Estrogen response element sequence impacts the conformation and transcriptional activity of estrogen receptor α 1 Supported by NIH R01 DK 53220 and a University of Louisville School of Medicine Research Grant to C.M.K. 1

Estrogens play a critical role in mammary gland development, bone homeostasis, reproduction, and the pathogenesis of breast cancer by activating estrogen receptors (ERs) alpha and beta. Ligand-activated ER stimulates the expression of target proteins by interacting with specific DNA sequences: estrogen response elements (EREs). We have demonstrated that the ERE sequence and the nucleotide sequences flanking the ERE impact ERalpha binding affinity and transcriptional activation. Here, we examined whether the sequence of the ERE modulates ERalpha conformation by measuring changes in sensitivity to protease digestion. ERalpha, occupied by estradiol (E2) or 4-hydroxytamoxifen (4-OHT), was incubated with select EREs and digested by chymotrypsin followed by a Western analysis with antibodies to ERalpha. ERE binding increased the sensitivity of ERalpha to chymotrypsin digestion. We found both ligand-specific and ERE-specific differences in ERalpha sensitivity to chymotrypsin digestion. The ERE-mediated increase in ERalpha sensitivity to chymotrypsin digestion correlates with E2-stimulated transcriptional activity from the same EREs in transiently transfected cells. Transcriptional activity also correlates with the affinity of ERalpha-ERE binding in vitro. Our results support the hypothesis that the ERE sequence acts as an allosteric effector, altering ER conformation. We speculate that ERE-induced alterations in ERalpha conformation modulate interaction with co-regulatory proteins.

[1]  P. Chambon,et al.  Widely spaced, directly repeated PuGGTCA elements act as promiscuous enhancers for different classes of nuclear receptors , 1995, Molecular and cellular biology.

[2]  B. O’Malley,et al.  Coactivator and corepressor regulation of the agonist/antagonist activity of the mixed antiestrogen, 4-hydroxytamoxifen. , 1997, Molecular endocrinology.

[3]  G. Puca,et al.  Characterization and epitope mapping of a new panel of monoclonal antibodies to estradiol receptor , 1993, Steroids.

[4]  A nuclear receptor corepressor modulates transcriptional activity of antagonist-occupied steroid hormone receptor. , 1998, Molecular endocrinology.

[5]  C. Klinge,et al.  Estrogen receptor interaction with co-activators and co-repressors☆ , 2000, Steroids.

[6]  B. Katzenellenbogen,et al.  Structural analysis of covalently labeled estrogen receptors by limited proteolysis and monoclonal antibody reactivity. , 1987, Biochemistry.

[7]  C. Klinge,et al.  Binding of type II nuclear receptors and estrogen receptor to full and half-site estrogen response elements in vitro. , 1997, Nucleic acids research.

[8]  F. Peale,et al.  Cooperative estrogen receptor interaction with consensus or variant estrogen responsive elements in vitro. , 1992, Cancer research.

[9]  Zbigniew Dauter,et al.  Molecular basis of agonism and antagonism in the oestrogen receptor , 1997, Nature.

[10]  B. O’Malley,et al.  Sequence and Characterization of a Coactivator for the Steroid Hormone Receptor Superfamily , 1995, Science.

[11]  W. Chin,et al.  Different DNA Elements Can Modulate the Conformation of Thyroid Hormone Receptor Heterodimer and Its Transcriptional Activity* , 1996, The Journal of Biological Chemistry.

[12]  S. Cowley,et al.  A comparison of transcriptional activation by ERα and ERβ , 1999, The Journal of Steroid Biochemistry and Molecular Biology.

[13]  C. Klinge,et al.  Chicken Ovalbumin Upstream Promoter-Transcription Factor Interacts with Estrogen Receptor, Binds to Estrogen Response Elements and Half-Sites, and Inhibits Estrogen-induced Gene Expression* , 1997, The Journal of Biological Chemistry.

[14]  G. Hager,et al.  The Glucocorticoid Receptor Is Tethered to DNA-bound Oct-1 at the Mouse Gonadotropin-releasing Hormone Distal Negative Glucocorticoid Response Element* , 1999, The Journal of Biological Chemistry.

[15]  John H. White,et al.  Function of Directly Repeated Half-sites as Response Elements for Steroid Hormone Receptors (*) , 1996, The Journal of Biological Chemistry.

[16]  T. Hutchens,et al.  Proteins associated with untransformed estrogen receptor in vitro. Perturbation of hydrophobic interactions induces alterations in quaternary structure and exposure of the DNA-binding site. , 1987, Biochemistry.

[17]  Jeffrey A. Lefstin,et al.  Allosteric effects of DNA on transcriptional regulators , 1998, Nature.

[18]  G. Greene,et al.  Estrogen Response Elements Function as Allosteric Modulators of Estrogen Receptor Conformation , 1998, Molecular and Cellular Biology.

[19]  B. Katzenellenbogen,et al.  Mechanistic aspects of estrogen receptor activation probed with constitutively active estrogen receptors: correlations with DNA and coregulator interactions and receptor conformational changes. , 1997, Molecular endocrinology.

[20]  C. Klinge,et al.  The aryl hydrocarbon receptor (AHR)/AHR nuclear translocator (ARNT) heterodimer interacts with naturally occurring estrogen response elements , 1999, Molecular and Cellular Endocrinology.

