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Histone H3 thiol reactivity as a probe of nucleosome structure Wong, Norman Tse Ngon, 1978

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HISTONE H3 THIOL REACTIVITY AS A PROBE OF NUCLEOSOME STRUCTURE by NORMAN TSE NGON(WONG B.Sc, University of B r i t i s h Columbia, 1976. A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF THE FACULTY OF GRADUATE STUDIES Department of Biochemistry The University of B r i t i s h Columbia We accept th i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA October, 1978 MASTER OF SCIENCE xn (c) Norman In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f the r e q u i r e m e n t s f o r an advanced degree at the U n i v e r s i t y o f B r i t i s h C o lumbia, I agree t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u rposes may be g r a n t e d by the Head o f my Department o r by h i s r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d ' t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l 1 not be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . Department o f Biochemistry The U n i v e r s i t y o f B r i t i s h Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1WS Date Oct. 5, 1978. ABSTRACT Nucleosomes were prepared from trout t e s t i s n u clei by micrococcal nuclease digestion. The r e a c t i v i t y toward 3 N- [ethyl- Hlmaleimide (NEM) of the single sulfhydryl group of histone H3 i n the nucleosomes was studied under a variety of conditions. Under conditions of low io n i c strength, there i s neg l i g i b l e reaction of nucleosomes with NEM, suggesting that the c y s t e i n y l residue of H3 i s buried. Complete denaturation of nucleosomes i n 6 M guanidinium chloride leads to reaction of 2 moles of NEM per mole of nucleosomes, i n agreement with the expected presence of 2 moles of H3 per p a r t i c l e . Exposure of nucleosomes to 2 M NaCI or 1 M MgCl 2 leads to exposure of the t h i o l group. At higher Mg + + concentrations, the t h i o l group remains exposed, but i n NaCI solutions, as the s a l t concentration i s increased beyond 2 M, the t h i o l group returns to an inaccessible state. The r e a c t i v i t y of nucleosome t h i o l groups i s r e l a t i v e l y unaffected by urea to approximately 5 M. Between 5 and 8 M urea, a rapid increase i n t h i o l r e a c t i v i t y indicates a cooperative unfolding of the nucleosome core. When added together, urea and s a l t act i n a cooperative manner to expose the H3 sulfhydryl group. Mixtures of oligonucleosomes have also been studied under d i f f e r e n t conditions. They were found to behave i n a s i m i l a r f a s h i o n to monomers i n 6 M guanidine, but t h e i r t h i o l s r e a c t more s l o w l y than those of monomers i n hig h s a l t . Removal of the amino-terminal r e g i o n s of the core h i s t o n e s by t r y p t i c d i g e s t i o n has no n o t i c e a b l e e f f e c t on the a c c e s s i b i l i t y o f nucleosome t h i o l groups. I t i s concluded t h a t the carb o x y - t e r m i n a l r e g i o n of H3 c o n t a i n i n g Cys 110 i s masked mainly by h i s t o n e - h i s t o n e i n t e r a c t i o n s i n the octameric core complex, and i s l o c a t e d i n a r e g i o n which i s r e l a t i v e l y i n s e n s i t i v e to the p e r t u r b a t i o n s induced by t r y p s i n or low c o n c e n t r a t i o n s of urea. Nucleosomes r e c o n s t i t u t e d i n the presence of a s u l f -h y d r y l r e d u c i n g agent were i n d i s t i n g u i s h a b l e from n a t i v e p a r t i c l e s i n t h e i r r e a c t i v i t y to NEM i n low s a l t b u f f e r s , i n 2 M NaCl and i n 6 M guanidine h y d r o c h l o r i d e . These s t u d i e s i n d i c a t e t h a t the degree of exposure of H3 s u l f h y d r y l groups i n nucleosomes can be e f f e c t i v e l y monitored u s i n g NEM. The ca r b o x y - t e r m i n a l r e g i o n of H3 c o n t a i n i n g Cys 110 seems to be l o c a t e d i n a r e l a t i v e l y s t a b l e r e g i o n of the nucleosome core, perhaps a t the i n t e r f a c e between h e t e r o t y p i c tetramers. - i i i -TABLE OF CONTENTS Page ABSTRACT i TABLE OF CONTENTS i i i LIST OF TABLES v i LIST OF FIGURES v i ACKNOWLEDGEMENT v i i INTRODUCTION 1 I. E u k a r y o t i c chromatin 1 I I . Nucleosome s t r u c t u r e 2 I I I . I n t e r n a l arrangement of h i s t o n e s 5 IV. The a r g i n i n e - r i c h h i s t o n e s 6 V. The present i n v e s t i g a t i o n 8 MATERIALS AND METHODS 9 I. M a t e r i a l s and A b b r e v i a t i o n s 9 (a) M a t e r i a l s 9 (b) A b b r e v i a t i o n s 9 I I . P r e p a r a t i o n of Nucleosomes 10 (a) M i c r o c o c c a l nuclease d i g e s t i o n 10 (b) P r e p a r a t i o n of i n v i t r o l a b e l l e d nucleosomes 11 I I I . Reaction of nucleosomes with N-ethylmaleimide 12 (a) Reaction o f monomers i n non-dena t u r i n g s o l u t i o n s 12 (b) Reaction of monomers i n den a t u r i n g s o l u t i o n s 12 (c) Reaction of nucleosome oligomers .... 12 IV. T r y p s i n d i g e s t i o n o f nucleosomes 13 V. R e c o n s t i t u t i o n - o f nucelosomes 13 - i v -Page VI. DNase I digestion of native and reconstituted nucleosomes 14 VII. Gel electrophoresis of nucleosomes 14 (a) 15% SDS-polyacrylamide slab gel electrophoresis for analysis of nucleosomal proteins 14 (b) Non-denaturing 3% polyacrylamide gel electrophoresis of DNA 15 (c) Denaturing 99% formamide, 6% polyacrylamide gel electrophoresis of DNA 16 RESULTS 17 Isolation of nucleosomes 17 Sulfhydryl r e a c t i v i t y of native nucleosomes . 17 Ef f e c t of s a l t s on sulfhydryl r e a c t i v i t y .... 21 (a) E f f e c t of guanidinium chloride 21 (b) E f f e c t of sodium chloride on t h i o l r e a c t i v i t y 23 (c) E f f e c t of magnesium chloride on t h i o l r e a c t i v i t y 25 Ef f e c t of urea on sulfhydryl r e a c t i v i t y 27 Cooperative e f f e c t of NaCl and urea on nucleosome conformation 27 Sulfhydryl r e a c t i v i t y of nucleosome oligomers 3 0 Tryptic digestion of nucleosomes 30 Reconstitution of nucleosomes 35 DISCUSSIONS 39 Carboxy-terminal region of H3 i n native nucleosomes 39 Ef f e c t of sal t s on the structure of H3 40 Ef f e c t of urea on nucleosomes 4 3 Thiol r e a c t i v i t y of nucleosome oligomers .... 44 Trypsin digestion of nucleosomal proteins ... 45 - V -Page R e c o n s t i t u t i o n of nucleosomes 47 C o n c l u s i o n 4 9 REFERENCES 5 0 - v i -LIST OF TABLES Page Table I Reaction of trypsin-digested mononucleosomes with N-ethylmaleimide 3 3 LIST OF FIGURES Figure 1. Bio-Gel A-5M column p r o f i l e of nucleosomes from 30 minutes of micrococcal nuclease digestion 18 2. SDS-polyacrylamide gel electrophoresis of the monomer peak f r a c t i o n from an A-5M column 19 3. Quantitation of [3H] counts from NEM-label l e d nucleosomal proteins 20 4. Time course of reaction of nucleosomes with NEM i n denaturing and non-denaturing conditions 22 5. E f f e c t of varying molarity of Gdn*HCl on t h i o l r e a c t i v i t y of nucleosomes 24 6. E f f e c t of io n i c strength on t h i o l r e a c t i v i t y of nucleosomes 26 7. E f f e c t of urea concentration on the t h i o l r e a c t i v i t y of nucleosomes 28 8. Synergistic e f f e c t of s a l t and urea on exposure of nucleosome t h i o l groups 29 9. Sulfhydryl r e a c t i v i t y of oligomers i n denaturing and non-denaturing solutions .... 31 10. E f f i c i e n c y of trypsin digestion 34 11. Time course of incorporation of [3H]-NEM into reconstituted nucleosomes 36 12. DNase I digestion of nucl e i , nucleosome monomers and reconstituted nucleosomes 38 - v i i -ACKNOWLEDGEMENT I wish to thank Dr. Peter Candido for his guidance, comments and supervision during the course of study. Jim Davie requires special mention for his constructive comments, helpful discussions and assistance. I would also l i k e to acknowledge the Medical Research Council for i t s support of these studies i n the form of a studentship. - 1 -INTRODUCTION I. E u k a r y o t i c chromatin Despite t h e i r g r e a t v a r i e t y and complexity, a l l eukary-o t i c organisms c o n t a i n i n the nucleus a n u c l e o p r o t e i n complex, the chromatin. This chromosomal m a t e r i a l c o n s i s t s of DNA, h i s t o n e s , nonhistones p r o t e i n s and a s m a l l amount of RNA. The s t r u c t u r e of the chromatin i s maintained by the i n t e r a c t i o n s among DNA and h i s t o n e s . Although the r o l e s of the components i n chromatin are not f u l l y e s t a b l i s h e d , d i f f e r e n t f u n c t i o n s have been assigned to v a r i o u s components on the b a s i s of p r e v i o u s experiments (1). The h i s t o n e s , b a s i c p r o t e i n s of f i v e major c l a s s e s , are p r e s e n t i n approximately equal weight w i t h DNA i n chromatin. They are thought to be important i n m a i n t a i n i n g chromatin s t r u c t u r e and to a c t as a "coarse" c o n t r o l of gene a c t i v i t y . In s p i t e of the l i t t l e v a r i e t y i n type, h i s t o n e s can be c o v a l e n t l y m o d i f i e d by p h o s p h o r y l a t i o n , a c e t y l a t i o n , m e t h y l a t i o n , e t c . (2), probably to modulate DNA-histone and h i s t o n e - h i s t o n e i n t e r a c t i o n s to b r i n g about changes i n chromatin s t r u c t u r e and f u n c t i o n . The nonhistone p r o t e i n s , a heterogeneous p o p u l a t i o n of d i f f e r e n t p r o t e i n