The Experts below are selected from a list of 294 Experts worldwide ranked by ideXlab platform
Robert A. Bambara - One of the best experts on this subject based on the ideXlab platform.
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Perspective Nuclear Receptor Signaling | The Open Access Journal of the Nuclear Receptor Signaling Atlas
2013Co-Authors: Jing Huang, Russell Hilf, Robert A. Bambara, Tao Qiao, Mesut MuyanAbstract:A tale of two Estrogen receptors (ERs): how differential ER-Estrogen Responsive Element interactions contribute to subtype-specific transcriptional response
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The Effects of Estrogen-Responsive Element- and Ligand-Induced Structural Changes on the Recruitment of Cofactors and Transcriptional Responses by ERα and ERβ
Molecular endocrinology (Baltimore Md.), 2002Co-Authors: Mark D. Driscoll, Jing Huang, Sumedha Bhagat, Russell Hilf, Robert A. Bambara, Mesut MuyanAbstract:Estrogen signaling is mediated by ERα and -β. ERs are converted from an inactive form to a transcriptionally active state through conformational changes induced by ligand and Estrogen-Responsive Element (ERE) sequences. We show here that ERα and ERβ bind to an ERE independently from ER ligands. We found that although the binding affinity of ERβ for an ERE is 2-fold lower than that of ERα, both ERs use the same nucleotides for DNA contacts. We show that both EREs and ligands are independent modulators of ER conformation. Specifically, the ERE primarily determines the receptor-DNA affinity, whereas the structure of the ER ligand dictates the affinity of ER for particular cofactors. We found that the ligand-dependent cofactor transcriptional intermediary factor-2, through a distinct surface, also interacts with ERα preferentially and independently of ligand. The extent of interaction, however, is dependent upon the ER-ERE affinity. In transfected cells, ERα is more transcriptionally active than ERβ. The ERE ...
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an explanation for observed Estrogen receptor binding to single stranded Estrogen Responsive Element dna
Molecular Endocrinology, 1999Co-Authors: Mark D. Driscoll, Russell Hilf, Ganesan Sathya, Layla F Saidi, Michael S Demott, Robert A. BambaraAbstract:Estrogen-inducible genes contain an enhancer called the Estrogen response Element (ERE), a double-stranded inverted repeat. The Estrogen receptor (ER) is generally thought to bind to the double-stranded ERE. However, some reports provide evidence that an ER homodimer can bind a single strand of the ERE and suggest that single-stranded ERE binding is the preferred binding mode for ER. Since these two models describe quite different mechanisms of receptor action, we have attempted to reconcile the observations. Analyzing DNA structure by nuclease sensitivity, we found that two identical molecules of a single strand of DNA containing the ERE sequence can partially anneal in an antiparallel manner. Bimolecular annealing produces double-stranded inverted repeats, with adjacent unannealed tails. The amount of annealing correlates exactly with the ability of ER to bind bimolecular EREs. Either strand of an ERE could anneal to itself in a way that would bind ER. We conclude that ER binds only the annealed double-stranded ERE both in vitro and in vivo. (Molecular Endocrinology 13: 958‐968, 1999)
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differential impact of flanking sequences on estradiol vs 4 hydroxytamoxifen liganded Estrogen receptor binding to Estrogen Responsive Element dna
