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.     

Glucocorticoid binding in the hen oviduct

[³H]Triamcinolone acetonide (15nm) was incubated with cytosol (150000g fraction) prepared from oviducts of egg-laying hens. The extent of steroid binding, as determined by charcoal assays, was greatest between 2–4h at 4°C. A similar time curve was obtained when cytosol preparations were first fractionated with (NH₄)₂SO₄ before labelling. The addition of 10mm-Na₂MoO₄ or 10mm-ATP during the incubation of hen oviduct cytosol with [³H]triamcinolone acetonide lowered the extent of steroid binding. The presence of glycerol (20%), however, increased the extent of [³H]triamcinolone acetonide binding in cytosol fractions from chick (330%) and hen (160%) oviducts. The [³H]triamcinolone acetonide–receptor complex was stable for over 4h at 4°C, but dissociated rapidly at 37°C, exhibiting a half-life of about 10min. The presence of 10mm-Na₂MoO₄ and 10mm-ATP or both had a small protective effect on the dissociation of [³H]triamcinolone acetonide–receptor complex. The receptor from hen oviduct showed significant affinity for unlabelled triamcinolone acetonide, cortisol, compound R5020 and dihydrotestosterone and, to a lesser extent, for oestradiol, oestrone and progesterone. Diethylstilboestrol treatment of immature chicks appeared to induce a more specific binder, which showed affinity for unlabelled triamcinolone acetonide, cortisol and compound R5020 only. Scatchard analysis of [³H]triamcinolone acetonide binding in hen oviduct cytosol revealed a K_d value of 6.4nm. The steroid–receptor complex sedimented as a 7–8S and a 4S entity on low-salt (0.01m-KCl)- and high-salt (0.3m-KCl)-containing sucrose gradients respectively. The cytosol [³H]triamcinolone acetonide–receptor complex showed no affinity for ATP–Sepharose or DNA–cellulose, but acquired this ability on heat activation (23°C, 40min). The data indicate the avian oviduct possesses a high-affinity binding molecule that fulfils the criteria of a glucocorticoid receptor.     

Distribution of estrogen receptors in subfractions of hen oviduct chromatin.

Hen oviduct chromatin was digested with DNase II and separated into two fractions. The MgCl2 insoluble chromatin fraction (43% of the total DNA) was enriched in nucleosome-like particles, which sedimented at 11 S and contained 185 base pairs of DNA. The MgCl2 soluble chromatin fraction (5% of the total DNA) was characterized by 5 S and 14 S peaks in sucrose gradients; Estrogen receptors in the chromatin fractions were labelled with [3H] estradiol using the steroid exchange assay. The concentration of receptors in the MgCl2 soluble chromatin was 4;5 times higher than that in the MgCl2 insoluble chromatinmin sucrose gradient analysis the 11 S particles displayed a negligible specific radioactivity suggesting that estrogen receptors mainly bind to extranucleosomal chromatin.     

Acceptor sites for the oestrogen receptor in hen oviduct chromatin.

