BIOCHIMIE, 1975, 57, 1307-1313.
Transcription of chromatin i om mouse fibroblasts. Claude R m s s ( ' ) , Michel Cn~HN, Denise PAULIN and F r a n c o i s Gnos ~.
l n s t i t u t Pasteur, D~partement de Biologie mol~culaire, Paris, (') I n s t i t u t du Radium, Orsay. (3-7-1975). Abstract. - - Chromatin purified from mouse fibroblasts can be fractionated after shearing by s.edimen:tation in a sucrose gradient into an extended > and a compact c heavy • component. Further purification of these cla.ssical~y described components van be achieved by a second cycle of centrifugation of the light and heavy compon~ents, through an equilibrium density gradient of metrizamide. The light component l~urificd from sucrose gradien, t set~iments faster (peak I) on metrizamide than its heavy counterpart (peak II). Template activity for DNA d.irected RNA synthesis in the presence of E. colt RNA polymerase is uegligib~e in peak l,I bat very pronounced in the peak I fracfio.n. The difference in template activity appears to be cormeeted with differences in propagation rat.her than initiation rates. Comparison of gel electrophoresis pa¢terns of proteins indicate that the active subcomponent includes high mol~ecular weight components not present in the inactive one, but that its histone conten~ is somewhat lower. Using a very highly sensitive automatic recording apparatus for the measurement of melting profiles, no clear cut difference has been observed in the behaviour of active and inactive chromatin subcomponents nor in, that of their total DNA.
INTRODUCTION. Interest has r e c e n t l y been focused on the mec h a n i s m s w h e r e b y n u c l e a r p r o t e i n s r e s t r i c t or i n d u c e gene e x p r e s s i o n in e u k a r y o t i ¢ cells, at the c h r o m a t i n level. E v i d e n c e has been p r e s e n t e d that the a c t i v i t y of t r a n s c r i p t i o n units is i n f l u e n c e d by the a v a i l a b i l i t y of active genetic sites to the RNA p o l y m e r a s e s w h i c h is g o v e r n e d , at least to some extent, by asso,ciation b e t w e e n the DNA and the c h r o m o s o m a l p r o t e i n s [1]. In t h e f r a m e of a long-range study of the c o r r e lation b e t w e e n the role of D NA as a template for RNA s y n t h e s i s a n d the nature of the p r o t e i n assortmen,t p r e s e n t on the c h r o m a t i n , w e h a v e f r a c t i o n a t e d , as a first step, the c h r o m a t i n f r o m t r a n s f o r m e d mouse fibrohlasts into ¢ ac¢ive )) and ¢ i n a c t i v e >> c o m p o n e n t s . As in the o t h e r systems [2], s h e a r e d c h r o m a t i n d e c o m p o s e s on s u c r o s e g r a d i e n t s into t w o f r a c t i o n s w h i c h e x h i b i t differ e n t template activities in d i r e c t i n g in vitro RNA synthesis. F u r t h e r p u r i f i c a t i o n can h o w e v e r be a c h i e v e d by a s e p a r a t i o n into a ¢light>> a n d f r a c t i o n t h r o u g h e q u i l i b r i u m density g r a d i e n t c e n t r i f u g a t i o n against a c o n c e n t r a t e d solution of m e t r i z a m i d e by a t e c h n i q u e w h i c h w e have a d a p t e d from R i e k w o o d , Hell and B i r n i e [3]. The p r e s e n t p a p e r r e p o r t s on the p r o p e r t i e s of these ¢ light ~ a n d ¢ h e a v y >> c h r o m a t i n f r a c t i o n s ~> To whom aH correspondence should be addressed.
w i t h r e g a r d to t h e i r t e m p l a t e a c t i v i t y in the p r e s e n c e of RNA p o l y m e r a s e , t h e i r p r o t e i n content and t e m p e r a t u r e d e p e n d e n t m e l t i n g c h a r a c teristics.
MATERIALS AND MIETHODS. C E L L LINES.
Our s t a r t i n g m a t e r i a l w ~ a cell line of fibroblaeds 3T3 t r a n s f o r m e d w i t h w i l d - t y p e p o l y o m a v i r u s as o b t a i n e d f r o m Dr. T. B e n j a m i n , w h i c h is r e f e r r e d to as PY6. T h e line c o n t a i n s t w o p o l y o m a - i n t e g r a t e d genomes in the c e l l u l a r DNA. It is c o m p l e t e l y p o l y o m a - v i r u s free, and does not p r o d u c e such virus d u r i n g c u l t i v a t i o n [4]. Cells w e r e g r o w n in D u l b e c c o [5] m o d i f i e d Eagle's m e d i u m , s u p p l e m e n t e d w i t h 10 p e r cent fetal calf serum. T h e ceil cultures w e r e t r e a t e d p e r i o d i c a l l y w i t h k a n a m y c i n an.d an anti-PPLO agent (Gibco catalog n ° 522), and c h e c k e d e v e r y 6 to 8 w e e k s for m y c o p l a s m a c o n t a m i n a t i o n [6]. PtAD IOACTIVE LAIKELLIN G.
