Binding of glucocorticoid receptors to mammary chromatin acceptor sites

002%4731/87$3.00+ 0.00 Copyright 0 1987Pergamon Journals Ltd

J. steroid Biochem.Vol. 28, No. 6, pp. 581-586, 1987 Printed in Great Britain. All rights reserved

BINDING OF GLUCOCORTICOID RECEPTORS TO MAMMARY CHROMATIN ACCEPTOR SITES M. F. RUH, R. K. SINGH, T. S. RUH and G. SHYAMALA*~ Department of Physiology, St Louis University School of Medicine, St Louis, MO 63104, U.S.A. and *Lady Davis Institute for Medical Research, Sir Mortimer B. Davis-Jewish General Hospital, and Department of Medicine, McGill University, Montreal, Quebec, Canada H3T 1E2 (Received 12 December

1986)

have recently characterized the interaction of mouse mammary estrogen receptors (ER) with mammary chromatin acceptor sites and demonstrated that ER from estrogen resistant lactating mammary glands do not bind to chromatin. In this study we have characterized the chromatin binding of the glucocorticoid receptor from mouse mammary glands isolated from nulliparous and lactating mice in order to better understand the relationship between receptor binding to chromatin and steroidogenic sensitivity of the tissue. Mammary chromatin was linked covalently to cellulose and deproteinized sequentially by O-8 M Gdn-HCl. Binding to intact chromatin as well as to chromatin deproteinized by Gdn-HC1 was determined using partial& purified [3H]dexamethasone labelled glucocor&oid-receptor complexes (GR) obtained bv fractionation on DEAE-cellulose columns. The binding of 13H1GR from mammary glanhs of nullip&ous mice to chromatin fractions from the same tissue-revealed maximal binding activity (acceptor sites) on chromatin previously extracted with 5-6M Gdn-HCl. Binding of t3H]GR was of high affinity (& = 0.2 nM) and saturable. A simultaneous comparison of the chromatin binding patterns for [3H]ER and [3H]GR isolated from mammary glands of nulliparous mice revealed that the chromatin subfractions obtained with 4-6 M Gdn-IW1 extraction contained acceptor sites for both [-‘H]ER and [3H]GR; however, while the [3H]ER bound to a 4.5 M and a 5.5 M site, the [3]GR bound a 5 M and a 6 M site. Competition experiments supported the steroid receptor specificity of the chromatin acceptor sites. Thus, the 4-6 M chromatin fractions contain distinct acceptor sites for the glucocorticoid receptor and for the estrogen receptor. In addition our studies reveal that the binding patterns of [‘H]GR isolated from mammary glands of nulliparous and lactating mice to their homologous chromatin is essentially similar. Thus, in contrast to estrogen receptors, glucocorticoid receptors from lactating mammary glands are able to effectively bind to chromatin acceptor sites which supports our previous suggestion that the estrogenic insensitivity of lactating mouse mammary glands may at least be in part due to the impeded interaction of ER with chromatin acceptor sites. Summary-We

INTRODUCTION

We previously examined the relationship between mammary estrogenic sensitivity and ability of mammary estrogen receptors (ER) to bind to specific high affinity sites on mammary chromatin. We found that the unresponsiveness of lactating mammary glands to estradiol coexists with the inability of estrogen receptors in these tissues to interact with specific high affinity sites on chromatin [l]. In addition, we demonstrated that the partially purified receptor preparation from lactating mammary glands contained an inhibitory factor which not only inhibited the chromatin binding of mammary ER but also binding of ER to chromatin in other systems [I]. Mouse mammary glands are a target organ for both estrogens and glucocorticoids [2] and, thus, in addition to ER, contain glucocorticoid receptors (GR) [3,4]. However, during lactation when mammary glands are insensitive to estradiol, the tissue is Worrespondence to: Dr G. Shyamala, Lady Davis Institute for Medical Research, Sir Mortimer B. Davis-Jewish Research Genera1 Hospital, 3755 Cote Sainte Catherine Road, Montreal, Quebec, Canada H3T lE2.

still responsive to glucocorticoids [2]. Therefore, if the interaction of steroid receptor with specific sites on chromatin is necessary for the expression of steroidogenie effects in lactating mammary glands, the binding of GR (in contrast to ER) to its high affinity sites on mammary chromatin would be expected to occur without any impediment. Therefore, we examined whether, mammary GR can bind to mammary chromatin with high affinity, and if so, whether these interactions are altered in lactating mammary glands. Therefore, to further verify that in lactating mammary glands the impeded interaction of ER with its chromatin binding sites was indeed related to the estrogenic insensitivity of the tissue, in the present studies we have examined the interaction of GR with chromatin isolated from mammary glands of both estrogen sensitive nulliparous and estrogen insensitive lactating mice. EXPERIMENTAL Animals

