Determinants for DNA-binding site recognition by the glucocorticoid receptor.

The glucocorticoid receptor binds with high specificity to glucocorticoid response elements, discriminating them from other closely related binding sites. Three amino acids in the recognition alpha-helix of the DNA-binding domain of the receptor are primarily responsible for this specific DNA binding activity. In this study we analyze in detail how these residues determine the specific DNA binding by studying a series of mutant glucocorticoid receptor DNA-binding domains containing all combinations of glucocorticoid and estrogen receptor-specific residues at these positions. Statistical analysis of the results enables us to create models describing the association between amino acids and base pairs. Several strategies appear to be used in accomplishing discrimination between the glucocorticoid and estrogen response elements. Single residues (i.e., Val-443 in the glucocorticoid receptor and Glu-439 in the estrogen receptor) appear to form both positive contacts with specific base pairs in the cognate binding site and negative contacts in the non-cognate site. In the glucocorticoid receptor Ser-440 is pleiotropically negative for all sites tested but the negative effect is stronger for the estrogen response element thus contributing to binding site discrimination. Furthermore, combinations of amino acids appear to act synergistically, most often causing a reduction in binding to non-cognate sites.     

Hormonal regulation of vitellogenin genes: an estrogen-responsive element in the Xenopus A2 gene and a multihormonal regulatory region in the chicken II gene

Expression of the vitellogenin genes in avian and amphibian liver is regulated by estrogens. The DNA elements mediating estrogen induction of the various vitellogenin genes of chicken and Xenopus encompass one or more copies of a 13-mer palindromic sequence called the estrogen-responsive element (ERE). Here we show that upon incubation with the purified estrogen receptor (ER) from calf uterus the Xenopus vitellogenin A2 gene yields a DNase-I footprint over the ERE between -331 and -319. This element does not mediate the response to glucocorticoids or progestins in T47D cells. The three guanine residues in each half of the palindrome are protected against methylation by dimethylsulfate after incubation with ER, but not with glucocorticoid (GR) or progesterone (PR) receptors. In contrast, the chicken vitellogenin II gene exhibits multihormonal regulation by estrogens, progestins, and glucocorticoids in T47D and MCF7 cells. Regulation is mediated by the DNA region between -721 and -591 that contains four binding sites for hormone receptors, as demonstrated by DNase-I footprints and methylation protection experiments. The two distal and most proximal binding sites are recognized by ER, GR, and PR, whereas the central binding site is only bound by ER and GR. At suboptimal concentrations, estrogens and progestins or glucocorticoids act synergistically. In experiments using a DNA fragment containing an ERE adjacent to a glucocorticoid-responsive element/progesterone-responsive element, ER and PR bind synergistically to their corresponding sites, perhaps explaining the functional synergism of both hormones. Thus, two very different regulatory elements are used to mediate estrogen induction of related genes in chickens and amphibians.     

Computer-based search for steroid and DNA binding sites on estrogen and glucocorticoid receptors

The primary amino acid sequences of proteins that are receptors for estrogen, glucocorticoids, and ouabain were compared with each other using computer programs designed to detect and quantify similarities between proteins. Three regions of similarity between the estrogen receptor (ER) and the glucocorticoid receptor (GR) were identified. On the ER, residues 173-250, 323-395, and 426-458 are similar to residues 409-486, 540-612, and 644-676, respectively, on the GR. The ALIGN computer analysis of these segments on the ER and the GR gave comparison scores that were 16.8, 13.7, and 6.8 standard deviations higher, respectively, than that obtained with a comparison of randomized sequences of these proteins. The probability of getting these scores by chance is less than 10(-60), 10(-40), and 10(-11), respectively. Others have proposed that the segment on the ER and GR that is nearest their amino terminus (e.g. residues 173-250 of the ER) is part of their DNA binding domain and that the other two similar segments on each receptor, which are closer to their carboxy terminus, are part of their steroid binding domain. Here, we present evidence to support both of these hypotheses. First, an Align computer analysis indicates that residues 323-395 of the ER and residues 570-612 of the GR contain a region that is similar to a part of the alpha-subunit of the (Na+ + K+)ATPase that is hypothesized to bind the steroid ouabain. This similarity provides additional support for the proposed location of the steroid binding site on the ER, GR, and (Na+ + K+)ATPase. Second, a computer search of the protein sequence database revealed that protamine, a DNA binding protein, has some similarity to residues 255-281 of the ER, which are thought to be part of the DNA binding domain in the ER. Further, we find that residues 276-281 of the ER contain a structure that has been found at the nucleotide binding domain of some protein kinases. If this region on the ER binds ATP, then it may be involved in phosphorylation/dephosphorylation of the ER, which is thought to be important in its mechanism of action.     

