Purified Progesterone Receptor Research Articles

Interplay of steroid hormone receptors and transcription factors on the mouse mammary tumor virus promoter

The mouse mammary tumor virus (MMTV) promoter, that responds to glucocorticoids and progestins, contains a complex hormone response element (HRE) in the long terminal repeat (LTR) region covered by a phased nucleosome. Hormone treatment leads to alterations in chromatin structure that make the HRE region more accessible to digestion by Dnase I and permit binding of transcription factors, including nuclear factor I (NFI), immediately downstream of the HRE. NFI acts as a basal transcription factor on the MMTV promoter in vitro but competes with the hormone receptors in terms of binding to free DNA. In uninduced chromatin, the precise positioning of the DNA double helix on the surface of the histone octamer precludes binding of NFI to its cognate sequence while still allowing recognition of the HRE by the hormone receptors. We postulate that receptor binding to the nucleosomally organized MMTV promoter disrupts the chromatin structure enabling NFI binding and subsequent formation of a stable transcription complex. Whether the receptor remains bound to DNA during induction or is displaced by NFI is not conclusively known, but our evidence supports a “hit and run” mechanism. NFI is not the only factor involved in hormonally induced transcription of the MMTV promoter. Two degenerated octamer motifs located immediately upstream of the TATA box are recognized by the ubiquitous transcription factor OTF-1 (Oct-1, NFIII), and are also important. In vitro, mutations in these motifs do not influence basal transcription, but completely abolish the stimulatory effect of purified progesterone receptor. Progesterone receptor bound to the HRE facilitates binding of OTF-1 to the two octamer motifs. Thus, OTF-1 is a natural mediator of progesterone induction of the MMTV promoter and acts through cooperation with the hormone receptor for binding to DNA.

A method for characterization of endogenous ligands to orphan receptors belonging to the steroid hormone receptor superfamily—Isolation of progesterone from pregnancy plasma using progesterone receptor ligand-binding domain

An analytical method is described whereby progesterone is isolated from pregnancy plasma on the basis of the high affinity and specificity of the progesterone receptor for its ligand. Partially purified progesterone receptor ligand-binding domain, expressed as a protein A fusion protein in Escherichia coli, is incubated with a neutral steroid fraction obtained by extraction and ion-exchange chromatography of human late-pregnancy plasma. The incubated sample is passed through two Lipidex 1000 (lipophilic gel) beds. The first, at 4°C, separates the specific ligand-fusion protein complex from nonspecifically bound and unbound compounds, and the second, at 40°C, separates the specific ligand from the protein. Elution of the second bed with methanol yields a fraction containing specific ligand that can be characterized by gas chromatography-mass spectrometry. This methodology may be valuable for identification of endogenous ligands to orphan receptors of the steroid hormone receptor superfamily.

Ubiquitous transcription factor OTF-1 mediates induction of the MMTV promoter through synergistic interaction with hormone receptors

Steroid hormones induce transcription from the mouse mammary tumor virus (MMTV) promoter by complex mechanisms requiring binding of the hormone receptors to the hormone responsive element (HRE) of the long terminal repeat region. Here we show that the MMTV promoter contains two degenerated octamer motifs immediately upstream of the TATA box that together bind OTF-1 (Oct-1, NFIII) with an affinity similar to the octamer consensus. In transfection experiments, mutation of these octamer motifs interferes with the hormonal response of the MMTV promoter. In vitro, these mutations do not influence basal transcription but completely abolish the stimulatory effect of purified progesterone receptor. Progesterone receptor and glucocorticoid receptor bound to the HRE facilitate binding of OTF-1 to the two octamer motifs. Thus, OTF-1 is a natural mediator of hormonal induction of the MMTV promoter and acts through cooperation with the hormone receptors for binding to DNA.

The steroid-dependent regulatory element in the ovalbumin gene does not function as a typical steroid response element.

