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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,  U n i v e r s i t y o f B r i t i s h Columbia, 1976.  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE xn THE FACULTY OF GRADUATE STUDIES Department o f B i o c h e m i s t r y The U n i v e r s i t y o f B r i t i s h  Columbia  We accept t h i s t h e s i s as conforming t o the r e q u i r e d  standard  THE UNIVERSITY OF BRITISH COLUMBIA October, 1978  (c) Norman  In p r e s e n t i n g an  this  thesis  in partial  advanced degree at t h e U n i v e r s i t y  the  Library  shall  I f u r t h e r agree for  make i t f r e e l y  that permission  h i s representatives.  of  this  written  thesis  The  University  Biochemistry of British  2 0 7 5 Wesbrook P l a c e V a n c o u v e r , Canada V6T 1WS  Date  gain  Oct.  5,  1978.  Columbia  that  copying o f t h i s  thesis  by t h e Head o f my D e p a r t m e n t o r  I t i s understood' that  f o r financial  I agree  f o r r e f e r e n c e and s t u d y .  f o rextensive  permission.  Department o f  of B r i t i s h Columbia,  available  s c h o l a r l y p u r p o s e s may be g r a n t e d  by  f u l f i l m e n t o f the requirements f o r  shal1  copying or p u b l i c a t i o n  n o t be a l l o w e d w i t h o u t  my  ABSTRACT Nucleosomes were prepared from t r o u t t e s t i s n u c l e i by m i c r o c o c c a l nuclease d i g e s t i o n .  The r e a c t i v i t y toward  3  N- [ e t h y l - Hlmaleimide (NEM) o f the s i n g l e s u l f h y d r y l group of h i s t o n e H3 i n the nucleosomes was s t u d i e d under a v a r i e t y of c o n d i t i o n s . Under c o n d i t i o n s o f low i o n i c s t r e n g t h , t h e r e i s n e g l i g i b l e r e a c t i o n o f nucleosomes w i t h NEM, suggesting the c y s t e i n y l r e s i d u e o f H3 i s b u r i e d .  that  Complete d e n a t u r a t i o n  of nucleosomes i n 6 M guanidinium c h l o r i d e leads t o r e a c t i o n of 2 moles o f NEM per mole o f nucleosomes, i n agreement w i t h the expected presence o f 2 moles of H3 per p a r t i c l e . of nucleosomes t o 2 M NaCI or 1 M MgCl the t h i o l group.  At higher M g  + +  2  Exposure  leads t o exposure o f  c o n c e n t r a t i o n s , the t h i o l  group remains exposed, but i n NaCI s o l u t i o n s , as the s a l t c o n c e n t r a t i o n i s i n c r e a s e d beyond 2 M, the t h i o l group r e t u r n s to an i n a c c e s s i b l e  state.  The r e a c t i v i t y o f nucleosome t h i o l groups i s r e l a t i v e l y u n a f f e c t e d by urea t o approximately 5 M.  Between 5 and 8 M  urea, a r a p i d i n c r e a s e i n t h i o l r e a c t i v i t y i n d i c a t e s a c o o p e r a t i v e u n f o l d i n g o f the nucleosome c o r e .  When added  together, urea and s a l t a c t i n a c o o p e r a t i v e manner t o expose the H3 s u l f h y d r y l group. Mixtures o f oligonucleosomes have a l s o been s t u d i e d under d i f f e r e n t c o n d i t i o n s .  They were found t o behave i n a  similar thiols  f a s h i o n t o monomers i n 6 M g u a n i d i n e , r e a c t more  s l o w l y than  those  Removal o f t h e a m i n o - t e r m i n a l h i s t o n e s by t r y p t i c the  accessibility  concluded Cys in  regions  o f nucleosome t h i o l  core  which i s r e l a t i v e l y  groups.  r e g i o n o f H3  containing  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  complex, and i s l o c a t e d i n a r e g i o n  insensitive  to the perturbations  by t r y p s i n o r low c o n c e n t r a t i o n s  reducing  particles  of urea.  in  a g e n t were i n d i s t i n g u i s h a b l e f r o m  i n their  on  Itis  Nucleosomes r e c o n s t i t u t e d i n t h e p r e s e n c e o f a hydryl  salt.  o f the core  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  110 i s masked m a i n l y  induced  o f monomers i n h i g h  t h a t the carboxy-terminal  the octameric  but t h e i r  reactivity  t o NEM  2 M N a C l and i n 6 M g u a n i d i n e  sulf-  native  i n low s a l t b u f f e r s ,  hydrochloride.  These s t u d i e s i n d i c a t e t h a t t h e degree o f exposure o f H3  sulfhydryl  g r o u p s i n n u c l e o s o m e s c a n be  m o n i t o r e d u s i n g NEM.  The c a r b o x y - t e r m i n a l  c o n t a i n i n g Cys 110 seems t o be l o c a t e d i n a s t a b l e r e g i o n o f the nucleosome c o r e , i n t e r f a c e between h e t e r o t y p i c  effectively  r e g i o n o f H3 relatively  perhaps a t the  tetramers.  - iii  -  TABLE OF CONTENTS Page ABSTRACT  i  TABLE OF CONTENTS  i i i  L I S T OF TABLES  v i  L I S T OF FIGURES  v i  ACKNOWLEDGEMENT  v i i  INTRODUCTION  1  I.  Eukaryotic  chromatin  1  II.  Nucleosome  structure  2  I n t e r n a l arrangement o f h i s t o n e s  5  The a r g i n i n e - r i c h h i s t o n e s  6  The p r e s e n t  8  III. IV. V.  investigation  MATERIALS AND METHODS I.  II.  III.  IV. V.  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 (b) A b b r e v i a t i o n s P r e p a r a t i o n o f Nucleosomes (a) M i c r o c o c c a l n u c l e a s e d i g e s t i o n (b) P r e p a r a t i o n o f i n v i t r o l a b e l l e d nucleosomes  9 9 9 9 10 10 11  R e a c t i o n o f nucleosomes w i t h N-ethylmaleimide (a) R e a c t i o n o f monomers i n n o n denaturing solutions (b) R e a c t i o n o f monomers i n d e n a t u r i n g solutions (c) R e a c t i o n o f n u c l e o s o m e o l i g o m e r s ....  12 12  T r y p s i n d i g e s t i o n o f nucleosomes  13  R e c o n s t i t u t i o n - o f nucelosomes  13  12 12  - iv Page VI. VII.  DNase I d i g e s t i o n o f n a t i v e and r e c o n s t i t u t e d nucleosomes  14  Gel e l e c t r o p h o r e s i s of nucleosomes (a) 15% SDS-polyacrylamide s l a b g e l electrophoresis f o r analysis of nucleosomal p r o t e i n s (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 o f DNA (c) Denaturing 99% formamide, 6% polyacrylamide g e l electrophoresis o f DNA  14 14 15 16  RESULTS Isolation  17 o f nucleosomes  17  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 .  17  E f f e c t o f s a l t s on s u l f h y d r y l r e a c t i v i t y (a) E f f e c t o f guanidinium c h l o r i d e (b) E f f e c t o f sodium c h l o r i d e on thiol reactivity (c) E f f e c t o f magnesium c h l o r i d e on thiol reactivity  21 21  ....  23 25  E f f e c t o f urea on s u l f h y d r y l r e a c t i v i t y  27  Cooperative e f f e c t o f NaCl and urea on nucleosome conformation  27  S u l f h y d r y l r e a c t i v i t y o f nucleosome oligomers  30  T r y p t i c d i g e s t i o n of nucleosomes  30  Reconstitution  35  o f nucleosomes  DISCUSSIONS Carboxy-terminal r e g i o n o f H3 i n n a t i v e nucleosomes  39 39  E f f e c t o f s a l t s on the s t r u c t u r e of H3  40  E f f e c t o f urea on nucleosomes  43  T h i o l r e a c t i v i t y o f nucleosome oligomers ....  44  T r y p s i n d i g e s t i o n o f nucleosomal p r o t e i n s  45  ...  -  V  -  Page Reconstitution Conclusion REFERENCES  o f nucleosomes  47 49 50  - viLIST OF TABLES Page Table I Reaction o f t r y p s i n - d i g e s t e d mononucleosomes w i t h N-ethylmaleimide  33  LIST OF FIGURES Figure 1.  B i o - G e l A-5M column p r o f i l e o f nucleosomes from 30 minutes o f m i c r o c o c c a l nuclease digestion  18  SDS-polyacrylamide g e l e l e c t r o p h o r e s i s of the monomer peak f r a c t i o n from an A-5M column  19  Q u a n t i t a t i o n of [ H] counts from NEMl a b e l l e d nucleosomal p r o t e i n s  20  Time course o f r e a c t i o n o f nucleosomes w i t h NEM i n denaturing and non-denaturing conditions  22  E f f e c t o f v a r y i n g m o l a r i t y o f Gdn*HCl on t h i o l r e a c t i v i t y of nucleosomes  24  E f f e c t o f i o n i c s t r e n g t h on t h i o l r e a c t i v i t y o f nucleosomes  26  7.  E f f e c t o f urea c o n c e n t r a t i o n on the t h i o l r e a c t i v i t y of nucleosomes  28  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 o f nucleosome t h i o l groups  29  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 ....  31  10.  E f f i c i e n c y of t r y p s i n digestion  34  11.  Time course o f i n c o r p o r a t i o n o f [ H]-NEM i n t o r e c o n s t i t u t e d nucleosomes  36  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  38  2.  3. 4.  5. 6.  9.  12.  3  3  - viiACKNOWLEDGEMENT I wish t o thank Dr. Peter Candido f o r h i s guidance, comments and s u p e r v i s i o n d u r i n g the course o f study.  Jim  Davie r e q u i r e s s p e c i a l mention f o r h i s c o n s t r u c t i v e comments, h e l p f u l d i s c u s s i o n s and a s s i s t a n c e . I would a l s o l i k e t o acknowledge the Medical C o u n c i l f o r i t s support studentship.  Research  o f these s t u d i e s i n the form o f a  - 1 INTRODUCTION I.  Eukaryotic Despite  otic  o f DNA, RNA.  the  The  i n the  nucleus  among DNA  the  chromatin  and  histones. not  chromatin.  They a r e  equal  t o a c t as  gene a c t i v i t y .  In  histones  covalently modified  can  acetylation,  be  s p i t e of the  methylation,  D N A - h i s t o n e and  etc.  histone-histone  (2),  by  the  r o l e s of  components  f i v e major c l a s s e s ,  "coarse"  by  DNA  in  in  maintaining  c o n t r o l of  variety in  type,  phosphorylation,  probably  to modulate  i n t e r a c t i o n s to b r i n g  about  function.  nonhistone p r o t e i n s , a heterogeneous p o p u l a t i o n  different  protein species,  interphase  chromatin.  p r o t e i n s are  not  are present  Although the  understood,  there  i n small  f u n c t i o n s of are  to i n c r e a s e  of  amounts i n nonhistone  several lines  t o 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  p r o t e i n s were f o u n d  of  (1).  