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Molecular cloning and characterization of human estrogen receptor [beta]cx: a potential inhibitor ofestrogen action in human
Introduction
Materials And Methods
Screening of human cDNA and genomic library
Plasmid construction
DNA sequence and analysis
Northern blot analysis of various human tissues
Cell transfection and whole cell extracts preparation
Antibody preparation and western blot analysis
Binding assay of estrogen to estrogen receptors
Gel shift assay
Chloramphenicol acetyltransferase (CAT) assay
Glutathione S-transferase (GST) pull-down assay
Results
Isolation of ER[beta]cx cDNA
The C-terminal ER[beta]cx region is derived from a unique exon 3[prime]-downstream of the human genomic ER[beta]
Expression of ER[beta]cx in various human tissues
Immunoblot of human cultured cells
Estrogen binding activities of ER[alpha], ER[beta] and ER[beta]cx
DNA binding ability of ER[alpha], ER[beta] and ER[beta]cx
Lack of transcriptional activity of ER[beta]cx and lack of interaction of ER[beta]cx with the cofactor TIF1[alpha]
Transcriptional activity of ER[beta]cx in the presence of ER[alpha] or ER[beta]
Discussion
Acknowledgements
References
Molecular cloning and characterization of human estrogen receptor [beta]cx: a potential inhibitor ofestrogen action in human
DDBJ/EMBL/GenBank accession no. AB006589
ABSTRACT
INTRODUCTION
Estrogen plays crucial roles in sexual development and the reproductive cycle (1). The estrogen receptor (ER), a member of the steroid/nuclear receptor superfamily, mediates this action by ligand-dependent binding to the estrogen response element (ERE), which exists in the enhancer region of target genes, regulating their transcription directly (2-4).
It has been shown that members of the nuclear receptor superfamily, such as RAR, RXR and TR, have multiple subtypes and isoforms (5-7). In the case of the ER, we and others have reported several isoforms that were mostly derived from alternative splicing (8,9). Most of these isoforms lost estrogen-dependent transactivation ability and some of them showed various effects on estrogen signaling, such as inhibitory effects on the wild-type ER (9,10).
In order to understand the mechanism of estrogen action and ER regulation of gene transcription, it is important to isolate and characterize novel subtypes and/or isoforms of the ER. Recently, another ER subtype, ER[beta] (11), has been identified from rat prostate and, therefore, the classical ER is renamed ER[alpha]. ER[beta] has been characterized by its distinct properties, including tissue localization and transactivation properties (12,13). The DNA binding domain (DBD) of ER[beta] is highly homologous with that of ER[alpha], implying that both ER[alpha] and ER[beta] share the same DNA response element. Subsequently, human ER[beta] (14,15) and mouse ER[beta] (16) were isolated.
In this study, we screened human cDNA libraries with an ER[alpha] cDNA probe to identify novel ER subtypes and isoforms. We have obtained three independent ER[beta]-related clones, including a novel isoform, designated ER[beta]cx.
In view of the structural and functional similarities among several nuclear receptor isoforms (17-19), some isoforms generated by alternative splicing of the C-terminal region may suppress ligand-dependent transactivation by canonical receptors. To test this possibility, we have investigated the effect of ER[beta]cx on transcriptional regulation by wild-type ER[alpha] and ER[beta] using a transfection system. Biochemical properties of ER[beta]cx are also described.
MATERIALS AND METHODS
Screening of human cDNA and genomic library
A human testis [lambda]ZAPII cDNA library (5.0 × 105 plaques) constructed in pBK-CMV (Stratagene) was screened with a 32P-labeled DBD fragment of rat ER[alpha] cDNA (20) encoding amino acids 177-281. The plaque-transferred filters were hybridized with the probe for 18 h at 63°C in 5× SSC, 0.5% (w/v) blocking agent (Amersham). The filters were washed for 15 min at room temperature in 2× SSC, 0.1% SDS twice and then exposed to X-ray film. Further screening was repeated until a single positive signal was obtained. The PCR-amplified DNA fragments specific for ER[beta]cx (nt 2667-3229) and specific for ER[beta] (nt 2690-2918) (15) were used as probes to isolate the genomic clones from a human genomic DNA library (Japanese Cancer Research Resources Bank).