The Journal of Steroid Biochemistry and Molecular Biology, 1993Co-Authors: Jennifer H Anolik, Robert A. Bambara, Carolyn M Klinge, Russell HilfAbstract:Abstract The mechanism by which antiEstrogens antagonize the ability of Estrogen receptor (ER) to induce the transcription of Estrogen-regulated genes is only partially understood. To examine the effect of Estrogen Responsive Element (ERE) stereoalignment and flanking sequences on estradiol-liganded ER (E 2 -ER)-ERE and antiEstrogen-liganded ER (4-hydroxytamoxifen-liganded ER or 4-OHT-ER)-ERE binding, several dimeric EREs, containing a perfect inverted repeat (5′-GGTCAgagTGACC-3′) but lacking the AT-rich flanking sequences typical of highly Estrogen-Responsive promoters, were cloned into a plasmid vector. The ERE centers of symmetry were spaced 1.5, 2.0, 3.0, 6.4 and 6.7 helical turns apart. E 2 -ER and 4-OHT-ER binding to these constructs was specific and saturable, but orientation-independent and, in contrast to our earlier work with E 2 -ER binding to AT-rich EREs, not cooperative. The affinity of E 2 -ER binding decreased as the distance between adjacent EREs was increased, suggesting that E 2 -ER binding to closely spaced EREs is more stable ( K d =0.38, 0.58, 0.83, 1.23, and 0.96 nM, respectively, for the above spacings). In contrast, the affinity of 4-OHT-ER binding increased with increased ERE spacing ( K d =2.90, 4.79, 1.39, 1.77, and 0.92 nM, respectively). The presence of AT-rich sequences flanking the ERE increased the binding affinity of E 2 -ER and 4-OHT-ER, an increase reflected in slower dissociation rates of ER from these EREs. The AT-rich sequence also enhanced the binding capacity of E 2 -ER but not 4-OHT-ER. Since the binding capacity of 4-OHT-ER is identical with or without an AT-rich region, we suggest that flanking sequences are more important in stabilizing E 2 -ER binding and may be critical for cooperative binding to stereoaligned EREs.
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antiEstrogen liganded Estrogen receptor interaction with Estrogen Responsive Element dna in vitro
The Journal of Steroid Biochemistry and Molecular Biology, 1992Co-Authors: Carolyn M Klinge, Robert A. Bambara, Russell HilfAbstract:Abstract The mechanism whereby antiEstrogens alter the ability of the Estrogen receptor (ER) to enhance transcription of Estrogen-regulated genes is largely unknown. The effect that selected Estrogenic and antiEstrogenic ligands have on binding of ER to specific DNA sequences, Estrogen Responsive Elements (EREs) has been quantitated. No differences in purification properties of calf uterine ER liganded with 4-hydroxytamoxifen (4-OHT-ER), ICI 164,384 (ICI 164,384-ER) or estradiol (E2-ER) were detected. A microtiter well plate assay was employed in which liganded ER bound to plasmid DNA is preferentially retained compared to free liganded ER. Binding of E2-ER, 4-OHT-ER, or ICI 164,384-ER was measured to plasmids containing or lacking a 38bp consensus ERE in vitro. The EREs tested contain an inverted repeat (5′- CAGGTCA GAG TGACCTG -3′). Both E2-ER and 4-OHT-ER showed similar high affinity specific binding (Kd = 0.24 and 0.16 nM, respectively) to one copy of the ERE. ICI 164,384-ER did not bind to plasmids containing one ERE. At saturation, however, 4-OHT-ER binding was about 50% of that observed for E2-ER. When the plasmid contained 3 or 4 tandem copies of the ERE, binding E2-ER, 4-OHT-ER, and ICI 164,384-ER binding was measurable. E2-ER bound in a cooperative manner as suggested by convex Scatchard plots and Hill coefficients > 1.5. In contrast, 4-OHT-ER binding displayed much reduced cooperativity, and ICI 164,384-ER did not display cooperative binding. From these results, we propose that the conformation of ER induced by 4-OHT reduces its binding capacity to this consensus ERE without altering its affinity of binding. Furthermore, higher order protein-protein interactions between antiEstrogen-liganded ER bound to DNA differ from those of E2-ER bound to ERE.
Russell Hilf - One of the best experts on this subject based on the ideXlab platform.