little  c h a n g e s i n c h r o m a t i n s t r u c t u r e and  these  to v a r i o u s  important a  amount  established,  weight with  t h o u g h t t o be  c h r o m a t i n s t r u c t u r e and  evidence  fully  experiments  i n approximately  a small  Although the  histones, b a s i c p r o t e i n s of  present  nucleoprotein  i s maintained  f u n c t i o n s have b e e n a s s i g n e d  the b a s i s of p r e v i o u s  The  a  a l l eukary-  T h i s chromosomal m a t e r i a l c o n s i s t s  components i n c h r o m a t i n a r e  The  complexity,  n o n h i s t o n e s p r o t e i n s and  structure of  different  are  g r e a t v a r i e t y and  chromatin.  histones,  interactions  on  their  organisms c o n t a i n  complex,  the  chromatin  of  them:  i n amount i n g e n e t i c a l l y  - 2 active tissues and  alter  DNA c o n f i g u r a t i o n  regulation II.  (3), i n d u c e t r a n s c r i p t i o n o f a c t i v e genes (3-4)  i s y e t t o be e l u c i d a t e d .  Nucleosome  structure  A major advance was  the discovery  1973-4  (6-9).  on c h r o m a t i n  The most c o n v i n c i n g  found t h a t  capacity  i n research  studies  (6, 8 ) .  e v i d e n c e emerged  an endogenous n u c l e a s e o f r a t l i v e r  to cleave  chromatin t o m u l t i p l e s  s u b s e q u e n t l y c o n f i r m e d by N o l l  same t i m e , Woodcock  (10) and O l i n s  appearance o f a "beads-on-a-string" in  e l e c t r o n micrographs.  subunit  i n chromatin  morphological Since  t o study  ational  This  observation  (8). A t about t h e & Olins  (9) o b s e r v e d t h e  structure  from  chromatin  and  studies. o f t h e nucleosome,  investigators  and DNA a r e a r r a n g e d w i t h i n t h e  E x p e r i m e n t s were done t o m e a s u r e t h e DNA c o n t e n t  (11-13), t o d e f i n e  and  o f a subunit of  Thus, t h e p r e s e n c e o f a r e p e t i t i v e  began t o a s k how t h e h i s t o n e s  nucleosomes  has t h e  i s e s t a b l i s h e d b o t h by b i o c h e m i c a l  the discovery  nucleosomes.  from  H e w i s h and B u r g o y n e  a b o u t 200 b a s e p a i r s o f DNA i n l e n g t h . was  structure  o f t h e "nucleosomes" or " v - b o d i e s " i n  nuclease d i g e s t i o n (6)  ( 5 ) . However, t h e mode o f  the r e l a t i v e  (14), t o f i n d  l o c a t i o n s o f DNA i n t h e  the stoichiometry  the propinquity  changes o f h i s t o n e s  o f histones  of histones  (15-7),  (16-23) and c o n f o r m -  under a v a r i e t y o f c o n d t i o n s  (24, 25).  -  The  3  -  f i r s t measurements of the DNA  values of 205 base p a i r s (8) and  u n i t s i z e gave  180-230 bp  (26).  These  experiments were done by f i r s t d i g e s t i n g the chromatin i n t o fragments c o n s i s t i n g of m u l t i p l e s of a u n i t s i z e by commerc i a l l y a v a i l a b l e m i c r o c o c c a l nuclease;  then the  fragments  were r e s o l v e d by g e l e l e c t r o p h o r e s i s and  their mobilities  were compared to DNA  s i z e s thus*  obtained tissues.  standards.  v a r i e d from 140-220 bp i n d i f f e r e n t organisms Since i n some experiments the DNA  d i f f e r e n t sources were analyzed v a r i a t i o n i n s i z e was now  The DNA  not due  fragments from  on the same g e l , t h i s  to experimental  error.  g e n e r a l l y agreed t h a t monomers of chromatin  by m i l d d i g e s t i o n c o n t a i n DNA  prepared  c r o s s - l i n k i n g studies  I t has been found t h a t a l l of the four  h i s t o n e s , H2A,  H2B,  H3  and H4  (15).  smaller  are r e q u i r e d 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 the e l e c t r o n microscope  H5.  of the nucleosome has been d e r i v e d  from r e c o n s t i t u t i o n experiments and (15-7).  8 particles in  The appearance of a c r o s s -  l i n k e d octamer of h i s t o n e s observed by Thomas and (16-7) f u r t h e r confirmed  the e x i s t e n c e of two  Romberg  each of  f o r u s m a l l h i s t o n e s . T h i s s t o i c h i o m e t r y of h i s t o n e s obtained  from chromatin of d i f f e r e n t sources  d e v i a t i o n s of 30-50% are f r e q u e n t l y obtained, to  inaccuracy  (27);  fragments  of 140-170 bp bound by whole h i s t o n e s with Hi or h i s t o n e content  It i s  segments of 180-205 bp  f u r t h e r d i g e s t i o n of these monomers y i e l d s DNA  The  and  i n measurements.  the  was  ( 2 8 ) ; however, probably  due  The has  role o f Hi i n association with  been under i n v e s t i g a t i o n by v a r i o u s w o r k e r s .  assumed t h a t o n l y one H i m o l e c u l e n u c l e o s o m e and i t i s e i t h e r  associated with  the o u t s i d e o f the nucleosomes.  w i t h DNA was s u p p o r t e d  by W h i t l o c k  It is  i s associated with  r e g i o n o f 30-45 bp o r i t i n t e r a c t s w i t h on  t h e nucleosome  t h e DNA  each spacer  the nucleosomal  The a s s o c i a t i o n  DNA  of Hi  & Simpson who d e m o n s t r a t e d  t h e e x p o s u r e o f DNA upon t h e r e m o v a l o f H i i n 0.7 M NaCI ( 2 9 ) . Nevertheless,  s i n c e HI i s t h e o n l y h i s t o n e t o d i s s o c i a t e  t h e n u c l e o s o m e s i n low s a l t  concentrations  susceptible  to proteolysis,  many r e s u l t s were g r e e t e d  suspicion.  C u r r e n t view f a v o r s t h e s u g g e s t i o n  from  (30) and i s most with  t h a t HI c r o s s -  l i n k s between n u c l e o s o m e s and t h a t H i i s n o n - e s s e n t i a l i n maintaining  t h e b a s i c n u c l e o s o m e s t r u c t u r e ( 2 7 ) , however, H i  may be i n v o l v e d i n t h e f o r m a t i o n o f h i g h o r d e r s t r u c t u r e of  chromatin  (3 2 ) .  Extensive digestion the  o f t h e n u c l e o s o m e monomer  "trimmed" monomer o f 140 bp i n l e n g t h .  product with  140 bp o f DNA and e i g h t h i s t o n e s  H2A, H2B, H3 and H4)' i s t e r m e d remaining types The be  DNA w h i c h i s v a r i a b l e  i s termed t h e " l i n k e r "  earliest roughly  This  physical  spherical  degradation  (two e a c h o f  the "core p a r t i c l e "  and t h e  i n length i n d i f f e r e n t  as i t c o n n e c t s  s t u d i e s suggested  cell  the nucleosomes.  the core p a r t i c l e to  and a b o u t 110 R i n d i a m e t e r ( 3 3 ) .  Neutron s c a t t e r i n g d a t a has r e c e n t l y the c o r e p a r t i c l e  t o be a f l a t  of  (34-5).  11 x 11 x 6 nm  produces  shown t h a t t h e shape o f  cylinder with o v e r a l l  The DNA was f o u n d  dimension  t o be e i t h e r  -  folded  or  s u p e r c o i l e d on  More i n f o r m a t i o n provided  by  particles ments o f  been under  intense  histones  with  contact with  be  various  the  core  measure-  neutron  an  the  of  Hi  adjacent  the  the  t o be  histones  others  and  the DNA.  respect  chemical and  involved  i n any  agents  trimers  each crosslinks  homopolymer.  c r o s s l i n k e d o c t a m e r s and  one.  Five of  the  have a l s o b e e n  c a n n o t be  the  nucleosome  is in  ten p o s s i b l e dimers found, but  r u l e d out,  as  the  they  i n small  Studies  of  the  a s s o c i a t i o n p r o p e r t i e s of histones  revealed  the  tetramer  core  particle  amounts  to  experiments,  of  form a  particle  of and  histones with  i n d i c a t e t h a t the  histones  DNA  location  to  core  aim  crosslinking  relative  Hi molecules  small  of  the  the  A n a l y s i s of dimers  of m u l t i p l e s of  four  the  36).  been  present  solution define  to  histones within  were c r o s s l i n k e d by  of polymeric  occurrence  the  found not  other  formation  from the  isolate  i n v e s t i g a t i o n , with  (16-23).  H i was  formation  the  were s t u d i e d m a i n l y by  formed d e f i n e s  except  to  now  (34,  histones  r e l a t i o n s h i p s among t h e  light  histone.  s h a p e has  results  t h e mechanism o f p a c k a g i n g  i n which the UV  nucleosome  X-ray d i f f r a c t i o n  gave s i m i l a r  l o c a t i o n s of  each other  o r by  of the  were a b l e  (37).  arrangement of  The  may  form  crystals  intermolecular  The  e t a l . who  I n t e r n a l arrangement o f  elucidating  thus  outside  data.  The has  Finch  the  the  about nucleosome c o r e  in crystal  scattering Ill.  5 -  (31).  (H3)2(H4) ,  (38-9),  2  e.g.  by  w h i c h may  in  itself  inducing supercoiling  - 6 of the DNA,  while the H2A-H2B dimer may  the complete nucleosome  (40).  The  a i d s t a b i l i z a t i o n of  r o l e s of H2A  and H2B  were  supported by r e p o r t s t h a t the two h i s t o n e s can a l t e r the t i c a l a c t i v i t y and conformation E a r l y attempts  of l o c a l r e g i o n s of DNA  op(41).  to demonstrate the octamer i n s o l u t i o n  have been u n s u c c e s s f u l because of i t s i n s t a b i l i t y  i n low  i o n i c s t r e n g t h ; however, hexamers, tetramers, dimers and even h e t e r o t y p i c tetramers comprising each of H2A, have a l l been found  (16-7, 42).  H2B,  H3 and  Recently Chung e t a l . were  able to i s o l a t e the core complex i n h i g h s a l t s and do on i t (43).  H4  analysis  They showed t h a t the octamer e x i s t s i n e q u i l i b r i u m  w i t h a h e t e r o t y p i c tetramer i n 2 M NaCI. The c o n s e r v a t i v e sequences o f H3 and H4 i n f e r e n c e t h a t the tetramer the b a s i c f o l d of DNA s t a b i l i z a t i o n purpose.  (H3) (H4) 2  2  i s important  i n the core w h i l e H2A H2A  and H2B  l e a d to the  may  and H2B  have o t h e r  f u n c t i o n s as w e l l s i n c e they are r e q u i r e d f o r the of the n a t i v e nucleosomes. probably due  i n defining are f o r specific  formation  V a r i a t i o n i n the l i n k e r r e g i o n i s  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 s i m i l a r i n terms of a r g i n i n e  content, sequence c o n s e r v a t i o n , s a l t e l u t i o n from DNA,  e t c . They  were found to a s s o c i a t e i n s o l u t i o n t o form a tetramer which i s most important  i n packaging  the core DNA  c o i l i n g of DNA  (28, 44-6).  The  t e c t s DNA  (H3) (H4) 2  and i n d u c i n g super2  tetramer a l s o p r o -  a g a i n s t nuclease d i g e s t i o n i n a manner s i m i l a r to t h a t  found  f o r core Biroc  7 -  particles  (47).  