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Structural rearrangement of histone-H1-depleted chromatin during thermal denaturation
392
Biochimica et Biophysica Acta, 610 (1980) 392--399
© Elsevier/North-Holland Biomedical Press
BBA 99768 S T R U C T U R A L R E A R R A N G E M E N T OF HISTONE-H1-DEPLETED CHROMATIN DURING THERMAL DENATURATION
STEPHAN I. DIMITROV, IRINA R. TSANEVA, ILIYA G. PASHEV and GEORGE G. MARKOV * Institute of Molecular Biology, Bulgarian Academy of Sciences, 1113 Sofia (Bulgaria)
(Received June 6th, 1980) Key words: Chromatin; Denaturation; Psoralen; Nucleosome sliding; Histone HI; Nucleopro tein
Summary The influence of thermal denaturation on the nucleosomal structure of histone-Hl
Introduction
Histone dissociation and redistribution has always been considered in the interpretation of the melting profile of chromatin, b u t direct evidence for or * To whom correspondence
should be addressed.
393 against structural rearrangement of the nucleoprotein has not been presented so far. Some indications for histone migration could be found in the earlier works on chromatin melting [1--3]. The possibility was suggested that under certain conditions histones could dissociate from denatured DNA and bind doublestranded DNA, thus increasing its thermal stability [3]. Previous work from this laboratory, however, presented evidence against dissociation of histones during melting of chromatin [4]. Dissociation-reassociation is not the only mechanism for histone redistribution. The latter could result from lateral movement of core histones along DNA. Such 'sliding' of histones was reported during fragmentation of chromatin [5,6], precipitation with MnCI~ [6], ultracentrifugation in high salt [7], increased ionic strength alone [8] or increased ionic strength at 37°C [9], and binding of ethidium bromide [ 10]. It was recently shown with chromatin-DNA hybrid molecules that at 37°C and under ionic conditions presumed to be physiological core histones moved onto DNA while retaining the nucleosome structure [11]. The aim of this work was to investigate the nucleosomal structure of histoneHI-depleted chromatin during thermal denaturation. To this end, partially denatured histone-Hl-depleted chromatin was used, the DNA of which was cross-linked with psoralen to ensure a complete renaturation of DNA upon cooling. Evidence is presented which shows a reorganization of the repeating structure of the nucleoprotein. These data support our previous electron microscopic results [12] showing that nucleosomal histones slide along DNA during thermal denaturation at low ionic strength. Materials and Methods
Isolation of nuclei and chromatin. Rat liver nuclei were isolated by the method of Blobel and Potter [13]. The nuclear pellet was centrifuged through 1 M sucrose containing 10 mM Tris-HC1, pH 7, 10 mM MgC12 and 1 mM phenylmethylsulphonylfluoride. To isolate chromatin, the final nuclear pellet was suspended in a buffer solution of 10 mM Tris-HC1, pH 7.5, 0.34 M sucrose, 1 mM CaC12 and 1 mM phenylmethylsulphonylfluoride (DNA concentration 1.5--2 mg/ml). The suspension was incubated with micrococcal nuclease (Worthington, 0.3 unit per A~60 unit of DNA) for 30 min in an ice bath. EDTA was added to stop the reaction and, after centrifugation at 4500 × g for 5 min, the pellet was suspended in 0.25 mM EDTA, pH 8. Preparation of histone-Hl-depleted chromatin. Histone H1 was selectively removed from chromatin by treatment with the cation-exchange resin AG 50WX2 (Fluka, Switzerland) following the protocol of Thoma and Koller [ 14]. The resin was prepared as described elsewhere [15] and equilibrated with 50 mM sodium phosphate buffer, pH 7/300 mM NaC1. Equal volumes of chromatin and buffer solution of 100 mM phosphate/600 mM NaC1 were mixed under gentle stirring. The resin was then added to the chromatin solution (0.2 ml sedimented resin per 10 A260 units of chromatin) and, after stirring for 1 h the sample was centrifuged at 600 × g for 5 min. The supernatant was used after dialysis overnight against 0.25 mM EDTA, pH 8. Photochemical cross-linking of DNA with psoralen (4,5',8-trimethylpsoralen). Histone-Hl-depleted chromatin in 0.25 mM EDTA was adjusted to a con-
394 centration of 2 A260 units/ml and DNA was covalently cross-linked with psoralen as described elsewhere [16]. The sample was irradiated with long wavelength ultraviolet light for 10 min at a drug concentration of 10 #g/ml. The procedure was repeated and the u n b o u n d psoralen was removed by dialysis against 0.25 mM EDTA, pH 8. Thermal denaturation. The melting curves were recorded at 260-nm using a Unicam SP 1800 s p e c t r o p h o t o m e t e r adapted for melting studies in this laboratory. The integral melting curves were differentiated numerically for intervals of 4°C. Gel electrophoresis of DNA fragments. DNA from the nuclease-digested nucleoprotein was isolated by pronase treatment and chromatography on hydroxyapatite [17]. Electrophoresis of DNA fragments was performed in 2% agarose slab gels. Ethidium bromide was used to stain the gels. Results The nucleoprotein used in our experiments was histone-Hl-depleted chromatin with psoralen cross-linked DNA. The m e t h o d employed for the selective dis-
start
H3 H2b t--12a ta4
a
b
a
b
~¢
Fig. 1. 15% p o l y a c r y l a m i d e / 0 . 1 % s o d i u m d o d e c y l s u l f a t e slab gel e l e c t r o p h o r e s i s of h i s t o n e s i s o l a t e d f r o m r a t liver h i s t o n e - H l - d e p l e t e d c h r o m a t i n (a) a n d f r o m r a t liver n u c l e i (b). Slot a was o v e r l o a d e d to s h o w t h a t h i s t o n e H1 w a s c o m p l e t e l y r e m o v e d a n d t h a t t h e c o r e histories w e r e p r e s e n t in e q u i m o l a r a m o u n t s . Fig. 2. A g a r o s e slab gel e l e c t r o p h o r e s i s o f D N A f r a g m e n t s o b t a i n e d u p o n d i g e s t i o n w i t h m i c r o c o c c a l n u c l e a s e o f r a t liver n u c l e i (a), h i s t o n e - H l - d e p l e t e d c h r o m a t i n (b), a n d p s o r a l e n - t r e a t e d h i s t o n e - H 1 d e p l e t e d c h r o m a t i n (c).
