Proc. Natl. Acad. Sci. USA Vol. 73, No. 6, pp. 1954-1958, June 1976
Biochemistry
Acceptor proteins in rat androgenic tissue chromatin (nonhistone proteins/chromatin phosphorylation/5a-dihydrotestosterone chromatin binding) LEOKADIA KLYZSEJKO-STEFANOWICZ*, JEN-FU CHIUt, YU-HUI TSAIt, AND LUBOMIR S. HNiLICAt Department of Biochemistry, The University of Texas System Cancer Center, M.D. Anderson Hospital and Tumor Institute, Houston, Texas 77025
Communicated by Sidney P. Colowick, April 9, 1976
ABSTRACT Fractionation of chromatin into urea-soluble chromosomal nonhistone proteins (UP), histones (HP), and DNA-associated nonhistone proteins (NP) revealed that the NP fraction from testicular and prostatic chromatin contains organ-specific acceptors for complexes of 5a-dihydrotestosterone (17j3-hydroxy-5a-androstan-3-one) and its receptor. This acceptor capacity of androgenic tissue chromatin could be transferred to chromatins from non-target tissues with the NP fraction of DNA-associated proteins. Phosphorylation of chromatin enhanced its hormone-receptor binding capacity. According to current views, early steps in the action of steroid hormones on their target tissues involve the association of the steroid hormone with cytoplasmic receptor, transport of this complex from cytoplasm into the nucleus, and, finally, its association with specific acceptor sites on chromatin (1-4). This association alters the transcriptional controls and allows the synthesis of new mRNA species. The new mRNAs are eventually translated into new proteins that are often specific for the hormone-stimulated tissue (1, 3, 5, 6). The target cell nuclear acceptor is selective in that the nuclei of responsive cells accept more hormone-cytosol protein complex than the nuclei of tissues refractory to the action of steroid hormones (7, 8). Recent years have seen the accumulation of considerable information concerning the steroid hormone-cytoplasmic receptor interactions in different types of tissue. After the hormone interacts with cytoplasmic receptor, the hormone-protein complex moves to the nucleus, where it is incorporated into chromatin. Liao and Fang (1) coined the term "acceptor protein" for the nuclear acceptor molecule. The acceptor protein fraction was isolated from prostate (9) and found to be heat labile and tissue specific, and could be transferred from the chromatin of one tissue to another with the fraction of chromosomal nonhistone proteins (3, 7). We report here the results of our studies on chromatin acceptor(s) in rat prostate and testis.
MATERIALS AND METHODS Prostatp, testis, liver, and pancreas from male Sprague-Dawley rats (125-150 g) were homogenized in ice-cold 0.25 M sucrose, 5 mM MgCl2, and 2 mM 2-mercaptoethanol in 20 mM Tris-HCI buffer, pH 6.8. The homogenate was centrifuged at 800 X g for 10 min. The crude nuclear pellet was purified by rehomoAbbreviations: SSC, standard saline citrate (150 mM NaCl, 15 mM sodium citrate); UP, chromosomal nonhistone proteins soluble in urea at low ionic strength; HP, histones; NP, DNA-binding chromosomal nonhistone protein fraction; DHT, 5a-dihydrotestosterone (17f,-
hydroxy-5a-androstah-3-one). * Present address: Institute of Biochemistry and Biophysics, University of Lodz, 90-237 Lodz, Poland. t Present address: Department of Biochemistry, Vanderbilt University School of Medicine,.Nashville, Tennessee 37232. Direct reprint requests to L.S.H. at this address. t Present address: Program in Reproductive Biology and Endocrinology, The University of Texas Medical School at Houston, Houston, Texas 77025.
genization in 2.2 M sucrose, 25 mM KC1, 5 mM MgCl2 in 50 mM Tris.HCl buffer, pH 7.8, and centrifuged at 100,000 X g (max) for 1 hr (10). The isolation of chromatin and criteria for its purity were described previously (11). Isolated chromatin was fractionated into the bulk of chromosomal nonhistone proteins (UP), histones (HP), and DNA-binding nonhistone proteins (NP). At each fractionation step a partially deproteinized chromatin was obtained which was devoid of the concerned chromosomal protein fractions, i.e., UC (devoid of UP), HC (devoid of UP and HP), and NC (devoid of UP, HP, and NP). Details of the fractionation procedure were described elsewhere (12). DNA was reconstituted with the protein fractions UP, HP, and NP isolated from rat testis, prostate, liver, or pancreatic chromatin. The reconstitution was accomplished by mixing the interacting components in 2.5 M NaCl, 5.0 M urea, 50 mM Tris-HCl buffer, pH 8.0, at a ratio of DNA:UP:HP:NP = 1:1: 1:0.2 and decreasing the NaCl concentration by a slow dialysis against 5.0 M urea in 50 mM Tris.HCl buffer (13). Finally, the urea was removed by dialysis against either 0.1 X SSC (SSC = standard saline citrate: 150 mM NaCl, 15 mM sodium citrate), or 10 mM Tris-HCl buffer, pH 7.6. The reconstituted chromatins were used in hormone acceptor experiments. Preparation and Interactions of 3H-Labeled 5a-Dihydrotestosterone-Cytoplasmic Receptor Complexes. Tritium-labeled 5a-dihydrotestosterone ([3H]DHT, 50 mCi/,gmol, New England Nuclear) was complexed with cytosol following the method of Spelsberg et al. (14) with some modifications. After incubation with labeled hormone, the cytosol-hormone complex was fractionated with (NH4)2SO4 (0-33% saturation). The active fraction was freed from unbound hormone by gel -filtration on a Sephadex G-25 column (1 X 24 cm). Sucrose density gradient analysis of the purified [3H]DHT-receptor complex showed that essentially all the radioactivity was associated with the receptor protein peak sedimenting at approximately 8 S. This is in agreement with the reports from other laboratories (15, 16). In our system, we were able to detect only a small amount of 3.5S receptor complex, probably because of the (NH4)2SO4 fractionation of the initial extract (16). The sucrose density gradient centrifugation also revealed that the (NH4)2SO4 fractionation followed by gel filtration in Sephadex G-25 removed essentially all the unbound [3H]DHT. In agreement with Fang and Liao (7) and Mainwaring and Peterken (15), the binding of our [3H]DHT complex increased when the incubation was performed at 250 instead of 40. However, because of a better stability of the [3H]DHT complexes at 40, all our studies were done at this temperature (14). Samples of native, reconstituted, or partially deproteinized chromatins (UC, HC, or NC), each containing 100 jig of DNA, were incubated with 0.6 mg of [3H]DHT-receptor complex in a total volume of 1 ml containing 150 mM NaCl, 0.7 mM EDTA, 3 mM Tris-HCl, pH 7.4, at 40 for 1 hr. After incubation, the samples were diluted with 30 ml of cold 150 mM NaCl, 10 1954
Proc. Natl. Acad. Sci. USA 73 (1976)
Biochemistry: Klyzsejko-Stefanowicz et al.
