UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Studies on the chromatin-bound histone deacetylase of HeLa cells Hay, Colin William 1983

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
831-UBC_1983_A1 H39.pdf [ 7.42MB ]
Metadata
JSON: 831-1.0095859.json
JSON-LD: 831-1.0095859-ld.json
RDF/XML (Pretty): 831-1.0095859-rdf.xml
RDF/JSON: 831-1.0095859-rdf.json
Turtle: 831-1.0095859-turtle.txt
N-Triples: 831-1.0095859-rdf-ntriples.txt
Original Record: 831-1.0095859-source.json
Full Text
831-1.0095859-fulltext.txt
Citation
831-1.0095859.ris

Full Text

STUDIES ON THE CHROMATIN-BOUND HISTONE DEACETYLASE OF HeLa CELLS  by  COLIN WILLIAM HAY B.Sc., The U n i v e r s i t y o f Aberdeen, 1978 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN THE FACULTY OF GRADUATE STUDIES DEPARTMENT OF  BIOCHEMISTRY  We a c c e p t t h i s t h e s i s as conforming to the required  standard  THE UNIVERSITY OF BRITISH COLUMBIA September 1983 © C o l i n W i l l i a m Hay, 1983  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 of  requirements f o r an advanced degree at the  the  University  o f B r i t i s h Columbia, I agree t h a t the L i b r a r y s h a l l make it  f r e e l y a v a i l a b l e f o r reference  and  study.  I further  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 copying of t h i s t h e s i s for  s c h o l a r l y purposes may  department or by h i s or her  be granted by the head o f representatives.  my  It is  understood t h a t copying or p u b l i c a t i o n of t h i s t h e s i s for  f i n a n c i a l gain  s h a l l not be  allowed without my  permission.  Department o f  Biochemistry  The U n i v e r s i t y of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada  V6T  1Y3  Date  September 1, 1983  written  ABSTRACT  The reversible acetylation of histones i s thought to play a role i n chromatin processing, including t r a n s c r i p t i o n , r e p l i c a t i o n and r e p a i r . Studies on the acetyltransferases, responsible for acetylating the nucleosomal core histones, have resulted i n characterization of these enzymes.  However, very l i t t l e i s known about the properties and  d i s t r i b u t i o n of histone deacetylase. The reversible i n h i b i t i o n of histone deacetylase by butyrate was employed to permit studies on the chromatin-bound histone deacetylase of HeLa c e l l s using endogenous [ H]-acetyl l a b e l l e d polynucleosomes 3  containing the enzyme.  These were prepared i n the presence of 50mM  butyrate and histone deacetylase was assayed upon removal of the inhibitor.  It was found that active enzyme i s present only i n association  with a high molecular weight complex.  This deacetylase-containing complex  i s r e l a t i v e l y resistant to digestion with micrococcal nuclease. a c t i v i t y i s found on mononucleosomes or oligonucleosomes.  No  Up to 90% of  l a b e l l e d acetyl groups are removed from histone deacetylase complexes incubated i n the absence of butyrate, i n d i c a t i n g that denaturation of the histone deacetylase i s kept to a minimum using the techniques developed i n t h i s study.  Free histones are a poor substrate under these conditions, but  histones i n mononucleosomes are deacetylated when they are incubated with histone deacetylase complex.  Histone deacetylase remains bound to t h i s  complex i n 1-2 M NaCl and does not dissociate from i t during i t s reaction  iii  w i t h a c e t y l a t e d core hisones.  Under t y p i c a l n u c l e a s e d i g e s t i o n c o n d i t i o n s ,  the h i s t o n e d e a c e t y l a s e complex c o n t a i n s DNA w i t h a s i z e d i s t r i b u t i o n of 5-11  k i l o b a s e p a i r s and a v a r i e t y o f n o n h i s t o n e p r o t e i n s .  Comparison o f  t h e p r o t e i n c o m p o s i t i o n o f h i s t o n e d e a c e t y l a s e complexes w i t h t h a t of n u c l e a r m a t r i x p r e p a r a t i o n s shows some s i m i l a r i t i e s . r e s u l t s on the chromatographic  b e h a v i o u r , the DNA  Taken t o g e t h e r , the  fragment s i z e s , and  the  p r o t e i n c o m p o s i t i o n o f the d e a c e t y l a s e complex suggest t h a t p r o t e i n - p r o t e i n i n t e r a c t i o n s may  be i m p o r t a n t i n m a i n t a i n i n g i t s s t r u c t u r e and a l s o i n the  b i n d i n g o f the d e a c e t y l a s e i t s e l f t o the complex L a t e r r e s e a r c h e f f o r t s were concerned h i s t o n e d e a c e t y l a s e complex. neocuproine  w i t h c h a r a c t e r i z a t i o n of the  The e f f e c t of J3-mercaptoethanol  on h i s t o n e d e a c e t y l a s e was  examined i n v i e w o f the f a c t t h a t  these reagents a r e known t o d i s r u p t chromosome s c a f f o l d s . h i s t o n e d e a c e t y l a s e complex p a r t i a l l y d i s s o c i a t e s i n 10 B-mercaptoethanol, presence o f 10 mM  and  HeLa c e l l  mM  r e s u l t i n g i n a l o s s of non-histone p r o t e i n s . J3-mercaptoethanol  The  d u r i n g the p a r t i a l 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 o f HeLa c e l l n u c l e i , r e s u l t s i n a v e r y low y i e l d o f h i s t o n e d e a c e t y l a s e complex, w i t h a c o r r e s p o n d i n g l y l a r g e i n c r e a s e i n the production of s m a l l oligonucleosomes  and mononucleosomes.  Histone  d e a c e t y l a s e a c t i v i t y on endogenous l a b e l l e d h i s t o n e i s s t r o n g l y i n h i b i t e d by e i t h e r 1 o r 10 mM  J3-mercaptoethanol  o r 3 mM n e o c u p r o i n e .  The l o s s o f  h i s t o n e d e a c e t y l a s e a c t i v i t i e s i s n o t due t o an i n a c t i v a t i o n o f t h e enzyme, but appears t o be a consequence o f the d i s r u p t i o n o f the s t r u c t u r e of t h e h i s t o n e d e a c e t y l a s e complex.  H i s t o n e H4 i n h i s t o n e d e a c e t y l a s e complex  prepared from HeLa c e l l n u c l e i by 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 was more  iv  h i g h l y a c e t y l a t e d than H4 i n b u l k nucleosomes. o f the DNA  R e s t r i c t i o n enzyme a n a l y s i s  a s s o c i a t e d w i t h the h i s t o n e d e a c e t y l a s e complex r e v e a l e d n e i t h e r  an enrichment  nor d e p l e t i o n of major s a t e l l i t e sequences i n t h i s m a t e r i a l .  I n v i e w o f t h e s e f i n d i n g s , h i s t o n e d e a c e t y l a s e appears t o be a s s o c i a t e d w i t h a h i g h m o l e c u l a r weight c h r o m a t i n complex w h i c h may a c e t y l group t u r n o v e r .  be a s i t e of r a p i d  V  TABLE OF CONTENTS  Page ABSTRACT  1  1  TABLE OF CONTENTS  v  LIST OF TABLES  x  LIST OF FIGURES ACKNOWLEDGEMENTS DEDICATION  xi x  i  i  i  xiv  INTRODUCTION  1  I  The Histones  2  II  Histone Modifications  9  a. Acetylation  9  b. Methylation  H  c. Phosphorylation  12  d. ADP-ribosylation  13  e. Ubiquitin Attachment  14  f. Glycosylation.  15  III  Review of Histone Acetylation  15  IV  The Nucleosome  18  a. Hi stone-Hi stone Interactions  19  b. The Structure of the Nucleosome  20  c. The Position of Histone HI  21  vi  d. The Shape of the Nucleosome  21  e. Higher Order Packing  22  V  Heterogeneity i n Chromatin Structure  22  VI  Nuclear Skeletal Structure  24  VII  The Present Investigation  27  EXPERIMENTAL PROCEDURES  30  Abbreviations  30  Materials  30  C e l l Culture Conditions  31  Labelling of HeLa C e l l s with [ H]-acetate  32  Micrococcal Nuclease Digestion  32  Preparation of Histones  33  Isokinetic Sucrose Gradient Centrifugation  34  Column Chromatography  34  3  P r e c i p i t a t i o n of Nucleosomes and Histone Deacetylase Complex for Enzyme Assays  34  Histone Deacetylase Assays  35  Precipation of Nucleosomes and Histone Deacetylase Complex  37  DNA Extraction  37  Agarose Gel Electrophoresis  38  SDS Polyacrylamide Gel Electrophoresis  39  Acid-Urea Polyacrylamide Gel Electrophoresis  39  Two-Dimensional Gel Electrophoresis  40  S i l v e r Staining of Polyacrylamide Gels  41  vii  Polyacrylamide Gel Scans  41  Nuclear Matrix Preparation  42  Preparation of High Mobility Group Proteins  42  Acid Hydrolysis  44  Amino Acid Analysis  44  R e s t r i c t i o n Enzyme Digestion  44  Nucleosome Reconstitution  45  Protein Assays  46  RESULTS PART A I  4  THE CHARACTERIZATION OF HISTONE DEACETYLASE  7  47  Development of a Physiological Assay System for Histone Deacetylase  47  a. Sucrose Gradient Centrifugation  47  b.  Isolation of Histone Deacetylase A c t i v i t y Using a Bio-Gel A-5m Column  51  c. Substrate Preference of Chromatin-Bound Histone Deacetylase II  D i s t r i b u t i o n of Histone Deacetylase i n Chromatin  III  Effect of Butyrate on the D i s t r i b u t i o n of Histone Deacetylase  IV  V  51 56  59  Effect of Salt Concentration on Histone Deacetylase Distribution  61  a. Chromatography Using a Bio-Gel A-5m Column  61  b. Chromatography Using a Bio-Gel A-50m Column  61  Effect of Nuclease Digestion on Histone Deacetylase Distribution  65  viii  VI  a. Reduced Nuclease Digestion  65  b. Redigestion of Histone Deacetylase Complex  67  c. Extensive Nuclease Digestion  69  Characteristics of Chromatin bound Histone D e a c e t y l a s e . . . .  72  a. Time Course  72  b. Effect of Assay Volume on the Deacetylase Reaction VII  74  Effect of High M o b i l i t y Group Proteins 14  and 17 on  Histone Deacetylase  74  a. Amino Acid Analysis of HMG 14 and HMG 17  76  b. The Effect of HeLa HMG 14 and HMG 17 on Histone Deacetylase  80  c. The Effect of Calf Thymus HMG 14 and HMG 17 on Histone Deacetylase  80  CHARACTERIZATION OF HISTONE DEACETYLASE COMPLEX  83  I  The Histone Content of Histone Deacetylase Complex  83  II  R e s t r i c t i o n Enzyme Analysis of Histone Deacetylase  PART B  Complex DNA III  IV  88  Comparison of Histone Deacetylase Complex and Nuclear Matrix  90  a. Histone Deacetylase A c t i v i t y of Nuclear Matrix  92  b. Protein Composition of Histone Deacetylase Complex and Nuclear Matrix  93  Effect of  fl-mercaptoethanol  and Neocuproine on Histone  Deacetylase Complex  95  a. Histone Deacetylase A c t i v i t y of Treated Complex  95  b. Physical Properties of Treated Complex  99  ix  DISCUSSION  105  I  Histone Deacetylase Assay  105  II  D i s t r i b u t i o n of Histone Deacetylase i n Chromatin  107  III  Characteristics of Chromatin Bound Histone D e a c e t y l a s e . . . .  Ill  IV  Histone Deacetylase Complex  114  BIBLIOGRAPHY  126  LIST OF TABLES  Table  Page  1.  Histone nomenclature  2.  I n h i b i t i o n of histone deacetylase by free histone  3.  Use of mononucleosomes as a substrate for  3 54  histone deacetylase  55  4.  Effect of assay volume on histone deacetylase  75  5.  Amino acid composition of isolated HMG proteins  77  6.  Effect of HeLa HMG 14 and HMG 17 on histone deacetylase  79  7.  Effect of C-mercaptoethanol and neocuproine on histone deacetylase complex  8.  98  Effect of fi-mercaptoethanol on histone deacetylase  100  xi  LIST OF FIGURES  Figure  Page  1.  The amino acid sequence of trout t e s t i s histone HI  4  2.  The amino acid sequence of bovine histone H2A  5  3.  The amino acid sequence of bovine histone H2B  6  4.  The amino acid sequence of bovine histone H3  7  5.  The amino acid sequence of bovine histone H4  8  6.  Time course of histone deacetylase i n nuclei  48  7.  Isoskinetic sucrose gradient p r o f i l e s of nucleosomes and histone deacetylase a c t i v i t y  8.  Fractionation of micrococcal nuclease digest products on a Bio-Gel A5m column  9.  52  Fractionation of microccocal nuclease digest products of HeLa chromatin on a Bio-Gel A-50m column  10.  50  58  D i s t r i b u t i o n of histone deacetylase i n chromatin fragments from butyrate-treated versus untreated Hela c e l l s  11.  Effect of s a l t concentration on histone deacetylase distribution  12.  62  Histone content of histone deacetylase complex prepared i n the presence of 2.0 M NaCl  13.  60  64  Effect of reduced nuclease digestion on the d i s t r i b u t i o n of histone deacetylase  66  xii 14.  Polynucleosomes  redigested with microccocal nuclease  r e t a i n t h e i r endogenous histone deacetylase activity 15.  68  Effect of the extent of nuclease digestion on the d i s t r i b u t i o n of histone deacetylase i n chromatin  70  16.  Time course of chromatin-bound histone deacetylase  73  17.  SDS polyacrylamide gel electrophoresis  of HMG 14  and HMG 17 18.  78  Effect of c a l f thymus HMG 14 and HMG 17 on histone deacetylase a c t i v i t y  19.  81  Acid urea gel p r o f i l e s of histone deacetylase complex and nucleosomes  20.  86  Optical scans of histone H4 on acid urea polyacrylamide gels  87  21.  Two-dimensional g e l electrophoresis  22.  R e s t r i c t i o n enzyme digestion of histone deacetylase complex DNA and genomic DNA  23.  of histones  94  SDS polyacrylamide gel p r o f i l e s of fractions from a Bio-Gel A-50m column  25.  Fractionation of B-mercaptoethanol-treated  96 histone  deacetylase complex on a Bio-Gel A-50m column 26.  91  SDS polyacrylamide gel p r o f i l e s of HeLa c e l l nuclear matrix preparations  24.  89  103  SDS polyacrylamide gel p r o f i l e s of histone deacetylase complex  104  xiii  ACKNOWLEDGEMENTS  I w i s h t o e x p r e s s my g r a t i t u d e t o my s u p e r v i s o r , Dr. P e t e r Candido, f o r h i s p a t i e n t s u p e r v i s i o n throughout  my graduate  studies.  I appreciate  h i s comments, encouragement and t h e g r e a t i n t e r e s t w h i c h he a l w a y s expressed. In a d d i t i o n , I w i s h t o thank the many people i n t h e B i o c h e m i s t r y Department who have a s s i s t e d my r e s e a r c h e f f o r t s and c a r e e r and the members o f Dr. Candido's l a b o r a t o r y who h e l p e d c r e a t e a p l e a s a n t atmosphere i n w h i c h t o work, i n p a r t i c u l a r R. Kay f o r h i s v a l u a b l e s u g g e s t i o n s . a l s o l i k e t o thank D. Bunyak f o r t y p i n g t h i s  I should  thesis.  I a l s o w i s h t o acknowledge t h e f i n a n c i a l support o f t h e Canadian  i M e d i c a l Research C o u n c i l .  DEDICATION  to  My P a r e n t s who have a l w a y s g i v e n t h e i r f u l l support t o my e d u c a t i o n  and  My B r o t h e r , Graeme  1  INTRODUCTION  H i s t o n e s a r e t h e major a r c h i t e c t u r a l p r o t e i n s o f c h r o m a t i n as they complex w i t h DNA t o form nucleosomes, t h e fundamental r e p e a t i n g u n i t of chromatin.  structural  Two o f each o f t h e h i s t o n e s H2A, H2B, H3 and H4 combine t o  f o r m a c o r e , around w h i c h a r e wrapped 146 bp o f DNA.  The r e g i o n o f a DNA  s t r a n d w h i c h j o i n s a d j a c e n t nucleosome c o r e p a r t i c l e s i s termed t h e l i n k e r DNA. The  h i s t o n e s c a n undergo a v a r i e t y o f p o s t - t r a n s l a t i o n a l  modifications  i n c l u d i n g a c e t y l a t i o n , p h o s p h o r y l a t i o n , m e t h y l a t i o n and A D P - r i b o s y l a t i o n .  The  enzymatic a c e t y l a t i o n of the nucleosomal h i s t o n e s a t s p e c i f i c l y s y l residues i n t h e a m i n o - t e r m i n a l p o r t i o n i s r a p i d and r e v e r s i b l e .  Each a c e t y l a t i o n o f a  h i s t o n e l y s y l residue r e s u l t s i n the n e u t r a l i z a t i o n of a p o s i t i v e charge, w h i c h reduces t h e i o n i c i n t e r a c t i o n between t h e h i s t o n e s and t h e n e g a t i v e l y charged phosphate backbone o f t h e DNA and i s thought t o a l t e r t h e c o n f o r m a t i o n of t h e nucleosome, p o s s i b l y t h e r e b y i n c r e a s i n g enzymes o r r e g u l a t o r y  proteins.  t h e a c c e s s i b i l i t y o f t h e DNA t o  The r a p i d a d d i t i o n and removal o f h i s t o n e  a c e t y l groups i s brought about by t h e a c t i o n o f s e v e r a l acetyltransferases  and h i s t o n e d e a c e t y l a s e ,  histone  respectively.  2  I  The Histones  Histones were f i r s t recognized i n 1884 (1) but i t was not u n t i l recently that the function of t h i s group of basic proteins was c l a r i f i e d . The histones have been studied for nearly a century, with well over  1,500  papers i n the l i t e r a t u r e ; as a r e s u l t , several different nomenclatures have been assigned to the histones.  Table 1 contains a summary of the  nomenclature and molecular weights of the histones.  Many histones have  been sequenced (2,3,4,5,214,215) and Figures 1 to 5 show the primary structure of the five histones. the figure legends. of d i s t i n c t domains.  The sources of the histones are shown i n  An examination of these sequences reveals the presence The amino-terminal portions of the four nucleosomal  core histones (H2A, H2B, H3 and H4) contain a very high proportion of basic residues and are the primary s i t e s of i n t e r a c t i o n with DNA. The carboxy-terminal portions of the nucleosomal core histones contain most of the hydrophobic and a c i d i c amino a c i d s , and possess a globular t e r t i a r y structure.  Histone HI also contains an uneven d i s t r i b u t i o n of residues and  consists of three regions; however, the p o l a r i t y i s the opposite to that of the core histones, the carboxy-terminal half having a r a t i o of basic to a c i d i c residues of 15:1.  There i s a short hydrophobic region at the  r e l a t i v e l y basic amino-terminal portion and the remainder of the histone  is  comprised of a globular hydrophobic region (6,7). The histones display a remarkable evolutionary conservation of t h e i r amino acid sequences, implying that precise binding of histones to each other and to the DNA i s absolutely essential chromatin.  