s p e c i e s , are p r e s e n t i n small amounts i n i n t e r p h a s e chromatin. Although the f u n c t i o n s of nonhistone p r o t e i n s are not understood, there are s e v e r a l l i n e s of evidence to i n d i c a t e r e g u l a t o r y r o l e s f o r some o f them: these p r o t e i n s were found to i n c r e a s e i n amount i n g e n e t i c a l l y - 2 -a c t i v e t i s s u e s (3), induce t r a n s c r i p t i o n of a c t i v e genes (3-4) and a l t e r DNA c o n f i g u r a t i o n (5). However, the mode o f r e g u l a t i o n i s y e t to be e l u c i d a t e d . I I . Nucleosome s t r u c t u r e A major advance i n r e s e a r c h on chromatin s t r u c t u r e was the d i s c o v e r y of the "nucleosomes" or "v-bodies" i n 1973-4 (6-9). The most c o n v i n c i n g evidence emerged from nuclease d i g e s t i o n s t u d i e s (6, 8). Hewish and Burgoyne (6) found t h a t an endogenous nuclease of r a t l i v e r has the c a p a c i t y to c l e a v e chromatin to m u l t i p l e s o f a subuni t of about 200 base p a i r s o f DNA i n l e n g t h . T h i s o b s e r v a t i o n was subsequently confirmed by N o l l (8). At about the same time, Woodcock (10) and O l i n s & O l i n s (9) observed the appearance of a "beads-on-a-string" s t r u c t u r e from chromatin i n e l e c t r o n micrographs. Thus, the presence of a r e p e t i t i v e subunit i n chromatin i s e s t a b l i s h e d both by b i o c h e m i c a l and mor p h o l o g i c a l s t u d i e s . Since the d i s c o v e r y o f the nucleosome, i n v e s t i g a t o r s began to ask how the h i s t o n e s and DNA are arranged w i t h i n the nucleosomes. Experiments were done to measure the DNA content (11-13), to d e f i n e the r e l a t i v e l o c a t i o n s o f DNA i n the nucleosomes (14), to f i n d the s t o i c h i o m e t r y of h i s t o n e s (15-7), and to study the p r o p i n q u i t y o f h i s t o n e s (16-23) and conform-a t i o n a l changes of h i s t o n e s under a v a r i e t y o f condtions (24, 25). - 3 -The f i r s t measurements of the DNA unit size gave values of 205 base pairs (8) and 180-230 bp (26). These experiments were done by f i r s t digesting the chromatin into fragments consisting of multiples of a unit size by commer-c i a l l y available micrococcal nuclease; then the fragments were resolved by gel electrophoresis and t h e i r m o b i l i t i e s were compared to DNA standards. The DNA sizes thus* obtained varied from 140-220 bp i n d i f f e r e n t organisms and tissues. Since i n some experiments the DNA fragments from d i f f e r e n t sources were analyzed on the same gel, t h i s v a r i a t i o n i n size was not due to experimental error. It i s now generally agreed that monomers of chromatin prepared by mild digestion contain DNA segments of 180-205 bp (27); further digestion of these monomers yi e l d s DNA fragments of 140-170 bp bound by whole histones with Hi or H5. The histone content of the nucleosome has been derived from reconstitution experiments and c r o s s - l i n k i n g studies (15-7). It has been found that a l l of the four smaller histones, H2A, H2B, H3 and H4 are required i n equal molar r a t i o to generate the c h a r a c t e r i s t i c 125 8 p a r t i c l e s i n the electron microscope (15). The appearance of a cross-linked octamer of histones observed by Thomas and Romberg (16-7) further confirmed the existence of two each of the foru small histones. This stoichiometry of histones was obtained from chromatin of d i f f e r e n t sources (28); however, deviations of 30-50% are frequently obtained, probably due to inaccuracy i n measurements. The r o l e o f Hi i n a s s o c i a t i o n w i t h the nucleosome has been under i n v e s t i g a t i o n by v a r i o u s workers. I t i s assumed t h a t only one H i molecule i s a s s o c i a t e d w i t h each nucleosome and i t i s e i t h e r a s s o c i a t e d with the DNA spacer r e g i o n o f 30-45 bp or i t i n t e r a c t s with the nucleosomal DNA on the o u t s i d e o f the nucleosomes. The a s s o c i a t i o n of Hi with DNA was supported by Whitlock & Simpson who demonstrated the exposure of DNA upon the removal of Hi i n 0.7 M NaCI (29). Neve r t h e l e s s , s i n c e HI i s the onl y h i s t o n e to d i s s o c i a t e from the nucleosomes i n low s a l t c o n c e n t r a t i o n s (30) and i s most s u s c e p t i b l e to p r o t e o l y s i s , many r e s u l t s were greeted with s u s p i c i o n . Current view f a v o r s the suggestion t h a t HI c r o s s -l i n k s between nucleosomes and t h a t Hi i s n o n - e s s e n t i a l i n ma i n t a i n i n g the b a s i c nucleosome s t r u c t u r e (27), however, H i may be i n v o l v e d i n the formation of hig h order s t r u c t u r e of chromatin (3 2). E x t e n s i v e d i g e s t i o n of the nucleosome monomer produces the "trimmed" monomer of 140 bp i n l e n g t h . T h i s d e g r a d a t i o n product w i t h 140 bp of DNA and e i g h t h i s t o n e s (two each of H2A, H2B, H3 and H4)' i s termed the "core p a r t i c l e " and the remaining DNA which i s v a r i a b l e i n l e n g t h i n d i f f e r e n t c e l l types i s termed the " l i n k e r " as i t connects the nucleosomes. The e a r l i e s t p h y s i c a l s t u d i e s suggested the core p a r t i c l e to be roughly s p h e r i c a l and about 110 R i n diameter (33). Neutron s c a t t e r i n g data has r e c e n t l y shown t h a t the shape of the core p a r t i c l e to be a f l a t c y l i n d e r w i t h o v e r a l l dimension of 11 x 11 x 6 nm (34-5). The DNA was found to be e i t h e r - 5 -f o l d e d or s u p e r c o i l e d on the o u t s i d e of the nucleosome (34, 36). More i n f o r m a t i o n about nucleosome core shape has now been provided by F i n c h e t a l . who were able to i s o l a t e the core p a r t i c l e s i n c r y s t a l form (37). X-ray d i f f r a c t i o n measure-ments of the c r y s t a l s gave s i m i l a r r e s u l t s to neutron s c a t t e r i n g d ata. I l l . I n t e r n a l arrangement of h i s t o n e s The arrangement of the h i s t o n e s w i t h i n the core p a r t i c l e has been under i n t e n s e i n v e s t i g a t i o n , with the aim of e l u c i d a t i n g the mechanism of packaging of the DNA and the i n t e r m o l e c u l a r r e l a t i o n s h i p s among the h i s t o n e s and DNA. The l o c a t i o n s of the v a r i o u s h i s t o n e s w i t h r e s p e c t to each other were s t u d i e d mainly by c r o s s l i n k i n g experiments, i n which the h i s t o n e s were c r o s s l i n k e d by chemical agents or by UV l i g h t (16-23). A n a l y s i s of dimers and t r i m e r s thus formed d e f i n e s the r e l a t i v e l o c a t i o n of each h i s t o n e . Hi was found not to be i n v o l v e d i n any c r o s s l i n k s except w i t h other Hi molecules to form a homopolymer. The formation of m u l t i p l e s of c r o s s l i n k e d octamers and the formation of polymeric Hi i n d i c a t e t h a t the nucleosome i s i n c o n t a c t w i t h an adjacent one. F i v e of the ten p o s s i b l e dimers from the four small h i s t o n e s have a l s o been found, but the occurrence of the others cannot be r u l e d out, as they may be p r e s e n t i n small amounts (31). S t u d i e s of the a s s o c i a t i o n p r o p e r t i e s of h i s t o n e s i n s o l u t i o n r e v e a l e d the tetramer ( H 3 ) 2 ( H 4 ) 2 , which may i t s e l f d e f i n e the core p a r t i c l e (38-9), e.g. by i n d u c i n g s u p e r c o i l i n g - 6 -of the DNA, while the H2A-H2B dimer may aid s t a b i l i z a t i o n of the complete nucleosome (40). The roles of H2A and H2B were supported by reports that the two histones can a l t e r the op-t i c a l a c t i v i t y and conformation of l o c a l regions of DNA (41). Early attempts to demonstrate the octamer i n solution have been unsuccessful because of i t s i n s t a b i l i t y i n low i o n i c strength; however, hexamers, tetramers, dimers and even heterotypic tetramers comprising each of H2A, H2B, H3 and H4 have a l l been found (16-7, 42). Recently Chung et a l . were able to i s o l a t e the core complex i n high s a l t s and do analysis on i t (43). They showed that the octamer exists i n equilibrium with a heterotypic tetramer i n 2 M NaCI. The conservative sequences of H3 and H4 lead to the inference that the tetramer (H3) 2(H4) 2 i s important i n defining the basic f o l d of DNA i n the core while H2A and H2B are for s t a b i l i z a t i o n purpose. H2A and H2B may have other s p e c i f i c functions as well since they are required for the formation of the native nucleosomes. Variation i n the l i n k e r region i s probably due to the v a r i a t i o n of Hi sequence i n d i f f e r e n t organisms (1). IV. The arginine-rich histones Histones H3 and H4 are similar i n terms of arginine content, sequence conservation, s a l t e l u t i o n from DNA, etc. They were found to associate i n solution to form a tetramer which i s most important i n packaging the core DNA and inducing super-c o i l i n g of DNA (28, 44-6). The (H3) 2(H4) 2 tetramer also pro-tects DNA against nuclease digestion i n a manner si m i l a r to that - 7 -found f o r core p a r t i c l e s (47). B i r o c and Reeder (25) have examined the r e a c t i o n of H4 t y r o s i n e s w i t h i o d i n e i n Xenopus. When i o d i n a t e d Xenopus H4 were d i g e s t e d with t r y p s i n and e l e