395
11 60
1
^
,,,,""' •
5'0
100 Time (rain)
160
/
//, 40
50
60 70 8'0 eb Temperature (°C)
Fig. 3. K i n e t i c s o f d i g e s t i o n w i t h m i c r o c o c c a l n u c l e a s e o f d i f f e r e n t p r e p a r a t i o n s o f h i s t o n e - H l - d e p l e t e d c h r o m a t i n : ' n a t i v e ' n u c l e o p r o t e i n ( o ) ; p s o r a l e n - t r e a t e d n u c l e o p r o t e i n (o); p s o r a l e n - t r e a t e d n u c l e o p r o t e i n , p r e h e a t e d a t 7 2 (~), 8 2 (A) a n d 9 8 ° C ( × ) . D i g e s t i o n w a s carried o u t a t 3 7 ° C in the p r e s e n c e o f 1 m M C a C I 2 / 1 0 m M Tris-HCI, p H 7 . 6 , u s i n g f o u r e n z y m e u n i t s ( W o r t h i n g t o n , N F C P ) p e r A 2 6 0 u n i t o f n u c l e o protein. Fig. 4. N o r m a l i z e d d e r i v a t i v e m e l t i n g c u r v e s o f p s o r a l e n - t r e a t e d h i s t o n e - H l - d e p l e t e d non-heated; • p r e h e a t e d a t 8 2 o C.
chromatin: ......
,
sociation of histone H1 from chromatin was efficient in removing this histone (Fig. 1) while preserving the DNA repeat length of the chromatin fragments (Fig. 2). Cross-linking of DNA with psoralen did not change the size of the DNA repeat either (Fig. 2) and ensured a complete renaturation of DNA after thermal denaturation of the nucleoprotein. Partial thermal denaturation and renaturation o f psoralen-treated histoneHI-depleted chromatin. Kinetics o f digestion with micrococcal nuclease. Nucleoprotein preparations were heated at low ionic strength (0.25 mM EDTA, pH 8) at selected temperatures, chilled on ice, and aliquots were digested with micrococcal nuclease for various times. The kinetics of digestion is shown in Fig. 3. In agreement with previous data [16], DNA in the psoralen-treated nucleoprotein was degraded at a reduced rate as compared to the non-treated histone-HI-depleted chromatin. When the cross-linked material was heated at 72, 82 and 98°C, the rate of hydrolysis of DNA increased with increasing the temperature at which the sample had been preheated. It should be noted, however, that the quantity of DNA accessible to the nuclease remained unchanged: a plateau level was always observed, showing that no more than 60% of DNA was rendered acid-soluble, a figure found for the 'native' histone-Hl-depleted chromatin. Melting curves o f psoralen treated histone-Hl-depleted chromatin after partial thermal denaturation and renaturation. Fig. 4 shows the melting curve of psoralen-treated nucleoprotein preheated at 82°C and renatured. The profile is markedly different from that of the non-heated material: a, the second transition at 72°C disappeared and the DNA hyperchromicity lost in this peak was
396
a
b
c
d
a
b
c
Fig. 5. A g a r o s e s l a b gel e l e c t r o p h o r e s l s o f D N A f r a g m e n t s o b t a i n e d u p o n d i g e s t i o n w i t h m i c r o c o c c a l n u clease o f p s o r a l e n - t r e a t e d h i s t o n e - H l - d e p l e t e d c h r o m a t i n , p r e h e a t e d a t 7 2 ° C . D i g e s t i o n waS c a r r i e d o u t a t 3 7 ° C ( t w o e n z y m e u n i t s p e r A 2 6 0 u n i t o f n u c l e o p r o t e i n ) f o r 8, 2 5 , a n d 3 5 r a i n (slots b, c, a n d d, respectively). S l o t a is a m i c r o c o c c a i n u c l e a S e d i g e s t o f r a t liver n u c l e i u s e d as f r a g m e n t size m a r k e r s . T h e n u m b e r s (in b a s e p a i r s ) o n t h e l e f t side r e f e r t o t h e size o f t h e m a r k e r b a n d s . T h e l e n g t h o f t h e s e m a r k e r s w a s d e t e r m i n e d u s i n g d i g e s t s o f m o u s e s a t e l l i t e D N A w i t h E c o R I I a n d ~bX 1 7 4 D N A w i t h H a e I I I . T h e n u m b e r s (in b a s e p a i r s ) o n t h e r i g h t s i d e r e f e r t o t h e size o f D N A f r a g m e n t s d i s c u s s e d i n t h e t e x t . Fig. 6. A g a r o s e s l a b gel e l e c t r o p h ' o r e s i s o f D N A f r a g m e n t s o b t a i n e d u p o n d i g e s t i o n o f h i s t o n e - H l - d e p l e t e d c h r o m a t i n w i t h m i c r o c o c c a l n u c l e a s e ( f o u r e n z y m e u n i t s p e r A 2 6 0 u n i t o f n u c l e o p r o t e i n ) f o r 5, 2 0 , a n d 3 5 m i n (slots a, b , a n d c, r e s p e c t i v e l y ) .