T h e D u l b e c c o Eagle m e d i u m was d i l u t e d t w o fold w i t h E a r l e solution (Institut Pasteur, c a t a l o g n ° 72.473) and sup~plemented w i t h a m i x t u r e of 15 a m i n o acids labelled w i t h 14C at 1 p~Ci p e r ml (C.E.A., F r a n c e , spe,cific radioaeAivity 100 m C i / mM). S u b c o n f l u e n t cultures w e r e g r o w n in this
1308
C. Reiss, M. Crdpin, D. Paulin a n d F. Gros.
m e d i u m for r a d i o a c t i v e labelling a n d harvested after 20 h. GEL ELECTROPHORESIS. The p r o t e i n s in the c h r o m a t i n fractions were analyzed b y gel electrophoresis. Each fraction was dissociated by h e a t i n g at 56°C for 10 ran, i n the p r e s e n c e of 2 p e r cen~ SDS a n d 1 per cent ~-mercaptoethanol. Electrophoresis proceeded for 4 hrs at 4,0 nlA through 1 p e r cent SDS, 15 per cent p o l y a c r y l a m i d slab gels a c c o r d i n g to the method of L a e m m l i [7] a n d Studier [8]. ISOLATION OF CHROMATIN. Samples of 109 cells were w a s h e d three times with the following buffer : 1.5 mM MgCa2, 140 mM NaC1 and 2.0 mM Tris HCI at pH 6.5. Cytoplasmic m e m b r a n e s were disrupted, as m o n i t o r e d by phase c o n t r a s t microscopy, after a d d i n g 0.5 per cent N o n i d e t (NP40) dissolved i n the same buffer. The nuclei were separated out b y centrifugation at 900 g for 10 m n at 4°C. The n u c l e a r pellet was w a s h e d twice with 75 mM NaCI a n d 25 mM EDTA, a n d then t r a n s f e r r e d into 10 ml of a 5 mM Tris s o l u t i o n at pH 6.5. The nuclei were then disrupted in a Potter h o m o g e n i z e r (20 strokes) a n d the solution centrifuged for 29 m n at 104 g. After two a d d i t i o n a l h o m o g e n i z a t i o n a n d centrifugation cycles, the final c h r o m a t i n pellet was s u s p e n d e d i n 8 ml Tris pH 8.5. TRANSCRIPTION ASSAY.
The s t a n d a r d assay m i x t u r e (0,25 ml) contain e d : 50 mM Tris H,CI pH 7.9; 10 m ~ MgCI2 ; 300 mM KC1 ; 0.4 mM CTP, ATP, G T P ; 0.1 mM (~H) U T P (specific r a d i o a c t i v i t y 20 Ci/M) a n d c h r o m a t i n at a c o n c e n t r a t i o n such that the optical d e n s i t y at 260 n m was 0.1. The reaction was started by a d d i n g 10 u n i t s of E. coli RNA polymerase. I n c u b a t i o n was c a r r i e d out at 37°C for the times specified in the legends of the figures. Acid-insoluble r a d i o a c t i v i t y was collected on a nitrocellulose filter a n d d e t e r m i n e d by a scintillation counter. To m e a s u r e the i n i t i a t i o n of RNA synthesis 73~p GTP or 7:~2p ATP was used (specific r a d i o a c t i v i t y 20,0.0 mCi/mM). In order to reduce the b a c k g r o u n d of the u n s p e c i f i c 32p i n c o r p o r a t i o n i n acid insoluble m a t e r i a l nitrocellulose filters were washed w i t h a TCA solution c o n t a i n i n g s o d i u m phosphate 0.65 M ; p y r o p h o s p h a t e , 0.02.5 M. Radioactivity b a c k g r o u n d was substracted. THERMAL DENATURATION EXPERIMENTS.
The m e l t i n g b e h a v i o r was m o n i t o r e d be measur i n g the optical density (OD) at 26,0 m n for DNA,
BIOCHIMIE, 1975, 57, n ° 11-12.
a n d at 260 n m and 32~0 n m for c h r o m a t i n w i t h a Zeiss PMQII spectrophotometer. The samples were heated in rooter-jacketed quartz cells (Hellma, model 160 QS). The t e m p e r a t u r e was kept constant to w i t h i n + 10 -2 C by a L a n d a u l t r a t h e r m o s t a t model S 8. Melting curves were o b t a i n e d t h r o u g h a temperature-time program, i n w h i c h the temperature is increased stepwise a n d kept c o n s t a n t u n t i l thermal a n d phase e q u i l i b r i a are reached, before the data are recorded automatically. As discussed elsewhere [9], this p r o c e d u r e is an imp r o v e m e n t over s t a n d a r d methods, especially with regard to the r e p r o d u c i b i l i t y a n d details of the m e l t i n g process. The fine structure i n the melting curves is emphasized by plotting the data differentially ( d O D / d T vs. T) on a c o m p u t e r - p e r i p h e ral Benson III tracer after smoothing a n d differ e n t i a t i n g the r a w data on a U n i v a c 1110 computer (Centre de calcul, Universit6 Paris-Sud, Orsay). RESULTS AND DISCUSSION. The s u s p e n s i o n of c h r o m a t i n , labelled i n its p r o t e i n moiety by [14C] amino acids, was treated for 4 m n at 4°C in a Virtis apparatus with a p o w e r setting at 40 W. The sheared c h r o m a t i n was then layered on a 5 - 40 p e r cent stLcrose gradient and centrifuged for 14 h at 10~ g. T w e n t y five fractions were collected. The optical density and r a d i o a c t i v i t y were m e a s u r e d on each. The s e d i m e n t a t i o n profile shown in figure 1 exhibits two peaks of c o m p a r a b l e m a g n i t u d e corr e s p o n d i n g to a heavy (H) r a p i d l y s e d i m e n t i n g and a light (L) slowly s e d i m e n t i n g c o m p o n e n t s of chromatin. The s e p a r a t i o n is a p p a r e n t i n the optical density at 2'8,0 n m (upper graph) and in the r a d i o a c t i v i t y of the p r o t e i n s (lower graph). A good c o i n c i d e n c e was observed between the OD and radioactivity d i s t r i b u t i o n profiles. Similar profiles were observed u n d e r analogous conditions by Mc Carthy el aI. for c h r o m a t i n from a n entirely different source, viz., from Drosophila melanogaster embryo cells [10]. The light fractions 11 to 15 and the heavy fractions 17 to 23 were c o m b i n e d into a c o m p o n e n t L and a c o m p o n e n t H, respectively, dialyzed separately against 0.1 SSC buffer, and c o n c e n t r a t e d by centrifugation for 20 h at 10~ g. The eL>> a n d (
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FIG. I. - - Two components are resolved in sheared chromatin, after sedimentation, in the sucrose density gradient, by their radioactiuity and optical density at 280 nm. Fractions. 11 to 15 and 17 to 23 are combined., respectively, into components L and H for the second fractionation shown in Figure 2.
