Distribution of estrogen receptors in hen oviduct chromatin fractions in the course of DNAase II digestion

109

Biochimica et Biophysica Acta, 474 (1977) 109--116 © Elsevier/North-Holland Biomedical Press

BBA 98806

DISTRIBUTION OF E S T R O G E N R E C E P T O R S IN HEN OVIDUCT CHROMATIN FRACTIONS IN THE COURSE OF DNAase II DIGESTION

K. HEMMINKI and M. VAUHKONEN

Department of Medical Chemistry, University of Helsinki, Siltavuorenpenger 10A, SF-O0170 Helsinki 17 (Finland) (Received April 5th, 1976)

Summary Hen oviduct chromatin was digested with DNAase II and two fractions were isolated: MgC12-insoluble chromatin and MgC12-soluble chromatin. The former contained 14 and 50% of the total DNA after a digestion time of 3 and 30 min, respectively. The fraction was characterized in sucrose gradients b y a peak sedimenting at 11 S. In the course of DNAase digestion this fraction lost most of its estrogen receptors as assayed by [3H]estradiol exchange reaction. The specific radioactivity of chromatin was particularly low in the 11 S region. The MgC12-soluble chromatin contained at most 5.1% of the total DNA. In sucrose gradients the fraction displayed peaks at 4 S and 14 S. After a 30 min DNAase digestion the specific radioactivity of chromatin in this fraction exceeded that of the MgCl~-insoluble fraction 7.7 fold. Material sedimenting at 14 S and at larger S values was enriched in estrogen receptors The results suggest that estrogen receptors are unevenly distributed on hen oviduct chromain.

Introduction Recent evidence indicates that the bulk of eukaryotic chromatin is composed of c o m p a c t bead-like structures (nucleosomes or p-bodies) connected by filaments of DNA [1--5]. DNAases are thought to nick primarily the extranucleosomal stretches of DNA, which can be applied for the purification of nucleosomes. They are reported to contain 120--200 base pairs of DNA [4,6, 7], an octet of histones [8] and some nonhistone proteins. The function of the nucleosomes remains to be established (see ref. 5). Less information has accumulated on the extracleosomal DNA. According to electron microscopic observations it appears to be of varable length. Some nucleosomes have been reported to be connected to each other with less than 20 base pairs of extended DNA [9]. By contrast, relatively extended sequences o f 'naked' DNA as large as 4000 base pairs have been described [5,10]. Bonnet and cowerkers have

110 digested liver chromatin with DNAase II and have obtained particles of discrete sizes other than nucleosomes or their multimers [11]. Some of these particles have been suggested to derive from transcriptionally active chromatin. In this study the action of DNAase II on hen oviduct chromatin is investigated. After various periods of enzyme action t w o chromatin fractions are isolated with MgCl: precipitation [11]. One of the fractions is enriched and the other impoverished in nucleosomes. The distribution of estrogen receptors in the fractions is followed in the course of enzyme action a n d analyzed on sucrose gradients. Materials and Methods Oviduct tissue was homogenized in 1 mM MgC12 (about 20 ml]g of oviduct) using 15 strokes of a loosely fitting Teflon-glass homogenizer. The homogenate was centrifuged at 700 × g for 10 min. The pellet was homogenized in 0.1 M NaC1 containing 1 mM MgCl~ (20 ml/g starting material) as above and passed through four layers of cheesecloth. The filtrate was centr.ifuged at 700 × g for 10 min. The pellet was homogenized in 0.1 M NaC1/1 mM MgCl~ containing 0.6 M sucrose, passed through four layers of cheesecloth and centrifuged. The pellet was homogenized in 2 M sucrose containing 1 mM MgCI~ and 10 mM HEPES, pH 7.0 (10 ml/g starting material} with three strokes; the homogenate was layered over an equal volume of 2 M sucrose containing MgC12 and HEPES; the tubes were centrifuged in a Spinco SW-25 rotor at 55 000 × g for 30 min. The nuclear pellet was washed three times with 0.15 M NaC1. The first wash contained 0.1% Triton X-100. Purified nuclei {about 1 mg of DNA) were lysed in 3 ml of 10 mM NaHSO3 containing 0.2 mM EDTA, pH 6.5 and chromatin was fractionated largely as described by Bonner et al. [12]. The tubes were transferred onto water bath at 37°C, 150 units of DNAase II {EC 3.1.4.6; HDAC, Worthington) were added and the incubation was contained for various periods of time; 8 ml of 50 mM Tris, pH 8.0 were added and the mixture was centrifuged at 10 000 × g for 10 min. The 10 000 × g supernatant was made 3 mM with respect to MgCl~ and was kept on ice for 30 min followed by centrifugation at 10 000 × g for 15 min to collect the MgCl~-insoluble chromatin [12]. The chromatin remaining in the 10 000 × g supernatant was collected at 100 000 × g for 2 h and designated as the M gCl~-soluble chrom atin [ 12 ].