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Perspective Nuclear Receptor Signaling | The Open Access Journal of the Nuclear Receptor Signaling Atlas
2013Co-Authors: Jing Huang, Russell Hilf, Robert A. Bambara, Tao Qiao, Mesut MuyanAbstract:A tale of two Estrogen receptors (ERs): how differential ER-Estrogen Responsive Element interactions contribute to subtype-specific transcriptional response
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The Effects of Estrogen-Responsive Element- and Ligand-Induced Structural Changes on the Recruitment of Cofactors and Transcriptional Responses by ERα and ERβ
Molecular endocrinology (Baltimore Md.), 2002Co-Authors: Mark D. Driscoll, Jing Huang, Sumedha Bhagat, Russell Hilf, Robert A. Bambara, Mesut MuyanAbstract:Estrogen signaling is mediated by ERα and -β. ERs are converted from an inactive form to a transcriptionally active state through conformational changes induced by ligand and Estrogen-Responsive Element (ERE) sequences. We show here that ERα and ERβ bind to an ERE independently from ER ligands. We found that although the binding affinity of ERβ for an ERE is 2-fold lower than that of ERα, both ERs use the same nucleotides for DNA contacts. We show that both EREs and ligands are independent modulators of ER conformation. Specifically, the ERE primarily determines the receptor-DNA affinity, whereas the structure of the ER ligand dictates the affinity of ER for particular cofactors. We found that the ligand-dependent cofactor transcriptional intermediary factor-2, through a distinct surface, also interacts with ERα preferentially and independently of ligand. The extent of interaction, however, is dependent upon the ER-ERE affinity. In transfected cells, ERα is more transcriptionally active than ERβ. The ERE ...
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an explanation for observed Estrogen receptor binding to single stranded Estrogen Responsive Element dna
Molecular Endocrinology, 1999Co-Authors: Mark D. Driscoll, Russell Hilf, Ganesan Sathya, Layla F Saidi, Michael S Demott, Robert A. BambaraAbstract:Estrogen-inducible genes contain an enhancer called the Estrogen response Element (ERE), a double-stranded inverted repeat. The Estrogen receptor (ER) is generally thought to bind to the double-stranded ERE. However, some reports provide evidence that an ER homodimer can bind a single strand of the ERE and suggest that single-stranded ERE binding is the preferred binding mode for ER. Since these two models describe quite different mechanisms of receptor action, we have attempted to reconcile the observations. Analyzing DNA structure by nuclease sensitivity, we found that two identical molecules of a single strand of DNA containing the ERE sequence can partially anneal in an antiparallel manner. Bimolecular annealing produces double-stranded inverted repeats, with adjacent unannealed tails. The amount of annealing correlates exactly with the ability of ER to bind bimolecular EREs. Either strand of an ERE could anneal to itself in a way that would bind ER. We conclude that ER binds only the annealed double-stranded ERE both in vitro and in vivo. (Molecular Endocrinology 13: 958‐968, 1999)
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differential impact of flanking sequences on estradiol vs 4 hydroxytamoxifen liganded Estrogen receptor binding to Estrogen Responsive Element dna
The Journal of Steroid Biochemistry and Molecular Biology, 1993Co-Authors: Jennifer H Anolik, Robert A. Bambara, Carolyn M Klinge, Russell HilfAbstract:Abstract The mechanism by which antiEstrogens antagonize the ability of Estrogen receptor (ER) to induce the transcription of Estrogen-regulated genes is only partially understood. To examine the effect of Estrogen Responsive Element (ERE) stereoalignment and flanking sequences on estradiol-liganded ER (E 2 -ER)-ERE and antiEstrogen-liganded ER (4-hydroxytamoxifen-liganded ER or 4-OHT-ER)-ERE binding, several dimeric EREs, containing a perfect inverted repeat (5′-GGTCAgagTGACC-3′) but lacking the AT-rich flanking sequences typical of highly Estrogen-Responsive promoters, were cloned into a plasmid vector. The ERE centers of symmetry were spaced 1.5, 2.0, 3.0, 6.4 and 6.7 helical turns apart. E 2 -ER and 4-OHT-ER binding to these constructs was specific and saturable, but orientation-independent and, in contrast to our earlier work with E 2 -ER binding to AT-rich EREs, not cooperative. The affinity of E 2 -ER binding decreased as the distance between adjacent EREs was increased, suggesting that E 2 -ER binding to closely spaced EREs is more stable ( K d =0.38, 0.58, 0.83, 1.23, and 0.96 nM, respectively, for the above spacings). In contrast, the affinity of 4-OHT-ER binding increased with increased ERE spacing ( K d =2.90, 4.79, 1.39, 1.77, and 0.92 nM, respectively). The presence of AT-rich sequences flanking the ERE increased the binding affinity of E 2 -ER and 4-OHT-ER, an increase reflected in slower dissociation rates of ER from these EREs. The AT-rich sequence also enhanced the binding capacity of E 2 -ER but not 4-OHT-ER. Since the binding capacity of 4-OHT-ER is identical with or without an AT-rich region, we suggest that flanking sequences are more important in stabilizing E 2 -ER binding and may be critical for cooperative binding to stereoaligned EREs.
