Characterization of the chromatin acceptor sites for the avian oviduct progesterone receptor using monoclonal antibodies

Monoclonal antibodies (MAb) against the chromatin acceptor sites for the avian oviduct progesterone receptor were prepared with highly purified hen oviduct acceptor proteins reconstituted to hen DNA. Addition of the MAbs to a cell-free assay blocked progesterone receptor from chick oviduct (PRov) binding to native-like acceptor sites on nucleoacidic protein (NAP) representing a partially deproteinized chromatin, which has been shown to be enriched in these binding sites. However, the antibodies do not block PRov binding to pure DNA, nor do they affect the receptor itself. Estrogen receptor binding to NAP was not inhibited, supporting a receptor specificity of the PRov acceptor sites as reported previously from direct competition studies. These data support earlier studies showing that (1) the reconstituted PRov acceptor sites resemble the native sites, (2) the acceptor sites are receptor specific, and (3) the PRov binding sites of NAP are different from those of pure DNA. While some animal-species specificity in the PRov binding inhibition was observed, no tissue specificity was seen. Direct binding of the antibodies to native acceptor sites was demonstrated in an enzyme-linked immunosorbent assay (ELISA) system. The antibodies showed little recognition of free acceptor protein or DNA alone, indicating specificity for the protein-DNA complex. A partial evolutionary conservation of the nuclear acceptor sites for PRov was shown by the fact that about 50% of the inhibition seen with hen NAP was obtained with NAPs from several other species, and this partial cross-reactivity of the MAbs with the same NAPs from other animal species was also seen in the ELISA.     

Binding of [3H]estradiol- and [3H]H1285-receptor complexes to rabbit uterine chromatin

In the present study we investigated the binding characteristics of estrogen and antiestrogen-receptor complexes to rabbit uterine chromatin. Activated or nonactivated estrogen receptors were partially purified by DEAE-cellulose chromatography using low (1 mM) or high (10 mM) concentrations of sodium molybdate. Activated [³H]estradiol-receptor complexes showed enhanced binding to chromatin acceptor sites unmasked by 1 M, 4 M and 6 M guanidine hydrochloride. We also examined the chromatin-binding characteristics of the estrogen receptors when bound by the high-affinity triphenylethylene antiestrogen, H1285. The acceptor site activity for the [³H]H1285-receptor complexes was markedly decreased at sites unmasked by 4 M and 6 M guanidine hydrochloride. Further, the nonactivated receptor complexes showed very low binding to deproteinized chromatin. The estrogen-receptor chromatin-acceptor sites were tissue specific and saturable. These chromatin acceptor sites differ in their affinity and capacity (number of binding sites per cell) for the estrogen- and antiestrogen-receptor complexes. Thus, we suggest that the differences in the physiological and physicochemical properties of estrogens and antiestrogens may be related to their differential interaction with uterine chromatin subfractions.     

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.     

Characterization of a purified chromatin acceptor protein (receptor binding factor 1) for the avian oviduct progesterone receptor

The specific, high-affinity binding of the avian oviduct progesterone receptor (PR) with target-cell nuclei and chromatin has been shown to involve DNA complexed with specific chromatin acceptor proteins. One of these chromatin acceptor proteins has been partially purified and found to be a small hydrophobic protein with a broad pI of 5.0-6.0 [Goldberger, A., & Spelsberg, T. C., (1988) Biochemistry 27, 2103-2109]. This paper describes the final purification over 100,000-fold to apparent homogeneity of this candidate PR acceptor protein, termed the receptor binding factor 1 (RBF-1). When the avian genomic DNA is bound by RBF-1, saturable, high-affinity (KD approximately 2 x 10(-9) M) binding sites for PR are generated. RBF-1 has a unique, hydrophobic N-terminal sequence. The PR binding to the RBF-1-DNA complexes is shown to be dependent on an intact activated PR with which excess nonradiolabeled PR can compete. By use of a new, highly specific monoclonal antibody (mAb) to the RBF-1 with Western immunoblotting, RBF-1 was shown to be localized in the nucleus and to be tissue and species specific. Selective removal of the chromatin proteins containing RBF-1 results in the loss of the highest affinity class of PR binding sites. A second class of residual PR binding sites remains in the nucleoacidic protein (NAP), a complex of proteins more tightly bound to the DNA. This class of PR binding activity has been classified as the RBF-2. The RBF-1 is estimated to be 0.03% of the total chromatin protein with about 1.2 x 10(5) molecules/diploid cell.     