and  Reeder  (25)  tyrosines with  iodine  i n Xenopus.  were d i g e s t e d four By  with  t r y p s i n and  heavily labelled  assuming the  tryptic  same H4  thymus, t h e y were a b l e in  the  reactivity  From a n a l y s i s o f under v a r i o u s tyrosines  of  the  chromatin.  indicating histone  reactivity  t h a t the  when t h e  the  o f the  are  One  ionic  c o n d i t i o n f o r the  tyr  88 i s involved  the  with  of  tyrosines peptide. be  analyzed.  residues of  the to  went up  histone  f o r the  d i s s o c i a t i o n of H i ,  cysteines  vertebrates, (50).  are  found  s u c h as  (48-9).  and  fishes,  and  protection 88,  0.5  H4,  cysteine  Hyde and  Walker  NaCI,  or  that  is  Hi.  vertebrates, i n lower  one  cysteine  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  s u l f h y d r y l reagents.  M  moieties  In h i g h e r  only  fold,  inferring  f o r e a c h m o l e c u l e o f H3;  chicken  NaCI  histone-  tyrosines, tyr  that contains  native  5-10  c h r o m a t i n and  i n i n t e r a c t i o n between HI  only  four  into 2 M  i o d i n e t o maximum a t  i t s amino a c i d s e q u e n c e  using  the  3.5,  c o n f o r m a t i o n a l . c h a n g e upon d i s s o c i a t i o n o f  i s the  present  four  put  H4  calf  attached  reactivities  s u p e r c o i l i n g of  tyrosine residues.  H3  in  tyrosine  histones  the  H4  obtained.  tryptic  c h r o m a t i n was  a l l of  the  by  each of  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  e x p o s e d by  were  t h e y showed t h a t two  its reactivity  two  spots  a t pH  i n d i v i d u a l tyrosine could  increases  within  peptide  to a s s i g n  However, i f t h e  5 M urea or both,  of the  electrophoresed  s e q u e n c e i n Xenopus as  conditions,  are b u r i e d  r e a c t i o n of  When i o d i n a t e d Xenopus  amino a c i d s e q u e n c e t o a l a b e l l e d  Thus t h e  or  have examined t h e  (24)  label  is H3  studied  calf  thymus H3  by  reacting  ditho-bis-[2-nitrobenzoic They f o u n d t h a t reactive well put  as  8  only  -  whole c h r o m a t i n w i t h acid], a specific thiol  one  of  the  two  under normal c o n d i t i o n s . the  total  into salt  cysteine The  r e a c t i v i t y increased  Candido  gible reaction  (51)  also  between t r o u t  testis  e n h a n c e d when t h e  These r e s u l t s i n d i c a t e reactivity V.  The The  as  the  present  c o n c e n t r a t i o n s and  the  H3  n u c l e o s o m e s were  and  thiol  thiol in  consequently,  p e p t i d e s or  employed  g r o u p s w i t h NEM  histone-DNA  reaction  the  thiol  structure.  different  r e a c t i v i t y of also  by  trypsin-  analyzed.  s u l f h y d r y l reagent I t can  radioactive  be  reaction  reported. of  The  of  with  analyzed  by  labelling.  nucleosome  r e s u l t s are  histone-histone  nucleosome.  H3  structure.  studies.  the  i n the  salt  signify alterations in  proteins.  a detailed picture  interactions  testis  changes i n nucleosome  i n these  are  r e a c t i v i t y toward  including  The  r e a c t i v i t y may  t h i s t h e s i s , d a t a on  formulating  studying  nucleosomes are  s p e c t r o p h o t o m e t r i c methods o r was  of  p-hydroxy  denatured.  groups i n t r o u t  N-ethylmaleimide i s a u s e f u l  In  was  is negli-  The  even nucleosome  urea s o l u t i o n s .  thiol  c o n f o r m a t i o n and  free cysteine,  condition.  conditions,  reconstituted  Changes i n t h e  latter  as  chromatin  there  i n v e s t i g a t i o n concerns the  nucleosomes under v a r i o u s  The  reaction  investigation  present  and  the  is  n u c l e o s o m e s and  possibility  a p r o b e o f H3  N-ethylmaleimide of  digested  as  groups of  found t h a t  mercurobenzoate under n o n - d e n a t u r i n g greatly  rate  reagent.  solutions.  Maher and  was  5,5'-  useful and  - 9 MATERIALS AND I. M a t e r i a l s (a)  METHODS  and A b b r e v i a t i o n s  Materials N a t u r a l l y maturing  Sun V a l l e y  trout  t e s t e s were o b t a i n e d f r o m  T r o u t Farm, M i s s i o n , B. C.  Micrococcal  (E.C.3.1.4.7.),  deoxyribonuclease I, trypsin  soybean  inhibitor,  trypsin  from Sigma.  Labelled  filters  used  Reeve A n g e l .  N-ethylmaleimide from  Sequencing  Na-[l-  were p u r c h a s e d  concentration  cells  1 4  Aldrich  grade heptane used t o Pierce  C ] - a c e t a t e and aqueous c o u n t i n g  f r o m Amersham.  M i n i c o n B15  were f r o m Amicon C o r p .  Glass  fibre  f o r N - e t h y l m a l e i m i d e a s s a y s were o b t a i n e d f r o m A c r y l a m i d e was f r o m M a t h e s o n , Coleman and B e l l ;  N, N ' - m e t h y l e n e - b i s - a c r y l a m i d e f r o m B i o - R a d ; (N,N,N',N'-tetramethyl-ethylenediamine) All  New  3  N - e t h y l m a l e i m i d e was o b t a i n e d f r o m  C h e m i c a l s Company. scintillant  purchased  N - e t h y l - [ H ] - m a l e i m i d e was f r o m  C h e m i c a l Co., W i s c o n s i n . labelled  (E.C. 3.4.4.4.),  d i t h i o t h r e i t o l were  E n g l a n d N u c l e a r and u n l a b e l l e d  dissolve  nuclease  and TEMED  f r o m Ames Company.  o t h e r c h e m i c a l s and r e a g e n t s were o f t h e h i g h e s t  or reagent grade.  Distilled  w a t e r was u s e d  purity  f o ra l l solutions.  (b) A b b r e v i a t i o n s EDTA: SDS:  ethylenediamine-tetraacetic sodium d o d e c y l  TMKS b u f f e r : sucrose  acid  sulfate  Tris-HCl  (50 mM,  pH 7 . 4 ) , M g C l  (0.25 M) , and B - m e r c a p t o e t h a n o l  2  (15 mM) .  (1 mM),  - 10 Tris-EDTA b u f f e r :  Tris-HCl  (lOmM, pH 7.5), and EDTA  (0.7 mM). PBS b u f f e r :  NaCI (0.14 M) , KCI (27 mM) , NaaHPO., (8 mM) ,  (1.5 mM), C a C l  KH2PO4  NEM:  2  (0.9 mM), MgCl  2  (0.5 mM), pH 7.2.  N-ethylmaleimide  Gdn«HCl:  guanidinium c h l o r i d e  TCA-tungstate:  10% t r i c h l o r o a c e t i c a c i d , and 0.5%  sodium t u n g s t a t e , pH 2.0. DTT:  dithiothreitol  DNase I:  deoxyribonuclease I  I I . P r e p a r a t i o n o f Nucleosomes (a)'  M i c r o c o c c a l nuclease d i g e s t i o n  Nucleosomes were prepared as d e s c r i b e d by Davie and Candido  (53) w i t h some m o d i f i c a t i o n s .  N u c l e i were i s o l a t e d homogenizing  from 6-8 g o f t r o u t t e s t e s by  i n TMKS b u f f e r i n a Waring Blendor f o r 2 minutes.  A f t e r 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, the p e l l e t was resuspended i n TMKS b u f f e r and homogenization and c e n t r i f u g a t i o n repeated.  The n u c l e i i n the p e l l e t were  i n 6 ml o f TMKS b u f f e r c o n t a i n i n g 1 mM C a C l  2  suspended  (5 x 1 0  8  ml), and d i g e s t e d w i t h m i c r o c o c c a l nuclease a t a f i n a l t r a t i o n o f 300 u n i t s / m l f o r 30 minutes a t 37°.  nuclei/ concen-  The r e a c t i o n  was stopped by adding EDTA t o 10 mM, and p l a c i n g t h e mixture on i c e .  The mixture was c e n t r i f u g e d a t 12,000 x g f o r 10  minutes and the supernatant was d i s c a r d e d . and multimers were r e l e a s e d by v i g o r o u s  Chromatin monomers  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  EDTA b u f f e r .  The  chromatin  subunits  To was  a t 12,000 x  through  equilibrated  multimer  column  with Tris-EDTA b u f f e r . o f 8 ml/hr  peak f r a c t i o n s were p o o l e d use.  40 A 6 o  About  units  2  The  homogenized  i n PBS  homogenate was  buffer  filtered  trout  4.4  ml),  ml  1.2  acetate  PBS  buffer,  ml  ml Waymouth's medium (50 u C i / m l ) .  The  stored  at  added and  testes  The  monomer  and until  5 A 6o  units  2  were m i n c e d  and  ( 5 3 ) , and  the l a b e l l e d c e l l s  t h e y were s t o r e d  then  c e l l s were  0.3  ml  suspended (100  sodium  1-[  units 1 4  C]-  incubated i n a gyratory  c o l l e c t e d by  When t h e c e l l s at  The  centri-  penicillin-streptomycin  25 ml  PBS  buffer  centrifugation were n o t  used  -80°.  N u c l e o s o m e monomers were p r e p a r e d by d i g e s t i o n  (52)  run  a t -8 0°  c h e e s e c l o t h and  m i x t u r e was  3,000 x g f o r 10 m i n u t e s .  immediately,  x 2 cm)  testis.  w a t e r b a t h a t 16 ° f o r 4 h r , a f t e r w h i c h t i m e was  supernatant  i n a g l a s s - t e f l o n homogenizer.  through  0.6  of  l a b e l l e d nucleosomes  f u g e d a t 3,000 x g f o r 10 m i n u t e s . in  The  o f monomer and  (b) P r e p a r a t i o n o f i n v i t r o grams o f f r e s h  multimers)  c o l u m n was  a t 4°. and  the  (107 cm  o f o l i g o m e r were o b t a i n e d p e r gram o f t r o u t  Three  Tris-  g.  a B i o - G e l A-5M  overnight at a flow rate  further  30 m i n u t e s  s e p a r a t e t h e monomers f r o m m u l t i m e r s ,  passed  of  (monomers and  were c o l l e c t e d i n t h e s u p e r n a t a n t a f t e r centrifugation  i n 6 ml  from  the l a b e l l e d  w i t h m i c r o c o c c a l n u c l e a s e as d e s c r i b e d  above.  cells  - 12 III.  Reaction o f nucleosomes w i t h (a)  N-ethylmaleimide  Reaction of monomers i n non-denaturing  solution  F i v e m i c r o l i t r e s o f N - e t h y l - [ H]maleimide (80 mCi/mmol) 3  c o n t a i n i n g 1.25 y C i were added t o 0.5 ml o f nucleosome monomers (2 A  2 6 0  EDTA b u f f e r .  /ml)  a t ambient temperature  F i f t y m i c r o l i t r e a l i q u o t s were taken a t time  i n t e r v a l s and put i n t o TCA-tungstate reaction.  (^22°) i n T r i s -  a t 0° t o stop the  The a c i d p r e c i p i t a b l e m a t e r i a l was  on g l a s s f i b r e f i l t e r s  collected  and washed three times w i t h TCA-  t u n g s t a t e , and once each with e t h a n o l and e t h e r . d i s c s were d r i e d and counted scintillant. of  The f i l t e r  i n 5 ml of aqueous counting  C o n t r o l r e a c t i o n s , i n which the c o n c e n t r a t i o n s  NEM and nucleosomes were v a r i e d , showed t h a t the reagent  was not l i m i t i n g under these c o n d i t i o n s . (b) One A to  Reaction o f nucleosome monomers i n d e n a t u r i n g solutions 2 6 0  u n i t of nucleosome monomers was  0.25 ml i n a Minicon B15 c e l l  concentrated  and added t o the a p p r o p r i -  ate amount o f s a l t , urea or Gdn«HCl.  