It is believed that steroid hormones induce differential gene expression in target cells by combining with specific cytoplasmic receptor proteins that subsequently translocate to the nuclear compartment where they interact with a limited number of acceptor sites (1). Although the nature of these nuclear acceptor sites is still unclear, it is well established that almost all steroid receptors described are able to combine with DNA (2-4). Andre and Rochefort (5) have demonstrated that the affinity of the estradiol receptor protein for DNA is increased by estrogen binding, analogous to the ligand-induced DNA binding property of cyclic AMP receptor protein, a prokaryotic gene regulatory protein (6). Several lines of evidence suggest that steroid receptor proteins, particularly estradiol and glucocorticoid receptor proteins, contain two distinct sites, one for the steroid hormone and the other for DNA. Limited proteolysis of the steroid receptor protein complexes destroys the DNA binding site without affecting the steroid binding site (7-9). In addition, compounds such as pyridoxal phosphate (10) and aurintricarboxylic acid (ATA) (11) have been shown to inhibit DNA binding of glucocorticoid and estradiol receptor proteins, respectively.Studies reported from this laboratory have shown that estradiol-receptor complexes (E2R) of mouse uterus and kidney bind to oligo(dT)-cellulose. The validity of this interaction as a model for the DNA binding property of estradiol-receptors has been well established (12). Using oligo(dT)-cellulose binding as an assay for DNA interaction, we studied the inhibitory effect of the dye Cibacron blue F3GA (CB) in order to characterize the DNA recognition site. CB, a sulfonated polyaromatic dye, has been shown to bind to several proteins that interact with nucleotide substrates, nucleotide coenzyme ligands, and polynucleotides (13)(14)(15)(16)(17)(18). In the case of enzymes that have polynucleotide binding sites in addition to nucleotide substrates, the dye interacts with the polynucleotide binding site preferentially (15,17,18). In the present study we have shown that CB interacts specifically with the DNA binding domain of the mouse uterine E2R. MATERIALS AND METHODS[3H]Estradiol (145 Ci/mol; 1 Ci = 3.7 X 1010 becquerels) was obtained from Amersham. Unlabeled estradiol was a product of Steraloids, Inc. (Wilton, NH). Olido(dT)-, oligo(dC)-, and oligo(dA)-celluloses were products of P-L Biochemicals. Calf thymus DNA and heparin were obtained from Sigma. Poly-L-glutamic acid was a product of Miles-Yeda (Rehovoth, Israel), and CB was a gift from CIBA (Basel, Switzerland). (vol/vol) glycerol with a Polytron P20ST homogenizer. The homogenate was centrifuged in a Beckman J-21 centrifuge with JA-20 rotor at 12,000 X g for 10 min, and the supernatant was recentrifuged at 300,000 X g for 60 min in a Beckman preparative ultracentrifuge (L-75) with a 75 Ti rotor. The supernatant fraction is referred to as the "cytosol." Testosterone (1.2 ,uM)

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The relative capacities of oligodeoxynucleotides, covalently linked to cellulose, to bind estradiol receptor complexes (E2R) of mouse uterine cytosol have been shown to follow the order oligo(dG) > oligo(dT) 2 oligo(dC) >> oligo(dA).The E2R oligo(dT)-cellulose-binding reaction is more sensitive to Cibacron blue F3GA than is ER oligo(dG)-cellulose or ofigo(dC)cellulose binding. Preformed ERoligo(dT) or ohigo(dC)-cellulose complexes are dissociated more readily by lower concentrations of KCI or Cibacron blue F3GA than is the E2%Roligo(dG)cellulose complex. Preincubation of ER at 370C results in a rapid loss of binding ability towards oligo(dT) and oligo(dC)cellulose, while up to 90% of the binding ability to oligo(dG-cellulose is retained. On the basis of the differential sensitivities of E2R to temperature and the inhibition by Cibacron blue F3GA of the binding reaction, it is suggested that the polynucleotide-binding domain consists of two types of subsites, one of which has a higher affinity for oligo(dG) residues and the other of which recognizes oligo(dT), oligo(dC), and, to a lesser extent, oligo(dA).Of the components necessary for nuclear binding of estradiol-receptor complexes (E2R), DNA, alone or in cooperation with specific chromosomal proteins, appears to be of prime importance. Cytosol receptor complexes from various target tissues are DNA-binding proteins and, even with crude preparations, it is apparent that the receptor proteins contain at least two distinct domains: the binding sites for steroid ligands and those for polydeoxynucleotides. The separateness of these sites was demonstrated for E2R and other steroid receptors by studying the effects of limited proteolysis (1). Under these conditions, DNA binding was abolished without affecting retention of the steroid. Subsequent studies with a series of inhibitors, including pyridoxal 5-phosphate (2-4), aurintricarboxylic acid (5), and Cibacron blue F3GA (CB) (6) showed loss of nuclear, DNA, and oligodeoxynucleotide binding without affecting the steroid-binding site.While these experiments indicated that a polydeoxynucleotide binding domain exists in E2R, they offered little information about what elements of DNA were recognized by the receptors. Despite some early reports, the binding to DNA of E2R is neither saturable nor specific as to the source of the DNA, at least under current experimental conditions (7-9). Yet the process is apparently not random, because preferences in binding have been reported for double-stranded DNA rather than single-stranded DNA (10), poly(dA-dT) rather than other synthetic polymers (11), and bromodeoxyuridine-substituted DNA rather than unsubstituted DNA (12). Furthermore, Simons (13) demonstrated by competition equilibrium experiments that glucocorticoid receptors bind preferentially to natural DNAs rather than to synthetic polydeoxynucleotides.Another approach to study the specificities of the polynucleotide domain of E2R has been the use of oligodeoxynucleotide-celluloses, in which the oligomer is covalently...

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During purification of E2R using oligo(dT)-cellulose chromatography, a receptor accessory factor (RAF) was identified in the cytosol of mouse kidney. This factor stimulates the binding of purified E2R to oligo(dT)-, oligo(dC)-, and oligo(dA)-cellulose as well as to DNA cellulose. It is a heat-stable, trypsin-resistant protein with an apparent molecular weight of between 10 and 30,000 daltons. Although structurally unrelated, similar stimulation of oligonucleotide binding was seen with calf thymus histones and, to a lesser extent, egg white lysozyme. Individual histones, especially H2a, H2B, and H3, also facilitate rebinding of purified E2R to oligo(dT)-cellulose, while H1 is less effective. Furthermore, histones stabilize the holoreceptor during sedimentation at 4 degrees and 12 degrees C. The N- and C-terminal half molecules of H2b were generated by cyanogen bromide-mediated cleavage and the N-terminal half was found to duplicate the effects of the parent molecule, both in binding and holoreceptor stabilization. These data suggest that the in vivo binding of E2R to DNA can be modulated by accessory proteins of cytosol and nuclear origin.