Female Balb/c mice were from our own colony. Nulliparous mice were between 2-3 months of age

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and where indicated had been ovariectomized l-2 wk prior to use. Lactating mice were used between days 7 and 10 postpartum. Chemicals

[6,7-3H]dexamethasone (sp. act. 46 Ci/mmol) was purchased from New England Nuclear (Boston, 17/?-(2,4,6,7) [3H]estradio1 (sp. act. MA). 91 Ci/mmol) was purchased from Amersham (Arlington Heights, IL). Ultrapure guanidine hydrochloride (Gdn-HCl) and sucrose were from Schwarz/Mann (Spring Valley, NY), DEAE-cellulose (DE-52) was from Whatman, Inc. (Clifton, NJ), and Cellex-410 and protein assay reagent were from BioRad Laboratories (Richmond, CA). All other chemicals were analytical grade. Preparation and labeling of glucocorticoid receptors in mammary cytoplasmic extracts

The cytoplasmic extracts were preparated in PG buffer (5 mM sodium phosphate, 10 mM monothioglycerol, and 10% glycerol; pH 7.4 at 23°C) containing 20 mM dithiothreitol according to procedures previously described [S]. Immediately after preparation, the cytoplasmic extracts were either used as is or after being frozen in liquid nitrogen and stored at - 70°C. Aliquots of extracts were incubated for 3 h at 4°C with 50 nM of [3H]dexamethasone and then treated with 0.4 M KC1 for another hour. Subsequently cytosols were diluted to a final concentration of 50 mM KC1 with PG buffer containing 20 mM sodium molybdate (PGM) and loaded onto a pre-equilibrated DEAE-cellulose column (packed vol= 6 ml). After extensive washing with PGM buffer, the column was washed with 50 ml of 50 mM KC1 and finally receptor complexes were eluted by 0.4 M KC1 in a single step. Aliquots (50 ~1) were counted in 4ml scintillation fluid [0.4% (w/v) Omnifluor/25% (v/v) Triton X-l 14 in xylene plus 7% (v/v) water] at 46% efficiency using a Beckman LS-7500 scintillation counter. The peak fraction containing [‘Hldexamethasone-receptor complexes was used for the chromatin-cellulose binding assay. The final KC1 concentration in the peak fraction was 0.3 M. Preparation and labeling of estrogen receptors mammary cytopIasmic extracts

in

Cytoplasmic extracts were prepared and labeled with [‘Hlestradiol exactly as described previously [I]. These extracts were exposed to 0.4 M KC1 and fractioned on DEAE-cellulose exactly as described above. Preparation of chromatin -cellulose

The procedure for preparing chromatin-cellulose from lactating and nulliparous mouse mammary glands was exactly the same as described previously [ 11. Briefly, mouse mammary glands were blended and homogenized in 0.5 M sucrose-TKM

al.

buffer (50 nM Tris-25 mM KCl-2 mM MgCl?. pH 7.5) and centrifuged at 27,000g for 15 min in a Sorvall GSA rotor. The resulting pellet was homogenized in 1.8 M sucrose-TKM buffer and centrifuged at 27,000g for 60 min. The nuclear pellet was first treated with 0.2% Triton X-100-0.5 M sucrose followed by homogenization in a Dounce homogenizer with 80mM NaCl-20mM EDTA (pH 6.3) and finally treated with 0.35 M NaCl. The chromatin was linked covalently to washed cellulose (Cellex-410) by exposure to 6 x 106erg/cm2 U.V. light. Chromatincellulose from lactating and nulliparous mammary glands contained 20 pg DNA/mg resin as determined by the diphenylamine method [6]. Chromatin -cellulose binding assay