The r e a c t i o n mixture  was brought t o 0.5 ml by a d d i t i o n of Tris-EDTA  b u f f e r , and  the NEM r e a c t i o n c a r r i e d out as d e s c r i b e d above. (c)  Reaction o f nucleosome oligomers w i t h  NEM  Nucleosome oligomers were c o l l e c t e d from the excluded peak a f t e r s e p a r a t i o n of m i c r o c o c c a l nuclease d i g e s t products on a B i o - G e l A-5M column.  One A  2 6 0  u n i t of  oligonucleosomes  was subjected t o t h i o l a n a l y s i s 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 as d e s c r i b e d above.  - 13 IV.  Trypsin  digestion  Nucleosomes of  o f nucleosomes  (2 A o / m l )  were i n c u b a t e d w i t h  2 6  t r y p s i n i n Tris-EDTA buffer  To s t o p t h e r e a c t i o n , added the  a t 20°, f o r t h e d e s i r e d  0.5 ml o f t h e i n c u b a t i o n  t o 10 y l o f s o y b e a n t r y p s i n  mixture c h i l l e d  to 0°.  After  above.  At  2 6 0  /ml)  was c h e c k e d  n u c l e o s o m e s , 1.8 m l o f n u c l e o s o m e s  1  A  m i x t u r e was  30 m i n u t e s , t h e NEM  When t h e 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  (1.5  period.  i n h i b i t o r (1 mg/ml) and  r e a c t i o n was p e r f o r m e d a s m e n t i o n e d  using[ ^C]-labelled  10 ug/ml  was added  t o 200 m i c r o l i t r e s o f t r y p s i n  (70 u g / m l ) .  t i m e 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 t a k e n o u t  and m i x e d 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 c o u n t e d i n 5 m l o f aqueous counting  scintillant  described V.  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  above.  Reconstitution  o f nucleosomes  Monomer n u c l e o s o m e s in DTT  Tris-EDTA buffer  (2 m l , 1.6 A  containing  5 hr.  against each,  The r e c o n s t i t u t e d  three  1 litre  2 6 0  /ml)  were  dissociated  2 M N a C l , 5 M u r e a a n d 1 mM  ( o r 10 M u r e a and 1 mM D T T ) .  removed by d i a l y s i s a g a i n s t for  d i s c s as  The s a l t  1 litre  and u r e a  o f T r i s - E D T A + 1 mM DTT  n u c l e o s o m e s were t h e n  changes o f T r i s - E D T A b u f f e r  t o remove t h e r e d u c i n g a g e n t .  were  dialyzed f o r 3 hr  R e a c t i o n s w i t h NEM were  t h e n p e r f o r m e d u n d e r d e n a t u r i n g and n o n - d e n a t u r i n g c o n d i t i o n s as  described  above.  - 14 VI.  DNase I d i g e s t i o n o f n a t i v e and r e c o n s t i t u t e d nucleosomes Nucleosomes  (20 A 6 o / m l ) were p r e i n c u b a t e d i n Tris-EDTA 2  b u f f e r a t 37° f o r 10 minutes.  A f t e r the i n c u b a t i o n  period,  the s o l u t i o n was made 2.7 mM i n MgCl + 5.4 mM NaCI t o complex 2  the EDTA and t o provide  the Mg : and Na* , c o n c e n t r a t i o n s ++  f o r optimal  DNase I d i g e s t i o n .  DNase I (1 mg/ml) was then added t o a  f i n a l concentration  o f 0.02 mg/ml and the nucleosomes were  digested  f o r 5 minutes a t 37°.  p l a c i n g the mixture on i c e .  The r e a c t i o n was stopped by  The nuclease d i g e s t e d  nucleosomes  were then analyzed on d e n a t u r i n g p o l y a c r y l a m i d e g e l s . VII.  G e l e l e c t r o p h o r e s i s o f nucleosomes (a)  15% SDS-polyacrylamide s l a b g e l e l e c t r o p h o r e s i s f o r a n a l y s i s o f nucleosomal p r o t e i n s  F i f t e e n per cent polyacrylamide-sodium dodecyl s u l f a t e s l a b g e l s were made using The  f o l l o w i n g volumes o f stock  (30.0 100  a modified  Laemmli procedure (52).  s o l u t i o n s : - 15 ml o f I  g acrylamide, 0.4 g N,N -methylenebisacrylamide i n 1  ml o f H 0 ) , 0.15 ml of I I (20 mg ammonium p e r s u l f a t e 2  f r e s h l y d i s s o l v e d i n 2 ml H 0 ) , 0.3 ml o f I I I (10 g SDS i n 2  100  ml o f water) and 7.5 ml o f IV (1.5 M T r i s - C l , pH 8.8) -  were combined w i t h 10  1 o f TEMED and 7.0 5 ml o f water, and  polymerized i n 1.5 mm s l a b s under t - b u t a n o l .  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% g l y c e r o l and 0.002% bromophenol b l u e .  The g e l was r u n a t 15 m i l l i a m p e r e s f o r 7-8  hr i n 0.05 M T r i s , p H  8.0, 0.384 M g l y c i n e and 0.1% SDS.  the g e l was t o be d i s s e c t e d and s o l u b i l i z e d , 0.6% N,N'd i a l l y l t a r t a r d i a m i d e was used as c r o s s l i n k e r i n s t e a d o f  When  bisacrylamide stained  (54).  After  e l e c t r o p h o r e s i s , t h e g e l was  i n Coomassie B r i l l i a n t  Blue  i n methanol:acetic  w a t e r , 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% m e t h a n o l 7.5%  acetic (b)  N o n - d e n a t u r i n g 3% p o l y a c r y l a m i d e p h o r e s i s o f DNA  were a n a l y z e d (55).  and o l i g o m e r i c f r a c t i o n s  7.5  The  acid,  f o l l o w i n g volumes  pH  7.8,  ml o f 20% a c r y l a m i d e  bisacrylamide,  a c r y l a m i d e was Nucleosome  f r o m A-5M  columns  solution  4 0 y l o f TEMED was  and t h e n p r o t e a s e  K mixture  EDTA, 2% SDS)  was  added  d i g e s t i o n was  carried was  and a p p l i e d d i r e c t l y  water.  Bands  added  precipitated  (50 0 yg/ml  10%  ml o f w a t e r and t h e mm  i n 10 mM  Protease  K,  slabs. MgCl2,  10  mM  The  o u t a t 37° f o r 1 h r , a f t e r w h i c h t h e made 4% SDS, to the g e l .  30 mM  EDTA, 20%  glycerol  The g e l s were p r e r u n  c o n d i t i o n s were 50 V f o r 10  f o l l o w e d by 100 V f o r 4 h r . (10 yg/ml)  36.6  ml o f  to d i g e s t the p r o t e i n s .  100 V f o r 3 h r and r u n n i n g  bromide  0.4  N,N'-methylene-  u n d e r t - b u t a n o l i n 1.5  samples were f i r s t  d i g e s t i o n mixture  with  M  M sodium a c e t a t e ) ,  (acrylamide:  ml o f 10% SDS,  polymerized  solutions:  ( t e a b u f f e r = 0.04  EDTA, 0.02  - were combined  Then  of stock  TEA b u f f e r 2 mM  1 9 : 1 ) , 0.5  ammonium p e r s u l f a t e and d e a e r a t e d .  gel electro-  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  - 5 ml o f l O x c o n c e n t r a t e d Tris-acetic  and  acid.  Monomeric  Loening  acid:  min,  G e l s were s t a i n e d i n e t h i d i u m  f o r 10 min and d e s t a i n e d b r i e f l y i n  were v i s u a l i z e d  at  u n d e r UV  light.  - 16 (c)  Denaturing 99% formamide, 6% 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 o f DNA  These g e l s were d e s c r i b e d by Staynov e t a l . (56) . formamide was s t i r r e d  w i t h Dowex 50W-X8 (3 g/100 ml)  1 h r , and was then f i l t e r e d  and used the same day.  of acrylamide, 0.36 g o f N,N'-methylenebisacrylamide  99%  for 2.04 g and  80  1 o f TEMED were d i s s o l v e d i n 4 0 ml o f formamide, which  was  then f i l t e r e d .  The g e l was made 2 0 mM i n phosphate  and 0.12% i n ammonium p e r s u l f a t e by adding 0.8 ml o f M sodium phosphate persulfate.  pH 7.0, c o n t a i n i n g 50 mg o f ammonium  A 15 x 15 x 0.15 cm s l a b g e l was poured.  Nucleosome samples were f i r s t d i s s o l v e d i n M NaCl, 2.5 M urea; they were then p r e c i p i t a t e d o v e r n i g h t i n 2 volumes of  ethanol.- The p r e c i p i t a t e was taken up i n formamide  ( c o n t a i n i n g 20 mM sodium phosphate,  pH 7.0, 20% sucrose,  0.005% bromophenol b l u e ) , heated t o 100 , c o o l e d on i c e , and e l e c t r o p h o r e s e d i n formamide-20 mM phosphate for  5 hours.  above.  a t 160 V  Gels were s t a i n e d and d e s t a i n e d as d e s c r i b e d  -  17 -  RESULTS I s o l a t i o n o f Nucleosome N u c l e o s o m e s were i s o l a t e d and  Methods.  The  excluded  oligomeric  A typical  A-5M p r o f i l e  f r a c t i o n was p o o l e d  fraction,  to contain  SDS p o l y a c r y l a m i d e  i n Materials  i s shown i n f i g u r e  as i l l u s t r a t e d .  only  1.  to constitute the  and t h e monomer f r a c t i o n was  f r o m t h e peak f r a c t i o n s was f o u n d  as d e s c r i b e d  obtained  The monomer  t r a c e s o f H i when a n a l y z e d  on 15%  slab gels  (Figure  2).  t o mean t h a t most o f t h e DNA  "tails"  o f t h e p a r t i c l e s had  been removed  (11, 5 7 ) .  p r o t e i n s were o b s e r v e d oligomeric from dimers  fraction  T h i s was  fraction  taken  V e r y low amounts o f n o n - h i s t o n e as r e p o r t e d  before  (52, 5 8 ) . The  i s a mixture of oligonucleosomes  (about 50%),  trimers  ranging  ( a b o u t 30%) t o m u l t i m e r s ,  as o b s e r v e d on a 3% n o n - d e n a t u r i n g a c r y l a m i d e  slab g e l  (not  shown). Sulfhydryl The  reactivity  of native  i n c o r p o r a t i o n o f NEM l a b e l  degree o f a c c e s s i b i l i t y within  nucleosomes  of the t h i o l  t h e nucleosome c o r e .  i s a measure o f t h e g r o u p i n t h e H3 m o l e c u l e s  The m o d i f i c a t i o n o f H3 was  c o n f i r m e d by a n a l y s i s o f t h e N E M - t r e a t e d n u c l e o s o m a l under d e n a t u r i n g  conditions  on 15% S D S - p o l y a c r y l a m i d e  More t h a n 80% o f t h e i n c o r p o r a t e d histone histones  H3  (Figure  3).  are probably  amino and i m i d a z o l e  gels.  l a b e l was a s s o c i a t e d  The c o u n t s a s s o c i a t e d  with  due t o s i d e r e a c t i o n s o f NEM  groups  proteins  i n these p r o t e i n s (59).  