Uterine Estrogen Receptor Interaction with EstrogenResponsive DNA Sequences in Vitro: Effects of Ligand Binding on Receptor-DNA Complexes
Receptor Biology Section Laboratory of Reproductive and Developmental Toxicology National Institute of Environmental Health Sciences Research Triangle Park, North Carolina 27709
Interaction between the mouse estrogen receptor (ER) and its responsive element (ERE) were examined in vitro using tissue extracts and an oligonucleotide containing a Vitellogenin A2 ERE (VRE) in a gel retardation assay. Three specific complexes were formed when nuclear extracts were prepared from estrogen agonist-treated uteri or when ligandfree ER from cytosol was used. ER protein was associated with the three complexes, as demonstrated by their ability to bind anti-ER antibody H222. Specific complexes were formed only when double stranded nonheat-denatured VRE was used. Mutation of the ERE in VRE abolished specific binding. The complexes formed by nuclear extracts were qualitatively identical when obtained from mice treated with a variety of estrogenic compounds with different potencies. Nuclear extracts obtained from mice treated with an estrogen antagonist LY117018 formed complexes that migrated slower than the complexes formed by the other estrogenic compounds examined. The dissociation rates (k^) and equilibrium dissociation constants (Kd) of the ERVRE complexes formed by estradiol- or estriolbound ER or ligand-free ER were measured and were found to be very similar, although the ligandfree specific ER complexes associate and dissociate less rapidly than those of estradiol- or estriolbound ER. In addition, one of the specific complexes formed by the estriol nuclear extract dissociated more slowly than the equivalent estradiol complex. Heat activating ligand-free ER did not increase its binding to VRE. It appears that the ligand-binding domain of the ER does not exert its regulatory effects at the level of sequence-specific DNA binding, since its occupancy does not alter binding to the ERE. The subtle differences we observed in association and dissociation of multiple complexes when unoccupied ER and ER bound to ligands of varying
potencies are compared may reflect differences in ER association with other protein factors that govern transcriptional activity. (Molecular Endocrinology 4: 276-286, 1990) INTRODUCTION Estrogen and its many agonists exert a variety of effects on their target tissues. These effects are mediated at the molecular level by the estrogen receptor (ER), a molecule with high affinity and specificity for binding both the estrogen ligand and specific estrogenresponsive enhancer (ERE) DNA sequences adjacent to estrogen-responsive genes. The human ER expressed in transfected cell lines has been shown to undergo a hormone-induced dimerization that allows it to bind ERE in vitro (1), which supports the model of steroid hormone action in which ligand binding increases a steroid receptor's affinity for specific enhancer sequences and, thus, activates transciption. An ongoing interest in our laboratory has been the physiological and biochemical activities of estrogen agonists. We have previously studied the relationship between ER binding and hormonal activities of steroidal (2) and stilbesterol (3, 4) estrogens. It has been shown that the impeded estrogen, estriol (E3), produces only a transient occupancy of the nuclear ER. In addition, recently we have shown that treatment with estradiol (E2) resulted in formation of a nuclear specific heterologous doublet form of the ER, which was maintained for several hours (4). We observed that the weak estrogens (E3 or 16aE2) initially produced, but did not maintain, this doublet form of the ER, which was lost after 30 min (5). Because of this observation and in light of the current model of estrogen action, we were interested in determining whether weak estrogens differ from more potent estrogens in their ability to confer high affinity binding of the ER to its enhancer sequence. To examine this, we characterized the interaction between ER bound to various estrogens and a synthetic oligonucleotide con-
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Sylvia W. Curtis and Kenneth S. Korach
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Effects of Ligand on ER-ERE Complexes
estrogen (6). This sequence allowed only complex D to form after incubation with ENE (Fig. 1, lane 9) as well as LNE and CNE (lanes 10 and 11), indicating that formation of complexes A, B, and C requires a functional enhancer sequence, and that the nonspecific nonER-containing complex D is formed by either a nonspecific DNA-binding protein or a protein that binds the flanking sequences of the oligonucleotides. The specificity for a functional ERE was confirmed by showing that ER-VRE complexes could not be displaced with a 100-fold excess of unlabeled NRE (not shown). ER Binds Double Stranded VRE