DNA binding specificity of mutant glucocorticoid receptor DNA-binding domains

Mutation of a small number of amino acids in the DNA-binding domain of the estrogen receptor to the corresponding sequence of the glucocorticoid receptor switches the specificity of the receptor in transactivation assays (Mader, S., Kumar, V., de Verneuil, H., and Chambon, P. (1989) Nature 338, 271-274). We have made the corresponding reciprocal mutations in the context of the glucocorticoid receptor DNA-binding domain and studied the binding of wild type and mutant purified proteins to palindromic glucocorticoid and estrogen response elements as well as to elements of intermediate sequence, using gel mobility shift assays. We show here that a protein with two altered amino acids binds glucocorticoid and estrogen response elements with a low but equal affinity, whereas a protein with an additional changed residue has a high affinity for estrogen response elements but still retains a considerable affinity for glucocorticoid response elements. Using binding sites of intermediate sequence we have further characterized the interaction with DNA. The in vitro DNA binding results are confirmed by in vivo transactivation assays in yeast. Finally we suggest a testable model for amino acid/base pair interactions involved in recognition by the glucocorticoid receptor DNA-binding domain of its target sequence.     

Assembly of a glucocorticoid receptor complex prior to DNA binding enhances its specific interaction with a glucocorticoid response element

Gel retardation analysis with full- and half-palindromic sequences using partially purified glucocorticoid receptor (GR) resulted in GR-glucocorticoid response element (GRE) species of identical mobilities, suggesting that formation of the dimeric GR protein complex is not catalyzed by DNA binding. These results are in contrast to the behavior of the isolated DNA binding domain of the glucocorticoid receptor where dimerization occurred on the GRE. Density gradient centrifugation of cytosolic GR resulted in two forms, a 4 S peak characteristic of the monomeric GR and a fraction which sediments at 6 S which is consistent with the observed size of the dimeric GR. These two forms were found to differ in their ability to bind to specific DNA sequences with the 6 S species having a higher affinity for a GRE. Taken together our results are consistent with a two-step model for hormone-induced transformation of GR: dissociation of the multimeric untransformed complex and dimerization of the GR to yield a high affinity DNA binding species.     

Molecular cloning and characterization of rat estrogen receptor cDNA.

A cDNA clone of rat uterus estrogen receptor (ER) has been isolated and sequenced. This clone contains a complete open reading frame encoding 600 amino acid residues which is 5 and 11 amino acids larger than the corresponding molecules of human and chicken, respectively. The molecular weight of this protein is calculated to be 67,029. When this clone was ligated to the pSV2 vector and transfected into COS7 cells, a protein was produced that had the same affinity to estrogen as rat uterus ER. This sequence shows 88% homology with human ER; 528 amino acids are identical and 14 amino acids are conservative substitutions. The comparison of rat, human and chicken ER sequences indicate the presence of three highly conserved regions suggesting that these regions play important roles in ER function. The putative DNA-binding domain is completely identical in rat, human and chicken. The C-terminal half region which is thought to be the estrogen binding domain is also highly conserved and is rich in hydrophobic amino acid residues. Southern blot analysis of genomic DNA with ER cDNA as a probe has shown that related sequences are present in the genome.     

Efficient binding of glucocorticoid receptor to its responsive element requires a dimer and DNA flanking sequences

A combination of the gel retardation assay and interference by hydroxyl radical modification (missing nucleoside technique) was used to analyze the interaction of the glucocorticoid receptor (GR) with various glucocorticoid responsive elements (GRE). Short oligonucleotides containing the 15-bp GRE and 1 to 3 flanking base pairs on each side, are bound with very low affinity. The same GREs, when positioned in the center of a large DNA fragment (40-50 bp), show high affinity for the receptor. However, when the GRE is positioned at the border of a 54-bp fragment, the affinity of the GR for the GRE decreases markedly. The DNA binding affinity increases linearly with each added flanking base pair and optimal binding is observed with 8-10 flanking bp. Thus, the nonconserved DNA sequences flanking the GRE contribute significantly to the free energy of receptor binding to DNA. Using larger DNA fragments (greater than 100 bp) and a smaller form of the receptor (40 kD), two retarded complexes are found that correspond to monomeric and homodimeric receptor DNA complexes. The DNA-binding domain of the GR (20 kD), expressed in bacteria, binds to the GRE as a monomer as well as a dimer and can form heterodimers with the native 94-kD GR. Insertion or deletion of one single base pair between the two halves of the GRE reduces the affinity for the homodimeric form of the native GR, and inhibits the function of the GRE in gene transfer experiments, suggesting that a dimer of the GR is the functional entity that binds to the GRE.     