n i n g 60 p e r cent give rise to a c l e a r l y r e s o l v e d p e a k IT at 1.20 g/cma. U n d e r such c o n d i t i o n s a l o w e r d e n s i t y p e a k (1.18 g/cm~) a p p e a r s in b o t h e h r o m a t i n s in eqttaI amount. This m a t e r i a l m i g h t be n u c l e o p r o t e i n s w i t h a h i g h n u c l e i c a c i d : p r o tein ratio i s o l a t e d after s h e a r i n g of the c h r o m a t i n . P a r t s I a n d II w e r e collected s e p a r a t e l y a n d dialyze,d a g a i n s t 0.1 SSJC buffer. S e p a r a t i o n of the sucrose gradient purified component H into I and II m a y be a s c r i b e d to an i n c o m p l e t e r e s o l u t i o n , in t h e step f o l l o ~ , i n g the f r a c t i o n a t i o n s h o w n in figure 1, of the t w o p r o d u c t s i n d u c e d b y s h e a r i n g . F r o m its t e m p l a t e a c t i v i t y to b e d e s c r i b e d i n t h e f o l l o w i n g s e c t i o n , w e can c o n c l u d e t h a t p a r t I is t h e s a m e m a t e r i a l as c o m p o n e n t L. W e t a k e p a r t II to be t h e p u r i f i e d a n d p r o p e r h e a v y start i n g m a t e r i ~ H i n t h e s e n s e of f i g u r e 1. T h i s suggests t h a t t h e d i s t r i b u t i o n of L i n figure 1 is i n h e r e n t l y w i d e r t h a n t h e di,stribution of H.
BIOCHIMIE, 1 9 7 5 ,
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Fro. 2. - - Sedimentation profiles in a metrizamide density gradient of the components L (© © ) and H (I I ) derived by the procedure described in figure 1 ; d~ensities of peak I and II are respectively 1,24 g/am3 and 1720 g/am3.
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Fro. 3. - - Scanning of autoradiographs of some of the polffacrylamide gel slabs of the sedimentation fractions labelled by their number shown in figure 1. Pep,tide position m,arkers are indicated by the molecul, ar weights (× 1'0-3). They are bovine serum a.lbumin (68), heavy chain of gamma-globulin, (5.0) and F1 h,istone. Fraction I ( . ) ; fraction II ( - - - - - - ) .
C. Reiss, M. Cr~pin, D. Paulin and F. Gros.
1310
TRANSCRIPTION OF CttROMATIN.
T h e t e m p l a t e activities of the c h r o m a t i n fractions w e r e m e a s u r e d b y the rate of i n c o r p o r a t i o n of (~H)-UT~P into RNA, in the p r e s e n c e of E. colt RNA p o l y m e r a s e p r e p a r e d a c c o r d i n g to Burgess a n d T r a v e r s [11], a n d of u n l a b e l l e d ATP, CTP a n d GTP u n d e r the c o n d i t i o n s given by M u r p h y
s e p a r a t i o n of c h r o m a t i n into active a n d i n a c t i v e c o m p o n e n t s than the sucrose gradient, simil.ar t r a n s c r i p t i o n e x p e r i m e n t s w e r e p e r f o r m e d on
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Flc,. 4. - - Template activity of chromatin components L and H, as measured through SH-UMP incorporation as a function of time, and for increasing concentrations of chromalin. T h e n u m b e r s ~0.5 ; 1.0 ; 2.0) r e f e r to u n i t s of op~ieal~ d e n s i t i e s at 260 (O C] ~ ) and, fra,ction H ( 0 • A).
nm.