RESULTS Detection of ER-VRE in Nuclear Extracts Nuclear extracts were combined with a 32P-labeled 56basepair synthetic oligonucleotide that contained the Xenopus vitellogenin A2 ERE (VRE), and protein-DNA complexes formed were separated from free VRE on native polyacrylamide gel electorphoresis (PAGE). Four retarded VRE complexes (A, B, C, and D) were detected after VRE was added to a 0.4-M KCI uterine nuclear extract from E2-treated mice (ENE). Complexes A, B, and C were eliminated by competition with a 10O-fold excess of unlabeled VRE (Fig. 1, lanes 1 and 2), while complex D remained, indicating that only A, B, and C are formed by a sequence-specific interaction with protein in ENE. Nuclear extract obtained from lung (LNE), a nontarget tissue containing no ER, formed only complex D, which could not be eliminated with a 100-fold excess of unlabeled VRE (lanes 3 and 4), suggesting that this complex was nonspecific. A similar result was seen when ER was immunoabsorbed from uterine control nuclear extracts (CNE) before incubation with VRE (lane 5). These results indicate that formation of complexes A, B, and C requires the presence of ER. To determine whether complexes A, B, and C contain ER, ENE was incubated with anti-ER antibody (H222 or H226) before the addition of labeled VRE. If A, B, and C complexes contain ER protein, then binding of antibody to ER will result in a larger complex and, thus, a shift to a slower migrating complex. As can be seen in Fig. 1, lanes 6 and 8, complexes A, B, and C were replaced by a single slower migrating complex. Preincubation of ENE with anti-Mac3, a control monoclonal antibody of the same type as H222, had no effect on the migration of complexes A, B, and C (lane 7), indicating that the shift seen with H222 and H226 is caused by specific ER-antibody binding. In addition, a Western blot of a VRE-binding reaction probed with H222 showed ER protein bands comigrating with the bands of complexes B and C (Fig. 2). ER protein coincident with band A was not observed, but was at a level below detectability with the antibody. To show that the complexes were specific to an ERE, ENE was incubated with 32P-labeled NRE, a sequence identical to VRE with two-point mutations that have been shown to render the sequence nonresponsive to
Recent studies have indicated that ER binds selectively to the coding strand of a heat-denatured 250-basepair fragment of the rat PRL enhancer (7). We wished to examine whether the multiple complexes formed with the uterine ENE resulted from the ER binding selectively to either strand of the VRE. VRE was heat denatured immediately before incubation with ENE. As can be seen in Fig. 3, lane 2, four complexes are formed (A', B', C , and D'), but only A' and B' appear to comigrate with complexes formed by double stranded VRE (complexes B and C, respectively). Complexes B', C , and D' are formed by CNE (lane 3), indicating that ER is required only for the formation of complex A'. This indication is confirmed when ENE is preincubated with H222, resulting in the decreased mobility of complex A' only, showing that it alone contains ER (lane 4). It should be noted that heat-denatured VRE does contain some double stranded VRE (not shown), which may be responsible for the formation of A'. Addition of heatdenatured VRE to LNE allows the formation of complexes C and D' only (lane 5). Complex B' may contain some tissue-specific single strand DNA-binding proteins that are not found in LNE. However, it is doubtful that the factors responsible for formation of nonspecific complexes C or D' have any significance to estrogen responsiveness, as incubation with heat-denatured NRE results in the formation of nonspecific complexes C and D' (lanes 6 and 7). To rule out the possibility that ER somehow "melts" VRE and binds to a specific strand, DNA was isolated from complexes B and C formed with unmelted VRE. It contains both strands of VRE in a double stranded form (Fig. 4). There was not enough complex A present to isolate and analyze its VRE. ER-VRE Can Be Formed by ER Bound to Other Estrogens To determine if there are any differences in the abilities of various estrogenic compounds to allow ER to bind its enhancer sequence, nuclear extracts were prepared from animals treated with potent estrogen agonist (E2), weak estrogen agonists (E3 and 16a-E2), stilbesterol compounds [diethylstilbestrol (DES), indenestrol-A or B, and pseudo-DES], or an estrogen antagonist (LY117018). As can be seen in Fig. 5, all of the agonistic
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taining the ERE sequence from the vitellogenin A2 gene. We examined the affinity and dissociation rates of the complexes formed by this interaction. These studies failed to detect an effect of ligand potency on ER interaction with ERE. In addition, ligand-free receptor exhibited high affinity for binding ERE, raising the possibility that the interaction between ER and its enhancer is not dependent on hormone, and that hormonal effects are mediated at another level, such as interaction between the ER and transcription factors or nuclear acceptor sites.