Molecular Dynamics Simulation in Solvent of the Estrogen Receptor Protein DNA Binding Domain in Complex with a Non-Consensus Estrogen Response Element DNA Sequence

Abstract

We investigated protein/DNA interactions, using molecular dynamics simulations computed between a 10 Angstom water layer model of the estrogen receptor (ER) protein DNA binding domain (DBD) amino acids and DNA of a non-consensus estrogen response element (ERE) consisting of 29 nucleotide base pairs. This ERE nucleotide sequence occurs naturally upstream of the Xenopus laevis Vitelligenin AI gene. The ER DBD is encoded by three exons. Namely, exons 2 and 3 which encode the two zinc binding motifs and a sequence of exon 4 which encodes a predicted alpha helix. We generated a computer model of the ER DBD using atomic coordinates derived from the average of 30 nuclear magnetic resonance (NMR) spectroscopy coordinate sets. Amino acids on the carboxyl end of the ER DBD were disordered in both X-ray crystallography and NMR determinations and no coordinates were reported. This disordered region includes 10 amino acids of a predicted alpha helix encoded in exon 4 at the exon 3/4 splice junction. These amino acids are known to be important in DNA binding and are also believed to function as a nuclear translocation signal sequence for the ER protein. We generated a computer model of the predicted alpha helix consisting of the 10 amino acids encoded in exon 4 and attached this helix to the carboxyl end of the ER DBD at the exon 3/4 splice junction site. We docked the ER DBD model within the DNA major groove halfsites of the 29 base pair non-consensus ERE and flanking nucleotides. We constructed a solvated model with the ER DBD/ERE complex surrounded by a ten Angstrom water layer and conducted molecular dynamics simulations. Hydrogen bonding interactions were monitored. In addition, van der Waals and electrostatic interaction energies were calculated. Amino acids of the ER DBD DNA recognition helix formed both direct and water mediated hydrogen bonds at cognate codon-anticodon nucleotide base and backbone sites within the ERE DNA right major groove halfsite. Amino acids of the ER DBD exon 4 encoded predicted alpha helix formed direct and water mediated H-bonds with base and backbone sites of their cognate codon-anticodon nucleotides within the minor grooves flanking the ERE DNA major groove halfsites. These interactions together induced bending of the DNA into the protein.     

1H NMR studies of DNA recognition by the glucocorticoid receptor: complex of the DNA binding domain with a half-site response element.

The complex of the rat glucocorticoid receptor (GR) DNA binding domain (DBD) and half-site sequence of the consensus glucocorticoid response element (GRE) has been studied by two-dimensional 1H NMR spectroscopy. The DNA fragment is a 10 base-pair oligonucleotide, 5'd(GCTGTTCTGC)3'.5'd-(GCAGAACAGC)3', containing the stronger binding GRE half-site hexamer, with GC base pairs at each end. The 93-residue GR-DBD contains an 86-residue segment corresponding to residues 440-525 of the rat GR. Eleven NOE cross peaks between the protein and DNA have been identified, and changes in the chemical shift of the DNA protons upon complex formation have been analyzed. Using these protein-DNA contact points, it can be concluded that (i) the "recognition helix" formed by residues C460-E469 lies in the major groove of the DNA; (ii) the GR-DBD is oriented on the GRE half-site such that residues A477-D481, forming the so-called D-loop, are available for protein-protein interaction in the GR-DBD dimer on the intact consensus GRE; and (iii) the 5-methyl of the second thymine in the half-site and valine 462 interact, confirming indirect evidence [Truss et al. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 7180-7184; Mader et al. (1989) Nature 338, 271-274] that both play an important role in GR-DBD DNA binding. These findings are consistent with the model proposed by H?rd et al. [(1990) Science 249, 157-160] and the X-ray crystallographic complex structure determined by Luisi et al. [(1991) Nature 352, 497-505].     