1955
Table 1. [3H]DHT binding affinity of prostate, liver, and testis chromatin fractions Liver
Prostate
Testis
Chromatin fractions
Exp. no. 1
Exp. no. 2
Exp. no. 1
Exp. no. 2
Exp. no. 1
Exp. no. 2
Native chromatin Dehistonized chromatin UC (less UP) HC (less UP and HP) NC (less UP, HP, and NP)
0.50 0.85 0.97 1.71 0.49
1.04 1.87
0.60 1.22 1.32 3.34 0.74
1.32 2.69 2.92 5.20 2.47
0.28 0.31 0.25 0.31 0.32
0.44 0.47 0.46 0.55 0.48
2.60 1.05
Native or fractionated chromatin (100 gg of DNA) from each tissue was used in all experiments. Exp. no. 1 was done with 0.3 mg of total protein (about 45% saturation) of [3H]DHT-receptor complex and Exp. no. 2 was done with 0.6 mg of total protein (about 80% saturation) of [3H]DHT-receptor complex in the incubation mixture; the amount of bound [3HJDHT is expressed in nmol/g of DNA.
mM MgCl2, and centrifuged at 100,000 X g (max) for 8 hr. The pellets were washed twice by resuspending them vigorously in 30 ml of the same medium and centrifuging them at the indicated speed for 8 hr. The washed precipitate was again resuspended in the same medium and collected on Millipore filters (0.45 ttm pore size, 24 mm diameter). The filters were washed twice with 20 ml of 150 mM NaCl, 10 mM Mg9l2, dried, and measured for radioactivity content. Samples (1 ml of reconstituted chromatin with [3H]DHTreceptor complex) were analyzed by density gradient centrifugation. Samples were layered over linear 5-20% (wt/vol) sucrose density gradients in 2 mM dithiothreitol, 0.1 mM EDTA, 10 mM Tris-HCl, pH 7.4, and centrifuged in a Spinco SW 40 rotor at 37,000 rpm for 8 hr at 4°. Phosphorylation of Chromatin In Vivo and In Vitro. For the in vivo labeling of chromosomal proteins, rats were injected intraperitoneally with 2 mCi/100 g body weight of carrier-free [32P]orthophosphate and sacrificed 60 min after the injection. The individual tissues were processed immediately. The in vitro labeling of chromatin using its endogenous phosphoprotein kinases was accomplished by incubation in 16 tIM [y-32P]ATP (0.23 Ci/mmol), 20 mM MgCl2, and 100 mM NaCl in 80 mM Tris-HCI buffer, pH 7.5, at 37° for 10 min (17). The reaction was terminated by addition of 1% Na-pyrophosphate and the samples were extensively washed with 1% Napyrophosphate solution in 0.01 X SSC, pH 7.0, to remove free
1). This agrees with the 2000-6000 sites reported by other investigators (7, 20). The binding affinities of [3H]DHT-receptor complex to the partially deproteinized chromatin preparations UC, HC, and NC are listed in Table 1. After the UP and HP proteins were removed the residual chromatin fraction HC exhibited receptor affinity 250-557% that of the native prostatic or testicular chromatin. However, the binding affinity for the [3H]DHT-receptor complex was markedly decreased after the removal of NP proteins, leaving nearly pure DNA fraction NC. In contrast, the [3H]DHT-receptor binding to liver chromatin was not significantly altered by the removal of UP and HP fractions. This suggests that the NP fraction of testis and prostate contains acceptor protein(s) for the [3H]DHT-receptor complex. This fraction represents less than 5% of the total chromatin protein and its electrophoretic heterogeneity is shown in Fig. 2.
If the NP fraction contains the acceptor proteins for [3H]DHT-receptor complex, the binding affinity of chromatin for such complex should be transferrable from one tissue to another by reconstituting liver chromatin devoid of its own NP fraction with the NP proteins from androgenic target tissue chromatin.
I0
[y-32P]ATP.
The isolated UP, HP, and NP protein fractions were phosphorylated in vitro by a partially purified protein kinase from rat liver (18). The reaction mixture contained 20 ,umol of Tris-HCI, pH 7.5,4 nmol of [-y-32P]ATP (0.23 Ci/mmol), 5 ,Amol of MgGI2, 25 ,umol of NaCl, 50 ,ug of protein fraction, and 10 ,ug of partially purified kinase. The final volume was 0.25 ml. The reaction was terminated after incubation at 370 for 10 min by addition of 10% CCG3COOH. The precipitate was washed three times with cold 5% CCG3COOH. The [32P]phosphate content of the UP, HP, and NP fractions from all experiments was determined by the method of Kleinsmith et al. (19). RESULTS The prostatic and testicular chromatin exhibited significantly higher affinity for the [3H]DHT-receptor complex than hepatic or pancreatic chromatin (Fig. 1). This confirms the results of other investigators (15, 16). The binding was temperature dependent, and the bound [3HJDHT-receptor complex could be almost completely removed from chromatin by extraction with 0.4 M KCI. Because of better selectivity, our binding experiments were performed at approximately 80% saturation. At this saturation level, there were approximately 4700 [3H]DHTreceptor binding sites per nucleus equivalent of DNA (Table
z 0
I
E
c
1
2
5a-(3HJDHT-receptor (mg) FIG. 1. [3H]DHT-receptor complex binding affinities of prostatic (0), testicular (0), pancreatic (-), and hepatic (A) chromatins. Chromatin (200 wg of DNA) was incubated with appropriate amounts
of [3H]DHT-receptor complex in 0.15 M NaCl, 0.7 mM EDTA in 3 mM Tris-HCl buffer, pH 7.4, for 1 hr at 4° (1 ml final volume). After incubation, the samples were washed and collected as described in
the Materials and Methods.
1956
Biochemistry: Klyzsejko-Stefanowicz et al.
Proc. Natl. Acad. Sci. USA 73 (1976) Table 2.
[
3HI DHT binding affinity of reconstituted chromatin
Bound DHT (nmol/g of DNA)
Fractions E
I
C«1\N Vt
§ 0.3 co
0.2
0.22 1.37 1.27 0.29 0.66 0.57 0.20 1.10 0.81 0.26
DNA (rat spleen or testis) DNA + NPp DNA + NPT DNA + NPL DNA + UPL + HPL + NPP DNA + UPL + HPL + NPT
DNA+UPT +HPT +NPL
10
20
40
30
Gel
50
60
70
Reconstituted prostate chromatin Reconstituted testis chromatin Reconstituted liver chromatin
80
length(mm) b
0.5
0.4
E §0.3
0.2
0.1
10
20
30
60 50 40 Gel length (mm)
70
80
C
0.4
0 0
co
1
2
3
5
6
7
8
Gel length (cm)
FIG. 2. Optical scans of polyacrylamide gel electropherograms of (a) rat testis UP, (b) rat testis HP, and (c) rat testis NP. Two hundred micrograms of UP, 75 Mug of HP, and 100 Mug of NP were dialyzed against sample buffer (0.1 M sodium phosphate buffer, 0.1% sodium dodecyl sulfate, 0.1% fl-mercaptoethanol, and 8 M urea, pH 8.0) with two changes, and then applied to 10% polyacrylamide gels containing 0.1% sodium dodecyl sulfate and 4 M urea. Electrophoresis was performed at 8 mA per tube for 8 hr. The gels were stained and scanned as described previously (10).