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antiEstrogen liganded Estrogen receptor interaction with Estrogen Responsive Element dna in vitro
The Journal of Steroid Biochemistry and Molecular Biology, 1992Co-Authors: Carolyn M Klinge, Robert A. Bambara, Russell HilfAbstract:Abstract The mechanism whereby antiEstrogens alter the ability of the Estrogen receptor (ER) to enhance transcription of Estrogen-regulated genes is largely unknown. The effect that selected Estrogenic and antiEstrogenic ligands have on binding of ER to specific DNA sequences, Estrogen Responsive Elements (EREs) has been quantitated. No differences in purification properties of calf uterine ER liganded with 4-hydroxytamoxifen (4-OHT-ER), ICI 164,384 (ICI 164,384-ER) or estradiol (E2-ER) were detected. A microtiter well plate assay was employed in which liganded ER bound to plasmid DNA is preferentially retained compared to free liganded ER. Binding of E2-ER, 4-OHT-ER, or ICI 164,384-ER was measured to plasmids containing or lacking a 38bp consensus ERE in vitro. The EREs tested contain an inverted repeat (5′- CAGGTCA GAG TGACCTG -3′). Both E2-ER and 4-OHT-ER showed similar high affinity specific binding (Kd = 0.24 and 0.16 nM, respectively) to one copy of the ERE. ICI 164,384-ER did not bind to plasmids containing one ERE. At saturation, however, 4-OHT-ER binding was about 50% of that observed for E2-ER. When the plasmid contained 3 or 4 tandem copies of the ERE, binding E2-ER, 4-OHT-ER, and ICI 164,384-ER binding was measurable. E2-ER bound in a cooperative manner as suggested by convex Scatchard plots and Hill coefficients > 1.5. In contrast, 4-OHT-ER binding displayed much reduced cooperativity, and ICI 164,384-ER did not display cooperative binding. From these results, we propose that the conformation of ER induced by 4-OHT reduces its binding capacity to this consensus ERE without altering its affinity of binding. Furthermore, higher order protein-protein interactions between antiEstrogen-liganded ER bound to DNA differ from those of E2-ER bound to ERE.
Stephen Safe - One of the best experts on this subject based on the ideXlab platform.
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Printed in U.S.A. Copyright © 1998 by The Endocrine Society Estrogen-Induced c-fos Protooncogene Expression in MCF-7 Human Breast Cancer Cells: Role of Estrogen Receptor Sp1 Complex Formation*
2013Co-Authors: Renqin Duan, Weston Porter, Stephen SafeAbstract:17�-Estradiol (E 2) induces c-fos protooncogene expression in MCF-7 human breast cancer cells, and previous studies in HeLa cells identified an imperfect palindromic Estrogen-Responsive Element (�1212 to �1200) that was required for trans-activation. In contrast, the Estrogen-Responsive Element was not required for E 2 Responsiveness in MCF-7 cells, and using a series of constructs containing wildtype (pF1) and mutant 5�-flanking sequences (�1220 to �1155) from the c-fos protooncogene promoter in transient transfection assays, it was shown that a GC-rich motif (5�-GGGGCGTGG) containing an imperfect Sp1-binding site was required for hormone-induced activity. This sequence also bound Sp1 protein in gel mobility shift assays, and coincubation with the Estrogen receptor (ER) enhanced Sp1-DN