For the measurement of binding activity of [‘H]GR (or [3H]ER) to intact and partially deproteinized chromatin-cellulose, a batch assay was used. Chromatin-cellulose powder (eqiv. to 2Opg DNA) obtained from the different tissues was added to 1.5 ml microfuge tubes and extracted with 1 ml extraction buffer (10 mM Tris, 5 mM sodium bisulfite and 0.1 M 2-mercaptoethanol, pH 8.5 at 22°C) containing O-8 M Gdn-HCl at 22°C for 30 min. Tubes were mixed every 10 min. The chromatin-cellulose pellet was washed free of Gdn-HCl by mixing with 1 ml cold TE-buffer (2 mM Tris-HCl, 0.1 mM EDTA and 0.5 mM PMSF, pH 7.4 at 4°C) followed by centrifugation at 80006 for 15 s in a Beckman Microfuge-B and repeating the washing two more times. Aliquots of the DEAE-peak fraction containing 0.39pmol [3H]GR were mixed with the O-8 M Gdn-HCl extracted chromatin cellulose slurry in a final volume of 1 ml (PG buffer) and incubated for 60min at 4’C. The final KC1 concentration was

maintained at 150 mM per tube. The unbound receptor complexes were removed by three washings with PG buffer containing 1OOmM KCl. Finally, radioactivity was extracted with 1 ml absolute ethanol for 15 min and counted in 4 ml scintillation fluid (0.4% (w/v) Omnifluor, 25% (v/v) Triton X-l 14 in xylene). DNA in the remaining pellet was also determined for each assay tube by hydrolyzing the pellet in 0.5 M perchloric acid at 90°C for 30 min and using the diphenylamine method [6]. The receptor binding to chromatin-cellulose was corrected for intact chromatin binding and blank cellulose binding and expressed as bound receptor (pmol) per mg DNA. Saturation and Scatchard analysis

Various aliquots (SCrSOOpl) of the partially purified [3H]GR (0.72 pmol/ml) were added in triplicate to chromatin
Mammary glucocorticoid receptors

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RESULTS

Binding of mammary mammary chromatin

g~u~ocortico~d receptors

to

The binding of 13H]GR to partially deproteinized chromat~n. Removal of non-histone nuclear proteins with Gdn-HCI is useful for the in vitro unmasking of

steroid receptor specific-acceptor sites on the chromatin including the sites for mammary ER [l]. Therefore, in these experiments similar to those we previously described for mammary ER, we prepared chromatin-ceilulose from mammary glands of nulliparous and lactating mice and extracted those chromatins with varying concentrations of Gdn-HCl. The binding of f3H]GR to each of the residual chromatin fractions was then examined. Maximal binding occurred with chromatin subfractions extracted by 5-7 M Gdn-HCl. In order to define more exactly the chromatin binding pattern, extractions were performed with half molar increments of Gdn-HCl between 4-8 M Gdn-HCI. As shown in Fig. 1, two peaks of activity were resolved, one occurring with chromatin extracted with 5 M Gdn-HCl and the other 6 M Gdn-HCI. Figure 1 also shows that under identical experimental conditions, t3H]GR from lac-

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Fig. 2. Comparison of the binding of mammary [‘HJGR with mammary [3H]ERto partially deproteinized mammary

chromatin. Mammary 13H]GR or [3H]ER were partially purified and incubate at a cun~ntration of 0.28 pmol per chromatin binding assay as in Fig. I. The data were corrected for intact chromatin-cellulose binding which was l = 0.68 and 0 = 1.40pmol/mg DNA. The data represent the mean + SEM of two experiments performed in triplicate. tating mammary glands bound to mammary chromatin from lactating mice with a binding pattern similar to that obtained from nulliparous mice. Next, to determine whether mammary ER and GR bound to discrete chromatin subfractions, a simultaneous comparison of t3H]ER and [3H]GR binding to various chromatin fractions from nulliparous mouse mammary glands was performed using half molar increments of Gdn-HCl. As shown in Fig. 2, [3H]GR bound maximally to chromatin subfractions extracted with 5 M and 6 M Gdn-HCl as also seen in Fig. 1, while 13H]ER bound maximaIIy to chromatin extracted with 4.5 M and 5.5 M Gdn-HCl. Saturation analysis of mammary glucocorticoid receptor binding to chromatin acceptor sites. To

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Fig. 1. Binding of mammary 13H]GR to partially deproteinized mammary chromatin. Mammary [3H]GR from lactating (O----0) or nulliparous mice (0-O) were partially purified by DEAE-ceilulose chromatography. Chromatin cellulose equivalent to 20 pg DNA was extracted with O-8 M Gdn-HCI to obtain deproteinized chromatin fractions and then washed to remove the Gdn-HCI. Subseouentlv these chromatin subfractions prepared from mammary chromatin of lactating (Q-o)br nulliparous mice (+--0) were incubated with 0.39 pmol of partially purified [%I]GR to determine the acceptor site binding activity. The data were corrected for intact chromatincellulose binding which was (a) = 0.72 and (0) = 0.50 pmol/mg DNA. The data represent the mean k SEM of three exueriments nerformed in dudicate.