i n the functioning of  Histones H3 and H4 are among the most conserved of a l l proteins  with histone H4 from the cow and the pea d i f f e r i n g by only two conservative  3  Table 1. Histone nomenclature  Contemporary Nomenclature  Alternative Nomenclature v  Molecular Weight  Amino Terminus  e y lysine r i c h , I , F l , KAP  21,500  Acetyl-serine  H  1  r  H  2  A  l y s i n e r i c h , I l b l , F2a2, LAK  14,004  Acetyl-serine  H  2  B  l y s i n e r i c h , IIb2, F2b, KAS  13,774  Proline  H 3  arginine r i c h , I I I , F3, ARE  15,324  Alanine  H A  arginine r i c h , IV, F 2 a l , GRK  11,282  Acetyl-serine  4  AcMa-Glu-Val-Ma-Pro-Ala-Pro-Ala-Ala-Ala-Ala-Pro-Ala-Lys-Ala1 5 10 15 Pro-Lys-Lys-Lys-Ala-Ala-Ala-Lys-Pro-Lys-Lys-Ser-Gly-Pro-Ala20 25 30 Val-Gly-Glu-LeiJ-Ala-Gly-Lys-Ala-Val-Ala-Ala-Ser-Lys-Glu-Arg35 40 45 Ser-Gly-Val-Ser-Leu-Ala-Ala-Leu-Lys-Lys-Ser-Leu-Ala-Ala-Gly50 55 60 Gly-Tyr-Asp-Val-Glu-Lys-Asn-Asn-Ser-Arg-Val-Lys-Ile-Ala-Val65 70 75 Lys-Ser-Leu-Val-Thr-Lys-Gly-Thr-l£U-Val-Glu-Thr-Lys-Gly-Thr80 85 90 Gly-Ala-Ser-Gly-Ser-Phe-Lys-Leu-Asn-Lys-Lys-Ala-Val-Glu-Ala95 100 105 Lys-Lys-Pro-Ala-Lys-Lys-Ala-Ala-Ala-Pro-Lys-Ala-Lys-Lys-Val110 115 120 Ala-Ala-Lys-Lys-Pro-Ala-Ala-Ala-Lys-Lys-Pro-Lys-Lys-Val-Ala125 130 135 Ala-Lys-Lys-Ala-Val-Ala-Ala-Lys-Lys-Ser-Pro-Lys-Lys-Ala-Lys140 145 150 Lys-Prc>-Ma-Thr-Prci-Lys-Lys-Ma-Ma-Lys-Ser-Pro-Lys^Lys-Ala155 160 165 Thr-Lys-Ala-Ala-Lys-Pro-Lys-Ala-Ala-Lys-Pro-Lys-Lys-Ala-Ala170 175 180 Lys-Ser-Pro-Lys-Lys-Val-Lys-Lys-Pro-Ala-Ala-Ala-Lys-Lys-COOH 185 190 194  F i g u r e 1.  The amino a c i d  sequence o f t r o u t  t e s t i s h i s t o n e HI  (216)  Ac-Ser-Gly-Arg-Gly-Lys-Gln-Gly-Gly-Lys-Ala-Arg-Ala-Lys-Ala-Lys1 5 10 15  Thr-Arg-Ser-Ser-Arg-Ala-Gly-Leu-Gln-Phe-Pro-Val-Gly-Arg-Val20 25 30  His-Arg-Leu-Leu-Arg-Lys-Gly-Asn-Tyr-Ala-Glu-Arg-Val-Gly-Ala35 40 45  Gly-Ala-Pro-Val-Tyr-Leu-Ala-Ala-Val-Leu-Glu-Tyr-Leu-Thr-Ala50 55 60  Glu-Ile-Leu-Glu-Leu-Ala-Gly-Asn-Ala-Ala-Arg-Asp-Asn-Lys-Lys65 70 75 Thr-Arg-Ile-Ile-Pro-Arg-His-Leu-Gln-Leu-Ala-Ile-Arg-Asn-Asp80 85 90  Glu-Glu-Leu-Asn-Lys-Leu-Leu-Gly-Lys-Val-Thr-Ile-Ala-Gln-Gly 95 100 105  Gly-Val-Leu-Pro-Asn-Ile-Gln-Ala-Val-Leu-Leu-Pro-Lys-Lys-Thr110 115 120  Glu-Ser-His-His-Lys-Ala-Lys-Gly-Lys-COOH 125 129  Figure 2.  The amino acid sequence of bovine histone H2A (214)  HoN-Pro-Gln-Pro-Ala-Lys-Ser-Ala-Pro-Ala-Pro-Lys-Lys-Gly-Ser-Lys1 5 10 15  Lys-Ala-Val-Thr-Lys-Ala-Gln-Lys-Lys-Asp-Gly-Lys-Lys-Arg-Lys20 25 30  Arg-Ser-Arg-Lys-Glu-Ser-Tyr-Ser-Val-Tyr-Val-Tyr-Lys-Val-Leu35 40 45 Lys-Gln-Val-His-Pro-Asp-Thr-Gly-Ile-Ser-Ser-Lys-Ala-Met-Gly50 55 60  Ile-Met-Asn-Ser-Phe-Val-Asn-Asp-Ile-Phe-Glu-Arg-Ile-Ala-Gly65 70 75  Glu-Ala-Ser-Arg-Leu-Ala-His-Tyr-Asn-Lys-Arg-Ser-Thr-Ile-Thr80 85 90 Ser-Arg-Glu-Ile-Gln-Thr-Ala-Val-Arg-Leu-Leu-Leu-Pro-Gly-Glu95 100 105 Leu-Ala-Lys-His-Ala-Val-Ser-Glu-Gly-Thr-Lys-Ala-Val-Thr-Lys 110 115 120  Tyr-Thr-Ser-Ser-Lys-COOH 125  Figure 3.  The amino acid sequence of bovine histone H2B (4).  H N-Ala-Arg-Thr-Lys-Gln-Thr-Ala-Arg-Leu-Ser-Thr-Gly-Gly-Lys-Ala1 5 10 15 2  Pro-Arg-Lys-Gln-Leu-Ala-Thr-Lys-Ala-Ala-Arg-Lys-Ser-Ala-Pro20 25 30  Ala-Thr-Gly-Gly-Val-Lys-Lys-Pro-His-Arg-Tyr-Arg-Pro-Gly-Thr35 40 45  Val-Ala-Leu-Arg-Glu-Ile-Arg-Arg-Tyr-Gln-Lys-Ser-Thr-Glu-Leu50 55 60  Leu-Ile-Arg-Lys-Leu-Pro-Phe-Gln-Arg-Leu-Val-Arg-Glu-Ile-Ala65 70 75 Gln-Asp-Phe-Lys-Thr-Asp-Leu-Arg-Phe-Gln-Ser-Ser-Ala-Val-Met80 85 90  Ala-Leu-Gln-Glu-Ala-Cys-Glu-Ala-Tyr-Leu-Val-Gly-Leu-Phe-Glu95 100 105  Asp-Thr-Asn-Leu-Cys-Ala-Ile-His-Ala-Lys-Arg-Val-Thr-Ile-Met110 115 120  Pro-Lys-Asp-Ile-Gln-Leu-Ala-Arg-Arg-Ile-Arg-Gly-Glu-Arg-Ala-COOH 125 130 135  Figure 4.  The amino acid sequence of bovine histone H3 (215).  Ac-Ser-Gly-Arg-Gly-Lys-Gly-Gly-Lys-Gly-Leu-Gly-Lys-Gly-Gly-Ala1 5 10 15 Lys-Arg-His-Arg-Lys-Val-Leu-Arg-Asp-Asn-Ile-Gln-Gly-Ile-Thr20 25 30 Lys-Pro-Ala-Ile-Arg-Arg-Leu-Ala-Arg-Arg-Gly-Gly-Val-Lys-Arg35 40 45  Ile-Ser-Gly-Leu-Ile-Tyr-Glu-Glu-Thr-Arg-Gly-Val-Leu-Lys-Val50 55 60 Phe-Leu-Glu-Asn-Val-Ile-Arg-Asp-Ala-Val-Thr-Tyr-Thr-Glu-His65 70 75 Ala-Lys-Arg-Lys-Thr-Val-Thr-Ala-Met-Asp-Val-Val-Tyr-Ala-Leu80 85 90  Lys-Arg-Gln-Gly-Arg-Thr-Leu-Tyr-Gly-Phe-Gly-Gly-COOH 95 100 102  Figure 5.  The amino acid sequence of bovine histone H4 (3)  9  amino acid replacements (8).  S i m i l a r l y , histone H3 from the cow and the  pea d i f f e r by only four amino acids and two of the changes are conservative (9).  Histones H2A and H2B are less highly conserved and show differences  between different vertebrates  (10).  hybrids since t h e i r histone-histone  They can be considered as evolutionary binding regions are highly conserved  while other regions are more v a r i a b l e .  Histone HI i s not required for the  maintenance of the nucleosome core structure and i s the least conserved of the histones.  Nevertheless,  i t too contains regions of conserved sequence;  a section of approximately 75 residues i s homologous between rat l i v e r HI and trout HI (11).  II  Histone Modifications  The histones are subject to a variety of post-translational modifications which a l t e r the charge and structure of amino acid residues i n the highly basic amino-terminal portions of the core histones and i n both the amino- and carboxy-terminal portions of HI. involve group substitutions  The modifications  such as a c e t y l a t i o n , phosphorylation,  methylation and ADP-ribosylation, and are sequence s p e c i f i c .  Although  these enzymatic reactions w i l l be considered separately, i t should be emphasized here that multiple forms of modification may occur within the same histone molecule, thereby creating a large number of species whose fundamental properties may vary.  a.  Acetylation  There are two d i s t i n c t forms of enzymatic histone acetylation.  The  10  f i r s t type results i n the addition of an acetyl group to the 2-amino group of the amino-terminal serine of histones HI, H2A, and H4.  The reaction  occurs i n the cytoplasm very shortly after synthesis and i s i r r e v e r s i b l e . In contrast, the second form of histone acetylation i s readily reversible (12,13) and occurs within the nucleus.  This reaction involves  modifications of the e-amino groups of s p e c i f i c l y s y l residues i n the basic amino-terminal portion of the nucleosomal core histones, and H4.  H2A, H2B, H3  N - a c e t y l a t i o n converts a basic l y s y l residue to a neutral 6  a c e t y l - l y s i n e and thereby reduces the net positive charge of the amino-terminal region by one.  Histones H2B, H3 and H4 contain 4 s i t e s of  acetylation while H2A contains a single l y s y l residue which can be reversibly acetylated.  The s i t e s of acetylation i n histone H4 are l y s y l  residues 5, 8, 12 and 16.  Thus histones H2B, H3 and H4 can exist i n m u l t i -  acetylated forms with the o v e r a l l positive charge i n the amino-terminal region varying from +5 to +1. The acetyl groups on the l y s y l residues turn over r a p i d l y , and different populations of core histones are modified with metabolically active acetate at different rates (13).  Both the acetylation and  deacetylation of histones are carried out by enzymes within the nucleus, namely, the histone acetyltransferases respectively.  and histone  deacetylase,  Attempts have been made to i s o l a t e these enzymes from  various tissues and t h e i r c h a r a c t e r i s t i c s w i l l be discussed i n a l a t e r section. The n e u t r a l i z a t i o n of the positive charges at the amino-terminal portions of the histones by acetylation i s thought to reduce the i o n i c interactions between the nucleosomal core histones and the negatively  11  charged DNA which i s wrapped around the histones.  Reduced i o n i c  interactions between the histones and the DNA could result i n an altered nucleosome conformation and increased a c c e s s i b i l i t y of the DNA to enzymes and other proteins.  Recent studies suggest that histone acetylation may  affect primarily the higher order structure of chromatin, i . e . the nucleosome chain into the 30 nm f i b r e (91).  folding of  As a consequence,  histone  acetylation has been considered as a possible mechanism for a l t e r i n g chromatin structure and regulating t r a n s c r i p t i o n a l a c t i v i t y , DNA r e p l i c a t i o n and DNA r e p a i r .  The possible functions of histone acetylation  w i l be reviewed i n a l a t e r section.  b.  Methylation  Histones H3 and H4 can undergo methylation at the e-amino group of s p e c i f i c l y s y l residues after histone synthesis (14).  The methylation of a  l y s y l residue i s i r r e v e r s i b l e and does not r e s u l t i n a change i n charge, but rather produces an increase i n b a s i c i t y and hydrophobicity.  Both  histones H3 and H4 are methylated i n the amino-terminal region, H4 containing one methylation s i t e at l y s y l 20 (15,16,17).  Histone H3 has  four l y s y l residues which are methylated d i f f e r e n t i a l l y , l y s y l residues 9 and 27 are the two most common s i t e s of methylation, while residues 4 and 36 are methylated less frequently  (18).  The immediate methyl group donor i s S-adenosylmethionine (19) and the reaction i s catalyzed by methylases within the nucleus (20).  Examination  of the methylase i n rat brain chromatin has shown that i t only methylates H3 and H4 when i t i s chromatin bound, but soluble preparations show loss of  12  s u b s t r a t e s p e c i f i c i t y and even a c i d - i n s o l u b l e p r o t e i n s o f c a l f n u c l e i can be methylated  thymus  i n v i t r o t o form mono- and d i m e t h y l l y s i n e s ( 2 1 ) .  The m o d i f i e d l y s y l r e s i d u e s c a n c o n t a i n one t o t h r e e methyl groups and i n many cases i t i s the d i m e t h y l l y s i n e which predominates ( 2 2 ) .  F o r example,  h i s t o n e H4 i s m o d i f i e d mainly as the N - d i m e t h y l l y s i n e i n E h r l i c h 6  tumour c e l l s (15).  ( 2 3 ) , c a l f thymus ( 2 4 ) , carp t e s t i s  (25) and t r o u t  ascites  testis  L i k e w i s e , h i s t o n e H3 i s m o d i f i e d m a i n l y as N - d i m e t h y l l y s i n e , 6  a l t h o u g h a l l t h r e e forms a r e o f t e n p r e s e n t .  However, m e t h y l a t i o n of the  s u s c e p t i b l e l y s y l r e s i d u e s need not o c c u r as about one q u a r t e r o f c a l f thymus h i s t o n e H3 i s unmodified  and c a l f thymus H4 c o n t a i n s f r e e  lysyl  r e s i d u e s , as w e l l as m o d i f i e d forms, a t p o s i t i o n 20 ( 8 ) . The b i o l o g i c a l s i g n i f i c a n c e o f h i s t o n e m e t h y l a t i o n remains u n r e s o l v e d , as m e t h y l a t i o n does not c o r r e l a t e w i t h an i n c r e a s e i n DNA t r a n s c r i p t i o n , DNA s y n t h e s i s o r w i t h h i s t o n e s y n t h e s i s ( 2 3 ) .  The changes i n b a s i c i t y and  h y d r o p h o b i c i t y r e s u l t i n g from h i s t o n e m e t h y l a t i o n suggest  t h a t changes i n  the i n t e r a c t i o n s between h i s t o n e s and o t h e r chromatin components may o c c u r , perhaps as a requirement  c.  f o r chromatin c o n d e n s a t i o n and m i t o s i s (22,26,27).  Phosphorylation  P h o s p h o r y l a t i o n o f s e r i n e and t h r e o n i n e r e s i d u e s i s a major r e v e r s i b l e post-translational modification of a l l histones. histones a r e phosphorylated HI can be p h o s p h o r y l a t e d  The nucleosomal  i n the b a s i c amino-terminal  core  p o r t i o n and h i s t o n e  i n both the amino- and c a r b o x y - t e r m i n a l r e g i o n s .  H i s t o n e H3 i s p h o s p h o r y l a t e d  a t s e r y l r e s i d u e s 10 and 28 w h i l e h i s t o n e H2B  can be p h o s p h o r y l a t e d a t s e r y l r e s i d u e s 6, 14, 32 and 36.  13  Various histone kinases have been described and a l l u t i l i z e ATP as the phosphoryl group donor.  Kinases have been reported which are cAMP  dependent (206), cAMP independent (207,  28) or Ca+ dependent (29). 2  kinases are s p e c i f i c for c e r t a i n histones,  Some  e.g. HeLa c e l l s contain a  nuclear kinase which only phosphorylates histone H3 (30) and c a l f thymus also contains an H3 s p e c i f i c kinase which phosphorylates the protein at a threonine residue (31).  Histones are dephosphorylated by histone  phosphatases such that phosphate groups on different histones have different half l i v e s  (32).  The function of histone phosphorylation remains unclear.  There i s  evidence of a relationship between phosphorylation of histones HI and H3 and chromatin condensation p r i o r to mitosis (32,33,34,35,36). N-phosphorylation can also occur at the h i s t i d y l residue of histone H4 to form N -phosphorylhistidine (37) and at the e-amino of a l y s y l 3  residue i n HI.  Two c y c l i c nucleotide independent kinases capable of  N-phosphorylation have been isolated from regenerating l i v e r (37) and Walker 256 carcinosarcoma c e l l s (38).  It has been suggested that the  N-phosphorylation of histones HI and H4 i s involved with DNA r e p l i c a t i o n (39).  d.  Adenosine-Diphospho-Ribosylation (ADP-ribosylation)  Histone HI i s extensively modified by the covalent attachment of ADP-ribose groups to the amino- and carboxy-portions of the protein (40). In contrast, the core histones are ADP-ribosylated to a much lesser extent.  The ADP-ribose group i s linked to a glutamyl residue i n histone HI  14  of rat l i v e r (41,42,43). The ADP-ribose moieties can be linked by ribose to ribose (1'—)2') bonds (44,45) to form polymers of 1-65 groups i n length (46).  The  ADP-ribose polymers are formed by the enzyme poly (ADP-ribose) polymerase which has been p u r i f i e d to near homogeneity from rat l i v e r (47), thymus (48), (52).  bovine  c a l f thymus (49,50), pig thymus (51) and ascites tumour c e l l s  A l l have an absolute requirement for DNA and u t i l i z e NAD as the  source of ADP-ribose.  The enzyme i s l o c a l i z e d within the internucleosomal  l i n k e r regions of HeLa c e l l chromatin (53).  However, i t i s not equally  distributed i n HeLa c e l l chromatin as i t i s present only on c e r t a i n subsets of nucleosomes (54).  1  The poly (ADP-ribose) chains can be degraded by poly (ADP-ribose) glycohydrolase and the lengths of the ADP-ribose polymers within a p a r t i c u l a r tissue i s thought to p a r t l y depend upon the a c t i v i t y of poly (ADP-ribose) glycohydrolase (55). ADP-ribose polymer  Phosphodiesterase can also degrade the  (56).  Various functions have been suggested for ADP-ribosylation, including the regulation of c e l l u l a r growth (57), (59) and DNA repair (60).  gene expression (58),  DNA synthesis  ADP-ribosylation i s also thought to be involved  i n chromosome condensation (61,62).  e.  Ubiquitin Attachment  Histone H2A can be modified by the covalent attachment of one molecule of u b i q u i t i n .  The u b i q u i t i n moiety i s attached through a glycylglycine  bridge to lysine 119 of H2A v i a an isopeptide bond (196) and the modified  15  histone i s referred to as A24 or uH2A.  Ubiquitin i s a very highly  conserved protein; human and bovine u b i q u i t i n are i d e n t i c a l and contain 74 amino acids (197,198). The precise function of this modification of H2A i s unclear.  A  selective arrangement of A24 has been observed i n the Drosophila genome. Approximately one i n two nucleosomes of the transcribed copia and heat shock protein 70 genes i n unshocked cultured c e l l s contains A24 whereas less than one i n 25 nucleosomes of nontranscribed s a t e l l i t e DNA contains A24 (199).  On the other hand, the replacement of H2A i n chromatin with A24  does not a l t e r the structure of i n d i v i d u a l nucleosomes or the digestion pattern of chromatin with DNase I (200).  Ubiquitin may f a c i l i t a t e  histone  degradation as i t i s i d e n t i c a l to the ATP-dependent proteolysis factor of rabbit reticulocyte lysates (201).  f.  Glycosylation  Only recently has the glycosylation of histones been established. Studies with Tetrahymena have shown that a l l f i v e histones contain fucose so that a minimum of 1 i n 1000 nucleosomes contains a fucosylated H2A molecule (63).  A l l f i v e histones also contain mannose.  If these results  are duplicated i n other organisms, histones w i l l have to be c l a s s i f i e d as glycoproteins.  Ill  Review of Histone Acetylation  The transfer of acetate from acetyl-CoA to the e-amino groups of  16  specific l y s y l residues of the core histones i s catalyzed by several histone acetyltransferases. CoA binding (64).  Histone binding i s a prerequisite for a c e t y l -  The different histone acetyltransferases are c l a s s i f i e d  primarily by t h e i r subcellular l o c a l i z a t i o n and their substrate preference with respect to histone c l a s s .  Histone acetyltransferase A from r a t  hepatoma cultured c e l l nuclei acetylates the core histones with a preference of H4>H2A=H2B>H3. The substrate preference i s altered i f the histones are free i n solution (65).  Acetyltransferase A from c a l f  thymus nuclei i s released from chromatin by mild micrococcal nuclease digestion and i t acetylates histone H3 least well when the l a t t e r present i n nucleosomes  (66).  is  Histone acetyltransferase B i s a cytoplasmic  enzyme which i s s p e c i f i c for histone H4 (67).  A t h i r d isozyme,  acetyltransferase DB (DNA binding) has been prepared from bovine lymphocytes (68) and from the nuclei of African green monkey kidney c e l l s infected with SV40 (69).  The enzyme has a s i m i l a r histone preference as  acetyltransferase A, but has a very high a f f i n i t y for DNA. Acetyl groups are cleaved from histones by histone  deacetylase.  However, very l i t t l e i s known about the properties and d i s t r i b u t i o n of enzyme within chromatin.  this  Histone deacetylase a c t i v i t y has been shown i n  c a l f thymus (70,71) and i n rat l i v e r and Novikoff hepatoma (72).  Attempts  to purify histone deacetylase have been largely unsuccessful as i s o l a t i o n results i n losses i n substrate s p e c i f i c i t y and enzyme a c t i v i t y .  For  example, the p a r t i a l p u r i f i c a t i o n of histone deacetylase from c a l f thymus nuclei results i n s i g n i f i c a n t change i n the r a t i o of the deacetylation rates of histones H3 and H4 and a complete loss of deacetylase against chromatin-bound histones,  activity  the physiological substrate (73).  An  17  a c i d i c protein which removes acetyl groups from free histones H4 and H3 has been p a r t i a l l y p u r i f i e d from c a l f thymus (74).  