Reconstitution of nativelike nuclear acceptor sites of the avian oviduct progesterone receptor: evidence for involvement of specific chromatin proteins and specific DNA sequences

A specific fraction of avian oviduct chromosomal proteins can be reannealed to pure avian DNA to reconstitute nativelike specific nuclear binding sites (acceptor sites) for the oviduct progesterone receptor (PR). These specific nuclear binding sites represent the difference between the binding to the reconstituted NAP and that to pure DNA. The specific fraction of chromatin protein which contains the acceptor activity, fraction CP-3, is very tightly bound to hen DNA in a complex termed nucleoacidic protein (NAP). Removal of the CP-3 fraction from NAP results in a loss of specific PR binding sites. Resins containing chromatin adsorbed to hydroxylapatite are used as a rapid method to isolate the CP-3 fraction. Reconstitution of the CP-3 fraction to DNA by the described method involving a regressing gradient of 6-0 M guanidine hydrochloride (Gdn-HCl) results in a reconstituted NAP which displays specific PR binding sites identical with those in native (undissociated) NAP and whole chromatin. Optimal conditions and potential problems for reconstituting these nucleoproteins are described. Only partially purified receptor preparations were used in these cell-free binding analyses since they have been shown to bind with similar properties and patterns as the nuclear binding in vivo. Therefore, the binding of PR to the reconstituted NAPs was demonstrated to be receptor dependent, saturable, and of high affinity. Further, the pattern of binding to the reconstituted sites mimics those which are observed in vivo. Thus, nonfunctional receptors that cannot translocate and bind to the nuclear acceptor sites in vivo also failed to bind to the acceptor sites on the reconstituted NAPs generated by the acceptor proteins. In contrast, the binding to pure DNA does not reflect these receptor differences in receptor bindings. Specific binding of PR to reconstituted NAP can be reversed by again removing the protein fraction. Moreover, the specific binding can be destroyed by proteases and protected by protease inhibitors, indicating that acceptor activity is proteinaceous in nature. The reconstitution of the activity is both a concentration-dependent and time-dependent process. During the reconstitution, acceptor activity appears to reconstitute on the DNA when the Gdn-HCl concentration reaches 2.0 M. By use of the reconstitution method as an assay for acceptor activity, the activity in the CP-3 fraction was shown by molecular sieve chromatography to elute in a relatively broad molecular weight range between 13 000 and 25 000. The activity also focuses in isoelectric focusing resins with apparent pI's of 5.2 and 6.4.(ABSTRACT TRUNCATED AT 400 WORDS)     

Binding of estradiol receptor complexes to isolated human breast chromatin

Summary The interaction of estradiol-receptor complexes and isolated human breast tumor chromatin was studied under equilibrium conditions.The estradiol-receptor complexes bound specifically to the chromatin of hormone dependent tumors and showed a single class of binding sites with a Ka of 0.96 × 10¹⁰ m ^–1 and a binding capacity of 1.5 pmoles/mg DNA. The binding was a temperature-dependent process and involved a transformation of the receptor protein.The heat-activated hormone-receptor com-plex was more active than the 8S form in the binding phenomenon. The specific interaction of estradiol-receptor complex with isolated chromatin was saturable and sensitive to conditions of temperature and ionic strength. Furthermore under optimal conditions no acceptor sites were detected in chromatin of hormone independent tumors.Dedicated to ProfessorLuis F. Leloir on the occasion of his 70th birthday.     

