Advertisement

Equilibrium Binding of Estrogen Receptor with DNA Using Fluorescence Anisotropy*

Open AccessPublished:November 28, 1997DOI:https://doi.org/10.1074/jbc.272.48.30405
      Interaction of estrogen receptor (ER) with DNA sequences known as estrogen response elements (ERE) is required for estrogen regulation of the expression of target genes. To characterize the affinity and specificity of ER interaction with ERE sequencesin vitro under equilibrium conditions, fluorescence anisotropy assays were performed using recombinant, purified ER and a fluorescein-labeled 35-base pair oligonucleotide bearing an idealized palindromic ERE. In buffer containing 100 mm KCl, the baculovirus-expressed, purified human ER bound with similar affinity to the consensus ERE and a mutant ERE with a single base pair change per half-site. Above 225 mm KCl, ER exhibited discrimination between the consensus and mutated ERE targets. Between 225 and 275 mm KCl, binding to the consensus ERE was independent of salt concentration and occurred with an equilibrium dissociation constant (K d ) of 1.8 ± 0.6 nm, whereas binding to the mutant ERE was not detected at ER concentrations below 100 nm under the same conditions. At 300 mm KCl, the K d for the consensus ERE increased approximately 25-fold, suggesting complex salt concentration dependence. Both estrogen-occupied and unoccupied ER bound to the consensus ERE sequence with similar affinity, indicating that estrogen affects ER activity at a step other than DNA binding. Unlike the full-length ER, the recombinant DNA binding domain of ER did not discriminate between the consensus and mutated ERE sequences even at buffer salt concentrations greater than 200 mm NaCl, suggesting that ER sequences outside the DNA binding domain may be important in promoting specific binding.
      As a member of the superfamily of steroid hormone receptors, the estrogen receptor (ER)
      The abbreviations used are: ER, estrogen receptor; ERE, estrogen response element; E2, 17β-estradiol; ER-E2, estradiol-occupied estrogen receptor; vitERE, vitellogenin estrogen response element; mutERE, mutated estrogen response element; bp, base pair(s); DBD, estrogen receptor DNA binding domain; DTT, dithiothreitol; F-vitERE, fluorescein-labeled vitellogenin estrogen response element; F-mutERE, fluorescein-labeled mutated estrogen response element; BSA, bovine serum albumin.
      1The abbreviations used are: ER, estrogen receptor; ERE, estrogen response element; E2, 17β-estradiol; ER-E2, estradiol-occupied estrogen receptor; vitERE, vitellogenin estrogen response element; mutERE, mutated estrogen response element; bp, base pair(s); DBD, estrogen receptor DNA binding domain; DTT, dithiothreitol; F-vitERE, fluorescein-labeled vitellogenin estrogen response element; F-mutERE, fluorescein-labeled mutated estrogen response element; BSA, bovine serum albumin.
      is a 67-kDa nuclear protein that regulates transcription for genes involved in cellular differentiation, development of the female reproductive system, and homeostasis (
      • Evans R.M.
      , ,
      • Lundeen S.
      • Anderson I.
      • Gray W.
      • Gorski J.
      ). ER binds to specific DNA sequences designated estrogen response elements (ERE), which are present in promoters of target genes. The consensus ERE is a 13-base pair (bp) palindromic sequence, consisting of inverted repeats of the half-sites 5′-GGTCA-3′ separated by a 3-bp spacer, found in the 5′-flanking region of the Xenopus and chicken vitellogenin A2 genes (
      • Walker P.
      • Germond J.-E.
      • Brown-Luedi M.
      • Givel F.
      • Wahli W.
      ,
      • Klein-Hitpass L.
      • Schorpp M.
      • Wagner U.
      • Ryffel G.U.
      ,
      • Klein-Hitpass L.
      • Ryffel G.U.
      • Heitlinger E.
      • Cato A.C.B.
      ). ER consists of conserved structural and functional domains (Fig.1) for DNA-binding in region C, nuclear localization in region D, and hormone-binding in region E (
      • Krust A.
      • Green S.
      • Argos P.
      • Kumar V.
      • Walter P.
      • Bornert J.-M.
      • Chambon P.
      ,
      • Kumar V.
      • Green S.
      • Stack G.
      • Berry M.