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I a n d H, w i t h the E. colt RNA p o l y t h e e x a m p l e s h o w n in figure 6, the s u p p o r t p r o p a g a t i o n and i n i t i a t i o n of i n c u b a t i o n are c o m p a r e d for diffe-
L
The k i n e t i c s s h o w n in figure 4 i n d i c a t e that the t e m p l a t e a c t i v i t y for exogenous E. colt RNA p o l y m e r a s e is a s s o c i a t e d p r i m a r i l y w i t h the e h r o m a t i n c o m p o n e n t . This is consistent w i t h the firrdings of D o e n e c k e an,d Mc Garthy [10] that e n d o g e n o u s RNA p o l y m e r a s e a c t i v i t y is a s s o c i a t e d w i t h the s l o w l y s e d i m e n t i n g f r a c t i o n L In an a t t e m p t to m e a s u r e the n u m b e r of RNA c h a i n s init i a t e d b y E. colt R~.A p o l y m e r a s e , t h e rate of (vmp)-G'Pp i n c o r p o r a t i o n was d e t e r m i n e d as a f u n c t i o n of t i m e for both c o m p o n e n t s (fig. 5). The n u m b e r o:f i n i t i a t e d c h a i n s in L is h i g h e r t h a n in H. Still, if j u d g e d r e l a t i v e to L, the i n i t i a t i o n rate w i t h H i.s c l e a r l y h i g h e r t h a n the p r o p a g a t i o n rate s h o w n in figure 4, since c o m p o n e n t H, although of v e r y l o w a c t i v i t y for t r a n s c r i p t i o n , possesses o n l y t w o fold less > s t r u c t u r e , t h e h y p o t h e s i s t h a t d e s t a b i l i z i n g p r o t e i n s a r e rem o v e d u p o n f r a c t i o n a t i o n c a n n o t be r e j e c t e d , e s p e c i a l l y s i n c e a t h i r d s p e c i e s is p r o d u c e d , a l o n g w i t h H a n d L ( f r a c t i o n s 1-10, l e f t s i d e of fig. 1). Second, the comparison between sheared and unsheared fractionated samples shows that part i a l destabi,lization of t h e I~NA t e m p l a t e t a k e s p l a c e in the f r a c t i o n s t h e m s e l v e s . It m a y be t h a i the satel,lite D N A is m a i n l y responsi~hle f o r t h i s finding, which would infer that specific destabiliz a t i o n p r o t e i n s are a t t a c h e d to t h e A T - r i c h c o m p o n e n t of t h e DNA.
Acknowledgements. This ~vo~k was s,upport.ed by grants from the Ddldgallon h ]~a Puecherche Seientifique et Technique, the Centre National de la Recherche S.cientifique, the Commissariat h l'Energie Atomique, the Ligue Natiohale Frangaise centre le Cancer, and the Fondation pour la Recherche l~ddieale Franqaise. R~SUM]~. On peut, apr~s l'avoir fragment6e et soumise h eentrifugation centre un gradient tie saeeharose, fractionner l,a ehromatine native de flbroblaste de souris
BIOCHIMIE, 1975, 57, n ° 11-12.
1313
en deux eomposantes : ldg~re et lourde. Une purification plus pouss~e ,peut ~tre r~alis~e en soume¢lane ees fractions h un nouveau cycle de centrifugation h l'dqui,Hbre centre un gradient de m~trizamide : d a n s ces conditions~ le com,posant ldg.er provenant d~ gradient de saccharose s6dimente plus vile, sans doute pJaree que son, degrd d'hydratation est plus dlev6 que celui du composant ]ourd. Le pic H (dquivalent du pic lourc~ en gradient (be saeeharose) ne peut servir de matrice pour la syn.thbse de RNA en prdsence de polym6rase d'E. colt et des substrats addquats. Le pic I e s t par centre tr~s actif. La difference parait lide au taux de propagation de l'enzyme plut6t qu'h sou t a u x d'initiation. Apr~s examen des prot6ines par 6lectrophorbse sur gel ti'acrylamide, il apparait que le p,ic I (actif) renferme Flus de protdines d,e haul poids moldculaire mats un. p.eu moins d'h~stones (principalement H0 que le pic II (inactif). L'emploi d'une tech~n,ique automatique de mesure, des temp.dratures de fusion, technique ayan,t une haute sensihilit6, ~'a pas permis de rdvdler de diffdrence significative dan, s les profils de d~naturation thermique des deux fractions de ehroma.tine ou des DNA qu'el,les renferment. REFERENCES. 1. Axel, R., Cedar, H. a Felsenfe]d, G. (1973) Cold Spring Harbor Syrup. Quant. Biol., 38, 773-783. 2. M~urphT, E. C. Jr., Hall, S. H., Shephe.rd, J. H. a Weiser, R. S. (1-973) Biochem., 12, 3843-9853. 3. Rickwood, D., H,ell, A. & Birnie, G. (1973) FEBS Letters, 33, 221-2~24. 4. K,amen, B., Lindstrom, D. M., Shure, H. a Old, R. W. (1,9,74), Cold Spring Harb. Syrup. Quant. Biol., 39, 187-19'8. 5. D ul,becco, R. (]97{)) Nature, 227, 802-806. 6. ~ayfliek, L. (1960) Nature, 185, 7:83-784. 7. Laemmli, U. (1970) Nature, 227, 680-685. 8. Studier, F. 0973) J. Mol. Biol., 79, 237-24.8. 9. R~iss, C. a Michel, F. (1974) Analytical Biochem., 6~, ¢9'4-508. 10. MIc Carthy, B., Nishiura, J., Doenecke, D., Nasser, D. Johnson, C. (1973) Cold Spring Harbor Syrup. Quant. Biol., 38, 763~783. 11. Burgess, R. a Travers, A. (1971) in Procedures in Nucleic Acid Research, edited by G. L. Cantoni and D. R. D~vies, H'arper and Row ]Publishers, New York, 2 , 85~-863. 12. Rickwood, D., Hell, A., M,alcolm, S., Birnie, G., Mac Gill.ivray & Paul, J. (19'74) Biochem. Biophys. Acta, 353, 35.3-361. 13. Clark, R. ~ Felsenfeld, G. (1971) Nature New Biol., 229, 10,1-106. 14. Rild, R. & Van Holde, K. (1973) J. Biol. Chem., 248, 108@-1083.