Binding of [3H]triamcinolone acetonide-receptor complexes to chromatin from the B-cell leukemia line,BCL1

The binding characteristics of partially purified glucocorticoid receptor complexes from hormone sensitive, non-differentiating BCL₁ cells to sequentially deproteinized BCL₁ chromatin-cellulose was investigated. [³H]Triamcinolone acetonide (TA)-receptor complexes were purified (approx. 30-fold) from DEAF-cellulose columns by salt elution which allowed receptor activation only in the absence of molybdate. Addition of 10 mM molybdate completely blocked salt activation. The binding pattern of the activated [³H]TA-receptor complexes to chromatin-cellulose extracted with 0–8 M guanidine hydrochloride revealed three regions of increased binding activity (acceptor sites), at 2, 5 and 7 M guanidine hydrochloride. Acceptor site binding was markedly reduced for chromatin extracted with 3, 6 and 8 M guanidine hydrochloride. Non-activated receptor complexes demonstrated very low binding to deproteinized chromatin. It was also shown that chromatin binding required glucocortical receptors and that free ligand or ligand bound to other proteins did not bind significantly to chromatin. In addition, binding of [³H]TA-receptor complexes to partially deproteinized chromatin was competable by unlabeled TA-receptor complexes. Scatchard analysis demonstrated that chromatin from non-differentiating BCL₁ cells possesses multiple, high-affinity binding sites which differ in their affinity for the glucocorticoid receptor. Partially deproteinized chromatin from lipopolysaccharide-stimulated BCL₁ cells demonstrated a different pattern of receptor binding, i.e., receptor binding was significantly greater to chromatin previously extracted with 6–8 M guanidine hydrochloride. These results suggest that differentiation alters the state of chromatin and the interaction of non-histone protein/DNA acceptor sites with glucocorticoid receptors. These alterations may play a role in the acquisition of hormone resistance.     

Tissue Differences in Antigenic Properties of Non-Histone Protein-DNA Complexes

THAT DNA and histones are not tissue specific^1,2 implies that other components of chromatin may be responsible for the tissue specific control of eukaryotic gene expression. We now report studies of the antigenic properties of non-histone protein-DNA complexes isolated in an undissociated state from native chromatin and compare these with the properties of native chromatin. Because DNA is a very weak immunogen³, the antigenic determinants in these preparations should be principally caused by the protein components.     

Effect of non-histone proteins on thermal transition of chromatin and of DNA.

The effect of chromatin non-histone protein on DNA and chromatin stability is investigated by differential thermal denaturation method. 1) Chromatin (rat liver) yields a multiphasic melting profile. The major part of the melting curve of this chromatin is situated at temperatures higher than pure DNA, with a distinct contribution due to nucleosomes melting. A minor part melts at temperatures lower than DNA which may be assigned to chromatin non-histone protein-DNA complex which destabilized DNA structure. 2) Heparin which extracts histones lowers the melting profile of chromatin and one observes also a contribution with a Tm lower that of pure DNA. In contrast, extraction on non-histone proteins by urea supresses the low Tm peak. 3) Reconstitution of chromatin non-histone protein-DNA complexes confirms the existence of a fraction of chromatin non-histone protein which lowers the melting temperature when compared to pure DNA. It is concluded that chromatin non-histone proteins contain different fractions of proteins which are causing stabilizing and destabilizing effect on DNA structure.     

Interaction of non-histone proteins with DNA and chromatin from Drosophila and mouse cells. Specificity, isolation and analysis of complexes.

The specificity of the binding of purified non-histone proteins to DNA has been investigated through two types of experiments. Using a nitrocellulose filter assay at a low protein/DNA ratio, the binding of mouse non-histone proteins to mouse DNA was twice as great as the binding of mouse non histone protein to Drosophila DNA. The reverse experiment using Drosophila non-histone protein confirmed the interpretation that some protein . DNA complexes were specific. Protein . DNA complexes isolated by gel filtration chromatography indicated that 20% or 10% of the non-histone protein was bound to homologous or heterologous DNA respectively. Purified non-histone proteins bound with lower efficiency (15%) than unpurified but with higher specificity to soluble chromatin than to naked DNA. This binding did not result from an exchange between chromatin non-histone proteins and purified non-histone proteins added in excess. DNA-bound and chromatin-bound proteins were analysed on polyacrylamide gels. Whereas no major qualitative differences were observed with DNA-bound proteins, some proteins bound to homologous mouse chromatin were different from those bound to heterologous Drosophila chromatin. These results suggest a possible role of DNA-bound non-histone proteins in the regulation of gene expression.     