gained in the first transition; and b, the first transition was shifted by 7--8°C to a lower temperature. The shifting was more expressed when the preheating was carried out at higher temperature. These changes clearly show that when the nucleoprotein had been denatured at a given temperature and renatured, the portion of DNA which melted as unprotected DNA increased. In our case, the melting temperature of the first transition was a b o u t 50°C, while at the same ionic strength (0.25 mM EDTA) the purified DNA melted at a b o u t 47°C (not shown). One should remember, however, that DNA in the psoralen-treated material is cross-linked, e.g. its melting temperature is higher than that of the noncross-linked DNA [ 18]. DNA fragment pattern generated upon micrococcal nuelease digestion o f partially denatured and renatured histone-Hl-depleted chrornatin. The structure of the psoralen-treated nucleoprotein after partial thermal denaturation
397 was also studied by sizing the DNA fragments obtained upon digestion with micrococcal nuclease. The DNA electrophoretic profiles are shown in Fig. 5. At very early times of digestion the repeating pattern was clearly seen but the bands were broader and the background increased. At late times of digestion the very high background obscured the bands. Nevertheless, two bands were observed in the broad dimer zone: one of 360--370 base pairs, corresponding to the dimer length of the control nuclear digest, and another band of shorter fragment length (about 290--300 base pairs). The appearance of the 'tight' dimer was obviously due to the preheating of the nucleoprotein since the dimer length of the non-heated material did not show any changes under the conditions of prolonged digestion with micrococcal nuclease (Fig. 6). Discussion
The influence of thermal denaturation on the nucleosomal structure of histone-Hi-depleted chromatin was studied with partially denatured nucleoprotein preparations in which the complete reannealing of DNA upon cooling was ensured by preliminary cross-linking with psoralen. Complete renaturation of DNA was an obligatory prerequisite of our experimental approach since the presence of single-stranded DNA would seriously affect both the thermal denaturation profile of the nucleohistone and its sensitivity to nucleases. Under our conditions of psoralen treatment (see Materials and Methods), the renaturation of DNA was always more than 98% as estimated by the regaining of the original absorbance at 260 nm and by the $1 nuclease assay. Therefore, in this paper, by partially denatured histone-Hl-depleted chromatin one should understand histone-Hl-depleted chromatin which had undergone denaturation at a certain temperature but in which the double-strandedness of DNA had been fully recovered after renaturation. The electron microscopic picture of partially denatured histone-Hl-depleted chromatin shows definite structural changes during denaturation [ 1 2 ] - - l o n g stretches of nucleosome-free DNA filaments together with closely packed material. As already discussed, the obvious explanation is that sliding of nucleosomes takes place during melting, but the electron microscopic evidence could also be interpreted as unfolding of nucleosomes in some regions of the nucleoprotein and aggregation of material in others, both due to DNA and protein denaturation. We present here additional evidence in order to obtain a clear explanation of the structural changes in chromatin during melting. The kinetics of digestion with micrococcal nuclease of partially denatured histone-Hl-depleted chromatin (Fig. 3) shows that a, the rate of DNA degradation increases with increasing the temperature at which the nucleohistone has been denatured; and b, the hydrolysis of DNA is restricted to the same extent as in the 'native' histone-Hl-depleted chromatin -- about 60% of DNA was rendered acid-soluble. This finding supports previous data that no dissociation of histones takes place during denaturation [4]. The increased rate of DNA degradation; however, cannot be interpreted unequivocally. It could reflect a lateral movement ('sliding') of the histone octamers along DNA which would generate long regions of histone-free DNA. Another possibility is that the denaturation of histones affects their interaction with DNA, thus rendering DNA more accessible to the enzyme.
