BIOCHIMIE, 1975, 57, 1301-1306.
A double coil chromatin sub-unit model. S t a n l e y Bm~M.
DOpartement de Biologie Moldculaire, lnstitut Pasteur, 75015 France and Institut du Radium, Facult~ des Sciences, 91400 Orsay, France. (30-10-1975). Summary. - - A model is proposed for the structure of DNA in e h r o m a t i n sub-units. Each s u b - u n i t is proposed to c o n t a i n two t u r n s of an i n n e r coil, With a pitch of about 40 A and an e x t e r n a l d i a m e t e r of 70 A. Around the i n n e r coil is wound, i n opposite b a n d e d n e s s , a slightly larger a m o u n t of DNA at a d i a m e t e r of about 150 A. The total contour length consistent w i t h the electron m i c r o g r a p h s and X-ray scattering is 600-700 A, or about 200 base pairs. It is suggested t h a t the i n n e r coil is p r o t e i n rich and contains all of the histories except H1, vchieh is associated w i t h the outer coil. The double-coil model is consistent w i t h previous biochemical and biophysical studies of c h r o m a t i n . The existence of 200 and 100 base pair digestion f r a g m e n t s and a 6 to 1 DNA c o m p a c t i o n are readily explained. This model is based upon the electron microscopic observation of replicas of frozen c h r o m a t i n and X-ray and n e u t r o n scattering. Structural details of 25 A are preserved and visualized b y the freeze fracture electron microscopy techniques employed. INTRODUCTION. A n e w t y p e of c h r o m a t i n s u b - u n i t s t r u c t u r a l m o del w i l l b e p r e s e n t e d h e r e . It w i l l b e s h o w n t h a t t h i s m o d e l is a r e a s o n a b l e i n t e r p r e t a t i o n o f t h e e l e c t r o n m i c r o g r a p h s a n d X-ray s c a t t e r i n g of hydrated chromatin. More extensive electron micrographs aud X-ray calculations will be published e l s e w h e r e [1, 2]. Previous electron microscopy studies have been m a d e on c h r o m a t i n solutions after d e h y d r a t i o n by e i t h e r o r g a n i c s o l v e n t s [3, 4, 5] o r b y a i r d r y i n g
[fi, 7]. A i r d r y i n g avoi, ds i m m e r s i o n i n n o n aqueous media, but the enormous surface tension forces arising upon dehydration flatten and denature macromolecnles. A su,b-structure along the f i b e r s w a s o b s e r v e d i n e l e c t r o n m i c r o g r a p h s of chromalin critical point dried from amyl acetate [3, 4]. H o w e v e r , n o b e a d s s e p a r a t e d b y a l i n k e r DNA [6, 7] w e r e o b s e r v e d i n t h i s w o r k [3, 4]. F r e e z e f r a c t u r e e l e c t r o n m i c r o g r a p h s of f r o z e n hydrated c h r o m a t i n s o l u t i o n s s h o w l i n e a r a r r a y s o f v e r y c l o s e l y p a c k e d s u b - u n i t s (see fig. 1 a n d 2). T h e s e p a r a t i o n of s u b - u n i t s , w h i c h is l e s s t h a n
Fro. l. - - Freeze fractured e h r o m a t i n f r o m calf t h y m u s (× 500,000). The shado'wing was at 45 ° with plat i n u m and carbon. C h r o m a t i n is black in the p h o t o g r a p h s and the direction of shadow is indicated by a large arrow. The s u b - u n i t n a t u r e of the c h r o m a t i n is quite visible. A p e r i p h e r a l coil 25 A thick (15{) A e x t e r n a l diameter) is seen about m o s t of the sub-units. Note t h a t the loops m a r k e d ¢ L ~> can be followed for more t h a u 300 A. The outer coil is often hidden behind or obseurred over the core. On the u p p e r side of the p h o t o g r a p h s it is i n the s h a d o w of the core. In m a n y views the c h r o m a t i n fiber appears to be a projection of a coil. In the center of the p h o t o g r a p h three regions, w h i c h seem to be a coil of sub-units shadowed f r o m the side, are parallel to the small a r r o w s whose s e p a r a t i o n is 400 A. The fibre regularly appears above (arrow) and t h e n disappears belo~v the ice matrix. Such an up and d o w n repeat of 400 A is only consistent w i t h a helix of this pitch.
! 302
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L
Fro. 2. - - Freeze f r a c t u r e d calf t h y m u s e h r o m a t i n ( × 730,000). T h e core p l u s o u t e r coil s u b - u n i t s t r u c t u r e is clear in m o s t views. At ¢ L >> it is o n b o t h sides of the core. T h e fiber h a s t h e a p p e a r a n c e of a c o n t i g u o u s l i n e a r a r r a y of s u b - u n i t s . P a r t s a r e n o t v i s i b l e w h e n t h e y a r e b e l o w the ice m a t r i x o r o b s c u r e d b y s h a d o w i n g effects. A h i g h e r o r d e r s t r u c t u r e c o m p a t i b l e w i t h a 400 to 500 • p i t c h coil I l l ] is visible. T h e a r r o w s are p a r a l l e l to w h a t s e e m s to be helical gyres, a n d indicate r e g i o n s a s s o c i a t e d w i t h long w h i t e s h a d o w s . T h i s i m p l i e s t h a t t h e y a r e p r o j e c t i n g u p in t h e ice. H a l f w a y b e t w e e n a r r o w s a r e r e g i o n s of s h o r t o r no s h a d o w s h o w i n g t h a t the m a t e r i a l is l o w e r d o w n .
BIOCHIMIE, 1975, 57, n ° 11-12.
A double coil c h r o m a t i n sub-unit model.
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30 ~, b e c o m e s slightly l a r g e r after r e m o v a l of histone H1.
figures 1 a n d 2 illustrate the h i g h e r o r d e r heli,cal a r r a n g e m e n t of sub-units.