90
Transcription of chromatin i om mouse fibroblasts. Claude R m s s ( ' ) , Michel Cn~HN, Denise PAULIN and F r a n c o i s Gnos ~.
l n s t i t u t Pasteur, D~partement de Biologie mol~culaire, Paris, (') I n s t i t u t du Radium, Orsay. (3-7-1975). Abstract. - - Chromatin purified from mouse fibroblasts can be fractionated after shearing by s.edimen:tation in a sucrose gradient into an extended > and a compact c heavy • component. Further purification of these cla.ssical~y described components van be achieved by a second cycle of centrifugation of the light and heavy compon~ents, through an equilibrium density gradient of metrizamide. The light component l~urificd from sucrose gradien, t set~iments faster (peak I) on metrizamide than its heavy counterpart (peak II). Template activity for DNA d.irected RNA synthesis in the presence of E. colt RNA polymerase is uegligib~e in peak l,I bat very pronounced in the peak I fracfio.n. The difference in template activity appears to be cormeeted with differences in propagation rat.her than initiation rates. Comparison of gel electrophoresis pa¢terns of proteins indicate that the active subcomponent includes high mol~ecular weight components not present in the inactive one, but that its histone conten~ is somewhat lower. Using a very highly sensitive automatic recording apparatus for the measurement of melting profiles, no clear cut difference has been observed in the behaviour of active and inactive chromatin subcomponents nor in, that of their total DNA.
INTRODUCTION. Interest has r e c e n t l y been focused on the mec h a n i s m s w h e r e b y n u c l e a r p r o t e i n s r e s t r i c t or i n d u c e gene e x p r e s s i o n in e u k a r y o t i ¢ cells, at the c h r o m a t i n level. E v i d e n c e has been p r e s e n t e d that the a c t i v i t y of t r a n s c r i p t i o n units is i n f l u e n c e d by the a v a i l a b i l i t y of active genetic sites to the RNA p o l y m e r a s e s w h i c h is g o v e r n e d , at least to some extent, by asso,ciation b e t w e e n the DNA and the c h r o m o s o m a l p r o t e i n s [1]. In t h e f r a m e of a long-range study of the c o r r e lation b e t w e e n the role of D NA as a template for RNA s y n t h e s i s a n d the nature of the p r o t e i n assortmen,t p r e s e n t on the c h r o m a t i n , w e h a v e f r a c t i o n a t e d , as a first step, the c h r o m a t i n f r o m t r a n s f o r m e d mouse fibrohlasts into ¢ ac¢ive )) and ¢ i n a c t i v e >> c o m p o n e n t s . As in the o t h e r systems [2], s h e a r e d c h r o m a t i n d e c o m p o s e s on s u c r o s e g r a d i e n t s into t w o f r a c t i o n s w h i c h e x h i b i t differ e n t template activities in d i r e c t i n g in vitro RNA synthesis. F u r t h e r p u r i f i c a t i o n can h o w e v e r be a c h i e v e d by a s e p a r a t i o n into a ¢light>> a n d f r a c t i o n t h r o u g h e q u i l i b r i u m density g r a d i e n t c e n t r i f u g a t i o n against a c o n c e n t r a t e d solution of m e t r i z a m i d e by a t e c h n i q u e w h i c h w e have a d a p t e d from R i e k w o o d , Hell and B i r n i e [3]. The p r e s e n t p a p e r r e p o r t s on the p r o p e r t i e s of these ¢ light ~ a n d ¢ h e a v y >> c h r o m a t i n f r a c t i o n s ~> To whom aH correspondence should be addressed.
w i t h r e g a r d to t h e i r t e m p l a t e a c t i v i t y in the p r e s e n c e of RNA p o l y m e r a s e , t h e i r p r o t e i n content and t e m p e r a t u r e d e p e n d e n t m e l t i n g c h a r a c teristics.
MATERIALS AND MIETHODS. C E L L LINES.
Our s t a r t i n g m a t e r i a l w ~ a cell line of fibroblaeds 3T3 t r a n s f o r m e d w i t h w i l d - t y p e p o l y o m a v i r u s as o b t a i n e d f r o m Dr. T. B e n j a m i n , w h i c h is r e f e r r e d to as PY6. T h e line c o n t a i n s t w o p o l y o m a - i n t e g r a t e d genomes in the c e l l u l a r DNA. It is c o m p l e t e l y p o l y o m a - v i r u s free, and does not p r o d u c e such virus d u r i n g c u l t i v a t i o n [4]. Cells w e r e g r o w n in D u l b e c c o [5] m o d i f i e d Eagle's m e d i u m , s u p p l e m e n t e d w i t h 10 p e r cent fetal calf serum. T h e ceil cultures w e r e t r e a t e d p e r i o d i c a l l y w i t h k a n a m y c i n an.d an anti-PPLO agent (Gibco catalog n ° 522), and c h e c k e d e v e r y 6 to 8 w e e k s for m y c o p l a s m a c o n t a m i n a t i o n [6]. PtAD IOACTIVE LAIKELLIN G.