Stability of the ligand-estrogen receptor interaction depends on estrogen response element flanking sequences and cellular factors

To determine whether accessory proteins mediate the ligand- and DNA sequence-dependent specificity of estrogen receptor (ER) interaction with DNA, the binding of partly purified vs highly purified bovine ER to various estrogen response elements (EREs) was measured in the presence of different ER ligands. Partly purified estradiol-liganded ER (E₂-ER) binds cooperatively to stereoaligned tandem EREs flanked by naturally occurring AT-rich sequences, with a stoichiometry of one E₂-ER dimer per ERE. In contrast, highly purified E₂-ER binds with a 10-fold lower affinity and non-cooperatively to EREs flanked by the AT-rich region. Moreover, the binding stoichiometry of highly purified E₂-ER was 0.5 E₂-ER dimer, or one monomer per ERE, independent of the ERE flanking sequence. Interestingly, the binding of ER liganded with the antiestrogen 4-hydroxytamoxifen (4-OHT-ER) was non-cooperative with an apparent stoichiometry of 0.5 4-OHT-ER dimer per ERE, regardless of ER purity or ERE flanking sequence. We recently showed that when 4-OHT-ER binds DNA, one molecule of 4-OHT dissociates from the dimeric 4-OHT-ER-ERE complex, accounting for the reduced apparent binding stoichiometry. In contrast, ER covalently bound by tamoxifen aziridine (TAz) gave an ERE binding stoichiometry of one TAz-ER dimer per ERE, and TAz-ER binds cooperatively to multiple AT-rich EREs, regardless of the purity of the receptor. We have obtained evidence that purification of ER removes an accessory protein(s) that interacts with ER in a sequence- and/or DNA conformational-dependent manner, resulting in stabilization of E₂, but not 4-OHT, in the ligand binding domain when the receptor binds to DNA. We postulate that retention of ligand by ER maintains the receptor in a conformation necessary to achieve high-affinity, cooperative ERE binding.     

Functional interaction of hybrid response elements with wild-type and mutant steroid hormone receptors.

Steroid hormone receptors can be divided into two subfamilies according to the structure of their DNA binding domains and the nucleotide sequences which they recognize. The glucocorticoid receptor and the progesterone receptor (PR) recognize an imperfect palindrome (glucocorticoid responsive element/progesterone responsive element [GRE/PRE]) with the conserved half-sequence TGTYCY, whereas the estrogen receptor (ER) recognizes a palindrome (estrogen responsive element) with the half-sequence TGACC. A series of symmetric and asymmetric variants of these hormone responsive elements (HREs) have been tested for receptor binding and for the ability to mediate induction in vivo. High-resolution analysis demonstrates that the overall number and distribution of contacts with the N-7 position of guanines and with the phosphate backbone of various HREs are quite similar for PR and ER. However, PR and glucocorticoid receptor, but not ER, are able to contact the 5'-methyl group of thymines found in position 3 of HREs, as shown by potassium permanganate interference. The ER mutant HE84, which contains a single amino acid exchange, Glu-203 to Gly, in the knuckle of ER, creates a promiscuous ER that is able to bind to GRE/PREs by contacting this thymine. Elements with the sequence GGTCAcagTGTYCT that represent hybrids between an estrogen response element and a GRE/PRE respond to estrogens, glucocorticoids, and progestins in vivo and bind all three wild-type receptors in vitro. These hybrid HREs could serve to confer promiscuous gene regulation.     

Determinants of target gene specificity for steroid/thyroid hormone receptors

The molecular specificity of the receptors for steroid and thyroid hormones is achieved by their selective interaction with DNA binding sites referred to as hormone response elements (HREs). HREs can differ in primary nucleotide sequence as well as in the spacing of their dyadic half-sites. The target gene specificity of the glucocorticoid receptor can be converted to that of the estrogen receptor by changing three amino acids clustered in the first zinc finger. Remarkably, a single Gly to Glu change in this region produces a receptor that recognizes both glucocorticoid and estrogen response elements. Further replacement of five amino acids in the stem of the second zinc finger transforms the specificity to that of the thyroid hormone receptor. These findings localize structural determinants required for discrimination of HRE sequence and half-site spacing, respectively, and suggest a simple pathway for the coevolution of receptor DNA binding domains and hormone-responsive gene networks.     