F r e e z e f r a c t u r e a n d freeze e t c h i n g e l e c t r o n m i c r o s c o p y are c~arried out on s h a d o w e d r e p l i c a s of cleaved frozen solutions (It is cogent to note h e r e t h a t f r e e z i n g in liqui,d n i t r o g e n w i t h subsequent t h a w i n g causes no o b s e r v a b l e change in the c h r o m a t i n X-ray [4] o r n e u t r o n s c a t t e r i n g [2] patterns). W i t h the freeze f r a c t u r e t e c h n i q u e the molecules a r e n e v e r d e h y d r a t e d , w h i l e for freeze e t c h i n g a p a r t of the ice m a t r i x is s u b l i m e d before sh,adowing. Only a s t a b i l i z e d h e a v y m e t a l m o u l d is e x a m i n e d in the el.ectron m i c r o s c o p e . The use of a m o u l d has the i m p o r t a n t a d v a n t a g e of b e i n g quite r e s i s t a n t to electron b e a m d a m a g e but limits the r e s o l u t i o n to about 20 A.
The sub-units themselves a p p e a r to consist of a central dense 90 ± 20 A b y 70 ± 10 A core. This c e n t r a l p a r t is s u r r o u n d e d by an outer loop of DNA h a v i n g an e x t e r n a l d i a m e t e r of 150 __+ 15 A a n d a c o n t o u r length of about 400 A (see figures 1, 2 a n d 3). This outer loop w h o s e t h i c k n e s s varies between 2'0 a n d 32 ~ often looks 1,ike a coil in p r o j e c t i o n (as i n d i c a t e d in figures 1 a n d 3). It is not a l w a y s visible w h e n it is b e h i n d o r over the c e n t r a l core, or in its shado~v.
METHODS.
Chromatin preparation. Calf t h y m u s c h r o m a t i n w a s p r e p a r e d b y t w o different methods. F o r total chroimatin, a m o d i f i c a t i o n of the m e t h o d of Zalta et al. [8] was usual,ly e m p l o y e d . H o w e v e r the final d i s p e r s a l w a s in 1 mLM NaC1. C h r o m a t i n for h i s t o n e HI d e p l e t i o n w a s p r e p a r e d a c c o r d i n g to P a n y m i n et at. [9]. S u b s e q u e n t l y H~I a n d most o~ the non h i s t o n e p r o t e i n s w e r e d e p l e t e d at .6 ,M NaCI w i t h a r e s i n [10].
Electron microscopy. C h r o m a t i n was u s u a l l y fixed b y the a d d i t i o n of g l u l a r a l d e h y d e (pH 7) to .2 p e r cent. But, fixed a n d unfixed m a t e r i a l gave v e r y s i m i l a r electro,n m i c r o s c o p e images and X-ray s c a t t e r i n g p a t t e r n s [1, 2]. Solutions at a c o n c e n t r a t i o n of a few m g / m l in 1 mM NaCt w e r e frozen in l i q u i d f r e o n at - - 1 8 0 ° C . F r e e z e fractur i n g w a s c a r r i e d out in a Blazers (301E) a p p a r a t u s at --15,0.°C. Some p r e p a r a t i o n s w e r e e t c h e d at --I10°C for 2 rain at 2 X l f f a torr. S h a d o w i n g w a s w i t h c a r b o n - p l a t i n u m a n d then carbon. After c l e a n i n g in clorox, the r e s u l t i n g rcplic.as w e r e e x a m i n e d in a Siemens 101 e l e c t r o n m i c r o s c o p e at p l a t e m a g n i f i c a t i o n s of 40 a n d 80,00.0 X. RESU,LTS AND DISCUSSION.
Electron Microscopy. At l o w r e s o l u t i o n the elect r o n m i c r o g r a p h s of h y d r a t e d c h r o m a t i n r e p l i c a s a r e s o m e w h a t s i m i l a r to those p r e v i o u s l y o b t a i n e d w i t h c r i t i c a l p o i n t d r y i n g [3, 4]. H o w e v e r , t h e y c o n t a i n a great deal m o r e i n f o r m a t i o n . The new m ' i c r o g r a p h s s u p p o r t the q u a t e r n a r y 400 to 500 A p i t c h s u p e r c o i l i n g p r o p o s e d for c h r o m a t i n from n e u t r o n small angle s c a t t e r i n g [11]. Although the e l e c t r o n m i c r o s c o p y of o u r freeze f r a c t u r e d c h r o m a t i n w i l l be p r e s e n t e d in detail elsewhere, [1] BIOCHIMIE, 1975,
57, n °
11-12.
H1 d e p l e t e d c h r o m a t i n exhibits a s i m i l a r ultras t r u c t u r e (Some of o u r e a r l i e r freeze e t c h i n g mie r o g r a p h s are s h o w n in r e f e r e n c e [12]). F r e e z e e t c h i n g m i c r o g r a p h s , su~ch as figure 3, have a d e c e p t i v e l y large a p p a r e n t s e p a r a t i o n b e t w e e n sub-units, due to e m b e d d i n g in the i.ce m a t r i x . In c o m p l e t e l y e x p o s e d r e g i o n s the s e p a r a t i o n is on the o r d e r of 50 A o r less. A c o r e - o u t e r coil configur a t i o n is even m o r e e v i d e n t after H1 d e p l e t i o n . The outer coil can s o m e t i m e s be o b s e r v e d to b r i d g e n e i g h b o r i n g subunits, as in figure 3, a n d c o n t o u r lengths of 4.00 to 450 A can be followed. In general, the t h i c k n e s s of the o u t e r loop a p p e a r s to be about 5 A less after remov,al of H1 but this is at the l i m i t of the technique. The c e n t r a l coil has the aspect of a t i g h t l y fold e d or c o i l e d 25 A t h i c k t h r e a d of D,NA. W h e n the o r i e n t a t i o n w i t h r e s p e c t to t,he s h a d o w i n g d i r e c tion is f a v o r a b l e , the c e n t r a l core a p p e a r s to consist of a,bout two t u r n s of a 40 A p i t c h 70-80 A outer d i a m e t e r coil (fig. 3). It s h o u l d be stressed t h a t the c o r e - o u t e r coil u~t r a s t r u e t u r e is seen in the vast m a j o r i t y of the e l e c t r o n m i c r o g r a p h s of frozen c h r o m a t i n r e p l i cas. T h r o u g h - f o c u s series of m i e r o g r a p h s do not a4ter this b a s i c configuration. W i t h r e s p e c t to conv e n t i o n a l t e c h n i q u e s for e l e c t r o n m i c r o s c o p y , the outer loop s h o u l d be quite fragile. Consequently, d e h y d r a t i o n or the s u r f a c e t e n s i o n forces generat e d by d r y i n g for c o n v e n t i o n a I e l e c t r o n m i c r o s c o p y p r e p a r a t i o n s m i g h t flatten it a g a i n s t the inner core and r e n d e r it i m p e r c e p t i b l e a n d r e s u l t in a k n o b b y a p p e a r a n c e . This might e x p l a i n somew h a t the v a r i a b l e d i a m e t e r s c~bserved for chrom a t i n b y different t e c h n i q u e s . Values b e t w e e n 70 a n d 150 ~ have been p u b l i s h e d [7, 13].