Plasmid construction
The ER[beta] cl 61-1 cDNA insert was cloned into the pCXN2 expression vector (21) and into pGEX4T-2 (Pharmacia) to construct pCXN2-hER[beta]cx and GST-hER[beta]cx respectively. pCXN2-hER[alpha], pCXN2-hER[beta], GST-hER[alpha], GST-hER[beta] and ERE-GCAT were constructed as described (15,22). All constructs were verified by sequencing.
DNA sequence and analysis
The nucleotide sequences were determined by sequencing both strands of alkaline denatured plasmid DNA using the BcaBest sequencing kit (TaKaRa Co.). The obtained DNA sequence was compiled and analyzed using the DNASIS computer programs (Hitachi Co.).
Northern blot analysis of various human tissues
Human multiple tissue northern blots of poly(A)+ RNA were purchased from Clontech. These blots were probed with 32P-labeled DNA fragments of ER[alpha] (2.1 kb EcoRI-digested fragment of HEG0), ER[beta] (nt 2690-2918), ER[beta]cx (nt 2667-3229) and glyceraldehyde 3-phosphate dehydrogenase (G3PDH), as an internal control, according to the manufacturer's instructions.
Cell transfection and whole cell extracts preparation
Cultured cells were maintained in Dulbecco's modified Eagle's medium (DMEM) without phenol red, supplemented with 10% dextran-coated charcoal-stripped fetal calf serum (23). Samples of 5 × 105 cells in 10 cm Petri dishes were transfected with a total of 20 µg plasmids using calcium phosphate (24). Cells were harvested 36 h after transfection and whole cell extracts were prepared by freeze-thawing and diluted in 100 µl TEG buffer (10 mM Tris, pH 7.5, 1.5 mM EDTA, 10% glycerol).
Antibody preparation and western blot analysis
To detect the specific ER[beta] and ER[beta]cx proteins, rabbit polyclonal antibodies against synthesized peptides of the C-terminal region of ER[beta] (CSPAEDSKSKEGSQNPQSQ) and ER[beta]cx (MKMETLLPEATMEQ) respectively were prepared as described elsewhere (25) and purified on affinity columns bound with each synthetic peptide. Whole cell extracts were fractionated on SDS-7.5% polyacrylamide gels under reducing conditions. Twenty micrograms of protein were then subjected to western blot analysis using the anti-ER[beta]- and anti-ER[beta]cx specific antibodies respectively as described, using the chemiluminescence-based ECL detection system (Amersham) according to the manufacturer's instructions.
Binding assay of estrogen to estrogen receptors
Ten microgram aliquots of whole cell extracts were incubated at 4°C for 16 h with various concentrations of [2.4.6.7-3H]E2 (91 Ci/mmol) (Amersham) in the presence and absence of a 150-fold excess of radioinert E2. Free [2.4.6.7-3H]E2 was removed by dextran-coated charcoal adsorption and the bound form was quantified using a liquid scintillation counter. Specific binding was then calculated for each concentration of radiolabeled E2 used. The data were analyzed by the method of Scatchard (26).
Gel shift assay
Double-stranded oligonucleotide (37 bp, 5[prime]-GTCCAAAGTCAGGTCACAGTGACCTGATCAAAGTT-3[prime], consensus ERE underlined) (27) was end-labeled with [32P]dCTP using the Klenow fragment. An appropriate volume (4-20 µg protein) of cell extract was incubated with 2 µg poly(dI·dC) on ice for 15 min in a 15 µl reaction mix of 10 mM Tris-HCl (pH 7.5), 1 mM DTT, 10% (v/v) glycerol, 100 mM KCl. Radiolabeled probe (~104 c.p.m.) was added to the reaction mixture and incubated at room temperature for 15 min. ER-DNA complex and free probe were then separated on a 4% polyacrylamide gel run for 2 h at 4°C in 0.5× TBE buffer.