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Fig. 1. VRE Binding Activity in Nuclear Extracts ENE or LNE from E2-treated mice or nuclear extracts that were ER depleted by immunoabsorbtion (CNE) were incubated with 32 P-labeled VRE (lanes 1-8) or NRE, which contains a mutant ERE (lanes 9-11). Reactions 2 and 4 also contained a 100-fold molar excess of uniabeled VRE. Reactions were carried out as described in Materials and Methods, and free VRE was separated from VRE-protein complexes by electrophoresis through a nondenaturing polyacrylamide gel. ENE was preincubated with monoclonal antibodies H222 in lane 6, H226 in lane 8, or control antibody anti-Mac3 in lane 7. Lane 12 contains VRE alone.
1
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Fig. 2. Western Blot of ENE and VRE ENE was reacted with uniabeled VRE (lane 1) or 32P-labeled VRE (lane 2) and separated on native PAGE. Lane 1 was electroblotted to nitrocellulose and probed with H222.
compounds produced complexes that are qualitatively identical, despite the variation in their biological potency (lanes 1-7). It appears that at the level of interaction with isolated vitellogenln ERE, the potency of estrogenic agonists has no discemable effect. However, the estrogen antagonist LY117018 (lane 9) produced complexes with slower mobilities than those of the estrogen agonists. Ligand-Free ER Binds VRE Because nuclear extracts from animals treated with various estrogen agonists appeared to form the same
complexes with VRE, we were interested in examining the effect of ligand binding on the ability of the ER protein to bind DNA. Ligand-free ER found in cytosol preparations from ovariectomized mouse uterus (before or after concentration with 40% ammonium sulfate) was incubated with VRE, which resulted in the formation of five complexes (A, B, C, D, and E; Fig. 6, lanes 1, 3, and 6). The ER in the cytosol was shown to be 100% unoccupied by [3H]E2 exchange binding assay. Only complexes A, B, and C, similar to those formed by the uterine ENE (lane 2), could be shifted to a slower migrating form after preincubation with H222 (Fig. 6, lane 4), which indicated that only complexes A, B, and C contain ER. In addition, incubation with NRE, which contains a mutation in its ERE sequence, resulted in the formation of complexes D and E only (lane 5), indicating that these are nonspecific complexes that do not require a functional ERE sequence to bind the DNA. To determine if binding of hormone could increase binding to VRE, a 40% ammonium sulfate fraction of uterine cytosol (UC) was incubated at 4 C with 37 nM E2 for 2 h. This treatment did not result in increased VRE binding and may have caused a slight decrease in VRE binding (compare lanes 6 and 8). It appears that the VRE binding seen in vitro with UC is not hormone dependent. Earlier studies have shown that ligand-free ER isolated from cytosol could be heat treated to a form termed activated ER that has an increased affinity for DNA cellulose (8). To examine whether this activation has an effect on ER binding to VRE, UC was heated for 15 min to 30 C before incubation with VRE. This
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H222 + ENE CNE ENE LNE ENE LNE + + + + Heat Denatured + +
Fig. 3. Binding to Heat-Denatured VRE ENE (lanes 1, 2, 4, and 6), CNE (lane 3), or LNE (lanes 5 and 7) was reacted with VRE (lane 1), VRE that had been heat denatured (lanes 2-5), or NRE that was heat denatured (lanes 6 and 7). ENE in lane 4 was preincubated with H222. Lanes 8 and 9 contain VRE and heat-denatured VRE alone, respectively.