The X-ray scattering from a core and shell. A dense c e n t r a l c o r e w i t h a less dense r i n g (shell) is the r a d i a l p r o j e c t i o n of a tight i n n e r coil w i t h an outer loop. Such a r a d i a l d i s t r i b u t i o n of mass is v e r y c o m p a t i b l e w i t h the small angle X-ray a n d n e u t r o n s c a t t e r i n g f r o m c h r o m a t i n solutions.
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Small angle X-ray and n e u t r o n s c a t t e r i n g show two Gaussian cross section regions. The one at smaller angles yields a cross section radius of g y r a t i o n of about 52 X, and the s e c o n d one gives 30 & [3, 4]. The s c a t t e r i n g c o r r e s p o n d i n g to the s e c o n d Gaussi,an regio.n ~ a s s h o w n some l i m e ago to be consistent w i t h a tight 45 ,~ p i t c h 3,0 X r a d i u s of g y r a t i o n coil c o n t a i n i n g about two t h i r d s of the total mass [3, 4]. The experimenta,1 55 A cross section r a d i u s of g y r a t i o n w o u l d c o r r e s p o n d to an outer r a d i u s of about 150 X. This value and the 45 X p i t c h central core p a r a m e t e r s agree very well w i t h the electron m i c r o g r a p h s . The c o n ~ a r i s o n can be c a r r i e d out in the other sense by cal, cu~lating the X-ray s c a t t e r i n g expected f r o m the s t r u c t u r e o b s e r v e d in the m i c r o g r a p h s . I n d e e d , the c a l c u l a t e d c u r v e s s h o w all of the imp o r t a n t features of the X-ray s c a t t e r i n g i n c l u d i n g the m o n o t o n i c decrease in i n t e n s i t y until about 40 ~ [4, 2]. AII p r e v i o u s m o d e l calculations based u p o n coils, coils c o n t a i n i n g solid p r o t e i n cores
and various other elongated forms gave v e r y sharp m a x i m a in this s c a t t e r i n g region [3, 4]. Thus, the X-ray scattering p r o v i d e s i n d e p e n d e n t s u p p o r t for the core-outer coil ultrastructure. F o r a double col,1 m o d e l the X-ray s c a t t e r i n g mass p e r unit length ~vould be d e r i v e d from the i n n e r cross section region instead of the outer one p r e v i o u s l y e m p l o y e d [41. A value greater than 22(~0 daltons /X wou~d then be calculated, giving a total DNA c o m p a c t i o n of 5.3 to 1. A 7:1 compaction has been d e t e r m i n e d by Griffith [14] from his electron m i c r o g r a p h s . Together, the outer and i n n e r coils of the freeze f r a c t u r e d sub-units have a DNA contour length of 6.0~) to 700 X. Then, about 600 3, of DNA is p a c k e d into a d i m e n s i o n of about 100 A.
Model Building. At this p o i n t a m o d e l can be constructed. It is a p p a r e n t that in o r d e r to h a v e the same c e n t e r of mass the outer and i n n e r coils nmst have opposite handedness. A v e r y rough mo-
Fro. 3. - - Freeze etched calf thymus ehromatin shadowed at 35 ° (× 650,000). The sub-unit nature is obvious ; however, the sub-unit separation is exaggerated due to embedding in the ice-matrix. Note, for example, that sub-units A and B are partially embedded. The outer loop is quite distinct about most sub-units and has an outer diameter of 150 to 180 A. The loops of sub-units C and D can be seen to cross over the center of the core. Double arrows, separated by 80 A, mark inner coils which appear to be two turns of a 40 to 50 A pitch coil.
BIOCHIMIE, 1975, 57, n ° 11-12.
A double coil c h r o m a t i n s u b - u n i t model. del can be o b t a i n e d by t w i s t i n g a flexible tube or r u b b e r b a n d about a rigid i n n e r support. Two t u r n s of h a n d e d n e s s opposite to the t w i s t i n g arise about the s u p p o r t a n d t h e n the tu~aing s p o n t a n e ously loops back over itself i n the opposite direction (to m i n i m i z e the stress). I1 has been previously noted that an over or u n d e r t u r n i n g of the DNA s e c o n d a r y structure could be r e s p o n s i b l e for the t e r t i a r y super coiling [5]. At present, we are not yet able to define the exact relative o r i e n t a t i o n of n e i g h b o r i n g subunits. If the core axes are almost p~arallel to the fibre direction, then an outer loop m a y w i n d over a n d be shared by two n e i g h b o r i n g sub-units a n d have an a p p r o x i m a t e p i t c h of about 2'40 X. Figure 4 depicts two s u b u n i t s in this o r i e n t a t i o n w h i c h appears to agree best w i t h most images. If the axes of n e i g h b o r i n g cores are almost p e r p e n d i c u l a r to the fibril, one outer loop with a p i t c h
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r i n g DNA folds i n s t e a d of coils m i g h t also be consistent w i t h the results. More precise i n f o r m a tion on the n a t u r e of the coils a n d the i n t e r - u n i t o r i e n t a t i o n s should be o b t a i n a b l e from a detailed analysis of the images a n d from the n e u t r o n scattering. No direct evidence for or against k i n k i n g [16] is observed however, a k i n k m a y well occur w h e r e the h a n d e d n e s s of the coiling changes sign.