Chloramphenicol acetyltransferase (CAT) assay
The CAT assay was performed as described (28). Briefly, 5 × 105 COS-7 cells were transfected with a total of 15 µg DNA. Two micrograms of ERE-GCAT reporter plasmid were co-transfected with the indicated amounts of receptor expression vectors. All assays were performed in the presence of 2 µg PCH110 (Pharmacia), a [beta]-galactosidase expression vector used as an internal control to normalize for variations in transfection efficiency. The total amounts of DNA and expression vectors for transfection were adjusted using pGEM3Zf (Promega) and pCXN2 respectively. After 12 h incubation with calcium phosphate-precipitated DNA, the cells were washed with fresh medium and incubated for an additional 24 h in the presence or absence of 10-7 M E2. Whole cell extracts were prepared by freeze-thawing and assayed for CAT after normalization for [beta]-galactosidase activity.
Figure 1. Genomic organization of ER[beta]cx. (A) Restriction map and the sites of alternative exons for ER[beta] and ER[beta]cx. The exons encoding the C-terminal-specific region of ER[beta] (L4) and ER[beta]cx (Lcx) are schematically represented, with each intron-exon junction indicated below. The pyrimidine-rich branch site (CACTGCA) is double underlined and a putative conserved branch point sequence is indicated by an asterisk. The sites for restriction enzymes in the ER[beta] genomic region around the specific exons are abbreviated as follows: B, BamHI; E, EcoRI. (B) Comparison of the alternative splicing of the 3[prime]-end among nuclear receptor isoforms. Exons and introns are schematically represented by open boxes and solid lines respectively. The numbers and names of exons are in open boxes. (C) Sequence alignment of nuclear receptor proteins. The central conserved acidic amino acid and two pairs of hydrophobic amino acids in the AF-2 core region are boxed. Amino acid numbers are indicated and terminal amino acids are indicated by a star. The alternative splicing junctions are indicated by a solid bar. ER[beta]cx and TIF1[alpha] (29) proteins were synthesized in vitro using the TnT-coupled reticulocyte lysate system (Promega). GST, GST-hER[alpha], GST-hER[beta] and GST-hER[beta]cx proteins were induced, solubilized, estimated as of equal quality on Coomassie stained gels and bound to glutathione beads following the manufacturer's instructions (Pharmacia LKB). After binding to glutathione beads, GST fusion proteins were preincubated for 30 min in 500 µl NETN buffer minus NP-40 in the absence (-) or presence (+) of 10-7 M E2. Then, 15 µl of the suspension were incubated with 1-2 µl appropriate 35S-labeled, in vitro translated protein for 1 h in 500 µl NETN (20 mM Tris-HCl, pH 7.5, 100 mM KCl, 0.7 mM EDTA, 0.5% NP-40, 1 mM phenylmethylsulfonyl floride). Following incubation, the beads were washed three times with NETN. Bound proteins were eluted with 20 µl 1× SDS-PAGE buffer (50 mM Tris-HCl, pH 6.8, 100 mM dithiothreitol, 2% SDS, 0.1% bromophenol blue, 10% glycerol) and electrophoretically separated on a SDS-7.5% polyacrylamide gel.