The histone deacetylase has  an approximate molecular weight of 160,000 and binds to chromatin.  Other  studies on histone deacetylase have used whole nuclei as a source of enzyme, with histones as substrate (75).  Shearing or sonication of c a l f  thymus chromatin has been used to release histone deacetylase a c t i v i t y i n experiments where chemically acetylated histones were used as substrate (76) .  The histone deacetylase s o l u b i l i z e d from c a l f thymus chromatin could  deacetylate added free a c e t y l - l a b e l l e d histones, whereas the chromatinbound histone deacetylase reacted poorly with t h i s substrate.  Histone  deacetylase a c t i v i t y has been located s p e c i f i c a l l y i n the nuclei of Physarum and sonication i n high s a l t i s required to release the enzyme (77) .  The enzyme has not been purified further. M i l l i m o l a r concentrations of butyrate r e v e r s i b i l y i n h i b i t histone  deacetylase by acting as a non-competitive i n h i b i t o r (78,79,80).  Butyrate  does not affect the rate of transfer of acetyl groups from acetyl-CoA to the histone l y s y l residues by the acetyltransferases.  As a consequence,  butyrate causes the dynamic equilibrium between acetylation and deacetylation to s h i f t ,  resulting i n an accumulation of the hyperacetylated  forms of the core histones (81).  While the exact function of histone  acetylation remains unclear, i t has been proposed that a reduction i n the p o s i t i v e charge of the acetylated core histones may result i n a concomitant increase i n the a c c e s s i b i l i t y of the DNA to s p e c i f i c nuclear enzymes or regulatory proteins.  Increased histone acetylation has been reported to  increase the a c c e s s i b i l i t y of histone H3 to the nuclear calcium dependent H3 kinase  (82).  18  High l e v e l s of histone acetylation have been associated with t r a n s c r i p t i o n a l l y competent chromatin i n fractionation experiments (83,84,85,86,87) and with nuclei that are t r a n s c r i p t i o n a l l y very a c t i v e , such as those of yeast (88) and the macronuclei of Tetrahymena (89). The acetate turnover i n histone H4 during the c e l l cycle has been examined i n Physarum macroplasmodia and an increase of [ H]-acetate 3  incorporation i s observed i n S phase (77).  Relatively high incorporations  of acetate were noted i n tetraacetylated H4 i n S phase and late G2 phase (M + 7 hours), c o r r e l a t i n g with high l e v e l s of tetraacetylated H4 at these times. It has also been suggested that histone acetylation may f a c i l i t a t e DNA r e p l i c a t i o n or repair since increasing the l e v e l of histone acetylation i n human fibroblasts leads to an increase i n UV-induced DNA repair (90). Recently i t has been proposed that a l l of the chromatin i n a c e l l may experience cycles of acetylation and deacetylation which may open up the chromatin for inspection and repair (91).  IV  The Nucleosome  An important c h a r a c t e r i s t i c of chromatin structure i s the t i g h t l y packed state of the DNA; up to a 10,000 fold contraction i s required to f o l d DNA molecules into chromosomes.  The discovery of nucleosomes  (92,93,94) has given insight into the i n i t i a l folding of DNA. The nucleosome represents the elemental repeating s t r u c t u r a l unit of chromatin and consists of two copies each of the core histones (95),  namely, histones  H2A, H2B, H3 and H4, around which are wrapped about 200 bp of DNA. A single molecule of histone HI i s associated with the nucleosome.  19  a.  Histone-Histone Interactions  Investigations into the interactions of histones i n chromatin (96) and i n isolated nucleosomes (97) using zero-length crosslinkers (98) have shown that H2A-H2B and H3-H4 dimers, and (H3-H4) crosslinked.  2  tetramers are p r e f e r e n t i a l l y  Histone H2B has been found to contain separate binding s i t e s  for H2A and H4.  The results are consistent with the formation of two  tetrameric complexes of H2A, H2B, H3, and H4 within the nucleosome C i r c u l a r dichroism, laser Raman (101) and infrared spectroscopy  (99).  (102)  studies have shown that the core histones possess a high content of a h e l i x with l i t t l e or no 6 sheet.  The carboxy-terminal portions of the  histones are involved i n histone-histone interactions and are generally considered to be i n a globular conformation (103).  The spectral properties  of a fluorescent group attached to methionine 84 i n histone H4 has indicated an apolar environment for t h i s group within nucleosomes (100). In contrast to the carboxy-terminal portions of the histone,  the  amino-terminal portions exist i n a random c o i l conformation i n the absence of DNA (104,105).  This finding i s supported by the observation that the  amino-terminal portions are highly susceptible to trypsin digestion, while the globular regions are not (106,208).  More recent studies using  a r g i n i n e - s p e c i f i c protease digestion of intact chromatin showed that one or more s i t e s i n the amino-terminal region of each core histone are accessible on the nucleosome surface (107).  Digestion of nucleosomes with t r y p s i n  indicates that 20-30 residues can be removed from the histone amino-terminal portions without causing major unfolding of the nucleosome cores (108).  20  b.  The Structure of the Nucleosome  The nucleosome core i s complexed with 146 bp of DNA (105,209) which wraps around the histones i n 1 3/4 turns i n a l e f t handed supercoil (109,210).  This complex i s often referred to as the core nucleosome.  The  DNA was f i r s t shown to be on the outside of the p a r t i c l e by digestion with DNase I (110).  The s i t e s of maximum a c c e s s i b i l i t y to DNase I occur at  intervals of 10.4 bp i n the nucleosomes (111).  This i s the same as the  p i t c h of the DNA which i s estimated to be 10.33-10.40 bp (112) from the digestion of DNA with DNase II and micrococcal nuclease, as well as with DNase I .  A l l of the nucleases digest at s i t e s 30, 60, 80 and 110 bp from  the 5' end of the nucleosomal core DNA (113,114,115,116). A stretch of DNA, referred to as l i n k e r DNA, joins adjacent core nucleosomes.  While a l l eukaryotes appear to have 146 bp wrapped around the  histone core (117,118,119), the l i n k e r DNA varies greatly i n length i n different organisms.  The variable length of the l i n k e r DNA results i n  different nucleosomal repeat length which vary from 154 bp i n Aspergillus to 241 bp i n sea urchin sperm.  However, the majority of eukaryotes have  between 185-200 bp of DNA associated with each nucleosome (120). Electron microscopy has shown that, under appropriate conditions of spreading, the chromatin fibres contain regularly spaced nucleosomes linked by t h i n filaments of l i n k e r DNA (121,122) and the analogy of "beads on a string" i s frequently used to describe the appearance of nucleosomes.  Mild  digestion of chromatin with micrococcal nuclease results i n the cleavage of the l i n k e r DNA and the production of mononucleosomes and polynucleosomes containing varying numbers of nucleosome repeat u n i t s .  21  c.  The P o s i t i o n of Histone HI  Histone HI i s not part of the core nucleosome, but i s generally regarded as being associated with about 35-40 bp of the l i n k e r DNA (123). Recently, i t has been proposed that histone HI i s responsible for bringing together the strands of DNA entering and leaving the core nucleosome. Electron microscopy has shown that only i n the presence of HI do the two strands of DNA leave the nucleosome at the same point (124).  Micrococcal  nuclease digestion of Hl-depleted chromatin gives r i s e to s t r u c t u r a l rearrangements i n d i c a t i n g nucleosome s l i d i n g (125). The p i t c h of the DNA h e l i x i n a nucleosome i s 2.8 nm (126) and t h i s  is  very s i m i l a r to the diameter of the globular domain of HI which i s 2.9 nm. The carboxy-terminal domain of HI i s most l i k e l y involved i n the condensation of the nucleofilament and i t s basic and hydrophilic nature suggests that i t interacts i o n i c a l l y with the DNA (211).  d.  The Shape of the Nucleosome  Low-angle neutron scattering studies of nucleosomes i n solution have shown that the DNA i s on the outside of the p a r t i c l e (127,128).  The DNA i s  2.2 nm thick and has a p i t c h of 2.8 nm while the histone core has an approximate diameter of 7.0 nm and i s 3.5 nm thick (129).  High-angle  neutron scattering studies (129), electron microscopy (130) and X-ray d i f f r a c t i o n studies (126) are consistent with the core nucleosome being wedge shaped and having dimensions of 11.0 x 11.0 x 5.7 nm.  Data from  X-ray, neutron and l a s e r l i g h t scattering and NMR studies (129) suggest a  22  substructure consisting of two face to face disc-shaped heterotypic tetramers each surrounded by a DNA annulus. have a sedimentation coefficient  e.  The core nucleosome  histone  particles  of 11 S (131).  Higher Order Packing  The 11.0 nm nucleosome represents the most extended form of chromatin and further compaction results i n the formation of a 30.0 nm diameter f i b r e (132,133,134).  Histone HI i s thought to play a role i n the aggregation of  nucleosomes and chicken erythrocyte oligomers (trimers to 20mers) are able to interact with each other through HI and H5 (an avian erythrocyte analogue of HI) to form p a r t i c l e s 30.0 nm i n diameter (135). A l t e r n a t i v e l y , nucleosomes are thought to be arranged i n a solenoid to form a 25-30 nm thick chromatin f i b r e (136,133,137). 50 nucleosomes,  At a length of about  polynucleosomes fold to form a f i b r e which corresponds to  8-10 turns of a solenoid with 5-6 nucleosomes per turn (138).  It i s  still  unclear how the chromatin f i b r e i s organized to form metaphase chromosomes.  However, X-ray d i f f r a c t i o n studies of chicken erythrocyte  chromosomes show a 40 nm p e r i o d i c i t y due to a structure that i s d i r e c t l y related to the 30 nm side by side packing of chromatin fibres (139).  V  Heterogeneity i n Chromatin Structure  There i s increasing evidence that s t r u c t u r a l changes i n chromatin occur i n vivo, i n association with functional changes.  The nucleosomes of  t r a n s c r i p t i o n a l l y active chromatin are more rapidly excised by micrococcal  23  nuclease (140,141) and t h e i r DNA i s more rapidly degraded by DNase I (142,143,144).  The s e n s i t i v i t y of the ovalbumin gene i n oviduct nuclei to  micrococcal nuclease and to endogenous nuclease has been shown to p a r a l l e l the estrogen dependent t r a n s c r i p t i o n of the ovalbumin gene i n the immature chicken oviduct (145,146). Furthermore, nucleosomes are highly heterogeneous i n structure due to postsynthetic modifications of the histones (described e a r l i e r ) . Non-histone proteins such as the High Mobility Group (HMG) proteins have been reported to be closely associated with the nucleosomes from a c t i v e l y transcribed chromatin (147 - 152).  Other non-histone chromatin proteins  have been i s o l a t e d from swine s k e l e t a l muscle, l i v e r and v e n t r i c l e .  These  proteins have molecular weights of 35-45K and appear to be important i n the maturation of mRNA (153). also vary.  The nucleosome repeat length of chromatin can  Sea urchin sperm before f e r t i l i z a t i o n possesses the largest  nucleosome repeat length for any chromatin, but by the time the f e r t i l i z e d egg has reached the b l a s t u l a stage, the repeat length has shortened considerably (212). The question of how nucleosomes are arranged r e l a t i v e to DNA sequences i s s t i l l unresolved.  Nucleosomes could be arranged randomly or  a l t e r n a t i v e l y , they might be located i n unique positions with respect to DNA primary structure.  The l a t t e r arrangement has been termed "phasing"  and could t h e o r e t i c a l l y be triggered by sequence s p e c i f i c DNA binding proteins (154).  An example of precise register between nucleosomes and DNA  has been reported for tRNA genes i n chicken embryos, where t r a n s c r i p t i o n of the tRNA genes examined depended on nucleosome position (155).  An ordered  nucleosome alignment has been reported i n the hsp (heat shock protein) 70  24  and hsp 80 genes i n Drosophila (156) and nucleosomes are precisely positioned i n the non-transcribed spacer of the histone genes i n Drosophila (157).  Nucleosome phasing can also give r i s e to chromatin heterogeneity as  the nucleosome phasing may vary with gene function.  A non-random  nucleosome arrangement i s found i n the inactive constant-region gene of the immunoglobulin kappa l i g h t chain of mouse l i v e r , whereas this ordered arrangement i s l o s t i n the active form of the gene i n a myeloma (158).  VI  Nuclear Skeletal Structure  It i s becoming evident that chromatin i n the interphase nucleus  is  constrained i n an ordered conformation, i n which the chromatin takes the form of loops which are anchored to a protein framework (159,160,161). Upon d i s s o c i a t i o n of the histones, the DNA forms a number of  independently  constrained loops or domains which exhibit negative s u p e r h e l i c i t y .  The  lengths of the loops varies from 60 Kbp i n mouse L c e l l s to 150 Kbp i n bovine l i v e r or hamster (162). Several different protein complexes have been isolated and proposed as the matrix to which the loops of chromatin are attached.  A nucleoskeletal  complex which has DNA and protein as the major s t r u c t u r a l elements has been found during a l l stages of the HeLa c e l l cycle except mitosis (163).  On  the other hand, the chromosome scaffold present during mitosis contains many of the proteins found i n the nucleoskeleton.  A structure, termed the  nuclear matrix, has been isolated from rat l i v e r nuclei after  treatment  with nuclease and high concentrations of NaCl, and consists of large protein and DNA matrixes (164).  Further evidence that a protein framework  25  i s involved i n the organization of chromatin fibres comes from the finding that high molecular weight protein scaffolds  can be derived from metaphase  chromosomes upon extraction of the histones (165).  The resistance of these  structures to RNase and high concentrations NaCl suggests that the attachment of DNA i s not mediated by DNA-RNA hybrids and may involve d i r e c t protein-DNA contacts. A variety of predominant polypeptides are found i n nuclear matrixes (163,164,165).  nucleosome-free  Several proteins have molecular weights  which correspond closely to the molecular weights of the lamins, l o c a l i z e d i n the peripheral lamina of the nucleus (166).  proteins  The lamina i s  visualized i n the electron microscope as a homogeneous structure l y i n g between the nuclear membrane and the peripheral chromatin.  Taken together,  these results have led to the proposal that i n interphase, chromosomal DNA i s attached to the peripheral lamina of the nucleus as loops which are compacted into nucleosomes and higher order conformations which extend  into  the nuclear i n t e r i o r (167). The question of whether chromatin processing i s associated with nuclear skeletal structures has been investigated mainly by examining nuclear matrix preparations.  P u l s e - l a b e l l i n g studies with thymidine  indicate that DNA i s synthesized on or near the nuclear matrix i n rat l i v e r and that new DNA migrates outwards (168). for bovine l i v e r (169,170).  Similar results have been found  Studies on DNA r e p l i c a t i o n have been c a r r i e d  out on Plasmodia of Physarum polycephalum, i n which a l l of the nuclei of a single Plasmodium go through mitosis and S phase i n complete synchrony. P u l s e - l a b e l l i n g experiments indicate that the origins of replicons are bound to the nuclear matrix during the entire c e l l cycle and that  26  r e p l i c a t i o n points are bound to the matrix i n S phase but are released from the binding s i t e s after termination of r e p l i c a t i o n (171).  This work  implies that the nuclear matrix contains specific binding s i t e s through which chromatin moves during r e p l i c a t i o n . There i s growing evidence for a non-random d i s t r i b u t i o n of the unique sequences within chromatin loops, a c t i v e l y transcribed genes being p r e f e r e n t i a l l y associated with the nuclear matrix.  For example,  the  ovalbumin gene i s associated with the nuclear matrix i n chicken oviduct c e l l s , but not i n chicken l i v e r c e l l s i n which the gene i s t r a n s c r i p t i o n a l l y inactive (172).  S i m i l a r l y , nuclear matrix DNA from SV40  infected 3T3 c e l l s has an enrichment of the SV40 sequences r e l a t i v e to the t o t a l DNA (173).  The a,  J3 and X globin genes i n HeLa c e l l s are not  randomly attached to the nuclear matrix:  the a globin gene l i e s close to  the point of attachment of the chromatin loops to the matrix (174).  The  suggestion that t r a n s c r i p t i o n i s associated with the nuclear matrix i s further supported by the observation that hnRNA i s tenaciously  bound to the  matrix of Friend c e l l nuclei (175) and nascent hnRNA transcripts are formed i n the nuclear matrix of the rat endothelium (176). The nuclear matrix has also been proposed as a s i t e for hormone binding (176,177). However, despite clear indications that at least some chromatin processing occurs within the nuclear matrix, there i s a paucity of information on the presence of specific structures.  enzyme a c t i v i t i e s i n these  27  VII  The Present Investigation  Histone acetylation has been the focus of considerable attention because of i t s possible role i n chromatin processing, including t r a n s c r i p t i o n , r e p l i c a t i o n and r e p a i r .  The acetyltransferases,  responsible  for acetylating the nucleosomal core histones, have been f a i r l y w e l l characterized.  