As shown in Table 2, the NP protein fraction indeed determined
the binding affinity of chromatin for hormone-receptor complex. The binding activities of reconstituted chromatins of UP and DNA or HP and DNA were also studied; only marginal binding could be detected. In agreement with studies in other systems (21-23), DNA alone still showed some binding affinity, probably contributed to either by incomplete removal of the NP proteins from the NC pellet or by nonspecific binding of type A receptor to free DNA (15, 23). O'Malley and associates (8, 23) have shown that the chick oviduct cytoplasmic progesterone receptor consists of two 4S components separable by DEAE-cellulose chromatography.
Pure DNA or reconstituted chromatin (100 Ag of DNA) was incubated with 0.6 mg (total protein) of [3H]DHT-receptor complex in incubation mixture. The subscripts indicate the tissue origin of the fractions (P = prostate, T = testis, L = liver).
Both these components (A and B) exhibited high affinity and specificity for progesterone, and both associated with isolated chick oviduct nuclei. The type A receptor-progesterone complex was not specific in its in vitro binding. It associated indiscriminately with free DNA. The type B receptor-progesterone complex associated with chromatin and its binding was specific for the target tissue. Since the administration of hormones was shown to enhance the phosphorylation of nuclear nonhistone proteins in experimental animals (24, 25), the binding affinity of [3H]DHTreceptor complex to phosphorylated androgenic chromatin was analyzed. Chromatin phosphorylated in vitro exhibited binding affinity 143-277% that of the nonphosphorylated samples (Table 3). To investigate whether the NP fraction of chromatin contains phosphoproteins, chromatin samples were phosphorylated both in vivo and in vitro and fractionated into UP, HP, and NP fractions (Table 4). Surprisingly, the NP fraction was not phosphorylated either in vivo or in vitro and, therefore, does not contain a natural phosphoprotein. The increased binding affinity of the DHT-receptor complex to phosphorylated chromatin is probably attributable to conformational changes Table 3. DHT binding activity of phosphorylated androgenic chromatin Exp. no. 1 Fraction Native testicular chromatin Phosphorylated testicular chromatin Native prostatic chromatin Phosphorylated prostatic chromatin
Exp. no. 2
Bound DHT
(%)
Bound DHT
(%)
1.04
(100)
0.61
(100)
1.50
(143)
1.33 0.6
(217) (100)
1.66
(277)
Chromatin (1 mg of DNA) was incubated in a 10 ml reaction mixture containing 200 jmol of Tris*HCl, pH 7.5, 40 nmol of [y-32P]ATP, 50 Mmol of MgCl2, and 250 jimol of NaCl for 10 min at chromatin was washed 370. After incubation the phosphorylated three times with 1% Na-pyrophosphate in 0.01 x SSC, pH 7.0, and then three times with 0.01 x SSC, pH 7.0, to remove the pyrophosphate. Control or phosphorylated chromatin (100 jig of DNA) was incubated with [3H]DHT-receptor complex (0.3 mg of protein) in the incubation mixture described in Fig. 1. The amount of bound [3H]DHT is expressed in nmol/g of DNA.
Proc. Natl. Acad. Sci. -USA 73 (1976)
Biochemistry: Klyzsejko-Stefanowicz et al.
1957
Table 4. Phosphorylation of testicular UP, HP, and NP chromatin proteins
Isolated fractions in vitro
Chromatin In vivo
In vitro
Protein fraction
(activity %)
(activity %)
(pmol of 32P/mg of protein)
(activity %)
Casein UP HP NP
73 26
83 17 0.1
39.0 7.8 5.7 0.01
58 42 0.1
0.1
Details of the in vivo and in vitro phosphorylation and fractionation procedures were described in the Materials and Methods. The numbers are either 32p activity percentages of total chromatin protein or pmol of 32p per mg of protein.
of chromatin resulting from its phosphorylation, allowing the NP proteins to become more readily accessible to the [3H]DHT-receptor complex.
DISCUSSION Early experiments showed that the acceptor sites of the hormone-cytosol receptor complex are firmly associated with the nucleus. When the nuclear components were fractionated, the acceptor sites remained largely associated with chromatin (26, 27). Using selective dehistonization of chromatin and reconstitution of hybrid chromatins containing chromosomal proteins from target and non-target tissues, Spelsberg et al. (28) demonstrated that the ability of chromatin to accept progesterone-receptor complex depends on the cellular source of chromosomal nonhistone proteins. More specifically, the progesterone-receptor complex acceptor sites appeared to be associated with a fraction of chromosomal nonhistone proteins from chick oviduct. This fraction, termed AP3, was represented mostly by high-molecular-weight proteins and its electrophoretic pattern was more complex than that of the testicular or prostatic proteins NP (8, 14, 28, 29). Our data indicate that binding of the cytoplasmic receptorhormone complex is at least partially influenced by the phosphorylation of some nonhistone proteins in fraction UP. Both phosphorylation and removal of this protein fraction resulted in a comparable increase in the [3H]DHT-receptor accepting ability of the treated chromatin. Additional increase of. the hormone-receptor binding can be achieved by the removal of histones (HP). The residual chromatin (HG) then contains only the DNA-binding proteins (NP) and DNA itself. Although this residual chromatin (HG) contains only about 3-5% of the original chromatin protein content, it has maximum ability to bind the [3H]DHT-receptor complex. However, this increased binding capacity is limited to chromatin of target cells. When only the histones were removed, the [3H]DHT-receptor binding increased by a fraction attributable to histone "inhibition." The dehistonized chromatin, which contains most of the chromosomal nonhistone proteins (including most of the UP fraction), is still significantly restricted with respect to its full capacity to accept the [3H]DHT-receptor complex. These observations are supported by the in vitro reconstitution experiments (Table 2). The deliberate selection of less than saturating binding assay conditions was based on our experience that, when in excess, the DHT-receptor complex associated with chromatin less selectively. This resembles the selectivity of other bioassays dependent on the interactions of large macromolecular complexes, e.g., in complement fixation the highest specificity is frequently attained at low antigen to antibody ratios. As be seen in Table 1 the [3H]DHT-receptor binding increase after the removal of UP or HP proteins was essentially the same can
seen