Distribution of Histones. With regard to the histone localization, it seems that h i s t o n e H1 is b o u n d to the outer coil a n d m a y be p a r t i a l l y i n v o l v e d i n m a i n t a i n i n g c o n f o r m a t i o n . After H1 is r e m o v e d the basic s u b - u n i t s t r u c t u r e is conserved b u t the particles may b e s o m e w h a t larger. It is r e a s o n a b l e to suggest that the r e m a i n i n g four histones are associated w i t h the central coil, I n s u c h a scheme the p r o t e i n to DNA ratio i n the core w o u l d be somewhat larger t h a n 2: 1. P r o t e i n cores about w h i c h 10~)A d i a m e t e r DNA he,lices are w r a p p e d have been c o n s i d e r e d to e x p l a i n the X-ray [17] a n d n e u t r o n s c a t t e r i n g [lS]. However, an i n n e r coil of DNA i n t i m a t e l y b o u n d to twice its weight of p r o t e i n w o u l d agree m u c h better w i t h the neutron s c a t t e r i n g on dilute solutions [2]. The neutron scattering from c o n c e n t r a t e d c h r o m a f i n gels [18] w o u l d be compatible w i t h a DN,A rich shell about a p r o t e i n rich i n n e r coil. Comparison of the double coil model to other results. Although o n l y a general o u t l i n e of a possible s t r u c t u r e for c h r o m a t i n s u b - u n i t s has been presented here, the model allows i n t e r p r e t a t i o n of m a n y e x p e r i m e n t a l results. A most i m p o r t a n t finding to e x p l a i n is the p r e s e n c e of 170 to 200 base p a i r fragments in b r i e f DNAse digests [15, 16] a n d a 100 base p a i r one i n the ¢ limit digests >> [21, 22]. The former woul, d result from the total double coil u n i t w h i l e the latter could come from the compact i n n e r coil. In a s i m i l a r fashion, the compaction ratios of 6 : 1 a n d 3 : 1 often cited i n the literature [14, 17, 22] wou~d also be exp~lainable by the double coil model.
Fro. 4. - - A rough wire model approximating the DNA of two double coil sub-units in the orientation parallel to the fiber axis. The wire fibril, proportional to a 20 A thickness, makes two turns with a 40 A pitch and then coils over itself and a neighboring sub-unit.
of about 120 A may be associated w i t h each subunit. This w o u l d then be one t u r n of a P a r d o n , W E k i n s and R i c h a r d s s u p e r coil. [15]. I n a n y case, the s u b u n i t a n d especially the outer loop geometry must be s o m e w h a t distorted to accommodate the t e r t i a r y structure. A s t r u c t u r e featu-
BIOCHIMIE, 1975, 57, n ° 11-12.
!Diffraction reflections at 110 A i n wet gels a n d at 80 X i n dried c h r o m a t i n [15, 18] are i m p o r t a n t in the literature. U p o n c o n c e n t r a t i o n a fiber of double coil sub-units w o u l d first pack so that a n outer Ioop touches a n e i g h b o r i n g core, giving a s e p a r a t i o n of 1~I0 X (75 + 35 X). U p o n d e h y d r a tion the outer coil might collapses a n d contact of 70 to 80 A d i a m e t e r cores w o u l d be possible. Finally, the r e c e n t results on SV40 c h r o m a t i n [23] i m p l y i n g that there is only one effective sup e r coil t u r n per s u b - u n i t eliminates m a n y types of s u b - u n i t model structures. Interestingly, the
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d o u b l e c o i l of t w o i n n e r t u r n s p l u s a n o u t e r o n e of o p p o s i t e h a n d e d n e s s has one effective turn p e r sub-unit.
A cdcno~wledgmen ls. I a m grateful to Dr. L. Chevance and Mr. S. Kouprach for t h e i r suggestions and technical assistance. Financial s u p p o r t w a s provided by the Centre National de la Recherche Seientifique and the D~l~gation G6n6rale h la Recherche Seientifique et Technique. An account of this w o r k ~was presented at the British Society for Cell Biology meeting or Chromatin in Gtasgow (Sept. 1975). REFERENCES. 1. 2. 3. 4. 5. 6. 7.
Bram, S. & Koupraeh, S. in preparation. Bandy, P., Brarfi, S. ,& Vastel, D. in preparation. Bram, S. (1968) Thesis University of Wisconsin. Brain, S. & Ris, H. (1971) J. Mol. Biol., 55, 325-336. Bram, S. (1972) Biochimie, 54, 1005-1011. Woodcock, C. L. F. (1973) J. Cell Biol., 54, 368. Olins, D. E. & Olins, A. L. (1974) Science, 183, 330332.
BIOCHIMIE, 1975, 57, n ° 11-12.
8. Zalta, J., Zalta, J. P. ~ Simard, R. (1971) J. Celt. Biol., 51, 563-568. 9. Panyim, S., Bilik, B. ~ Chalkley, R. (1971) J. Biol. Chem., 246, 281-300. 10. Bolund, L. H. • Johns, E. W. (1973) Enr. J. Biochem., 35, 533-546. 11. B r a m , S., Butler-Browne, G., Baudy, P. & tbel, K. (1975) Proe. Natl. Acad. Sci. USA, 72, 1043. 12. Brain, S., Hellio, R. • Koupraeh, S. (1975) C. R. Aead. Sci., 281, 847-849. 13. Slayter, H. S., Sinh, T. G., Adler, A. J. ~ F a s m a n (1972) Biochemistry, ]J1, 3044-3054. 14. Griffith, J. D. (1975) Science, 187, 1202-1203. 15. Pardon, J. F., Wilkins, M. H. F. & Richards, B. M. (1967) Nature, 215, 508-509. 16. Crick, F. H. C. & Klug, A. (1975) Nature, 255, 530534. 17. Kornberg, R. D. (1974) Science, 184, 868-871. 18. Baldwin, J. P., Boseley, P. G., Bradbury, E. M. & Ibel, M. (1975) Nature, 253, 245-249. 19. Hewish, R. D. & Bnrgogne, L. A. (1973) Biochem. Biophgs. Res. Comm., 52, 504-510. 20. Noll, M. (1974) Nature, 251, 249-251. 21. Clark, R. J..& Felsenfeld, G. (1974) Biochemistry, 13, 3622-3628. 22. Sarasrabnddhe, C. a Van Hold, K. (19743 J. Biol. Chem., 249, 152-156. 23. Germond, J. E., Hirt, B., Gross-Bellard, M. & Chambon, P. (1975) Proc. Natl. Acad. Sci. USA, 72, 1843-1847.