On the other hand, very l i t t l e i s known about the  properties and d i s t r i b u t i o n of histone deacetylase.  The reversible  i n h i b i t o r y effect of butyrate on histone deacetylase a c t i v i t y (78,79,80) has been used to f a c i l i t a t e investigations into the k i n e t i c s of histone acetylation (178,179,180), but our knowledge of the histone  deacetylase  i t s e l f i s scant, despite i t s obvious relevance to histone acetyl group turnover. The major goal of the work reported i n t h i s thesis was to study histone deacetylase from HeLa c e l l n u c l e i (a human c e l l l i n e ) and to develop a p h y s i o l o g i c a l l y meaningful assay system which would provide information on the i n t e r a c t i o n between histone deacetylase and i t s normal substrate i . e .  i n vivo assembled histones i n chromatin.  Another major goal  was to determine the d i s t r i b u t i o n of the enzyme within chromatin. Advantage was taken of the non-competitive i n h i b i t i o n of histone deacetylase to develop procedures for the i s o l a t i o n of histone i n association with endogenous [ H]-acetate l a b e l l e d chromatin. 3  deacetylase The  reversible nature of the butyrate i n h i b i t i o n of histone deacetylase allowed enzyme a c t i v i t y to be assayed after removal of the sodium butyrate from the system. As mentioned e a r l i e r , histone deacetylase preparations commonly lose  28  the a b i l i t y t o d e a c e t y l a t e chromatin-bound h i s t o n e s and t h i s has l i m i t e d s t u d i e s on the enzyme.  The  severely  i n v i v o assembled h i s t o n e d e a c e t y l a s e -  c h r o m a t i n complex d e s c r i b e d i n t h i s r e p o r t p e r m i t t e d the e l u c i d a t i o n o f many o f the c h a r a c t e r i s t i c s o f h i s t o n e d e a c e t y l a s e .  Free h i s t o n e s were  found t o be a p o o r s u b s t r a t e , but h i s t o n e s i n t h e i r p h y s i o l o g i c a l conformation  i n nucleosomes were d e a c e t y l a t e d r a p i d l y .  Up t o 90% of the  l a b e l l e d a c e t y l groups were removed from h i s t o n e s i n c h r o m a t i n by h i s t o n e d e a c e t y l a s e , showing t h a t d e n a t u r a t i o n of the enzyme had been kept t o a minimum u s i n g the i s o l a t i o n t e c h n i q u e s developed  i n this  study.  The d i s t r i b u t i o n of h i s t o n e d e a c e t y l a s e i n c h r o m a t i n was  examined by  d i g e s t i n g endogenous a c e t a t e l a b e l l e d HeLa c e l l n u c l e i w i t h m i c r o c o c c a l n u c l e a s e w h i c h p r e f e r e n t a i l l y d i g e s t s c h r o m a t i n a t the l i n k e r DNA m i x t u r e s o f mononucleosomes and o l i g o n u c l e o s o m e s .  to y i e l d  Chromatin f r a c t i o n s were  i s o l a t e d i n the p r e s e n c e o f b u t y r a t e from such d i g e s t s by  isokinetic  g r a d i e n t c e n t r i f u g a t i o n and column chromatography and assayed f o r h i s t o n e d e a c e t y l a s e a c t i v i t y a f t e r the removal o f the i n h i b i t o r . d e a c e t y l a s e was  Active histone  found o n l y i n a s s o c i a t i o n w i t h a h i g h m o l e c u l a r  n u c l e a s e r e s i s t a n t complex and no a c t i v i t y was  weight,  found on mononucleosomes o r  oligonucleosomes. C h a r a c t e r i z a t i o n of the h i g h m o l e c u l a r w e i g h t h i s t o n e d e a c e t y l a s e complex became the p r i m a r y c o n c e r n o f l a t e r r e s e a r c h e f f o r t s .  Under  t y p i c a l 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 c o n d i t i o n s , the h i s t o n e d e a c e t y l a s e complex c o n t a i n e d DNA of non-histone the m a i n t a i n a n c e  w i t h a s i z e d i s t r i b u t i o n o f 5-11  proteins.  K bp, and a v a r i e t y  P r o t e i n - p r o t e i n i n t e r a c t i o n s were i m p l i c a t e d i n  o f the s t r u c t u r e and the p r o t e i n c o m p o s i t i o n and p h y s i c a l  p r o p e r t i e s o f t h i s h i s t o n e d e a c e t y l a s e complex were compared w i t h those of  29  nuclear matrix. The  s o l u b l e c h r o m a t i n p r e p a r a t i o n d e s c r i b e d i n t h i s t h e s i s has  p r o v i d e d a u s e f u l system f o r s t u d y i n g h i s t o n e d e a c e t y l a s e and  answering  some o f the q u e s t i o n s r e g a r d i n g i t s p r o p e r t i e s and o r g a n i z a t i o n .  30  EXPERIMENTAL PROCEDURES  Abbreviations  ACS:  Aqueous c o u n t i n g  bis-acrylamide: bp:  scintillant.  N,N'-Methylene-bis-acrylamide.  base p a i r  BSA: DNA:  Bovine serum a l b u m i n Deoxyribonucleic acid  DNase I : D e o x y r i b o n u c l e a s e I EDTA:  ( E t h y l e n e d i n i t r i l o ) t e t r a c e t i c a c i d (disodium s a l t )  EGTA:  Ethyleneglycol-bis-(B-aminoethylether) N,N'-tetracetlc acid  HMG:  H i g h m o b i l i t y group  PMSF:  Phenylmethylsulphonyl f l u o r i d e  P r o t e i n a s e K: RNA:  Ribonucleic acid  RNase: SDS:  Ribonuclease  Sodium d o d e c y l s u l p h a t e  TEMED: Tris:  T r i t i r a c h i u m album p r o t e a s e  N,N,N',N'-Tetramethylethylenediamine Tris(hydroxymethyl)aminomethane  Materials  A l l c h e m i c a l s were o b t a i n e d c o m m e r c i a l l y and were o f t h e h i g h e s t  31  purity or reagent grade.  Special reagents were obtained as follows:  micrococcal nuclease ( E . C . 3.1.4.7) from Sigma; DNase I ( E . C . 3.1.4.6) from Boehringer Mannheim; RNase A ( E . C . 3.1.4.22) from BRL; EcoRI from BRL; H a e l l l from BRL; TaqI from BRL; Mspl from BRL;proteinase K from Sigma; BSA for r e s t r i c t i o n enzyme digests from BRL; BSA for histone deacetylase complex p r e c i p i t a t i o n from Calbiochem; acrylamide from Bio-Rad Laboratories; bis-acrylamide  from Eastman Kodak; TEMED from Bio-Rad  Laboratories; urea from Schwartz/Mann; agarose from BRL; hexylene glycol from Eastman Kodak; ACS from Amersham/Searle; NSC tissue s o l u b i l i z e r  from  Amersham/Searle; Bio-Gel A-5m and Bio-Gel A-50m from Bio-Rad Laboratories; HeLa c e l l s from The American Type Culture C o l l e c t i o n ; f e t a l bovine serum from GIBCO; Eagle's minimum e s s e n t i a l medium from GIBCO, penicillin/streptomycin  from GIBCO; Amphotericin from GIBCO and  [ H]-acetate (sodium s a l t ) spec. act. >500 mCi/mmol from New England 3  Nuclear.  C e l l Culture Conditions  HeLa c e l l s derived from the s t r a i n o r i g i n a l l y cultured by Gey et a l . (218)  were grown i n monolayer culture and passaged i n a medium comprising  95% Eagle's minimum e s s e n t i a l medium containing non-essential amino acids, 5% Fetal bovine serum, 100 u n i t s / ml of penicillin-streptomycin 0.00013% Amphotericin. bottles at 37°C.  and  Cultures of 100 ml were grown i n 3.8 1 r o l l e r  32  L a b e l l i n g of HeLa C e l l s w i t h [ H ] - a c e t a t e 3  HeLa c e l l s , grown t o near c o n f l u e n c e , were h a r v e s t e d u s i n g a rubber policeman, 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 700 xg f o r 7 minutes and taken up i n complete medium.  Butyric acid  f i n a l c o n c e n t r a t i o n o f 10 mM  ( n e u t r a l i s e d w i t h NaOH) was  added t o a  and the c e l l s were i n c u b a t e d f o r 15 minutes a t  37°C w i t h s h a k i n g t o a l l o w the b u t y r a t e t o e n t e r the c e l l s and histone deacetylation.  inhibit  The c e l l s were then l a b e l l e d i n a 2 hour  incubation  w i t h 150 u C i / m l o f [ H ] - a c e t a t e (sodium s a l t , spec. act.>500 3  mCi/mmol; New  England N u c l e a r ) and c e n t r i f u g e d a t 700 xg f o r 7 minutes.  The c e l l s were then taken up i n f r e s h medium c o n t a i n i n g 10 mM 8 mM  sodium a c e t a t e and excess l a b e l was  incubation.  b u t y r a t e and  chased out i n a 6 hour  A f t e r c e n t r i f u g a t i o n a t 2000 xg f o r 10 minutes, the c e l l s were  f r o z e n r a p i d l y and s t o r e d a t  -80°C.  M i c r o c o c c a l Nuclease D i g e s t i o n  F r o z e n (-80°C) c e l l p e l l e t s were homogenized g e n t l y i n 4 volumes o f TMKS (0.05 M T r i s - H C l , pH 7.4, mM  sodium b u t y r a t e , 0.1 mM  sample was  2  KC1,  0.25  M s u c r o s e , 40  PMSF) i n a g l a s s - T e f l o n hand homogenizer.  c e n t r i f u g e d a t 3,000 xg f o r 7 minutes and the p e l l e t  rehomogenized suspended  3 mM M g C l , 25 mM  and c e n t r i f u g e d as b e f o r e .  i n 3 volumes o f TMKS made 1 mM  was  The r e s u l t i n g n u c l e a r p e l l e t i n CaC^,  the n u c l e a r s u s p e n s i o n was mixed w i t h 0.99  The  was  a 10 u l a l i q u o t of  ml o f a s o l u t i o n c o n t a i n i n g 5 M  u r e a and 2 M NaCl, and the absorbance a t 260 nm was measured, w i t h corrections for turbidity.  M i c r o c o c c a l n u c l e a s e was  added t o the n u c l e a r  33  suspension at 2.5 &260  u  n  i  t  s  ° f enzyme/A2go of nuclei and, unless  stated otherwise, digestion was carried out for 5 minutes at 2 5 ° C . Digestion was terminated by the addition of 0.1 M EGTA (pH 7.4)  to a f i n a l  concentration of 3 mM, the nuclear suspension was c h i l l e d on i c e , centrifuged at 5,000 xg for 10 minutes and the supernatant c o l l e c t e d . nuclei were lysed with Buffer A (10 mM T r i s - H C l pH 7.4,  The  0.2 mM EDTA, 50 mM  sodium butyrate), containing 0.1 mM PMSF, on i c e for 30 minutes, centrifuged at 12,000 xg for 20 minutes and this supernatant combined with the f i r s t .  Preparation of Histones  Histones were isolated from HeLa c e l l s by extraction with ^ S O ^ (191).  Frozen ( - 8 0 ° C ) c e l l p e l l e t s (10 -10 s  2 ml TMK (10 mM T r i s - H C l , pH 8.0, glass-Teflon hand homogenizer.  c e l l s ) were homogenized i n  s  2 mM M g C l , 25 mM KC1) using a 2  The sample was centrifuged at 3,000 xg f o r  10 minutes and the p e l l e t was rehomogenized and centrifuged as before. nuclear p e l l e t was homogenized i n 2 ml of 10 mM T r i s - H C l (pH 7.4), onto 3 ml of 1 M sucrose i n 10 mM T r i s - H C l , 16,000 xg for 20 minutes.  The  layered  (pH 7.4) and centrifuged at  The gelatinous chromatin p e l l e t was extracted by  the addition of 0.3 ml of 0.4 N ^ S O ^ , for 15 minutes on i c e .  The  mixture was centrifuged at 12,000 xg for 10 minutes and the supernatant was added to 4 volumes of 95% ethanol at - 2 0 ° C .  Histones were precipitated  overnight at - 2 0 ° C and collected by centrifugation at 5,000 xg for 10 minutes.  The histone p e l l e t was dissolved i n 0.3 ml of 0.1 M acetic a c i d ,  dialyzed against the same solution for 2 hours and l y o p h i l i z e d .  34  Isokinetic Sucrose Gradient Centrifugation  Nucleosomes were sedimented through 10-28.3% (w/v) i s o k i n e t i c sucrose gradients (184) prepared for a p a r t i c l e density of 1.5 g/ml (185). gradients contained 10 mM T r i s - H C l , (pH 7.4),  The  50 mM butyrate, 0.1 mM PMSF  and centrifugation was carried out i n a Beckman SW41 rotor at 25,000 rpm for 18 hours at 4 ° C . Fractions were collected by upward displacement with 50% (w/v)  sucrose.  Column Chromatography  The products of p a r t i a l micrococcal nuclease digestion of HeLa c e l l nuclei were fractionated using either a Bio-Gel A-5m column (1.6 cm x 90 cm) with a flow rate of 3.5 ml/h or a Bio-Gel A-50m column (1.5 cm x 90 cm) with a flow rate of 2.4 m l / h . T r i s - H C l , pH 7.4,  The column contained buffer A (10 mM  0.7 mM EDTA, 50 mM sodium butyrate) and a l l procedures  were performed at 4 ° C .  P r e c i p i t a t i o n of Nucleosomes and Histone Deacetylase Complex for Enzyme As says  Histone deacetylase complex was prepared by micrococcal nuclease digestion of HeLa nuclei as described above and equal aliquots were made 100 mM i n NaCl by the addition of 4.0 M NaCl.  P r e c i p i t a t i o n was carried  out on ice for 30 minutes and the p e l l e t collected by centrifugation at 16,000 xg for 15 minutes.  The supernatants were aspirated and the p e l l e t s  were taken up i n Buffer B (10 mM T r i s - H C l , pH 7.8,  20 mM NaCl).  35  Histone Deacetylase  Assays  Histone deacetylase a c t i v i t y was determined by measuring the release of  [ H]-acetate from [ H ] - a c e t y l - l a b e l l e d histones. 3  3  Four different  procedures were employed depending upon the methods used to prepare the enzyme. For assays of whole n u c l e i , HeLa nuclei were isolated as described above (see Micrococcal Nuclease Digestion) except that butyrate was omitted.  The nuclei were suspended i n 2 volumes of Buffer C (10 mM  T r i s - H C l , pH 7.8,  3 mM M g C l  2>  20 mM NaCl) and divided into aliquots  (200  Vii), one of which was boiled for 1 minute to act as a background control.  Each aliquot was incubated with an equal amount of  [ H]-acetyl-labelled 3  histones (usually 40,000 cpm) at 22°C for 4 hours,  and the reaction was terminated by b o i l i n g for 1 minute.  Histone  deacetylase a c t i v i t y i n unlabelled nucleosome f r a c t i o n s , obtained by i s o k i n e t i c gradient centrifugation, was also measured by the release of [ H]-acetate from added free labelled histones. 3  Gradient fractions were  microdialyzed at 4°C against Buffer B and assayed as described above for nuclei. Fractions collected from Bio-Gel columns or from i s o k i n e t i c  sucrose  gradients which had been loaded with nucleosomes containing endogenous [ H]-acetyl labelled histones were assayed for histone deacetylase 3  a c t i v i t y by microdialysis against Buffer B at 4 ° C , followed by incubation at 22°C for 4 hours without further additions. stopped by b o i l i n g for 1 minute.  The enzyme reaction was  In other experiments, histone deacetylase  complex with a c e t y l - l a b e l l e d histones was precipitated with 100 mM NaCl and  36  assayed d i r e c t l y i n Buffer B.  The d i a l y s i s or p r e c i p i t a t i o n step was  required to separate nucleosomes from the sodium butyrate present i n the buffers used to prepare them. Samples of [ H]-acetate l a b e l l e d histone deacetylase complex which 3  were used to investigate  the effects of neocuproine were dialyzed for 2  hours against buffer A which was either unmodified or contained 3 mM neocuproine.  A l t e r n a t i v e l y , equal aliquots of acetate labelled histone  deacetylase complex were either untreated, boiled for 1 minute or made 1 mM or 10 mM i n B-mercaptoethanol.  Sodium butyrate was removed from a l l  samples of histone deacetylase complex by d i a l y s i s for 3 hours at 4°C against buffer B which had either nothing added or contained the same concentration of reagent as the sample of histone deacetylase complex being dialyzed.  Histone deacetylase a c t i v i t y was measured by incubating the  samples for 2 hours at 22°C and terminating the reaction by b o i l i n g for 1 minute. The t o t a l cpm present i n a sample of l a b e l l e d histone  deacetylase  complex was determined by dissolving an aliquot i n 1.0 ml of NCS tissue s o l u b i l i z e r and counting i n 10 ml ACS. Released [ H]-acetate was 3  extracted and counted i n the same manner for a l l of the assay systems.  The  incubation mixtures were a c i d i f i e d with 0.05 volumes of concentrated HC1 and 4 volumes of ethyl acetate were added.  The resulting 2 phase system  was thoroughly mixed, centrifuged at 1,000 xg for 5 minutes and the ethyl acetate phase c o l l e c t e d .  The extraction was repeated and the pooled ethyl  acetate phases were counted i n 10 ml of ACS .  37  P r e c i p i t a t i o n of Nucleosomes and Histone Deacetylase Complex  Appropriate Bio-Gel A-50m column fractions containing histone deacetylase complexes or nucleosomes were pooled and concentrated under nitrogen using an Amicon concentrator f i t t e d with a YM10 membrane.  The  concentrate was made 0.2 M i n sodium acetate and 50 ug ultrapure bovine serum albumin was added as c a r r i e r .  The samples were precipitated  overnight at - 2 0 ° C upon the addition of 6 volumes of -20°C ethanol.  The  precipitates were collected by centrifugation at 16,000 xg for 15 minutes and the p e l l e t s washed i n - 2 0 ° C ethanol and collected by centrifugation as before.  The p e l l e t s were dried i n vacuo for 10 minutes at room temperature  and used for SDS polyacrylamide gel electrophoresis, acid urea gel electrophoresis or the preparation of the associated DNA.  DNA Extraction  Appropriate Bio-Gel A-50m column fractions containing nucleosomes were pooled and concentrated under nitrogen using an Amicon concentrator f i t t e d with a YM10 membrane.  The concentrated nucleosomes were made 0.2 M i n  sodium acetate and precipitated overnight at - 2 0 ° C upon the addition of 6 volumes of ethanol.  Precipitated nucleosomes were collected by  centrifugation at 16,000 xg for 15 minutes.  The p e l l e t was washed i n  - 2 0 ° C ethanol and collected by centrifugation as before.  The nucleosome  p e l l e t was dried i n vacuo for 10 minutes at room temperature, taken up i n 200 u l of proteinase K buffer (0.2 M T r i s - H C l , pH 8.0,  0.2 M EDTA, 0.5%  SDS) and digested with 100 Pg proteinase K overnight at 50°C.  DNA was  38  extracted 4 times with phenol:chloroform (1:1), 4 times with chloroform, twice with water - saturated ether, and precipitated with 2 volumes of ethanol at - 7 0 ° C for 15 minutes. containing 50 mM T r i s - H C l ,  pH 8.0,  The p e l l e t was taken up i n buffer 50 mM EDTA and digested with 100 ug/  ml of pre-boiled RNase A for 45 minutes at 3 7 ° C .  The RNase A digest was  made 0.1 M i n NaCl by the addition of 4.0 M NaCl and predigested was added to a f i n a l concentration of 100 ug/ ml.  pronase  Pronase digestion was  carried out for 45 minutes at 37°C and the DNA was phenol extracted and precipitated i n ethanol at - 7 0 ° C as described above.  Agarose Gel Electrophoresis  Electrophoresis of DNA was carried out on horizontal slab gels of 0.9% or 0.75% agarose. acid (pH 8.0),  The 0.9% agarose gels contained 40 mM Tris-phosphoric  20 mM sodium phosphate, 1 mM EDTA, 0.1% SDS and were run at  80 mA u n t i l bromophenol blue marker dye had moved approximately 14 cm.  