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)     

Binding of [3H]triamcinolone acetonide-receptor complexes to chromatin from the B-cell leukemia line, BCL1

The binding characteristics of partially purified glucocorticoid receptor complexes from hormone sensitive, non-differentiating BCL1 cells to sequentially deproteinized BCL1 chromatin-cellulose was investigated. [3H]Triamcinolone acetonide (TA)-receptor complexes were purified (approx. 30-fold) from DEAE-cellulose columns by salt elution which allowed receptor activation only in the absence of molybdate. Addition of 10 mM molybdate completely blocked salt activation. The binding pattern of the activated [3H]TA-receptor complexes to chromatin-cellulose extracted with 0-8 M guanidine hydrochloride revealed three regions of increased binding activity (acceptor sites), at 2, 5 and 7 M guanidine hydrochloride. Acceptor site binding was markedly reduced for chromatin extracted with 3, 6 and 8 M guanidine hydrochloride. Non-activated receptor complexes demonstrated very low binding to deproteinized chromatin. It was also shown that chromatin binding required glucocorticoid receptors and that free ligand or ligand bound to other proteins did not bind significantly to chromatin. In addition, binding of [3H]TA-receptor complexes to partially deproteinized chromatin was competable by unlabeled TA-receptor complexes. Scatchard analysis demonstrated that chromatin from non-differentiating BCL1 cells possesses multiple, high-affinity binding sites which differ in their affinity for the glucocorticoid receptor. Partially deproteinized chromatin from lipopolysaccharide-stimulated BCL1 cells demonstrated a different pattern of receptor binding, i.e., receptor binding was significantly greater to chromatin previously extracted with 6-8 M guanidine hydrochloride. These results suggest that differentiation alters the state of chromatin and the interaction of non-histone protein/DNA acceptor sites with glucocorticoid receptors. These alterations may play a role in the acquisition of hormone resistance.     

Interaction of the non-histone protein PS1 with some chromatin components

The binding of non-histone protein from mouse spleen chromatin located in the sites highly sensitive to micrococcal nuclease and DNA-ase I, to DNA and histones was studied. The binding of the DNA-protein complexes to nitrocellulose filters demonstrated the absence of protein binding to DNA. A highly selective binding of protein PS1 to histones H1 and H2A and to one of the non-histone proteins (presumably HMG 14) was revealed. It is concluded that protein PS1 is incorporated into chromatin by the protein-protein interactions.     

Modification of rat liver chromatin by N-methyl-N'-nitro-N-nitrosoguanidine or N-ethyl-N'-nitro-N-nitrosoguanidine and template activity for RNA synthesis by Escherichia coli RNA polymerase after reconstitution.

Rat liver chromatin was fractionated into DNA, histones and non-histone chromosomal proteins and each component was modified with N-methyl-l-N'-nitro-N-nitrosoguanidine of N-ethyl-N'-nitrosoguanidine. The radioactivity of 14C-labeled alkyl or guanidino moieties of both compounds bound significantly to both histones and non-histone chromosomal proteins and the binding of N-methyl-N'-nitro-N-nitrosoguanidine was higher than N-ethyl-N'-nitro-N-nitrosoguanidine. However the binding of both compounds to DNA was very low and its significance was hard to evaluate. All of the three components, one of which was modified, were reconstituted into chromatin, then, [3H]UMP incorporation into acid insoluble material using Escherichia coli RNA polymerase (EC 2.7.7.6) was measured. Only with the reconstituted chromatin containing histones modified either by N-methyl-N'-nitro-N-nitrosoguanidine or N-ethyl-N'-nitro-N-nitrosoguanidine, the template activity increased drastically; i.e., about 10 or 5 times higher than that with the unmodified reconstituted chromatin, respectively. However, any remarkable alteration in the electrophoretic pattern of protein fraction of the reconstituted chromatin could not be found. The results obtained in this study are discussed in the context that the modified histones could give rise to change in the mutual interaction of chromosomal components during the reconstitution of chromatin accompanied with the increase of chromatine template activity.     