      • Jin J.-R.
      • Chambon P.
      ). Two transcriptional activation function domains, designated TAF-1 and TAF-2, are found in regions A/B and E, respectively (
      • Kumar V.
      • Green S.
      • Stack G.
      • Berry M.
      • Jin J.-R.
      • Chambon P.
      ,
      • Webster N.J.G.
      • Green S.
      • Jin J.R.
      • Chambon P.
      ,
      • Lees J.A.
      • Fawell S.E.
      • Parker M.G.
      ,
      • Tora L.
      • White J.
      • Brou C.
      • Tasset D.
      • Webster N.
      • Scheer E.
      • Chambon P.
      ). The physiological ligand for ER is 17β-estradiol (E2), which diffuses into target cells and binds to ER. Binding of E2to ER induces conformational changes in the receptor (
      • Hansen J.C.
      • Gorski J.
      ,
      • Fritsch M.
      • Leary C.M.
      • Furlow J.D.
      • Ahrens H.
      • Schuh T.J.
      • Mueller G.C.
      • Gorski J.
      ,
      • Fritsch M.
      • Anderson I.
      • Gorski J.
      ) and dissociation of receptor-associated proteins including hsp90 and p59 (
      • Joab I.
      • Radanyi C.
      • Renoir M.
      • Buchou T.
      • Catalli M.-G.
      • Binart N.
      • Mester J.
      • Baulieu E.-E.
      ,
      • Sanchez E.R.
      • Faber L.E.
      • Henzel W.J.
      • Pratt W.B.
      ), leading to the assembly of an active transcriptional complex responsible for the positive and negative regulation of target genes.
      Figure thumbnail gr1
      Figure 1The structural and functional domains of human ER. Domains A/B and E contain transcriptional activation function sequences, designatedTAF-1 and TAF-2. The highly conserved DNA binding domain is located in region C. Domain D is known as the hinge region and also contains the antigenic site for antibody ER712. The ligand binding domain is found in region E.
      One of the principal requirements for E2-induced regulation of target genes is the specific association of ER with the ERE. The ability of unpurified ER from rat uterine cytosolic extracts to discriminate the consensus ERE from other DNA sequences was examined by using an avidin-biotin complexed with DNA assay to quantify the binding interaction (
      • Murdoch F.E.
      • Grunwald K.A.A.
      • Gorski J.
      ). These assays indicated that ER recognized the vitellogenin ERE (vitERE) with an affinity that was three orders of magnitude greater than that for a mutated form of the ERE (mutERE) bearing 1 base change per half-site in a salt-dependent manner. This unpurified ER also bound to the vitERE with an affinity that was five orders of magnitude greater than that for plasmid DNA. The highest level of discrimination was found with binding buffer containing physiological levels of salt (
      • Murdoch F.E.
      • Grunwald K.A.A.
      • Gorski J.
      ). The ability of purified ER to discriminate between ERE sequences in the absence of other proteins at various salt concentrations has not been examined in such detail under equilibrium conditions.
      The amino acid sequences in the DNA binding domain (DBD) of ER that are necessary for specific interaction of the ER with ERE have been located in the N-terminal zinc finger (
      • Danielsen M.
      • Hinck L.
      • Ringold G.M.
      ,
      • Mader S.
      • Kumar V.
      • de Verneuil H.
      • Chambon P.
      ) and verified by the crystal structure of the DBD bound to the ERE (
      • Schwabe J.W.R.
      • Chapman L.
      • Finch J.T.
      • Rhodes D.
      ). Although a great deal of evidence has been accrued for the ER DBD, there are several functional differences noted between the DBD fragment and the full-length receptor. The DBD binds to the ERE with lower affinity than the full-length ER (
      • Nardulli A.M.
      • Lew D.
      • Erijman L.
      • Shapiro D.J.
      ). The DNA bending induced by ER DBD binding was found to be 34°, rather than the 56° bending detected for the full-length receptor (
      • Nardulli A.M.
      • Shapiro D.J.
      ,
      • Nardulli A.M.
      • Greene G.L.
      • Shapiro D.J.
      ). Studies using a construct consisting of the DBD from region C plus sequences from region D indicated that this larger protein fragment bound to imperfect ERE sequences more stably than the DBD alone (
      • Mader S.
