Selectivity of antibodies to estrogen receptors α and β (ERα and ERβ) for detecting DNA-bound ERα and ERβ in vitro

doi:10.1016/S0039-128X(00)00109-4

Steroids

Volume 65, Issue 9, September 2000, Pages 505-512

https://doi.org/10.1016/S0039-128X(00)00109-4 Get rights and content

Abstract

Antibodies are widely used to detect estrogen receptor (ER) in ER-DNA complexes in electrophoretic mobility shift assays (EMSA). We compared the specificity of antibodies raised to different regions of ERα or ERβ for detecting recombinant human ERα (rhERα) and recombinant rat ERβ (rrERβ) when bound to a consensus estrogen response element (ERE). ERα–specific antibodies specifically slowed the migration of the ER-ERE complex by 32 to 84% and inhibited rhERα-ERE binding by 17 to 75%. None of antibodies to ERβ supershifted rhERα-ERE complex. Some ERα-specific antibodies increased whereas some decreased rrERβ-ERE binding. Anti-ERβ antibodies supershifted different amounts of the rrERβ-ERE complex. Our results indicate that supershift and inhibition of ER-ERE interaction with a specific antibody are equally reliable in the detection of rhERα and rrERβ. ERα antibody Ab10, antisera G20 and AT3B, and ERβ-antiserum Y19 offered the best discrimination between ERα and ERβ. Comparison of the peptide sequences against which various antibodies were raised indicate directions for new ERα and ERβ- specific antibody development. We conclude that a cognate ER antibody that retards the migration of the ER-ERE complex by at least 40% or inhibits ER-ERE interaction by at least 8% provides a reliable detection of a specific ER isoform in EMSA.

Introduction

Estrogen receptors α and β (ERα and ERβ) are members of the steroid/nuclear receptor superfamily that bind to specific sequences called estrogen response elements (EREs). Identification of ERα and ERβ in electrophoretic mobility shift assays (EMSA) relies on ‘supershift,’ i.e. the formation of a ERE-bound complex of ER with a specific antibody that has reduced migration compared to the receptor-ERE complex alone. ERα and ERβ may cross-react with antibodies because they share sequence homology [1], making identification of each isoform problematic. Antibodies to ER can also inhibit ER-ERE binding. Inhibition of ER-ERE binding in the presence of an isoform-specific ER antibody provides an alternative to supershift that can be used to identify an ER-containing band in EMSA.

In earlier work we observed that antibodies to the N- and C-terminal regions of ERα supershift the ERα-ERE complex in EMSA [2]. We also observed that antiserum AT3B, raised to a peptide corresponding to a region in the DNA binding domain (DBD) of ERα [3], inhibited ERα-ERE binding [4]. However, it is important to examine if AT3B cross-reacts with ERβ because there is a 95% aa homology in the DBD between ERα and ERβ [1].

Here we compared the impact of various ERα and ERβ antibodies on the interaction of ERα or ERβ with a consensus ERE. Our purpose was to evaluate what antibodies are most selective for each particular ER isoform and to determine the best epitopes of ERα and ERβ against which to develop new antibodies. Additionally, we offer criteria for the significance of supershift and inhibition of ER-ERE interaction: if an ERα or ERβ antibody retards the migration of its cognate ER-ERE complex by at least 40% or inhibits ER-ERE interaction by at least 8%, this antibody is reliable for detecting that ER isoform in EMSA.

Section snippets

Materials

17β-Estradiol (E₂) was purchased from Sigma (St. Louis, MO, USA). ERα monoclonal antibodies (MAbs) AER304, AER308, AER314, AER320, and Ab-10 were purchased from Neomarkers (Lab Vision Corp., Fremont, CA, USA). MAb H222 to ERα was a gift of Abbott Laboratories (Abbott Park, IL, USA). MAb N350 to actin was purchased from Amersham (Piscataway, NJ, USA). Polyclonal antisera Ab715 to ERα was supplied by the National Hormone and Pituitary Program [5]. Polyclonal antisera AT3B, 78–1, 78–3, 14A to ERα,

Effect of ERα antibodies on ERα–ERE complex formation and migration

Mutagenesis experiments demonstrated that the N-terminal zinc finger (CI, hERα aa 185–205) specifies ER binding to EREs while the C-terminal zinc finger (CII, hERα aa 221–245) is involved in nonspecific DNA binding [14], [15]. We previously reported that antiserum AT2A to the CII zinc finger enhanced, and antisera AT3A and AT3B, prepared to a peptide region immediately adjacent to the CII zinc finger (hERα aa 247–261), inhibited calf uterine ERα binding to EREc38 [4]. Here we confirmed that

Discussion

Since the detection of ER in an ER-ERE complex in EMSA relies on ER interaction with a receptor-specific antibody, the selectivity of this interaction is critical in interpreting data. Detection of ER-ERE complexes is important in analysis of ER expression and activity in crude cell extracts [17] and in human tumor samples [18], [19], [20]. Here we examined the effects of antibodies raised to different regions of ERα and ERβ on the formation and migration of ERα-ERE and ERβ-ERE complexes in

Acknowledgements

We thank Dr Peter C. Kulakosky for preparing ERα and ERβ and his review of this manuscript. We thank Dr Ronald Gregg for supplying normal mouse serum.