Partially purified hen oviduct oestrogen receptors, charged with [3H]oestradiol, were shown to specifically bind in vitro to purified hen oviduct chromatin. Maximal binding occurred within 60min at 0 degrees C in a Tris buffer containing 0.1 M-KCl and 0.5 mM-phenylmethanesulphonyl fluoride. The binding of the [3H]oestradiol-receptor complexes to intact purified chromatin was saturable, whereas the receptor binding to hen DNA remained linear. Saturation was further demonstrated by the minimal acceptor binding of receptor charged with [3H]oestradiol plus 200-fold oestradiol compared with [3H]oestradiol receptors at equal [3H]oestradiol concentrations. Scatchard analysis of [3H]oestradiol-receptor binding to chromatin above DNA levels gave indications of high-affinity binding with a low capacity. Further, the nuclear binding was tissue-specific since the binding to hen spleen chromatin was negligible. To further uncover the specific acceptor sites, proteins were removed from hen oviduct chromatin by increasing concentrations of guanidine hydrochloride (1-7M). Those residual fractions extracted with 3-7 M-guanidine hydrochloride had the highest acceptor activity (above DNA levels) with the peak activity uncovered by 5 M-guanidine hydrochloride. To further characterize the oestrogen-receptor acceptor sites, oviduct chromatin was bound to hydroxyapatite in the presence of 3 M-NaCl and then protein fractions were extracted sequentially with 1-7 M-guanidine hydrochloride. Each fraction was then reconstituted to pure hen DNA by reverse gradient dialysis. [3H]Oestradiol receptors were found to bind to the greatest degree to the fraction reconstituted from the 5 M-guanidine hydrochloride protein extract. Reconstituted nucleoacidic proteins (NAP) from combined 4-7 M-guanidine hydrochloride protein extracts showed saturable binding by [3H]-oestradiol receptors, whereas binding to hen DNA did not saturate. The high affinity, low capacity, and specificity of binding of oestrogen receptors to NAP was similar to that found in intact chromatin. Thus, chromatin acceptor proteins for the oestrogen receptor have been partially isolated and characterized in the hen oviduct and display properties similar to that reported for the acceptor proteins of the progesterone receptor.     

Estrogen receptor in hen oviduct chromatin, digested by micrococcal nuclease.

Nuclei from laying hen oviduct were prepared according to Hewish and Burgoyne i.e. in the presence of spermine and spermidine and in the absence of divalent cations and were then moderately digested by micrococcal nuclease. When the resulting chromatin was analysed by ultracentrifugation on a sucrose gradient, a peak of specific estradiol-binding sites was observed, sedimenting slightly faster (13-14 S) than the mononucleosomes (12 S). When the chromatin was centrifuged on a gradient containing heparin (5 microngram/ml) the sedimentation coefficient of the estradiol receptor peak shifted to 7-8 S; it returned to the 13-14 S position in the absence of heparin, when target organ chromatin was also present in the gradient. The preparation of the chromatin is described and the validity of the method to explore receptor localisation is discussed, as is the specificity of the receptor-DNA interaction.     

Studies on the stability of chromatin from hen oviduct

The stability of sheared chromatin from the magnum portion of hen oviduct has been examined during storage at 0 °C using several parameters. Chemical composition and ultraviolet spectra were unchanged after 1 month while changes in melting profiles were seen after 2 weeks. Gel electrophoretic patterns of histones indicated slight proteolysis after 4 weeks with no apparent degradation occurring at earlier times. Acidic protein gel patterns remained unchanged during the period of study. Rapid changes (within 1 to 3 days) were seen in chromatin size distributions as measured in sucrose gradients and in chromatin template activity. The mean chromatin size decreased with storage, an increasing percentage of the total population sedimenting as a 17S component. This change in chromatin size was accompanied by an increase in the amount of free protein floating on the top of gradients.Several experiments were conducted to investigate the nature of the chromatin changes occurring at early storage times. Changes in chromatin size and template activity could be attributed to neither proteolysis nor DNase activity. Diisopropylfluorophosphate, a known nuclear protease inhibitor, did not inhibit the rate of change in chromatin size with storage. Furthermore, no proteolysis was detectable by gel electrophoresis during storage times at which significant changes in chromatin size and template activity had occurred. When the size of chromatin DNA was examined, no changes were seen with storage nor were there differences in the size of DNA from fast and slow sedimenting chromatin fractions.When template activities of fast and slow sedimenting chromatin fractions were compared, the slow sedimenting fractions were found to be the most template active. The template activity of a slow sedimenting, high template activity fraction was examined with storage and was found to increase with time.     