Glutathione S-transferase (GST) pull-down assay
RESULTS
Isolation of ER[beta]cx cDNA
We screened the human testis cDNA library with a DNA probe generated from the coding sequence of the rat ER[beta] DBD and found 13 positive clones out of 200 000 plaques. Among them, three clones were ER[beta]-related and two were partial ER[alpha]s, as judged by sequence analysis. Restriction mapping and partial sequencing of the three ER[beta]-related clones indicated that all were derived from the same RNA. One of the three overlapping clones, cl 61-1, had the longest, and complete, open reading frame containing a poly(A)+ tail. Its nucleotide sequence and deduced amino acid sequence were determined, designated ER[beta]cx and deposited in the DNA Data Bank of Japan (accession no. AB006589). The predicted ER[beta]cx protein consists of 495 amino acids, with a calculated relative molecular mass (Mr) of 55.5 kDa, counted from the ATG codon at nt 1276, preceded by an in-frame stop codon at nt 1210. This putative first ATG codon is identical to that of the ER[beta] cDNA (15). Computer-assisted analysis and database searches reveal that ER[beta]cx encodes an A/B domain, DBD, hinge region and part of its ligand binding domain (LBD). It was found to be identical to the human ER[beta] cDNA except that the C-terminal 61 amino acids of ER[beta] were replaced by a unique 26 amino acid sequence in ER[beta]cx. Thus, ER[beta]cx is named for its C-terminal exchanged property.
Figure 2. Expression of the ER[alpha], ER[beta] and ER[beta]cx transcripts in various human tissues. Northern blots with poly(A)+ mRNA from various human tissues were hybridized with 32P-labeled DNA probes specific for each ER. Each lane contained 2 µg poly(A)+ mRNA and G3PDH was used as the internal control. Six independent clones, ~20 kb insert size on average, were isolated from the human genomic DNA library probed with the C-terminal-specific region of ER[beta] (nt 2690-2918) or ER[beta]cx (nt 2667-3229). The restriction map of the clones showed that these six genomic DNA fragments overlapped and were derived from the same region. Two DNA fragments digested with EcoRI, 5 and 1.8 kb in size, were hybridized with ER[beta]- and ER[beta]cx-specific probes respectively. These EcoRI-digested fragments were subcloned and sequenced. The 5 kb fragment included the ER[beta]-specific sequence as a single exon and its intron-exon junction was CCGTAG (Fig. Figure 3. Immunoblot analysis of ER[beta] and ER[beta]cx. (A) Determination of ER[beta] by immunoblot analysis of transfected cells. Twenty micrograms of transfected COS-7 cell extracts were resolved by SDS-PAGE and transferred to nitrocellulose. Blots were probed with the ER[beta]-specific polyclonal antibody (1:500). (B) Immunoblot analysis of transfected cells with the anti-ER[beta]cx antibody (1:500). The same volumes of the cell extracts were used as shown in (A). (C) Immunoblot analysis with the anti-ER[beta]cx antibody (1:500) in human cultured cells. COS-7 cells transfected with either 20 µg pCXN2 or 20 µg pCXN2-hER[beta]cx, HOS-TE85 (derived from human osteosarcoma), Saos-2 (derived from human osteosarcoma) and HEC-1 (derived from human endometrial carcinoma) cells were cultured and harvested. Twenty micrograms of whole cell extracts were used for the analysis. Comparison of the alternative splicing ends between ER[beta]cx and other nuclear receptor isoforms and their splicing junctions are shown in Figure Northern blot analysis was performed using DNA fragments of specific regions of ERs as probe. It revealed that an ~7 kb ER[beta]cx transcript was detected in testis, ovary, prostate and thymus. In contrast, ER[alpha] transcript was expressed in ovary and prostate and ER[beta] transcript was distributed in testis and ovary (Fig. Western blot analysis using the ER[beta]- and ER[beta]cx-specific polyclonal antibodies against their C-terminal amino acid residues respectively demonstrated the ER[beta] protein as 60 and 57 kDa bands (Fig. [3H]E2 radioligand binding assays were performed with whole cell extracts of transfected