Transcription of the chicken ovalbumin gene is induced both in vivo and in vitro by four classes of steroid hormones. Recent experiments identified a steroid-dependent regulatory element (SDRE) in the 5'-flanking region of the ovalbumin gene between -900 and -521. To characterize the regulatory properties of the SDRE more precisely, additional mutations were created in this region, and fusion genes prepared by linking the ovalbumin 5'-flanking region and promoter to the chloramphenicol acetyltransferase structural gene. When the ovalbumin-chloramphenicol acetyltransferase fusion genes were transfected into steroid-responsive primary oviduct cells, mutants lacking sequences between -900 and -732 were no longer responsive to estrogen, corticosterone, progesterone, or dihydrotestosterone. The SDRE did not confer steroid-dependent expression on the heterologous thymidine kinase promoter by itself but did in conjunction with the negative regulatory element identified between -350 and -100. This suggests that the two elements act as a single functional entity and that the SDRE is not behaving as a typical steroid response element. Gel shift analyses revealed that two SDRE.protein complexes were formed when nuclear protein extracts were derived from estrogen-treated chicken oviduct but that only one complex was formed with extracts from estrogen-withdrawn oviduct or from other tissues. Neither an estrogen response element oligomer nor a glucocorticoid/progesterone response element oligomer competed for either of the DNA.protein complexes. Partially purified progesterone receptor also did not bind to the SDRE. These data indicate that induction of the ovalbumin gene by steroid hormones requires complex interactions involving both the SDRE and the negative regulatory element.

Estrogen and progesterone receptor-binding sites on the chicken vitellogenin II gene: synergism of steroid hormone action.

The chicken vitellogenin II gene is transcriptionally activated by estrogens. In transient transfection experiments in human T47D cells that contain receptors for various steroids, we showed estradiol, progestin, and androgen responses of a chimeric chicken vitellogenin II construct. This construct consists of DNA sequences from -626 to -590 upstream of the start of transcription of the chicken vitellogenin gene linked to the herpes simplex virus thymidine kinase promoter driving the transcription of the bacterial chloramphenicol acetyltransferase gene. Treatment of the transfected T47D cells with a combination of estradiol and the progestin R5020 led to a superinduction of chloramphenicol acetyltransferase activity, showing a synergistic action of these two steroids. This synergism was not observed upon treatment of the transfected cells with estradiol and the androgen dihydrotestosterone. Using point mutations in the vitellogenin gene fragment, we showed in functional and in in vitro DNase I footprinting assays with a purified progesterone receptor that, for the synergistic action of estradiol and R5020 to occur, the progesterone receptor must be bound to the vitellogenin gene fragment. The progesterone receptor-binding site was localized at -610 to -590, close to the consensus sequence (-626 to -613) for estrogen receptor binding and function. We therefore demonstrate here that two different steroid hormones can be functionally synergistic through the interaction of their corresponding receptors with two different binding sites adjacent to one another.

Estrogen and progesterone receptor-binding sites on the chicken vitellogenin II gene: synergism of steroid hormone action.

The chicken vitellogenin II gene is transcriptionally activated by estrogens. In transient transfection experiments in human T47D cells that contain receptors for various steroids, we showed estradiol, progestin, and androgen responses of a chimeric chicken vitellogenin II construct. This construct consists of DNA sequences from -626 to -590 upstream of the start of transcription of the chicken vitellogenin gene linked to the herpes simplex virus thymidine kinase promoter driving the transcription of the bacterial chloramphenicol acetyltransferase gene. Treatment of the transfected T47D cells with a combination of estradiol and the progestin R5020 led to a superinduction of chloramphenicol acetyltransferase activity, showing a synergistic action of these two steroids. This synergism was not observed upon treatment of the transfected cells with estradiol and the androgen dihydrotestosterone. Using point mutations in the vitellogenin gene fragment, we showed in functional and in in vitro DNase I footprinting assays with a purified progesterone receptor that, for the synergistic action of estradiol and R5020 to occur, the progesterone receptor must be bound to the vitellogenin gene fragment. The progesterone receptor-binding site was localized at -610 to -590, close to the consensus sequence (-626 to -613) for estrogen receptor binding and function. We therefore demonstrate here that two different steroid hormones can be functionally synergistic through the interaction of their corresponding receptors with two different binding sites adjacent to one another.

Progesterone induction of metallothionein-IIA gene expression.

Expression of the metallothionein gene is known to be induced by glucocorticoids in a variety of cells. Here we show that in human cell lines containing functional progesterone receptors, the endogenous metallothionein-IIA (hMTIIA) gene is inducible by the synthetic progestins R5020 and medroxy-progesterone acetate. That this effect reflects a direct interaction with the metallothionein gene is supported by our finding that the partially purified progesterone receptor binds to the promoter region of the gene in vitro. The limits of the DNase I footprint and the guanine residues protected in methylation studies with the progesterone receptor are similar to those previously described for the glucocorticoid receptor. Thus, the hormone regulatory element of the human metallothionein-IIA gene can mediate regulation by both glucocorticoids and progestins, as does the hormone regulatory element of mouse mammary tumor virus.