A high-affinity calcium-stimulated ATPase activity in the hen oviduct shell gland

The hen oviduct shell gland is a highly active calcium-transporting epithelial tissue which is responsible for the mineralization of the egg shell. We have identified a calcium-stimulated ATPase present at high specific activity in membrane preparations from shell gland mucosal shavings. In the presence of optimal MgCl₂ (5 mm) and a Ca²⁺ buffer, ATP hydrolysis was stimulated by addition of low concentrations of free Ca²⁺ (K_0.5 ~0.4 μm); but not by similar concentrations of Mn²⁺, Zn²⁺, Co²⁺, or La²⁺. This stimulation was specific for ATP; there was little or no effect of Ca²⁺ on hydrolysis of ADP, AMP, GTP, ITP, or p-nitrophenyl phosphate. Calcium-stimulated ATPase activity was inhibited by chlorpromazine, trifluoperazine, and quercetin, as well as by sulfhydryl-blocking agents, but not by oligomycin or ouabain. No significant effect of calmodulin was observed. Finally, low concentrations of free Ca²⁺ (10 to 100 μm) in the presence or absence of Mg²⁺ stimulated transfer of ³²P from [γ-³²P]ATP to a 105,000 molecular weight shell gland membrane protein. This phosphoprotein was sensitive to hydrolysis by heating or by hydroxylamine treatment at acidic pH, and its formation was not inhibited by addition of K⁺. The specific activity of Ca²⁺-ATPase in total membrane preparations from laying hen shell gland ranged from 80 to 150 nmol/min/ mg protein, similar to or greater than levels found in purified plasma membrane fractions from a variety of tissues. No significant activity was found in membrane preparations from the magnum or isthmus regions of the oviduct, which are not involved in egg shell calcification. The characteristics of the Ca²⁺-ATPase, its high specific activity, and its preferential localization in the shell gland region of the oviduct suggest a role for an ATP-dependent calcium transport system in egg shell mineralization.     

Non-activated progesterone receptor extracted from nuclei of hen oviduct

When the progesterone receptor was extracted from nuclei of laying hen oviduct with 0.5 M sodium molybdate, a large, 7-8 S, form of the receptor was observed. This receptor form resembled non-activated cytoplasmic receptor not only in displaying the same sedimentation coefficient, but also in rapid dissociation rate of the hormone-receptor complex. This finding suggests that either activation may occur within the nuclear compartment, or that activation may be reversed under certain conditions.     

Role of constraint in catalysis and high-affinity binding by proteins

Using a model for catalysis of a dynamic equilibrium, the role of constraint in catalysis is quantified. The intrinsic rigidity of proteins is shown to be insufficient to constrain the activated complexes of enzymes, irrespective of the mechanism. However, when minimization of the surface excess free energy of water surrounding a protein is considered, model proteins can be designed with regions of sufficient rigidity. Structures can be designed to focus surface tension or hydrophobic attraction as compressive stress. A monomeric structure has a limited ability to concentrate compressive stress and constrain activated complexes. Oligomeric or multidomain proteins, with domains surrounding a rigid core, have unlimited ability to concentrate stress, provided there are at least four domains. Under some circumstances, four is the optimum number, which could explain the frequency of tetrameric enzymes in nature. The minimum compressive stress in oligomers increases with the square of the radius. For tetramers of similar size to natural enzymes, this stress agrees reasonably well with that needed to constrain the activated complex. A similar principle applies to high affinity binding proteins. The models explain the trigonal pyramidal shape of fibroblast growth factor and provide a basis for interpretation of protein crystal structures.     

Nuclear progesterone receptor in the hen pituitary and hypothalamus.

The existence of specific progesterone-binding sites with a high affinity and a limited capacity was demonstrated by [3H]progesterone exchange in the nuclear fractions of the pituitary and hypothalamus as well as in those of the oviduct magnum in the hen.     

Studies on the high-mobility-group non-histone proteins from hen oviduct.

Nuclear high-mobility-group (HMG) proteins were isolated from hen oviduct. These were proteins HMG-1, -2, -3, -14 and -17, which are equivalent to the classification of calf thymus HMG proteins. Hen oviduct proteins HMG-1 and -2 were individually isolated by HCIO4.extraction and CM-Sephadex chromatographic separation. Their mol.wts. were determined as 28 000 and 27 000, respectively. The proteins have a high content of acidic and basic amino acids. The association of proteins HMG-1 and -2 with the genome of hen oviduct nuclei was probed by a limited digestion with nucleases. Hen oviduct nuclei were incubated with deoxyribonuclease I or micrococcal nuclease until 10% of the DNA was digested. The nuclear suspension was centrifuged and the contents of proteins HMG-1 and -2 in the supernatant and sediment fractions were analysed by polyacrylamide-gel electrophoresis. HMG proteins were found to be preferentially released by micrococcal-nuclease digestion rather than by deoxyribonuclease I.     