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However, no supershift band was observed, probably because the interaction between the antibody and nuclear receptors prevented their binding to the ERE elements. In fact, this occurrence has previously been reported by others (Chen et al., 2007; Ou et al., 2003; Tyulmenkov and Klinge, 2000; Zatyka et al., 2002). Nevertheless, as both anti-ERα and anti-ERβ decreased the shift band intensity, we suggest that the presence of at least one of the ERs is required for the interaction with the ERETTR to occur.
Previous studies reported that 17 beta-estradiol (E2) is responsible for the up-regulation of transthyretin (TTR) expression via an estrogen receptor (ER)-dependent pathway in rat choroid plexus (CP) and liver. A computer-assisted homology search identified a putative estrogen-responsive element (ERE) in the 5′ flanking region of the human TTR (hTTR) gene (ERETTR), with the sequence aAGTCAAAGTGACCa, between − 3406 and − 3392 bp. Luciferase reporter assays and electrophoretic mobility shift (EMSA) and supershift analysis were carried out to investigate if E2 regulates TTR transcription via this putative ERE. Luciferase reporter assays in COS-7 cells were carried out with a plasmid construction where the TTR fragment containing the putative ERETTR was cloned in pGL2-promoter vector (pGL2-P) (pGL2-P/TTR), co-transfected with estrogen receptor α (ERα) and/or estrogen receptor β (ERβ) expression vectors. These assays demonstrated that, upon incubation with E2, one or both ERs (α and/or β) transactivate the reporter gene. The pGL2-P/TTR showed a significant transactivation of up to 6.8-fold, by E2, when co-transfected with ERβ, and up to 4-fold with ERα. Specific binding of ER (α and/or β) to ERETTR was demonstrated by EMSA and supershift assays confirmed the binding to ERα and/or ERβ. Our findings further suggest a mechanism underlying the regulation of TTR expression through the identification of a novel ERE in the TTR gene, which functions as an E2-dependent enhancer-like element.
Identification of two functional estrogen response elements complexed with AP-1-like sites in the human insulin receptor gene promoter
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This study was designed to explore the possible existence and location of estrogen response elements (EREs) in the human insulin receptor (hIR) gene promoter. Transfections of U-937 cells with the reported plasmids phIR(−1819)-GL2, phIR(−1473)-GL2, and phIR(−876)-GL2, that contain the −1819 to −271 bp fragment of the hIR promoter (wild-type promoter) and progressive 5′ deletions of this promoter, revealed that while the activity of the wild-type promoter, was repressed 36% by treatment with 17β-estradiol (E₂), the activities of progressive 5′ deletions of this promoter were reduced by 26% and by 0%, by this hormone. This suggests that E₂ needs the wild-type promoter for full transcriptional repression of this gene and it also suggests the presence of putative EREs in the region between −1819/−877 bp of this promoter. To identify these EREs we performed a computer search, using the SEQFIND programme developed in our laboratory, by homology with the consensus vit-ERE (5′GGTCAnnnTGACC3′) of the Xenopus vitellogenin A₂ gene promoter. The results of our search indicated no sequence identical to this consensus ERE, and neither was any sequence found to show 9 or 8 of the 10 bases of this consensus in this promoter. Nevertheless, a putative hIR ERE1 (5′AGTGAaacTGGCC3′) showing 7 bases of the consensus vit-ERE, and 10 bases of the optimal binding sequence ERE (5′CA/GGGTCAnnnTGACCT/CG3′), was identified between −1430/−1418 bp of the hIR promoter. An AP-1-like site was covering the 3′ half-element of this ERE; another AP-1-like site was overlapping the first AP-1-like site, and finally a third AP-1-like site was located beside to the 5′ half-element. In addition, another putative hIR ERE2 (5′GCTCCtagCAAAC3′) showing 5 bases of the consensus vit-ERE, and 9 bases of the optimal binding sequence ERE, was located upstream of the hIR promoter, between −1567/−1555 bp. An AP-1-like site was located downstream of the 3′ half-element of this ERE, and another AP-1-like site was beside the 5′ half-element. EMSA analysis using nuclear extracts of E₂-treated cells and natural sequences, including these putative EREs, indicated that ERβ – the only isoform expressed in U-937 cells – specifically recognized both EREs because ERβ–DNA complexes were efficiently competed by the corresponding unlabelled probe and supershifted by the anti-human ERβ (L-20) antibody. These data provide the first identification of EREs complexed with AP-1-like sites in the hIR promoter, which account for the transcriptional repression of the hIR gene mediated by ERβ in U-937 cells.
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^☆: Supported by NIH R01 DK 53220 and a University of Louisville School of Medicine Research Grant to C.M.K.

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Selectivity of antibodies to estrogen receptors α and β (ERα and ERβ) for detecting DNA-bound ERα and ERβ in vitro☆

Abstract

Introduction

Section snippets

Materials

Effect of ERα antibodies on ERα–ERE complex formation and migration

Discussion

Acknowledgements

Steroids

Steroids

Steroids

Mol Cell Endocrinol

J Steroid Biochem

J Biol Chem

J Steroid Biochem Mol Biol

J Biol Chem

J Steroid Biochem Mol Biol

J Steroid Biochem Mol Biol

J Steroid Biochem Mol Biol