c t r o p h o r e s e d a t pH 3.5, four h e a v i l y l a b e l l e d t r y p t i c p e p t i d e spots were ob t a i n e d . By assuming the same H4 sequence i n Xenopus as i n c a l f thymus, they were ab l e to a s s i g n each of the four t y r o s i n e s i n the amino a c i d sequence to a l a b e l l e d t r y p t i c p e p t i d e . Thus the r e a c t i v i t y o f i n d i v i d u a l t y r o s i n e c o u l d be analyzed. From a n a l y s i s of the r e a c t i v i t y o f the t y r o s i n e r e s i d u e s under v a r i o u s c o n d i t i o n s , they showed t h a t two of the four t y r o s i n e s are b u r i e d when the h i s t o n e s are a t t a c h e d to n a t i v e chromatin. However, i f the chromatin was put i n t o 2 M NaCI or 5 M urea or both, a l l of the r e a c t i v i t i e s went up 5-10 f o l d , i n d i c a t i n g t h a t the s u p e r c o i l i n g of the chromatin and h i s t o n e -h i s t o n e i n t e r a c t i o n s are both r e s p o n s i b l e f o r the p r o t e c t i o n of the t y r o s i n e r e s i d u e s . One of the t y r o s i n e s , t y r 88, i n c r e a s e s i t s r e a c t i v i t y w i t h i o d i n e to maximum at 0.5 M NaCI, the i o n i c c o n d i t i o n f o r the d i s s o c i a t i o n of H i , i n f e r r i n g t h a t t y r 8 8 i s i n v o l v e d i n i n t e r a c t i o n between HI and H4, or i s exposed by conformational.change upon d i s s o c i a t i o n of H i . H3 i s the o n l y h i s t o n e t h a t c o n t a i n s c y s t e i n e m o i e t i e s w i t h i n i t s amino a c i d sequence (48-9). In higher v e r t e b r a t e s , two c y s t e i n e s are found f o r each molecule of H3; i n lower v e r t e b r a t e s , such as c h i c k e n and f i s h e s , o n l y one c y s t e i n e i s present (50). I t i s t h e r e f o r e easy to s p e c i f i c a l l y l a b e l H3 by u s i n g s u l f h y d r y l reagents. Hyde and Walker (24) s t u d i e d - 8 -c a l f thymus H3 by r e a c t i n g whole chromatin w i t h 5,5'-d i t h o - b i s - [ 2 - n i t r o b e n z o i c a c i d ] , a s p e c i f i c t h i o l reagent. They found t h a t o n l y one of the two c y s t e i n e groups i s r e a c t i v e under normal c o n d i t i o n s . The r a t e of r e a c t i o n as w e l l as the t o t a l r e a c t i v i t y i n c r e a s e d as the chromatin was put i n t o s a l t s o l u t i o n s . Maher and Candido (51) a l s o found t h a t there i s n e g l i -g i b l e r e a c t i o n between t r o u t t e s t i s nucleosomes and p-hydroxy mercurobenzoate under non-denaturing c o n d i t i o n . The r e a c t i o n was g r e a t l y enhanced when the nucleosomes were denatured. These r e s u l t s i n d i c a t e the p o s s i b i l i t y of s t u d y i n g the t h i o l r e a c t i v i t y as a probe of H3 and even nucleosome s t r u c t u r e . V. The p r e s e n t i n v e s t i g a t i o n The p r e s e n t i n v e s t i g a t i o n concerns the r e a c t i v i t y toward N-ethylmaleimide of the H3 t h i o l groups i n t r o u t t e s t i s nucleosomes under v a r i o u s c o n d i t i o n s , i n c l u d i n g d i f f e r e n t s a l t c o n c e n t r a t i o n s and urea s o l u t i o n s . The r e a c t i v i t y of t r y p s i n -d i g e s t e d and r e c o n s t i t u t e d nucleosomes are a l s o analyzed. Changes i n the t h i o l r e a c t i v i t y may s i g n i f y a l t e r a t i o n s i n H3 conformation and consequently, changes i n nucleosome s t r u c t u r e . N-ethylmaleimide i s a u s e f u l s u l f h y d r y l reagent w i t h f r e e c y s t e i n e , p e p t i d e s or p r o t e i n s . I t can be analyzed by spectrophotometric methods or by r a d i o a c t i v e l a b e l l i n g . The l a t t e r was employed i n these s t u d i e s . In t h i s t h e s i s , data on the r e a c t i o n of nucleosome t h i o l groups w i t h NEM are r e p o r t e d . The r e s u l t s are u s e f u l i n f o r m u l a t i n g a d e t a i l e d p i c t u r e of h i s t o n e - h i s t o n e and histone-DNA i n t e r a c t i o n s i n the nucleosome. - 9 -MATERIALS AND METHODS I. M a t e r i a l s and A b b r e v i a t i o n s (a) M a t e r i a l s N a t u r a l l y maturing t r o u t t e s t e s were o b t a i n e d from Sun V a l l e y Trout Farm, M i s s i o n , B. C. M i c r o c o c c a l nuclease (E.C.3.1.4.7.), deoxyribonuclease I, t r y p s i n (E.C. 3.4.4.4.), soybean t r y p s i n i n h i b i t o r , d i t h i o t h r e i t o l were purchased from Sigma. L a b e l l e d N-ethyl-[ 3H]-maleimide was from New England Nuclear and u n l a b e l l e d N-ethylmaleimide from A l d r i c h Chemical Co., Wisconsin. Sequencing grade heptane used to d i s s o l v e l a b e l l e d N-ethylmaleimide was obtained from P i e r c e Chemicals Company. N a - [ l - 1 4 C ] - a c e t a t e and aqueous c o u n t i n g s c i n t i l l a n t were purchased from Amersham. Minicon B15 c o n c e n t r a t i o n c e l l s were from Amicon Corp. G l a s s f i b r e f i l t e r s used f o r N-ethylmaleimide assays were obtained from Reeve Angel. Acrylamide was from Matheson, Coleman and B e l l ; N, N'-methylene-bis-acrylamide from Bio-Rad; and TEMED (N,N,N',N'-tetramethyl-ethylenediamine) from Ames Company. A l l other chemicals and reagents were of the h i g h e s t p u r i t y or reagent grade. D i s t i l l e d water was used f o r a l l s o l u t i o n s . (b) A b b r e v i a t i o n s EDTA: e t h y l e n e d i a m i n e - t e t r a a c e t i c a c i d SDS: sodium dodecyl s u l f a t e TMKS b u f f e r : T r i s - H C l (50 mM, pH 7.4), MgCl 2 (1 mM), sucrose (0.25 M) , and B-mercaptoethanol (15 mM) . - 10 -Tris-EDTA buffer: Tris-HCl (lOmM, pH 7.5), and EDTA (0.7 mM). PBS buffer: NaCI (0.14 M) , KCI (27 mM) , NaaHPO., (8 mM) , K H 2 P O 4 (1.5 mM), CaCl 2 (0.9 mM), MgCl 2 (0.5 mM), pH 7.2. NEM: N-ethylmaleimide Gdn«HCl: guanidinium chloride TCA-tungstate: 10% t r i c h l o r o a c e t i c acid, and 0.5% sodium tungstate, pH 2.0. DTT: d i t h i o t h r e i t o l DNase I: deoxyribonuclease I II. Preparation of Nucleosomes (a)' Micrococcal nuclease digestion Nucleosomes were prepared as described by Davie and Candido (53) with some modifications. Nuclei were isola t e d from 6-8 g of trout testes by homogenizing i n TMKS buffer i n a Waring Blendor for 2 minutes. After centrifugation at 3,000 x g for 10 minutes, the p e l l e t was resuspended i n TMKS buffer and homogenization and c e n t r i -fugation repeated. The nuclei i n the p e l l e t were suspended i n 6 ml of TMKS buffer containing 1 mM CaCl 2 (5 x 10 8 n u c l e i / ml), and digested with micrococcal nuclease at a f i n a l concen-t r a t i o n of 300 units/ml for 30 minutes at 37°. The reaction was stopped by adding EDTA to 10 mM, and placing the mixture on i c e . The mixture was centrifuged at 12,000 x g for 10 minutes and the supernatant was discarded. Chromatin monomers and multimers were released by vigorous hand-homogenization - 11 -of the p e l l e t i n a g l a s s - t e f l o n homogenizer i n 6 ml of T r i s -EDTA b u f f e r . The chromatin subunits (monomers and multimers) were c o l l e c t e d i n the supernatant a f t e r 30 minutes of c e n t r i f u g a t i o n a t 12,000 x g. To separate the monomers from multimers, the supernatant was passed through a B i o - G e l A-5M column (107 cm x 2 cm) (52) e q u i l i b r a t e d w i t h Tris-EDTA b u f f e r . The column was run o v e r n i g h t a t a flow r a t e o f 8 ml/hr a t 4°. The monomer and multimer peak f r a c t i o n s were pooled and s t o r e d a t -8 0° u n t i l f u r t h e r use. About 40 A 2 6 o u n i t s of monomer and 5 A 2 6 o u n i t s of oligomer were obtained per gram of t r o u t t e s t i s . (b) P r e p a r a t i o n of i n v i t r o l a b e l l e d nucleosomes Three grams of f r e s h t r o u t t e s t e s were minced and homogenized i n PBS b u f f e r i n a g l a s s - t e f l o n homogenizer. The homogenate was f i l t e r e d through c h e e s e c l o t h and then c e n t r i -fuged a t 3,000 x g f o r 10 minutes. The c e l l s were suspended i n 4.4 ml PBS b u f f e r , 0.6 ml p e n i c i l l i n - s t r e p t o m y c i n (100 u n i t s ml), 1.2 ml Waymouth's medium ( 5 3 ) , and 0.3 ml sodium 1 - [ 1 4 C ] -a c e t a t e (50 uCi/ml). The mixture was incubated i n a g y r a t o r y water bath a t 16 ° f o r 4 hr, a f t e r which time 25 ml PBS b u f f e r was added and the l a b e l l e d c e l l s c o l l e c t e d by c e n t r i f u g a t i o n a t 3,000 x g f o r 10 minutes. When the c e l l s were not used immediately, they were s t o r e d at -80°. Nucleosome monomers were prepared from the l a b e l l e d c e l l s by d i g e s t i o n w i t h m i c r o c o c c a l nuclease as d e s c r i b e d above. - 12 -III. Reaction of nucleosomes with N-ethylmaleimide (a) Reaction of monomers i n non-denaturing solution Five m i c r o l i t r e s of N-ethyl-[ 3H]maleimide (80 mCi/mmol) containing 1.25 yCi were added to 0.5 ml of nucleosome monomers (2 A 2 6 0/ml) at ambient temperature (^22°) i n T r i s -EDTA buffer. F i f t y m i c r o l i t r e aliquots were taken at time inter v a l s and put into TCA-tungstate at 0° to stop the reaction. The acid p r e c i p i t a b l e material was co l l e c t e d on glass f i b r e f i l t e r s and washed three times with TCA-tungstate, and once each with ethanol and ether. The f i l t e r discs were dried and counted i n 5 ml of aqueous counting s c i n t i l l a n t . Control reactions, i n which the concentrations of NEM and nucleosomes