A double coil chromatin sub-unit model. S t a n l e y Bm~M.
DOpartement de Biologie Moldculaire, lnstitut Pasteur, 75015 France and Institut du Radium, Facult~ des Sciences, 91400 Orsay, France. (30-10-1975). Summary. - - A model is proposed for the structure of DNA in e h r o m a t i n sub-units. Each s u b - u n i t is proposed to c o n t a i n two t u r n s of an i n n e r coil, With a pitch of about 40 A and an e x t e r n a l d i a m e t e r of 70 A. Around the i n n e r coil is wound, i n opposite b a n d e d n e s s , a slightly larger a m o u n t of DNA at a d i a m e t e r of about 150 A. The total contour length consistent w i t h the electron m i c r o g r a p h s and X-ray scattering is 600-700 A, or about 200 base pairs. It is suggested t h a t the i n n e r coil is p r o t e i n rich and contains all of the histories except H1, vchieh is associated w i t h the outer coil. The double-coil model is consistent w i t h previous biochemical and biophysical studies of c h r o m a t i n . The existence of 200 and 100 base pair digestion f r a g m e n t s and a 6 to 1 DNA c o m p a c t i o n are readily explained. This model is based upon the electron microscopic observation of replicas of frozen c h r o m a t i n and X-ray and n e u t r o n scattering. Structural details of 25 A are preserved and visualized b y the freeze fracture electron microscopy techniques employed. INTRODUCTION. A n e w t y p e of c h r o m a t i n s u b - u n i t s t r u c t u r a l m o del w i l l b e p r e s e n t e d h e r e . It w i l l b e s h o w n t h a t t h i s m o d e l is a r e a s o n a b l e i n t e r p r e t a t i o n o f t h e e l e c t r o n m i c r o g r a p h s a n d X-ray s c a t t e r i n g of hydrated chromatin. More extensive electron micrographs aud X-ray calculations will be published e l s e w h e r e [1, 2]. Previous electron microscopy studies have been m a d e on c h r o m a t i n solutions after d e h y d r a t i o n by e i t h e r o r g a n i c s o l v e n t s [3, 4, 5] o r b y a i r d r y i n g
[fi, 7]. A i r d r y i n g avoi, ds i m m e r s i o n i n n o n aqueous media, but the enormous surface tension forces arising upon dehydration flatten and denature macromolecnles. A su,b-structure along the f i b e r s w a s o b s e r v e d i n e l e c t r o n m i c r o g r a p h s of chromalin critical point dried from amyl acetate [3, 4]. H o w e v e r , n o b e a d s s e p a r a t e d b y a l i n k e r DNA [6, 7] w e r e o b s e r v e d i n t h i s w o r k [3, 4]. F r e e z e f r a c t u r e e l e c t r o n m i c r o g r a p h s of f r o z e n hydrated c h r o m a t i n s o l u t i o n s s h o w l i n e a r a r r a y s o f v e r y c l o s e l y p a c k e d s u b - u n i t s (see fig. 1 a n d 2). T h e s e p a r a t i o n of s u b - u n i t s , w h i c h is l e s s t h a n
Fro. l. - - Freeze fractured e h r o m a t i n f r o m calf t h y m u s (× 500,000). The shado'wing was at 45 ° with plat i n u m and carbon. C h r o m a t i n is black in the p h o t o g r a p h s and the direction of shadow is indicated by a large arrow. The s u b - u n i t n a t u r e of the c h r o m a t i n is quite visible. A p e r i p h e r a l coil 25 A thick (15{) A e x t e r n a l diameter) is seen about m o s t of the sub-units. Note t h a t the loops m a r k e d ¢ L ~> can be followed for more t h a u 300 A. The outer coil is often hidden behind or obseurred over the core. On the u p p e r side of the p h o t o g r a p h s it is i n the s h a d o w of the core. In m a n y views the c h r o m a t i n fiber appears to be a projection of a coil. In the center of the p h o t o g r a p h three regions, w h i c h seem to be a coil of sub-units shadowed f r o m the side, are parallel to the small a r r o w s whose s e p a r a t i o n is 400 A. The fibre regularly appears above (arrow) and t h e n disappears belo~v the ice matrix. Such an up and d o w n repeat of 400 A is only consistent w i t h a helix of this pitch.
! 302
S. Brain.
L
Fro. 2. - - Freeze f r a c t u r e d calf t h y m u s e h r o m a t i n ( × 730,000). T h e core p l u s o u t e r coil s u b - u n i t s t r u c t u r e is clear in m o s t views. At ¢ L >> it is o n b o t h sides of the core. T h e fiber h a s t h e a p p e a r a n c e of a c o n t i g u o u s l i n e a r a r r a y of s u b - u n i t s . P a r t s a r e n o t v i s i b l e w h e n t h e y a r e b e l o w the ice m a t r i x o r o b s c u r e d b y s h a d o w i n g effects. A h i g h e r o r d e r s t r u c t u r e c o m p a t i b l e w i t h a 400 to 500 • p i t c h coil I l l ] is visible. T h e a r r o w s are p a r a l l e l to w h a t s e e m s to be helical gyres, a n d indicate r e g i o n s a s s o c i a t e d w i t h long w h i t e s h a d o w s . T h i s i m p l i e s t h a t t h e y a r e p r o j e c t i n g u p in t h e ice. H a l f w a y b e t w e e n a r r o w s a r e r e g i o n s of s h o r t o r no s h a d o w s h o w i n g t h a t the m a t e r i a l is l o w e r d o w n .
BIOCHIMIE, 1975, 57, n ° 11-12.
A double coil c h r o m a t i n sub-unit model.
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30 ~, b e c o m e s slightly l a r g e r after r e m o v a l of histone H1.
figures 1 a n d 2 illustrate the h i g h e r o r d e r heli,cal a r r a n g e m e n t of sub-units.