398 The thermal denaturation profile of the partially denatured histone-H1depleted chromatin is more informative in this respect. The first transition in the melting profile of histone-Hl-depleted chromatin (at about 52°C under out experimental conditions} is now ascribed to the melting of unprotected DNA [ 19]. The melting temperature of this transition, however, is higher than that of the protein-free DNA (about 47°C in 0.25 mM EDTA, pH 8) because the DNA helix is 'clamped' by the bound histones (h-h state, see Ref. 20). In the psoralen-treated material, the first transition is further shifted to a higher temperature (about 56°C) due to the cross-links introduced in DNA [18]. When such material was heated to a temperature above 68--70°C and its DNA allowed to renature, the profile of redenaturation (Fig. 4} showed that the unprotected DNA melted at the temperature of the protein-free DNA (in our experiments this temperature was 50°C instead of 47°C because DNA was cross-linked}. This finding cannot be attributed to the less efficient protection by the denatured histones only. Its explanation requires long regions of histone-free DNA as a result of the rearrangement of nucleosomes along the DNA fiber. Electron microscopic evidence has been obtained in support of this explanation. When partially denatured DNA in histone-Hl-depleted chromatin was fixed with glyoxal at elevated temperatures and spread for electron microscopy after pronase treatment, long regions of denatured DNA could be observed in the electron micrographs which exceeded, by far, the length of the internucleosomal linker (Tsaneva, I.R. and Tsanev, R., unpublished data}. Further evidence for the nature of the structural changes observed in histone-HI-depleted chromatin during thermal denaturation comes from the modified DNA repeat length after nuclease digestion of preheated nucleoprotein preparations. As Fig. 5 shows, a shortened repeat length of about 145--150 base pairs appeared at late times of digestion, superimposed on a very high background. The shorter DNA repeat length is well demonstrated with the dimer where two bands could be recognized: a shorter dimer of about 290 base pairs and a dimer of virtually normal length (about 360--370 base pairs). These results agree well with the recent reports on the rearrangement of nucleosomes in nucleoprotein preparations exposed to increased ionic strength alone [ 8], or increased ionic strength at 37°C [9], where the shortening of the DNA repeat length was used as a convincing evidence for a packaging of nucleosomes as a results of lateral movement of histone cores along DNA. In summary, several kinds of experiments with partially denatured histoneHI-depleted chromatin show that nucleosomes slide along DNA during thermal denaturation: a, electron microscopic observations of long stretches of nucleosome-free DNA alternating with clustered nucleosomes; b, increased rate of hydrolysis of DNA with micrococcal nuclease without change in the extent of degradation; c, part of DNA melts as a protein-free DNA; and d, shortening of the DNA repeat length upon nuclease digestion. At higher temperatures, the process of sliding is parallelled by protein denaturation. This results in more profound changes in the chromatin structure and accounts for the loss of nucleosome morphology at temperatures higher than 65°C, the high background of the DNA digestion pattern, and the loss of the transition at 72°C in the meltting profile of the preheated histone-Hl-depleted chromatin.
399
References 1 0 h l e n b u s c h , H.H., Olivera, B.M., T u a n , D. a n d D a v i d s o n , N. ( 1 9 6 7 ) J. Mol. Biol. 25, 2 9 9 - - 3 1 5 2 H e n s o n , P. a n d Walker, I.O. ( 1 9 7 0 ) Eur. J. B i o c h e m . 16, 5 2 4 - - 5 3 1 3 Subirana, J. ( 1 9 7 3 ) J. Mol. Biol. 74, 3 6 3 - - 3 8 6 4 Tsaneva, I. a n d Tsanev, R. ( 1 9 7 6 ) C o m p t . R e n d . A c a d . Bulg. Sci. 29, 1 0 4 7 - - 1 0 5 0 5 D o e n e c k e , D. a n d M c C a r t h y , B.J. ( 1 9 7 6 ) Eu.r.J. B i o c h e m . 64, 4 0 5 - - 4 0 9 6 Bloch, D.P. a n d Cedar, H. ( 1 9 7 6 ) Nucleic Acids Res. 3, 1 5 0 7 - - 1 5 1 9 7 S t e i n m e t z , M., Stzeeck, R. a n d Z a c h a u , H.G. ( 1 9 7 8 ) Eur. J. B i o c h e m . 8 3 , 6 1 5 - - 6 2 8 8 Weischet, W. ( 1 9 7 9 ) Nucleic A c i d s Res. 7, 2 9 1 - - 3 0 4 9 S p a d a f o r a , C., Oudet, P. a n d C h a m b o n , P. ( 1 9 7 9 ) Eur. J. B i o c h e m . 1 0 0 , 2 2 5 - - 2 3 5 10 E r a r d , M., Das, G.C., de Murcia, G., Mazen, A., P o u y e t , J., C h a m p a g n e , M. a n d D a u n e , M. ( 1 9 7 9 ) Nucleic Acids Res. 6, 3 2 3 1 - - 3 2 5 3 11 Beard, P. ( 1 9 7 8 ) Cell 15, 9 5 5 - - 9 6 7 12 Tsaneva, I., D i m i t r o v , S., Pashev, I. a n d Tsanev, R. ( 1 9 8 0 ) FEBS Lett. 1 1 2 , 1 4 3 - - 1 4 6 13 Blobel, G. a n d P o t t e r , V.R. ( 1 9 6 6 ) Science 1 5 4 , 1 6 6 2 - - 1 6 6 5 1 4 T h o m a , F. a n d KoUer, T. ( 1 9 7 7 ) Cell 1 2 , 1 0 1 - - 1 0 7 15 B o l u n d , E.W. a n d J o h n , C. ( 1 9 7 3 ) Eu~. J. B i o c h e m . 35, 5 4 6 - - 5 5 3 16 Cech, T. a n d P a r d u e , M.-L. ( 1 9 7 7 ) Cell 1 1 , 6 3 1 - - 6 4 0 17 Pashev, I.G., N e n c h e v a , M.M. a n d Markov, G.G. ( 1 9 7 8 ) Mol. Biol. Rep. 4, 1 4 3 - - 1 4 7 18 Chipev, C.C., D i m i t r o v , S. a n d S t a y n o v , D.Z. ( 1 9 8 0 ) Int. J. B i o c h e m . , in the press 19 Li, H.J. a n d B o n n e r , J. ( 1 9 7 1 ) B i o c h e m i s t r y 10, 1 4 6 1 - - 1 4 7 2 20 S t a y n o v , D.Z. ( 1 9 7 6 ) N a t u r e 2 6 4 , 5 2 2 - - 5 2 5
Biochimica et Biophysica Acta, 610 (1980) 392--399
© Elsevier/North-Holland Biomedical Press
BBA 99768 S T R U C T U R A L R E A R R A N G E M E N T OF HISTONE-H1-DEPLETED CHROMATIN DURING THERMAL DENATURATION
STEPHAN I. DIMITROV, IRINA R. TSANEVA, ILIYA G. PASHEV and GEORGE G. MARKOV * Institute of Molecular Biology, Bulgarian Academy of Sciences, 1113 Sofia (Bulgaria)
(Received June 6th, 1980) Key words: Chromatin; Denaturation; Psoralen; Nucleosome sliding; Histone HI; Nucleopro tein
Summary The influence of thermal denaturation on the nucleosomal structure of histone-Hl
Introduction
Histone dissociation and redistribution has always been considered in the interpretation of the melting profile of chromatin, b u t direct evidence for or * To whom correspondence
should be addressed.