were varied, showed that the reagent was not l i m i t i n g under these conditions. (b) Reaction of nucleosome monomers i n denaturing solutions One A 2 6 0 unit of nucleosome monomers was concentrated to 0.25 ml i n a Minicon B15 c e l l and added to the appropri-ate amount of s a l t , urea or Gdn«HCl. The reaction mixture was brought to 0.5 ml by addition of Tris-EDTA buffer, and the NEM reaction carried out as described above. (c) Reaction of nucleosome oligomers with NEM Nucleosome oligomers were coll e c t e d from the excluded peak after separation of micrococcal nuclease digest products on a Bio-Gel A-5M column. One A 2 6 0 unit of oligonucleosomes was subjected to t h i o l analysis with NEM under denaturing and non-denaturing conditions as described above. - 13 -IV. T r y p s i n d i g e s t i o n of nucleosomes Nucleosomes (2 A 2 6o/ml) were incubated w i t h 10 ug/ml of t r y p s i n i n Tris-EDTA b u f f e r a t 20°, f o r the d e s i r e d p e r i o d . To stop the r e a c t i o n , 0.5 ml of the i n c u b a t i o n mixture was added to 10 y l of soybean t r y p s i n i n h i b i t o r (1 mg/ml) and the mixture c h i l l e d to 0°. A f t e r 30 minutes, the NEM r e a c t i o n was performed as mentioned above. When the e f f i c i e n c y of t r y p s i n d i g e s t i o n was checked u s i n g [ 1 ^ C ] - l a b e l l e d nucleosomes, 1.8 ml of nucleosomes (1.5 A 2 6 0 / m l ) was added to 200 m i c r o l i t r e s of t r y p s i n (70 ug/ml). At time i n t e r v a l s , 200 m i c r o l i t r e a l i q u o t s were taken out and mixed w i t h 5 m i c r o l i t r e s o f t r y p s i n i n h i b i t o r (0.4 mg/ml). F i f t y m i c r o l i t r e samples were counted i n 5 ml of aqueous counting s c i n t i l l a n t a f t e r p r e c i p i t a t i o n onto f i l t e r d i s c s as d e s c r i b e d above. V. R e c o n s t i t u t i o n of nucleosomes Monomer nucleosomes (2 ml, 1.6 A 2 6 0 / m l ) were d i s s o c i a t e d i n Tris-EDTA b u f f e r c o n t a i n i n g 2 M NaCl, 5 M urea and 1 mM DTT (or 10 M urea and 1 mM DTT). The s a l t and urea were removed by d i a l y s i s a g a i n s t 1 l i t r e o f Tris-EDTA + 1 mM DTT fo r 5 hr. The r e c o n s t i t u t e d nucleosomes were then d i a l y z e d a g a i n s t three 1 l i t r e changes o f Tris-EDTA b u f f e r f o r 3 hr each, to remove the re d u c i n g agent. Reactions w i t h NEM were then performed under d e n a t u r i n g and non-denaturing c o n d i t i o n s as d e s c r i b e d above. - 14 -VI. DNase I digestion of native and reconstituted nucleosomes Nucleosomes (20 A 2 6 o/ml) were preincubated i n Tris-EDTA buffer at 37° for 10 minutes. After the incubation period, the solution was made 2.7 mM i n MgCl 2 + 5.4 mM NaCI to complex the EDTA and to provide the Mg++: and Na* , concentrations for optimal DNase I digestion. DNase I (1 mg/ml) was then added to a f i n a l concentration of 0.02 mg/ml and the nucleosomes were digested for 5 minutes at 37°. The reaction was stopped by placing the mixture on ic e . The nuclease digested nucleosomes were then analyzed on denaturing polyacrylamide gels. VII. Gel electrophoresis of nucleosomes (a) 15% SDS-polyacrylamide slab gel electrophoresis for analysis of nucleosomal proteins F i f t e e n per cent polyacrylamide-sodium dodecyl sulfate slab gels were made using a modified Laemmli procedure (52). The following volumes of stock solutions: - 15 ml of I (30.0 g acrylamide, 0.4 g N,N 1-methylenebisacrylamide i n 100 ml of H 20), 0.15 ml of II (20 mg ammonium persulfate freshly dissolved i n 2 ml H 20), 0.3 ml of III (10 g SDS i n 100 ml of water) and 7.5 ml of IV (1.5 M T r i s - C l , pH 8.8) -were combined with 10 1 of TEMED and 7.0 5 ml of water, and polymerized i n 1.5 mm slabs under t-butanol. Nucleosome samples were l y o p h i l i z e d and then heated i n 4% SDS, 0.125 M T r i s , pH 6.8, 10% B-mercaptoethanol, 20% gly c e r o l and 0.002% bromophenol blue. The gel was run at 15 milliamperes for 7-8 hr i n 0.05 M Tris,pH 8.0, 0.384 M glycine and 0.1% SDS. When the gel was to be dissected and s o l u b i l i z e d , 0.6% N,N'-dia l l y l t a r t a r d i a m i d e was used as crosslinker instead of b i s a c r y l a m i d e (54). A f t e r e l e c t r o p h o r e s i s , the g e l was s t a i n e d i n Coomassie B r i l l i a n t Blue i n methanol:acetic a c i d : water, 5:1:5, and d e s t a i n e d o v e r n i g h t i n 5% methanol and 7.5% a c e t i c a c i d . (b) Non-denaturing 3% p o l y a c r y l a m i d e g e l e l e c t r o -p h o r e s i s of DNA Monomeric and o l i g o m e r i c f r a c t i o n s from A-5M columns were analyzed i n 3% p o l y a c r y l a m i d e g e l s as d e s c r i b e d by Loening (55). The f o l l o w i n g volumes of stock s o l u t i o n s : - 5 ml of lOx concentrated TEA b u f f e r (tea b u f f e r = 0.04 M T r i s - a c e t i c a c i d , pH 7.8, 2 mM EDTA, 0.02 M sodium a c e t a t e ) , 7.5 ml of 20% acrylamide s o l u t i o n (acrylamide: N,N'-methylene-b i s a c r y l a m i d e , 19:1), 0.5 ml of 10% SDS, 0.4 ml of 10% ammonium p e r s u l f a t e - were combined with 36.6 ml of water and deaerated. Then 4 0 y l of TEMED was added and the acrylamide was polymerized under t - b u t a n o l i n 1.5 mm s l a b s . Nucleosome samples were f i r s t p r e c i p i t a t e d i n 10 mM MgCl2, and then protease K mixture (50 0 yg/ml Protease K, 10 mM EDTA, 2% SDS) was added to d i g e s t the p r o t e i n s . The d i g e s t i o n was c a r r i e d out a t 37° f o r 1 hr, a f t e r which the d i g e s t i o n mixture was made 4% SDS, 30 mM EDTA, 20% g l y c e r o l and a p p l i e d d i r e c t l y to the g e l . The g e l s were prerun at 100 V f o r 3 hr and running c o n d i t i o n s were 50 V f o r 10 min, fo l l o w e d by 100 V f o r 4 hr. Gels were s t a i n e d i n ethidium bromide (10 yg/ml) f o r 10 min and d e s t a i n e d b r i e f l y i n water. Bands were v i s u a l i z e d under UV l i g h t . - 16 -(c) Denaturing 99% formamide, 6% polyacrylamide gel electrophoresis of DNA These gels were described by Staynov et a l . (56) . 99% formamide was s t i r r e d with Dowex 50W-X8 (3 g/100 ml) for 1 hr, and was then f i l t e r e d and used the same day. 2.04 g of acrylamide, 0.36 g of N,N'-methylenebisacrylamide and 8 0 1 of TEMED were dissolved i n 4 0 ml of formamide, which was then f i l t e r e d . The gel was made 2 0 mM i n phosphate and 0.12% i n ammonium persulfate by adding 0.8 ml of M sodium phosphate pH 7.0, containing 50 mg of ammonium persulfate. A 15 x 15 x 0.15 cm slab gel was poured. Nucleosome samples were f i r s t dissolved i n M NaCl, 2.5 M urea; they were then precipitated overnight i n 2 volumes of ethanol.- The pre c i p i t a t e was taken up i n formamide (containing 20 mM sodium phosphate, pH 7.0, 20% sucrose, 0.005% bromophenol blue), heated to 100 , cooled on i c e , and electrophoresed i n formamide-20 mM phosphate at 160 V for 5 hours. Gels were stained and destained as described above. - 17 -RESULTS I s o l a t i o n of Nucleosome Nucleosomes were i s o l a t e d as d e s c r i b e d i n M a t e r i a l s and Methods. A t y p i c a l A-5M p r o f i l e i s shown i n f i g u r e 1. The excluded f r a c t i o n was pooled to c o n s t i t u t e the o l i g o m e r i c f r a c t i o n , and the monomer f r a c t i o n was obtained from the peak f r a c t i o n s as i l l u s t r a t e d . The monomer f r a c t i o n was found to c o n t a i n only t r a c e s of H i when analyzed on 15% SDS po l y a c r y l a m i d e s l a b g e l s (Figure 2). This was taken to mean t h a t most of the DNA " t a i l s " of the p a r t i c l e s had been removed (11, 57). Very low amounts of non-histone p r o t e i n s were observed as r e p o r t e d b e f o r e (52, 58). The o l i g o m e r i c f r a c t i o n i s a mixture of oligonucleosomes ranging from dimers (about 50%), t r i m e r s (about 30%) to multimers, as observed on a 3% non-denaturing acrylamide s l a b g e l (not shown). S u l f h y d r y l r e a c t i v i t y o f n a t i v e nucleosomes The i n c o r p o r a t i o n o f NEM l a b e l i s a measure of the degree of a c c e s s i b i l i t y of the t h i o l group i n the H3 molecules w i t h i n the nucleosome core. The m o d i f i c a t i o n of H3 was confirmed by a n a l y s i s of the NEM-treated nucleosomal p r o t e i n s under den a t u r i n g c o n d i t i o n s on 15% SDS-polyacrylamide g e l s . More than 80% of the i n c o r p o r a t e d l a b e l was a s s o c i a t e d w i t h h i s t o n e H3 (Figure 3). The counts a s s o c i a t e d with other h i s t o n e s are probably due to s i d e r e a c t i o n s o f NEM wit h amino and im i d a z o l e groups i n these p r o t e i n s (59). - 18 -I oligomers j monomers FRACTION NO., 4 ml F i g u r e 1. B i o - G e l A-5M column p r o f i l e o f nucleosomes from 30 minutes of m i c r o c o c c a l nuclease d i g e s t i o n . Trout t e s t i s n u c l e i were d i g e s t e d with m i c r o c o c c a l nuclease f o r 30 minutes as d e s c r i b e d i n " M a t e r i a l s and Methods". The d i g e s t i o n products were f r a c t i o n e d on a B i o - G e l A-5M column. The absorbance a t 260 nm o f each f r a c t i o n i s p l o t t e d v s . the f r a c t i o n number. F i g u r e 2. SDS-polyacrylamide g e l e l e c t r o p h o r e s i s o f the monomer peak f r a c t i o n from an A-5M column. One A2 e ou n i t of the monomer peak f r a c t i o n was l y o p h i l i z e d and then heated i n 40 y l o f sample b u f