T h e D u l b e c c o Eagle m e d i u m was d i l u t e d t w o fold w i t h E a r l e solution (Institut Pasteur, c a t a l o g n ° 72.473) and sup~plemented w i t h a m i x t u r e of 15 a m i n o acids labelled w i t h 14C at 1 p~Ci p e r ml (C.E.A., F r a n c e , spe,cific radioaeAivity 100 m C i / mM). S u b c o n f l u e n t cultures w e r e g r o w n in this
1308
C. Reiss, M. Crdpin, D. Paulin a n d F. Gros.
m e d i u m for r a d i o a c t i v e labelling a n d harvested after 20 h. GEL ELECTROPHORESIS. The p r o t e i n s in the c h r o m a t i n fractions were analyzed b y gel electrophoresis. Each fraction was dissociated by h e a t i n g at 56°C for 10 ran, i n the p r e s e n c e of 2 p e r cen~ SDS a n d 1 per cent ~-mercaptoethanol. Electrophoresis proceeded for 4 hrs at 4,0 nlA through 1 p e r cent SDS, 15 per cent p o l y a c r y l a m i d slab gels a c c o r d i n g to the method of L a e m m l i [7] a n d Studier [8]. ISOLATION OF CHROMATIN. Samples of 109 cells were w a s h e d three times with the following buffer : 1.5 mM MgCa2, 140 mM NaC1 and 2.0 mM Tris HCI at pH 6.5. Cytoplasmic m e m b r a n e s were disrupted, as m o n i t o r e d by phase c o n t r a s t microscopy, after a d d i n g 0.5 per cent N o n i d e t (NP40) dissolved i n the same buffer. The nuclei were separated out b y centrifugation at 900 g for 10 m n at 4°C. The n u c l e a r pellet was w a s h e d twice with 75 mM NaCI a n d 25 mM EDTA, a n d then t r a n s f e r r e d into 10 ml of a 5 mM Tris s o l u t i o n at pH 6.5. The nuclei were then disrupted in a Potter h o m o g e n i z e r (20 strokes) a n d the solution centrifuged for 29 m n at 104 g. After two a d d i t i o n a l h o m o g e n i z a t i o n a n d centrifugation cycles, the final c h r o m a t i n pellet was s u s p e n d e d i n 8 ml Tris pH 8.5. TRANSCRIPTION ASSAY.
The s t a n d a r d assay m i x t u r e (0,25 ml) contain e d : 50 mM Tris H,CI pH 7.9; 10 m ~ MgCI2 ; 300 mM KC1 ; 0.4 mM CTP, ATP, G T P ; 0.1 mM (~H) U T P (specific r a d i o a c t i v i t y 20 Ci/M) a n d c h r o m a t i n at a c o n c e n t r a t i o n such that the optical d e n s i t y at 260 n m was 0.1. The reaction was started by a d d i n g 10 u n i t s of E. coli RNA polymerase. I n c u b a t i o n was c a r r i e d out at 37°C for the times specified in the legends of the figures. Acid-insoluble r a d i o a c t i v i t y was collected on a nitrocellulose filter a n d d e t e r m i n e d by a scintillation counter. To m e a s u r e the i n i t i a t i o n of RNA synthesis 73~p GTP or 7:~2p ATP was used (specific r a d i o a c t i v i t y 20,0.0 mCi/mM). In order to reduce the b a c k g r o u n d of the u n s p e c i f i c 32p i n c o r p o r a t i o n i n acid insoluble m a t e r i a l nitrocellulose filters were washed w i t h a TCA solution c o n t a i n i n g s o d i u m phosphate 0.65 M ; p y r o p h o s p h a t e , 0.02.5 M. Radioactivity b a c k g r o u n d was substracted. THERMAL DENATURATION EXPERIMENTS.
The m e l t i n g b e h a v i o r was m o n i t o r e d be measur i n g the optical density (OD) at 26,0 m n for DNA,
BIOCHIMIE, 1975, 57, n ° 11-12.
a n d at 260 n m and 32~0 n m for c h r o m a t i n w i t h a Zeiss PMQII spectrophotometer. The samples were heated in rooter-jacketed quartz cells (Hellma, model 160 QS). The t e m p e r a t u r e was kept constant to w i t h i n + 10 -2 C by a L a n d a u l t r a t h e r m o s t a t model S 8. Melting curves were o b t a i n e d t h r o u g h a temperature-time program, i n w h i c h the temperature is increased stepwise a n d kept c o n s t a n t u n t i l thermal a n d phase e q u i l i b r i a are reached, before the data are recorded automatically. As discussed elsewhere [9], this p r o c e d u r e is an imp r o v e m e n t over s t a n d a r d methods, especially with regard to the r e p r o d u c i b i l i t y a n d details of the m e l t i n g process. The fine structure i n the melting curves is emphasized by plotting the data differentially ( d O D / d T vs. T) on a c o m p u t e r - p e r i p h e ral Benson III tracer after smoothing a n d differ e n t i a t i n g the r a w data on a U n i v a c 1110 computer (Centre de calcul, Universit6 Paris-Sud, Orsay). RESULTS AND DISCUSSION. The s u s p e n s i o n of c h r o m a t i n , labelled i n its p r o t e i n moiety by [14C] amino acids, was treated for 4 m n at 4°C in a Virtis apparatus with a p o w e r setting at 40 W. The sheared c h r o m a t i n was then layered on a 5 - 40 p e r cent stLcrose gradient and centrifuged for 14 h at 10~ g. T w e n t y five fractions were collected. The optical density and r a d i o a c t i v i t y were m e a s u r e d on each. The s e d i m e n t a t i o n profile shown in figure 1 exhibits two peaks of c o m p a r a b l e m a g n i t u d e corr e s p o n d i n g to a heavy (H) r a p i d l y s e d i m e n t i n g and a light (L) slowly s e d i m e n t i n g c o m p o n e n t s of chromatin. The s e p a r a t i o n is a p p a r e n t i n the optical density at 2'8,0 n m (upper graph) and in the r a d i o a c t i v i t y of the p r o t e i n s (lower graph). A good c o i n c i d e n c e was observed between the OD and radioactivity d i s t r i b u t i o n profiles. Similar profiles were observed u n d e r analogous conditions by Mc Carthy el aI. for c h r o m a t i n from a n entirely different source, viz., from Drosophila melanogaster embryo cells [10]. The light fractions 11 to 15 and the heavy fractions 17 to 23 were c o m b i n e d into a c o m p o n e n t L and a c o m p o n e n t H, respectively, dialyzed separately against 0.1 SSC buffer, and c o n c e n t r a t e d by centrifugation for 20 h at 10~ g. The eL>> a n d (
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FIG. I. - - Two components are resolved in sheared chromatin, after sedimentation, in the sucrose density gradient, by their radioactiuity and optical density at 280 nm. Fractions. 11 to 15 and 17 to 23 are combined., respectively, into components L and H for the second fractionation shown in Figure 2.