with  other with  - 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 n u c l e o s o m e s f r o m 30 m i n u t e s o f m i c r o c o c c a l n u c l e a s e d i g e s t i o n . T r o u t t e s t i s n u c l e i were d i g e s t e d w i t h m i c r o c o c c a l n u c l e a s e f o r 30 m i n u t e s as d e s c r i b e d i n " M a t e r i a l s and M e t h o d s " . The d i g e s t i o n p r o d u c t s were f r a c t i o n e d on a B i o - G e l A-5M c o l u m n . The a b s o r b a n c e a t 260 nm o f e a c h f r a c t i o n i s p l o t t e d v s . t h e 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 of the monomer p e a k f r a c t i o n f r o m an A-5M c o l u m n . One A2 e o u n i t o f t h e monomer p e a k f r a c t i o n was l y o p h i l i z e d a n d t h e n h e a t e d i n 40 y l o f sample b u f f e r . The s a m p l e was a p p l i e d t o a 15% S D S - p o l y a c r y l a m i d e s l a b g e l and r u n a s 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 C o o m a s s i e Blue.  - 20 -  9  O  X  FRACTION NO.  F i g u r e 3. L a b e l l i n g of H3 w i t h N-ethylmaleimide. Two A 2 6 0 u n i t s o f nucleosomes were l a b e l l e d w i t h [ H]N-ethylmaleimide i n 10 M urea a t ambient temperature f o r 5 h r . Tfie r e a c t i o n mixture was d i a l y z e d a g a i n s t Tris-EDTA b u f f e r and l y o p h i l i z e d . The l y o p h i l i z e d m a t e r i a l was taken up i n 100 y l and 50 y l 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. A f t e r electrophoresis-, the g e l was s t a i n e d 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% p e r i o d i c a c i d f o r 2 days a t room temperature. The s o l u b i l i z e d g e l 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 . 3  Hyde and W a l k e r one  21 -  (24) r e p o r t e d  o f t h e two c y s t e i n e s  under n o n - d e n a t u r i n g  the i n a c c e s s i b i l i t y o f  i n whole c h r o m a t i n o f c a l f  conditions.  I t i s thus i n t e r e s t i n g  to observe the behaviour o f the sole c y s t e i n e trout  testis  histone  nucleosomes i n extremely  T r i s - E D T A b u f f e r were e x p o s e d  i n c o r p o r a t i o n was v e r y 10 m i n u t e s .  This  was o b s e r v e d  side reactions  r a p i d , and e s s e n t i a l l y c o m p l e t e i n  benzoate a l s o  showed  experiments using  physical  studies  somes, h a d a l s o r e p o r t e d groups i n chicken are  of salts  the u n a v a i l a b i l i t y of the t h i o l  erythrocytes  on s u l f d y d r y l  (a)  reactivity  nucleosomes.  structure  These r e s u l t s  findings. reactivity i n the studies of  w i t h NEM: G d n « H C l , NaCI and M g C l . 2  E f f e c t of guanidinium  Guanidinium c h l o r i d e histones  p-hydroxymercuri-  (51). O l i n s e t al.(60)  T h r e e d i f f e r e n t s a l t s were u s e d H3 t h i o l  occur  o f t h e e f f e c t s o f u r e a on n u c l e o -  consistent with the present  Effect  The p o s s i b i l i t y  t h e H3 s u l f h y d r y l g r o u p s o f t r o u t  n u c l e o s o m e s t o be u n r e a c t i v e  their  o f denatured  c a n be r u l e d o u t a s t h e s e r e a c t i o n s  Previous  in  4 ) . The  r e a c t i o n may be due t o t r a c e s  much s l o w e r .  testis  When  t o NEM, an  (Figure  n u c l e o s o m e s o r o f f r e e H3 o r H3 f r a g m e n t s . of  residue i n  H3 u n d e r s i m i l a r c o n d i t i o n s .  low r e a c t i v i t y  thymus  chloride  (Gdn«HCl) i s known t o d i s s o c i a t e  f r o m DNA, and t o d e s t r o y i n histones  and p r o t e i n s  somes i n 6 M G d n « H C l were a l l o w e d  s e c o n d a r y and t e r t i a r y i n general.  When n u c l e o -  t o r e a c t w i t h NEM, a  - 22 -  I  i  r=  ft  TIME OF REACTION, MIN  F i g u r e 4. Time course o f r e a c t i o n o f nucleosomes i n d e n a t u r i n g and non-denaturing c o n d i t i o n s . Monomers were r e a c t e d w i t h [ HJNEM under the i n d i c a t e d c o n d i t i o n s as d e s c r i b e d i n M a t e r i a l s 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 o f 20 per mg DNA per ml a t 260 nm, and a DNA m o l e c u l a r weight o f 110,000. 3  maximum o f 2.2 5 minutes the  m o l e s NEM  (Figure  4).  p e r mole n u c l e o s o m e s was  monomer, and i n d i c a t e s t h a t are f u l l y  The e f f e c t of histones  f r o m DNA  with  were a l s o  per  o f Gdn*HCl  studied  on t h e r e l e a s e  and t h e r e s u l t s a r e  When n u c l e o s o m e s were e x p o s e d t o  the native  Gdn«HC1 c o n c e n t r a t i o n increased  per nucleosome  exposed.  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 , associated  with  i n 6 M Gdn-HCI t h e c y s t e i n e  of varying molarity  shown on f i g u r e 5.  within  T h i s maximum i s i n good a g r e e m e n t  e x p e c t e d p r e s e n c e o f two m o l e s o f H3  residues  bound  particles  e x c e e d e d 0.5  r a p i d l y and r e a c h e d  mole n u c l e o s o m e a t 1.2  negligible reactivity persisted until  M.  The  the  reactivity  t h e maximum o f two m o l e s  M.  This plateau  level  NEM  d i d not  change e v e n a t 6 M Gdn•HCI. (b)  Effect  Nucleosome in  o f s o d i u m c h l o r i d e on t h i o l sulfhydryl reactivity  2 M NaCl s o l u t i o n s .  rapid  was  reactivity  first  analyzed  The n u c l e o s o m e s e x h i b i t e d a m o d e r a t e l y  rate of reaction with  NEM,  attaining a ratio  of  mole r e a g e n t p e r mole n u c l e o s o m e s a t a p p r o x i m a t e l y minutes, hours  and r e a c h i n g  (Figure 4).  attributed  a maximum o f 1.6  This  to a d i r e c t  large  increase  unmasking  moles/mole  the  by. t h e r e m o v a l o f DNA.  possibility  induced  at  studies  are taken  may  be  change i n The  seems more p r o b a b l e when t h e r e s u l t s o f  concentration  three  of the c y s t e i n y l residue  or t o a conformational  core  3 0-4 0  in reactivity  upon r e m o v a l o f t h e DNA, histone  1.0  i n t o account  latter salt  (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 o f 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 o f [ H]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 o f Gdn*HCl concentration 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 o u t f o r 120 m i n u t e s . 3  When nucleosomes were p l a c e d i n v a r y i n g NaCI concent r a t i o n s 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 t h a t o f Gdn«HCl was o b t a i n e d .  The  r e a c t i v i t y o f nucleosomes d i d not i n c r e a s e u n t i l the s a l t c o n c e n t r a t i o n was above 1.0 M (Figure 6), when H3 s t a r t s t o d i s s o c i a t e from the DNA  (3 0).  The r e a c t i v i t y i n c r e a s e d  from  1.0 M t o 1.5 M, the c o n c e n t r a t i o n a t which h i s t o n e s a r e completely  dissociated.  The r e a c t i v i t y then s t a r t e d t o drop  at 1.75 M and reached a minimum o f 0.4 moles reagent per mole nucleosome a t 4 M. achieved  The maximum r e a c t i v i t y i s probably  when the h i s t o n e s d i s s o c i a t e from DNA, and before  any r e a s s o c i a t i o n occurs.  The h i s t o n e s may then r e a s s o c i a t e  to form h e t e r o t y p i c tetramers  and octamers a t 2 and 4 M  NaCI s o l u t i o n s r e s p e c t i v e l y (61). (c)  E f f e c t of magnesium c h l o r i d e on t h i o l  reactivity  NEM r e a c t i v i t y o f nucleosomes was a l s o examined i n MgCl  2  solutions of increasing concentration.  p r o f i l e t o t h a t o f Gdn'HCl was obtained and  6).  A similar  (compare f i g u r e s 4  The h i s t o n e s began t o d i s s o c i a t e from DNA a t an  i o n i c s t r e n g t h of 0.5 as monitored by an i n c r e a s e i n t h i o l reactivity. at  The r e a c t i v i t y reached a maximum of 2 moles/mole  r/2 = 1.2, which p e r s i s t s as the M g C l  increased.  2  c o n c e n t r a t i o n was  The s i m i l a r i t y i n the behaviour o f nucleosomes i n  Gdn*HCl and MgCl  s o l u t i o n s i s probably  2  l i k e Gdn*HC1, M g  + +  a l s o has a denaturing  nucleosomal p r o t e i n s  (62) .  due t o the f a c t t h a t e f f e c t on the  _ 26  -  I  ! i i  I I  i  \ 0  i  '  »  '  |  *  0.5  1.0  1.5  2.0  2.5  3.0  i  3.5  IONIC STRENGTH  F i g u r e 6. E f f e c t of i o n i c s t r e n g t h on t h i o l r e a c t i v i t y of nucleosomes. The nucleosomes were allowed to r e a c t w i t h [ H]NEM i n MgCl ( 8 ) and NaCl s o l u t i o n s ( • ) f o r 60 minutes. The s p e c i f i c a c t i v i t i e s are p l o t t e d as a f u n c t i o n of i o n i c s t r e n g t h of the s o l u t i o n s . 3  2  4.0  - 27 E f f e c t o f urea on s u l f h y d r y l  reactivity  F i g u r e 7 shows the e f f e c t o f i n c r e a s i n g urea  concen-  t r a t i o n s on the r e a c t i v i t y o f nucleosome t h i o l groups. 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  Very  5-6 M urea was  reached, where an abrupt t r a n s i t i o n began, centered a t approximately 8.5 M urea. e f f e c t on hydrophobic  Since urea has a d i s r u p t i v e  i n t e r a c t i o n s , the r e s u l t s  suggest  t h a t there i s a c o o p e r a t i v e u n f o l d i n g o f nucleosome s t r u c t u r e above 7 M urea, l e a d i n g t o f u l l of H3 t h i o l groups a t 9-10 M urea. demonstrated  exposure  O l i n s e t al. have  the same behaviour w i t h c h i c k e n e r y t h r o c y t e  nucleosomes i n urea s o l u t i o n s i n good agreement w i t h t h e i r  (60).  The p r e s e n t data a r e  results.  Cooperative e f f e c t o f NaCl and urea on nucleosome conformation Nucleosomes were exposed t o v a r i o u s combinations o f s a l t and urea c o n c e n t r a t i o n s , and the r e a c t i v i t y w i t h NEM was measured.  The r e s u l t s are summarized i n f i g u r e 8.  A  maximum o f 2 moles NEM per mole nucleosomes was a t t a i n e d when nucleosomes i n 6 M urea were f u r t h e r exposed t o 0.5 M NaCl  (Figure 8A).  Conversely, i f nucleosomes i n 0.5 M NaCl  were exposed to i n c r e a s i n g urea c o n c e n t r a t i o n s , a steady r i s e i n NEM i n c o r p o r a t e d was observed u n t i l the urea t r a t i o n reached 6 M (Figure 8B) .  concen-  Since n e i t h e r 0.'5 M NaCl  nor 6 M urea alone allows s i g n i f i c a n t i n c o r p o r a t i o n o f the l a b e l l e d NEM (Figures 6 and 7 ) , they must a c t c o o p e r a t i v e l y to a l t e r nucleosome conformation.  