f e r . The sample was a p p l i e d to a 15% SDS-polyacrylamide s l a b g e l and run as d e s c r i b e d i n M a t e r i a l s and Methods. The g e l was s t a i n e d i n Coomassie Blue. - 20 -9 O X FRACTION NO. Figure 3. Labelling of H3 with N-ethylmaleimide. Two A 2 6 0 units of nucleosomes were l a b e l l e d with [ 3H]N-ethylmaleimide in 10 M urea at ambient temperature for 5 hr. Tfie reaction mixture was dialyzed against Tris-EDTA buffer and l y o p h i l i z e d . The l y o p h i l i z e d material was taken up i n 100 y l and 50 y l was applied to a 15% SDS-polyacrylamide slab gel and run as described i n Materials and Methods. After electrophoresis-, the gel was stained and s l i c e d . The s l i c e s were s o l u b i l i z e d i n 3 ml of 2% periodic acid for 2 days at room temperature. The s o l u b i l i z e d gel s l i c e s were counted i n 10 ml of aqueous counting s c i n t i l l a n t . - 21 -Hyde and Walker (24) r e p o r t e d the i n a c c e s s i b i l i t y o f one o f the two c y s t e i n e s i n whole chromatin o f c a l f thymus under non-denaturing c o n d i t i o n s . I t i s thus i n t e r e s t i n g to observe the behaviour o f the s o l e c y s t e i n e r e s i d u e i n t r o u t t e s t i s h i s t o n e H3 under s i m i l a r c o n d i t i o n s . When nucleosomes i n Tris-EDTA b u f f e r were exposed to NEM, an extremely low r e a c t i v i t y was observed (Figure 4). The i n c o r p o r a t i o n was very r a p i d , and e s s e n t i a l l y complete i n 10 minutes. This r e a c t i o n may be due to t r a c e s o f denatured nucleosomes or of f r e e H3 or H3 fragments. The p o s s i b i l i t y o f s i d e r e a c t i o n s can be r u l e d out as these r e a c t i o n s occur much slower. Previous experiments u s i n g p-hydroxymercuri-benzoate a l s o showed the H3 s u l f h y d r y l groups of t r o u t t e s t i s nucleosomes to be u n r e a c t i v e (51). O l i n s e t al.(60) i n t h e i r p h y s i c a l s t u d i e s o f the e f f e c t s o f urea on nucleo-somes, had a l s o r e p o r t e d the u n a v a i l a b i l i t y o f the t h i o l groups i n ch i c k e n e r y t h r o c y t e s nucleosomes. These r e s u l t s are c o n s i s t e n t w i t h the present f i n d i n g s . E f f e c t of s a l t s on s u l f d y d r y l r e a c t i v i t y Three d i f f e r e n t s a l t s were used i n the s t u d i e s of H3 t h i o l r e a c t i v i t y with NEM: Gdn«HCl, NaCI and MgCl 2. (a) E f f e c t of guanidinium c h l o r i d e Guanidinium c h l o r i d e (Gdn«HCl) i s known to d i s s o c i a t e h i s t o n e s from DNA, and to d e s t r o y secondary and t e r t i a r y s t r u c t u r e i n h i s t o n e s and p r o t e i n s i n g e n e r a l . When nucl e o -somes i n 6 M Gdn«HCl were allowed to r e a c t w i t h NEM, a - 22 -I i r= ft TIME OF REACTION, MIN Figure 4. Time course of reaction of nucleosomes i n denaturing and non-denaturing conditions. Monomers were reacted with [3HJNEM under the indicated conditions as described i n Materials and Methods. The s p e c i f i c r e a c t i v i t y of monomers was estimated by assuming an absorbance of 20 per mg DNA per ml at 260 nm, and a DNA molecular weight of 110,000. maximum of 2.2 moles NEM per mole nucleosomes was bound w i t h i n 5 minutes (Figure 4). T h i s maximum i s i n good agreement w i t h the expected presence of two moles of H3 per nucleosome monomer, and i n d i c a t e s t h a t i n 6 M Gdn-HCI the c y s t e i n e r e s i d u e s are f u l l y exposed. The e f f e c t o f v a r y i n g m o l a r i t y of Gdn*HCl on the r e l e a s e of h i s t o n e s from DNA were a l s o s t u d i e d and the r e s u l t s are shown on f i g u r e 5. When nucleosomes were exposed to i n c r e a s i n g Gdn«HCl c o n c e n t r a t i o n s , n e g l i g i b l e r e a c t i v i t y a s s o c i a t e d with the n a t i v e p a r t i c l e s p e r s i s t e d u n t i l the Gdn«HC1 c o n c e n t r a t i o n exceeded 0.5 M. The r e a c t i v i t y i n c r e a s e d r a p i d l y and reached the maximum of two moles NEM per mole nucleosome at 1.2 M. T h i s p l a t e a u l e v e l d i d not change even a t 6 M Gdn•HCI. (b) E f f e c t o f sodium c h l o r i d e on t h i o l r e a c t i v i t y Nucleosome s u l f h y d r y l r e a c t i v i t y was f i r s t analyzed i n 2 M NaCl s o l u t i o n s . The nucleosomes e x h i b i t e d a moderately r a p i d r a t e of r e a c t i o n w i t h NEM, a t t a i n i n g a r a t i o o f 1.0 mole reagent per mole nucleosomes a t approximately 3 0-4 0 minutes, and r e a c h i n g a maximum of 1.6 moles/mole a t th r e e hours (Figure 4). T h i s l a r g e i n c r e a s e i n r e a c t i v i t y may be a t t r i b u t e d to a d i r e c t unmasking of the c y s t e i n y l r e s i d u e upon removal of the DNA, or t o a c o n f o r m a t i o n a l change i n the h i s t o n e core induced by. the removal of DNA. The l a t t e r p o s s i b i l i t y seems more probable when the r e s u l t s of s a l t c o n c e n t r a t i o n s t u d i e s are taken i n t o account (see below). - 24 -3 4 [Gdn HCl] , M F i g u r e 5. E f f e c t o f v a r y i n g m o l a r i t y of Gdn'HCl on t h i o l r e a c t i v i t y o f nucleosomes. The s p e c i f i c a c t i v i t y of [ 3H]NEM i n c o r p o r a t e d i s p l o t t e d as a f u n c t i o n of Gdn*HCl c o n c e n t r a t i o n i n Tris-EDTA b u f f e r . The r e a c t i o n s were c a r r i e d out f o r 120 minutes. When nucleosomes were placed i n varying NaCI concen-trations and the t h i o l r e a c t i v i t y analyzed, a completely d i f f e r e n t p r o f i l e from that of Gdn«HCl was obtained. The r e a c t i v i t y of nucleosomes did not increase u n t i l the s a l t concentration was above 1.0 M (Figure 6), when H3 sta r t s to dissociate from the DNA (3 0). The r e a c t i v i t y increased from 1.0 M to 1.5 M, the concentration at which histones are completely dissociated. The r e a c t i v i t y then started to drop at 1.75 M and reached a minimum of 0.4 moles reagent per mole nucleosome at 4 M. The maximum r e a c t i v i t y i s probably achieved when the histones dissociate from DNA, and before any reassociation occurs. The histones may then reassociate to form heterotypic tetramers and octamers at 2 and 4 M NaCI solutions respectively (61). (c) E f f e c t of magnesium chloride on t h i o l r e a c t i v i t y NEM r e a c t i v i t y of nucleosomes was also examined i n MgCl 2 solutions of increasing concentration. A similar p r o f i l e to that of Gdn'HCl was obtained (compare figures 4 and 6). The histones began to dissociate from DNA at an ionic strength of 0.5 as monitored by an increase i n t h i o l r e a c t i v i t y . The r e a c t i v i t y reached a maximum of 2 moles/mole at r/2 = 1.2, which p e r s i s t s as the MgCl 2 concentration was increased. The s i m i l a r i t y i n the behaviour of nucleosomes i n Gdn*HCl and MgCl 2 solutions i s probably due to the fact that l i k e Gdn*HC1, Mg + + also has a denaturing e f f e c t on the nucleosomal proteins (62) . _ 26 -I ! i i i I I i i \ ' » ' | * 0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 IONIC STRENGTH Figure 6. E f f e c t of i o n i c strength on t h i o l r e a c t i v i t y of nucleosomes. The nucleosomes were allowed to react with [3H]NEM i n MgCl 2 ( 8 ) and NaCl solutions(•) for 60 minutes. The s p e c i f i c a c t i v i t i e s are plotted as a function of i o n i c strength of the solutions. - 27 -Ef f e c t of urea on sulfhydryl r e a c t i v i t y Figure 7 shows the e f f e c t of increasing urea concen-trations on the r e a c t i v i t y of nucleosome t h i o l groups. Very l i t t l e change i n r e a c t i v i t y was seen u n t i l 5-6 M urea was reached, where an abrupt t r a n s i t i o n began, centered at approximately 8.5 M urea. Since urea has a disruptive e f f e c t on hydrophobic interactions, the re s u l t s suggest that there i s a cooperative unfolding of nucleosome structure above 7 M urea, leading to f u l l exposure of H3 t h i o l groups at 9-10 M urea. Olins et al. have demonstrated the same behaviour with chicken erythrocyte nucleosomes i n urea solutions (60). The present data are in good agreement with t h e i r r e s u l t s . Cooperative e f f e c t of NaCl and urea on nucleosome conformation Nucleosomes were exposed to various combinations of s a l t and urea concentrations, and the r e a c t i v i t y with NEM was measured. The re s u l t s are summarized i n figure 8. A maximum of 2 moles NEM per mole nucleosomes was attained when nucleosomes i n 6 M urea were further exposed to 0.5 M NaCl (Figure 8A). Conversely, i f nucleosomes i n 0.5 M NaCl were exposed to increasing urea concentrations, a steady r i s e i n NEM incorporated was observed u n t i l the urea concen-t r a t i o n reached 6 M (Figure 8B) . Since neither 0.'5 M NaCl nor 6 M urea alone allows s i g n i f i c a n t incorporation of the label l e d NEM (Figures 6 and 7), they must act cooperatively to a l t e r nucleosome conformation. In 2 M NaCl, only 3-4 M - 28 -t ' 1 T r [UREA], M F i g u r e 7. E f f e c t of urea c o n c e n t r a t i o n on the r e a c t i v i t y of nucleosomes with NEM. Nucleosomes i n s o l u t i o n s of d i f f e r e n t urea c o n c e n t r a t i o n s were r e a c t e d with[ 3H]NEM f o r 150 minutes. The s p e c i f i c a c t i v i t y i s p l o t t e d as a f u n c t i o n of urea m o l a r i t y . D i f f e r e n t symbols r e p r e s e n t d i f f e r e n t monomer