n i n g 60 p e r cent give rise to a c l e a r l y r e s o l v e d p e a k IT at 1.20 g/cma. U n d e r such c o n d i t i o n s a l o w e r d e n s i t y p e a k (1.18 g/cm~) a p p e a r s in b o t h e h r o m a t i n s in eqttaI amount. This m a t e r i a l m i g h t be n u c l e o p r o t e i n s w i t h a h i g h n u c l e i c a c i d : p r o tein ratio i s o l a t e d after s h e a r i n g of the c h r o m a t i n . P a r t s I a n d II w e r e collected s e p a r a t e l y a n d dialyze,d a g a i n s t 0.1 SSJC buffer. S e p a r a t i o n of the sucrose gradient purified component H into I and II m a y be a s c r i b e d to an i n c o m p l e t e r e s o l u t i o n , in t h e step f o l l o ~ , i n g the f r a c t i o n a t i o n s h o w n in figure 1, of the t w o p r o d u c t s i n d u c e d b y s h e a r i n g . F r o m its t e m p l a t e a c t i v i t y to b e d e s c r i b e d i n t h e f o l l o w i n g s e c t i o n , w e can c o n c l u d e t h a t p a r t I is t h e s a m e m a t e r i a l as c o m p o n e n t L. W e t a k e p a r t II to be t h e p u r i f i e d a n d p r o p e r h e a v y start i n g m a t e r i ~ H i n t h e s e n s e of f i g u r e 1. T h i s suggests t h a t t h e d i s t r i b u t i o n of L i n figure 1 is i n h e r e n t l y w i d e r t h a n t h e di,stribution of H.
BIOCHIMIE, 1 9 7 5 ,
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Fro. 2. - - Sedimentation profiles in a metrizamide density gradient of the components L (© © ) and H (I I ) derived by the procedure described in figure 1 ; d~ensities of peak I and II are respectively 1,24 g/am3 and 1720 g/am3.
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Fro. 3. - - Scanning of autoradiographs of some of the polffacrylamide gel slabs of the sedimentation fractions labelled by their number shown in figure 1. Pep,tide position m,arkers are indicated by the molecul, ar weights (× 1'0-3). They are bovine serum a.lbumin (68), heavy chain of gamma-globulin, (5.0) and F1 h,istone. Fraction I ( . ) ; fraction II ( - - - - - - ) .
C. Reiss, M. Cr~pin, D. Paulin and F. Gros.
1310
TRANSCRIPTION OF CttROMATIN.
T h e t e m p l a t e activities of the c h r o m a t i n fractions w e r e m e a s u r e d b y the rate of i n c o r p o r a t i o n of (~H)-UT~P into RNA, in the p r e s e n c e of E. colt RNA p o l y m e r a s e p r e p a r e d a c c o r d i n g to Burgess a n d T r a v e r s [11], a n d of u n l a b e l l e d ATP, CTP a n d GTP u n d e r the c o n d i t i o n s given by M u r p h y
s e p a r a t i o n of c h r o m a t i n into active a n d i n a c t i v e c o m p o n e n t s than the sucrose gradient, simil.ar t r a n s c r i p t i o n e x p e r i m e n t s w e r e p e r f o r m e d on
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Flc,. 4. - - Template activity of chromatin components L and H, as measured through SH-UMP incorporation as a function of time, and for increasing concentrations of chromalin. T h e n u m b e r s ~0.5 ; 1.0 ; 2.0) r e f e r to u n i t s of op~ieal~ d e n s i t i e s at 260 (O C] ~ ) and, fra,ction H ( 0 • A).
