Nuclear matrix localization and specific matrix DNA binding by receptor binding factor 1 of the avian oviduct progesterone receptor

A chromatin acceptor protein for the avian oviduct progesterone receptor (PR), termed receptor binding factor 1 (RBF-1), has recently been shown to (1) be a component of the nuclear binding sites (acceptor sites) for PR and (2) generate high-affinity binding sites (termed the RBF-1 class of sites) on avian genomic DNA [Schuchard et al. (1991) Biochemistry 30, 4535-4542]. A second class of sites and its associated protein (termed RBF-2) were also identified. This paper demonstrates that RBF-1 and also the PR nuclear binding sites are localized in the oviduct nuclear matrix. RBF-1 is found in abundance in the nuclear matrix of liver but only in traces in the nuclear matrix of spleen. Extraction of the nuclear matrix with 4.0 M Gdn-HCl results in the complete removal of RBF-1 as occurs with whole chromatin. Interestingly, a second class of specific PR binding, termed RBF-2, remains on the nuclear matrix after the removal of all RBF-1. Southern blot analysis indicates that the nuclear matrix DNA contains sequences homologous with the 5'-flanking domains of the rapidly steroid regulated c-myc and c-jun protooncogenes and the beta-actin gene, but not genomic sequences of the late sex steroid regulated gene, ovalbumin, or the alpha-actin gene. A specific, small region in the 5'-flanking domain of the c-myc gene appears to be associated with the nuclear matrix. Southwestern blot analysis using partially purified RBF-1 shows a marked affinity and specificity of the RBF-1 for the nuclear matrix DNA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Enrichment of a second class of native acceptor sites for the avian oviduct progesterone receptor as intact chromatin fragments

Several classes of specific progesterone receptor (PR) nuclear binding sites (acceptor sites) have previously been identified in avian oviduct chromatin on the basis of different binding affinities. Recently, two classes of acceptor proteins (AP) that are associated with these binding sites in the avian oviduct have been identified. These APs were termed receptor binding factors (RBF-1 and -2), and one (RBF-1) has been purified [Schuchard et al. (1991) Biochemistry 30, 4535-4542]. The RBF-1 is associated with the highest affinity class of sites in the intact chromatin, and the RBF-2 is associated with the second highest affinity class of sites. The PR binding sites and their associated RBF-2 protein remain with the residual chromatin fraction following extraction by 4 M Gdn-HCl. This Gdn-HCl-treated chromatin has been termed nucleoacidic protein (NAP). This paper describes the 200-fold enrichment of the native RBF-2 class of PR acceptor sites beginning with the DNase I digestion of NAP to obtain DNase-resistant fragment (NAPf) containing approximately 150 bp of DNA. The PR binding sites are further enriched by high-performance or fast protein liquid chromatography and chromatofocusing. Anti-RBF-1/RBF-2 protein antibodies identify antigens that coelute with the PR binding activity. Hybridization analysis of the DNAf from the enriched NAPf demonstrates sequence homologies with the nuclear matrix DNA as well as with genomic sequences of the rapid steroid responding nuclear protooncogenes c-myc and c-jun. However, comparative analyses of the whole genomic DNA with the nuclear matrix DNA indicate that the RBF-2 (NAPf) is largely nonnuclear matrix.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nuclear binding of progesterone in chick oviduct. Multiple binding sites in vivo and transcriptional response.

1. Varied doses of labelled or unlabelled progesterone were injected into immature chicks which had previously been stimulated with oestrogen. The concentrations of nuclear bound [3H]progesterone were correlated with the effects of the hormone on endogenous RNA polymerase I and II activities in isolated oviduct nuclei. 2. The extent of nuclear localization of [3H]progesterone in oviduct (a progesterone target tissue) was shown to be much greater than in lung (non-target tissue). The conccentration of bivalent cations in solvents used in the nuclei isolations has a marked effect on the amount of bound hormone in the nuclei. 3. Evidence for the existence of several classes of binding sites for progesterone in the oviduct nuclei is given. These classes represent about 1000) 10000 and 100000 molecules of the hormone per cell nucleus and are saturated by injecting approx. 10, 100 and 1000 mug of progesterone respectively. 4. When saturation of the first (highest affinity) class of nuclear sites occurs, a marked inhibition in RNA polymerase II (but not RNA polymerase I) activity was observed. When the second class of sites was saturated, alterations in both RNA polymerase I and II activities were observed. Binding to the third class of nuclear binding sites was not accompained by further changes in polymerase activity. It is suggested that the first two classes of nuclear binding sites may represent functional sites for progesterone action in the chick oviduct.     