393 against structural rearrangement of the nucleoprotein has not been presented so far. Some indications for histone migration could be found in the earlier works on chromatin melting [1--3]. The possibility was suggested that under certain conditions histones could dissociate from denatured DNA and bind doublestranded DNA, thus increasing its thermal stability [3]. Previous work from this laboratory, however, presented evidence against dissociation of histones during melting of chromatin [4]. Dissociation-reassociation is not the only mechanism for histone redistribution. The latter could result from lateral movement of core histones along DNA. Such 'sliding' of histones was reported during fragmentation of chromatin [5,6], precipitation with MnCI~ [6], ultracentrifugation in high salt [7], increased ionic strength alone [8] or increased ionic strength at 37°C [9], and binding of ethidium bromide [ 10]. It was recently shown with chromatin-DNA hybrid molecules that at 37°C and under ionic conditions presumed to be physiological core histones moved onto DNA while retaining the nucleosome structure [11]. The aim of this work was to investigate the nucleosomal structure of histoneHI-depleted chromatin during thermal denaturation. To this end, partially denatured histone-Hl-depleted chromatin was used, the DNA of which was cross-linked with psoralen to ensure a complete renaturation of DNA upon cooling. Evidence is presented which shows a reorganization of the repeating structure of the nucleoprotein. These data support our previous electron microscopic results [12] showing that nucleosomal histones slide along DNA during thermal denaturation at low ionic strength. Materials and Methods
Isolation of nuclei and chromatin. Rat liver nuclei were isolated by the method of Blobel and Potter [13]. The nuclear pellet was centrifuged through 1 M sucrose containing 10 mM Tris-HC1, pH 7, 10 mM MgC12 and 1 mM phenylmethylsulphonylfluoride. To isolate chromatin, the final nuclear pellet was suspended in a buffer solution of 10 mM Tris-HC1, pH 7.5, 0.34 M sucrose, 1 mM CaC12 and 1 mM phenylmethylsulphonylfluoride (DNA concentration 1.5--2 mg/ml). The suspension was incubated with micrococcal nuclease (Worthington, 0.3 unit per A~60 unit of DNA) for 30 min in an ice bath. EDTA was added to stop the reaction and, after centrifugation at 4500 × g for 5 min, the pellet was suspended in 0.25 mM EDTA, pH 8. Preparation of histone-Hl-depleted chromatin. Histone H1 was selectively removed from chromatin by treatment with the cation-exchange resin AG 50WX2 (Fluka, Switzerland) following the protocol of Thoma and Koller [ 14]. The resin was prepared as described elsewhere [15] and equilibrated with 50 mM sodium phosphate buffer, pH 7/300 mM NaC1. Equal volumes of chromatin and buffer solution of 100 mM phosphate/600 mM NaC1 were mixed under gentle stirring. The resin was then added to the chromatin solution (0.2 ml sedimented resin per 10 A260 units of chromatin) and, after stirring for 1 h the sample was centrifuged at 600 × g for 5 min. The supernatant was used after dialysis overnight against 0.25 mM EDTA, pH 8. Photochemical cross-linking of DNA with psoralen (4,5',8-trimethylpsoralen). Histone-Hl-depleted chromatin in 0.25 mM EDTA was adjusted to a con-
394 centration of 2 A260 units/ml and DNA was covalently cross-linked with psoralen as described elsewhere [16]. The sample was irradiated with long wavelength ultraviolet light for 10 min at a drug concentration of 10 #g/ml. The procedure was repeated and the u n b o u n d psoralen was removed by dialysis against 0.25 mM EDTA, pH 8. Thermal denaturation. The melting curves were recorded at 260-nm using a Unicam SP 1800 s p e c t r o p h o t o m e t e r adapted for melting studies in this laboratory. The integral melting curves were differentiated numerically for intervals of 4°C. Gel electrophoresis of DNA fragments. DNA from the nuclease-digested nucleoprotein was isolated by pronase treatment and chromatography on hydroxyapatite [17]. Electrophoresis of DNA fragments was performed in 2% agarose slab gels. Ethidium bromide was used to stain the gels. Results The nucleoprotein used in our experiments was histone-Hl-depleted chromatin with psoralen cross-linked DNA. The m e t h o d employed for the selective dis-
start
H3 H2b t--12a ta4
a
b
a
b
~¢
Fig. 1. 15% p o l y a c r y l a m i d e / 0 . 1 % s o d i u m d o d e c y l s u l f a t e slab gel e l e c t r o p h o r e s i s of h i s t o n e s i s o l a t e d f r o m r a t liver h i s t o n e - H l - d e p l e t e d c h r o m a t i n (a) a n d f r o m r a t liver n u c l e i (b). Slot a was o v e r l o a d e d to s h o w t h a t h i s t o n e H1 w a s c o m p l e t e l y r e m o v e d a n d t h a t t h e c o r e histories w e r e p r e s e n t in e q u i m o l a r a m o u n t s . Fig. 2. A g a r o s e slab gel e l e c t r o p h o r e s i s o f D N A f r a g m e n t s o b t a i n e d u p o n d i g e s t i o n w i t h m i c r o c o c c a l n u c l e a s e o f r a t liver n u c l e i (a), h i s t o n e - H l - d e p l e t e d c h r o m a t i n (b), a n d p s o r a l e n - t r e a t e d h i s t o n e - H 1 d e p l e t e d c h r o m a t i n (c).