Chemical and antigenic properties of pure 108,000 molecular weight chick progesterone receptor.

Previous purifications of the progesterone receptor have yielded inadequate amounts of pure protein along with significant amounts of a nonreceptor contaminant. We have taken advantage of the high yield provided by an affinity chromatography method for partial purification and after the incorporation of additional steps, we obtained purified progesterone receptor devoid of detectable contaminants and suitable for chemical analysis. A polyclonal antibody was obtained using the pure receptor as the antigen. The antibody was specific for progesterone-binding receptor. Tissue distribution of cross-reacting material, analyzed by immunoblotting, confirmed the presence of the receptor protein only in the two tissues where progesterone binding has been described in the chick: the oviduct and the bursa of Fabricius. It was absent in receptor-negative tissues such as liver and lung. The receptor was cleaved with cyanogen bromide and trypsin to obtain fragments that were partially sequenced.

Immunocytochemical localization of progesterone receptor in the guinea pig central nervous system

The distribution of neurons containing progesterone receptors in the brain of guinea pigs ovariectomized and primed by estradiol was investigated immunohistochemically using monoclonal antibodies to the progesterone receptor. We found that the picric acid paraformaldehyde perfusion provided satisfactory conditions of fixation for visualizing the progesterone receptor in sections of frozen tissue. Among the different techniques of immunocytochemical detection used, the indirect antibody peroxidase-antiperoxidase method gave the best results. The immunostained neurons were mainly located in two specific regions of the hypothalamus: the preoptic area and the mediobasal hypothalamus. Within the preoptic region, the majority of immunoreactive cells were present in the nucleus preopticus periventricularis particularly at the level of the pars suprachiasmatica. Within the mediobasal hypothalamus, immunostained neurons were found in the nucleus periventricularis, arcuatus, ventromedialis and premamillaris. Differences in the intensity of immunoreactivity appeared from one region to another. A marked cellular heterogeneity was observed: in each neuroanatomical structure, labeled cells alternated with non-labeled cells. The receptor, even in absence of progesterone, was localized in the nucleus. The nucleolus was not stained and only neurons were labeled. There was no progesterone receptor immunoreactivity in other regions of the brain, especially in the amygdala, hippocampus and cortex where biochemical studies have shown the presence of a non-estrogen regulated protein binding the progestin [ 3H]R 5020. Control experiments with antibody pretreated with purified progesterone receptor or with mouse receptor-unrelated monoclonal antibody did not show fluorescent or immunoreactive cells.

Molecular Cloning of the Chicken Progesterone Receptor

To define the functional domains of the progesterone receptor required for gene regulation, complementary DNA (cDNA) clones encoding the chicken progesterone receptor have been isolated from a chicken oviduct lambda gt11 cDNA expression library. Positive clones expressed antigenic determinants that cross-reacted with six monospecific antibodies derived from two independent sources. A 36-amino acid peptide sequence obtained by microsequencing of purified progesterone receptor was encoded by nucleotide sequences in the longest cDNA clone. Analysis of the amino acid sequence of the progesterone receptor deduced from the cDNA clones revealed a cysteine-rich region that was homologous to a region found in the estrogen and glucocorticoid receptors and to the avian erythroblastosis virus gag-erb-A fusion protein. Northern blot analysis with chicken progesterone receptor cDNA's indicated the existence of at least three messenger RNA species. These messages were found only in oviduct and could be induced by estrogens.

Interaction of oestrogen and progesterone receptors with specific subfractions of laying-hen oviduct chromatin.

Salt (NaCl)-extracted nuclear oestrogen receptor from hen oviduct was incubated with salt-depleted oviduct chromatin and dialysed to low salt. The oestrogen receptor (re)associated with chromatin to form a 13-14S-sedimenting fraction, as found in 'native' chromatin, and saturation of this interaction was obtained for very low receptor concentrations (approx. 0.04 nM). Similarly, purified progesterone receptor from chick oviduct cytosol associated with depleted chromatin to form an 11-12S-sedimenting fraction, as in 'native' chromatin; this interaction tended towards saturation for much higher concentrations of progesterone receptor (approx. 8 nM) than that observed for oestrogen receptor. When the two receptors were incubated with depleted chromatin from hen kidney or erythrocytes, their s values were as for oviduct chromatin. However, no saturation of these interactions was seen, even for high concentrations of receptor. Steroid-hormone receptors can therefore bind in vitro to particular subfractions of non-target-tissue chromatin, but with a much lower affinity than to target-tissue chromatin.