In 2 M NaCl, o n l y 3-4 M  -  t  28  -  '  1  T  r  [UREA], M  F i g u r e 7. E f f e c t o f u r e a c o n c e n t r a t i o n on t h e r e a c t i v i t y o f n u c l e o s o m e s w i t h NEM. Nucleosomes i n s o l u t i o n s o f d i f f e r e n t u r e a c o n c e n t r a t i o n s were r e a c t e d w i t h [ H ] N E M f o r 150 m i n u t e s . 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 a s 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 . 3  -  0.2  0.4  [NaCl], M  0.6  0  29  2  -  4  6  [Urea] , M  0  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 u r e a on e x p o s u r e o f nucleosome t h i o l groups. N u c l e o s o m e s were e x p o s e d t o v a r i o u s c o m b i n a t i o n s o f N a C l and u r e a c o n c e n t r a t i o n s f o r 6 0 m i n , and t h e r e a c t i o n w i t h [ H]NEM was c a r r i e d o u t f o r 24 h r a t 2 2 ° . I n p a n e l A, t h e r e a c t i o n i n 6 M u r e a i s p l o t t e d as a f u n c t i o n o f NaCl c o n c e n t r a t i o n . I n B and C, t h e r e a c t i o n i n 0.5 M and 2 M N a C l , 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 o f urea concentration. 3  - 30 u r e a was  sufficient  nucleosomal Sulfhydryl The  to a l l o w complete  t h i o l g r o u p s w i t h NEM reactivity  excluded  negligible  oligomers  o f an A-5M  amount o f monomers, was  u n d e r d e n a t u r i n g and  of  (Figure 8C).  o f nucleosome  fraction  reaction  column, which c o n t a i n s  allowed  non-denaturing  to react with  conditions.  Figure 9  shows t h e e x t e n t o f r e a c t i o n o f o l i g o n u c l e o s o m e s in  10 mM  NaCl  T r i s , p H 7.4  + 6 M u r e a and  + 0.7  t h a t o f monomers, e x c e p t  in  2 M NaCl,  after 1.2  effect  o n l y 0.5  60 m i n u t e s ,  Tryptic  o f NaCl SH  similar,  and  NEM  i f not  of  oligomers  identical  i n 2 M NaCl.  u r e a was  2 M  to,  Again,  observed.  However,  g r o u p s p e r mole n u c l e o s o m e r e a c t e d  ( F i g u r e 3).  vs.  approximately  The  reason  found  that  for  this  i s unclear. nucleosomes  S a h a s r a b u d d h e and isolated  by  Van  Holde  approximately  5-7S  (63)  nuclease d i g e s t i o n  change i n s e d i m e n t a t i o n v e l o c i t y  was  with  6 M urea,  behaviour  using oligonucleosomes,  digestion of  particles  very  The  f o r the r e s u l t s  SH/mole u s i n g monomers  behaviour  EDTA, 2 M N a C l ,  6 M Gdn*HCl.  u n d e r t h e s e c o n d i t i o n s was  the s y n e r g i s t i c  mM  NEM  from  underwent a  upon d i g e s t i o n w i t h t r y p s i n .  ational  c h a n g e , i . e . , an u n f o l d i n g o f t h e p a r t i c l e .  and Van  Lente  subsequently  shift  i n molecular  and  (64)  to  This  weight,  of chromatin  therefore attributed  dramatic  a v a l u e o f 11S  a c c o m p a n i e d by o n l y a s m a l l d e c r e a s e was  chromatin  largely  showed t h a t  to a  conform-  tryptic  Weintraub digestion  leads to the l o s s of only the N-terminal  20-30  F i g u r e 9. Sulfhydryl r e a c t i v i t y of oligomers i n denaturing and n o n - d e n a t u r i n g s o l u t i o n s . R e a c t i v i t y of oligomers with NEM was d e t e r m i n e d i n t h e f o l l o w i n g s o l u t i o n s : •, 2 M NaCI and 6 M u r e a ; o, 6 M Gdn*HCl; A , 2 M NaCI; • , 6 M u r e a ; •, T r i s - E D T A b u f f e r .  amino that  acid the  residues  loss  of  conformational are  It in  and  the  reaction  could  in  of  during  NEM  buffer,  2  times.  various  trypsin  digestion  had  no  was  of  added  to  monomers M after  6  stoichiometric SH/mole NaCI (1.0  mole  at  treatment that  the  also  with  in  subjected  to  was  that  are  found of  present  histones  as  did  trypsin more  digestion only  (66-70),  in  the  t h i o l s  change  regions  80%  (Figure the  and  were  acetate  digestion than  3  of  the  10).  was  of after  that  basal If  Gdn-HCl  e s s e n t i a l l y  Table  label  in  2  M  digestion  I).  before To  under  confirm  such  monomers  were  conditions.  was  l o s t  e-acetyl  regions in  moles  (1.6-1.75  similar  effective  the  moles/mole  Since  I  M Gdn-HCl  buffer.  removed  amino-terminal  trypsin  on  labelled  under  r e a c t i v i t y  minutes  tryptic  1.2  synthesis.  Table  r e a c t i v i t y  after to  DNA  observed  occurs  The  (65) ,  altering  t h i o l  6  was  digestion,  (Figure  f - i ^ . i -  It  in  Tris-EDTA  compared  trypsin)  vitro  in  for  or  60  effect  expected.  not  amino-terminal  conditions,  minutes  of  minutes  60  NEM  trypsin  reaction  nucleosomes),  solution  to  or  regions  vivo  t r y p s i n .  after  M NaCI  noticeable  in  monitor  with  a  these  mechanism  incorporated  for  Since  to  suggested  trigger  transcription  digestion  digestion  r e a c t i v i t y  could  a  interest  trypsin  This  acetylated  provided  of  Tris-EDTA  regions  nucleosomes.  following  amount  histones.  extensively  therefore  nucleosomes  shows  in  conformation  was  core  basic  change  modifications  nucleosome  the  these  phosphorylated  these  of  32 -  of  at  30-40  groups the  cleaving  core these  It  - 33 -  ' Table I Reaction of t r y p s i n - d i g e s t e d  mononucleosomes  w i t h N-ethylmaleimide  mTime^ of Digestion (min)  Reactivity i n J  Tris-EDTA  2 M NaCl  (moles N-ethylmaleimide/mole  6 M Gdn•HCI 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 d i g e s t e d f o r 0-30 min w i t h t r y p s i n were allowed to r e a c t with NEM  f o r 6 0 min i n .the i n d i c a t e d s a l t s o l u t i o n s a t  ambient temperature.  The a c i d - p r e c i p i t a b l e f r a c t i o n was  as d e s c r i b e d i n M a t e r i a l s and Methods.  counted  - 34 -  Time of digestion min  F i g u r e 10. Efficiency of trypsin digestion. [ C]~ l a b e l l e d n u c l e o s o m e s were d i g e s t e d w i t h t r y p s i n f o r t h e a p p r o p r i a t e t i m e as d e s c r i b e d i n M a t e r i a l s and Methods. The p e r c e n t a g e o f l a b e l l e f t i n t h e 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 o f d i g e s t i o n . lk  -  35 -  regions  from the nucleosomes.  tryptic  d i g e s t i o n d o e s n o t c a u s e any  changes  i n the environment of the t h i o l  Reconstitution  The r e s u l t s show significant  o f nucleosomes  and DNA  (71-74). native  has b e e n r e p o r t e d  These r e c o n s t i t u t e d  monomers i n s t r u c t u r e  pattern  (74).  We  NaCl + 5 M urea monitored  (Figure  of  nucleosomes  using  nucleosomes  of  t h e H3  i n the  reaction  reconstituted  4 and  o f NEM  11).  that Exposure  l e d t o the  p e r mole o f  In o r d e r t o o b t a i n  particles,  high  yields  out the r e c o n s t i t u t i o n i n  a g e n t , e . g . 1 mM  agent y i e l d e d  t h i o l s was  conditions,  little  nucleosomes w i t h normal b e h a v i o u r towards  presence of a reducing the reducing  and  o f NaCl to 2 M markedly  nucleosomes t o 6 M Gdn«HCl  necessary to carry  of  from 2 M  a t i m e c o u r s e o f r e a c t i o n much l i k e  preparations.  reconstituted i t was  digestion  NEM.  showed v e r y  Addition  o f a l m o s t 2 moles  as w i t h n a t i v e  the  i n nuclease  p a r t i c l e s ( e . g . compare F i g u r e  reconstituted  NEM,  reactivity  11).  giving  incorporation  of  (15) and  the exposure of t h i o l s  particles, of native  nucleosomes resemble the  ( o r 10 M u r e a ) s o l u t i o n s by d i a l y s i s ,  the t h i o l  increased  from s a l t - d i s s o c i a t e d  by a number o f w o r k e r s  have r e c o n s t i t u t e d  Reconstituted w i t h NEM  conformational  groups.  The r e c o n s t i t u t i o n o f n u c l e o s o m e s histones  that  p a r t i c l e s i n which a  unreactive  p r e s u m a b l y due  DTT.  t o NEM  even under  t o f o r m a t i o n o f H3  Omission proportion denaturing  intermolecular  -  Co  2.0i  i  36  i  -  i  TIME OF REACTION, MIN  F i g u r e 11. Time c o u r s e o f i n c o r p o r a t i o n o f [ H ] N E M 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 o f 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 n o n - d e n a t u r i n g 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 t h e t i m e 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 t h e p r e s e n c e o f 1 mM DTT. Dashed l i n e , N u c l e o s o m e s r e c o n s t i t u t e d i n the absence o f r e d u c i n g agent. 3  disulfides  In o r d e r reconstituted the  native  digest gel.  11,  (Figure to  dashed  further  support  seen from  nucleosomes are clearly  figure 12,  that  particles  similar.  visible.  reconstituted and  hypothesis  o n e s , DNase I d i g e s t i o n was on the  f r o m n u c l e i , n u c l e o s o m e monomers  is  the  the by  that  n u c l e o s o m e s have a s i m i l a r c o n f o r m a t i o n  p r o d u c t s were a n a l y z e d As  line).  The  This  a f t e r the  99%  formamide,  gel pattern  of  ( n a t i v e ) , and  indicates that  6%  acrylamide  digest  products  reconstituted pattern  n u c l e o s o m e s can histones  from  nucleosomes resemble the  criteria.  to the  c h a r a c t e r i s t i c "ladder"  d i s s o c i a t i o n of  reconstituted several  a  p e r f o r m e d and  the  be  DNA  native  -  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 , n u c l e o s o m e monomers and r e c o n s t i t u t e d n u c l e o s o m e s . Nucleosomes were d i g e s t e d w i t h DNase I a n d a n a l y z e d o n 9 9 % f o r m a m i d e , 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 u n d e r UV l i g h t .  - 39  -  DISCUSSIONS Carboxy-terminal r e g i o n of H3 I t i s evident  i n n a t i v e nucleosomes  from the low  reactivity  of the H3  thiol  group i n n a t i v e nucleosomes t h a t the c y s t e i n e r e s i d u e H3  in trout testis  of  i s b u r i e d w i t h i n the p r o t e i n core.  the complete amino a c i d sequence of t r o u t t e s t i s  H3  has  been determined, the p o s i t i o n of the s i n g l e c y s t e i n e i s i n f e r r e d from comparative data from other unreactive  lower  vertebrates  (1).  