p r e p a r a t i o n s . - 29 -0.2 0.4 0.6 0 [NaCl], M 2 4 6 0 [Urea] , M 2 4 6 [Urea] , M F i g u r e 8. S y n e r g i s t i c e f f e c t o f s a l t and urea on exposure of nucleosome t h i o l groups. Nucleosomes were exposed to v a r i o u s combinations o f NaCl and urea c o n c e n t r a t i o n s f o r 6 0 min, and the r e a c t i o n with [ 3H]NEM was c a r r i e d out f o r 24 hr a t 22°. In panel A, the r e a c t i o n i n 6 M urea i s p l o t t e d as a f u n c t i o n of NaCl c o n c e n t r a t i o n . In B and C, the r e a c t i o n i n 0.5 M and 2 M NaCl, r e s p e c t i v e l y , i s p l o t t e d as a f u n c t i o n of urea c o n c e n t r a t i o n . - 30 -urea was s u f f i c i e n t to allow complete r e a c t i o n of nucleosomal t h i o l groups w i t h NEM (Figure 8 C ) . S u l f h y d r y l r e a c t i v i t y of nucleosome oligomers The excluded f r a c t i o n of an A-5M column, which c o n t a i n s n e g l i g i b l e amount of monomers, was allowed to r e a c t w i t h NEM under d e n a t u r i n g and non-denaturing c o n d i t i o n s . F i g u r e 9 shows the extent of r e a c t i o n of oligonucleosomes w i t h NEM i n 10 mM T r i s , p H 7.4 + 0.7 mM EDTA, 2 M NaCl, 6 M urea, 2 M NaCl + 6 M urea and 6 M Gdn*HCl. The behaviour of oligomers under these c o n d i t i o n s was very s i m i l a r , i f not i d e n t i c a l t o , t h a t o f monomers, except f o r the r e s u l t s i n 2 M NaCl. Again, the s y n e r g i s t i c e f f e c t o f NaCl and urea was observed. However, i n 2 M NaCl, o n l y 0.5 SH groups per mole nucleosome r e a c t e d a f t e r 60 minutes, u s i n g oligonucleosomes, vs. approximately 1.2 SH/mole u s i n g monomers (Figure 3). The reason f o r t h i s behaviour i s u n c l e a r . T r y p t i c d i g e s t i o n o f nucleosomes Sahasrabuddhe and Van Holde (63) found t h a t chromatin p a r t i c l e s i s o l a t e d by nuclease d i g e s t i o n underwent a dramatic change i n sedimentation v e l o c i t y from a v a l u e of 11S to approximately 5-7S upon d i g e s t i o n w i t h t r y p s i n . T h i s s h i f t was accompanied by o n l y a small decrease i n molecular weight, and was t h e r e f o r e a t t r i b u t e d l a r g e l y to a conform-a t i o n a l change, i . e . , an u n f o l d i n g of the p a r t i c l e . Weintraub and Van Lente (64) subsequently showed t h a t t r y p t i c d i g e s t i o n of chromatin leads to the l o s s of only the N-terminal 20-30 F i g u r e 9. S u l f h y d r y l r e a c t i v i t y o f oligomers i n d e n a t u r i n g and non-denaturing s o l u t i o n s . R e a c t i v i t y of oligomers w i t h NEM was determined i n the f o l l o w i n g s o l u t i o n s : •, 2 M NaCI and 6 M urea; o, 6 M Gdn*HCl; A , 2 M NaCI; • , 6 M urea; •, Tris-EDTA b u f f e r . - 32 -a m i n o a c i d r e s i d u e s o f t h e c o r e h i s t o n e s . T h i s s u g g e s t e d t h a t t h e l o s s o f t h e s e b a s i c r e g i o n s c o u l d t r i g g e r a c o n f o r m a t i o n a l c h a n g e i n n u c l e o s o m e s . S i n c e t h e s e r e g i o n s a r e p h o s p h o r y l a t e d a n d e x t e n s i v e l y a c e t y l a t e d i n v i v o ( 6 5 ) , t h e s e m o d i f i c a t i o n s c o u l d p r o v i d e d a m e c h a n i s m f o r a l t e r i n g n u c l e o s o m e c o n f o r m a t i o n d u r i n g t r a n s c r i p t i o n o r DNA s y n t h e s i s . I t w a s t h e r e f o r e o f i n t e r e s t t o m o n i t o r t h i o l r e a c t i v i t y i n n u c l e o s o m e s f o l l o w i n g d i g e s t i o n w i t h t r y p s i n . T a b l e I s h o w s t h e a m o u n t o f N E M i n c o r p o r a t e d a f t e r 60 m i n u t e s o f r e a c t i o n i n T r i s - E D T A b u f f e r , 2 M N a C I o r i n 6 M G d n - H C l a f t e r t r y p s i n d i g e s t i o n f o r v a r i o u s t i m e s . I t w a s o b s e r v e d t h a t t r y p s i n d i g e s t i o n h a d n o n o t i c e a b l e e f f e c t o n t h e b a s a l r e a c t i v i t y o f m o n o m e r s t o N E M i n T r i s - E D T A b u f f e r . I f G d n - H C l w a s a d d e d t o 6 M a f t e r t r y p s i n d i g e s t i o n , e s s e n t i a l l y s t o i c h i o m e t r i c r e a c t i o n o f t h e t h i o l s o c c u r s ( 1 . 6 - 1 . 7 5 m o l e s S H / m o l e n u c l e o s o m e s ) , a s e x p e c t e d . T h e r e a c t i v i t y i n 2 M N a C I s o l u t i o n a l s o d i d n o t c h a n g e a f t e r t r y p t i c d i g e s t i o n ( 1 . 0 m o l e a t 60 m i n u t e s c o m p a r e d t o 1.2 m o l e s / m o l e b e f o r e t r e a t m e n t w i t h t r y p s i n ) ( F i g u r e 3 a n d T a b l e I ) . T o c o n f i r m t h a t t h e a m i n o - t e r m i n a l r e g i o n s w e r e r e m o v e d u n d e r s u c h c o n d i t i o n s , i n v i t r o f - i ^ . i - a c e t a t e l a b e l l e d m o n o m e r s w e r e s u b j e c t e d t o t r y p s i n d i g e s t i o n u n d e r s i m i l a r c o n d i t i o n s . I t w a s f o u n d t h a t m o r e t h a n 8 0 % o f t h e l a b e l w a s l o s t a t 3 0 - 4 0 m i n u t e s o f d i g e s t i o n ( F i g u r e 1 0 ) . S i n c e e - a c e t y l g r o u p s a r e p r e s e n t o n l y i n t h e a m i n o - t e r m i n a l r e g i o n s o f t h e c o r e h i s t o n e s ( 6 6 - 7 0 ) , t r y p s i n w a s e f f e c t i v e i n c l e a v i n g t h e s e - 33 -' Table I Reaction of trypsin-digested mononucleosomes  with N-ethylmaleimide m- ^ Reactivity i n Time of J Digestion (min) Tris-EDTA 2 M NaCl 6 M Gdn•HCI (moles N-ethylmaleimide/mole monomer) 0 0.05 0.82 1.75 2 0.05 1.03 1.51 5 0.05 0/85 1.63 10 0.04 1.03 1.61 30 0.05 1.03 1.61 Nucleosomes digested for 0-30 min with trypsin were allowed to react with NEM for 6 0 min i n .the indicated s a l t solutions at ambient temperature. The acid-precipitable f r a c t i o n was counted as described i n Materials and Methods. - 34 -Time of digestion min F i g u r e 10. E f f i c i e n c y o f t r y p s i n d i g e s t i o n . [lkC]~ l a b e l l e d nucleosomes were d i g e s t e d w i t h t r y p s i n f o r the a p p r o p r i a t e time as d e s c r i b e d i n M a t e r i a l s and Methods. The percentage of l a b e l l e f t i n the a c i d - p r e c i p i t a t a b l e f r a c t i o n i s p l o t t e d a g a i n s t the time of d i g e s t i o n . - 35 -reg i o n s from the nucleosomes. The r e s u l t s show t h a t t r y p t i c d i g e s t i o n does not cause any s i g n i f i c a n t c o n f o r m a t i o n a l changes i n the environment of the t h i o l groups. R e c o n s t i t u t i o n of nucleosomes The r e c o n s t i t u t i o n of nucleosomes from s a l t - d i s s o c i a t e d h i s t o n e s and DNA has been r e p o r t e d by a number of workers (71-74). These r e c o n s t i t u t e d nucleosomes resemble the n a t i v e monomers i n s t r u c t u r e (15) and i n nuclease d i g e s t i o n p a t t e r n (74). We have r e c o n s t i t u t e d nucleosomes from 2 M NaCl + 5 M urea (or 10 M urea) s o l u t i o n s by d i a l y s i s , and monitored the t h i o l r e a c t i v i t y u s i n g NEM. R e c o n s t i t u t e d nucleosomes showed very l i t t l e r e a c t i o n w i t h NEM (Figure 11). A d d i t i o n of NaCl to 2 M markedly i n c r e a s e d the exposure of t h i o l s i n the r e c o n s t i t u t e d p a r t i c l e s , g i v i n g a time course of r e a c t i o n much l i k e t h a t of n a t i v e p a r t i c l e s (e.g. compare F i g u r e 4 and 11). Exposure of r e c o n s t i t u t e d nucleosomes to 6 M Gdn«HCl l e d to the i n c o r p o r a t i o n o f almost 2 moles of NEM per mole of p a r t i c l e s , as w i t h n a t i v e p r e p a r a t i o n s . In order t o o b t a i n h i g h y i e l d s of r e c o n s t i t u t e d nucleosomes w i t h normal behaviour towards NEM, i t was necessary to c a r r y out the r e c o n s t i t u t i o n i n the presence of a red u c i n g agent, e.g. 1 mM DTT. Omission of the r e d u c i n g agent y i e l d e d p a r t i c l e s i n which a p r o p o r t i o n o f the H3 t h i o l s was u n r e a c t i v e to NEM even under d e n a t u r i n g c o n d i t i o n s , presumably due to formation of H3 i n t e r m o l e c u l a r - 36 -Co 2.0i i i i TIME OF REACTION, MIN F i g u r e 11. Time course of i n c o r p o r a t i o n of[ 3H]NEM i n t o r e c o n s t i t u t e d nucleosomes. The s p e c i f i c a c t i v i t y of NEM i n c o r p o r a t e d i n t o r e c o n s t i t u t e d nucleosomes under d e n a t u r i n g and non-denaturing c o n d i t i o n s i s p l o t t e d a g a i n s t the time of r e a c t i o n . S o l i d l i n e s , nucleosomes r e c o n s t i t u t e d i n the presence of 1 mM DTT. Dashed l i n e , Nucleosomes r e c o n s t i t u t e d i n the absence of r e d u c i n g agent. d i s u l f i d e s (Figure 1 1 , dashed l i n e ) . In order to f u r t h e r support the hypothesis t h a t the r e c o n s t i t u t e d nucleosomes have a s i m i l a r conformation to the n a t i v e ones, DNase I d i g e s t i o n was performed and the d i g e s t products were analyzed on a 9 9 % formamide, 6% acrylamide g e l . As seen from f i g u r e 1 2 , the g e l p a t t e r n of d i g e s t products from n u c l e i , nucleosome monomers ( n a t i v e ) , and r e c o n s t i t u t e d nucleosomes are s i m i l a r . The c h a r a c t e r i s t i c "ladder" p a t t e r n i s c l e a r l y v i s i b l e . T h i s i n d i c a t e s t h a t nucleosomes can be r e c o n s t i t u t e d a f t e r the d i s s o c i a t i o n of h