F r e e z e f r a c t u r e a n d freeze e t c h i n g e l e c t r o n m i c r o s c o p y are c~arried out on s h a d o w e d r e p l i c a s of cleaved frozen solutions (It is cogent to note h e r e t h a t f r e e z i n g in liqui,d n i t r o g e n w i t h subsequent t h a w i n g causes no o b s e r v a b l e change in the c h r o m a t i n X-ray [4] o r n e u t r o n s c a t t e r i n g [2] patterns). W i t h the freeze f r a c t u r e t e c h n i q u e the molecules a r e n e v e r d e h y d r a t e d , w h i l e for freeze e t c h i n g a p a r t of the ice m a t r i x is s u b l i m e d before sh,adowing. Only a s t a b i l i z e d h e a v y m e t a l m o u l d is e x a m i n e d in the el.ectron m i c r o s c o p e . The use of a m o u l d has the i m p o r t a n t a d v a n t a g e of b e i n g quite r e s i s t a n t to electron b e a m d a m a g e but limits the r e s o l u t i o n to about 20 A.
The sub-units themselves a p p e a r to consist of a central dense 90 ± 20 A b y 70 ± 10 A core. This c e n t r a l p a r t is s u r r o u n d e d by an outer loop of DNA h a v i n g an e x t e r n a l d i a m e t e r of 150 __+ 15 A a n d a c o n t o u r length of about 400 A (see figures 1, 2 a n d 3). This outer loop w h o s e t h i c k n e s s varies between 2'0 a n d 32 ~ often looks 1,ike a coil in p r o j e c t i o n (as i n d i c a t e d in figures 1 a n d 3). It is not a l w a y s visible w h e n it is b e h i n d o r over the c e n t r a l core, or in its shado~v.
METHODS.
Chromatin preparation. Calf t h y m u s c h r o m a t i n w a s p r e p a r e d b y t w o different methods. F o r total chroimatin, a m o d i f i c a t i o n of the m e t h o d of Zalta et al. [8] was usual,ly e m p l o y e d . H o w e v e r the final d i s p e r s a l w a s in 1 mLM NaC1. C h r o m a t i n for h i s t o n e HI d e p l e t i o n w a s p r e p a r e d a c c o r d i n g to P a n y m i n et at. [9]. S u b s e q u e n t l y H~I a n d most o~ the non h i s t o n e p r o t e i n s w e r e d e p l e t e d at .6 ,M NaCI w i t h a r e s i n [10].
Electron microscopy. C h r o m a t i n was u s u a l l y fixed b y the a d d i t i o n of g l u l a r a l d e h y d e (pH 7) to .2 p e r cent. But, fixed a n d unfixed m a t e r i a l gave v e r y s i m i l a r electro,n m i c r o s c o p e images and X-ray s c a t t e r i n g p a t t e r n s [1, 2]. Solutions at a c o n c e n t r a t i o n of a few m g / m l in 1 mM NaCt w e r e frozen in l i q u i d f r e o n at - - 1 8 0 ° C . F r e e z e fractur i n g w a s c a r r i e d out in a Blazers (301E) a p p a r a t u s at --15,0.°C. Some p r e p a r a t i o n s w e r e e t c h e d at --I10°C for 2 rain at 2 X l f f a torr. S h a d o w i n g w a s w i t h c a r b o n - p l a t i n u m a n d then carbon. After c l e a n i n g in clorox, the r e s u l t i n g rcplic.as w e r e e x a m i n e d in a Siemens 101 e l e c t r o n m i c r o s c o p e at p l a t e m a g n i f i c a t i o n s of 40 a n d 80,00.0 X. RESU,LTS AND DISCUSSION.
Electron Microscopy. At l o w r e s o l u t i o n the elect r o n m i c r o g r a p h s of h y d r a t e d c h r o m a t i n r e p l i c a s a r e s o m e w h a t s i m i l a r to those p r e v i o u s l y o b t a i n e d w i t h c r i t i c a l p o i n t d r y i n g [3, 4]. H o w e v e r , t h e y c o n t a i n a great deal m o r e i n f o r m a t i o n . The new m ' i c r o g r a p h s s u p p o r t the q u a t e r n a r y 400 to 500 A p i t c h s u p e r c o i l i n g p r o p o s e d for c h r o m a t i n from n e u t r o n small angle s c a t t e r i n g [11]. Although the e l e c t r o n m i c r o s c o p y of o u r freeze f r a c t u r e d c h r o m a t i n w i l l be p r e s e n t e d in detail elsewhere, [1] BIOCHIMIE, 1975,
57, n °
11-12.
H1 d e p l e t e d c h r o m a t i n exhibits a s i m i l a r ultras t r u c t u r e (Some of o u r e a r l i e r freeze e t c h i n g mie r o g r a p h s are s h o w n in r e f e r e n c e [12]). F r e e z e e t c h i n g m i c r o g r a p h s , su~ch as figure 3, have a d e c e p t i v e l y large a p p a r e n t s e p a r a t i o n b e t w e e n sub-units, due to e m b e d d i n g in the i.ce m a t r i x . In c o m p l e t e l y e x p o s e d r e g i o n s the s e p a r a t i o n is on the o r d e r of 50 A o r less. A c o r e - o u t e r coil configur a t i o n is even m o r e e v i d e n t after H1 d e p l e t i o n . The outer coil can s o m e t i m e s be o b s e r v e d to b r i d g e n e i g h b o r i n g subunits, as in figure 3, a n d c o n t o u r lengths of 4.00 to 450 A can be followed. In general, the t h i c k n e s s of the o u t e r loop a p p e a r s to be about 5 A less after remov,al of H1 but this is at the l i m i t of the technique. The c e n t r a l coil has the aspect of a t i g h t l y fold e d or c o i l e d 25 A t h i c k t h r e a d of D,NA. W h e n the o r i e n t a t i o n w i t h r e s p e c t to t,he s h a d o w i n g d i r e c tion is f a v o r a b l e , the c e n t r a l core a p p e a r s to consist of a,bout two t u r n s of a 40 A p i t c h 70-80 A outer d i a m e t e r coil (fig. 3). It s h o u l d be stressed t h a t the c o r e - o u t e r coil u~t r a s t r u e t u r e is seen in the vast m a j o r i t y of the e l e c t r o n m i c r o g r a p h s of frozen c h r o m a t i n r e p l i cas. T h r o u g h - f o c u s series of m i e r o g r a p h s do not a4ter this b a s i c configuration. W i t h r e s p e c t to conv e n t i o n a l t e c h n i q u e s for e l e c t r o n m i c r o s c o p y , the outer loop s h o u l d be quite fragile. Consequently, d e h y d r a t i o n or the s u r f a c e t e n s i o n forces generat e d by d r y i n g for c o n v e n t i o n a I e l e c t r o n m i c r o s c o p y p r e p a r a t i o n s m i g h t flatten it a g a i n s t the inner core and r e n d e r it i m p e r c e p t i b l e a n d r e s u l t in a k n o b b y a p p e a r a n c e . This might e x p l a i n somew h a t the v a r i a b l e d i a m e t e r s c~bserved for chrom a t i n b y different t e c h n i q u e s . Values b e t w e e n 70 a n d 150 ~ have been p u b l i s h e d [7, 13].