395
11 60
1
^
,,,,""' •
5'0
100 Time (rain)
160
/
//, 40
50
60 70 8'0 eb Temperature (°C)
Fig. 3. K i n e t i c s o f d i g e s t i o n w i t h m i c r o c o c c a l n u c l e a s e o f d i f f e r e n t p r e p a r a t i o n s o f h i s t o n e - H l - d e p l e t e d c h r o m a t i n : ' n a t i v e ' n u c l e o p r o t e i n ( o ) ; p s o r a l e n - t r e a t e d n u c l e o p r o t e i n (o); p s o r a l e n - t r e a t e d n u c l e o p r o t e i n , p r e h e a t e d a t 7 2 (~), 8 2 (A) a n d 9 8 ° C ( × ) . D i g e s t i o n w a s carried o u t a t 3 7 ° C in the p r e s e n c e o f 1 m M C a C I 2 / 1 0 m M Tris-HCI, p H 7 . 6 , u s i n g f o u r e n z y m e u n i t s ( W o r t h i n g t o n , N F C P ) p e r A 2 6 0 u n i t o f n u c l e o protein. Fig. 4. N o r m a l i z e d d e r i v a t i v e m e l t i n g c u r v e s o f p s o r a l e n - t r e a t e d h i s t o n e - H l - d e p l e t e d non-heated; • p r e h e a t e d a t 8 2 o C.
chromatin: ......
,
sociation of histone H1 from chromatin was efficient in removing this histone (Fig. 1) while preserving the DNA repeat length of the chromatin fragments (Fig. 2). Cross-linking of DNA with psoralen did not change the size of the DNA repeat either (Fig. 2) and ensured a complete renaturation of DNA after thermal denaturation of the nucleoprotein. Partial thermal denaturation and renaturation o f psoralen-treated histoneHI-depleted chromatin. Kinetics o f digestion with micrococcal nuclease. Nucleoprotein preparations were heated at low ionic strength (0.25 mM EDTA, pH 8) at selected temperatures, chilled on ice, and aliquots were digested with micrococcal nuclease for various times. The kinetics of digestion is shown in Fig. 3. In agreement with previous data [16], DNA in the psoralen-treated nucleoprotein was degraded at a reduced rate as compared to the non-treated histone-HI-depleted chromatin. When the cross-linked material was heated at 72, 82 and 98°C, the rate of hydrolysis of DNA increased with increasing the temperature at which the sample had been preheated. It should be noted, however, that the quantity of DNA accessible to the nuclease remained unchanged: a plateau level was always observed, showing that no more than 60% of DNA was rendered acid-soluble, a figure found for the 'native' histone-Hl-depleted chromatin. Melting curves o f psoralen treated histone-Hl-depleted chromatin after partial thermal denaturation and renaturation. Fig. 4 shows the melting curve of psoralen-treated nucleoprotein preheated at 82°C and renatured. The profile is markedly different from that of the non-heated material: a, the second transition at 72°C disappeared and the DNA hyperchromicity lost in this peak was
396
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Fig. 5. A g a r o s e s l a b gel e l e c t r o p h o r e s l s o f D N A f r a g m e n t s o b t a i n e d u p o n d i g e s t i o n w i t h m i c r o c o c c a l n u clease o f p s o r a l e n - t r e a t e d h i s t o n e - H l - d e p l e t e d c h r o m a t i n , p r e h e a t e d a t 7 2 ° C . D i g e s t i o n waS c a r r i e d o u t a t 3 7 ° C ( t w o e n z y m e u n i t s p e r A 2 6 0 u n i t o f n u c l e o p r o t e i n ) f o r 8, 2 5 , a n d 3 5 r a i n (slots b, c, a n d d, respectively). S l o t a is a m i c r o c o c c a i n u c l e a S e d i g e s t o f r a t liver n u c l e i u s e d as f r a g m e n t size m a r k e r s . T h e n u m b e r s (in b a s e p a i r s ) o n t h e l e f t side r e f e r t o t h e size o f t h e m a r k e r b a n d s . T h e l e n g t h o f t h e s e m a r k e r s w a s d e t e r m i n e d u s i n g d i g e s t s o f m o u s e s a t e l l i t e D N A w i t h E c o R I I a n d ~bX 1 7 4 D N A w i t h H a e I I I . T h e n u m b e r s (in b a s e p a i r s ) o n t h e r i g h t s i d e r e f e r t o t h e size o f D N A f r a g m e n t s d i s c u s s e d i n t h e t e x t . Fig. 6. A g a r o s e s l a b gel e l e c t r o p h ' o r e s i s o f D N A f r a g m e n t s o b t a i n e d u p o n d i g e s t i o n o f h i s t o n e - H l - d e p l e t e d c h r o m a t i n w i t h m i c r o c o c c a l n u c l e a s e ( f o u r e n z y m e u n i t s p e r A 2 6 0 u n i t o f n u c l e o p r o t e i n ) f o r 5, 2 0 , a n d 3 5 m i n (slots a, b , a n d c, r e s p e c t i v e l y ) .