The  gels were stained with 10 ug/ml of ethidium bromide, and the DNA bands visualized under UV l i g h t .  Gels were photographed with a Polaroid camera.  The 0.75% agarose gels contained 89 mM T r i s - b o r i c acid (pH 8.3),  2.5  mM EDTA, 5 ug/ml ethidium bromide and were run at 50 v u n t i l the samples had entered the g e l and then at 80 v u n t i l the bromophenol blue marker dye had moved approximatly 12 cm. as above.  DNA bands were visualized and photographed  39  SDS Polyacrylamide Gel Electrophoresis  SDS slab gels (0.08 cm x 7.5 cm x 10 cm) containing either 10% or 15% polyacrylamide with a 4.5% stacking gel were prepared using the discontinuous buffer system of Laemmli (181).  The separating gels  contained either 10 or 15% acrylamide (acrylamide:bisacrylamide r a t i o of 30:0.4, w/w), 0.375 M T r i s - H C l , pH 8.8, ammonium persulphate.  0.1% SDS, 0.03% TEMED, 0.05%  The stacking gel contained 4.5% acrylamide  (acrylamide:bisacrylamide r a t i o of 30:0.8, w/w), 0.125 M T r i s - H C l pH 6.8, 0.1% SDS, 0.05% TEMED, 0.10% ammonium persulphate.  Samples were boiled for  1 minute i n 2 volumes of loading buffer (0.05 M T r i s - H C l , pH 6.8,  1.0% SDS,  0.16 M B-mercaptoethanol, 7.5% sucrose) and loaded onto the g e l s .  An  aliquot of bromophenol blue i n loading buffer was applied to a separate lane.  The gel running buffer contained 0.05 M T r i s pH ^ 8.3,  0.38 M  g l y c i n e , 0.1% SDS, and the gels were run at 25 v u n t i l the samples had entered the stacking gel and then at 100 v u n t i l the bromophenol blue marker dye reached the end of the separating g e l . The gels were stained with 0.25% Coomassie Blue i n a methanol and acetic acid system containing methanol:glacial acetic acid:water i n a r a t i o of 2:1:5  ( v / v ) , however, the r a t i o of components was 5:1:5  (v/v) for gels  containing samples of high mobility group proteins.  Acid-Urea Polyacrylamide Gel Electrophoresis  Acetic acid-urea slab gels (0.08 cm x 7.5 cm x 10 cm) were made according to the methods of Davie (182).  The separating gels contained 15%  40  acrylamide (acrylamide:bisacrylamide 30:0.8, w/w), 2.5 M urea, 0.9 M acetic a c i d , 0.5% TEMED, 0.125% ammonium persulphate. pre-electrophoresed acetic acid.  at 200 v for 2 h at 4°C with running buffer of 0.9 M  The stacking gel was poured after pre-electrophoresis  separating gel and consisted of 4.5% acrylamide 30:0.8, w/w),  The separating gel was  (acrylamide:bisacrylamide  2.5 M urea, 0.375 M potassium acetate pH 4.0,  0.125% ammonium persulphate.  of the  1.0% TEMED,  Samples were taken up i n loading buffer,  containing 2.5 M urea, 0.9 M acetic a c i d , 0.5 M J3-mercaptoethanol, 10% sucrose.  The running buffer consisted of 0.9 M acetic acid and  electrophoresis  was carried out at 100 v for 7 h at 4 ° C .  The gels were stained with 0.25% Coomassie Blue and destained described for SDS polyacrylamide gel  as  electrophoresis.  Two-Dimensional Polyacrylamide Gel Electrophoresis  Acetic acid-urea slab gels were run as described above and the lane of interest was cut out and rinsed for 30 minutes i n buffer containing 0.1 M T r i s - H C l , pH 7.4,  2.3% SDS, and 10% g l y c e r o l .  The rinsed gel was cut  longitudinally to y i e l d a 2 mm wide s l i c e which was applied horizontally to the stacking gel of a SDS polyacrylamide gel (0.08 cm x 7.5 cm x 10 cm) which was prepared as described before.  The separating gel contained 18%  acrylamide and the stacking gel contained 5% acrylamide. i n buffer (60 mM T r i s - H C l , pH 6.8,  Melted 1% agarose  2.3% SDS, 0.002% bromophenol blue) was  applied to the top of the stacking gel to seal the acid urea gel The gel running buffer contained 0.05 M Tris-pH ^ 8.3,  slice.  0.38M glycine,  0.1% SDS, and the gel was run at 50 v u n t i l the bromophenol blue had  41  entered the stacking gel and then at 80 v u n t i l the dye entered the separating g e l .  Electrophoresis was performed at 120 v u n t i l the  bromophenol blue reached the end of the g e l . The g e l was stained with s i l v e r .  S i l v e r Staining of Polyacrylamide Gels  S i l v e r staining was performed on c e r t a i n SDS polyacrylamide g e l s .  The  gel was washed by shaking overnight i n 50% methanol and stained by shaking for  15 minutes i n s i l v e r staining s o l u t i o n .  The staining solution was made  by dissolving 0.8 g AgN0 i n 4.0 ml H 0 and adding dropwise to 20 ml of 3  2  a mixture containing 95 mM NaOH and 0.52 M NH^OH.  The s i l v e r staining  solution was made up to 100 ml with 1^0 and used within 5 minutes.  The  gel was washed for 5 minutes i n 1^0 and gently shaken i n 200 ml developer (0.005% c i t r i c a c i d , 0.019% formaldehyde) u n t i l the stained proteins were clearly v i s i b l e .  The reaction was stopped by the addition of 50 ml  methanol.  Polyacrylamide Gel Scans  Both SDS and acid urea polyacrylamide gels were stained with Coomassie Blue and destained as described above.  The destained gels were placed  between two sheets of pre-washed d i a l y s i s membrane and dried i n vacuo at 60°C.  The dried gels were inserted into the gel c a r r i e r of a Beckman DU-8  spectrophotometer and the protein p r o f i l e s of appropriate lanes were determined by measuring the absorbance at 550 nm.  42  Nuclear Matrix Preparation  Nuclear matrix was isolated from HeLa c e l l nuclei which were prepared as described before (see Micrococcal Nuclease Digestion).  The nuclei were  digested with DNase I (10 units/mg of DNA) for 20 minutes at 0°C i n 0.25 M sucrose and TM buffer (20 mM T r i s - H C l , pH 7.4,  5 mM M g C l , 1 mM PMSF). 2  The digest was centrifuged at 1,000 xg for 15 minutes and the p e l l e t , containing the digested n u c l e i , was retained and extracted with NaCl according to the standard protocol for nuclear matrix preparation (164). A l l extractions were performed on i c e for 15 minutes and centrifugation was carried out at 1000 xg for 15 minutes. The DNase I digested nuclei were consecutively extracted one time with LM buffer (10 mM T r i s - H C l , pH 7.4,  0.2 mM M g C l , 1 mM PMSF) three times 2  with LM buffer containing 2.0 M NaCl and two times with LM buffer.  The  f i n a l p e l l e t contained the nuclear matrix.  Preparation of High Mobility Group Proteins  High Mobility Group (HMG) proteins were prepared from either HeLa or c a l f thymus chromatin according to the methods of Bhullar and Candido (183).  Frozen ( - 8 0 ° C ) HeLa c e l l p e l l e t s were homogenized i n 4 volumes of  TMK (10 mM T r i s - H C l , pH 8.0,  2 mM M g C l , 25 mM KC1, 0.1 mM PMSF) using a  g l a s s - t e f l o n hand homogenizer.  2  The sample was centrifuged at 3,000 xg f o r  7 minutes and the p e l l e t was rehomogenized and centrifuged as before. thymus was collected and frozen within 5 minutes of slaughter.  Calf  Frozen  ( - 8 0 ° C ) c a l f thymus was minced with scissors and homogenized i n 4 volumes  43  of PHMC (10 mM PIPES-HC1 pH 7.0,  1.0 M hexylene g l y c o l , 5 mM M g C l  C a C l , 0.1 mM PMSF) using a motor driven teflon pestle. 2  2>  1 mM  The sample was  f i l t e r e d through 8 layers of cheese c l o t h and centrifuged at 1,000 xg for 10 minutes.  The p e l l e t was resuspended i n the same buffer and  rehomogenized and centrifuged twice as described above, except that hand homogenization was used. The resulting HeLa or c a l f thymus nuclear p e l l e t s were treated identically.  The p e l l e t s were homogenized i n 4 volumes of T r i s buffer (10  mM T r i s - H C l , pH 7.4,  0.1 mM PMSF), layered onto 1 M sucrose i n T r i s buffer  and centrifuged at 17,000 xg for 20 minutes.  The translucent p e l l e t was  suspended i n T r i s buffer i n a f i n a l volume of 5 ml/g i n i t i a l wet weight of the t i s s u e , made 0.15 M i n ammonium sulphate and centrifuged at 17,000 xg for 15 minutes.  The supernatant was collected and 100% t r i c h l o r o a c e t i c  acid was added dropwise to a f i n a l concentration of 2%.  The solution was  kept on ice for 20 minutes and centrifuged at 17,000 xg for 20 minutes. The supernatant was taken and s o l i d ammonium sulphate was added to give a concentration of 55%.  The mixture was s t i r r e d on ice for 30 minutes and  centrifuged at 17,000 xg for 30 minutes.  The p e l l e t , which contained HMG 1  and HMG 2 and the supernatant were collected and s o l i d ammonium sulphate was added to the supernatant to a f i n a l concentration of 90%.  The  supernatant was s t i r r e d on ice for 30 minutes and HMG 14 and HMG 17 were collected by centrifugation at 17,000 xg for 1 hour.  The HMG p e l l e t s were  taken up i n 0.1 M NH^CO-j, dialyzed i n Spectrapor 3 d i a l y s i s tubing against the same buffer and l y o p h i l i z e d .  A l t e r n a t i v e l y , the HMG p e l l e t was  dialyzed against 20 mM NaCl i n buffer D (15 mM T r i s - H C l , pH 8.0,  6.0 M  urea, 5 mM C-mercaptoethanol) and the sample was applied to a Whatman CM52  44  column (2.8 cm x 40 cm) which was eluted with two successive l i n e a r gradients of NaCl i n buffer D to obtain separate fractions of HMG 14 and HMG 17 (213).  The f i r s t gradient was produced by mixing 250 ml each of  0.02 M and 0.20 M NaCl, and the second by mixing 250 ml each of 0.2 M and 0.60 M NaCl i n buffer D.  Acid Hydrolysis  50 ug samples of histones or HMG 14 and HMG 17 were dissolved i n 0.4 ml of 6 N HC1 and maintained at 110°C for 20 or 36 hours i n sealed glass vials.  The HC1 was evaporated under vacuum at 70°C.  Amino Acid Analysis  HMG 14 and HMG 17 proteins prepared from c a l f thymus were hydrolyzed i n 6 N HC1.  The hydrolyzate was analyzed on an automatic amino acid  analyzer using a single column system.  No corrections were made for  hydrolytic losses.  R e s t r i c t i o n Enzyme Digestion  R e s t r i c t i o n endonuclease digestion of purified DNA was carried out according to the manufacturer's (BRL) i n s t r u c t i o n s .  B r i e f l y , the DNA  samples were taken up i n the appropriate enzyme buffer and ultrapure BSA (BRL) was added to a f i n a l concentration of 100 ug/ml.  Aliquots of  r e s t r i c t i o n enzyme were added every two hours and digestion was carried out  45  for 4 - 1 2 Taql.  hours at 37°C with H a e l l l , EcoRI and Mspl, and at 65°C with  The reactions were terminated by the addition of 0.5 M EDTA to a  f i n a l concentration of 8 mM, and heating to 68°C for 5 minutes.  Nucleosome Reconstitution  A c e t y l - l a b e l l e d core histones were prepared from labelled HeLa c e l l s by NaCl extraction i n the presence of 100 mM sodium butyrate (186).  3 g  l a b e l l e d HeLa c e l l nuclei were prepared using the procedures described i n "Micrococcal Nuclease Digestion".  The nuclei were lysed i n 90 ml buffer E  (10 mM T r i s - c a c o d y l i c acid pH 7.2,  0.7 mM EDTA, 100 mM sodium butyrate, 1  mM PMSF), and s o l i d NaCl was added to give a concentration of 0.6 M and the n u c l e i were allowed to swell overnight at 4 ° C . The swollen nuclei were collected by centrifugation at 5,000 xg for 10 minutes and were transferred to 90 ml of buffer D containing 0.65 M NaCl and s t i r r e d gently for 24 hours at 4 ° C .  The mixture was centrifuged at 17,000 xg for 15 minutes and the  p e l l e t containing histone Hl-depleted chromatin, was taken up i n 80 ml buffer E and s t i r r e d gently for 6 hours at 4°C and centrifuged as before. The p e l l e t was recovered and mixed with 4 ml of buffer E containing 2 M NaCl and s t i r r e d gently overnight at 4 ° C .  The mixture was centrifuged at  170,000 xg f o r 24 hours i n a Beckman SW60Ti rotor and the histone f r a c t i o n was c o l l e c t e d . The histones were passed through a Sephadex G-25 column (1.0 cm x 30 cm) i n order to remove free  [ H]-acetate and concentrated using an Amicon 3  concentrator f i t t e d with a PM10 membrane.  The histones were heated to 60°C  for 10 minutes to inactivate any possible contaminating histone deacetylase.  46  Unlabelled histone deacetylase complex was isolated using a Bio-Gel A50m column containing 2.0 M NaCl and 3.0 ml a l i q u o t s , containing 37 ug of DNA, were mixed with the [ H]-acetyl labelled core histones at a 3  histone:DNA r a t i o of 0.9:1  (w/w).  The histones and DNA were reconstituted  by d i a l y s i s against buffer E containing successively lower i o n i c strength (187).  D i a l y s i s was carried out at 4°C for 15 minutes against 1.6 M NaCl,  15 minutes against 1.2 M NaCl, 1 hour each against 0.85 M, 0.75 M and 0.65 M NaCl, 15 minutes against 0.5 M NaCl, 15 minutes against 0.25 M NaCl and overnight against 50 mM NaCl.  F i n a l l y , the sodium butyrate was removed by  d i a l y s i s against buffer B for 4 hours.  Protein Assays  Protein concentrations were determined using Bio-Rad protein assay concentrate.  Samples were mixed with H^O to a f i n a l volume of 0.8 ml and  0.2 ml Bio-Rad protein assay concentrate was added.  The assay system was  thoroughly mixed and allowed to stand at room temperature for 10 minutes. The absorbance at 595 nm was measured and the protein concentration was determined by comparing the absorbance with those of standard solutions containing 0-20 ug of p r o t e i n .  47  RESULTS  PART A.  THE CHARACTERIZATION OF HISTONE DEACETYLASE  Incubations of Hela n u c l e i with free  [ H ] - a c e t y l - l a b e l l e d histones 3  at 22°C for varying periods of time resulted i n the release of up to about 25% of the [ H]-acetyl groups after 4 hours (Figure 6). 3  The rather slow  rate of enzymatic- deacetylation of histones observed with nuclei may be due to the time required for d i f f u s i o n of the free histones through the nucleus to the s i t e of deacetylation or for assembly of the added labelled histones into nucleosomes.  As a consequence, only a limited amount of information  on histone deacetylase can be obtained from assay systems u t i l i z i n g whole nuclei and therefore, efforts were made to develop a physiologically meaningful assay system.  I.  Development of a Physiological Assay System for Histone Deacetylase  a.  Sucrose Gradient Centrifugation  In order to examine the d i s t r i b u t i o n of histone deacetylase  in  chromatin, Hela nuclei were p a r t i a l l y digested with micrococcal nuclease the extent of 12-15% s o l u b i l i z a t i o n of the i n i t i a l ^oC),  a n <  * ^ t  to  i e  resulting nucleosomes were isolated by centrifugation through an i s o k i n e t i c sucrose gradient prepared for a p a r t i c l e density of 1.5 g/ml (185). Fractions were collected by upward displacement and sucrose was removed by  48  C O  I n c u b a t i o n Time ( h )  Figure 6.  Time course of histone deacetylase i n n u c l e i .  HeLa n u c l e i were prepared as described i n Experimental Procedures and incubated with added free d i f f e r e n t lengths of time.  [ H ] - a c e t y l - l a b e l l e d histones (40,000 cpm) f o r 3  The reactions were stopped by b o i l i n g for 1 minute  and released [ H]-acetate was extracted and counted. 3  49  microdialysis.  Gradient fractions were assayed for histone  a c t i v i t y by the addition of free a c e t y l - l a b e l l e d histones  deacetylase  (Figure 7A).  The  majority of enzyme a c t i v i t y was detected near the top of the gradients between free DNA and mononucleosomes, presumably representing dissociated enzyme.  A  second peak of enzyme a c t i v i t y was observed i n oligonucleosomes at the bottom of the gradients. The nuclease digestion and centrifugation were repeated i n the presence of butyrate with nuclei containing endogenous a c e t y l - l a b e l l e d histones and the sucrose gradient fractions were assayed for histone deacetylase upon removal of the butyrate by d i a l y s i s (Figure 7B).  In this case, a l l of the histone  deacetylase was detected i n high molecular weight material. finding that c e r t a i n HMG proteins can i n h i b i t nuclear histone  In view of the deacetylase  a c t i v i t y i n v i t r o (188), i t was possible that the lack of detectable a c t i v i t y on mononucleosomes was due to the presence of such an i n h i b i t o r .  The  endogenous deacetylase a c t i v i t y of micrococcal nuclease digests was examined on gradients run i n 0.4M NaCl, to remove HMG proteins (Figure 7C). Dinucleosomes sedimented more slowly i n t h i s case due to the lack of histone HI and non-histone chromatin proteins (189,190);  however, the histone  deacetylase p r o f i l e was i d e n t i c a l to the one seen i n gradients without NaCl. The above experiments indicate that histone deacetylase does not bind to mono- or dinucleosomes under the conditions t y p i c a l l y used i n sucrose gradient fractionations, and suggest that perhaps higher order chromatin structures are important i n binding this enzyme.  50  50a  Figure 7.  Isokinetic sucrose gradient p r o f i l e s of nucleosomes and histone deacetylase  activity.  He l a nuclei were p a r t i a l l y digested with micrococcal nuclease and nucleosomes were isolated by sedimentation through i s o k i n e t i c sucrose gradients i n a Beckman SW41 rotor at 25,000 rpm for 18 h r . A  260  [ H]-acetate, CPM released (XI0~ ).  ;  3  3  (A)  (-•—t-)  Free  a c e t y l - l a b e l l e d HeLa histones were added to gradient fractions which were incubated at 22°C for 4 hr and histone deacetylase a c t i v i t y was determined by extracting and counting the released [ H]-acetate. 3  (B)  Nucleosomes  were prepared from a c e t y l - l a b e l l e d HeLa nuclei using i d e n t i c a l procedures as i n A, except that a l l buffers contained 40 mM butyrate to i n h i b i t histone deacetylase.  The butyrate was removed from the gradient fractions  by d i a l y s i s and the fractions were incubated for 4 hr at 22°C.  t r i -  acetate, released from endogenous l a b e l l e d histones, was extracted and counted.  (C)  Endogenous a c e t y l - l a b e l l e d nucleosomes were prepared and  assayed for histone deacetylase a c t i v i t y as described i n B, except that the sucrose gradient contained 0.4 M NaCl i n order to remove non-histone chromosomal proteins.  51  b.  