1 Heat Denatured
i Fig. 4. Composition of VRE in Complexes B and C DNA was isolated from PAGE slices containing complexes B (lane 4), C (lane 3), or free VRE (lane 2). The DNA was loaded directly onto PAGE or heat treated before PAGE. Lane 1 contains VRE that was not reacted with ENE.
treatment had no effect on VRE binding (Fig. 6, lane 9), indicating no change in ER affinity for a specific DNA sequence. When UC was bound to E2 before heat treatment, all specific VRE binding was abolished (Fig. 6, lane 10), indicating a blockage of the DNA-binding site. The nonspecific bands were still detected and increased slightly in intensity. Sodium dodecyl sulfatePAGE (SDS-PAGE) analysis of the receptor protein in these preparations indicated that no proteolysis had occurred (data not presented), eliminating the possibility that heat treatment resulted in proteolysis of the ER to a non-DNA-binding form. Binding and heat activating
the preformed UC-VRE complex potentiate the formation of specific complexes by cytosol receptor (lane 11), indicating that VRE can be included in the structure formed when UC is bound to E2 and heated. Specific complexes are not formed when preformed NRE complexes are bound to hormone and heat treated (lane 13), showing that the increased binding requires a specific DNA sequence interaction. Heating the preformed UC-VRE complexes in the absence of E2 also potentiates formation of specific complexes (lane 12). Further analysis has revealed that the ligand-free ER associates with the VRE at a slower rate than ligand-
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Fig. 6. Ligand-Free ER-VRE-Binding Activity Ligand-free ER was obtained from UC and determined to be 100% unoccupied by [3H]E2 binding. Cytosol was reacted with VRE before (lane 1) or after (lanes 3, 4, and 6-12) concentration with 40% ammonium sulfate. Lane 2 contains ENE instead of cytosol, and reactions 5 and 13 contain the control DNA NRE instead of VRE. Reaction 4 was preincubated with H222. Reaction 7 contained a 100-fold excess of unlabeled VRE. Reactions containing E2 were incubated with 37 nM E2 for 2 h at 4 C before (lanes 8 and 10) or after (lanes 11 and 13) incubation with DNA. Heat-activated reactions were incubated for 15 min at 30 C before (lanes 9 and 10) or after (lanes 11-13) incubation with DNA.
bound ER, so that the potentiation seen upon heat activation in the presence of VRE may result from an increased association rate at an elevated temperature. These results indicate that ER is capable of associating with its enhancer sequence in the absence of hormone.
Dissociation Rates of ER-VRE Complexes To probe the quantitative effects of estrogen ligand on ER-VRE interaction, the dissociation rates of VRE complexes formed by ER bound to different estrogens were
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Fig. 5. VRE Complexes Formed by ER Bound to Various Estrogen Agonists and Antagonists Nuclear extract was prepared from mice that had been treated for 1 h with 20 /xg/kg E2 (lanes 1 and 8), DES (lane 2), indenestrolA (lane 5), indenestrol-B (lane 6), or pseudo-DES (lane 7); for 20 min with 20 ^g/kg E3 (lane 3) or 16a-E2 (lane 4); or for 30 min with 150 Mg/kg LY117018 (lane 9). Extracts were reacted with 32P-labeled VRE in the absence (-) or presence (+) of a 100-fold excess of unlabeled VRE.