gained in the first transition; and b, the first transition was shifted by 7--8°C to a lower temperature. The shifting was more expressed when the preheating was carried out at higher temperature. These changes clearly show that when the nucleoprotein had been denatured at a given temperature and renatured, the portion of DNA which melted as unprotected DNA increased. In our case, the melting temperature of the first transition was a b o u t 50°C, while at the same ionic strength (0.25 mM EDTA) the purified DNA melted at a b o u t 47°C (not shown). One should remember, however, that DNA in the psoralen-treated material is cross-linked, e.g. its melting temperature is higher than that of the noncross-linked DNA [ 18]. DNA fragment pattern generated upon micrococcal nuelease digestion o f partially denatured and renatured histone-Hl-depleted chrornatin. The structure of the psoralen-treated nucleoprotein after partial thermal denaturation
397 was also studied by sizing the DNA fragments obtained upon digestion with micrococcal nuclease. The DNA electrophoretic profiles are shown in Fig. 5. At very early times of digestion the repeating pattern was clearly seen but the bands were broader and the background increased. At late times of digestion the very high background obscured the bands. Nevertheless, two bands were observed in the broad dimer zone: one of 360--370 base pairs, corresponding to the dimer length of the control nuclear digest, and another band of shorter fragment length (about 290--300 base pairs). The appearance of the 'tight' dimer was obviously due to the preheating of the nucleoprotein since the dimer length of the non-heated material did not show any changes under the conditions of prolonged digestion with micrococcal nuclease (Fig. 6). Discussion
The influence of thermal denaturation on the nucleosomal structure of histone-Hi-depleted chromatin was studied with partially denatured nucleoprotein preparations in which the complete reannealing of DNA upon cooling was ensured by preliminary cross-linking with psoralen. Complete renaturation of DNA was an obligatory prerequisite of our experimental approach since the presence of single-stranded DNA would seriously affect both the thermal denaturation profile of the nucleohistone and its sensitivity to nucleases. Under our conditions of psoralen treatment (see Materials and Methods), the renaturation of DNA was always more than 98% as estimated by the regaining of the original absorbance at 260 nm and by the $1 nuclease assay. Therefore, in this paper, by partially denatured histone-Hl-depleted chromatin one should understand histone-Hl-depleted chromatin which had undergone denaturation at a certain temperature but in which the double-strandedness of DNA had been fully recovered after renaturation. The electron microscopic picture of partially denatured histone-Hl-depleted chromatin shows definite structural changes during denaturation [ 1 2 ] - - l o n g stretches of nucleosome-free DNA filaments together with closely packed material. As already discussed, the obvious explanation is that sliding of nucleosomes takes place during melting, but the electron microscopic evidence could also be interpreted as unfolding of nucleosomes in some regions of the nucleoprotein and aggregation of material in others, both due to DNA and protein denaturation. We present here additional evidence in order to obtain a clear explanation of the structural changes in chromatin during melting. The kinetics of digestion with micrococcal nuclease of partially denatured histone-Hl-depleted chromatin (Fig. 3) shows that a, the rate of DNA degradation increases with increasing the temperature at which the nucleohistone has been denatured; and b, the hydrolysis of DNA is restricted to the same extent as in the 'native' histone-Hl-depleted chromatin -- about 60% of DNA was rendered acid-soluble. This finding supports previous data that no dissociation of histones takes place during denaturation [4]. The increased rate of DNA degradation; however, cannot be interpreted unequivocally. It could reflect a lateral movement ('sliding') of the histone octamers along DNA which would generate long regions of histone-free DNA. Another possibility is that the denaturation of histones affects their interaction with DNA, thus rendering DNA more accessible to the enzyme.