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Estrogen-Induced c-fos Protooncogene Expression in MCF-7 Human Breast Cancer Cells: Role of Estrogen Receptor Sp1 Complex Formation
Endocrinology, 1998Co-Authors: Renqin Duan, Weston Porter, Stephen SafeAbstract:17Beta-estradiol (E2) induces c-fos protooncogene expression in MCF-7 human breast cancer cells, and previous studies in HeLa cells identified an imperfect palindromic Estrogen-Responsive Element (-1212 to -1200) that was required for trans-activation. In contrast, the Estrogen-Responsive Element was not required for E2 Responsiveness in MCF-7 cells, and using a series of constructs containing wild-type (pF1) and mutant 5'-flanking sequences (-1220 to -1155) from the c-fos protooncogene promoter in transient transfection assays, it was shown that a GC-rich motif (5'-GGGGCGTGG) containing an imperfect Sp1-binding site was required for hormone-induced activity. This sequence also bound Sp1 protein in gel mobility shift assays, and coincubation with the Estrogen receptor (ER) enhanced Sp1-DNA binding. E2 and 4'-hydroxytamoxifen, but not ICI 164,384, induced reporter gene activity in cells transiently transfected with pF1. E2 induced reporter gene activity in MDA-MB-231 breast cancer cells transiently cotransfected with pF1 and wild-type ER or variant ER in which the DNA-binding domain was deleted (HE11); plasmids expressing N-terminal or C-terminal domains of the ER containing activator function-1 or -2, respectively, were inactive in these assays. In contrast, only wild-type ER mediated 4'-hydroxytamoxifen-induced activity. Induction of c-fos protooncogene expression by E2 in MCF-7 cells is dependent on the formation of a transcriptionally active ER/Sp1 complex that binds to a GC-rich enhancer Element.
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Identification of a functional imperfect Estrogen-Responsive Element in the 5'-promoter region of the human cathepsin D gene.
Biochemistry, 1997Co-Authors: Fan Wang, Weston Porter, W. Xing, Trevor K. Archer, Stephen SafeAbstract:17beta-Estradiol (E2) induces cathepsin D gene expression in MCF-7 human breast cancer cells. Previous studies have identified an Sp1-imperfect Estrogen-Responsive Element (ERE) half-site [GGGCGG(N)23ACGGG] (-199 to -165) in the promoter region which forms an Sp1-Estrogen receptor (ER) complex and confers E2 Responsiveness on the corresponding Sp1-ERE-chloramphenicol acetyl transferase (CAT) construct. Further analysis of downstream regions of the promoter identified a CGCCC(N)3TGACC sequence (-119 to -107) which is homologous to the adenovirus major late promoter Element (MLPE) and binds the ER to form a retarded band in a gel electrophoretic mobility shift assay. The corresponding promoter-CAT construct is also E2-inducible. The MLPE resembles an imperfect palindromic ERE containing imperfect (5') and perfect (3') ERE half-sites; analysis of oligonucleotides with mutations in these half-sites shows that only the perfect ERE half-site is required for binding the ER, whereas both sites are required for transactivation. In vivo exonuclease III footprinting showed that treatment with E2 also enhanced binding at the MLPE site. Identification of this second functional enhancer sequence in the 5'-promoter region of cathepsin D is consistent with the increasingly complex cell-specific regulation of hormone-Responsive genes.