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Effects of Ligand on ER-ERE Complexes
min, complex B with a t./2 of 40-45 min, and complex C with an initial t./2 of 30 min, followed by a slower dissociation with a ty2 of 50 min. Affinity of ER for VRE
To determine whether complexes containing potent or weak estrogens have different affinities for VRE, the K, of E2- or E3-bound ER for VRE was measured. Increasing amounts of VRE were added, and specific binding was measured. Scatchard analysis (Fig. 9) showed that the Kc for ER bound to either E2 or E3 are virtually the same; the K
Receptor Biology Section Laboratory of Reproductive and Developmental Toxicology National Institute of Environmental Health Sciences Research Triangle Park, North Carolina 27709
Interaction between the mouse estrogen receptor (ER) and its responsive element (ERE) were examined in vitro using tissue extracts and an oligonucleotide containing a Vitellogenin A2 ERE (VRE) in a gel retardation assay. Three specific complexes were formed when nuclear extracts were prepared from estrogen agonist-treated uteri or when ligandfree ER from cytosol was used. ER protein was associated with the three complexes, as demonstrated by their ability to bind anti-ER antibody H222. Specific complexes were formed only when double stranded nonheat-denatured VRE was used. Mutation of the ERE in VRE abolished specific binding. The complexes formed by nuclear extracts were qualitatively identical when obtained from mice treated with a variety of estrogenic compounds with different potencies. Nuclear extracts obtained from mice treated with an estrogen antagonist LY117018 formed complexes that migrated slower than the complexes formed by the other estrogenic compounds examined. The dissociation rates (k^) and equilibrium dissociation constants (Kd) of the ERVRE complexes formed by estradiol- or estriolbound ER or ligand-free ER were measured and were found to be very similar, although the ligandfree specific ER complexes associate and dissociate less rapidly than those of estradiol- or estriolbound ER. In addition, one of the specific complexes formed by the estriol nuclear extract dissociated more slowly than the equivalent estradiol complex. Heat activating ligand-free ER did not increase its binding to VRE. It appears that the ligand-binding domain of the ER does not exert its regulatory effects at the level of sequence-specific DNA binding, since its occupancy does not alter binding to the ERE. The subtle differences we observed in association and dissociation of multiple complexes when unoccupied ER and ER bound to ligands of varying
potencies are compared may reflect differences in ER association with other protein factors that govern transcriptional activity. (Molecular Endocrinology 4: 276-286, 1990) INTRODUCTION Estrogen and its many agonists exert a variety of effects on their target tissues. These effects are mediated at the molecular level by the estrogen receptor (ER), a molecule with high affinity and specificity for binding both the estrogen ligand and specific estrogenresponsive enhancer (ERE) DNA sequences adjacent to estrogen-responsive genes. The human ER expressed in transfected cell lines has been shown to undergo a hormone-induced dimerization that allows it to bind ERE in vitro (1), which supports the model of steroid hormone action in which ligand binding increases a steroid receptor's affinity for specific enhancer sequences and, thus, activates transciption. An ongoing interest in our laboratory has been the physiological and biochemical activities of estrogen agonists. We have previously studied the relationship between ER binding and hormonal activities of steroidal (2) and stilbesterol (3, 4) estrogens. It has been shown that the impeded estrogen, estriol (E3), produces only a transient occupancy of the nuclear ER. In addition, recently we have shown that treatment with estradiol (E2) resulted in formation of a nuclear specific heterologous doublet form of the ER, which was maintained for several hours (4). We observed that the weak estrogens (E3 or 16aE2) initially produced, but did not maintain, this doublet form of the ER, which was lost after 30 min (5). Because of this observation and in light of the current model of estrogen action, we were interested in determining whether weak estrogens differ from more potent estrogens in their ability to confer high affinity binding of the ER to its enhancer sequence. To examine this, we characterized the interaction between ER bound to various estrogens and a synthetic oligonucleotide con-
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276
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Effects of Ligand on ER-ERE Complexes
estrogen (6). This sequence allowed only complex D to form after incubation with ENE (Fig. 1, lane 9) as well as LNE and CNE (lanes 10 and 11), indicating that formation of complexes A, B, and C requires a functional enhancer sequence, and that the nonspecific nonER-containing complex D is formed by either a nonspecific DNA-binding protein or a protein that binds the flanking sequences of the oligonucleotides. The specificity for a functional ERE was confirmed by showing that ER-VRE complexes could not be displaced with a 100-fold excess of unlabeled NRE (not shown). ER Binds Double Stranded VRE