The X-ray scattering from a core and shell. A dense c e n t r a l c o r e w i t h a less dense r i n g (shell) is the r a d i a l p r o j e c t i o n of a tight i n n e r coil w i t h an outer loop. Such a r a d i a l d i s t r i b u t i o n of mass is v e r y c o m p a t i b l e w i t h the small angle X-ray a n d n e u t r o n s c a t t e r i n g f r o m c h r o m a t i n solutions.
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S. Bram.
Small angle X-ray and n e u t r o n s c a t t e r i n g show two Gaussian cross section regions. The one at smaller angles yields a cross section radius of g y r a t i o n of about 52 X, and the s e c o n d one gives 30 & [3, 4]. The s c a t t e r i n g c o r r e s p o n d i n g to the s e c o n d Gaussi,an regio.n ~ a s s h o w n some l i m e ago to be consistent w i t h a tight 45 ,~ p i t c h 3,0 X r a d i u s of g y r a t i o n coil c o n t a i n i n g about two t h i r d s of the total mass [3, 4]. The experimenta,1 55 A cross section r a d i u s of g y r a t i o n w o u l d c o r r e s p o n d to an outer r a d i u s of about 150 X. This value and the 45 X p i t c h central core p a r a m e t e r s agree very well w i t h the electron m i c r o g r a p h s . The c o n ~ a r i s o n can be c a r r i e d out in the other sense by cal, cu~lating the X-ray s c a t t e r i n g expected f r o m the s t r u c t u r e o b s e r v e d in the m i c r o g r a p h s . I n d e e d , the c a l c u l a t e d c u r v e s s h o w all of the imp o r t a n t features of the X-ray s c a t t e r i n g i n c l u d i n g the m o n o t o n i c decrease in i n t e n s i t y until about 40 ~ [4, 2]. AII p r e v i o u s m o d e l calculations based u p o n coils, coils c o n t a i n i n g solid p r o t e i n cores
and various other elongated forms gave v e r y sharp m a x i m a in this s c a t t e r i n g region [3, 4]. Thus, the X-ray scattering p r o v i d e s i n d e p e n d e n t s u p p o r t for the core-outer coil ultrastructure. F o r a double col,1 m o d e l the X-ray s c a t t e r i n g mass p e r unit length ~vould be d e r i v e d from the i n n e r cross section region instead of the outer one p r e v i o u s l y e m p l o y e d [41. A value greater than 22(~0 daltons /X wou~d then be calculated, giving a total DNA c o m p a c t i o n of 5.3 to 1. A 7:1 compaction has been d e t e r m i n e d by Griffith [14] from his electron m i c r o g r a p h s . Together, the outer and i n n e r coils of the freeze f r a c t u r e d sub-units have a DNA contour length of 6.0~) to 700 X. Then, about 600 3, of DNA is p a c k e d into a d i m e n s i o n of about 100 A.
Model Building. At this p o i n t a m o d e l can be constructed. It is a p p a r e n t that in o r d e r to h a v e the same c e n t e r of mass the outer and i n n e r coils nmst have opposite handedness. A v e r y rough mo-
Fro. 3. - - Freeze etched calf thymus ehromatin shadowed at 35 ° (× 650,000). The sub-unit nature is obvious ; however, the sub-unit separation is exaggerated due to embedding in the ice-matrix. Note, for example, that sub-units A and B are partially embedded. The outer loop is quite distinct about most sub-units and has an outer diameter of 150 to 180 A. The loops of sub-units C and D can be seen to cross over the center of the core. Double arrows, separated by 80 A, mark inner coils which appear to be two turns of a 40 to 50 A pitch coil.
BIOCHIMIE, 1975, 57, n ° 11-12.
A double coil c h r o m a t i n s u b - u n i t model. del can be o b t a i n e d by t w i s t i n g a flexible tube or r u b b e r b a n d about a rigid i n n e r support. Two t u r n s of h a n d e d n e s s opposite to the t w i s t i n g arise about the s u p p o r t a n d t h e n the tu~aing s p o n t a n e ously loops back over itself i n the opposite direction (to m i n i m i z e the stress). I1 has been previously noted that an over or u n d e r t u r n i n g of the DNA s e c o n d a r y structure could be r e s p o n s i b l e for the t e r t i a r y super coiling [5]. At present, we are not yet able to define the exact relative o r i e n t a t i o n of n e i g h b o r i n g subunits. If the core axes are almost p~arallel to the fibre direction, then an outer loop m a y w i n d over a n d be shared by two n e i g h b o r i n g sub-units a n d have an a p p r o x i m a t e p i t c h of about 2'40 X. Figure 4 depicts two s u b u n i t s in this o r i e n t a t i o n w h i c h appears to agree best w i t h most images. If the axes of n e i g h b o r i n g cores are almost p e r p e n d i c u l a r to the fibril, one outer loop with a p i t c h
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r i n g DNA folds i n s t e a d of coils m i g h t also be consistent w i t h the results. More precise i n f o r m a tion on the n a t u r e of the coils a n d the i n t e r - u n i t o r i e n t a t i o n s should be o b t a i n a b l e from a detailed analysis of the images a n d from the n e u t r o n scattering. No direct evidence for or against k i n k i n g [16] is observed however, a k i n k m a y well occur w h e r e the h a n d e d n e s s of the coiling changes sign.