either at a low (approximately 45%) or high (approximately 80%) saturation level. It was shown by Buller et al. (30) and by Spelsberg et al. (31) that chick oviduct chromatin contains two or more classes of affinity sites for progesterone-receptor complex. It can be speculated that these various affinity sites correlate to the site-blocking ability of various chromosomal proteins. One level may depend on the proteins contained in the fraction UP, another on the histones, and the third upon the absolute chromatin acceptor capacity determined by the NP proteins. The cells could then regulate their quantitative and perhaps qualitative response to the hormone by manipulating the appropriate chromosomal proteins restricting the full capacity of the NP fraction. Protein phosphorylation may play an important role in this process. This study was supported by the USPHS Grant CA-07746 and the Robert A. Welch Foundation Grant G 138. The excellent technical assistance of Mrs. Mildred Hunt is greatfully acknowledged. The authors extend their sincere appreciation to Drs. F. Chytil, W. M. Mitchell, and D. N. Orth for their valuable suggestions during the preparation of this manuscript. 1. Liao, S. & Fang, S. (1969) "Receptor-proteins for androgens and the mode of action of androgens on gene transcription in ventral prostates," Vitam. Horm. 27, 17-90. 2. Wang, T. Y. & Nyberg, L. M. (1974) "Androgen receptors in the nonhistone protein fractions of prostatic chromatin," Int. Rev. Cytol. 39, 1-33. 3. O'Malley, B. W. & Means, A. R. (1974) "Female steroid hormones and target cell nuclei," Science 183, 610-620. 4. Jensen, E. V., Suzuki, T., Kawashima, T., Stumpf, W. E., Jungblut, P. W. & De Sombre, E. R. (1968) "A two step mechanism for the interaction of estradiol with rat uterus," Proc. Natl. Acad. Sci. USA 59,632-638. 5. Hamilton, T. H. (1968) "Control by estrogen of genetic transcription and translation," Science 161, 649-661. 6. Jensen, E. V. & De Sombre, E. R. (1972) "Mechanism of action of the female sex hormones," Annu. Rev. Biochem. 41, 203-230. 7. Fang, S. & Liao, S. (1971) "Androgen receptors. Steroid- and tissue-specific retention of al7 fl-hydroxy-5a-androstan-3-oneprotein complex by the cell nuclei of ventral prostate," J. Biol. Chem. 246, 16-24. 8. O'Malley, B. W., Spelsberg, T. C., Schrader, W. T., Chytil, F. & Steggles, A. W. (1972) "Mechanisms of interaction of a hormone-receptor complex with the genome of a eukaryotic target cell," Nature 235, 141-144. 9. Tymoczo, J. L. & Liao, S. (1971) "Retention of an androgenprotein complex by nuclear chromatin aggregates: Heat labile factors," Biochim. Biophys. Acta 252,607-611. 10. Whilhelm, J. A., Ansevin, A. T., Johnson, A. W. & Hnilica, L. S. (1972) "Proteins of chromatin in genetic restriction. IV. Coinparision of histone and nonhistone proteins of rat liver nucleolar and extranucleolar chromatin," Biochim. Biophys. Acta 272,
220-230.
1958
Biochemistry: Klyzsejko-Stefanowicz et al.
11. Blobel, G. & Potter, V. R. (1966) "Nuclei from rat liver: Isolation method that combines purity with high yield," Science 154, 1662-1665. 12. Chiu, J. F., Hunt, M. & Hnilica, L. S. (1975) "Tissue-specific DNA-protein complexes during azo-dye hepatocarcinogenesis," Cancer Res. 35, 913-919. 13. Bekhor, I., Kung, G. M. & Bonner, J. (1969) "Sequence-specific interaction of DNA and chromosomal protein," J. Mol. Biol. 39, 351-364. 14. Spelsberg, T. C., Steggles, A. W. & O'Malley, B. W. (1971) "Progesterone-binding components of chick oviduct. III. Chromatin acceptor sites," J. Biol. Chem. 246, 4188-4197. 15. Mainwaring, W. I. P. & Peterken, B. M. (1971) "A reconstituted cell-free system for the specific transfer of steroid receptor complexes into nuclear chromatin isolated from rat ventral prostate gland," Biochem. J. 125, 285-295. 16. Steggles, A. W., Spelsberg, T. C., Glasser, S. R. & O'Malley, B. W. (1971) "Soluble complexes between steriod hormones and target-tissue receptors bind specifically to target-tissue chromatin," Proc. Nat!. Acad. Sci. USA 68, 1749-1482. 17. Chiu, J. F., Craddock, C., Getz, S. & Hnilica, L. S. (1973) "Nonhistone chromatin protein phosphorylation during azo-dye carcinogenesis," FEBS Lett. 33, 247-250. 18. Kamiyama, M., Dastugue, B. & Kruh, J. (1971) "Action of phosphoproteins and protein kinase from rat liver chromatin on RNA synthesis," Biochem. Biophys. Res. Commun. 44, 13451350. 19. Kleinsmith, L. J., Allfrey, V. G. & Mrisky, A. E. (1966) "Phosphoprotein metabolism in isolated lymphocyte nuclei," Proc. Natl. Acad. Sci. USA 55, 1182-1189. 20. Mainwaring, W. I. P. & Irving, R. (1973) "The use of deoxyribonucleic acid-cellulose chromatography and isoelectric focusing for the characterization and partial purification of steroid-receptor complexes," Biochem. J. 134, 113-127. 21. Toft, D. (1972) "The interaction of uterine estrogen receptors
Proc. Natl. Acad. Sci. USA 73 (1976) with DNA," J. Steroid Biochem. 3,515-522. 22. King, R. J. B. & Gordon, J. (1972) "Involvement of DNA in the receptor mechanism for uterine oestradiol receptor," Nature New Biol. 240, 185-187. 23. O'Malley, B. W. & Schrader, W. T. (1972) "Progesterone receptor components: Identification of subunits binding to the target cell-genome," J. Steroid Biochem. 3, 617-629. 24. Ahmed, K. & Ishida, H. (1971) "Effects of testosterone on nuclear phosphoproteins of rat ventral prostate," Mol. Pharmacol. 7, 323-327. 25. Ahmed, K. & Wilson, M. J. (1975) "Chromatin-associated protein phosphokinases of rat ventral prostate," J. Biol. Chem. 250, 2370-2375. 26. King, R. J. B., Gordon, J., Cowan, D. M. & Inman, D. R. (1966) "The intranuclear location of [6,7-3H1-oestradiol-17a in dimethylbenzanthracene-induced rat mammary adenocarcinoma and other tissues," J. Endocrinol 36, 139-150. 27. Shyamala, G. & Gorski, J. (1969) "Estrogen receptors in the rat uterus. Studies on the interaction of cytosol and nuclear binding sites," J. Biol. Chem. 244, 1097-1103. 28. Spelsberg, T. C., Steggles, A. W., Chytil, F. & O'Malley, B. W. (1972) "Progesterone-binding components of chick oviduct. V. Exchange of progesterone-binding capacity from target to nontarget tissue chromatins," J. Biol. Chem. 247, 1368-1374. 29. Spelsberg, T. C., Mitchell, W. M., Chytil, F., Wilson, E. M. & O'Malley, B. W. (1973) "Chromatin of the developing chick oviduct: Changes in the acidic proteins," Biochim. Biophys. Acta 312,765-778. 30. Buller, R. E., Schrader, W. T. & O'Malley, B. W. (1975) "Progesterone binding components of chick oviduct. IX. The kinetics of nuclear bindings," J. Biol. Chem. 250, 809-818. 31. Pikler, G., Webster, R. & Spelsberg, T. C. (1975) "Specific binding site for progesterone in the oviduct nucleus," 57th Annual Meeting of the Endocrine Society, New York, June 18-20, abstr. 28.