RESULTS Detection of ER-VRE in Nuclear Extracts Nuclear extracts were combined with a 32P-labeled 56basepair synthetic oligonucleotide that contained the Xenopus vitellogenin A2 ERE (VRE), and protein-DNA complexes formed were separated from free VRE on native polyacrylamide gel electorphoresis (PAGE). Four retarded VRE complexes (A, B, C, and D) were detected after VRE was added to a 0.4-M KCI uterine nuclear extract from E2-treated mice (ENE). Complexes A, B, and C were eliminated by competition with a 10O-fold excess of unlabeled VRE (Fig. 1, lanes 1 and 2), while complex D remained, indicating that only A, B, and C are formed by a sequence-specific interaction with protein in ENE. Nuclear extract obtained from lung (LNE), a nontarget tissue containing no ER, formed only complex D, which could not be eliminated with a 100-fold excess of unlabeled VRE (lanes 3 and 4), suggesting that this complex was nonspecific. A similar result was seen when ER was immunoabsorbed from uterine control nuclear extracts (CNE) before incubation with VRE (lane 5). These results indicate that formation of complexes A, B, and C requires the presence of ER. To determine whether complexes A, B, and C contain ER, ENE was incubated with anti-ER antibody (H222 or H226) before the addition of labeled VRE. If A, B, and C complexes contain ER protein, then binding of antibody to ER will result in a larger complex and, thus, a shift to a slower migrating complex. As can be seen in Fig. 1, lanes 6 and 8, complexes A, B, and C were replaced by a single slower migrating complex. Preincubation of ENE with anti-Mac3, a control monoclonal antibody of the same type as H222, had no effect on the migration of complexes A, B, and C (lane 7), indicating that the shift seen with H222 and H226 is caused by specific ER-antibody binding. In addition, a Western blot of a VRE-binding reaction probed with H222 showed ER protein bands comigrating with the bands of complexes B and C (Fig. 2). ER protein coincident with band A was not observed, but was at a level below detectability with the antibody. To show that the complexes were specific to an ERE, ENE was incubated with 32P-labeled NRE, a sequence identical to VRE with two-point mutations that have been shown to render the sequence nonresponsive to
Recent studies have indicated that ER binds selectively to the coding strand of a heat-denatured 250-basepair fragment of the rat PRL enhancer (7). We wished to examine whether the multiple complexes formed with the uterine ENE resulted from the ER binding selectively to either strand of the VRE. VRE was heat denatured immediately before incubation with ENE. As can be seen in Fig. 3, lane 2, four complexes are formed (A', B', C , and D'), but only A' and B' appear to comigrate with complexes formed by double stranded VRE (complexes B and C, respectively). Complexes B', C , and D' are formed by CNE (lane 3), indicating that ER is required only for the formation of complex A'. This indication is confirmed when ENE is preincubated with H222, resulting in the decreased mobility of complex A' only, showing that it alone contains ER (lane 4). It should be noted that heat-denatured VRE does contain some double stranded VRE (not shown), which may be responsible for the formation of A'. Addition of heatdenatured VRE to LNE allows the formation of complexes C and D' only (lane 5). Complex B' may contain some tissue-specific single strand DNA-binding proteins that are not found in LNE. However, it is doubtful that the factors responsible for formation of nonspecific complexes C or D' have any significance to estrogen responsiveness, as incubation with heat-denatured NRE results in the formation of nonspecific complexes C and D' (lanes 6 and 7). To rule out the possibility that ER somehow "melts" VRE and binds to a specific strand, DNA was isolated from complexes B and C formed with unmelted VRE. It contains both strands of VRE in a double stranded form (Fig. 4). There was not enough complex A present to isolate and analyze its VRE. ER-VRE Can Be Formed by ER Bound to Other Estrogens To determine if there are any differences in the abilities of various estrogenic compounds to allow ER to bind its enhancer sequence, nuclear extracts were prepared from animals treated with potent estrogen agonist (E2), weak estrogen agonists (E3 and 16a-E2), stilbesterol compounds [diethylstilbestrol (DES), indenestrol-A or B, and pseudo-DES], or an estrogen antagonist (LY117018). As can be seen in Fig. 5, all of the agonistic
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taining the ERE sequence from the vitellogenin A2 gene. We examined the affinity and dissociation rates of the complexes formed by this interaction. These studies failed to detect an effect of ligand potency on ER interaction with ERE. In addition, ligand-free receptor exhibited high affinity for binding ERE, raising the possibility that the interaction between ER and its enhancer is not dependent on hormone, and that hormonal effects are mediated at another level, such as interaction between the ER and transcription factors or nuclear acceptor sites.