determine whether the binding of mammal GR to mammary chromatin was saturable, increasing concentrations of mammary [3H]GR from nulliparous or lactating mice were incubated with 5 M Gdn-HCI extracted chromatin~IIulose prepared from the respective mammary glands, Le. nulliparous or lactating mice. The binding data thus obtained were analyzed by Scatchard plot [7] which yielded a Kd of 1.8 x IO-” M for the interaction of [3H]GR with mammary chromatin from nulliparous mammary glands and a Kd of 1.4 x lo-” M for chromatin from lactating mammary glands (Fig. 3). The concentration of 5 M acceptor sites for [‘H]GR on the chromatin from nulliparous and lactating mice was approx 8500 sites/cell and 6500 sites/cell, respectively. Steroid receptor specljicity of mammary glucocorticoid receptor binding to chromatin acceptor

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RECEPTOR

COMPLEXES

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Fig. 3. Saturation analyses of the binding of mammary [)H]GR to mammary chromatin acceptor sites. Mammary chromatin cellulose equivalent to 20~18 DNA prepared from either nulliparous (a---0) or lactating (O---O) mice were extracted with 5 M Gdn-HCl prior to incubation with varying concentrations of [‘H]GR iSO /II of receptor preparation = 0.36 pmol) prepared from mammary glands of nulliparous or lactating mice. Protein content per assay tube was kept constant using heat-treated cytosol fractions obtained by DEAE chromatography. Each value was corrected for intact chromatin binding. Dissociation constants (&) were calculated by Scatchard analysis. The data represent the mean of two experiments performed in duplicate. d’R

sites. In the

following

experiments

we examined

whether the interaction of f3HJGR with mammary chromatin subfractions was specific for GR. Mammary chromatin-cellulose from nulliparous mice (extracted with 5 M or 6 M Gdn-HCl) was incubated with 13H]GR plus a 2-fold excess (by protein content) of either heat-treated cytosol fraction or radioinert GR, and competition with [3H]GR was determined. These results shown in Fig. 4 clearly demonstrate that radioinert GR was abie to compete with [‘H]GR for binding to chromatin acceptor sites. In addition, the 50-60s decreased [‘H]GR binding indicates that approx 80-90% of the acceptor sites are specific for [‘H]GR since the theoretical maximum for a 2-fold excess of GR would be a 67% decrease. Figure 4 also demonstrates that in contrast to radioinert GR complexes, radioinert mammary ER complexes do not compete signi~~antly for the binding of 13H]GR to mammary chromatin. Binding of mammary f3H]GR to chromatin acceptor sites of various tissues. Previous studies in our labora-

tory have demonstrated that ER will bind with high affinity to chromatin from several target tissues from the same species [l]. In the present studies mouse mammary [‘H]GR bound partially deproteinized mouse mammary gland, uterine and spleen ehromatin (Fig. 5), all these tissues being known target tissues for glucocorticoids. The differences in the amount of [3H]GR binding to these various chromatin preparations most probably reflects the responsiveness of these tissues to glucocorticoids. For example, spleen contains cells of the lymphoid system in various states of differentiation and glucocorticoid responsiveness. Whereas resting lymphocytes are re-

G’R

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Fig. 4 Steroid receptor specificity for the binding of mammary [-‘H]GR to mammary chromatin acceptor sites. Chromatin cellulose prepared from mammary glands of nulliparous mice was extracted with 5 M (A) or 6 M (B) Gdn-HCl and then washed to remove Gdn-HCl. Mammary radioinert estrogen receptor (ER) and glucocorticoid receptor (GR) were prepared by incubating mammary cytosol from nulliparous mice with radioinert estradiol or dexamethasone, respectively. Subsequently the chromatin preparation was incubated with 0.26 pmol (5 M site) or 0.24 pmol (6 M site) of [‘HJGR/ZOicg DNA in the presence of a 2-fold excess of these various radioinert steroid-

receptor complexes or heat-treated cytosol fractions (dR). All assay tubes had a constant protein concentration of 370pg/tube. The data have been corrected for intact chromatin binding and represent the mean + SEM of two experiments performed in triplicate.