Biochemistry
Acceptor proteins in rat androgenic tissue chromatin (nonhistone proteins/chromatin phosphorylation/5a-dihydrotestosterone chromatin binding) LEOKADIA KLYZSEJKO-STEFANOWICZ*, JEN-FU CHIUt, YU-HUI TSAIt, AND LUBOMIR S. HNiLICAt Department of Biochemistry, The University of Texas System Cancer Center, M.D. Anderson Hospital and Tumor Institute, Houston, Texas 77025
Communicated by Sidney P. Colowick, April 9, 1976
ABSTRACT Fractionation of chromatin into urea-soluble chromosomal nonhistone proteins (UP), histones (HP), and DNA-associated nonhistone proteins (NP) revealed that the NP fraction from testicular and prostatic chromatin contains organ-specific acceptors for complexes of 5a-dihydrotestosterone (17j3-hydroxy-5a-androstan-3-one) and its receptor. This acceptor capacity of androgenic tissue chromatin could be transferred to chromatins from non-target tissues with the NP fraction of DNA-associated proteins. Phosphorylation of chromatin enhanced its hormone-receptor binding capacity. According to current views, early steps in the action of steroid hormones on their target tissues involve the association of the steroid hormone with cytoplasmic receptor, transport of this complex from cytoplasm into the nucleus, and, finally, its association with specific acceptor sites on chromatin (1-4). This association alters the transcriptional controls and allows the synthesis of new mRNA species. The new mRNAs are eventually translated into new proteins that are often specific for the hormone-stimulated tissue (1, 3, 5, 6). The target cell nuclear acceptor is selective in that the nuclei of responsive cells accept more hormone-cytosol protein complex than the nuclei of tissues refractory to the action of steroid hormones (7, 8). Recent years have seen the accumulation of considerable information concerning the steroid hormone-cytoplasmic receptor interactions in different types of tissue. After the hormone interacts with cytoplasmic receptor, the hormone-protein complex moves to the nucleus, where it is incorporated into chromatin. Liao and Fang (1) coined the term "acceptor protein" for the nuclear acceptor molecule. The acceptor protein fraction was isolated from prostate (9) and found to be heat labile and tissue specific, and could be transferred from the chromatin of one tissue to another with the fraction of chromosomal nonhistone proteins (3, 7). We report here the results of our studies on chromatin acceptor(s) in rat prostate and testis.
MATERIALS AND METHODS Prostatp, testis, liver, and pancreas from male Sprague-Dawley rats (125-150 g) were homogenized in ice-cold 0.25 M sucrose, 5 mM MgCl2, and 2 mM 2-mercaptoethanol in 20 mM Tris-HCI buffer, pH 6.8. The homogenate was centrifuged at 800 X g for 10 min. The crude nuclear pellet was purified by rehomoAbbreviations: SSC, standard saline citrate (150 mM NaCl, 15 mM sodium citrate); UP, chromosomal nonhistone proteins soluble in urea at low ionic strength; HP, histones; NP, DNA-binding chromosomal nonhistone protein fraction; DHT, 5a-dihydrotestosterone (17f,-
hydroxy-5a-androstah-3-one). * Present address: Institute of Biochemistry and Biophysics, University of Lodz, 90-237 Lodz, Poland. t Present address: Department of Biochemistry, Vanderbilt University School of Medicine,.Nashville, Tennessee 37232. Direct reprint requests to L.S.H. at this address. t Present address: Program in Reproductive Biology and Endocrinology, The University of Texas Medical School at Houston, Houston, Texas 77025.
genization in 2.2 M sucrose, 25 mM KC1, 5 mM MgCl2 in 50 mM Tris.HCl buffer, pH 7.8, and centrifuged at 100,000 X g (max) for 1 hr (10). The isolation of chromatin and criteria for its purity were described previously (11). Isolated chromatin was fractionated into the bulk of chromosomal nonhistone proteins (UP), histones (HP), and DNA-binding nonhistone proteins (NP). At each fractionation step a partially deproteinized chromatin was obtained which was devoid of the concerned chromosomal protein fractions, i.e., UC (devoid of UP), HC (devoid of UP and HP), and NC (devoid of UP, HP, and NP). Details of the fractionation procedure were described elsewhere (12). DNA was reconstituted with the protein fractions UP, HP, and NP isolated from rat testis, prostate, liver, or pancreatic chromatin. The reconstitution was accomplished by mixing the interacting components in 2.5 M NaCl, 5.0 M urea, 50 mM Tris-HCl buffer, pH 8.0, at a ratio of DNA:UP:HP:NP = 1:1: 1:0.2 and decreasing the NaCl concentration by a slow dialysis against 5.0 M urea in 50 mM Tris.HCl buffer (13). Finally, the urea was removed by dialysis against either 0.1 X SSC (SSC = standard saline citrate: 150 mM NaCl, 15 mM sodium citrate), or 10 mM Tris-HCl buffer, pH 7.6. The reconstituted chromatins were used in hormone acceptor experiments. Preparation and Interactions of 3H-Labeled 5a-Dihydrotestosterone-Cytoplasmic Receptor Complexes. Tritium-labeled 5a-dihydrotestosterone ([3H]DHT, 50 mCi/,gmol, New England Nuclear) was complexed with cytosol following the method of Spelsberg et al. (14) with some modifications. After incubation with labeled hormone, the cytosol-hormone complex was fractionated with (NH4)2SO4 (0-33% saturation). The active fraction was freed from unbound hormone by gel -filtration on a Sephadex G-25 column (1 X 24 cm). Sucrose density gradient analysis of the purified [3H]DHT-receptor complex showed that essentially all the radioactivity was associated with the receptor protein peak sedimenting at approximately 8 S. This is in agreement with the reports from other laboratories (15, 16). In our system, we were able to detect only a small amount of 3.5S receptor complex, probably because of the (NH4)2SO4 fractionation of the initial extract (16). The sucrose density gradient centrifugation also revealed that the (NH4)2SO4 fractionation followed by gel filtration in Sephadex G-25 removed essentially all the unbound [3H]DHT. In agreement with Fang and Liao (7) and Mainwaring and Peterken (15), the binding of our [3H]DHT complex increased when the incubation was performed at 250 instead of 40. However, because of a better stability of the [3H]DHT complexes at 40, all our studies were done at this temperature (14). Samples of native, reconstituted, or partially deproteinized chromatins (UC, HC, or NC), each containing 100 jig of DNA, were incubated with 0.6 mg of [3H]DHT-receptor complex in a total volume of 1 ml containing 150 mM NaCl, 0.7 mM EDTA, 3 mM Tris-HCl, pH 7.4, at 40 for 1 hr. After incubation, the samples were diluted with 30 ml of cold 150 mM NaCl, 10 1954
Proc. Natl. Acad. Sci. USA 73 (1976)
Biochemistry: Klyzsejko-Stefanowicz et al.