      • Kumar V.
      • de Verneuil H.
      • Chambon P.
      ). Thus, these results suggest that protein sequences other than those present in the DBD may be important for high affinity binding of the ER to the ERE.
      The role of E2 in the ability of ER to bind to DNA has been controversial. Previous models of E2 action suggested that hormone binding induced interaction of ER with DNA. Several studies using gel shift analysis and quantitative DNA binding assays have indicated that E2-occupied and unoccupied ER from rat uterine extracts bind to ERE sequences with similar affinity (
      • Murdoch F.E.
      • Meier D.A.
      • Furlow J.D.
      • Grunwald K.A.A.
      • Gorski J.
      ,
      • Furlow J.D.
      • Murdoch F.E.
      • Gorski J.
      ). Ligand-independent binding of human ER to ERE sequences has also been reported in transiently transfected cells (
      • Reese J.C.
      • Katzenellenbogen B.S.
      ) and in whole cells containing endogenous ER (
      • Reese J.C.
      • Katzenellenbogen B.S.
      ). However, it has also been shown that human ER expressed from a recombinant baculovirus and produced byin vitro transcription/translation requires E2for DNA binding (
      • Beekman J.M.
      • Allan G.F.
      • Tsai S.Y.
      • Tsai M.-J.
      • O'Malley B.W.
      ).
      In these studies, we used fluorescence anisotropy to examine the specificity and affinity of the ER-ERE interaction under equilibrium conditions. The ability of the full-length purified ER and ER DBD to discriminate between the consensus ERE and the mutERE was compared under various salt conditions. Since ER has been reported to interact with the ERE as either a dimer (
      • Schwabe J.W.R.
      • Chapman L.
      • Finch J.T.
      • Rhodes D.
      ,
      • Klein-Hitpass L.
      • Tsai S.Y.
      • Greene G.L.
      • Clark J.H.
      • Tsai M.-J.
      • O'Malley B.W.
      ,
      • Fawell S.E.
      • Lees J.A.
      • White R.
      • Parker M.G.
      ) or a monomer (
      • Furlow J.D.
      • Murdoch F.E.
      • Gorski J.
      ), the data were fitted using either a simple binding model of a single, structural ER unit (monomer or dimer) or a cooperative model of DNA-induced ER dimerization. The role of E2 in ER binding to the ERE was also examined using this equilibrium technique.

      DISCUSSION

      We have used fluorescence anisotropy to examine the interaction of the full-length ER and ER DBD with consensus and mutated ERE sequences under equilibrium binding conditions. Our results indicate that theXenopus ER DBD bound indiscriminately to the fluorescein-labeled vitERE and mutERE sequences with identical salt concentration dependence in these solution conditions, suggesting that sequences outside the DBD may be important for specific binding of the ER DBD. Several previous studies indicated that the ER DBD forms specific dimeric complexes with the consensus ERE and monomeric complexes with imperfect ERE sequences. The 66-amino acid zinc finger core of the DBD has been shown to contain the sequences necessary for homodimerization and ER recognition of the ERE sequence (
      • Mader S.
      • Kumar V.
      • de Verneuil H.
      • Chambon P.
      ,
      • Kumar V.
      • Chambon P.
      ). TheXenopus ER DBD has been observed to exist as a monomer in solution by column chromatography and fluorescence polarization measurements of the rotational relaxation times (
      • Nardulli A.M.
      • Lew D.
      • Erijman L.
      • Shapiro D.J.
      ). PurifiedXenopus ER DBD formed a single complex with the consensus ERE over a range of 18 to 3600 nm ER DBD added in gel shift assays (
      • Nardulli A.M.
      • Lew D.
      • Erijman L.
      • Shapiro D.J.
      ). Mixing different amounts of the ER DBD and another ER truncation mutant, which also retained DNA binding ability, resulted in the formation of gel-shifted complexes intermediate in size between the DBD·DNA complex and the ER truncation mutant-DNA complex, suggesting that ER DBD bound as a dimer to the ERE (
      • Mader S.
      • Chambon P.
      • White J.H.
      ). ER DBD complexed as a monomer with an imperfect ERE containing a double base pair change in one arm of the palindrome, as determined in gel shift assays (
      • Mader S.
      • Chambon P.