Distribution of Histones. With regard to the histone localization, it seems that h i s t o n e H1 is b o u n d to the outer coil a n d m a y be p a r t i a l l y i n v o l v e d i n m a i n t a i n i n g c o n f o r m a t i o n . After H1 is r e m o v e d the basic s u b - u n i t s t r u c t u r e is conserved b u t the particles may b e s o m e w h a t larger. It is r e a s o n a b l e to suggest that the r e m a i n i n g four histones are associated w i t h the central coil, I n s u c h a scheme the p r o t e i n to DNA ratio i n the core w o u l d be somewhat larger t h a n 2: 1. P r o t e i n cores about w h i c h 10~)A d i a m e t e r DNA he,lices are w r a p p e d have been c o n s i d e r e d to e x p l a i n the X-ray [17] a n d n e u t r o n s c a t t e r i n g [lS]. However, an i n n e r coil of DNA i n t i m a t e l y b o u n d to twice its weight of p r o t e i n w o u l d agree m u c h better w i t h the neutron s c a t t e r i n g on dilute solutions [2]. The neutron scattering from c o n c e n t r a t e d c h r o m a f i n gels [18] w o u l d be compatible w i t h a DN,A rich shell about a p r o t e i n rich i n n e r coil. Comparison of the double coil model to other results. Although o n l y a general o u t l i n e of a possible s t r u c t u r e for c h r o m a t i n s u b - u n i t s has been presented here, the model allows i n t e r p r e t a t i o n of m a n y e x p e r i m e n t a l results. A most i m p o r t a n t finding to e x p l a i n is the p r e s e n c e of 170 to 200 base p a i r fragments in b r i e f DNAse digests [15, 16] a n d a 100 base p a i r one i n the ¢ limit digests >> [21, 22]. The former woul, d result from the total double coil u n i t w h i l e the latter could come from the compact i n n e r coil. In a s i m i l a r fashion, the compaction ratios of 6 : 1 a n d 3 : 1 often cited i n the literature [14, 17, 22] wou~d also be exp~lainable by the double coil model.
Fro. 4. - - A rough wire model approximating the DNA of two double coil sub-units in the orientation parallel to the fiber axis. The wire fibril, proportional to a 20 A thickness, makes two turns with a 40 A pitch and then coils over itself and a neighboring sub-unit.
of about 120 A may be associated w i t h each subunit. This w o u l d then be one t u r n of a P a r d o n , W E k i n s and R i c h a r d s s u p e r coil. [15]. I n a n y case, the s u b u n i t a n d especially the outer loop geometry must be s o m e w h a t distorted to accommodate the t e r t i a r y structure. A s t r u c t u r e featu-
BIOCHIMIE, 1975, 57, n ° 11-12.
!Diffraction reflections at 110 A i n wet gels a n d at 80 X i n dried c h r o m a t i n [15, 18] are i m p o r t a n t in the literature. U p o n c o n c e n t r a t i o n a fiber of double coil sub-units w o u l d first pack so that a n outer Ioop touches a n e i g h b o r i n g core, giving a s e p a r a t i o n of 1~I0 X (75 + 35 X). U p o n d e h y d r a tion the outer coil might collapses a n d contact of 70 to 80 A d i a m e t e r cores w o u l d be possible. Finally, the r e c e n t results on SV40 c h r o m a t i n [23] i m p l y i n g that there is only one effective sup e r coil t u r n per s u b - u n i t eliminates m a n y types of s u b - u n i t model structures. Interestingly, the
1306
S. Brain.
d o u b l e c o i l of t w o i n n e r t u r n s p l u s a n o u t e r o n e of o p p o s i t e h a n d e d n e s s has one effective turn p e r sub-unit.
A cdcno~wledgmen ls. I a m grateful to Dr. L. Chevance and Mr. S. Kouprach for t h e i r suggestions and technical assistance. Financial s u p p o r t w a s provided by the Centre National de la Recherche Seientifique and the D~l~gation G6n6rale h la Recherche Seientifique et Technique. An account of this w o r k ~was presented at the British Society for Cell Biology meeting or Chromatin in Gtasgow (Sept. 1975). REFERENCES. 1. 2. 3. 4. 5. 6. 7.
Bram, S. & Koupraeh, S. in preparation. Bandy, P., Brarfi, S. ,& Vastel, D. in preparation. Bram, S. (1968) Thesis University of Wisconsin. Brain, S. & Ris, H. (1971) J. Mol. Biol., 55, 325-336. Bram, S. (1972) Biochimie, 54, 1005-1011. Woodcock, C. L. F. (1973) J. Cell Biol., 54, 368. Olins, D. E. & Olins, A. L. (1974) Science, 183, 330332.
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8. Zalta, J., Zalta, J. P. ~ Simard, R. (1971) J. Celt. Biol., 51, 563-568. 9. Panyim, S., Bilik, B. ~ Chalkley, R. (1971) J. Biol. Chem., 246, 281-300. 10. Bolund, L. H. • Johns, E. W. (1973) Enr. J. Biochem., 35, 533-546. 11. B r a m , S., Butler-Browne, G., Baudy, P. & tbel, K. (1975) Proe. Natl. Acad. Sci. USA, 72, 1043. 12. Brain, S., Hellio, R. • Koupraeh, S. (1975) C. R. Aead. Sci., 281, 847-849. 13. Slayter, H. S., Sinh, T. G., Adler, A. J. ~ F a s m a n (1972) Biochemistry, ]J1, 3044-3054. 14. Griffith, J. D. (1975) Science, 187, 1202-1203. 15. Pardon, J. F., Wilkins, M. H. F. & Richards, B. M. (1967) Nature, 215, 508-509. 16. Crick, F. H. C. & Klug, A. (1975) Nature, 255, 530534. 17. Kornberg, R. D. (1974) Science, 184, 868-871. 18. Baldwin, J. P., Boseley, P. G., Bradbury, E. M. & Ibel, M. (1975) Nature, 253, 245-249. 19. Hewish, R. D. & Bnrgogne, L. A. (1973) Biochem. Biophgs. Res. Comm., 52, 504-510. 20. Noll, M. (1974) Nature, 251, 249-251. 21. Clark, R. J..& Felsenfeld, G. (1974) Biochemistry, 13, 3622-3628. 22. Sarasrabnddhe, C. a Van Hold, K. (19743 J. Biol. Chem., 249, 152-156. 23. Germond, J. E., Hirt, B., Gross-Bellard, M. & Chambon, P. (1975) Proc. Natl. Acad. Sci. USA, 72, 1843-1847.