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Fig. 1. VRE Binding Activity in Nuclear Extracts ENE or LNE from E2-treated mice or nuclear extracts that were ER depleted by immunoabsorbtion (CNE) were incubated with 32 P-labeled VRE (lanes 1-8) or NRE, which contains a mutant ERE (lanes 9-11). Reactions 2 and 4 also contained a 100-fold molar excess of uniabeled VRE. Reactions were carried out as described in Materials and Methods, and free VRE was separated from VRE-protein complexes by electrophoresis through a nondenaturing polyacrylamide gel. ENE was preincubated with monoclonal antibodies H222 in lane 6, H226 in lane 8, or control antibody anti-Mac3 in lane 7. Lane 12 contains VRE alone.
1
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Fig. 2. Western Blot of ENE and VRE ENE was reacted with uniabeled VRE (lane 1) or 32P-labeled VRE (lane 2) and separated on native PAGE. Lane 1 was electroblotted to nitrocellulose and probed with H222.
compounds produced complexes that are qualitatively identical, despite the variation in their biological potency (lanes 1-7). It appears that at the level of interaction with isolated vitellogenln ERE, the potency of estrogenic agonists has no discemable effect. However, the estrogen antagonist LY117018 (lane 9) produced complexes with slower mobilities than those of the estrogen agonists. Ligand-Free ER Binds VRE Because nuclear extracts from animals treated with various estrogen agonists appeared to form the same
complexes with VRE, we were interested in examining the effect of ligand binding on the ability of the ER protein to bind DNA. Ligand-free ER found in cytosol preparations from ovariectomized mouse uterus (before or after concentration with 40% ammonium sulfate) was incubated with VRE, which resulted in the formation of five complexes (A, B, C, D, and E; Fig. 6, lanes 1, 3, and 6). The ER in the cytosol was shown to be 100% unoccupied by [3H]E2 exchange binding assay. Only complexes A, B, and C, similar to those formed by the uterine ENE (lane 2), could be shifted to a slower migrating form after preincubation with H222 (Fig. 6, lane 4), which indicated that only complexes A, B, and C contain ER. In addition, incubation with NRE, which contains a mutation in its ERE sequence, resulted in the formation of complexes D and E only (lane 5), indicating that these are nonspecific complexes that do not require a functional ERE sequence to bind the DNA. To determine if binding of hormone could increase binding to VRE, a 40% ammonium sulfate fraction of uterine cytosol (UC) was incubated at 4 C with 37 nM E2 for 2 h. This treatment did not result in increased VRE binding and may have caused a slight decrease in VRE binding (compare lanes 6 and 8). It appears that the VRE binding seen in vitro with UC is not hormone dependent. Earlier studies have shown that ligand-free ER isolated from cytosol could be heat treated to a form termed activated ER that has an increased affinity for DNA cellulose (8). To examine whether this activation has an effect on ER binding to VRE, UC was heated for 15 min to 30 C before incubation with VRE. This
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H222 + ENE CNE ENE LNE ENE LNE + + + + Heat Denatured + +
Fig. 3. Binding to Heat-Denatured VRE ENE (lanes 1, 2, 4, and 6), CNE (lane 3), or LNE (lanes 5 and 7) was reacted with VRE (lane 1), VRE that had been heat denatured (lanes 2-5), or NRE that was heat denatured (lanes 6 and 7). ENE in lane 4 was preincubated with H222. Lanes 8 and 9 contain VRE and heat-denatured VRE alone, respectively.