Effects of calcium on the chick oviduct progesterone receptor

The chick oviduct cytosol progesterone receptor can be transformed to a small form (R s = 21 A ̊ , S 20, w: 2.9 ) denoted “mero-receptor” by incubation in the presence of Ca 2+ [8]. In the molybdate-free cytosol all the progestin binding components could be completely transformed to mero-form by 1 h treatment with 100 mM Ca 2+ at 0°C. If EDTA was secondarily added, the ligand was rapidly released. If molybdate (20 mM) containing cytosol was incubated with Ca 2+, no radioactivity was found in the meroposition on the Agarose A 0.5 m column, but the bound steroid sedimented at 2.9 S in sucrose gradients containing Ca 2+ (and no molybdate). When 20 nM molybdate was added to cytosol containing receptor activated by 0.3 M KCl, complete mero-transformation by Ca 2+ was obtained also by the gel filtration criterion, indicating that molybdate does not inhibit the mero-transforming factor. Ligand-free progesterone receptor could also be completely converted to mero-form by endogenous cytosolic transforming factor and calcium. The transforming factor was completely inactivated, when cytosol was run through Agarose A 0.5 m gel. Mero-transformation was found to be irreversible. The purified progesterone receptor subunit 110 K (B) was partially converted to smaller forms by calcium alone (100 mM, 0°C, 1 h) whereas addition of a small amount of cytosol allowed complete conversion to mero-form.

Purification of chick oviduct progesterone receptor apoprotein

The 8S progesterone receptor from chick oviduct has been partially purified (500-fold) as the apoprotein lacking hormone. The procedure employs polyethylene glycol precipitation, phosphocellulose, DNA cellulose, DEAE cellulose, hydroxylapatite and ethyl agarose column chromatography. As shown for the native cytosolic receptor, the purified apoprotein can be dissociated into A and B subunits. Qualitative and quantitative analysis of subunit structure shows no difference between purified and cytosolic native progesterone receptor. The A subunit dissociated from the purified receptor maintains its DNA binding activity as shown by the “Western” protein blotting technique and sedimentation velocity analysis. Moreover, purified progesterone receptor has steroid binding specificity very close to the native material. The purified progesterone receptor is free of DNase and RNase contaminating activities and represents a useful tool for in vitro studies of hormone action.

Photoaffinity Labeling of Steroid Binding Proteins with Unmodified Ligands

Photoactivation of the alpha,beta-unsaturated ketones of natural and synthetic steroid molecules by light of lambda greater than or equal to 330 nm allows their covalent attachment to steroid-binding proteins. The general validity of this method is demonstrated with two steroid hormone receptors and the steroid-binding protein uteroglobin. Progesterone can be covalently attached to the partially purified progesterone receptor and to uteroglobin, and comigrates with the binding proteins upon electrophoresis in polyacrylamide gels containing sodium dodecyl sulfate. Similarly the synthetic glucocorticoid triamcinolone acetonide can be covalently bound to the partially purified glucocorticoid of rat liver. This method allows the identification of steroid hormone receptors after electrophoresis in polyacrylamide gels containing sodium dodecyl sulfate. Labeling with radioactive steroids is specific since it can be prevented by the addition of an excess of non-radioactive ligand. Digestion of the labeled binding proteins with trypsin or chymotrypsin yields a defined pattern of radioactive peptides, demonstrating that covalent attachment takes place at specific binding sites.

Steroid receptor binding to nuclei: Effect of assay conditions on the integrity of chromatin

Various incubation conditions previously used in cell-free assays of steroid receptor binding to nuclei were evaluated on the basis of their effect on the integrity of the nuclear chromatin. Incubation of nuclei for 60 min in whole cytosol containing no added salt at 25°C resulted in significant DNA degradation and some proteolysis. This deterioration in the integrity of the chromatin increased its capacity to bind the [ 3H]-progesterone-receptor complex, possibly via exposure of previously “masked” acceptor sites. Decreasing the temperature and/or increasing the salt concentration reduced or eliminated these changes in the chromatin. Incubation of nuclei in an ammonium sulfate-fraction of the cytosol, which contained the partially purified progesterone receptor, resulted in no detectable deterioration of the chromatin integrity irrespective of the temperature (⩽25°C) or the added salt concentration (0–0.15 M KCl). These results suggest that some of the conflicting reports on the nature of the nuclear acceptor sites for steroid receptors could be due to the use of inappropriate assay conditions, which permit degradation of the nucleo-protein and result in altered levels of steroid receptor binding. (Steroid/ Progesterone/Nuclei/Chromatin.)