1955
Table 1. [3H]DHT binding affinity of prostate, liver, and testis chromatin fractions Liver
Prostate
Testis
Chromatin fractions
Exp. no. 1
Exp. no. 2
Exp. no. 1
Exp. no. 2
Exp. no. 1
Exp. no. 2
Native chromatin Dehistonized chromatin UC (less UP) HC (less UP and HP) NC (less UP, HP, and NP)
0.50 0.85 0.97 1.71 0.49
1.04 1.87
0.60 1.22 1.32 3.34 0.74
1.32 2.69 2.92 5.20 2.47
0.28 0.31 0.25 0.31 0.32
0.44 0.47 0.46 0.55 0.48
2.60 1.05
Native or fractionated chromatin (100 gg of DNA) from each tissue was used in all experiments. Exp. no. 1 was done with 0.3 mg of total protein (about 45% saturation) of [3H]DHT-receptor complex and Exp. no. 2 was done with 0.6 mg of total protein (about 80% saturation) of [3H]DHT-receptor complex in the incubation mixture; the amount of bound [3HJDHT is expressed in nmol/g of DNA.
mM MgCl2, and centrifuged at 100,000 X g (max) for 8 hr. The pellets were washed twice by resuspending them vigorously in 30 ml of the same medium and centrifuging them at the indicated speed for 8 hr. The washed precipitate was again resuspended in the same medium and collected on Millipore filters (0.45 ttm pore size, 24 mm diameter). The filters were washed twice with 20 ml of 150 mM NaCl, 10 mM Mg9l2, dried, and measured for radioactivity content. Samples (1 ml of reconstituted chromatin with [3H]DHTreceptor complex) were analyzed by density gradient centrifugation. Samples were layered over linear 5-20% (wt/vol) sucrose density gradients in 2 mM dithiothreitol, 0.1 mM EDTA, 10 mM Tris-HCl, pH 7.4, and centrifuged in a Spinco SW 40 rotor at 37,000 rpm for 8 hr at 4°. Phosphorylation of Chromatin In Vivo and In Vitro. For the in vivo labeling of chromosomal proteins, rats were injected intraperitoneally with 2 mCi/100 g body weight of carrier-free [32P]orthophosphate and sacrificed 60 min after the injection. The individual tissues were processed immediately. The in vitro labeling of chromatin using its endogenous phosphoprotein kinases was accomplished by incubation in 16 tIM [y-32P]ATP (0.23 Ci/mmol), 20 mM MgCl2, and 100 mM NaCl in 80 mM Tris-HCI buffer, pH 7.5, at 37° for 10 min (17). The reaction was terminated by addition of 1% Na-pyrophosphate and the samples were extensively washed with 1% Napyrophosphate solution in 0.01 X SSC, pH 7.0, to remove free
1). This agrees with the 2000-6000 sites reported by other investigators (7, 20). The binding affinities of [3H]DHT-receptor complex to the partially deproteinized chromatin preparations UC, HC, and NC are listed in Table 1. After the UP and HP proteins were removed the residual chromatin fraction HC exhibited receptor affinity 250-557% that of the native prostatic or testicular chromatin. However, the binding affinity for the [3H]DHT-receptor complex was markedly decreased after the removal of NP proteins, leaving nearly pure DNA fraction NC. In contrast, the [3H]DHT-receptor binding to liver chromatin was not significantly altered by the removal of UP and HP fractions. This suggests that the NP fraction of testis and prostate contains acceptor protein(s) for the [3H]DHT-receptor complex. This fraction represents less than 5% of the total chromatin protein and its electrophoretic heterogeneity is shown in Fig. 2.
If the NP fraction contains the acceptor proteins for [3H]DHT-receptor complex, the binding affinity of chromatin for such complex should be transferrable from one tissue to another by reconstituting liver chromatin devoid of its own NP fraction with the NP proteins from androgenic target tissue chromatin.
I0
[y-32P]ATP.
The isolated UP, HP, and NP protein fractions were phosphorylated in vitro by a partially purified protein kinase from rat liver (18). The reaction mixture contained 20 ,umol of Tris-HCI, pH 7.5,4 nmol of [-y-32P]ATP (0.23 Ci/mmol), 5 ,Amol of MgGI2, 25 ,umol of NaCl, 50 ,ug of protein fraction, and 10 ,ug of partially purified kinase. The final volume was 0.25 ml. The reaction was terminated after incubation at 370 for 10 min by addition of 10% CCG3COOH. The precipitate was washed three times with cold 5% CCG3COOH. The [32P]phosphate content of the UP, HP, and NP fractions from all experiments was determined by the method of Kleinsmith et al. (19). RESULTS The prostatic and testicular chromatin exhibited significantly higher affinity for the [3H]DHT-receptor complex than hepatic or pancreatic chromatin (Fig. 1). This confirms the results of other investigators (15, 16). The binding was temperature dependent, and the bound [3HJDHT-receptor complex could be almost completely removed from chromatin by extraction with 0.4 M KCI. Because of better selectivity, our binding experiments were performed at approximately 80% saturation. At this saturation level, there were approximately 4700 [3H]DHTreceptor binding sites per nucleus equivalent of DNA (Table
z 0
I
E
c
1
2
5a-(3HJDHT-receptor (mg) FIG. 1. [3H]DHT-receptor complex binding affinities of prostatic (0), testicular (0), pancreatic (-), and hepatic (A) chromatins. Chromatin (200 wg of DNA) was incubated with appropriate amounts
of [3H]DHT-receptor complex in 0.15 M NaCl, 0.7 mM EDTA in 3 mM Tris-HCl buffer, pH 7.4, for 1 hr at 4° (1 ml final volume). After incubation, the samples were washed and collected as described in
the Materials and Methods.
1956
Biochemistry: Klyzsejko-Stefanowicz et al.
Proc. Natl. Acad. Sci. USA 73 (1976) Table 2.
[
3HI DHT binding affinity of reconstituted chromatin
Bound DHT (nmol/g of DNA)
Fractions E
I
C«1\N Vt
§ 0.3 co
0.2
0.22 1.37 1.27 0.29 0.66 0.57 0.20 1.10 0.81 0.26
DNA (rat spleen or testis) DNA + NPp DNA + NPT DNA + NPL DNA + UPL + HPL + NPP DNA + UPL + HPL + NPT
DNA+UPT +HPT +NPL
10
20
40
30
Gel
50
60
70
Reconstituted prostate chromatin Reconstituted testis chromatin Reconstituted liver chromatin
80
length(mm) b
0.5
0.4
E §0.3
0.2
0.1
10
20
30
60 50 40 Gel length (mm)
70
80
C
0.4
0 0
co
1
2
3
5
6
7
8
Gel length (cm)
FIG. 2. Optical scans of polyacrylamide gel electropherograms of (a) rat testis UP, (b) rat testis HP, and (c) rat testis NP. Two hundred micrograms of UP, 75 Mug of HP, and 100 Mug of NP were dialyzed against sample buffer (0.1 M sodium phosphate buffer, 0.1% sodium dodecyl sulfate, 0.1% fl-mercaptoethanol, and 8 M urea, pH 8.0) with two changes, and then applied to 10% polyacrylamide gels containing 0.1% sodium dodecyl sulfate and 4 M urea. Electrophoresis was performed at 8 mA per tube for 8 hr. The gels were stained and scanned as described previously (10).