      • White J.H.
      ). D region amino acids adjacent to the DBD were found to stabilize the ER fragment binding to imperfect ERE sequences (
      • Mader S.
      • Chambon P.
      • White J.H.
      ). Xenopus ER DBD was shown to recognize the consensus ERE with higher affinity than imperfect ERE sequences bearing one or two nucleotide changes in competition gel shift assays (
      • Chang T.-C.
      • Nardulli A.M.
      • Lew D.
      • Shapiro D.J.
      ). In our fluorescence anisotropy assays, which did not require physical separation of free and DNA-bound protein, the Xenopus ER DBD (amino acids 171–281) recognized the palindromic vitERE and mutERE sequences with similar affinity, perhaps due to detection of lower affinity complexes. These results indicate that sequences or conformation present in the full-length ER protein may contribute to specificity of binding.
      Our observations also indicate that purified, full-length human ER-E2 binds nonspecifically to vitERE and mutERE in buffer containing 100 mm KCl as demonstrated by both gel shift analysis and fluorescence anisotropy titrations (Figs. 2 and 3). Even in the 10–30 nm physiological range of ER concentration, ER did not discriminate between the consensus and mutated ERE sequences in the fluorescence anisotropy assays. The large anisotropy change observed from unbound to bound oligonucleotide and the lack of a well defined plateau even at high ER concentrations suggested that the purified ER was aggregating on the DNA under these salt conditions. Recent studies have indicated that baculovirus-expressed mouse ER binds to the consensus ERE in an oligomeric form larger than a dimer in buffer containing 100 mmsalt.
      I. Anderson and J. Gorski, unpublished data.
      Further experimentation is needed to decipher the oligomeric state of the purified ER bound to DNA at 100 mm KCl, but it is likely that protein-protein interactions are occurring at this salt concentration.
      In buffer conditions that included salt concentrations equal to or greater than 200 mm KCl, ER exhibited discrimination between the consensus and mutant ERE sequences. From 225 to 275 mm KCl, ER-E2 bound to the F-vitERE but not to the F-mutERE over the protein concentration range examined. Increasing the salt concentration has been shown to decrease the apparent affinity for protein-DNA complexes (
      • Record Jr., M.T.
      • Lohman T.M.
      • de Haseth P.
      ,
      • Record Jr., M.T.
      • Ha J.-H.
      • Fisher M.A.
      ). However, in these studies, the mode of binding shifted from a nonspecific oligomeric mode to a specific mode between 100 and 200 mm KCl, likely involving dimeric complexes, given the changes in the values of the anisotropy of the complexes. Between 200 and 275 mm KCl, little change in affinity was observed. However, a further increase in the salt concentration to 300 mm KCl resulted in a shift in the binding curves to higher protein concentration. A drastic reduction in ER·ERE complex formation at salt concentrations above physiological levels was previously observed with rat uterine ER (
      • Murdoch F.E.
      • Grunwald K.A.A.
      • Gorski J.
      ). This unusual salt dependence strongly suggests that salt may be affecting protein-DNA and protein-protein interactions differentially over this range.
      In the salt concentration range between 225 and 275 mm, where the anisotropy profiles were characterized by well defined plateaus, testing of both simple and cooperative binding models revealed that the data did not provide the precision necessary for discriminating between these two possibilities. If a cooperative binding model had consistently described the data better than a simple model, this would have provided strong evidence for dimer binding. Unfortunately, our data are equally consistent with models of noncooperative binding of two monomers, simple binding of one monomer, simple binding of a preformed dimer, or cooperative binding of two monomers.
      In our studies, ER-E2 was able to discriminate between vitERE and mutERE only at salt concentrations greater than 200 mm KCl, which was slightly higher than the optimal salt concentrations found for the unpurified rat uterine ER (
      • Murdoch F.E.
      • Grunwald K.A.A.
      • Gorski J.
      ). The equilibrium dissociation constant measured in buffer conditions containing 225 to 275 mm KCl was 1.8 ± 0.6 nm, which was about fivefold greater than the value of 390 ± 40 pm obtained for unpurified, E2-occupied, rat uterine ER binding to the ERE (
      • Murdoch F.E.
      • Meier D.A.
      • Furlow J.D.
      • Grunwald K.A.A.