COS-7 cells. A representative result of the saturation test is shown in Figure Figure 4. Comparison of E2 binding activities among ER[alpha], ER[beta] and ER[beta]cx. Whole cell extract of COS-7 cells transfected with ER[alpha], ER[beta] or ER[beta]cx was incubated for 16 h with 0.1-1 nM [2,4,6,7-3H]E2 in the presence or absence of various concentrations of unlabeled E2. Unbound E2 was removed by centrifugation and the receptor-bound radioactivity was measured with a liquid scintillation counter. Only one representative result is shown from the three independent experiments showing similar results. Whole cell extracts were prepared from COS-7 cells transfected with ER expression vectors and used for gel shift assay. The ER[alpha]-DNA complexes, which were blocked by cold ERE probe, were detected as a distinct band (Fig. Figure 5. DNA binding abilities of ER[alpha], ER[beta] and ER[beta]cx. (A) Whole cell extracts of COS-7 cells transfected with the indicated ER expression vectors were analyzed for their ability to bind the 32P-labeled ERE. ER[alpha] and ER[beta] bind to the ERE and competition is shown by adding an increasing amount of cold ERE (lanes 1-8). ER[beta]cx-DNA complexes were not detected (lanes 9-12). (B) DNA binding of ER[alpha] is inhibited in the presence of ER[beta]cx. A constant amount (4 µg) of whole cell extracts of COS-7 cells transfected with pCXN2-hER[alpha] was applied to lanes 1-4, with increasing amounts (0, 4, 8 or 16 µg) of whole cell extracts transfected with pCXN2-hER[beta]cx. Sixteen micrograms of this extract alone were run in lane 5. The amount in each lane was equalized with untransfected cell extracts. Upon stimulation of cells transfected with ER[alpha] or ER[beta] expression vectors with 10-7 M E2, CAT activity from the ERE-GCAT construct increased 25- and 9-fold respectively, whereas it did not change in the case of ER[beta]cx (Fig. Figure 6. Transcriptional activity of ER[beta]cx and its inability to interact with a cofactor, TIF1[alpha]. (A) Transactivation of the ERE-GCAT reporter by ER[alpha], ER[beta] and ER[beta]cx. COS-7 cells were transfected with 2 µg ERE-GCAT reporter plasmid and 10 ng wild-type ER expression vectors respectively. The transfected cells were incubated for 24 h in the presence (+) or absence (-) of 10-7 M E2 and CAT activities were normalized relative to [beta]-galactosidase activity expressed from the PCH110 internal control vector and are reported as means ± SD, calculated from three independent experiments. CAT activities are indicated as the amounts of fold induction. (B) Lack of interaction between ER[beta]cx and TIF1[alpha]. The GST pull-down assay was performed as indicated except that GST fusion proteins, each estimated as of equal quality on Coomassie stained gels (data not shown), were preincubated for 30 min in 500 µl NETN buffer minus NP-40 in the presence (+) or absence (-) of 10-7 M E2; 20% of the input is shown in lane 1. When ER[alpha] or ER[beta] was co-transfected with ER[beta]cx, ER[beta]cx rather inhibited ligand-induced transactivation by ER[alpha] (Fig. Figure 7. Dominant negative activity of ER[beta]cx in the ER[alpha] signaling pathway. (A) Dose-dependent inhibition by ER[beta]cx of ER[alpha] transactivation. The cells were transfected with 0.1 µg ER[alpha] expression vector and ER[beta]cx expression vector at the indicated amounts (0.01-1 µg) in the presence (+) or absence (-) of 10-7 M E2. CAT activities are reported as means ± SD, calculated from three independent experiments. (B) ER[beta]cx did not influence transactivation by ER[beta]. The experimental conditions were as in (A) except that the cells were transfected with 0.1 µg ER[beta] expression vector instead of ER[alpha] expression vector. (C) GST pull-down assay. ER[beta]cx protein was synthesized in vitro using the TnT-coupled reticulocyte lysate system (Promega); 20% of the input is shown in lane 1. After binding of GST-hER[alpha], GST-hER[beta] and GST proteins, each estimated as of equal quality on Coomassie stained gels (data not shown), to glutathione beads, 15 µl suspension