Isolation of Histone Deactylase A c t i v i t y Using a Bio-Gel A-5m Column  Endogenous [ H]-acetate-labelled HeLa c e l l nuclei were p a r t i a l l y 3  digested with micrococcal nuclease and the digest products were separated a Bio-Gel A-5m column, which had a molecular weight exclusion l i m i t of 5,000,000.  Nuclease digestion and column chromatography were carried out  i n the presence of 50 mM butyrate and the fractions were assayed for histone deacetylase a c t i v i t y after d i a l y s i s as before. column i s shown i n Figure 8.  A p r o f i l e of the  The p r o f i l e s of histone deacetylase  and absorbance at 260 nm did not coincide.  The histone  activity  deacetylase  a c t i v i t y was d i s t i n c t l y skewed to the high molecular weight side of the excluded absorbance peak. Therefore, the histone deacetylase a c t i v i t y appeared to be associated with a high molecular weight structure.  c.  Substrate Preference of Chromatin-Bound Histone Deacetylase  In order to examine the substrate preference of native histone deacetylase, mixing experiments were performed using the high molecular weight form of the enzyme, and various forms of histone substrate. Chromatin complexes containing histones, [ H]-acetate, 3  labelled endogenously with  and deacetylase were prepared by micrococcal nuclease  digestion of l a b e l l e d nuclei and isolated by chromatography on a Bio-Gel A-5m column i n the presence of 50 mM butyrate.  They were then mixed with  equal amounts of either free l a b e l l e d HeLa histones,  free unlabelled HeLa  histones or free hyper-acetylated unlabelled HeLa histones as shown i n Table 2.  The added free l a b e l l e d histones contained an equal number of  52  CO  X 200  o Q  ?  n  -o ?  73 100  A A Q vt (D 0.  X  o  I  CJ  50  100  150  V o l u m e (ml)  Figure 8.  Fractionation of micrococcal nuclease digest products on a  Bio-Gel A-5m column.  [ H]-acetyl-labelled 3  HeLa n u c l e i were p a r t i a l l y digested with  micrococcal nuclease and the digest products were separated using a Bio-Gel A-5m column e q u i l i b r i a t e d with 50 mM butyrate.  Fractions of 1 ml were  c o l l e c t e d and the butyrate was removed by d i a l y s i s and the  histone  deacetylase a c t i v i t y was detected by" extracting and measuring the  free  [ H]-acetate released from the endogenous histones after incubation at 3  22°C for 4 hours ( (xlO~ ). 3  ) A  2 6 Q  ;  (  •) [ H]-acetate, CPM released 3  53  [ H]-acetyl groups as were present i n the endogenous l a b e l l e d histones of 3  the chromatin complexes. butyrate by d i a l y s i s .  The reaction was i n i t i a t e d by the removal of  Each aliquot of l a b e l l e d chromatin complex contained  213,000 cpm and complexes incubated alone released 88% of t h e i r [ H]-acetyl groups. 3  The presence of free unlabelled histones caused 11%  i n h i b i t i o n of the deacetylase a c t i v i t y , while hyper-acetylated unlabelled histones caused 46% i n h i b i t i o n .  This i n h i b i t i o n could have occurred as a  result of non-productive binding between free histone and histone deacetylase, or by the p r e f e r e n t i a l release of unlabelled acetyl groups from the added free histone.  However, when free a c e t y l - l a b e l l e d histones  were mixed with the enzyme complexes, the release of radioactive acetate was also i n h i b i t e d by 40% (Table 2).  This suggests that the f i r s t of the  above alternatives i s the correct one, i . e .  the chromatin bound histone  deacetylase must interact non-productively with free histones.  Histone  deacetylase assays were also carried out with reduced amounts of a l l three types of added free histones and i t was found that the i n h i b i t i o n of the histone deacetylase was only half as great when half as much free histones were added to the assays.  Therefore, the i n h i b i t i o n of histone  deacetylase  by free histones appeared to be concentration dependent. To determine whether nucleosome structure i s important for productive binding between histones and histone deacetylase, high molecular weight chromatin complexes l a b e l l e d endogenously with [ H]-acetate were isolated 3  as above and incubated either alone, with l a b e l l e d mononucleosomes containing an equal number of [ H]-acetyl groups, or with unlabelled 3  mononucleosomes as shown i n Table 3.  The mononucleosomes were prepared by  p a r t i a l micrococcal nuclease digestion of either a c e t y l - l a b e l l e d or  54  Table 2  I n h i b i t i o n of histone deacetylase by free histone  Percent Inhibition of Histone Added Free Histone  Deacetylase  Unlabelled histone  11  Unlabelled hyper-acetylated histone  46  Labelled hyper-acetylated histone  40  Chromatin-bound histone deacetylase with endogenous  [ H]-acetyl-labelled  histones was prepared from labelled HeLa nuclei by p a r t i a l digestion with micrococcal nuclease and i s o l a t i o n of the polynucleosomes on a Bio-Gel A-5m column i n the presence of 50 mM butyrate.  HeLa histones were prepared as  described i n Experimental Procedures and equal amounts of either unlabelled histone, unlabelled hyper-acetylated histone or labelled hyper-acetylated histone (containing an equal number of counts as the labelled polynucleosomes) were added.  Butyrate was removed by d i a l y s i s and the polynucleosomes were  incubated for 4 hours at 22°C.  The reaction was stopped by b o i l i n g and the  released [ H]-acetate was extracted and counted. 3  55  Table 3  Use of mononcleosomes as a substrate for histone  Assay System  deacetylase  [ H]-Acetate, CPM Released 3  Chromatin complex  25,539  Chromatin complex + labelled mononucleosomes  35,636  Chromatin complex + unlabelled mononucleosomes  20,400  Both unlabelled and a c e t y l - l a b e l l e d HeLa nuclei were p a r t i a l l y digested with micrococcal nuclease and nucleosomes were isolated using a Bio-Gel A-5m column i n the presence of 50 mM butyrate.  Either endogenously labelled  mononucleosomes (containing an equal number of counts as the labelled chromatin complex) or unlabelled mononucleosomes were added to equal aliquots of endogenous l a b e l l e d chromatin complex (containing 40,000 cpm).  Butyrate  was removed by d i a l y s i s and deacetylase a c t i v i t y was assayed by incubation at 22°C for 4 hours.  The reaction was stopped by b o i l i n g and the free acetate  was extracted and counted.  56  unlabelled HeLa nuclei and isolated by chromatography on i d e n t i c a l Bio-Gel A-5m columns containing 50 mM butyrate.  The addition of labelled  mononucleosomes produced an increase i n the amount of [ H]-acetate released, 3  and the addition of unlabelled mononucleosomes  (present at a lower  concentration than the labelled mononucleosomes) amount of released  [ H]-acetate. 3  resulted i n a decrease i n the  This shows that histones must be i n t h e i r  physiological conformation as nucleosome complexes i n order to serve as efficient  II.  substrates for the chromatin-bound histone deacetylase.  The D i s t r i b u t i o n of Histone Deacetylase i n Chromatin  To examine more closely the d i s t r i b u t i o n of histone deacetylase a c t i v i t y i n chromatin fragments, nuclease digests were separated on a Bio-Gel A-50m column (50,000,000 molecular weight exclusion l i m i t ) .  Fractions were taken  for DNA extraction and analysis on 0.9% and 0.75% agarose gels, and the gel patterns are shown i n the insert to Figure 9.  DNA fragment lengths were  determined using TaqI digests of pBR322 and H i n d l l l digests of lambda DNA as markers.  The excluded peak from the A-50m column (Figure 9C) contained DNA  which ranged from 11-5 Kbp i n length, corresponding to chromatin fragment lengths of 55-25 nucleosomes (Figure 9A).  In three separate preparations, the  DNA from peak I polynucleosomes gave size d i s t r i b u t i o n s of 11-5 Kbp, 10-6 Kbp and 15.5-5 Kbp, the l a s t preparation being the result of the shorter nuclease digestion conditions used.  The DNA from the polynucleosomes i n peak II ranged  from 10.0 - 4.2 Kbp, corresponding to chromatin fragments 50 - 21 nucleosomes i n length while peak III contained DNA of approximately 4.0 Kbp (20 nucleosomes) to 200 base pairs i n length.  Peak IV contained free  which have migrated off the end of the gel shown i n Figure 9.  nucleotides  An A-50m column  57  therefore provides good resolution of chromatin fragments over a broad size range.  Peak I of the p r o f i l e , however, contains DNA which overlaps i n size  with the DNA of peak I I , which i s included by the column.  This suggested that  peak I contains polynucleosomes which are either aggregated, or contain additional non-histone proteins, or both. The p o s s i b i l i t y that peak I might consist of non-specific chromatin aggregates i s rendered u n l i k e l y by the fact that the proportion of A2gQ-absorbing material i n peak I i s unaffected by running the columns i n 1-2 M NaCl (see below).  Under these conditions, histone dissociation occurs,  and d i s s o l u t i o n of such aggregates would be expected.  Furthermore, the  fractions corresponding to peak I exhibit no t u r b i d i t y .  The protein  composition of the Bio-Gel A-50m chromatin fractions i s discussed i n d e t a i l below.  Since polynucleosomes from both region I (excluded) and regions  II-III  (included) of the column contain a variety of non-histone proteins, t h e i r presence alone does not explain the difference i n chromatographic properties between the two classes of polynucleosomes.  The most l i k e l y explanation i s  that the polynucleosomes of peak I are associated with each other through protein-protein interactions involving some specific component(s) of  this  f r a c t i o n . Micrococcal nuclease digests of [ H ] - a c e t y l - l a b e l l e d chromatin 3  were then fractionated on a Bio-Gel A-50m column i n the presence of butyrate and the fractions were assayed for endogenous histone deacetylase a c t i v i t y as before.  A p r o f i l e of such a column i s shown i n Figure 10A.  a c t i v i t y was found i n the i n i t i a l peak. mononucleosomes (regions II and I I I ) , sucrose gradients (Figure 7B).  A l l of the enzyme  No a c t i v i t y was detected i n o l i g o - or  confirming the results obtained with  The p r o f i l e s of histone deacetylase  and absorbance at 260 nm i n the f i r s t peak coincided.  activity  58  58a  Figure 9.  Fractionation of micrococcal nuclease digest products of HeLa chromatin on a Bio-Gel A-50m column.  HeLa nuclei were p a r t i a l l y digested with micrococcal nuclease and nucleosomes were isolated using a Bio-Gel A-50m column (1.5 x 90 cm) with a flow rate of 2.4 m l / h r .  DNA was extracted from the column fractions  and analyzed on a horizontal slab gel of 0.75% agarose i n Tris-borate buffer (pH 8.3) (B).  (A) or 0.9% agarose i n Tris-phosphate buffer (pH 8.0)  The gel was stained with 10 ug/ml of ethidium bromide and the DNA  bands were v i s u a l i z e d under UV l i g h t .  Approximate DNA fragment sizes  were determined using a Taq I digest of pBR322 (lane 4 of panel B) and a H i n d l l l digest of lambda DNA (panel A) as markers. (A)  DNA from peak I of a Bio-Gel A-50m column; (B)  indicated regions of the column p r o f i l e ; (C) A-50m column at 260 nm.  DNA from the  Absorbance p r o f i l e of  the  The data i n (B) and (C) are from the same column  run; the g e l i n (A) i s from a separate but i d e n t i c a l column run.  59  III  Effect of Butyrate on the D i s t r i b u t i o n of Histone Deacetylase  HeLa c e l l s were normally l a b e l l e d with [ H]-acetate for 2 hours and 3  chased with unlabelled acetate for 6 hours i n the presence of 10 mM butyrate i n order to increase the s p e c i f i c a c t i v i t y of the acetylated histones.  Butyrate has manifold effects on different c e l l lines including  alterations i n protein synthesis (219,220,222), changes i n protein modification (221,223,225) and causes a reversible decrease i n condensed chromatin clumps i n HeLa c e l l s (224).  In order to rule out the p o s s i b i l i t y  that the deacetylase d i s t r i b u t i o n seen i n the above experiments might be an a r t i f a c t of the butyrate treatment, a s i m i l a r experiment was done with c e l l s labelled i n the absence of butyrate. labelled with 150 uCi/ml of [ H]-acetate. 3  HeLa c e l l s were normally The medium used for  l a b e l l i n g HeLa c e l l s i n the absence of butyrate contained 300 uCi/ml of [ H]-acetate, to compensate for the expected turnover of acetyl groups 3  under these conditions.  A l a b e l l i n g time of 2 hours was used and the c e l l s  were frozen rapidly without an intervening cold chase.  Nucleosomes were  prepared from the c e l l s by standard procedures and were separated on a Bio-Gel A-50m column.  Butyrate was present during the preparation and  fractionation of the chromatin digest to prevent loss of the i n vivo incorporated l a b e l . before.  Fractions were then assayed for histone deacetylase as  As seen i n Figure 10B, the results were exactly the same as for  c e l l s labelled i n the presence of butyrate (Figure 10A), the  histone  deacetylase being found only i n the high molecular weight material excluded by the column.  This c h a r a c t e r i s t i c d i s t r i b u t i o n of histone deacetylase  therefore occurs under normal culture conditions and i s not caused or influenced by exposure of the c e l l s to butyrate.  This chromatin f r a c t i o n  60a  Figure 10.  D i s t r i b u t i o n of histone deacetylase i n chromatin fragments from butyrate treated versus untreated HeLa c e l l s  HeLa c e l l s were l a b e l l e d i n vivo with [ H]-acetate (sodium s a l t , 3  spec. act. > 500 mCi/mol) for 2 h r .  Labeled nuclei were prepared from  the c e l l s and p a r t i a l l y digested with micrococcal nuclease.  The nuclease  digest products were fractionated on a Bio-Gel A-50m column equilibrated with buffer containing 50 mM butyrate.  Fractions of 1 ml were c o l l e c t e d ,  dialyzed to remove the butyrate, and incubated for 4 hr at 2 2 ° C . released  The  [ H]-acetate was extracted and counted as described i n 3  Experimental Procedures. released (xlO ) . 3  (A)  (  ) ^^Q', (••••-  ) [ H]-acetate, CPM 3  HeLa c e l l s were labelled with 150 pCi/ml of  [ H]-acetate for 2 hr i n medium containing 10 mM butyrate, and then 3  incubated a further 6 hr i n unlabelled medium containing 8 mM sodium acetate as well as 10 mM butyrate. 300 uCi/ml of  (B)  HeLa c e l l s were l a b e l l e d with  [ H]-acetate for 2 hr with no butyrate present, and 3  without a cold chase.  61  containing the deacetylase a c t i v i t y w i l l be referred to as the "histone deacetylase complex".  IV  Effect of Salt Concentration on Histone Deacetylase D i s t r i b u t i o n  a.  Chromatography Using a Bio-Gel A-5m Column  The effect of s a l t concentration on the d i s t r i b u t i o n of histone deacetylase was i n i t i a l l y examined by fractionating micrococcal nuclease digests of [ H ] - a c e t y l - l a b e l l e d 3  HeLa c e l l nuclei using a Bio-Gel A-5m  column i n the presence of sodium butyrate and varying concentrations of NaCl.  Column fractions were assayed for histone deacetylase a c t i v i t y i n  the normal manner after d i a l y s i s to remove f i r s t the NaCl, and then the butyrate.  The r e s u l t s of fractionating nuclease digests i n the presence of  0.5 M NaCl, 1.0 M NaCl and 2.0 M NaCl are shown i n Figure 11.  Histone  deacetylase a c t i v i t y was found i n the high molecular weight material i n a l l cases.  Although 2.0 M NaCl causes the d i s s o c i a t i o n of histones from  chromatin, histones were detected i n a l l of the nucleosome fractions from the Bio-Gel A-5m column by SDS polyacrylamide gel electrophoresis.  These  histones most probably arose by aggregation i n the high s a l t .  b.  Chromatography Using a Bio-Gel A-50m Column  In view of the presence of histones, and possibly other aggregates, i n the i n i t i a l peak from the Bio-Gel A-5m column, the fractionation of nuclease digests of labelled HeLa c e l l nuclei was repeated using a Bio-Gel A-50m column containing 1.0 M NaCl and 2.0 M NaCl.  The results i n the case  62  62a  Figure 11.  Effect of s a l t concentration on histone deacetylase d i s t r i b u t i o n .  Endogenous l a b e l l e d HeLa n u c l e i were p a r t i a l l y digested with micrococcal nuclease and the r e s u l t i n g products were separated on a Bio-Gel A-50m column containing butyrate and different concentrations of NaCl.  Fractions were  assayed for histone deacetylase a c t i v i t y after d i a l y s i s to remove the NaCl and butyrate.  Free [ H]-acetate was collected and counted (A) 0.5 M NaCl; (B) 1.0 3  M NaCl; (C) 2.0 M NaCl.  63  of 1.0 M NaCl were the same as those shown i n Figure 10A, i . e .  histone  deacetylase was associated with the high molecular weight excluded peak. Therefore, the removal of s a l t dissociable non-histone chromosomal proteins at concentrations of NaCl greater than 0.35 M did not a l t e r the p r o f i l e . As expected, no\histone deacetylase a c t i v i t y was detected i n the excluded fractions from the Bio-Gel A-50m column run i n 2.0 M NaCl, since the polynucleosomal DNA present i n the void peak did not contain histones as shown by SDS polyacrylamide gel electrophoresis  (Figure 12).  Since the  assays of deacetylase a c t i v i t y i n these experiments depended on the release of labelled acetate from histones present i n the chromatin f r a c t i o n s , was necessary to reconstitute  it  labelled histones with the polynucleosomal  DNA i n order to assay for histone deacetylase a c t i v i t y i n the 2 M NaCl profile. As the presence of histone HI during nucleosome reconstitution by s a l t d i a l y s i s results i n p r e c i p i t a t i o n of the complexes (186),  [ H]-acetyl3  labelled core histones were prepared from labelled HeLa c e l l s by NaCl extraction i n the presence of 100 mM sodium butyrate.  The Hl-depleted  chromatin was prepared by washing l a b e l l e d HeLa nuclei with 0.6 M NaCl for 24 hours with 100 mM butyrate present.  