As shown in Table 2, the NP protein fraction indeed determined
the binding affinity of chromatin for hormone-receptor complex. The binding activities of reconstituted chromatins of UP and DNA or HP and DNA were also studied; only marginal binding could be detected. In agreement with studies in other systems (21-23), DNA alone still showed some binding affinity, probably contributed to either by incomplete removal of the NP proteins from the NC pellet or by nonspecific binding of type A receptor to free DNA (15, 23). O'Malley and associates (8, 23) have shown that the chick oviduct cytoplasmic progesterone receptor consists of two 4S components separable by DEAE-cellulose chromatography.
Pure DNA or reconstituted chromatin (100 Ag of DNA) was incubated with 0.6 mg (total protein) of [3H]DHT-receptor complex in incubation mixture. The subscripts indicate the tissue origin of the fractions (P = prostate, T = testis, L = liver).
Both these components (A and B) exhibited high affinity and specificity for progesterone, and both associated with isolated chick oviduct nuclei. The type A receptor-progesterone complex was not specific in its in vitro binding. It associated indiscriminately with free DNA. The type B receptor-progesterone complex associated with chromatin and its binding was specific for the target tissue. Since the administration of hormones was shown to enhance the phosphorylation of nuclear nonhistone proteins in experimental animals (24, 25), the binding affinity of [3H]DHTreceptor complex to phosphorylated androgenic chromatin was analyzed. Chromatin phosphorylated in vitro exhibited binding affinity 143-277% that of the nonphosphorylated samples (Table 3). To investigate whether the NP fraction of chromatin contains phosphoproteins, chromatin samples were phosphorylated both in vivo and in vitro and fractionated into UP, HP, and NP fractions (Table 4). Surprisingly, the NP fraction was not phosphorylated either in vivo or in vitro and, therefore, does not contain a natural phosphoprotein. The increased binding affinity of the DHT-receptor complex to phosphorylated chromatin is probably attributable to conformational changes Table 3. DHT binding activity of phosphorylated androgenic chromatin Exp. no. 1 Fraction Native testicular chromatin Phosphorylated testicular chromatin Native prostatic chromatin Phosphorylated prostatic chromatin
Exp. no. 2
Bound DHT
(%)
Bound DHT
(%)
1.04
(100)
0.61
(100)
1.50
(143)
1.33 0.6
(217) (100)
1.66
(277)
Chromatin (1 mg of DNA) was incubated in a 10 ml reaction mixture containing 200 jmol of Tris*HCl, pH 7.5, 40 nmol of [y-32P]ATP, 50 Mmol of MgCl2, and 250 jimol of NaCl for 10 min at chromatin was washed 370. After incubation the phosphorylated three times with 1% Na-pyrophosphate in 0.01 x SSC, pH 7.0, and then three times with 0.01 x SSC, pH 7.0, to remove the pyrophosphate. Control or phosphorylated chromatin (100 jig of DNA) was incubated with [3H]DHT-receptor complex (0.3 mg of protein) in the incubation mixture described in Fig. 1. The amount of bound [3H]DHT is expressed in nmol/g of DNA.
Proc. Natl. Acad. Sci. -USA 73 (1976)
Biochemistry: Klyzsejko-Stefanowicz et al.
1957
Table 4. Phosphorylation of testicular UP, HP, and NP chromatin proteins
Isolated fractions in vitro
Chromatin In vivo
In vitro
Protein fraction
(activity %)
(activity %)
(pmol of 32P/mg of protein)
(activity %)
Casein UP HP NP
73 26
83 17 0.1
39.0 7.8 5.7 0.01
58 42 0.1
0.1
Details of the in vivo and in vitro phosphorylation and fractionation procedures were described in the Materials and Methods. The numbers are either 32p activity percentages of total chromatin protein or pmol of 32p per mg of protein.
of chromatin resulting from its phosphorylation, allowing the NP proteins to become more readily accessible to the [3H]DHT-receptor complex.
DISCUSSION Early experiments showed that the acceptor sites of the hormone-cytosol receptor complex are firmly associated with the nucleus. When the nuclear components were fractionated, the acceptor sites remained largely associated with chromatin (26, 27). Using selective dehistonization of chromatin and reconstitution of hybrid chromatins containing chromosomal proteins from target and non-target tissues, Spelsberg et al. (28) demonstrated that the ability of chromatin to accept progesterone-receptor complex depends on the cellular source of chromosomal nonhistone proteins. More specifically, the progesterone-receptor complex acceptor sites appeared to be associated with a fraction of chromosomal nonhistone proteins from chick oviduct. This fraction, termed AP3, was represented mostly by high-molecular-weight proteins and its electrophoretic pattern was more complex than that of the testicular or prostatic proteins NP (8, 14, 28, 29). Our data indicate that binding of the cytoplasmic receptorhormone complex is at least partially influenced by the phosphorylation of some nonhistone proteins in fraction UP. Both phosphorylation and removal of this protein fraction resulted in a comparable increase in the [3H]DHT-receptor accepting ability of the treated chromatin. Additional increase of. the hormone-receptor binding can be achieved by the removal of histones (HP). The residual chromatin (HG) then contains only the DNA-binding proteins (NP) and DNA itself. Although this residual chromatin (HG) contains only about 3-5% of the original chromatin protein content, it has maximum ability to bind the [3H]DHT-receptor complex. However, this increased binding capacity is limited to chromatin of target cells. When only the histones were removed, the [3H]DHT-receptor binding increased by a fraction attributable to histone "inhibition." The dehistonized chromatin, which contains most of the chromosomal nonhistone proteins (including most of the UP fraction), is still significantly restricted with respect to its full capacity to accept the [3H]DHT-receptor complex. These observations are supported by the in vitro reconstitution experiments (Table 2). The deliberate selection of less than saturating binding assay conditions was based on our experience that, when in excess, the DHT-receptor complex associated with chromatin less selectively. This resembles the selectivity of other bioassays dependent on the interactions of large macromolecular complexes, e.g., in complement fixation the highest specificity is frequently attained at low antigen to antibody ratios. As be seen in Table 1 the [3H]DHT-receptor binding increase after the removal of UP or HP proteins was essentially the same can
seen