Oestrogen and progesterone receptors in chick oviduct chromatin after administration of oestradiol, progesterone or anti-oestrogen.

Oestrogen-primed and withdrawn chicks were injected with oestradiol benzoate, progesterone, and/or the anti-oestrogens tamoxifen and 4-hydroxytamoxifen. Oestrogen receptors were studied in oviduct chromatin solubilized by mild digestion of purified nuclei with micrococcal nuclease. After a single injection of oestradiol benzoate, ultracentrifugation on sucrose gradients of chromatin extracts labelled with [3H]-oestradiol showed two peaks of oestradiol binding sites, sedimenting at 13--14 S and 7--8 S. After repeated injections of oestradiol benzoate, the 13--14 S peak increased more than the 7--8 S peak. After injection of anti-oestrogen alone or together with oestradiol benzoate, no [3H]oestradiol-binding or 4-hydroxy[3H]tamoxifen-binding peaks were detected in the chromatin. Injection of progesterone also produced an increase of the 13--14 S and 7--8 S chromatin oestradiol receptor. Progesterone receptor could only by detected in chromatin early after progesterone administration, and it sedimented in density gradients with the 12 S mononucleosome fraction. Tamoxifen injected together with progesterone gave higher levels of 13--14 S oestrogen binding sites than did progesterone alone. The presence of a 13--14 S peak of oestrogen binding sites in hormonal situations which promote a biological response in the chick oviduct, and the absence of this peak after administration of anti-oestrogens, suggest that this subfraction of chromatin contains elements involved in gene regulation.     

Chicken oviduct progesterone receptor: Location of specific regions of high-affinity binding in cloned DNA fragments of hormone-responsive genes

We have used a DNA-cellulose competitive binding assay to measure the extent of displacement of the chicken oviduct progesterone-receptor complex from calf thymus DNA-cellulose by purified cloned fragments of genomic DNA. Several DNA fragments from hormonally responsive genes coding for egg-white proteins were found to be efficient competitors for either crude or partially purified receptor complexes when compared with calf thymus DNA. Data obtained with deletion mutations constructed in vitro allowed delineation of a specific region necessary for strong competition, located 250–300 bp upstream from the mRNA startsite of the oval-bumin gene. Sequence homologies with this 5′-upstream region were observed in other fragments of the ovalbumin, conalbumin, ovomucoid, X and Y genes, which were also efficient competitors. Based on a comparison of such sequences of homology, a consensus sequence that may constitute a region binding progesterone-receptor complex has been constructed: ATC_TT^CCATT_T^ATCTG_T^GTTGTA. The results suggest that specific double-stranded DNA sequences are recognized by. the oviduct progesterone-receptor complex in vitro, and are relevant to the question of whether specific DNA sequences are directly involved as genomic binding sites for steroid receptors.     

Iodination of the progesterone receptor from hen oviduct spares the DNA-binding domain

The progesterone receptor from hen oviduct is isolated as a complex of two subunits, A and B. The A protein binds one molecule of progesterone and also binds to DNA with high affinity. The native A protein can be labeled with iodine with no loss of DNA binding activity. Limited Staphylococcus aureus V8 protease digestion of the labeled preparation results in a number of DNA-binding and non-DNA-binding fragments of the receptor. The progesterone-binding domain contains iodine label. However, two low-molecular-weight DNA-binding fragments do not contain iodine label, indicating a lack of susceptible tyrosine residues near the DNA-binding site of the native receptor. The labeled receptor and its fragments will facilitate studies of the isolated DNA-binding and progesterone-binding domains of the hen A protein as well as of the activity of the native receptor in the presence and absence of hormone.