Other techniques Endogenous estrogen receptors in the chromatin fractions were labelled by an estrogen exchange assay of Anderson et al. [13]. Nuclei were purified and washed as described above. They were lysed in 4 ml of 2 mM NaHSO3, pH 7.0 and divided into t w o aliquots, which were made 18 nM with respect to 17/~[ 3H ] estradiol {45 Ci/mmol, Radiochemical Centre, Amersham ). For the estimation of nonspecific binding sites, one aliquot additionally contained 1.8/aM nonradioactive estradiol [13]. The samples were incubated at 37°C for 30 min, which was sufficient for nearly complete (over 80%} exchange. DNAase II digestion and fractionation of chromatin ensued as above. However, it was necessary to remove free h o r m o n e before sedimentation of the MgCl~-soluble

111 chromatin. This was done by adding 0.05 vol. of dextran~oated charcoal (3% charcoal and 0.1% dextran}. The solution was shaken at 0°C for 5 rain and the charcoal was removed by centrifugation at 10 000 × g for 10 rain. The supernatant was used for the purification of chromatin fractions as above. The results of the exchange assays were given after the subtraction of the nonspecific radioactivity, which amounted to about 15% of the radioactivity measured at 18 nM estradiol. The chromatin fractions suspended in 3 ml of 5 mM Na~EDTA, pH 7.6 were analysed on 5--24% isokinetic sucrose gradients (30 ml) containing 1 mM Na~EDTA and 10 mM Tris pH 8.0. Centrifugation was done at 63 000 × g for about 18 h in a Spinco SW-25.1 rotor. Gradient absorbance profiles were measured with an ISCO absorbances monitor. Bovine serum albumin was run in parallel tubes as a referei~ce protein (4.5 S) in the estimation of sedimentation coefficients. The chromatin fractions were prepared for polyacrylamide gel electrophoresis using RNAase and proteinase K digestion followed by chloroform and isoamyl alcohol extraction [14]. Polyacrylamide gel electrophoresis was carried out according to Loening [15] using xylene cyanol FF and bromphenol blue as molecular weight standards [16]. The double-strandedness of the preparations was monitored by the extent of hyperchromicity exhibited on denaturation. The samples showed on average a 28% hyperchromicity. Results

Hen oviduct nuclei were lysed and digested with DNAase II to investigate the distribution of estrogen receptors in the subfractions of chromatin. After 3 rain of DNAase II digestion 14% of the total nuclear DNA was recovered in the MgCl~-insoluble chromatin fraction (Table I}. At this digestion time 2.8% of DNA was collected in the MgCl~-soluble chromatin fraction. The recovery of DNA in the MgC12-insoluble chromatin fraction increased at longer digestion times: 45 and 50% of the total DNA were obtained in the fraction after a diges-

TABLE I RECOVERY OF DNA AND PROTEINfDNA RATIOS OF HEN OVIDUCT CHROMATIN FRACTIONS IN T H E C O U R S E O F D N A a s e II D I G E S T I O N H e n o v i d u c t n u c l e i w e r e l y s e d in 1 0 m M N a H S O 3 c o n t a i n i n g 0 . 2 r a m E D T A , p H 6 . 5 . D N A a s e II w a s a d d e d at 5 u n i t s / A 2 6 0 n m u n i t a n d t h e d i g e s t i o n c o n t i n u e d f o r t h e i n d i c a t e d p e r i o d s o f t i m e . M e a n s + SE of 4--5 experiments.