In vitro interaction of 63-nickel(II) with chromatin and DNA from rat kidney and liver nuclei

The interaction of nickel(II) with chromatin was studied in vitro and in isolated nuclei from rat liver and kidney. Nickel(II) bound to chromatin, polynucleosomes (DNA + histone octamer protein complex), and to deproteinized DNA both in intact nuclei and in vitro. The amount of nickel(II) bound depended on the concentration of nickel(II), the presence of chromosomal proteins and the binding sites on DNA which provide a stable coordination environment for nickel(II). The binding of nickel(II) to chromatin and to DNA in whole nuclei was much slower than in vitro indicating that assessibility of the DNA binding sites was influenced by the presence of the nuclear membrane, nuclear matrix and nuclear proteins and/or by the condensed nuclear structure of chromatin. Since DNA containing bound nickel(II) was isolated from chromatin, nickel(II) directly interacted with stable binding sites on the DNA molecule in chromatin. Nickel(II) was associated with the histone and non-histone nuclear proteins as well as the DNA in rat liver and kidney chromatin. Nickel(II) was found to bind to calf thymus histones in vitro. Nickel(II)-nuclear protein and -DNA interactions were investigated by gel electrophoretic analysis of in vitro incubation products. Although nickel-histone and nickel-non-histone protein interactions were completely disrupted by the electrophoretic conditions, fluorography revealed the presence of inert nickel(II)-DNA and/or nickel(II)-DNA-protein complexes.     

The binding of 3H-labelled oestradiol- and progesterone-receptor complexes to hypothalamic chromatin of male and female sheep.

Chromatin isolated from hypothalamic nuclei of sexually mature entire male and female sheep was linked to cellulose in u.v. light. The saturation binding of 3H-labelled oestrogen- and progesterone-receptor complexes, prepared by (NH4)2SO4 precipitation from the 105000g supernatant of hypothalamic cytosol, was then measured in vitro in 0.15m-KCl. Saturation binding was also measured after extraction of histones and masking acidic proteins. Salt + urea was observed to be more effective than guanidine hydrochloride in unmasking receptor acceptor sites, and the binding of labelled receptor complexes to dehistonized unmasked chromatin was shown to be largely resistant to 0.4m-KCl extraction. Whereas extents of receptor-complex binding were similar to published values for comparable preparations of hen oviduct chromatin, no sex-related difference was observed. However, binding of progesterone-receptor to chromatin was greater than that of oestradiol-receptor. Binding also increased more after removal of histones and masking acidic proteins, suggesting the presence of a greater number of progesterone-receptor acceptor sites in hypothalamic chromatin than of estradiol-receptor acceptor sites. The failure to demonstrate a sex-related difference in oestradiol-receptor binding to hypothalamic chromatin in vitro is discussed.     

Nuclear binding of progesterone in hen oviduct. Role of acidic chromatin proteins in high-affinity binding.

The multiple classes of binding sites for the progesterone-receptor complex in hen oviduct muclei were found to be of chromatin origin. The highest-affinity, and presumably most physiologically important class, is localized in oviduct chromatin and contains approx. 6000-10000 sites per nucleus. None of these sites is detected in spleen chromatin. Two new techniques were used for assaying rapidly the binding of steroid-receptor complexes to soluble deoxyribonucleoproteins in vito. The extent of high-affinity binding by the nucleo-acidic protein fraction from spleen chromatin is as great as that by the nucleo-acidic protein from oviduct chromatin. Consequently the tissue-specific nuclear binding of the progesterone receptor is found not to be a consequence of the absence of the nuclear binding sites (acceptors) from chromatin of non-target tissue (spleen), but rather a result of complete masking of these sites. In the target-tissue (oviduct) chromatin, approx. 70% of the high-affinity acceptor sites are also masked. Acidic proteins, and not histones, appear to be responsible for the masking of these acceptor sites. In addition, acidic proteins represent (or at least are an essential part of) these high-affinity sites in the oviduct nucleus. Pure DNA displays a few high-and many low-affinity binding sites. In support of previous work with immature chicks, the acidic protein fraction of the nucleo-acidic results thus support the hypotheis that protein complexed with DNA, and not DNA alone, represent the high-affinity binding sites for the steroid-receptor complexes in nuclear chromatin. The lower-affinity classes of binding sites may represent DNA and/or other nuclear components.