sponsive to glucocorticoids, differentiated immunoglobulin-secreting cells are no longer sensitive to glucocorticoids but still retain the same quantity of glucocorticoid receptors [8-IO]. DISCUSSION

The purpose of the present studies was to examine the interaction of mouse mammary GR with its chromatin binding sites in order to better understand the relationship between mammary steroid receptors and mammary steroid responsiveness. In previous studies we demonstrated a positive relationship between high affinity binding of mammary ER to chromatin and mammary estrogenic sensitivity in nulliparous mice [l]. However, mammary ER from estrogen-insensitive lactating mice did not bind chromatin. In the present study we found that the mam-

Mammary glucocorticoid receptors

MMG

MU

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Fig. 5. Binding of [‘H]GR to chromatin from various target tissues. Chromatin cellulose esuivalent to 20 ticg DNA prepared from mouse mammary glands (Ml@), uteri (MU) and spleen (MS) were extracted with 5 M Gdn-HCI before incubation with [‘HJGR (0.28 pmol) prepared from mouse mammary glands. The data have been corrected for binding to intact chromatin and represent the average of three experiments.

mary glands of both nulliparous and lactating mice which are responsive to glucorticoids [2], also have specific high affinity nuclear binding sites for GR.

More importantly, in lactating mammary glands specific binding of GR to chromatin subfractions occurred without any impediment. Thus, the inhibitor(s) of ER interaction with its chromatin binding sites present in lactating mammary glands and identified in our previous studies [I] does not appear to have a very significant effect on the binding of GR to its chromatin binding sites in these tisssues. Previous studies in our laboratory [I I-151 and other laboratories [l&18] have demonstrated that intact as well as partially deproteinized chromatin contains saturable, high affinity binding sites for steroid hormone receptors. These nuclear acceptor sites are composed of specific acceptor proteins bound to DNA [18]. Removal of masking protein [19] exposes these acceptor sites (acceptor protein/DNA complexes) whereas removal of these tightly bound acceptor proteins from DNA results in the loss of acceptor activity. Acceptor site binding has been shown to require intact, functional, activated receptor and demonstrated steroid receptor specificity. Of special significance, specific acceptor sites have been reconstituted by reanneailing acceptor proteins to DNA, thus permitting the detection and isolation of specific acceptor proteins and the enrichment of specific DNA sequences essential for these acceptor sites[l6, 171. In the present studies we have identified two populations of chromatin binding sites for GR, one revealed by extraction of chromatin with 5 M Gdn-HCl and the other with 6 M Gdn-HCl. Similarly, two populations of chromatin binding sites also appear to be present for ER in mammary glands but these seem to be distinct from those which bind GR;

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the two populations of ER binding sites are revealed after extraction of chromatin with 4.5 M and 5.5 M Gdn-HCl, respectively. That there are different populations of nuclear acceptors for ER versus GR was previously reported [20] and is confirmed by our competition studies. The presence of multiple populations of chromatin binding sites for ER and GR is not unique to mammary glands since multiple chromatin binding sites for ER have also been detected in hen oviduct [I 11, rabbit uterus f 121, calf uterus [ 131 and MCF-7 cells [14], and multipie chromatin binding sites for GR have been demonstrated in the B lymphocyte cell line, BCL, [ 151.At present the precise physiological significance for the presence of multiple chromatin binding sites for receptors revealed in tiitro is not clear. It has been suggested[l4] that ER can interact with several sets of chromatin sites which are responsible (directly or indirectly) for the activation of a variety of genes, which include those for growth, progesterone receptor, plasminogen activator and the 52K protein. The assumption that not all estrogensensitive sites are the same is supported by studies, using antiestrogen-resistant cell lines [14]. It is worthwhile to emphasize that in the lactating estrogen-insensitive mammary gland the binding of ER does not occur to any of the Gdn-HCl extracted chromatin subfractions. Thus, the previous studies and the observed unimpeded interaction of mammary GR with its chromatin binding sites in lactating glucocorticoid-sensitive mammary glands supports our previous suggestion that a positive relationship exists between steroid sensitivity of mammary glands and the ability of mammary steroid receptors to bind chromatin acceptor sites. Therefore, the estrogenic insensitivity of lactating mammary glands may at least in part arise from the inability of ER in this tissue to bind with high affinity to specific acceptor sites in chromatin. ~ck~o~~~edge~e~~~-These studies were supported by grants from the Medical Research Council of Canada (GS) and NIH RR05388 (TSR).

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