were incubated with 1-2 µl of the appropriate 35S-labeled, in vitro translated protein for 1 h in 500 µl NETN buffer. Bound proteins were eluted and electrophoretically separated on a SDS-7.5% polyacrylamide gel. In this study we have identified an endogenous variant of ER[beta] and named it ER[beta]cx. Among three in-frame ATG codons of ER[beta]cx, located at nt 1276, 1412 and 1435, preceded by an in-frame stop codon at nt 1210, the first and second ATG codons conform to the Kozak consensus sequence (32). This first ATG codon is identical to that of ER[beta], recently reported (15), encoding the additional 53 amino acids in its A/B domain compared with the previously published ER[beta] sequence (14). The open reading frame of ER[beta]cx encodes a protein of 495 amino acid residues with a calculated molecular weight of 55.5 kDa (calculated from the first methionine). ER[beta]cx is identical to ER[beta] for the first 469 amino acids, but differs from ER[beta] in replacement of the last 61 amino acids of the latter by a unique 26 amino acid sequence. The 61 amino acids of ER[beta] include the AF-2 core, which is essential for ligand-dependent transcriptional activation, including cofactor binding (29-31), and their substitution suggests that ER[beta]cx may lose its transactivation capacity (see below). We have shown that alternative splicing of the human ER[beta] primary transcript produces a novel isoform, ER[beta]cx, differing at the last exon. Both the ER[beta]- and ER[beta]cx-specific intron-exon junctions obeyed the splice consensus sequence (33); AG nucleotides at the end of each intron followed by the exon sequences. Moreover, the branch site is also conserved at ~30 nt upstream of the splice site of the ER[beta]cx-specific exon (Lcx), retaining the target A nucleotide (34). Although distinct splicing mechanisms between Lcx and the ER[beta]-specific exon (L4) (35) have yet to be elucidated, we have also shown that ER[beta]cx inhibits E2-dependent transactivation by wild-type ER[alpha]. Similar cases exist for other nuclear receptors, such as hGR[beta] (17), hTR[alpha]2 (18) and rVDR1 (19). These are also alternative splicing variants substituting part of the last exon, including the AF-2 core, by unique sequences (see Fig. In the case of ER[alpha], although the corresponding C-terminal spliced isoform remains to be identified, several other isoforms have been reported. The ER [Delta]E3 isoform, which harbors a deletion of exon 3 encoding the second zinc-finger of the DBD, especially inhibits E2-dependent transcription activation in a dominant negative fashion when it is co-transfected with the wild-type ER and a reporter plasmid (10). We previously reported that the ER[alpha] [Delta]exon 4/5 isoform also inhibits wild-type ER[alpha] (9). Recently, ER[beta] isoforms other than ER[beta]cx have been reported, although they were not characterized for their transactivation properties (36,37). Northern blot analysis showed that in some human tissues, such as ovary and testis, expression of ER[alpha], ER[beta] and ER[beta]cx overlapped. Co-localization of these ERs may play some role in reproduction, at least in ovary and testis. It was reported that both ER[alpha] and ER[beta] mRNAs are co-expressed in some human breast tumors, suggesting their involvement in malignancy (38). Although expression of ER[beta] was not confirmed specifically in human prostate, ER[beta]cx was still detectable where expression of ER[alpha] and ER[beta]cx overlapped. In these organs, ER[alpha]/ER[beta]cx heterodimer formation and their transcriptional suppression may be of physiological importance. The specificity of the anti-ER[beta] and anti-ER[beta]cx antibodies was verified by immunoblotting of whole cell extracts of COS-7 cells transfected with each ER expression vector (Fig. Unlike ER[alpha] and ER[beta], ER[beta]cx lacks ligand binding ability and does not have or has only very low ERE binding ability, resulting in the loss of ligand-dependent transactivation ability. In the case of ER[alpha], the 521-528 amino acid region of exon 