either at a low (approximately 45%) or high (approximately 80%) saturation level. It was shown by Buller et al. (30) and by Spelsberg et al. (31) that chick oviduct chromatin contains two or more classes of affinity sites for progesterone-receptor complex. It can be speculated that these various affinity sites correlate to the site-blocking ability of various chromosomal proteins. One level may depend on the proteins contained in the fraction UP, another on the histones, and the third upon the absolute chromatin acceptor capacity determined by the NP proteins. The cells could then regulate their quantitative and perhaps qualitative response to the hormone by manipulating the appropriate chromosomal proteins restricting the full capacity of the NP fraction. Protein phosphorylation may play an important role in this process. This study was supported by the USPHS Grant CA-07746 and the Robert A. Welch Foundation Grant G 138. The excellent technical assistance of Mrs. Mildred Hunt is greatfully acknowledged. The authors extend their sincere appreciation to Drs. F. Chytil, W. M. Mitchell, and D. N. Orth for their valuable suggestions during the preparation of this manuscript. 1. Liao, S. & Fang, S. (1969) "Receptor-proteins for androgens and the mode of action of androgens on gene transcription in ventral prostates," Vitam. Horm. 27, 17-90. 2. Wang, T. Y. & Nyberg, L. M. (1974) "Androgen receptors in the nonhistone protein fractions of prostatic chromatin," Int. Rev. Cytol. 39, 1-33. 3. O'Malley, B. W. & Means, A. R. (1974) "Female steroid hormones and target cell nuclei," Science 183, 610-620. 4. Jensen, E. V., Suzuki, T., Kawashima, T., Stumpf, W. E., Jungblut, P. W. & De Sombre, E. R. (1968) "A two step mechanism for the interaction of estradiol with rat uterus," Proc. Natl. Acad. Sci. USA 59,632-638. 5. Hamilton, T. H. (1968) "Control by estrogen of genetic transcription and translation," Science 161, 649-661. 6. Jensen, E. V. & De Sombre, E. R. (1972) "Mechanism of action of the female sex hormones," Annu. Rev. Biochem. 41, 203-230. 7. Fang, S. & Liao, S. (1971) "Androgen receptors. Steroid- and tissue-specific retention of al7 fl-hydroxy-5a-androstan-3-oneprotein complex by the cell nuclei of ventral prostate," J. Biol. Chem. 246, 16-24. 8. O'Malley, B. W., Spelsberg, T. C., Schrader, W. T., Chytil, F. & Steggles, A. W. (1972) "Mechanisms of interaction of a hormone-receptor complex with the genome of a eukaryotic target cell," Nature 235, 141-144. 9. Tymoczo, J. L. & Liao, S. (1971) "Retention of an androgenprotein complex by nuclear chromatin aggregates: Heat labile factors," Biochim. Biophys. Acta 252,607-611. 10. Whilhelm, J. A., Ansevin, A. T., Johnson, A. W. & Hnilica, L. S. (1972) "Proteins of chromatin in genetic restriction. IV. Coinparision of histone and nonhistone proteins of rat liver nucleolar and extranucleolar chromatin," Biochim. Biophys. Acta 272,
220-230.
1958
Biochemistry: Klyzsejko-Stefanowicz et al.
11. Blobel, G. & Potter, V. R. (1966) "Nuclei from rat liver: Isolation method that combines purity with high yield," Science 154, 1662-1665. 12. Chiu, J. F., Hunt, M. & Hnilica, L. S. (1975) "Tissue-specific DNA-protein complexes during azo-dye hepatocarcinogenesis," Cancer Res. 35, 913-919. 13. Bekhor, I., Kung, G. M. & Bonner, J. (1969) "Sequence-specific interaction of DNA and chromosomal protein," J. Mol. Biol. 39, 351-364. 14. Spelsberg, T. C., Steggles, A. W. & O'Malley, B. W. (1971) "Progesterone-binding components of chick oviduct. III. Chromatin acceptor sites," J. Biol. Chem. 246, 4188-4197. 15. Mainwaring, W. I. P. & Peterken, B. M. (1971) "A reconstituted cell-free system for the specific transfer of steroid receptor complexes into nuclear chromatin isolated from rat ventral prostate gland," Biochem. J. 125, 285-295. 16. Steggles, A. W., Spelsberg, T. C., Glasser, S. R. & O'Malley, B. W. (1971) "Soluble complexes between steriod hormones and target-tissue receptors bind specifically to target-tissue chromatin," Proc. Nat!. Acad. Sci. USA 68, 1749-1482. 17. Chiu, J. F., Craddock, C., Getz, S. & Hnilica, L. S. (1973) "Nonhistone chromatin protein phosphorylation during azo-dye carcinogenesis," FEBS Lett. 33, 247-250. 18. Kamiyama, M., Dastugue, B. & Kruh, J. (1971) "Action of phosphoproteins and protein kinase from rat liver chromatin on RNA synthesis," Biochem. Biophys. Res. Commun. 44, 13451350. 19. Kleinsmith, L. J., Allfrey, V. G. & Mrisky, A. E. (1966) "Phosphoprotein metabolism in isolated lymphocyte nuclei," Proc. Natl. Acad. Sci. USA 55, 1182-1189. 20. Mainwaring, W. I. P. & Irving, R. (1973) "The use of deoxyribonucleic acid-cellulose chromatography and isoelectric focusing for the characterization and partial purification of steroid-receptor complexes," Biochem. J. 134, 113-127. 21. Toft, D. (1972) "The interaction of uterine estrogen receptors
Proc. Natl. Acad. Sci. USA 73 (1976) with DNA," J. Steroid Biochem. 3,515-522. 22. King, R. J. B. & Gordon, J. (1972) "Involvement of DNA in the receptor mechanism for uterine oestradiol receptor," Nature New Biol. 240, 185-187. 23. O'Malley, B. W. & Schrader, W. T. (1972) "Progesterone receptor components: Identification of subunits binding to the target cell-genome," J. Steroid Biochem. 3, 617-629. 24. Ahmed, K. & Ishida, H. (1971) "Effects of testosterone on nuclear phosphoproteins of rat ventral prostate," Mol. Pharmacol. 7, 323-327. 25. Ahmed, K. & Wilson, M. J. (1975) "Chromatin-associated protein phosphokinases of rat ventral prostate," J. Biol. Chem. 250, 2370-2375. 26. King, R. J. B., Gordon, J., Cowan, D. M. & Inman, D. R. (1966) "The intranuclear location of [6,7-3H1-oestradiol-17a in dimethylbenzanthracene-induced rat mammary adenocarcinoma and other tissues," J. Endocrinol 36, 139-150. 27. Shyamala, G. & Gorski, J. (1969) "Estrogen receptors in the rat uterus. Studies on the interaction of cytosol and nuclear binding sites," J. Biol. Chem. 244, 1097-1103. 28. Spelsberg, T. C., Steggles, A. W., Chytil, F. & O'Malley, B. W. (1972) "Progesterone-binding components of chick oviduct. V. Exchange of progesterone-binding capacity from target to nontarget tissue chromatins," J. Biol. Chem. 247, 1368-1374. 29. Spelsberg, T. C., Mitchell, W. M., Chytil, F., Wilson, E. M. & O'Malley, B. W. (1973) "Chromatin of the developing chick oviduct: Changes in the acidic proteins," Biochim. Biophys. Acta 312,765-778. 30. Buller, R. E., Schrader, W. T. & O'Malley, B. W. (1975) "Progesterone binding components of chick oviduct. IX. The kinetics of nuclear bindings," J. Biol. Chem. 250, 809-818. 31. Pikler, G., Webster, R. & Spelsberg, T. C. (1975) "Specific binding site for progesterone in the oviduct nucleus," 57th Annual Meeting of the Endocrine Society, New York, June 18-20, abstr. 28.