t h a t the c a r b o x y l  of the p r o t e i n i s f o l d e d i n such a  t h a t the t h i o l group i s masked and Hyde and Walker groups i n c a l f [2-nitrobenzoic the other  (24)  reported  thymus H3 was  t h a t one  i n a c c e s s i b l e to  a c i d ] i n chromatin a t low  t h i o l was  cysteine  unavailable  not  residue  to be at p o s i t i o n 110 end  The  Although  indicates way  for reaction. •  of the two  thiol  5,5'-dithiobis-  salt  concentrations;  found to be exposed to s o l v e n t .  They  i n f e r r e d from primary sequence a n a l y s i s t h a t the b u r i e d  thiol  group i s at p o s i t i o n 96.  the  protected  The  t h i o l group i n c a l f  present  data suggest t h a t  thymus H3 very  l i k e l y corresponds,  i n i t s p o s i t i o n i n the amino a c i d sequence, to the s u l f h y d r y l r e s i d u e of t r o u t t e s t i s  and  which i s l o c a t e d a t p o s i t i o n 110.  T h i s i s reasonable  the c y s t e i n e at p o s i t i o n 96 i n c a l f  chicken  single  thymus H3  r a r e amino a c i d s t h a t are not conserved i n the sequence of H3.  I t i s l i k e l y that t h i s  e s s e n t i a l f o r proper H3  erythrocyte  i s one  since of  primary  c y s t e i n e i s non-  o r i e n t a t i o n i n the nucleosomes.  the  H3,  - 40 The has  unreactive sulfhydryl  not  (i.e.  (51). as  as  2 moles r e a g e n t  Effect  the  ionic  nucleosomal divalent  the p r e s e n t  s t r u c t u r e of  75-7).  i o n s a t low  The  s t u d i e d by  ionic  of high  salt  Olins  a tetramer  (76)  octamer  investigation:  who  a t an  found  solubility  NaCl  and studies  analyses  (77).  nucleosome s t r u c t u r e  t h a t the  complex a t an ionic  by  i n hydrodynamic  predominant  ionic  strength of  s t r e n g t h o f 4,  s a l t s were employed  Gdn"HCI, N a C l and  Gdn«HCl have d e n a t u r i n g of nucleosomal  similar.  effects  ionic  obtained  s t r e n g t h , the  must t h e r e f o r e be  reactivity.  The  MgCl -  i n the  the  on p r o t e i n s and  o f two  present  Both M g  2  p r o t e i n s i n these  A maximal r e a c t i v i t y  n u c l e o s o m e s was  and  affected  predominates.  Three d i f f e r e n t  haviour  and  s t r u c t u r e of  c o n c e n t r a t i o n s on  (61)  while  t o be  strengths  s p e c i e s of the h i s t o n e core 2 was  be  H3  secondary  found  (75), e l e c t r o n microscopy  was  quantitative reaction  changes o f nucleosomes i n s o l u t i o n s o f  h i s t o n e s was  effect  one;  s t r e n g t h s have b e e n s t u d i e d i n v a r i o u s  (61,  The  p-hydroxymercuri-  p e r mole n u c l e o s o m e s ) c o u l d n o t  the  Conformational  laboratories  H3  f o r m e r method.  o f s a l t s on  different  testis  However, t h e p r e v i o u s method o f a n a l y s i s  sensitive  obtained with  this  group i n t r o u t  p r e v i o u s l y been d e m o n s t r a t e d u s i n g  benzoate was  -  salt  the  moles r e a g e n t  per  mole  strength.  h i s t o n e s are d i s s o c i a t e d by  be-  s o l u t i o n s are  a t a b o u t 1.2-1.5 i o n i c  denatured  and  + +  from the  the  salts  to achieve  plateau reaction with  these  s a l t s was  At DNA  maximal reached  - 41 at a lower i o n i c s t r e n g t h than w i t h NaCI.  This i s  probably due t o the f a c t t h a t both s a l t s are more e f f i c i e n t than N a  +  a t d i s p l a c i n g h i s t o n e s from DNA.  Furthermore,  r e a c t i o n maximum reached i n these s a l t s was i n NaCI s o l u t i o n s .  These r e s u l t s may  u n f o l d i n g e f f e c t of M g  + +  and Gdn  +  the  higher than t h a t  be a t t r i b u t e d to an  on h i s t o n e conformation a t  high s a l t concentrations. Nucleosomes i n NaCI s o l u t i o n s e x h i b i t a completely d i f f e r e n t behaviour i n the r e a c t i o n w i t h NEM. the t h i o l groups r e a c t w i t h NEM  a t a moderately r a p i d  a t t a i n i n g a r a t i o of 1.0 mole reagent/mole at approximately 30-40 min, moles/mole a t three hours  In 2 M NaCI, rate,  nucleosomes  and r e a c h i n g a miximum of  (Figure 4 ) .  1.6  T h i s i s much slower  than the r e a c t i o n i n Gdn*HCl which a t t a i n s the maximum of 2 moles/mole w i t h i n 5 minutes. h i s t o n e s are d i s s o c i a t e d  Under these c o n d i t i o n s , the  from the DNA  (30), and s e v e r a l  lines  of evidence suggest t h a t the n a t i v e s t r u c t u r e s of the h i s t o n e s are r e t a i n e d :  (i) s a l t - d i s s o c i a t e d h i s t o n e s r e a d i l y r e a s s o -  c i a t e w i t h DNA  to y i e l d normal-appearing  the s a l t has been removed (16) demonstrated  (11, 57);  nucleosomes once  ( i i ) Thomas and  Romberg  the e x i s t e n c e of h i s t o n e octamers i n  s a l t d i s s o c i a t e d h i s t o n e s , s i m i l a r to those observed i n n a t i v e chromatin;  ( i i i ) Weintraub  and Van Lente  (64)  observed  the same t r y p t i c h i s t o n e cores whether nucleosomes or  salt-  d i s s o c i a t e d h i s t o n e s were d i g e s t e d w i t h t r y p s i n .  possibi-  Two  l i t i e s can e x p l a i n the behaviour of H3 i n 2 M NaCI: e i t h e r  - 42 there i s 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 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 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 h i s t o n e octamer to two h e t e r o t y p i c tetramers  (4 2), with the H3  t h i o l s being present a t the boundary o f i n t e r a c t i n g  tetramers.  S t u d i e s on the r e a c t i v i t y of the H3 t h i o l group i n v a r y i n g s a l t c o n c e n t r a t i o n s f a v o r the l a t t e r of the above alternatives.  two  The r e a c t i v i t y o f H3 t h i o l groups i n c r e a s e s  from 1.0 M t o 1.5 M, the c o n c e n t r a t i o n at which the h i s t o n e s are completely  d i s s o c i a t e d , and f a l l s markedly a f t e r  r e a c h i n g 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 c o n t r o l nucleosomes a t low s a l t concentration. completely  Under these c o n d i t i o n s the h i s t o n e s are  d i s s o c i a t e d from the DNA;  t h e r e f o r e , the masking  of the t h i o l groups i n 4 M NaCI must be due to the formation of histone-histone i n t e r a c t i o n s .  These data are c o n s i s t e n t  w i t h a model i n which the t h i o l groups become exposed due t o d i s s o c i a t i o n of a h i s t o n e octamer a t 1.5-2.0 M NaCI, but become b u r i e d again upon r e f o r m a t i o n of an octameric s t r u c t u r e a t 3.5-4.0 M NaCI.  O l i n s (61), on the b a s i s of hydrodynamic  s t u d i e s , reached s i m i l a r c o n c l u s i o n s r e g a r d i n g i n t e r a c t i o n s over these s a l t  histone  concentrations.  The f a c t t h a t the maximum r e a c t i v i t y obtained  i n NaCI  s o l u t i o n s i s o n l y 1.6 moles SH/mole nucleosomes i s probably due t o the formation of H3-H3 i n t e r a c t i o n s which promote i n t e r m o l e c u l a r d i s u l f i d e formation  (see below, under  "recons-  t i t u t i o n " ) , thus g r a d u a l l y r e n d e r i n g the t h i o l s u n r e a c t i v e toward NEM.  T h i s process would be l e s s f a v o r a b l e w i t h  t o t a l l y denatured H3  i n Gdn'HCI s o l u t i o n s  of t h i s i n t e r p r e t a t i o n , Hyde and Walker  (6 M).  In support  (24) found t h a t both  c y s t e i n y l r e s i d u e s of a c i d - e x t r a c t e d (and t h e r e f o r e presumably denatured)  c a l f thymus H3 r e a c t e d r a p i d l y w i t h  5,5 -dithiobis1  [ 2 - n i t r o - b e n z o i c a c i d ] , but t h a t upon i n c u b a t i o n of the h i s t o n e i n 2 M NaCl, the r e a c t i o n r a t e s t e a d i l y  decreased.  That the r e a c t i o n of nucleosomes w i t h NEM  occurs much  more s l o w l y i n 2 M NaCl than i n 6 M Gdn«HCl probably the heterogeneous  reflects  nature of the 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  under these c o n d i t i o n s (4 2).  A gradual s h i f t i n the octamer-  tetramer e q u i l i b r i u m towards the tetramer, and/or the p o s s i b i lity  t h a t some of the s t r u c t u r e s  g r a d u a l t i t r a t i o n of the t h i o l  "breathe" may  allow the  groups.  E f f e c t of urea on nucleosomes O l i n s e t a l . demonstrated  the d i s r u p t i v e e f f e c t of urea  on chromatin, u s i n g hydrodynamic s t u d i e s (60).  They were able  to d i s t i n g u i s h non-cooperative changes i n s t r u c t u r e which were a t t r i b u t e d to t r a n s i t i o n s of the outer DNA-rich  shell  of the p a r t i c l e , and c o o p e r a t i v e changes between 5 and M urea, a t t r i b u t e d to changes i n the p r o t e i n c o r e .  10  The  e x i s t e n c e of the l a t t e r c o o p e r a t i v e e f f e c t s i s thus confirmed by our data on t h i o l r e a c t i v i t y .  In h i g h urea  i n f e r t h a t the h i s t o n e s , although s t i l l  (>8 M),  attached to  through b a s i c r e g i o n s , are completely denatured  we  DNA  l e a d i n g to  -  exposure  44  of the H3 t h i o l s .  -  A s i m i l a r scheme was  presented  by O l i n s e t a l . ( 6 0 ) . The i n f e r e n c e reached above was  further  by the s t u d i e s i n v a r i o u s combinations concentrations.  confirmed  of s a l t and  urea  The s a l t and urea were found to a c t syner-  g i s t i c a l l y i n i n c r e a s i n g the r e a c t i v i t y of H3 t h i o l s . nucleosomes were put i n 6 M urea and i n c o r p o r a t i o n of 2 moles NEM/mole was  0.5  When  M NaCl, the maximum  attained.  Since  n e i t h e r 6 M urea nor 0.5 M NaCl alone a l l o w s i g n i f i c a n t r e a c t i o n of NEM  w i t h H3 s u l f h y d r y l groups,  these agents must  a c t c o o p e r a t i v e l y to a l t e r nucleosome conformation. i s thus concluded t h a t i n nucleosomes the H3  sulfhydryl  groups a t p o s i t i o n 110 are p r o t e c t e d from NEM  by both  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 .  