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High passage T47D human breast cancer cells: altered endocrine and 2,3,7,8-tetrachlorodibenzo-p-dioxin Responsiveness
European journal of pharmacology, 1994Co-Authors: Patricia Fernandez, Robert C. Burghardt, Roger Smith, Kathy Nodland, Stephen SafeAbstract:Low passage (L) T47D cells cultured for up to 12 months in media containing fetal bovine serum, fetal bovine serum plus 1 nM 17 beta-estradiol and Estrogen-deficient media gave high passage cells denoted H, H(E+) and H(E-) cells, respectively, which exhibited differential Responsiveness to 17 beta-estradiol, various growth factors, tamoxifen, 2,3,7,8-tetrachlorodibenzo-p-dioxin, and their combinations. Moreover, the altered mitogen/antimitogen Responsiveness was paralleled by changes in hormone receptor levels, cellular architecture and ploidy. Estrogen receptor binding levels in the H, L and H(E+) cells varied from 36 to 155 fmol/mg protein; in contrast, the Estrogen receptor binding in H(E-) cells exhibited a time-dependent increase from 81 to 1229 fmol/mg after culturing in Estrogen-deficient media for approximately 12 months. Gel mobility shift assays of the nuclear Estrogen receptor extracts from high and low passage cells with 32P-labeled Estrogen Responsive Element showed that levels of the Estrogen receptor-Estrogen Responsive Element retarded band were lower in all the high passage cells compared to the low passage cells. These studies further illustrate the genetic instability of the T47D human breast cancer cell line and the resulting changes in mitogen and antimitogen Responsiveness. In addition, the high passage H(E-) cells which express high Estrogen receptor but low Estrogen Responsive Element binding represent a unique model system for investigating the cellular and molecular biology of breast cancer cells which appear to be Estrogen receptor-positive but are insensitive to antiEstrogens.
Yves Valotaire - One of the best experts on this subject based on the ideXlab platform.
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Synergism between a half‐site and an imperfect Estrogen‐Responsive Element, and cooperation with COUP‐TFI are required for Estrogen receptor (ER) to achieve a maximal Estrogen‐stimulation of rainbow trout ER gene
European journal of biochemistry, 1999Co-Authors: Fabrice G Petit, Yves Valotaire, Raphael Metivier, Farzad PakdelAbstract:In all oviparous, liver represents one of the main E2-target tissues where Estrogen receptor (ER) constitutes the key mediator of Estrogen action. The rainbow trout Estrogen receptor (rtER) gene expression is markedly up-regulated by Estrogens and the sequences responsible for this autoregulation have been located in a 0.2 kb upstream transcription start site within - 40/- 248 enhancer region. Absence of interference with steroid hormone receptors and tissue-specific factors and a conserved basal transcriptional machinery between yeast and higher eukaryotes, make yeast a simple assay system that will enable determination of important cis-acting regulatory sequences within rtER gene promoter and identification of transcription factors implicated in the regulation of this gene. Deletion analysis allowed to show a synergistic effect between an imperfect Estrogen-Responsive Element (ERE) and a consensus half-ERE to achieve a high hormone-dependent transcriptional activation of the rtER gene promoter in the presence of stably expressed rtER. As in mammalian cells, here we observed a positive regulation of the rtER gene promoter by the chicken ovalbumin upstream promoter-transcription factor I (COUP-TFI) through enhancing autoregulation. Using a point mutation COUP-TFI mutant unable to bind DNA demonstrates that enhancement of rtER gene autoregulation requires the interaction of COUP-TFI to the DNA. Moreover, this enhancement of transcriptional activation by COUP-TFI requires specifically the AF-1 transactivation function of ER and can be observed in the presence of E2 or 4-hydroxytamoxifen but not ICI 164384. Thus, this paper describes the reconstitution of a hormone-Responsive transcription unit in yeast in which the regulation of rtER gene promoter could be enhanced by the participation of cis-Elements and/or trans-acting factors, such as ER itself or COUP-TF.