i s t o n e s from DNA and t h a t the r e c o n s t i t u t e d nucleosomes resemble the n a t i v e p a r t i c l e s by s e v e r a l c r i t e r i a . - 38 -Native monomers Reconstituted Nuclei monomers 1 F i g u r e 1 2 . DNase I d i g e s t i o n o f n u c l e i , nucleosome monomers and r e c o n s t i t u t e d nucleosomes. Nucleosomes were d i g e s t e d w i t h DNase I and a n a l y z e d on 99% formamide, 6% p o l y -a c r y l a m i d e g e l as d e s c r i b e d i n M a t e r i a l s and Methods. The g e l s were s t a i n e d i n e t h i d i u m bromide and p h o t o g r a p h e d under UV l i g h t . - 39 -DISCUSSIONS Carboxy-terminal region of H3 i n native nucleosomes It i s evident from the low r e a c t i v i t y of the H3 t h i o l group i n native nucleosomes that the cysteine residue of H3 i n trout t e s t i s i s buried within the protein core. Although the complete amino acid sequence of trout t e s t i s H3 has not been determined, the position of the single cysteine residue i s i nferred from comparative data from other lower vertebrates to be at position 110 (1). The unreactive cysteine indicates that the carboxyl end of the protein i s folded i n such a way that the t h i o l group i s masked and unavailable for reaction. • Hyde and Walker (24) reported that one of the two t h i o l groups i n c a l f thymus H3 was inaccessible to 5,5'-dithiobis-[2-nitrobenzoic acid] i n chromatin at low s a l t concentrations; the other t h i o l was found to be exposed to solvent. They inferred from primary sequence analysis that the buried t h i o l group i s at position 96. The present data suggest that the protected t h i o l group i n c a l f thymus H3 very l i k e l y corresponds, in i t s position i n the amino acid sequence, to the single sulfhydryl residue of trout t e s t i s and chicken erythrocyte H3, which i s located at position 110. This i s reasonable since the cysteine at position 96 i n c a l f thymus H3 i s one of the rare amino acids that are not conserved i n the primary sequence of H3. I t i s l i k e l y that t h i s cysteine i s non-esse n t i a l for proper H3 orientation i n the nucleosomes. - 40 -The u n r e a c t i v e s u l f h y d r y l group i n t r o u t t e s t i s H3 has p r e v i o u s l y been demonstrated u s i n g p-hydroxymercuri-benzoate (51). However, the p r e v i o u s method of a n a l y s i s was not as s e n s i t i v e as the p r e s e n t one; q u a n t i t a t i v e r e a c t i o n ( i . e . 2 moles reagent per mole nucleosomes) c o u l d not be obtained w i t h the former method. E f f e c t of s a l t s on the s t r u c t u r e of H3 Conformational changes of nucleosomes i n s o l u t i o n s of d i f f e r e n t i o n i c s t r e n g t h s have been s t u d i e d i n v a r i o u s l a b o r a t o r i e s (61, 75-7). The secondary s t r u c t u r e of nucleosomal h i s t o n e s was found to be a f f e c t e d by NaCl and d i v a l e n t ions a t low i o n i c s t r e n g t h s i n hydrodynamic s t u d i e s (75), e l e c t r o n microscopy (76) and s o l u b i l i t y analyses (77). The e f f e c t of high s a l t c o n c e n t r a t i o n s on nucleosome s t r u c t u r e was s t u d i e d by O l i n s (61) who found t h a t the predominant s p e c i e s of the h i s t o n e core complex a t an i o n i c s t r e n g t h of 2 was a tetramer w h i l e a t an i o n i c s t r e n g t h of 4, the octamer predominates. Three d i f f e r e n t s a l t s were employed i n the p r e s e n t i n v e s t i g a t i o n : Gdn"HCI, NaCl and MgCl 2- Both M g + + and Gdn«HCl have d e n a t u r i n g e f f e c t s on p r o t e i n s and the be-haviour of nucleosomal p r o t e i n s i n these s a l t s o l u t i o n s are s i m i l a r . A maximal r e a c t i v i t y of two moles reagent per mole nucleosomes was obtained a t about 1.2-1.5 i o n i c s t r e n g t h . At t h i s i o n i c s t r e n g t h , the h i s t o n e s are d i s s o c i a t e d from the DNA and must t h e r e f o r e be denatured by the s a l t s to achieve maximal r e a c t i v i t y . The p l a t e a u r e a c t i o n w i t h these s a l t s was reached - 41 -at a lower ionic strength than with NaCI. This i s probably due to the fact that both s a l t s are more e f f i c i e n t than Na + at displacing histones from DNA. Furthermore, the reaction maximum reached i n these s a l t s was higher than that in NaCI solutions. These r e s u l t s may be attributed to an unfolding e f f e c t of Mg + + and Gdn + on histone conformation at high s a l t concentrations. Nucleosomes i n NaCI solutions exhibit a completely d i f f e r e n t behaviour i n the reaction with NEM. In 2 M NaCI, the t h i o l groups react with NEM at a moderately rapid rate, attaining a r a t i o of 1.0 mole reagent/mole nucleosomes at approximately 30-40 min, and reaching a miximum of 1.6 moles/mole at three hours (Figure 4). This i s much slower than the reaction i n Gdn*HCl which attains the maximum of 2 moles/mole within 5 minutes. Under these conditions, the histones are dissociated from the DNA (30), and several l i n e s of evidence suggest that the native structures of the histones are retained: (i) s a l t - d i s s o c i a t e d histones read i l y reasso-cia t e with DNA to y i e l d normal-appearing nucleosomes once the s a l t has been removed (11, 57); ( i i ) Thomas and Romberg (16) demonstrated the existence of histone octamers i n s a l t dissociated histones, similar to those observed i n native chromatin; ( i i i ) Weintraub and Van Lente (64) observed the same t r y p t i c histone cores whether nucleosomes or s a l t -dissociated histones were digested with trypsin. Two p o s s i b i -l i t i e s can explain the behaviour of H3 i n 2 M NaCI: either - 42 -there i s a d i r e c t unmasking of the c y s t e i n y l residue upon removal of the DNA,or a conformational change i n the histone core occurs as a r e s u l t of d i s s o c i a t i o n of the DNA. An example of the l a t t e r p o s s i b i l i t y would be d i s s o c i a t i o n of a histone octamer to two heterotypic tetramers (4 2), with the H3 t h i o l s being present at the boundary of interacting tetramers. Studies on the r e a c t i v i t y of the H3 t h i o l group i n varying s a l t concentrations favor the l a t t e r of the above two alte r n a t i v e s . The r e a c t i v i t y of H3 t h i o l groups increases from 1.0 M to 1.5 M, the concentration at which the histones are completely dissociated, and f a l l s markedly after reaching a maximum at 2.0 M. At 4.0 M, the r e a c t i v i t y approaches the low l e v e l seen i n control nucleosomes at low s a l t concentration. Under these conditions the histones are completely dissociated from the DNA; therefore, the masking of the t h i o l groups i n 4 M NaCI must be due to the formation of histone-histone interactions. These data are consistent with a model i n which the t h i o l groups become exposed due to d i s s o c i a t i o n of a histone octamer at 1.5-2.0 M NaCI, but become buried again upon reformation of an octameric structure at 3.5-4.0 M NaCI. Olins (61), on the basis of hydrodynamic studies, reached similar conclusions regarding histone interactions over these s a l t concentrations. The fact that the maximum r e a c t i v i t y obtained i n NaCI solutions i s only 1.6 moles SH/mole nucleosomes i s probably due to the formation of H3-H3 interactions which promote intermolecular d i s u l f i d e formation (see below, under "recons-t i t u t i o n " ) , thus gradually rendering the t h i o l s unreactive toward NEM. This process would be less favorable with t o t a l l y denatured H3 i n Gdn'HCI solutions (6 M). In support of t h i s interpretation, Hyde and Walker (24) found that both cy s t e i n y l residues of acid-extracted (and therefore presumably denatured) c a l f thymus H3 reacted rapidly with 5,5 1 - d i t h i o b i s -[2-nitro-benzoic acid], but that upon incubation of the histone in 2 M NaCl, the reaction rate steadily decreased. That the reaction of nucleosomes with NEM occurs much more slowly i n 2 M NaCl than i n 6 M Gdn«HCl probably r e f l e c t s the heterogeneous nature of the histone-histone interactions under these conditions (4 2). A gradual s h i f t i n the octamer-tetramer equilibrium towards the tetramer, and/or the p o s s i b i -l i t y that some of the structures "breathe" may allow the gradual t i t r a t i o n of the t h i o l groups. E f f e c t of urea on nucleosomes Olins et al . demonstrated the disruptive e f f e c t of urea on chromatin, using hydrodynamic studies (60). They were able to d i s t i n g u i s h non-cooperative changes i n structure which were attributed to t r a n s i t i o n s of the outer DNA-rich s h e l l of the p a r t i c l e , and cooperative changes between 5 and 10 M urea, attributed to changes i n the protein core. The existence of the l a t t e r cooperative effects i s thus confirmed by our data on t h i o l r e a c t i v i t y . In high urea (>8 M), we i n f e r that the histones, although s t i l l attached to DNA through basic regions, are completely denatured leading to - 44 -exposure of the H3 t h i o l s . A similar scheme was presented by Olins et al.