERα binding to estrogen response elements (EREs) (Klinge, 2000). Once bound to an ERE, interaction between specific motifs within E2-ERα and coactivator proteins, e.g. SRC-1 (Onate et al., 1995), or the TATA binding complex, e.g. TFIIB (Ing et al., 1992) and TATA-box binding protein (TBP) (Sadovsky et al., 1995), result in increased gene transcription (reviewed in McKenna et al., 1999, Klinge, 2000).
Occupation of ERα by selective estrogen receptor modulators (SERMs), e.g. tamoxifen (TAM), 4-hydroxy-TAM (4-OHT), or raloxifene (RAL), changes the interaction of ERα with coactivators. Whereas E2-occupied ERα interacts with specific coactivators, e.g. SRC-1, occupation of ERα by TAM or 4-OHT prevents interaction with these proteins (reviewed in McKenna et al., 1999, Klinge, 2000). Although antagonist-liganded ERα interacts with corepressors NCoR and SMRT (Smith et al., 1997, Zhang et al., 1998), there is no evidence that these corepressors are required for the antagonist activity of TAM. TAM-occupied ERα also interacts with the ER-selective coregulator REA that potentiates the antagonist activity of TAM and suppresses E2-induced transcriptional activity of ERα and ERβ (Montano et al., 1999). Similarly, the nuclear matrix protein HET/SEF-B shows a preferential interaction with TAM-occupied ERα and inhibits both E2- and TAM- stimulated transcription in HepG2 cells in which TAM is an agonist (Oesterreich et al., 2000). Although crystal structure studies show a different conformation of the LBD occupied by 4-OHT versus E2 or the synthetic estrogen diethylstilbestrol (DES) (Brzozowski et al., 1997, Shiau et al., 1998), specific details of the mechanisms of antagonist activity of 4-OHT remain to be elucidated.
In addition to ligand effects, Lefstin and Yamamoto proposed that response elements recognized by nuclear transcription factors, including members of the steroid/nuclear receptor superfamily, contain information that is interpreted by bound regulator factors (Lefstin and Yamamoto, 1998). Based on Lefstin and Yamamoto's model, we postulate that DNA acts as an allosteric ligand whose binding alters ERs' affinity for other ligands, such as coactivators or corepressors, and predict that the DBD of the ERα harbors activities in addition to DNA recognition. Indeed, the ERα DBD constitutes surfaces for both intramolecular and intermolecular protein–protein contacts (Chen et al., 1999). Thus, ERα-ERE binding should produce changes in ERα conformation that alter ERα interaction with other proteins.
To assess alterations in ERα conformation in the presence of various ERE sequences, we analyzed the sensitivity of ERα to α-chymotrypsin digestion. An earlier study showed that chymotrypsin or trypsin digestion of MCF-7 cell extracts resulted in different sizes of ERα fragments in cells treated with E2 versus tamoxifen (TAM) (Hutchens et al., 1987). This result was an early indication that the conformation of E2-occupied ERα is different from TAM-occupied ERα, a finding confirmed by X-ray crystal structure studies of purified ERα LBD occupied by 4-OHT versus E2 or DES (Brzozowski et al., 1997, Shiau et al., 1998). A more recent report showed that specific amino acid (aa) mutations that constitutively activate ERα result in a proteolytic digestion pattern similar to that of the E2-liganded wild-type (wt) ERα (Lazennec et al., 1997). These reports indicate that alterations in ERα conformation correlate with ERα agonist activity, whether induced by E2 binding or by alterations in aa sequence. Because ER binding to an ERE activates gene expression from that element, it is important to investigate if there is a similar correlation between ERE-driven transcription and altered ER conformation.
Two reports conflict on the effect of DNA binding on ERα conformation as measured by sensitivity of ERα to trypsin digestion (Fritsch et al., 1992, Wood et al., 1998). One report showed that rat uterine ERα conformation is unaffected by DNA binding (Fritsch et al., 1992). The other report showed that yeast-expressed, recombinant human (rh) ERα is more sensitive to trypsin or chymotrypsin digestion when bound to the Xenopus vitellogenin A2 ERE than when ERα was incubated with the ERE from the human pS2 gene, containing a single nucleotide change in the 3’ half-site (Wood et al., 1998). The latter is important because, although many investigations of ERα activity utilize the perfectly palindromic Xenopus vitellogenin A2 ERE, the promoters of most natural estrogen responsive genes contain one or more imperfect EREs and/or ERE half-sites (Anolik et al., 1995). No one has examined how the ligand affects ERα conformation in the presence of perfect versus imperfect EREs.
In the present study, we assessed changes in the conformation of ERα in the presence or absence of a consensus ERE, naturally occurring non-palindromic EREs, or indirect and direct repeats (IR or DR, respectively) of the ERE half-site. We report that ERE binding enhances the sensitivity of ERα to proteolysis by chymotrypsin. In contrast, incubation of ERα with DNA sequences that do not bind ERα does not alter ERα sensitivity to proteolysis. We detected differences in the sensitivity of ERα to chymotrypsin digestion in the presence of different EREs and some changes in the sizes of proteolytic fragments with E2 or 4-OHT. Although the affinity of ERα for the different EREs does not correlate with transcriptional activity in transiently transfected cells, the sensitivity to chymotrypsin digestion correlates with reporter gene expression. These results are consistent with a role for the sequence of the ERE as an allosteric regulator of ERα activity.
Section snippets
Preparation of ERE-containing OLIGOMERs
The sequences of EREs used are included in Table 1. The EREs, EREc38, PR-1148, pS2, and Fos-1211, were cloned into pGEM-7Zf(+) or pGL3-promoter (pro) luciferase reporter vector (Promega, Madison, WI) as described (Klinge et al., 1997a, Klinge et al., 1997b, Klinge et al., 1997c). ERE oligomers were prepared by restriction digestion of the pGL3-pro-luciferase plasmid DNA with HinfI and XhoI or by digestion with EcoRI and BamH1 from pGEM-7Zf(+) and electroelution (Klinge et al., 1996). The
ERE binding increases ERα susceptibility to chymotrypsin digestion
To investigate whether ERE binding induces conformational changes in ERα, rh ERα, occupied by either E2 or 4-OHT, was incubated with limited concentrations of chymotrypsin. Fig. 1 shows four representative ERα chymotrypsin digestion experiments. Western blot analysis was performed with Ab10, a C-terminal specific monoclonal antibody to ERα. Without chymotrypsin treatment, a prominent band was detected at approximately 66 kDa (Fig. 1A–D). This corresponds in size to intact ERα. Identical results
Discussion
ERα and ERβ are members of the nuclear receptor superfamily that mediate the complex signaling processes by which estrogens regulate physiological functions, including mammary gland differentiation, reproduction, cholesterol metabolism, and bone homeostasis. Binding of E2 within the hydrophobic core of the LBD activates the receptor and facilitates high-affinity ER binding to EREs. Although most natural EREs are not perfect palindromes, the majority of transcriptional activation studies have
Acknowledgements
We thank Rosemary L. Sims and Kelly E. Risinger, for their assistance in some of the experiments reported here, and Timothy L. Ramsey for assistance in calculating the MW of chymotryptic ERα fragments. We thank Dr Barbara J. Clark for her suggestions on this manuscript.
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Supported by NIH R01 DK 53220 and a University of Louisville School of Medicine Research Grant to C.M.K.