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synergism between a half site and an imperfect Estrogen Responsive Element and cooperation with coup tfi are required for Estrogen receptor er to achieve a maximal Estrogen stimulation of rainbow trout er gene
FEBS Journal, 1999Co-Authors: Fabrice G Petit, Yves Valotaire, Raphael Metivier, Farzad PakdelAbstract:In all oviparous, liver represents one of the main E2-target tissues where Estrogen receptor (ER) constitutes the key mediator of Estrogen action. The rainbow trout Estrogen receptor (rtER) gene expression is markedly up-regulated by Estrogens and the sequences responsible for this autoregulation have been located in a 0.2 kb upstream transcription start site within - 40/- 248 enhancer region. Absence of interference with steroid hormone receptors and tissue-specific factors and a conserved basal transcriptional machinery between yeast and higher eukaryotes, make yeast a simple assay system that will enable determination of important cis-acting regulatory sequences within rtER gene promoter and identification of transcription factors implicated in the regulation of this gene. Deletion analysis allowed to show a synergistic effect between an imperfect Estrogen-Responsive Element (ERE) and a consensus half-ERE to achieve a high hormone-dependent transcriptional activation of the rtER gene promoter in the presence of stably expressed rtER. As in mammalian cells, here we observed a positive regulation of the rtER gene promoter by the chicken ovalbumin upstream promoter-transcription factor I (COUP-TFI) through enhancing autoregulation. Using a point mutation COUP-TFI mutant unable to bind DNA demonstrates that enhancement of rtER gene autoregulation requires the interaction of COUP-TFI to the DNA. Moreover, this enhancement of transcriptional activation by COUP-TFI requires specifically the AF-1 transactivation function of ER and can be observed in the presence of E2 or 4-hydroxytamoxifen but not ICI 164384. Thus, this paper describes the reconstitution of a hormone-Responsive transcription unit in yeast in which the regulation of rtER gene promoter could be enhanced by the participation of cis-Elements and/or trans-acting factors, such as ER itself or COUP-TF.
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CHARACTERIZATION OF AN Estrogen-Responsive Element IMPLICATED IN REGULATION OF THE RAINBOW TROUT Estrogen RECEPTOR GENE
Journal of molecular endocrinology, 1995Co-Authors: Y. Le Drean, Gwendal Lazennec, Laurence Kern, Dany Saligaut, Farzad Pakdel, Yves ValotaireAbstract:We previously reported that the expression of the rainbow trout Estrogen receptor (rtER) gene is markedly increased by estradiol (E2). In this paper, we have used transient transfection assays with reporter plasmids expressing chloramphenicol acetyl transferase (CAT), linked to 5' flanking regions of the rtER gene promoter, to identify cis-Elements responsible for E2 inducibility. Deletion analysis localized an Estrogen-Responsive Element (ERE), at position +242, with one mutation on the first base compared with the consensus sequence. This Element confers Estrogen Responsiveness to CAT reporter linked to both the herpes simplex virus thymidine kinase promoter and the homologous rtER promoter. Moreover, using a 0.2 kb fragment of the rtER promoter encompassing the ERE and the rtER DNA binding domain obtained from a bacterial expression system, DNase I footprinting experiments demonstrated a specific protection covering 20 bp (+240/+260) containing the ERE sequence. Based on these studies, we believe that this ERE sequence, identified in the rtER gene promoter, may be a major cis-acting Element involved in the regulation of the gene by Estrogen.
Farzad Pakdel - One of the best experts on this subject based on the ideXlab platform.
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Synergism between a half‐site and an imperfect Estrogen‐Responsive Element, and cooperation with COUP‐TFI are required for Estrogen receptor (ER) to achieve a maximal Estrogen‐stimulation of rainbow trout ER gene
European journal of biochemistry, 1999Co-Authors: Fabrice G Petit, Yves Valotaire, Raphael Metivier, Farzad PakdelAbstract:In all oviparous, liver represents one of the main E2-target tissues where Estrogen receptor (ER) constitutes the key mediator of Estrogen action. The rainbow trout Estrogen receptor (rtER) gene expression is markedly up-regulated by Estrogens and the sequences responsible for this autoregulation have been located in a 0.2 kb upstream transcription start site within - 40/- 248 enhancer region. Absence of interference with steroid hormone receptors and tissue-specific factors and a conserved basal transcriptional machinery between yeast and higher eukaryotes, make yeast a simple assay system that will enable determination of important cis-acting regulatory sequences within rtER gene promoter and identification of transcription factors implicated in the regulation of this gene. Deletion analysis allowed to show a synergistic effect between an imperfect Estrogen-Responsive Element (ERE) and a consensus half-ERE to achieve a high hormone-dependent transcriptional activation of the rtER gene promoter in the presence of stably expressed rtER. As in mammalian cells, here we observed a positive regulation of the rtER gene promoter by the chicken ovalbumin upstream promoter-transcription factor I (COUP-TFI) through enhancing autoregulation. Using a point mutation COUP-TFI mutant unable to bind DNA demonstrates that enhancement of rtER gene autoregulation requires the interaction of COUP-TFI to the DNA. Moreover, this enhancement of transcriptional activation by COUP-TFI requires specifically the AF-1 transactivation function of ER and can be observed in the presence of E2 or 4-hydroxytamoxifen but not ICI 164384. Thus, this paper describes the reconstitution of a hormone-Responsive transcription unit in yeast in which the regulation of rtER gene promoter could be enhanced by the participation of cis-Elements and/or trans-acting factors, such as ER itself or COUP-TF.