Growth hormone and drug metabolism. Acute effects on nuclear ribonucleic acid polymerase activity and chromatin.

Adult male rats, subjected either to sham operation or to hypophysectomy and adrenalectomy were maintained for 10 days before treatment with growth hormone. Results of the acute effects of growth hormone on the rat liver nuclear RNA polymerase I (nucleolar) and II (nucleoplasmic) activities as well as the chromatin template capacity were then studied and compared with the growth-hormone effects on the drug metabolism described in the preceding paper (Wilson & Spelsberg, 1976). 2. Conditions for isolation and storage of nuclei for maintenance of optimal polymerase activities are described. It is verified that the assays for polymerase activities require a DNA template, all four nucleoside triphosphates, and a bivalent cation, and that the acid-insoluble radioactive product represents RNA. Proof is presented that under high-salt conditions DNA-like RNA (polymerase II) is synthesized, and that under low-salt conditions in the presence of alpha-amanitin, rRNA (polymerase I) is synthesized. 3. In the livers of hypophysectomized/adrenalectomized rats, growth hormone increases the activity of both RNA polymerase enzymes and the chromatin template capacity within 1h after treatment. The effects last for 12h in the case of polymerase II but for only 6h in the case of polymerase I. Sham-operated rats respond to growth hormone in a manner somewhat similar to that shown by hypophysectomized/adrenalectomized rats. These results, which demonstrate an enhancement of RNA polymerase I activity in response to growth hormone, support those from other laboratories. 4. Growth-hormone enhancement of the chromatin template capacity in the liver of hypophysectomized/adrenalectomized rats contrasts with previous reports. The growth-hormone-induced de-repression of the chromatin DNA could represent the basis of the growth-hormone-induced enhancement of RNA polymerase II activity in the hypophysectomized/adrenalectomized rats, although some effect of growth-hormone on the polymerase enzymes is still suggested.     

Analysis of the molecular species of the chick oviduct progesterone receptor using isoelectric focusing.

Conditions are described for the preparative isoelectric focusing in flat beds of Sephadex of the progesterone receptor from the chick oviduct. The method allows the fractionation of the receptor into two molecular species, one focusing at pI 6 and the other at pI 7 with good purification and recovery. The pI 6 and pI 7 receptor species were purified 2- and 26-fold, respectively. The assaying of the focused fractions with the charcoal binding method provides an accurate identification and quantitation of the [3H]progesterone receptor. The method is reproducible in recovery, quantitation, and resolution of the two receptor species. The receptor with an apparent pI of 6 sediments at approximately 4 S on linear sucrose gradients, while the receptor with an apparent pI of 7 sediments at approximately 3.5 S. On the basis of the sedimentation values and elution patterns from diethylaminoethyl (DEAE) chromatography, the pI 6 component is equivalent to the "B" receptor species and the pI 7 component is equivalent to the "A" receptor species described previously [schrader, W, T., 7 O'Malley, B. W. )1972) J. Biol. Chem. 241, 51--59].     

Rapid inhibition of c-myc gene expression by a glucocorticoid in the avian oviduct

The glucocorticoid dexamethasone (DEX) causes a rapid, reversible reduction in c-myc mRNA level in the oviducts of estrogen-treated, immature chickens. The c-myc mRNA level begins to decrease by 5 min after injection of 0.5 mg DEX, reaches a minimum of 10% of the control value by 30 min, and returns to 30-40% of the control value by 4 h post injection. This rapid effect of DEX on the c-myc mRNA level occurs in both diethylstilbestrol-stimulated and diethylstilbestrol-withdrawn oviducts. The effect is dose dependent, with reduction of the c-myc mRNA measured with as little as 10 micrograms DEX injection (0.03 micrograms/g BW). The effect of the steroid is gene specific with H2B histone mRNA displaying a significantly reduced response. The effect is also tissue specific with liver displaying an increase of 170% of control values in c-myc mRNA level by 30 min after injection of 0.5 mg DEX. The reduction of avian oviduct c-myc mRNA levels by DEX may play a role in glucocorticoid inhibition of cell proliferation in this tissue. The rapidity of the steroid effects on c-myc expression makes it likely that the steroid-induced reduction of c-myc mRNA levels represents a direct primary action of the steroid-receptor complex on the c-myc gene expression.