Length of digestion (rain)

Recovery of DNA % of the total DNA

MgCI 2-insoluble chromatin Nuclei

Protein/DNA ratio

MgCI 2-soluble chromatin

MgCI 2-insoluble chromatin

100

MgCl2-soluble chromatin

4.2±0.6

3

14.2±1.7

2.8±0.4

2.0±0.28

4.7±0.7

15

45.0±5.9

5.1±0.7

1.8±0.24

4.1±0.6

30

50.0±3.8

4.7±0.6

1.6±0.10

3.7±0.5

112

tion time of 15 and 30 min, respectively. By contrast, no such large increase in recovery was observed in the MgC12-soluble chromatin. At most, 5.1% of DNA was collected in this fraction. The protein/DNA ratios of the chromatin fractions decreased slightly in the course of DNAase II digestion (Table I). In the case of MgC12-insoluble chromatin the decrease in the protein/DNA ratios was from 2.0 to 1.6 between 3 and 30 min of digestion. The corresponding ratios for the MgCl~-soluble chromatin changed from 4.7 to 3.7. Estrogen receptors of hen oviduct nuclei were labelled with[3H]estradiol using the steroid exchange assay and fractionated after various periods of DNAase II action (Table II). The specific radioactivity of chromatin (cpm/mg DNA) decreased in the MgC12-insoluble chromatin and increased in the MgCl~soluble chromatin in the course of enzyme digestion. The decrease was 60% in the case of the MgCl~-insoluble chromatin as compared to the increase of 45% in the MgC12-soluble chromatin. At all digestion times measured the specific radioactivity of the MgCl:-soluble chromatin exceeded that of the MgCl~-insoluble chromatin. At 3 min digestion the difference between the specific radioactivities of the chromatin fractions was 2.1 fold and increased to a difference of 7.7 fold at 30 min digestion. The results may indicate a precursor/product relationship in the distribution of chromatin segments rich in estrogen receptors between the MgCl:-insoluble and the MgCl:-soluble chromatin fractions. At short incubation times the MgCl~-insoluble chromatin contains segments rich in estrogen receptors. In the course of digestion the segments are excised and removed into the MgCl~-insoluble chromatin fraction. The [3H]estradiol-labelled chromatin fractions prepared at different DNAase II digestion times were analysed on 5--24% isokinetic sucrose gradients. The sedimentation profile of the MgCl:-insoluble chromatin clearly changed in the course of enzyme action (Fig. 1). At 3 min digestion the bulk of the material was of large molecular weight ( ~ 2 0 S) and only a small peak of 11 S could be observed in the small molecular weight region of gradient. At 30 min digestion the sedimentation pattern was characterized by a prominent peak at 11 S and minor peaks at a b o u t 15 S and 18 S. The specific radioactivity of chromatin was monitored along the sucrose gradients (Fig. 1). At the two digestion times investigated the highest specific T A B L E II D I S T R I B U T I O N OF E S T R O G E N R E C E P T O R S IN T H E S U B F R A C T I O N S OF HEN O V I D U C T CHROM A T I N I N T H E C O U R S E O F D N A a s e II D I G E S T I O N E s t r o g e n r e c e p t o r s w e r e labelled w i t h | 3 H ] e s t r a d i o l using t h e s t e r o i d e x c h a n g e assay [ 1 3 ] . M e a n s -+ SE of 5 determinations. L e n g t h of d i g e s t i o n (rain)

Specific r a d i o a c t i v i t y of [ 3 H ] e s t r a d i o l ( c p m / m g D N A ) MgC12-insoluble c h r o m a t i n