      • Gorski J.
      ). Since purified ER bound rather indiscriminately and with a slightly lower affinity as compared with the unpurified rat uterine ER at physiological salt concentrations, the mechanism by which ER differentiates between imperfect ERE sequences may include participation of other nuclear proteins to increase formation of active ER complexes at appropriate target genes. Alternatively, some loss of specific DNA binding activity may result from the ER purification procedure. Aggregation phenomena of the DNA-bound ER at high protein concentrations precludes an independent measure of DNA binding activity. In addition, our results verified that E2-occupied and unoccupied ER bound with the same affinity to vitERE (Table II). These findings are similar to those obtained using ER from rat uterine cytosol (
      • Murdoch F.E.
      • Meier D.A.
      • Furlow J.D.
      • Grunwald K.A.A.
      • Gorski J.
      ,
      • Furlow J.D.
      • Murdoch F.E.
      • Gorski J.
      ). Thus, E2-binding to the ER affects another step in the transcription pathway, other than DNA binding, in support of the current model of estrogen action.
      In summary, our results indicate that ER sequences outside of the DBD may play an important role in promoting specific interactions with ERE sequences. Unlike the ER DBD, the full-length human ER specifically recognized the ERE sequence at buffer salt concentrations near physiological salt concentrations. Unoccupied and E2-occupied ER bound to the ERE sequence with similar affinity, verifying that ER binds to ERE sequences in the absence of estrogen. Moreover, our results suggest that factors other than the receptor itself may serve to provide binding specificity.

      Acknowledgments

      We thank Dr. Iain Anderson for construction of the recombinant baculovirus expressing mouse ER, Chris Bartley for purification of mouse ER, Dr. Michael Newton for assistance in error analysis, and Kathryn A. Holtgraver for editorial assistance.

      REFERENCES

        • Evans R.M.
        Science. 1988; 240: 889-895
        • Beato M.
        Cell. 1989; 56: 335-344
        • Lundeen S.
        • Anderson I.
        • Gray W.
        • Gorski J.
        Handbook of Endocrinology. 2nd Ed. CRC Press, Boca Raton, FL1996: 121-148
        • Walker P.
        • Germond J.-E.
        • Brown-Luedi M.
        • Givel F.
        • Wahli W.
        Nucleic Acids Res. 1984; 12: 8611-8626
        • Klein-Hitpass L.
        • Schorpp M.
        • Wagner U.
        • Ryffel G.U.
        Cell. 1986; 46: 1053-1061
        • Klein-Hitpass L.
        • Ryffel G.U.
        • Heitlinger E.
        • Cato A.C.B.
        Nucleic Acids Res. 1988; 16: 647-663
        • Krust A.
        • Green S.
        • Argos P.
        • Kumar V.
        • Walter P.
        • Bornert J.-M.
        • Chambon P.
        EMBO J. 1986; 5: 891-897
        • Kumar V.
        • Green S.
        • Stack G.
        • Berry M.
        • Jin J.-R.
        • Chambon P.
        Cell. 1987; 51: 941-951
        • Webster N.J.G.
        • Green S.
        • Jin J.R.
        • Chambon P.
        Cell. 1988; 54: 199-207
        • Lees J.A.
        • Fawell S.E.
        • Parker M.G.
        Nucleic Acids Res. 1989; 17: 5477-5488
        • Tora L.
        • White J.
        • Brou C.
        • Tasset D.
        • Webster N.
        • Scheer E.
        • Chambon P.
        Cell. 1989; 59: 477-487
        • Hansen J.C.
        • Gorski J.
        J. Biol. Chem. 1986; 261: 13990-13996
        • Fritsch M.
        • Leary C.M.
        • Furlow J.D.
        • Ahrens H.
        • Schuh T.J.
        • Mueller G.C.
        • Gorski J.
        Biochemistry. 1992; 31: 5303-5311
        • Fritsch M.
        • Anderson I.
        • Gorski J.
        Biochemistry. 1993; 32: 14000-14008
        • Joab I.
        • Radanyi C.
        • Renoir M.
        • Buchou T.
        • Catalli M.-G.
        • Binart N.
        • Mester J.
        • Baulieu E.-E.
        Nature. 1984; 308: 850-853
        • Sanchez E.R.