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I a n d H, w i t h the E. colt RNA p o l y t h e e x a m p l e s h o w n in figure 6, the s u p p o r t p r o p a g a t i o n and i n i t i a t i o n of i n c u b a t i o n are c o m p a r e d for diffe-
L
The k i n e t i c s s h o w n in figure 4 i n d i c a t e that the t e m p l a t e a c t i v i t y for exogenous E. colt RNA p o l y m e r a s e is a s s o c i a t e d p r i m a r i l y w i t h the e h r o m a t i n c o m p o n e n t . This is consistent w i t h the firrdings of D o e n e c k e an,d Mc Garthy [10] that e n d o g e n o u s RNA p o l y m e r a s e a c t i v i t y is a s s o c i a t e d w i t h the s l o w l y s e d i m e n t i n g f r a c t i o n L In an a t t e m p t to m e a s u r e the n u m b e r of RNA c h a i n s init i a t e d b y E. colt R~.A p o l y m e r a s e , t h e rate of (vmp)-G'Pp i n c o r p o r a t i o n was d e t e r m i n e d as a f u n c t i o n of t i m e for both c o m p o n e n t s (fig. 5). The n u m b e r o:f i n i t i a t e d c h a i n s in L is h i g h e r t h a n in H. Still, if j u d g e d r e l a t i v e to L, the i n i t i a t i o n rate w i t h H i.s c l e a r l y h i g h e r t h a n the p r o p a g a t i o n rate s h o w n in figure 4, since c o m p o n e n t H, although of v e r y l o w a c t i v i t y for t r a n s c r i p t i o n , possesses o n l y t w o fold less > s t r u c t u r e , t h e h y p o t h e s i s t h a t d e s t a b i l i z i n g p r o t e i n s a r e rem o v e d u p o n f r a c t i o n a t i o n c a n n o t be r e j e c t e d , e s p e c i a l l y s i n c e a t h i r d s p e c i e s is p r o d u c e d , a l o n g w i t h H a n d L ( f r a c t i o n s 1-10, l e f t s i d e of fig. 1). Second, the comparison between sheared and unsheared fractionated samples shows that part i a l destabi,lization of t h e I~NA t e m p l a t e t a k e s p l a c e in the f r a c t i o n s t h e m s e l v e s . It m a y be t h a i the satel,lite D N A is m a i n l y responsi~hle f o r t h i s finding, which would infer that specific destabiliz a t i o n p r o t e i n s are a t t a c h e d to t h e A T - r i c h c o m p o n e n t of t h e DNA.
Acknowledgements. This ~vo~k was s,upport.ed by grants from the Ddldgallon h ]~a Puecherche Seientifique et Technique, the Centre National de la Recherche S.cientifique, the Commissariat h l'Energie Atomique, the Ligue Natiohale Frangaise centre le Cancer, and the Fondation pour la Recherche l~ddieale Franqaise. R~SUM]~. On peut, apr~s l'avoir fragment6e et soumise h eentrifugation centre un gradient tie saeeharose, fractionner l,a ehromatine native de flbroblaste de souris
BIOCHIMIE, 1975, 57, n ° 11-12.
1313
en deux eomposantes : ldg~re et lourde. Une purification plus pouss~e ,peut ~tre r~alis~e en soume¢lane ees fractions h un nouveau cycle de centrifugation h l'dqui,Hbre centre un gradient de m~trizamide : d a n s ces conditions~ le com,posant ldg.er provenant d~ gradient de saccharose s6dimente plus vile, sans doute pJaree que son, degrd d'hydratation est plus dlev6 que celui du composant ]ourd. Le pic H (dquivalent du pic lourc~ en gradient (be saeeharose) ne peut servir de matrice pour la syn.thbse de RNA en prdsence de polym6rase d'E. colt et des substrats addquats. Le pic I e s t par centre tr~s actif. La difference parait lide au taux de propagation de l'enzyme plut6t qu'h sou t a u x d'initiation. Apr~s examen des prot6ines par 6lectrophorbse sur gel ti'acrylamide, il apparait que le p,ic I (actif) renferme Flus de protdines d,e haul poids moldculaire mats un. p.eu moins d'h~stones (principalement H0 que le pic II (inactif). L'emploi d'une tech~n,ique automatique de mesure, des temp.dratures de fusion, technique ayan,t une haute sensihilit6, ~'a pas permis de rdvdler de diffdrence significative dan, s les profils de d~naturation thermique des deux fractions de ehroma.tine ou des DNA qu'el,les renferment. REFERENCES. 1. Axel, R., Cedar, H. a Felsenfe]d, G. (1973) Cold Spring Harbor Syrup. Quant. Biol., 38, 773-783. 2. M~urphT, E. C. Jr., Hall, S. H., Shephe.rd, J. H. a Weiser, R. S. (1-973) Biochem., 12, 3843-9853. 3. Rickwood, D., H,ell, A. & Birnie, G. (1973) FEBS Letters, 33, 221-2~24. 4. K,amen, B., Lindstrom, D. M., Shure, H. a Old, R. W. (1,9,74), Cold Spring Harb. Syrup. Quant. Biol., 39, 187-19'8. 5. D ul,becco, R. (]97{)) Nature, 227, 802-806. 6. ~ayfliek, L. (1960) Nature, 185, 7:83-784. 7. Laemmli, U. (1970) Nature, 227, 680-685. 8. Studier, F. 0973) J. Mol. Biol., 79, 237-24.8. 9. R~iss, C. a Michel, F. (1974) Analytical Biochem., 6~, ¢9'4-508. 10. MIc Carthy, B., Nishiura, J., Doenecke, D., Nasser, D. Johnson, C. (1973) Cold Spring Harbor Syrup. Quant. Biol., 38, 763~783. 11. Burgess, R. a Travers, A. (1971) in Procedures in Nucleic Acid Research, edited by G. L. Cantoni and D. R. D~vies, H'arper and Row ]Publishers, New York, 2 , 85~-863. 12. Rickwood, D., Hell, A., M,alcolm, S., Birnie, G., Mac Gill.ivray & Paul, J. (19'74) Biochem. Biophys. Acta, 353, 35.3-361. 13. Clark, R. ~ Felsenfeld, G. (1971) Nature New Biol., 229, 10,1-106. 14. Rild, R. & Van Holde, K. (1973) J. Biol. Chem., 248, 108@-1083.
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