In the  of 0.5 M NaCl and 6 M urea, they may due  It  presence  become f u l l y exposed  to a) a r u p t u r e of some i o n i c histone-DNA i n t e r a c t i o n s  which d e - s t a b i l i z e s the nucleosome and allows the urea to e x t e n s i v e l y denature H3, or b) a c o n f o r m a t i o n a l change i n the nucleosome which exposes the s u l f h y d r y l groups without e x t e n s i v e d e n a t u r a t i o n of the h i s t o n e s . T h 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 i n  the a s s o c i a t i o n of H i and i n i n c l u s i o n of the DNA regions.  spacer  When such oligomers were s u b j e c t e d the same treatments  as nucleosome monomers, s i m i l a r r e s u l t s were o b t a i n e d .  This  i n d i c a t e s t h a t the a s s o c i a t i o n w i t h Hi and a longer spacer  r e g i o n has The  no s i g n i f i c a n t e f f e c t on the b u r i e d t h i o l s i n  carboxy-terminal  r e g i o n of H3  i s t h e r e f o r e probably  i n v o l v e d i n i n t e r a c t i o n s among adjacent was,  nucleosomes.  H3. not  There  however, a s l i g h t d i f f e r e n c e observed i n 2 M NaCI, i n  which the oligomers  r e a c t e d a t a f a s t e r r a t e but a t t a i n i n g  lower maximum l e v e l . unknown.  The  reason f o r t h i s d i s c r e p a n c y  Perhaps other p r o t e i n s present  dissociated histones.  is  i n t e r a c t with  the  Since q u a n t i t a t i v e r e a c t i o n of  t h i o l s of oligonucleosom.es occurs r e a c t i v i t y i n s a l t i s not due  i n 6 M Gdn* HCl,  the  the  to i n a c t i v a t i o n of the  lower NEM  itself. T r y p s i n d i g e s t i o n of nucleosomal p r o t e i n s S t u d i e s of the primary s t r u c t u r e s of the h i s t o n e s have r e v e a l e d t h a t the p o s i t i v e l y charge l y s y l and a r g i n y l r e s i d u e s are d i s t r i b u t e d i n c l u s t e r s near the ends of  the  p r o t e i n molecules  the  (1).  The  amino t e r m i n a l r e g i o n has  g r e a t e s t d e n s i t y of p o s i t i v e charge and was be the primary s i t e of i n t e r a c t i o n w i t h DNA.  thought to This region  i s more s u s c e p t i b l e to p r o t e o l y s i s than other r e g i o n s the h i s t o n e s .  Earlier  change i n sedimentation t r y p t i c d i g e s t i o n (63).  i n v e s t i g a t i o n s had  shown a dramatic  v e l o c i t y of the nucleosomes upon Subsequently, the  change accompanying t r y p s i n d i g e s t i o n was nuclease  conformational confirmed  d i g e s t i o n of t r y p s i n - t r e a t e d nucleosomes  Recently,  Whitlock and  the N H 2 ~ t e r m i n a l relatively  of  by (78).  S t e i n r e p o r t e d t h a t the removal of  h i s t o n e r e g i o n s w i t h t r y p s i n produces  small changes i n the f o l d i n g of core  particle  - 46 DNA  (79).  -  They suggested t h a t the c e n t r a l and  histone regions  COOH-terminal  are important i n forming the p r o t e i n  as w e l l as s t a b i l i z i n g the DNA  w i t h i n the core  core  particle  complex. In our experiments, t r y p s i n d i g e s t i o n 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  groups.  Since  the H3  thiol  s u l f h y d r y l groups are s i t u a t e d i n the  COOH-terminal r e g i o n , our r e s u l t s suggest t h a t even i f there i s any  l a r g e conformational  the amino-terminal r e g i o n ,  change induced upon removal of these changes must not a f f e c t the  environment around c y s t e i n e 110 Weintraub and Van  Lente  of H3 (64)  r e s i s t a n t r e g i o n i s not a f f e c t e d by by  ( i . e . the c a r b o x y l  end).  have shown t h a t the t r y p s i n 2 M NaCl, but  i s affected  6 M urea, which i n d i c a t e s t h a t the t r y p s i n r e s i s t a n c e i s  conferred  by i n t e r m o l e c u l a r  i n t e r a c t i o n s between h i s t o n e s .  Bohm e t a_l a l s o demonstrated, using n u c l e a r magnetic resonance, t h a t the r e s i d u e s required  f o r the  1 to 41 of H3 formation  and  of the c o r r e c t  i n t e r a c t i o n s i n the H3-H4 complex were d e s c r i b e d erythrocyte  1 to 37 of H4  by L i l l e y and  (39).  are  not  histone-histone Similar  T a t c h e l l (80)  for  properties  chicken  core p a r t i c l e s t r e a t e d w i t h t r y p s i n .  A l l evidence accumulated to date supports the of Whitlock and  suggestion  S t e i n t h a t the COOH-terminal r e g i o n i s respon-  s i b l e f o r the o r g a n i z a t i o n and the core p a r t i c l e . i n s t r u c t u r e , and  s t a b i l i z a t i o n of the DNA  This r e g i o n must be r e l a t i v e l y r e s i s t a n t to p r o t e o l y s i s .  The  in  constant  NH -terminal 2  - 47 regions, in  the  on  the  other  organization  -  hand, a r e  of  the  DNA  likely within  t o be the  non-essential  core;  they  may,  / however, have o t h e r  functions  chromatin structure  since  act with other and  kinases  for  core  proteins  (81)  (the  histone  Reconstitution  they are  s u c h as  relatively  histones  amino end  i s the  only  of  required  f o r the  The  r e c o n s t i t u t e d nucleosomes resemble the  results  (15)  I t has  and  the  thiol  behaves i n the  structures to obtain  necessary to c a r r y out a g e n t , e.g.  the  high  1 mM  unreactive  observation disulfides. reported  may  t o NEM be  due  These  native  y i e l d s of  that  the  to occur during  of  the  nucleo-  native  identical,  reconstituted  i n the  even under d e n a t u r i n g  Auto-oxidation  (74).  nucleosomes.  o f H3 cysteine  i t  was  presence of  Omission of  formation  (15).  monomers  of  i f not  the  i n which a p r o p o r t i o n  to  histones  reconstituted  t o w a r d s NEM,  dialysis DTT.  many  A* p a r t i c l e  native  as  have s i m i l a r , as  the  agent y i e l d e d p a r t i c l e s was  a l l four  a 125  same way  nucleosomes w i t h normal b e h a v i o u r  reducing  by  group of r e c o n s t i t u t e d  conditions.  n u c l e o s o m e s must t h e r e f o r e  In o r d e r  described  i n nuclease digestion pattern  monomers u n d e r v a r i o u s  conformational  been found t h a t  r e c o n s t i t u t i o n of  show t h a t  somes g e n e r a l l y  site  inter-  methylases  e x p e r i m e n t s have been r e p o r t e d  are  Our  free to  nucleosomes  71-78).  structure  native  acetylase,  (15,  in  the  modifications).  Reconstitution workers  i n maintaining  of  a  reducing the  H3  conditions.  thiols This  intermolecular residues  homogenization of c a l f  has  thymus  been (82).  - 48 Thus, although the H3 t h i o l s a t p o s i t i o n 110 do not form a d i s u l f i d e bond i n the n a t i v e nucleosomes, they are r e a d i l y o x i d i z e d once H3 i s d i s s o c i a t e d from DNA.  I t has r e c e n t l y  been shown t h a t dimers of H3 l i n k e d through c y s t e i n e  110  can be s u b s t i t u t e d f o r monomeric H3 i n r e c o n s t i t u t i o n experiments, and y i e l d nucleosomes which are i n d i s t i n g u i s h a b l e from the n a t i v e p a r t i c l e s by a v a r i e t y 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 t h a t the c y s t e i n e s i n the nucleosome;  they a l s o confirm  are c l o s e together  c r o s s l i n k i n g data  i n d i c a t i n g t h a t the two H3 are c l o s e t o each other  (16, 22).  The f a c t t h a t they do not form a d i s u l f i d e i n v i v o suggests t h a t e i t h e r the r e c o n s t i t u t e d p a r t i c l e s have been d i s t o r t e d s l i g h t l y to accommodate the H3 dimer, or t h a t the environment around r e s i d u e  110 i n the n a t i v e nucleosome  i s not conducive  to i o n i z a t i o n o f the t h i o l group, perhaps due to a low d i e l e c t r i c constant.  I f the l a t t e r were t r u e , the two  might be i n c l o s e contact  cysteines  and y e t remain reduced.  DNase I d i g e s t i o n products of r e c o n s t i t u t e d  nucleosomes  are s i m i l a r t o those of n a t i v e nucleosomes, i n d i c a t i n g t h a t the DNase I s e n s i t i v e s i t e s are a l s o r e c o n s t i t u t e d . assembly o f nucleosomes i s thus an i n t r i n s i c property the h i s t o n e s  and  DNA.  The of  -  49  -  Conclusion The  studies reported  e x p o s u r e o f H3  thiol  monitored using This  sensitive  groups i n nucleosomes can  a specific assay  histones  in isolated  fraction  (83-4),  may  may  thiol  be  i f full  carboxy-terminal  This region  r e g i o n may  be  of  i n core  Current  essential  "active"  studies of  thiol  particles  sulfhydryl  i n the  the  environment of  by  the  heteroty-  changes i n  digestion.  o r g a n i z a t i o n and  This  stabilization  specific  amino a c i d  a detailed  histone-DNA i n t e r a c t i o n  relative  (37)  residues  p i c t u r e of  histones  from s t u d i e s  investigation,  i n the  should  p o s i t i o n s of  Knowledge g a i n e d  i n the present  i n formulating  three-dimensional  X-ray d i f f r a c t i o n  of the  particles.  s u c h as  tryptic  to e l u c i d a t e the  the d e f i n i t i o n  DNA  s t a b l e region of  110  particles.  efforts  and  and  of  groups i s  i n t e r f a c e between  by  i n the  s t r u c t u r e o f n u c l e o s o m e s by  useful  the  c o n t a i n i n g Cys  i s unaffected  nucleosome s t r u c t u r e induced  histones,  Further  r e g i o n o f H3  located i n a r e l a t i v e l y  tetramers.  soon a l l o w  such as  i n the masking of the  nucleosome c o r e , perhaps a t the  DNA  amount  H3.  seems t o be  the  NEM.  the  thiol  of  effectively  o f c r o s s l i n k e d h i s t o n e octamers from c o r e  groups of  pic  fractions  conditions.  be  s u c h as  r e a c t i o n o f the  r e v e a l t h e r o l e o f DNA  The  reagent  used t o e s t i m a t e  nucleosomal  assumed u n d e r d e n a t u r i n g reactivity  here i n d i c a t e t h a t the degree  in  of  the  should  be  histone-histone  nucleosomes.  - 50 REFERENCES  1.  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