A review of regulation of gene expression by steroid hormone receptors

We have investigated the manner by which progesterone receptors act to induce initiation of RNA synthesis in a cell free system derived from chick oviduct. A method utilizing rifampicin enabled us to measure the formation of binary initiation complexes between RNA polymerase and chick oviduct chromatin and allowed for the quantitative assessment of RNA chain initiation sites. RNA chain propagation rates, and RNA chain size under conditions which prevent secondary chain reinitiations. Purified progesterone-receptor complex stimulated transcription of oviduct chromatin in vitro by promoting an increase of 3000 to 5000 additional sites for RNA chain initiation. These data showed that progesterone receptor can directly increase the number of RNA polymerase initiation sites in the chromatin template in the absence of a detectable change in either the rate of RNA propagation or the size of the RNA product. The stimulatory effect of the purified progesterone receptor was time dependent (T 1 2 of 15 min) and the concentration of receptor for half maximal stimulation of RNA chain initiation was ~5 × 10 −9M, a value which correlates directly with the receptor affinity for binding to chromatin. The stimulatory effect of the purified progesterone receptor appeared to be relatively specific for oviduct chromatin in comparison to non-target tissue chromatins or DNA. Utilizing a copy DNA probe to ovalbumin mRNA, a greater than ten-fold increase in transcription of the ovalbumin gene was detected after the incubation of progesterone receptor with oviduct chromatin. The data presented here show that steroid hormone receptor complexes can directly regulate aene transcription in vitro in a manner which mimics the events observed in vivo in target cells.

Progesterone-binding components of chick oviduct. In vitro effects of purified hormone-receptor complexes on the initiation of RNA synthesis in chromatin.

We have investigated the manner by which progesterone receptors act to induce initiation of RNA synthesis in a cell-free system derived from chick oviduct. A method utilizing rifampicin enabled us to measure the formation of binary initiation complexes between RNA polymerase and chick oviduct chromatin (Tsai, M.-J., Schwartz, R.J., Tsai S.Y., and O'Malley, B.W. (1975) J.Biol. Chem. 250, 5165-5174) and allowed for the quantitative assessment of RNA chain initiation sites, RNA chain propagation rates, and RNA chain size under conditions which prevent secondary chain reinitiations. We have measured the available initiation sites for transcription in oviduct chromatin prepared from chicks withdrawn from all hormone and then restimulated with a secondary injection of progesterone. Within 1/2 hour after administration of progesterone, the number of initiation sites increased from 8,700 sites/pg of chromatin DNA for the control to 15,500 sites. After 1 hour, the concentration of RNA polymerase needed to saturate chromatin binding sites was increased 60% in comparison to control values, while the number of initiation sites increased 160%. This rapid increment in transcriptional activity preceded temporally the induction of synthesis of ovalbumin mRNA. To test directly the effect of progesterone receptor on transcription, in vitro, a reconstituted cell-free system was employed which contained purified cytoplasmic progesterone-receptor complexes, Escherichia coli RNA polymerase, and chromatin prepared from hormonally withdrawn chick oviducts. Purified progesterone-receptor complex stimulated transcription of oviduct chromatin in vitro by promoting an increase of 3,000 to 5,000 additional sites for RNA chain initiation. These data showed that progesterone receptor can directly increase the number of RNA polymerase binding and initiation sites in the chromatin template in the absence of a detectable change in either the rate of RNA chain propagation or the size of the RNA product. The kinetics of progesterone-receptor stimulation of RNA synthesis in chromatin revealed a t1/2 of 15 min for this effect to occur. This value was identical with the optimal time required for binding of receptor to chromatin. The concentration of receptor required for half-maximal stimulation of RNA chain initiation was approximately 5 x 10(-9) M. This value agreed closely with our previously reported estimates of the affinity (Kd approximately 5 x 10(-9)M) of the progesterone-receptor complex for oviduct chromatin. The stimulatory effect of purified progesterone receptor appeared to be relatively specific for oviduct chromatin in comparison to nontarget tissue chromatins or chick DNA. The data presented here show that steroid hormone-receptor complex can directly regulate gene transcription in vitro in a manner which mimics the events observed in vivo in target cells.