1 Heat Denatured
i Fig. 4. Composition of VRE in Complexes B and C DNA was isolated from PAGE slices containing complexes B (lane 4), C (lane 3), or free VRE (lane 2). The DNA was loaded directly onto PAGE or heat treated before PAGE. Lane 1 contains VRE that was not reacted with ENE.
treatment had no effect on VRE binding (Fig. 6, lane 9), indicating no change in ER affinity for a specific DNA sequence. When UC was bound to E2 before heat treatment, all specific VRE binding was abolished (Fig. 6, lane 10), indicating a blockage of the DNA-binding site. The nonspecific bands were still detected and increased slightly in intensity. Sodium dodecyl sulfatePAGE (SDS-PAGE) analysis of the receptor protein in these preparations indicated that no proteolysis had occurred (data not presented), eliminating the possibility that heat treatment resulted in proteolysis of the ER to a non-DNA-binding form. Binding and heat activating
the preformed UC-VRE complex potentiate the formation of specific complexes by cytosol receptor (lane 11), indicating that VRE can be included in the structure formed when UC is bound to E2 and heated. Specific complexes are not formed when preformed NRE complexes are bound to hormone and heat treated (lane 13), showing that the increased binding requires a specific DNA sequence interaction. Heating the preformed UC-VRE complexes in the absence of E2 also potentiates formation of specific complexes (lane 12). Further analysis has revealed that the ligand-free ER associates with the VRE at a slower rate than ligand-
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Fig. 6. Ligand-Free ER-VRE-Binding Activity Ligand-free ER was obtained from UC and determined to be 100% unoccupied by [3H]E2 binding. Cytosol was reacted with VRE before (lane 1) or after (lanes 3, 4, and 6-12) concentration with 40% ammonium sulfate. Lane 2 contains ENE instead of cytosol, and reactions 5 and 13 contain the control DNA NRE instead of VRE. Reaction 4 was preincubated with H222. Reaction 7 contained a 100-fold excess of unlabeled VRE. Reactions containing E2 were incubated with 37 nM E2 for 2 h at 4 C before (lanes 8 and 10) or after (lanes 11 and 13) incubation with DNA. Heat-activated reactions were incubated for 15 min at 30 C before (lanes 9 and 10) or after (lanes 11-13) incubation with DNA.
bound ER, so that the potentiation seen upon heat activation in the presence of VRE may result from an increased association rate at an elevated temperature. These results indicate that ER is capable of associating with its enhancer sequence in the absence of hormone.
Dissociation Rates of ER-VRE Complexes To probe the quantitative effects of estrogen ligand on ER-VRE interaction, the dissociation rates of VRE complexes formed by ER bound to different estrogens were
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Fig. 5. VRE Complexes Formed by ER Bound to Various Estrogen Agonists and Antagonists Nuclear extract was prepared from mice that had been treated for 1 h with 20 /xg/kg E2 (lanes 1 and 8), DES (lane 2), indenestrolA (lane 5), indenestrol-B (lane 6), or pseudo-DES (lane 7); for 20 min with 20 ^g/kg E3 (lane 3) or 16a-E2 (lane 4); or for 30 min with 150 Mg/kg LY117018 (lane 9). Extracts were reacted with 32P-labeled VRE in the absence (-) or presence (+) of a 100-fold excess of unlabeled VRE.
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Effects of Ligand on ER-ERE Complexes
min, complex B with a t./2 of 40-45 min, and complex C with an initial t./2 of 30 min, followed by a slower dissociation with a ty2 of 50 min. Affinity of ER for VRE
To determine whether complexes containing potent or weak estrogens have different affinities for VRE, the K, of E2- or E3-bound ER for VRE was measured. Increasing amounts of VRE were added, and specific binding was measured. Scatchard analysis (Fig. 9) showed that the Kc for ER bound to either E2 or E3 are virtually the same; the K