8 is essential for ligand binding (39). The E2 binding inability of ER[beta]cx must be due to substitution of the corresponding L4 exon by the Lcx exon. However, we cannot exclude the possibility that ER[beta]cx has a much reduced ERE binding activity, because the gel shift assay may require a threshold level of affinity for gel shifted protein-DNA complexes (40). Furthermore, ER[beta]cx was incapable of interacting with TIF1[alpha]. It was suggested that TIF1[alpha] induces chromatin remodeling, thereby allowing nuclear receptors and other transactivators to bind to their cognate response elements (41). The lack of transcriptional activity of ER[beta]cx, as well as its much reduced ERE binding ability, might be partly due to its inability to bind cofactors, such as TIF1[alpha]. We have shown that the dominant negative activity of ER[beta]cx was especially against ER[alpha] transactivation, rather than that of ER[beta]. DNA binding by ER[alpha] was inhibited by ER[beta]cx, possibly due to formation of ER[alpha]/ER[beta]cx heterodimers. We also showed a preference for forming ER[alpha]/ER[beta]cx heterodimers rather than ER[beta]/ER[beta]cx heterodimers. Recently, Cowley et al. (42) reported that ER[alpha] homodimers and ER[alpha]/ER[beta] heterodimers bound to the ERE better than ER[beta] homodimers (~4-fold greater Kd), suggesting that the former are more functional than the latter under physio-logical conditions in vivo. Taken together, we assume that selective suppression of the ER[alpha] signaling pathway by ER[beta]cx may be due to the easier formation of transcriptionally inactive ER[alpha]/ER[beta]cx heterodimers that have no or much reduced DNA binding activity than that of ER[beta]/ER[beta]cx heterodimers. Considering the results of the GST pull-down assay, indicating ER[beta]/ER[beta]cx heterodimers, we cannot exclude the possibility that ER[beta]cx inhibits transactivation by ER[beta] beyond the experimental conditions described here. There are other possibilities for the inhibitory mechanism: (i) ER[beta]cx competes with ER[alpha] for ERE binding; (ii) specific transcriptional silencing, such as titering out cofactors and basal transcription factors essential for ER[alpha] transactivation (40,43-45). Based on our results that ER[beta]cx was hardly capable of binding to the ERE, competition for ERE binding is unlikely to be responsible for the dominant negative properties of ER[beta]cx against ER[alpha]. In addition, transcriptional squelching may be negligible in the experimental ranges, since suppression of ligand-induced transactivation was not observed even when 1 µg ER[alpha] or ER[beta] expression vector was added (data not shown). Our studies do not rule out the possibility that ER[beta]cx may act through a novel, as yet unknown estrogen-responsive pathway, through specific response elements, specific phosphorylation, novel ligand binding and so on. Further characterization of the physiological function of ER[beta]cx will provide more insights into the diverse effects of estrogens in vivo. We thank Dr Chambon for the gift of the TIF1[alpha] plasmid and Drs H.Toyoshima and S.Kato for helpful discussion. S.O. was supported by a JSPS Research Fellowship for Young Scientists. This work was supported in part by research grants from the Ministry of Education, Science and Culture of Japan and in part by CREST (Core Research for Evolutional Science and Technology) of the Japan Science and Technology Corporation.
The C-terminal ER[beta]cx region is derived from a unique exon 3[prime]-downstream of the human genomic ER[beta]
Expression of ER[beta]cx in various human tissues
Immunoblot of human cultured cells
Estrogen binding activities of ER[alpha], ER[beta] and ER[beta]cx
DNA binding ability of ER[alpha], ER[beta] and ER[beta]cx
Lack of transcriptional activity of ER[beta]cx and lack of interaction of ER[beta]cx with the cofactor TIF1[alpha]
Transcriptional activity of ER[beta]cx in the presence of ER[alpha] or ER[beta]
DISCUSSION
ACKNOWLEDGEMENTS
REFERENCES
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