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synergism between a half site and an imperfect Estrogen Responsive Element and cooperation with coup tfi are required for Estrogen receptor er to achieve a maximal Estrogen stimulation of rainbow trout er gene
FEBS Journal, 1999Co-Authors: Fabrice G Petit, Yves Valotaire, Raphael Metivier, Farzad PakdelAbstract:In all oviparous, liver represents one of the main E2-target tissues where Estrogen receptor (ER) constitutes the key mediator of Estrogen action. The rainbow trout Estrogen receptor (rtER) gene expression is markedly up-regulated by Estrogens and the sequences responsible for this autoregulation have been located in a 0.2 kb upstream transcription start site within - 40/- 248 enhancer region. Absence of interference with steroid hormone receptors and tissue-specific factors and a conserved basal transcriptional machinery between yeast and higher eukaryotes, make yeast a simple assay system that will enable determination of important cis-acting regulatory sequences within rtER gene promoter and identification of transcription factors implicated in the regulation of this gene. Deletion analysis allowed to show a synergistic effect between an imperfect Estrogen-Responsive Element (ERE) and a consensus half-ERE to achieve a high hormone-dependent transcriptional activation of the rtER gene promoter in the presence of stably expressed rtER. As in mammalian cells, here we observed a positive regulation of the rtER gene promoter by the chicken ovalbumin upstream promoter-transcription factor I (COUP-TFI) through enhancing autoregulation. Using a point mutation COUP-TFI mutant unable to bind DNA demonstrates that enhancement of rtER gene autoregulation requires the interaction of COUP-TFI to the DNA. Moreover, this enhancement of transcriptional activation by COUP-TFI requires specifically the AF-1 transactivation function of ER and can be observed in the presence of E2 or 4-hydroxytamoxifen but not ICI 164384. Thus, this paper describes the reconstitution of a hormone-Responsive transcription unit in yeast in which the regulation of rtER gene promoter could be enhanced by the participation of cis-Elements and/or trans-acting factors, such as ER itself or COUP-TF.
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CHARACTERIZATION OF AN Estrogen-Responsive Element IMPLICATED IN REGULATION OF THE RAINBOW TROUT Estrogen RECEPTOR GENE
Journal of molecular endocrinology, 1995Co-Authors: Y. Le Drean, Gwendal Lazennec, Laurence Kern, Dany Saligaut, Farzad Pakdel, Yves ValotaireAbstract:We previously reported that the expression of the rainbow trout Estrogen receptor (rtER) gene is markedly increased by estradiol (E2). In this paper, we have used transient transfection assays with reporter plasmids expressing chloramphenicol acetyl transferase (CAT), linked to 5' flanking regions of the rtER gene promoter, to identify cis-Elements responsible for E2 inducibility. Deletion analysis localized an Estrogen-Responsive Element (ERE), at position +242, with one mutation on the first base compared with the consensus sequence. This Element confers Estrogen Responsiveness to CAT reporter linked to both the herpes simplex virus thymidine kinase promoter and the homologous rtER promoter. Moreover, using a 0.2 kb fragment of the rtER promoter encompassing the ERE and the rtER DNA binding domain obtained from a bacterial expression system, DNase I footprinting experiments demonstrated a specific protection covering 20 bp (+240/+260) containing the ERE sequence. Based on these studies, we believe that this ERE sequence, identified in the rtER gene promoter, may be a major cis-acting Element involved in the regulation of the gene by Estrogen.