The plasticity of estrogen receptor-DNA complexes: binding affinity and specificity of estrogen receptors to estrogen response element half-sites separated by variant spacers

The consensus estrogen response element (cERE) contains a palindromic sequence of two 6-base pair (bp) half-sites separated by a spacer size of 3bp. This study investigates the extent to which estrogen receptors, ERalpha and ERbeta can bind target sequences not considered as conventional EREs. We determined the effect of spacer size (n=0-4) on the binding affinity and conformation of ERalpha and ERbeta in these complexes and the effect of HMGB1 on the complexation. We find (1) both receptors bind similarly and with progressively reduced affinity to cEREn, as n differs from 3; (2) however, both receptors bind as strongly to the cERE with no spacer (cERE0) as to cERE3; (3) HMGB1 enhances ER binding affinity in all complexes, resulting in strong and comparable binding affinities in all complexes examined; (4) the full-length ER binding differs strikingly from similar binding studies for the ER DNA binding domain (ERDBD), with the full-length ER dimer exhibiting strong binding affinity, enormous plasticity and retaining binding cooperativity as the spacer size varies; (5) both protease digestion profiles and monoclonal antibody binding assays indicate the conformation of the receptor in the ER/ERE complex is sensitive to the spacer size; (6) the ER/cERE0 complex appears to be singularly different than the other ER/cEREn complexes in binding and conformation. This multifaceted approach reinforces the notion of the plasticity in ER binding and leads to the hypothesis that in most cases, the minimum requirement for estrogen receptor binding is the ERE half-site, in which one or more cofactors, such as HMGB1, can cooperate to decrease ER binding specificity, while increasing its binding affinity.     

Structural requirements for high affinity ligand binding by estrogen receptors: a comparative analysis of truncated and full length estrogen receptors expressed in bacteria, yeast, and mammalian cells.

In order to better understand the structural requirements for effective high affinity binding of estrogens and antiestrogens by the human estrogen receptor (ER), a comparative study was undertaken in which we examined: 1) native ER from the MCF-7 ER-positive human breast cancer cell line; 2) full length ER expressed in yeast; 3) the ER hormone binding domain (amino acid residues 302-595) expressed in yeast; 4) a bacterially expressed protein A fusion product encoding a truncated ER (amino acid residues 240-595); and 5) a synthetic peptide encompassing amino acids 510-551 of the ER. The binding parameters studied included affinity, kinetics, structural specificity for ligands, and stability. Full length ER expressed in yeast was very similar to the MCF-7 ER in its affinity [dissociation constant (Kd), 0.35 +/- 0.05 nM], dissociation rate (t1/2, 3-4 h at 25 C), and structural specificity for both reversible and covalently attaching affinity ligands. While the truncated ER expressed in yeast was similar to MCF-7 ER in its specificity of ligand binding, it showed a slightly reduced affinity for estradiol (Kd, 1.00 +/- 0.17 nM). The bacterially expressed ER also had a lower affinity for estradiol (Kd, 1.49 +/- 0.16 nM), which may be due in part to an increase in the dissociation rate (t1/2, 0.5 h at 25 C). The attachment of covalent affinity ligands and structural specificity for a variety of reversible ligands was comparable in the bacterially expressed ER to that observed for the receptors expressed in MCF-7 cells and yeast.(ABSTRACT TRUNCATED AT 250 WORDS)     

Affinity of interactions between human glucocorticoid receptors and DNA: at physiologic ionic strength, stable binding occurs only with DNAs containing partially symmetric glucocorticoid response elements

Sucrose density gradient shift assays were adapted to permit determination of the affinity of interaction between human glucocorticoid receptors (GR) and DNA under conditions of DNA excess. Saturation analyses were performed to ascertain dissociation constants for the interaction of activated human GR with each of five DNA fragments. Centrifugation of GR-DNA complexes on sucrose gradients under nearly isotonic salt conditions revealed similar affinities with dissociation constants in the range of 2-16 nM for GR interaction with DNA fragments containing glucocorticoid response elements (GREs) exhibiting partial dyad symmetry. By contrast, GR exhibited virtually no affinity for non-GRE-containing DNA or for DNA containing only GRE half-sites. Additionally, GR showed evidence of multiple-site interaction with a DNA fragment containing two partially symmetric GREs, but interacted at only one site of an MMTV LTR DNA fragment containing a single partially symmetric GRE along with a cluster of three half-GREs. Together these data indicate that under physiologically relevant conditions, glucocorticoid receptors have high selectivity and affinity only for DNA containing specific partially symmetric GREs and further suggest that this high affinity for such DNA sites may be sufficient to account for the selective regulation of gene expression observed in glucocorticoid-responsive cells.