[21]  C. Klinge,et al.  Differential impact of flanking sequences on estradiol- vs 4-hydroxytamoxifen-liganded estrogen receptor binding to estrogen responsive element DNA , 1993, The Journal of Steroid Biochemistry and Molecular Biology.

[22]  C. Klinge,et al.  Dissociation of 4-hydroxytamoxifen, but not estradiol or tamoxifen aziridine, from the estrogen receptor as the receptor binds estrogen response element DNA , 1996, The Journal of Steroid Biochemistry and Molecular Biology.

[23]  Simak Ali,et al.  Phosphorylation of Human Estrogen Receptor α by Protein Kinase A Regulates Dimerization , 1999, Molecular and Cellular Biology.

[24]  L. Freedman,et al.  Granulocyte-macrophage colony-stimulating factor gene transcription is directly repressed by the vitamin D3 receptor. Implications for allosteric influences on nuclear receptor structure and function by a DNA element. , 1998, The Journal of biological chemistry.

[25]  J M Thornton,et al.  Assessment of conformational parameters as predictors of limited proteolytic sites in native protein structures. , 1998, Protein engineering.

[26]  C. Klinge,et al.  Comparison of tamoxifen ligands on estrogen receptor interaction with estrogen response elements , 1998, Molecular and Cellular Endocrinology.

[27]  G. Greene,et al.  Estrogen receptor affinity and location of consensus and imperfect estrogen response elements influence transcription activation of simplified promoters. , 1996, Molecular endocrinology.

[28]  R. Lanz,et al.  Nuclear receptor coregulators: cellular and molecular biology. , 1999, Endocrine reviews.

[29]  P. Coulson,et al.  Hydroxylapatite "batch" assay for estrogen receptors: increased sensitivity over present receptor assays. , 1976, Journal of steroid biochemistry.

[30]  Tamoxifen-bound estrogen receptor (ER) strongly interacts with the nuclear matrix protein HET/SAF-B, a novel inhibitor of ER-mediated transactivation. , 2000, Molecular endocrinology.

[31]  B. Katzenellenbogen,et al.  Editorial: A New Actor in the Estrogen Receptor Drama-Enter ER-β. , 1997, Endocrinology.

[32]  C. Klinge,et al.  hsp70 is not required for high affinity binding of purified calf uterine estrogen receptor to estrogen response element DNA in Vitro , 1997, The Journal of Steroid Biochemistry and Molecular Biology.

[33]  C. Klinge,et al.  Stability of the ligand-estrogen receptor interaction depends on estrogen response element flanking sequences and cellular factors , 1996, The Journal of Steroid Biochemistry and Molecular Biology.

[34]  D. McDonnell,et al.  The Estrogen Receptor ␤-isoform (er␤) of the Human Estrogen Receptor Modulates Er␣ Transcriptional Activity and Is a Key Regulator of the Cellular Response to Estrogens and Antiestrogens* , 2022 .

[35]  P. Jones,et al.  Activation of Transcription by Estrogen Receptor α and β Is Cell Type- and Promoter-dependent* , 1999, The Journal of Biological Chemistry.

[36]  C. Klinge,et al.  Cooperative binding of estrogen receptor to DNA depends on spacing of binding sites, flanking sequence, and ligand. , 1995, Biochemistry.

[37]  G. Sathya,et al.  Sequence Requirements for Estrogen Receptor Binding to Estrogen Response Elements* , 1998, The Journal of Biological Chemistry.

[38]  S. Hubbard,et al.  The structural aspects of limited proteolysis of native proteins. , 1998, Biochimica et biophysica acta.

[39]  B. Katzenellenbogen,et al.  Analysis of Estrogen Response Element Binding by Genetically Selected Steroid Receptor DNA Binding Domain Mutants Exhibiting Altered Specificity and Enhanced Affinity* , 1999, The Journal of Biological Chemistry.

[40]  Y. Sadovsky,et al.  Transcriptional activators differ in their responses to overexpression of TATA-box-binding protein , 1995, Molecular and cellular biology.

[41]  J. Katzenellenbogen,et al.  Analysis of the structural core of the human estrogen receptor ligand binding domain by selective proteolysis/mass spectrometric analysis. , 1995, Biochemistry.

[42]  K. Horwitz,et al.  The N-terminal Region of the Human Progesterone A-receptor , 2000, The Journal of Biological Chemistry.

[43]  E. Ariazi,et al.  Estrogen receptor binding to DNA: affinity for nonpalindromic elements from the rat prolactin gene. , 1995, Biochemistry.

[44]  David A. Agard,et al.  The Structural Basis of Estrogen Receptor/Coactivator Recognition and the Antagonism of This Interaction by Tamoxifen , 1998, Cell.

[45]  K. Umesono,et al.  The nuclear receptor superfamily: The second decade , 1995, Cell.

[46]  I. Anderson,et al.  DNA allosterically modulates the steroid binding domain of the estrogen receptor. , 1992, The Journal of biological chemistry.

[47]  C. Klinge,et al.  Antiestrogen-liganded estrogen receptor interaction with estrogen responsive element DNA in vitro , 1992, The Journal of Steroid Biochemistry and Molecular Biology.

[48]  B. Katzenellenbogen,et al.  An estrogen receptor-selective coregulator that potentiates the effectiveness of antiestrogens and represses the activity of estrogens. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[49]  B. O’Malley,et al.  Members of the steroid hormone receptor superfamily interact with TFIIB (S300-II). , 1992, The Journal of biological chemistry.