Nuclei 3

MgCl2-soluble c h r o m a t i n

39 2 0 0 ± 3 1 0 0 48 6 0 0 -+ 10 6 0 0

1 0 3 3 0 0 ± 11 0 0 0

15

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132200"+ 16600

30

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15300

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Fig. 1. S u c r o s e g r a d i e n t s e d i m e n t a t i o n of t h e MgC12-insoluble c h r o m a t i n p r e p a r e d a f t e r a 3 m i n ( A ) a n d 3 0 rnin (B) D N A a s e I I digestion. The c h r o m a t i n f r a c t i o n s w e r e s u s p e n d e d in 3 m l o f 5 m M E D T A p H 7.6 a n d Layered o v e r 5 - - 2 4 % s u c r o s e g r a d i e n t s . T h e g r a d i e n t s w e r e r u n a t 63 0 0 0 X g for 1 8 h. Approximate s e d i m e n t a t i o n c o e f f i c i e n t s w e r e d e t e r m i n e d using b o v i n e s e r u m a l b u m i n ( B S A ) as a s t a n d a r d . - - , absorbance at 254 n m ; . . . . . . . [ 3 H ] e s t r a d i o l r a d i o a c t i v i t y / A 2 6 0 n m u n i t in g r a d i e n t f r a c t i o n s ; , n o n s p e c i f i c [ 3 H ] e s t r a d i o l r a d i o a c t i v i t y / A 2 6 0 n m u n i t (see M e t h o d s ) . Fig. 2. S u c r o s e g r a d i e n t s e d i m e n t a t i o n of t h e MgC!2osoluble c h r o m a t i n p r e p a x e d a f t e r a 3 m i n ( A ) a n d 3 0 m i n (B) D N A a s e d i g e s t i o n . - - , absorbance at 245 nm; ....... [ 3 H ] e s t r a d i o l r a d i o a c t i v i t y / A 2 6 0 nm u n i t in g r a d i e n t f r a c t i o n s ; - - . --~ n o n s p e c i f i c [ 3 H ] e s t r a d i o l r a d i o a c t i v i t y [A 2 6 0 r i m u n i t .

radioactivities were recorded in the large molecular weight region of the gradient including the peak produced after 3 min digestion. By contrast, the 11 S peak displayed the lowest specific radioactivity of all fractions investigated. This indicated that in the course of DNAase digestion the segments of chromatin rich in estrogen receptors were selectively removed from the MgCl~-insoluble chromatin fractions. The radioactivity remaining at the top of the gradient appeared to be due mainly to free hormone because of its slow sedimentation rate and because it could be removed with activated charcoal [17]. The sedimentation pattern of the MgCl~-soluble chromatin revealed two major peaks of 5 S and 14 S as well as some minor peaks in the larger molecular weight region (Fig. 2). The major peaks remained at all digestion times tested. However, the 5 S peak increased in relation to the 14 S peak at longer digestion times. Analogously, more radioactivity was recovered in the 5 S region after longer digestion times. Other peaks of radioactivity were observed at about 14 S, 20 S and in the >20 S region independent of the digestion time. The size of DNA in the chromatin fractions was analysed on polyacrylamide gels. The size of DNA in the MgCl~-insoluble cromatin changed from a single large molecular weight peak (1700 base pairs, 1 rain digestion) to a series of peaks (Fig. 3). At 30 min the major DNA peak was about 180 base pairs and the minor peaks 420 and 630 base pairs. The MgCl:-soluble chromatin displayed a large molecular weight componer~t (over 2000 base pairs) at 3 min DNAase II digestion (Fig. 4). In the course of DNAase II action the component disappeared and relatively more material

114

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100

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Fig. 3. P o l y a c r y l a m i d e gels o f D N A s a m p l e s p u r i f i e d f r o m t h e MgCl2-insoluble c h r o m a t i n p r e p a r e d ( A ) after a 1 rain ( ), 3 rain ( . . . . . . ), 5 rain ( . . . . . . ) d i g e s t i o n o r (B) a f t e r a 15 m i n ( . . . . . . ) or 30 m i n ( ) d i g e s t i o n . T h e gels w e r e c a l i b r a t e d w i t h x y l e n e c y a n o l F F ( 4 5 0 base pairs) a n d b r o m p h e n o l b l u e ( 1 0 0 b a s e pairs). T h e f i g u r e s r e f e r t o t h e n u m b e r o f base pairs c a l e u l a t e d f r o m t h e d i s t a n c e o f m i g r a t i o n . Fig. 4. P o l y a c r y l a m i d e gels o f D N A s a m p l e s p u r i f i e d f r o m t h e MgC12-soluble c h r o m a t i n p r e p a r e d a f t e r a 3 min ( ), 15 m i n ( . . . . . . ) a n d 30 m i n ( . . . . . . ) d i g e s t i o n .