        • Faber L.E.
        • Henzel W.J.
        • Pratt W.B.
        Biochemistry. 1990; 29: 5145-5152
        • Murdoch F.E.
        • Grunwald K.A.A.
        • Gorski J.
        Biochemistry. 1991; 30: 10838-10844
        • Danielsen M.
        • Hinck L.
        • Ringold G.M.
        Cell. 1989; 57: 1131-1138
        • Mader S.
        • Kumar V.
        • de Verneuil H.
        • Chambon P.
        Nature. 1989; 338: 271-274
        • Schwabe J.W.R.
        • Chapman L.
        • Finch J.T.
        • Rhodes D.
        Cell. 1993; 75: 567-578
        • Nardulli A.M.
        • Lew D.
        • Erijman L.
        • Shapiro D.J.
        J. Biol. Chem. 1991; 266: 24070-24076
        • Nardulli A.M.
        • Shapiro D.J.
        Mol. Cell. Biol. 1992; 12: 2037-2042
        • Nardulli A.M.
        • Greene G.L.
        • Shapiro D.J.
        Mol. Endocrinol. 1993; 7: 331-340
        • Murdoch F.E.
        • Meier D.A.
        • Furlow J.D.
        • Grunwald K.A.A.
        • Gorski J.
        Biochemistry. 1990; 29: 8377-8385
        • Furlow J.D.
        • Murdoch F.E.
        • Gorski J.
        J. Biol. Chem. 1993; 268: 12519-12525
        • Reese J.C.
        • Katzenellenbogen B.S.
        Nucleic Acids Res. 1991; 19: 6595-6602
        • Reese J.C.
        • Katzenellenbogen B.S.
        Mol. Cell. Biol. 1992; 12: 4531-4538
        • Beekman J.M.
        • Allan G.F.
        • Tsai S.Y.
        • Tsai M.-J.
        • O'Malley B.W.
        Mol. Endocrinol. 1993; 7: 1266-1274
        • Klein-Hitpass L.
        • Tsai S.Y.
        • Greene G.L.
        • Clark J.H.
        • Tsai M.-J.
        • O'Malley B.W.
        Mol. Cell. Biol. 1989; 9: 43-49
        • Fawell S.E.
        • Lees J.A.
        • White R.
        • Parker M.G.
        Cell. 1990; 60: 953-962
        • Hill J.J.
        • Royer C.A.
        Methods Enzymol. 1997; 278: 390-416
        • Royer C.A.
        • Smith W.R.
        • Beechem J.M.
        Anal. Biochem. 1990; 191: 287-294
        • Royer C.A.
        • Beechem J.M.
        Methods Enzymol. 1992; 210: 481-505
        • Royer C.A.
        Anal. Biochem. 1993; 210: 91-97
        • Beechem J.M.
        Methods Enzymol. 1992; 210: 37-54
        • Furlow J.D.
        • Ahrens H.
        • Mueller G.C.
        • Gorski J.
        Endocrinology. 1990; 127: 1028-1032
        • Heyduk T.
        • Lee J.C.
        Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 1744-1748
        • Fernando T.
        • Royer C.
        Biochemistry. 1992; 31: 3429-3441
        • LeTilly V.
        • Royer C.A.
        Biochemistry. 1993; 32: 7753-7758
        • Kersten S.
        • Pan L.
        • Chambon P.
        • Gronemeyer H.
        • Noy N.
        Biochemistry. 1995; 34: 13717-13721
        • Lundblad J.R.
        • Laurance M.
        • Goodman R.H.
        Mol. Endocrinol. 1996; 10: 607-612
        • Record Jr., M.T.
        • Lohman T.M.
        • de Haseth P.
        J. Mol. Biol. 1976; 107: 145-158
        • Record Jr., M.T.
        • Ha J.-H.
        • Fisher M.A.
        Methods Enzymol. 1991; 208: 291-343
        • Kumar V.
        • Chambon P.
        Cell. 1988; 55: 145-156
        • Mader S.
        • Chambon P.
        • White J.H.
        Nucleic Acids Res. 1993; 21: 1125-1132
        • Chang T.-C.
        • Nardulli A.M.
        • Lew D.
        • Shapiro D.J.
        Mol. Endocrinol. 1992; 6: 346-354