(60). The inference reached above was further confirmed by the studies i n various combinations of s a l t and urea concentrations. The s a l t and urea were found to act syner-g i s t i c a l l y i n increasing the r e a c t i v i t y of H3 t h i o l s . When nucleosomes were put i n 6 M urea and 0.5 M NaCl, the maximum incorporation of 2 moles NEM/mole was attained. Since neither 6 M urea nor 0.5 M NaCl alone allow s i g n i f i c a n t reaction of NEM with H3 sulfhydryl groups, these agents must act cooperatively to a l t e r nucleosome conformation. I t i s thus concluded that i n nucleosomes the H3 sulfhydryl groups at position 110 are protected from NEM by both histone-histone and histone-DNA interactions. In the presence of 0.5 M NaCl and 6 M urea, they may become f u l l y exposed due to a) a rupture of some io n i c histone-DNA interactions which d e - s t a b i l i z e s the nucleosome and allows the urea to extensively denature H3, or b) a conformational change i n the nucleosome which exposes the sulfhydryl groups without extensive denaturation of the histones. Th i o l r e a c t i v i t y of nucleosome oligomers Oligonucleosomes d i f f e r from nucleosome monomers in the association of Hi and i n incl u s i o n of the DNA spacer regions. When such oligomers were subjected the same treatments as nucleosome monomers, similar r e s u l t s were obtained. This indicates that the association with Hi and a longer spacer region has no s i g n i f i c a n t e f f e c t on the buried t h i o l s i n H3. The carboxy-terminal region of H3 i s therefore probably not involved i n interactions among adjacent nucleosomes. There was, however, a s l i g h t difference observed i n 2 M NaCI, i n which the oligomers reacted at a faster rate but attaining lower maximum l e v e l . The reason for t h i s discrepancy i s unknown. Perhaps other proteins present i n t e r a c t with the dissociated histones. Since quantitative reaction of the t h i o l s of oligonucleosom.es occurs i n 6 M Gdn* HCl, the lower r e a c t i v i t y i n s a l t i s not due to i n a c t i v a t i o n of the NEM i t s e l f . Trypsin digestion of nucleosomal proteins Studies of the primary structures of the histones have revealed that the p o s i t i v e l y charge l y s y l and arginyl residues are d i s t r i b u t e d i n clusters near the ends of the protein molecules (1). The amino terminal region has the greatest density of p o s i t i v e charge and was thought to be the primary s i t e of i n t e r a c t i o n with DNA. This region i s more susceptible to proteolysis than other regions of the histones. E a r l i e r investigations had shown a dramatic change i n sedimentation v e l o c i t y of the nucleosomes upon t r y p t i c digestion (63). Subsequently, the conformational change accompanying trypsin digestion was confirmed by nuclease digestion of trypsin-treated nucleosomes (78). Recently, Whitlock and Stein reported that the removal of the NH2~terminal histone regions with trypsin produces r e l a t i v e l y small changes i n the folding of core p a r t i c l e - 46 -DNA (79). They suggested that the central and COOH-terminal histone regions are important i n forming the protein core as well as s t a b i l i z i n g the DNA within the core p a r t i c l e complex. In our experiments, trypsin digestion of the nucleosomes was found to have no e f f e c t on the r e a c t i v i t y of the t h i o l groups. Since the H3 sulfhydryl groups are situated i n the COOH-terminal region, our results suggest that even i f there i s any large conformational change induced upon removal of the amino-terminal region, these changes must not a f f e c t the environment around cysteine 110 of H3 ( i . e . the carboxyl end). Weintraub and Van Lente (64) have shown that the t r y p s i n -r e s i s t a n t region i s not affected by 2 M NaCl, but i s affected by 6 M urea, which indicates that the trypsin resistance i s conferred by intermolecular interactions between histones. Bohm et a_l also demonstrated, using nuclear magnetic resonance, that the residues 1 to 41 of H3 and 1 to 37 of H4 are not required for the formation of the correct histone-histone interactions i n the H3-H4 complex (39). Similar properties were described by L i l l e y and Tatchell (80) for chicken erythrocyte core p a r t i c l e s treated with trypsin. A l l evidence accumulated to date supports the suggestion of Whitlock and Stein that the COOH-terminal region i s respon-s i b l e for the organization and s t a b i l i z a t i o n of the DNA i n the core p a r t i c l e . This region must be r e l a t i v e l y constant i n structure, and r e s i s t a n t to proteolysis. The NH 2-terminal - 47 -r e g i o n s , on the other hand, are l i k e l y to be n o n - e s s e n t i a l i n the o r g a n i z a t i o n of the DNA w i t h i n the core; they may, / however, have other f u n c t i o n s i n m a i n t a i n i n g the n a t i v e chromatin s t r u c t u r e s i n c e they are r e l a t i v e l y f r e e to i n t e r -a c t w i t h o t h e r p r o t e i n s such as h i s t o n e s a c e t y l a s e , methylases and k i n a s e s (81) (the amino end i s the o n l y s i t e d e s c r i b e d f o r core h i s t o n e m o d i f i c a t i o n s ) . R e c o n s t i t u t i o n of nucleosomes R e c o n s t i t u t i o n experiments have been r e p o r t e d by many workers (15, 71-78). I t has been found t h a t a l l four h i s t o n e s are r e q u i r e d f o r the r e c o n s t i t u t i o n of a 125 A* p a r t i c l e (15). The r e c o n s t i t u t e d nucleosomes resemble the n a t i v e monomers i n s t r u c t u r e (15) and i n nuclease d i g e s t i o n p a t t e r n (74). Our r e s u l t s show t h a t the t h i o l group of r e c o n s t i t u t e d nucleo-somes g e n e r a l l y behaves i n the same way as t h a t of n a t i v e monomers under v a r i o u s c o n d i t i o n s . These r e c o n s t i t u t e d nucleosomes must t h e r e f o r e have s i m i l a r , i f not i d e n t i c a l , c o n f o r m a t i o n a l s t r u c t u r e s as the n a t i v e nucleosomes. In order to o b t a i n high y i e l d s of the r e c o n s t i t u t e d nucleosomes w i t h normal behaviour towards NEM, i t was necessary to c a r r y out the d i a l y s i s i n the presence of a r e d u c i n g agent, e.g. 1 mM DTT. Omission of the r e d u c i n g agent y i e l d e d p a r t i c l e s i n which a p r o p o r t i o n of the H3 t h i o l s was u n r e a c t i v e to NEM even under d e n a t u r i n g c o n d i t i o n s . T h i s o b s e r v a t i o n may be due to formation of H3 i n t e r m o l e c u l a r d i s u l f i d e s . A u t o - o x i d a t i o n of the c y s t e i n e r e s i d u e s has been r e p o r t e d to occur d u r i n g homogenization of c a l f thymus (82). - 48 -Thus, although the H3 t h i o l s at position 110 do not form a d i s u l f i d e bond i n the native nucleosomes, they are r e a d i l y oxidized once H3 i s dissociated from DNA. I t has recently been shown that dimers of H3 linked through cysteine 110 can be substituted for monomeric H3 i n reconstitution experiments, and y i e l d nucleosomes which are indistinguishable from the native p a r t i c l e s by a variety of c r i t e r i a (74). These r e s u l t s e s t a b l i s h that the cysteines are close together i n the nucleosome; they also confirm c r o s s l i n k i n g data in d i c a t i n g that the two H3 are close to each other (16, 22). The fact that they do not form a d i s u l f i d e i n vivo suggests that either the reconstituted p a r t i c l e s have been distorted s l i g h t l y to accommodate the H3 dimer, or that the environment around residue 110 i n the native nucleosome i s not conducive to i o n i z a t i o n of the t h i o l group, perhaps due to a low d i e l e c t r i c constant. If the l a t t e r were true, the two cysteines might be i n close contact and yet remain reduced. DNase I digestion products of reconstituted nucleosomes are similar to those of native nucleosomes, indicating that the DNase I sensitive s i t e s are also reconstituted. The assembly of nucleosomes i s thus an i n t r i n s i c property of the histones and DNA. - 49 -C o n c l u s i o n The s t u d i e s r e p o r t e d here i n d i c a t e t h a t the degree of exposure of H3 t h i o l groups i n nucleosomes can be e f f e c t i v e l y monitored u s i n g a s p e c i f i c t h i o l reagent such as NEM. This s e n s i t i v e assay may be used to estimate the amount of h i s t o n e s i n i s o l a t e d nucleosomal f r a c t i o n s such as the " a c t i v e " f r a c t i o n (83-4), i f f u l l r e a c t i o n o f the t h i o l groups i s assumed under d e n a t u r i n g c o n d i t i o n s . F u r t h e r s t u d i e s of t h i o l r e a c t i v i t y o f c r o s s l i n k e d h i s t o n e octamers from core p a r t i c l e s may r e v e a l the r o l e of DNA i n the masking of the s u l f h y d r y l groups of H3. The c a r b o x y - t e r m i n a l r e g i o n of H3 c o n t a i n i n g Cys 110 seems to be l o c a t e d i n a r e l a t i v e l y s t a b l e r e g i o n of the nucleosome core, perhaps at the i n t e r f a c e between h e t e r o t y -p i c tetramers. T h i s r e g i o n i s u n a f f e c t e d by changes i n nucleosome s t r u c t u r e induced by t r y p t i c d i g e s t i o n . T h i s r e g i o n may be e s s e n t i a l i n the o r g a n i z a t i o n and s t a b i l i z a t i o n of the DNA i n core p a r t i c l e s . C urrent e f f o r t s to e l u c i d a t e the t h r e e - d i m e n s i o n a l s t r u c t u r e of nucleosomes by X-ray d i f f r a c t i o n (37) should soon a l l o w the d e f i n i t i o n of the r e l a t i v e p o s i t i o n s of h i s t o n e s and DNA i n the p a r t i c l e s . 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