398 The thermal denaturation profile of the partially denatured histone-H1depleted chromatin is more informative in this respect. The first transition in the melting profile of histone-Hl-depleted chromatin (at about 52°C under out experimental conditions} is now ascribed to the melting of unprotected DNA [ 19]. The melting temperature of this transition, however, is higher than that of the protein-free DNA (about 47°C in 0.25 mM EDTA, pH 8) because the DNA helix is 'clamped' by the bound histones (h-h state, see Ref. 20). In the psoralen-treated material, the first transition is further shifted to a higher temperature (about 56°C) due to the cross-links introduced in DNA [18]. When such material was heated to a temperature above 68--70°C and its DNA allowed to renature, the profile of redenaturation (Fig. 4} showed that the unprotected DNA melted at the temperature of the protein-free DNA (in our experiments this temperature was 50°C instead of 47°C because DNA was cross-linked}. This finding cannot be attributed to the less efficient protection by the denatured histones only. Its explanation requires long regions of histone-free DNA as a result of the rearrangement of nucleosomes along the DNA fiber. Electron microscopic evidence has been obtained in support of this explanation. When partially denatured DNA in histone-Hl-depleted chromatin was fixed with glyoxal at elevated temperatures and spread for electron microscopy after pronase treatment, long regions of denatured DNA could be observed in the electron micrographs which exceeded, by far, the length of the internucleosomal linker (Tsaneva, I.R. and Tsanev, R., unpublished data}. Further evidence for the nature of the structural changes observed in histone-HI-depleted chromatin during thermal denaturation comes from the modified DNA repeat length after nuclease digestion of preheated nucleoprotein preparations. As Fig. 5 shows, a shortened repeat length of about 145--150 base pairs appeared at late times of digestion, superimposed on a very high background. The shorter DNA repeat length is well demonstrated with the dimer where two bands could be recognized: a shorter dimer of about 290 base pairs and a dimer of virtually normal length (about 360--370 base pairs). These results agree well with the recent reports on the rearrangement of nucleosomes in nucleoprotein preparations exposed to increased ionic strength alone [ 8], or increased ionic strength at 37°C [9], where the shortening of the DNA repeat length was used as a convincing evidence for a packaging of nucleosomes as a results of lateral movement of histone cores along DNA. In summary, several kinds of experiments with partially denatured histoneHI-depleted chromatin show that nucleosomes slide along DNA during thermal denaturation: a, electron microscopic observations of long stretches of nucleosome-free DNA alternating with clustered nucleosomes; b, increased rate of hydrolysis of DNA with micrococcal nuclease without change in the extent of degradation; c, part of DNA melts as a protein-free DNA; and d, shortening of the DNA repeat length upon nuclease digestion. At higher temperatures, the process of sliding is parallelled by protein denaturation. This results in more profound changes in the chromatin structure and accounts for the loss of nucleosome morphology at temperatures higher than 65°C, the high background of the DNA digestion pattern, and the loss of the transition at 72°C in the meltting profile of the preheated histone-Hl-depleted chromatin.
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References 1 0 h l e n b u s c h , H.H., Olivera, B.M., T u a n , D. a n d D a v i d s o n , N. ( 1 9 6 7 ) J. Mol. Biol. 25, 2 9 9 - - 3 1 5 2 H e n s o n , P. a n d Walker, I.O. ( 1 9 7 0 ) Eur. J. B i o c h e m . 16, 5 2 4 - - 5 3 1 3 Subirana, J. ( 1 9 7 3 ) J. Mol. Biol. 74, 3 6 3 - - 3 8 6 4 Tsaneva, I. a n d Tsanev, R. ( 1 9 7 6 ) C o m p t . R e n d . A c a d . Bulg. Sci. 29, 1 0 4 7 - - 1 0 5 0 5 D o e n e c k e , D. a n d M c C a r t h y , B.J. ( 1 9 7 6 ) Eu.r.J. B i o c h e m . 64, 4 0 5 - - 4 0 9 6 Bloch, D.P. a n d Cedar, H. ( 1 9 7 6 ) Nucleic Acids Res. 3, 1 5 0 7 - - 1 5 1 9 7 S t e i n m e t z , M., Stzeeck, R. a n d Z a c h a u , H.G. ( 1 9 7 8 ) Eur. J. B i o c h e m . 8 3 , 6 1 5 - - 6 2 8 8 Weischet, W. ( 1 9 7 9 ) Nucleic A c i d s Res. 7, 2 9 1 - - 3 0 4 9 S p a d a f o r a , C., Oudet, P. a n d C h a m b o n , P. ( 1 9 7 9 ) Eur. J. B i o c h e m . 1 0 0 , 2 2 5 - - 2 3 5 10 E r a r d , M., Das, G.C., de Murcia, G., Mazen, A., P o u y e t , J., C h a m p a g n e , M. a n d D a u n e , M. ( 1 9 7 9 ) Nucleic Acids Res. 6, 3 2 3 1 - - 3 2 5 3 11 Beard, P. ( 1 9 7 8 ) Cell 15, 9 5 5 - - 9 6 7 12 Tsaneva, I., D i m i t r o v , S., Pashev, I. a n d Tsanev, R. ( 1 9 8 0 ) FEBS Lett. 1 1 2 , 1 4 3 - - 1 4 6 13 Blobel, G. a n d P o t t e r , V.R. ( 1 9 6 6 ) Science 1 5 4 , 1 6 6 2 - - 1 6 6 5 1 4 T h o m a , F. a n d KoUer, T. ( 1 9 7 7 ) Cell 1 2 , 1 0 1 - - 1 0 7 15 B o l u n d , E.W. a n d J o h n , C. ( 1 9 7 3 ) Eu~. J. B i o c h e m . 35, 5 4 6 - - 5 5 3 16 Cech, T. a n d P a r d u e , M.-L. ( 1 9 7 7 ) Cell 1 1 , 6 3 1 - - 6 4 0 17 Pashev, I.G., N e n c h e v a , M.M. a n d Markov, G.G. ( 1 9 7 8 ) Mol. Biol. Rep. 4, 1 4 3 - - 1 4 7 18 Chipev, C.C., D i m i t r o v , S. a n d S t a y n o v , D.Z. ( 1 9 8 0 ) Int. J. B i o c h e m . , in the press 19 Li, H.J. a n d B o n n e r , J. ( 1 9 7 1 ) B i o c h e m i s t r y 10, 1 4 6 1 - - 1 4 7 2 20 S t a y n o v , D.Z. ( 1 9 7 6 ) N a t u r e 2 6 4 , 5 2 2 - - 5 2 5