The histones were heated to 60°C  for 10 minutes i n order to inactivate any possible contaminating histone deacetylase.  Unlabelled nuclease digests were chromatographed on a Bio-Gel  A-50m column containing 2.0 M NaCl and 3.0 ml aliquots of the excluded fraction,  (histone deacetylase complex) containing 37 ug of DNA, were  mixed with [ H ] - a c e t y l - l a b e l l e d core histones i n buffer containing 2.0 M 3  NaCl and 50 mM sodium butyrate at a histone:DNA r a t i o of 0.9:1 histone:DNA r a t i o of 0.9:1  (w/w).  A  (w/w) was chosen as reconstituted nucleosomes  precipitate i f the r a t i o i s greater than 1:1.  The mixtures were dialyzed  64  Figure 12.  The histone content of histone deacetylase complex prepared i n the  presence of 2.0M NaCl.  He l a nuclei were p a r t i a l l y digested with micrococcal nuclease and the digest products were isolated using a Bio-Gel A-50m column containing 2.0 M NaCl.  The excluded peak containing histone deacetylase complex was pooled,  and proteins examined by electrophoresis on a SDS 15% polyacrylamide g e l . Lane 1, HeLa histone deacetylase complex prepared using a Bio-Gel A-50m column e q u i l i b r i a t e d with 2.0 M NaCl; 2, HeLa histones.  65  against buffers of successively lower i o n i c strength as described i n Experimental Procedures.  Butyrate was removed from the reconstituted  material by d i a l y s i s and histone deacetylase a c t i v i t y was assayed as before.  Equal aliquots of [ H ] - a c e t y l - l a b e l l e d chromatin were incubated 3  with buffer alone to serve as controls.  It was found that 15% of l a b e l l e d  acetyl groups were released from the reconstituted histones.  Thus at least  some histone deacetylase remains bound to chromatin i n 2 M NaCl, strongly suggesting that the major interactions involved are non-ionic i n nature.  V.  Effect of Nuclease Digestion on Histone Deacetylase D i s t r i b u t i o n  HeLa n u c l e i were usually digested with micrococcal nuclease for 5 minutes as described i n Experimental Procedures.  In order to determine  whether the degree of nuclease digestion affected the d i s t r i b u t i o n of histone deacetylase i n chromatin, several experiments were performed.  a.  Reduced Nuclease Digestion  The p o s s i b i l i t y existed that the lack of observable histone deacetylase a c t i v i t y on mononucleosomes could have been due to extensive nuclease digestion or the presence of i n h i b i t o r y proteins, such as HMG proteins which can i n h i b i t nuclear histone deacetylase a c t i v i t y in_ v i t r o (188).  However, when n u c l e i were digested less extensively,  d i s t r i b u t i o n of histone deacetylase remained the same.  the  For example, a 30  second digest under the same conditions released only 15% as much DNA as did a 5 minute digestion and f r a c t i o n a t i o n of the products of a 30 second micrococcal nuclease digestion of l a b e l l e d HeLa nuclei on a Bio-Gel A-50m  66  Figure 13.  Effect of reduced nuclease digestion on the d i s t r i b u t i o n of  histone deacetylase.  Labelled HeLa n u c l e i were digested with micrococcal nuclease  (2.5  units of enzyme/A2kQ of n u c l e i ) for 30 seconds at 25°C and 15% as much DNA was released as i n a normal 5 minute d i g e s t i o n .  The digest products were  separated using a Bio-Gel A-50m column containing 0.4 M NaCl and 50 mM butyrate.  Fractions were assayed for histone deacetylase a c t i v i t y upon  removal of the butyrate and incubated for 4 hours at 22°C. [ H]-acetate was extracted and counted. 3  [ H]-acetate, CPM released ( x l O ) . 3  - 3  (  ) ^60'  (•  Free *)  67  column containing 0.4 M NaCl to dissociate any HMG proteins (Figure 13) produced a histone deacetylase p r o f i l e that was v i r t u a l l y i d e n t i c a l to that of Figure 10A.  A l l of the histone deacetylase a c t i v i t y was present i n the  histone deacetylase complex i n the f i r s t peak and no a c t i v i t y was detected i n oligonucleosomes or mononucleosomes.  The  P °fil r  e  w  a  s  also very  similar to that seen for the standard nuclease digestion, except that the y i e l d was lower.  b.  Redigestion of Histone Deacetylase Complex  The effect of extensive micrococcal nuclease digestion on the deacetylase d i s t r i b u t i o n within chromatin was examined i n the following experiment.  Endogenous a c e t y l - l a b e l l e d HeLa nuclei were digested with  micrococcal nuclease under standard conditions and the digest products were separated on a Bio-Gel A-5m column which was used i n place of a Bio-Gel A-50m column for speed and convenience as greater flow rates could be achieved.  It should be noted that i n contrast to the Bio-Gel A-50m  p r o f i l e , the histone deacetylase a c t i v i t y i s d i s t i n c t l y skewed to the high molecular weight side of the excluded absorbance peak from a Bio Gel A-5m column (Figure 14A).  The fractions containing histone deacetylase were  pooled after aliquots had been assayed.  The pooled material was made 1 mM  i n C a C l , 1 mM i n MgCl and 2.5 mM i n KC1 by the addition of lOOx 2  solution.  2  The pooled material was then redigested with micrococcal  nuclease (10  units of enzyme/A ^Q of deacetylase f r a c t i o n at 15°C  for 10 minutes).  The nuclease digestion was stopped by the addition of  2  M EGTA to a f i n a l concentration of 1 mM and the redigested deacetylase complex was reapplied to the same column.  histone  Fractions were  0.1  68  68a  Figure 14.  Polynucleosomes redigested with micrococcal nuclease r e t a i n t h e i r endogenous histone deacetylase a c t i v i t y .  A Bio-Gel A-5m column equilibrated with 50 mM butyrate was used to separate endogenous l a b e l l e d chromatin fragments.  Fractions of 1 ml were  collected and dialyzed to remove the butyrate and the histone deacetylase a c t i v i t y was detected by extracting and measuring the free  [ H]-acetate 3  released from the endogenous histones after a 4 hr incubation at 22°C. ( (A)  ) A  2 6 Q  ;  (•  •) [ H]-acetate, CPM released (xl0~ ) . 3  3  A c e t y l - l a b e l l e d HeLa nuclei were p a r t i a l l y digested with micrococcal  nuclease and the endogenous l a b e l l e d chromatin fragments were isolated by column chromatography.  The polynucleosome fractions containing histone  deacetylase were pooled after aliquots had been assayed.  (B)  The  polynucleosomes pooled i n A were redigested with micrococcal nuclease ^260  u n :  *-  t s  ° f enzyme/A ^Q of polynucleosomes) at 15°C for 10 min, 2  reapplied to the column and assayed i n the same way.  (10  assayed for histone deacetylase a c t i v i t y upon the removal of butyrate as before, and this p r o f i l e i s shown i n Figure 14B.  The absorbance and  histone deacetylase a c t i v i t y present i n the pooled fractions from the i n i t i a l digestion were recovered quantitatively i n the fractions from the second digestion.  The most obvious feature of the redigested material was  the s h i f t i n the r e l a t i v e positions of the peaks.  and histone  deacetylase  The two p r o f i l e s were not coincident i n the i n i t i a l digestion,  histone deacetylase a c t i v i t y being ahead of the absorbance peak;  the  however,  the a c t i v i t y and the absorbance peaks did coincide after redigestion of the complex under the above conditions.  The s p e c i f i c r a d i o a c t i v i t y of the  histones i n the mononucleosomes produced by redigestion of the l a b e l l e d deacetylase complexes was 3.2 times higher than that of the mononucleosomes produced i n the i n i t i a l digestion.  In spite of t h i s , no release of  [ H]-acetate from mononucleosomes was detected. 3  c.  Extensive Nuclease Digestion  Extensive nuclease digestions were also performed on whole n u c l e i i n order to study t h e i r effects on histone deacetylase d i s t r i b u t i o n . [ H ] - a c e t y l l a b e l l e d HeLa nuclei were divided into 2 equal aliquots and 3  one was p a r t i a l l y digested with micrococcal nuclease (2.5 enzyme/A2gQ of nuclei for 5 minutes at 2 5 ° C ) .  units of  The resulting digest was  fractionated on a Bio-Gel A-5m column and samples were assayed for histone deacetylase as shown i n Figure 15A.  The other aliquot of nuclei was  digested with 3 times the amount of micrococcal nuclease for 15 minutes at 25°C.  This digest was then treated i n the same way as the f i r s t aliquot  and the data are shown i n Figure 15B.  70a  Figure 15.  Effect of the extent of nuclease digestion on the d i s t r i b u t i o n of histone deacetylase i n chromatin.  Equal aliquots of HeLa n u c l e i containing  [ H]-acetyl-labelled 3  histones were digested to different extents with micrococcal nuclease. The digests were fractionated on Bio-Gel A-5m columns containing 50 mM butyrate.  Fractions of 1 ml were collected and dialyzed to remove  butyrate, and histone deacetylase a c t i v i t y was determined by incubating dialyzed fractions f o r 4 hr at 22°C and measuring released [ H]-acetate. 3  ( (xlO  (  ), lower curve, ^ 6 0 ' 3  ).  (A) (B)  ^* ~ ~  [ H]-acetate released (%); 3  [ H]-acetate, CPM released 3  Labelled n u c l e i were p a r t i a l l y digested with micrococcal  nuclease (2.5 ^260 25°C.  ) , upper curve,  u  n  i  t  s  ° f enzyme/A2^Q of nuclei) for 5 min. at  Labelled nuclei were extensively digested with micrococcal  nuclease (7.5  n n  ^-  t s  ° f enzyme/A^Q of nuclei) for 15 min at 2 5 ° C .  71  The d i s t r i b u t i o n of histone deacetylase a c t i v i t y i n the digest prepared by routine conditions (Figure 15A) was the same as for previously described experiments.  More extensive nuclease digestion resulted i n the  production of very large peaks of mononucleosomes and nucleotides but the histone deacetylase a c t i v i t y remained confined to the i n i t i a l peak. ^260  a n <  *  e  n  z  v  m  e  The  a c t i v i t y p r o f i l e s i n the polynucleosome peak were  coincident, as was the case for redigested polynucleosomes (Figure 14B) and for  the excluded peaks from Bio-Gel A-50m columns (Figure 10). A comparison of histone deacetylase a c t i v i t y between complexes  prepared by normal versus extensive digestion yielded the following results.  If the percent of [ H]-acetate released was used for 3  comparisons of enzyme a c t i v i t y , histone deacetylase complex prepared by normal digestion released 44.2% of t o t a l [ H]-acetyl groups whereas 3  complexes prepared by extensive digestion released 26.8% of t o t a l [ H]-acetyl groups, implying that some histone deacetylase had been l o s t 3  during digestion.  If enzyme a c t i v i t y was expressed as  cpm [ H]-acetate 3  released/A2gQ, i t was found that deacetylase complexes prepared by normal digestion released 2.5 x 10  s  cpm/A^^,  extensive digestion released 3.3 x 10  6  whereas those prepared by c p m / A „ , , an increase of zou n  approximately 30%. Mononucleosomes resulting from extensive digestion had a higher s p e c i f i c r a d i o a c t i v i t y than mononucleosomes prepared by normal digestion, but no endogenous histone deacetylase a c t i v i t y was observed. It can be seen from the inserts i n Figures 15A and 15B showing the percent of [ H]-acetate released across the deacetylase peak that the 3  complexes were heterogeneous with respect to histone deacetylase a c t i v i t y after normal digestion, and remained that way after more extensive  72  digestion.  A s i m i l a r heterogeneity was observed after redigestion of  deacetylase complexes, or with complexes isolated from Bio-Gel A-50m columns.  VI  Characteristics of Chromatin-Bound Histone Deacetylase  The high rate at which endogenous histone deacetylase cleaves acetyl groups from hyperacetylated histones necessitated the development of p a r t i c u l a r assay procedures i n order that enzyme c h a r a c t e r i s t i c s could be studied.  For routine assays,  [ H]-acetyl l a b e l l e d deacetylase 3  complexes  were precipitated on ice with 100 mM NaCl i n the presence of 50 mM butyrate as described i n d e t a i l i n Experimental Procedures.  Assays were started as  soon as the complexes were redissolved i n butyrate-free buffer.  The  p r e c i p i t a t i o n i n 100 mM NaCl was necessary to separate the histone deacetylase complex from the butyrate.  a.  Time Course  A time course for chromatin bound histone deacetylase i s shown i n Figure 16.  F i f t y percent of the [ H]-acetyl groups were released within  3 minutes.  This very rapid i n i t i a l release of acetyl groups was followed  3  by a more gradual release over the next 2-3 hours.  A small percentage of  chromatin bound [ H]-acetyl groups remained even after 15 hours of 3  incubation.  Acid hydrolysis of the a c e t y l - l a b e l l e d histones rendered a l l  of the l a b e l v o l a t i l e , indicating that i t was present as acetyl groups and that none had entered the polypeptide backbone during l a b e l l i n g . l i k e l y that the remaining [ H]-acetyl groups represent a-N-acetyl 3  It  is  73a  Figure 16.  Time course of chromatin-bound histone deacetylase  Histone deacetylase complex containing a c e t y l - l a b e l l e d histones was prepared from l a b e l l e d HeLa nuclei by p a r t i a l digestion with micrococcal nuclease, precipitated with 100 mM NaCl containing 50 mM butyrate, and taken up i n butyrate-free buffer at 22°C. b o i l i n g and the released  The reaction was stopped by  [ H]-acetate was extracted and counted. 3  74  groups of histones HI, H2A and H4 which are known to be metabolically stable.  An assay time of 3 minutes was chosen for further studies on the  enzyme, t h i s being a convenient point within the l i n e a r portion of the time course.  b.  Effect of Assay Volume on the Deacetylase Reaction  The question of whether histone deacetylase encounters the nucleosomal histone substrate by reversibly dissociating from the high molecular weight complex or remains attached to i t , was investigated i n the following manner.  P r e c i p i t a t e d , endogenous labelled deacetylase complexes were taken  up and assayed i n different volumes of buffer (Table 4).  Although a 30  fold difference i n assay volumes was employed, the release of [ H ] 3  acetate over a 3 minute period was e s s e n t i a l l y unaltered i n repeated experiments.  Therefore, d i l u t i o n had no s i g n i f i c a n t effect on histone  deacetylase a c t i v i t y , implying that the enzyme does not reversibly dissociate from i t s substrates during the course of the reaction.  VII  Effect of HMG 14 and HMG 17 on Histone Deacetylase  The addition of high mobility group (HMG) proteins 14 and 17 to Friend c e l l nuclei causes an i n h i b i t i o n of histone deacetylase a c t i v i t y when free a c e t y l - l a b e l l e d histones are used as the substrate (188).  Histone  deacetylase complex appeared to be a suitable system for investigating the effects of HMG 14 and HMG 17 on the enzyme.  75  Table 4  Effect of assay volume on histone  Assay Volume (pi)  deacetylase  [ H]-Acetate Released (%) 3  50  557*5  500  53.9  1500  56.1  Endogenously labelled histone deacetylase  complex was prepared by p a r t i a l  micrococcal nuclease digestion of labelled HeLa n u c l e i .  The histone  deacetylase complex was precipitated i n 100 mM NaCl containing 50 mM butyrate, taken up i n different volumes of butyrate-free buffer, and histone deacetylase a c t i v i t y was assayed for 3 minutes at 22°C.  The  reaction was stopped by b o i l i n g for 1 minute and the released labelled acetate was extracted and counted.  Each reaction contained 54,000 cpm.  76  a.  Amino Acid Analysis of HMG 14 and HMG 17  As an accurate estimate of the concentration of HMG 14 and HMG 17 present i n histone deacetylase assays was e s s e n t i a l ,  these proteins were  isolated from c a l f thymus and hydrolyzed i n 6 N HC1 as described i n Experimental Procedures.  The hydrolyzate was analyzed on an automatic  amino acid analyzer and the amino acid content i s shown i n Table 5. corrections were made for hydrolytic losses.  No  The preparation of c a l f  thymus HMG 14 and HMG 17 displayed the c h a r a c t e r i s t i c amino acid composition of the small HMG proteins namely, large amounts of l y s i n e , glutamate, alanine and proline with only trace amounts of the hydrophobic amino acids and no cysteine.  Comparison with the amino acid composition of  c a l f thymus HMG 14 (192) and HMG 17 (193) and examination of the HMG proteins by SDS polyacrylamide gel electrophoresis  showed that HMG 14 and  HMG 17, isolated from c a l f thymus by ammonium sulphate precipation, were present i n a r a t i o of approximately 1:8 (mol/mol).  This sample was used as  a standard for comparative SDS polyacrylamide gel electrophoresis of different c a l f thymus and HeLa protein preparations.  Preparation of HMG  proteins with ammonium sulphate involves the sequential p r e c i p i t a t i o n of HMG proteins with increasing concentrations of ammonium sulphate Experimental Procedures).  (see  It was found that a reduction i n the ammonium  sulphate concentration from 55% to 50% during the f i r s t p r e c i p i t a t i o n to remove HMG 1 and HMG 2 resulted i n an increased y i e l d of HMG 14; however, large amounts of HMG 1 and HMG 2 were also present.  Figure 17 shows the  SDS polyacrylamide gel p r o f i l e of a t y p i c a l preparation of HMG 14 and HMG 17.  Amino A c i d  C a l f Thymus HMG 14 + 17 P r e p a r e d by Ammonium:Sulphate Precipitation  C a l f Thymus HMG 14  C a l f Thymus HMG 17  C a l f Thymus HMG 14+17 (1:8 r a t i o )  Asp  9.5  8.3  12.0  11.3  Thr  3.9  4.1  1.2  1.8  Ser  3.5  8.0  2.3  3.4  Glu  10.9  17.2  10.5  11.9  Pro  12.1  8.1  12.9  11.9  Gly  9.4  6.4  11.2  10.2  Ala  17.8  14.6  18.4  17.7  0  0  Cys  o  0  Val  2.5  4.0  2.0  2.4  Met  trace  0.1  trace  trace  lieu  trace  0.3  trace  trace  Leu  1.4  2.0  1.0  1.2  Tyr  trace  0.2  trace  trace  Phe  trace  0.3  trace  trace  His  0.1  0.2  trace  trace  Lys  23.9  .21.1  24.3  23.6  Arg  5.0  5.4  4.1  4.4  77a  Table 5  Amino acid composition of i s o l a t e d HMG proteins  HMG 14 and HMG 17 were isolated from c a l f thymus by ammonium sulphate p r e c i p i t a t i o n and the amino acid composition was determined as described i n Experimental Procedures.  