accumulated in the 175 base pair region. At all incubation times DNA segments of a b o u t 90 base pairs were also detected. Discussion The specificity of binding and the distribution of steroid receptors on chromatin has been a matter of recent debate [18,19]. Some workers have reported specific, high affinity binding of the receptor by target tissue chromatins (see ref. 18) whereas others have been unable to detect high affinity binding by nuclei [19] or DNA [20]. However, it should be emphasized that such studies have been performed with exogenous steroid receptors. When the fractionation of endogenous chromatin-bound receptors on chromatin subfractions has been investigated, an uneven distribution of receptors in the subfractions has been reported [21]. In this study the action of DNAase II on hen oviduct chromatin was investigated. T w o fractions were isolated from the digestion mixture with MgCI~ precipitation. This m e t h o d has been used by Bonnet and coworkers to prepare the MgCl~-insoluble (template-inactive) and the MgCl~-soluble (templateactive) subfractions of chromatin [12]. It was shown accordingly in the present study using sucrose gradients and DNA gels that the MgCl:-insoluble chromatin was enriched in l l - S particles containing 180 base pairs of DNA in agreement with data on nucleosomes [5--7,22]. After a 30 min DNAase digestion the nucleosome-rich fraction contained 50% of the total nuclear DNA. The MgCI:soluble chromatin fraction amounted to no more than 5.1% of the total DNA.

115 The fraction was characterized with a protein/DNA ratio of 3.7 and two major sedimentation peaks of 4--5 S and 14 S in sucrose gradients. On DNA gels two major peaks of 90 and 175 base pairs were also observed. These data closely resemble the properties of the template-active chromatin of Bonnet and coworkers [11]. The distribution of estrogen receptors in the two fractions was studied by labelling the receptors with [3H]estradiol according to the steroid exchange assay [13]. After a 3 min DNAase II digestion the specific radioactivity of chromatin was slightly higher in the MgCl~-insoluble chromatin and much higher in the MgC12-soluble chromatin than in the whole nuclei. At longer DNAase digestion times the specific radioactivity of the MgCl:-insoluble chromatin decreased and that of the MgCl~-soluble chromatin increased. After a 30 min DNAase digestion the difference in specific radioactivities between the two chromatin fractions was 7.7 fold. Sucrose gradients of the MgCl:-insoluble chromatin revealed very low specific radioactivities in the 11--20 S region suggesting that nucleosomes bind a few estrogen receptor. The MgCl:-soluble chromatin displayed some radioactivity around the 14 S absorbance peak but the highest receptor concentrations were observed in material sedimenting at 19 and larger S values. The observed enrichment of estrogen receptors in the MgCl~-soluble chromatin in the course of DNAase II digestion may indicate that the receptors are differentially distributed on chromatin. It is of interest that the extent of receptor enrichment has been reported to depend on estrogen stimulation [23]. Furthermore, ovalbumin gene sequences appear to be enriched in the MgCI:soluble fraction [24]. However, the observed results may alternatively indicate the release of receptors from nucleosomes, caused by the enzyme, and subsequent attachment of the receptors to the MgCl:-soluble chromatin. A release of estrogen receptors from chromatin [25] and of nonhistone proteins from nucleosomes [26] after DNAase action has been described in the literature. We have tried to eliminate the latter alternative using 0.4 M NaC1 to extract loosely bound receptors from the chromatin fractions (Hemminki and Vauhkonen, unpublished results). The enrichment of estrogen receptors in the MgCl:-insoluble chromatin fraction remains unchanged after the salt extraction ruling out that weak ionic associations would cause the observed fractionation. However, the true binding of receptors to chromatin remains to be established. The results of this study suggest that estrogen receptors were bound to components of chromatin which were excised with DNAase II. Some of the initial digestion products were of larger molecular weight to be split in the course of DNAase action. The segments of chromatin rich in estrogen receptors were enriched in the MgCl:-soluble chromatin fraction. It contained 4.7% of the total DNA and 18% of the labelled estrogen receptors of the intact chromatin. This fraction of the receptor may be partially interesting as it remains tightly bound to chromatin, which could be an expected property of a gene repressor [27].

Acknowledgements The skilled technical assistance of Mrs. Kirsti Salmela and Mrs. Ulla Riihivaara

116

is appreciated. The study was supported by the National Research Council for Medical Sciences, Finland and the Sigrid Jus~lius Foundation. References 1 Hewish, D.R. and Burgoyne, L.A. (1973) Biochem. Biophys. Res. C ommun. 5 2 , 5 0 4 - - 5 1 0 2 0 l i n s , A.L. and Olins, D.E. (1974) Science 1 8 3 , 3 3 0 - - 3 3 2 3 Kornberg, R.D. and Thomas, J.O. (1974) Science 184, 865--868 4 Noll, M. (1974) Nature 2 5 1 , 2 4 9 - - 2 5 1 5 0 u d e t , P., G~oss-Bellard, M. and Chambon, P. (1975) Cell 4 , 2 8 1 - - 3 0 0 6 Axel, R. (1975) Biochemistry 14, 2921--2925 7 SoHner-Webb, B. and Felsenfeld, G. (1975) Biochemistry 14, 2 9 1 5 - - 2 9 2 0 8 Thomas, J.O. and Kornberg, R.D. (1975) Proc. Natl. Acad. Sci. U.S. 72, 2 6 2 6 - - 2 6 3 0 9 Finch, J.T., Noll, M. and Kornberg, R.D. (1975) Proc. Natl. Acad. Sci. U.S. 72, 3 3 2 0 - - 3 3 2 2 10 Varshavsky, A.J., Ilyin, Y.V. and Georgiev, G.P. (1974) Nature. 250, 602--606 I I Gottesfeld, J.M., Murphy, R.F, and Bonner, J. (1975) Proc. Natl. Acad. Sci. U.S. 72, 4 4 0 4 - - 4 4 0 8 12 Bonnet, J., Gottesfeld, J., Garrard, W., Billing, R. and Uphouse, L. (1975) in Methods in E n z y m o l o g y (O'Malley, B.W. and Hardman, J.G., eds.), Vol. XL, Part E, pp. 97--102, Academic Press, New York 13 Anderson, J.N., Clark, J.H. and Peck, Jr., E.J., (1972) Biochem. J. 1 2 6 , 5 6 1 - - 5 6 7 14 Noll, M., Thomas, J.O. and Kornberg, R.D. (1975) Science 187, 1203--1206 15 Loerdng, U.E. (1967) Biochem. J. 1 0 2 , 2 5 1 - - 2 5 7 16 Maniatis, T., Jeffrey, A. and van deSande, H. (1975) Biochemistry 3787--3794 17 H e m m i n k i , K. (1976) Steroid Biochem. 7 , 4 1 3 - - 4 1 8 18 O'Mailey, B.W. and Means, A.R. (1974) Science 1 8 3 , 6 1 0 - - 6 2 0 19 Chamness, G.C., Jennings, A.W. and McGuire, W.L. (1974) Biochemistry 13, 327--331 20 Y a m a m o t o , K. and Alberts, B. (1974) J. Biol. Chem. 249, 7076--8086 21 Sala-Trepat, J.M., Vallet-Strouve, C. and Rat, L. (1975) Proc. 10th FEBS Meet. Paris, p. 1398 22 Oosterhof, D.K., Hozier, J.C. and Rill, R.L. (1975) Proc. Natl. Acad. Sci. U.S. 7 2 , 6 3 3 - - 6 3 7 23 H e m m i n k i , K. (1976) Acta Endocrino1., in press 2 H e m m i n k i , K. and Vauhkonen, M. (1976) J. Steroid Biochem., in press 25 Harris, G.S. (1971) Nat. New Biol. 2 3 1 , 2 4 6 - - 2 4 8 26 Augenlicht, L.H. and Lipkin, M. (1975) Biochem. Biophys. Res. Commun. 7 0 , 5 4 0 - - 5 4 4 27 Y a m a m o t o , K. and Alberts, B. (1975) Cell 4, 301--310