No corrections were made for  hydrolytic losses. The amino acid compositions of c a l f thymus HMG 14 and HMG 17 have been published (192,193) and are included i n the Table. Since the amino acid composition of the isolated c a l f thymus HMG proteins i s from a mixture of HMG 14 and HMG 17 i n about a 1:8 mass r a t i o (Figure 17), approximately comparable amino acid values for this mixture have been l i s t e d .  78  Figure 17.  SDS polyacrylamide gel electrophoresis  of HMG 14 and HMG 17.  HMG 14 and HMG 17 were prepared from c a l f thymus nuclei and HeLa nuclei by extraction with 2% t r i c h l o r o a c e t i c acid and p r e c i p i t a t i o n with ammonium sulphate as described under Experimental Procedures. HMG 14 and HMG 17 were separated by column chromatography (183) standards.  Calf thymus and used as  Electrophoresis was carried out i n a gel containing 15%  acrylamide which was stained with Coomassie Blue.  Lane 1, HeLa core  histones; 2, c a l f thymus HMG 14; 3, c a l f thymus HMG 17; 4, c a l f thymus HMG 14 and HMG 17; 5, HeLa HMG 14 and HMG 17.  Table 6.  Effect of HeLa HMG 14 and HMG 17 on histone deacetylase.  HMG 14 a n d HMG 17 nucleosome r a t i o (mol/mol)  .0  [^-Acetate (%)  46.8  •1  48.0  2  46.5 45.2  .3  Released  Endogenous l a b e l l e d histone deacetylase complex was prepared by p a r t i a l micrococcal nuclease digestion of l a b e l l e d HeLa n u c l e i , and precipitated i n 400 mM NaCl containing 50 mM butyrate.  The HMG protein  depleted histone deacetylase complex was, taken up i n butyrate-free buffer containing d i f f e r e n t concentrations of HeLa HMG 14 and HMG 17, and histone deacetylase a c t i v i t y was assayed for 3 min at 22°C.  The reaction was  stopped by b o i l i n g for 1 minute and the released labelled acetate was extracted and counted.  Each reaction contained 90,000 cpm.  80  b.  Effect of HeLa HMG 14 and HMG 17 on Histone Deacetylase  The effect of HeLa HMG proteins 14 and 17 was investigated i n several experiments.  F i r s t , endogenous l a b e l l e d complexes were precipitated i n  either 100 mM NaCl, as usual, or 400 mM NaCl, i n order to determine whether the release of non-histone chromatin proteins (e.g. high mobility group proteins 14 and 17) would affect histone deacetylase a c t i v i t y .  Complexes  precipitated i n 100 mM NaCl released 53% of [ H]-acetyl groups i n 3 3  minutes compared to 51% for those precipitated i n 400 mM NaCl. P r e c i p i t a t i o n i n 400 mM NaCl therefore had no significant effect on histone deacetylase a c t i v i t y . A l t e r n a t i v e l y , the effect of the addition of HeLa HMG 14 and HMG 17 to HMG-depleted HeLa histone deacetylase complex was investigated. the very rapid deacetylation of histones,  In view of  the histone deacetylase assays  were carried out for 3 minutes at 4°C on samples of endogenous l a b e l l e d histone deacetylase complex which had been collected by p r e c i p i t a t i o n i n 0.4M NaCl.  The samples of precipitated deacetylase complex were taken up  i n butyrate-free buffer containing varying concentrations of HeLa HMG 14 and HMG 17.  The addition of HeLa HMG 14 and HMG 17 to HeLa histone  deacetylase complex at a HMG:nucleosome r a t i o of 1 to 3:1 (mol/mol) had no effect on histone deacetylase a c t i v i t y (Table 6).  c.  Effect of Calf Thymus HMG 14 and HMG 17 on Histone Deacetylase  The p o s s i b i l i t y existed that only a f r a c t i o n of the HMG proteins were i n solution or i n t h e i r correct conformation and so greatly increased concentrations of HMG proteins were used.  The limited y i e l d of these  81  0  1  0  1  1  1  1  1000  500  H M G : nucleosome ( m o l / m o l )  Figure' 18.  E f f e c t of c a l f thymus HMG 14 and HMG 17 on histone  deacetylase  activity.  Endogenous l a b e l l e d histone deacetylase complex was prepared by p a r t i a l digestion of HeLa n u c l e i with micrococcal nuclease, and equal aliquots were c o l l e c t e d by p r e c i p i t a t i o n i n 0.4 M NaCl containing 50 mM butyrate.  The HMG  protein depleted histone deacetylase complex was taken up i n butyrate-free buffer containing d i f f e r e n t concentrations of c a l f thymus HMG 14 and HMG 17, and deacetylase a c t i v i t y was assayed for 3 minutes at 4 ° C . The reaction was stopped by b o i l i n g for 1 minute and released [ H]-acetate was extracted and 3  counted.  Each reaction contained 80,000 cpm.  82  proteins from HeLa c e l l s necessitated  the use of c a l f thymus nuclei as the  source of HMG proteins. Endogenous l a b e l l e d histone deacetylase complex was prepared from HeLa nuclei by p a r t i a l digestion with micrococcal nuclease and aliquots were precipitated i n 0.4 M NaCl containing 50 mM butyrate.  The concentration of  nucleosomes i n samples of complex was determined by measuring the absorbance at 260 nm.  The HMG-depleted deacetylase complex was taken up i n  butyrate-free buffer containing different concentrations of c a l f thymus HMG 14 and HMG 17, and histone deacetylase a c t i v i t y was assayed for 3 minutes at 4°C .  The presence of c a l f thymus HMG 14 and HMG 17 at very much higher  concentrations caused an increase i n the release of [ H]-acetate (Figure 3  18).  As the histone deacetylase assays contained a wide range of  concentrations of c a l f thymus HMG 14 and HMG 17, the protein concentration was kept constant by the addition of BSA, which had no effect on histone deacetylase a c t i v i t y .  The increase i n the release of [ H]-acetate was 3  only observed when the HMG:nucleosome r a t i o was 500:1 (mol/mol) or greater, and therefore, disruption of nucleosome structure resulting i n increased a c c e s s i b i l i t y of the histones i s the most l i k e l y reason for an increase i n [ H]-acetate release. 3  A possible explanation for the varying effect of HMG 14 and HMG 17 i s that nuclei contain additional factors which are required to e l i c i t  the  i n h i b i t i o n of histone deacetylase by HMG 14 and HMG 17, and these factors are missing from histone deacetylase complex prepared by microccocal nuclease digestion of HeLa c e l l chromatin.  83  PART B  CHARACTERIZATION OF HISTONE DEACETYLASE COMPLEX  The observation that histone deacetylase was associated with a nuclease r e s i s t a n t , high molecular weight complex prompted investigations into the properties and c h a r a c t e r i s t i c s of t h i s complex.  The recent  findings that chromatin may be organized as large loops of DNA attached to a protein matrix (162) and that the nuclear matrix may serve as the s i t e for  several forms of chromatin processing including r e p l i c a t i o n  (168,169,170), t r a n s c r i p t i o n (175,176) and hormone binding (176,177) thus led  to a comparison of the histone deacetylase complex with the nuclear  matrix.  I.  The Histone Content of Histone Deacetylase Complexes  HeLa nuclei were p a r t i a l l y digested with micrococcal nuclease and the products of digestion were separated using a Bio-Gel A-50m column equilibrated with butyrate.  Appropriate fractions containing histone  deacetylase complex or nucleosomes were precipitated i n - 2 0 ° C ethanol as described i n Experimental Procedures and the histones were extracted by displacement with 15 mM spermine, 6.25 M urea, 0.9 M acetic acid and analyzed by acid urea polyacrylamide gel electrophoresis.  Examination of  the gels shown i n Figure 19 shows that exposure of HeLa c e l l s to 10 mM sodium butyrate for 8 hours results i n extensive hyperacetylation of the histones associated with the histone deacetylase complex.  Although both  histones H3 and H4 exist i n several acetylated forms, positive i d e n t i f i c a t i o n of the bands corresponding to the alternative forms of H3 was extremely d i f f i c u l t due to the presence of non-histone proteins i n the  84  same region of the acid urea g e l s .  For t h i s reason, only the regions of  the gels containing histone H4 were scanned.  The gel scan (Figure 20A)  revealed that tetracetylated H4 was the most abundant class of histone H4, 30% of the t o t a l histone H4 being i n this form.  T r i - , d i - and  monoacetylated H4 accounted for 27%, 23% and 16% of the t o t a l histone H4 respectively.  Very l i t t l e (4%) of histone H4 was unacetylated.  Comparison  of t h i s gel pattern with those of bulk histones from HeLa c e l l s exposed to butyrate (78) shows that the histones of the deacetylase complex from butyrate-treated c e l l s were acetylated to a greater degree than bulk histones from the same c e l l s . Examination of the histones from deacetylase complexes of c e l l s which had not been exposed to butyrate also showed H4 to be hyperacetylated r e l a t i v e to bulk H4 from the same c e l l s .  The corresponding acid urea gel  i s shown i n Figure 19A and the o p t i c a l scan i n Figure 20B.  The histones of  the o l i g o - and mononucleosomes produced along with histone  deacetylase  complex during the micrococcal nuclease digestion of HeLa c e l l n u c l e i were also examined and found to contain acetylated histones.  The major forms of  histone H4 i n the o l i g o - and mononucleosomes were the unacetylated and monoacetylated d e r i v a t i v e s .  A protein which had a s i m i l a r mobility to  tetracetylated histone H4 (arrow, Figure 19) was observed i n nucleosomes but not i n preparations of the histone deacetylase complex.  The molecular  weight of the protein was determined by two-dimensional gel electrophoresis (Figure 21).  The f i r s t dimension was acid urea 15% polyacrylamide (lane 4  of Figure 19A containing the histones from nucleosomes of butyrate-treated HeLa c e l l s ) and the second dimension was SDS 18% polyacrylamide gel electrophoresis.  The protein was c l e a r l y not a histone and had an  approximate molecular weight of 12K.  Samples of spermine and BSA which  85  were used i n the preparation of the histones were applied to a separate lane and found to be free of histone contamination. Histone deacetylase complex containing hyperacetylated histones was prepared from butyrate-treated HeLa c e l l s i n the normal manner and incubated at 22°C for 4 hours after d i a l y s i s to remove the butyrate, according to the standard procedures for assaying histone activity.  deacetylase  The resulting histones were analyzed by acid urea gel  electrophoresis (Figure 19B) and found to contain predominantly unacetylated and monoacetylated H4 (Figure 20C).  Incubation of o l i g o - and  mononucleosomes under the same conditions f a i l e d to a l t e r the degree of histone a c e t y l a t i o n .  These results confirm the conclusions from other  experiments, that histone deacetylase i s associated with a high molecular weight complex. It i s interesting to note the differences i n the positions of several non-histone proteins i n samples of histone deacetylase complex and nucleosomes from butyrate-treated HeLa c e l l s  (Figure 19, lanes 1 and 4 of  panel A, and panel B) and butyrate-untreated HeLa c e l l s  (Figure 19A, lanes  2 and 3), i n p a r t i c u l a r of a protein which migrates behind histone H3. These differences i n mobility during electrophoresis i n acid urea gels are most probably due to changes i n the net charge of the proteins and may r e f l e c t altered patterns of protein modification.  This observation lends  support to the view that butyrate has many effects on c e l l s  (219,220,221,  223,224), although as was shown e a r l i e r , exposure of HeLa c e l l s to butyrate does not a l t e r the d i s t r i b u t i o n of histone deacetylase i n chromatin.  86  Figure 19  Acid urea gel p r o f i l e s of histone deacetylase complex and  nucleosomes  Histones were prepared from histone deacetylase complex and nucleosomes as described i n Experimental Procedures and applied to two acid urea 15% polyacrylamide gels which were run according to the methods of Davie, (182). (A) Lane 1, histone deacetylase complex prepared from butyrate-treated HeLa c e l l s ; 2, histone deacetylase complex prepared from HeLa c e l l s which had not been exposed to butyrate; 3, o l i g o - and mononucleosomes prepared from HeLa c e l l s which had not been exposed to butyrate; 4, o l i g o - and mononucleosomes prepared from butyrate-treated HeLa c e l l s ; 5, bovine serum albumin and spermine which were used i n the preparation of the histones (see Experimental Procedures).  (B) Lane 1, histone deacetylase complex prepared from  butyrate-treated HeLa c e l l s and incubated at 22°C for 4 hours after removal of sodium butyrate using the standard procedures for histone deacetylase assays; 2, o l i g o - and mononucleosomes from butyrate-treated HeLa c e l l s .  87  87a  Figure 20.  Optical scans of histone H4 on acid urea polyacrylamide g e l s .  The acid urea gels shown i n Figure 19 were stained with Coomassie Blue and the portions containing histone H4 were scanned at 550 nm.  The  t e t r a - , t r i - , d i - , mono-, unacetylated forms of histone H4 are separated according to the presence of a c e t y l groups and form a series of bands with the least acetylated forms migrating furthest.  The positions of the  tetraacetylated and unacetylated forms of H4 are marked by Ac4 and AcO respectively.  (A)  Histone H4 from histone deacetylase complex prepared  from HeLa c e l l s which had been exposed to 10 mM butyrate for 8 hours; (B) Histone H4 from histone deacetylase complex prepared from HeLa c e l l s which had not been exposed to butyrate; (C)  Histone H4 from histone  deacetylase complex of butyrate-treated HeLa c e l l s ; the complex was incubated at 22°C for 4 hours upon removal of the butyrate.  88  II  R e s t r i c t i o n Enzyme Analysis of Histone Deacetylase Complex DNA  The DNA associated with the deacetylase complex was found to range from 5-11 Kbp, permitting r e s t r i c t i o n endonuclease analysis to be carried out.  The human genome has a number of s a t e l l i t e DNA components which  produce fragments of c h a r a c t e r i s t i c sizes when digested with p a r t i c u l a r r e s t r i c t i o n endonucleases.  Since the sizes of the fragments created by the  digestion of human s a t e l l i t e s II and III with H a e l l l and EcoRI have been published (194), histone deacetylase complex DNA and t o t a l HeLa c e l l DNA were digested with these enzymes i n order to ascertain i f these  satellite  sequences were s i g n i f i c a n t l y enriched or depleted i n the histone deacetylase complex. recognizes GGCC.  EcoRI recognizes the sequence GAATTC while H a e l l l  Two other r e s t r i c t i o n enzymes which recognize 4bp  sequences were also used:  Mspl which cuts at CCGG and TaqI which has the  recognition s i t e TCGA. DNA was prepared from both the deacetylase complex and HeLa c e l l nuclei by phenol extraction, and digested with the above r e s t r i c t i o n enzymes.  The digestion products were separated by electrophoresis on 0.75%  agarose gels and the results are shown i n Figure 22.  The undigested  genomic DNA (not shown) was considerably larger than 24 Kbp as i t migrated behind the 24 Kbp fragment of H i n d l l l digested lambda DNA.  Approximate DNA  fragment sizes were determined using a TaqI digest of pBR322 and a H i n d l l l digest of lambda DNA as markers.  H a e l l l digestion of either histone  deacetylase complex DNA or genomic DNA resulted i n a smear on which were superimposed several prominent bands.  These bands had approximate sizes of  1170, 1050, 830, 710, 530 and 400 bp and correspond to the fragments produced by H a e l l l digestion of human s a t e l l i t e  III.  89  Figure 21.  Two-dimensional gel electrophoresis  of histones  Butyrate-treated HeLa nuclei were p a r t i a l l y digested with micrococcal nuclease and the digest products were separated on a Bio-Gel A-50m column. Histones were extracted from the oligonucleosomes and examined by acid urea 15% polyacrylamide gel electrophoresis polyacrylamide gel electrophoresis stained with s i l v e r .  i n the f i r s t dimension and SDS 18%  i n the second dimension.  The gel was  The histones are i d e n t i f i e d and the protein with a  molecular weight of approximately 12K i s marked with an arrow.  90  Digestion of histone deacetylase complex DNA and t o t a l DNA with EcoRI also produced a smear and bands of approximately 1720, 1400, 1070 and 780 bp. The largest two were the same size as the fragments produced by EcoRI digestion of human s a t e l l i t e s II and I I I .  The TaqI and Mspl digests of DNA  from histone deacetylase complex and from HeLa nuclei yielded smears with no obvious bands. Close scrutiny of the gel patterns of the H a e l l l and EcoRI r e s t r i c t i o n digests of histone deacetylase complex DNA and HeLa genomic DNA f a i l e d to detect any s i g n i f i c a n t differences i n the proportion of s a t e l l i t e DNAs. The DNA associated with the deacetylase complex i s therefore not noticeably enriched or depleted i n s a t e l l i t e s II and I I I , and appears to be of high complexity.  Ill  Comparison of the Histone Deacetylase Complex and the Nuclear Matrix  Several high molecular weight complexes from nuclei have been reported (163,164,165) and a l l have been found to consist of protein and DNA. One of the most studied of these complexes i s the nuclear matrix, which i s prepared by nuclease digestion of n u c l e i at 0°C followed by extraction with 2.0 M NaCl (164).  Since assays of histone deacetylase require the presence  of histone substrate, the time of DNase I digestion was reduced from 30 to 20 minutes to ensure that s u f f i c i e n t histones were retained.  These  histones remained bound to the nuclear matrix despite repeated extraction with 2 M NaCl.  The existence i n matrix preparations of t i g h t l y bound core  histones which are resistant to extraction with 2 M NaCl has been described i n the l i t e r a t u r e (164).  kbp •23.7 ^9.5 ^6.7 •2.3 -2.0  kbp  1.4 1.3 0.6H 0.4-J 0.3  91a  Figure 22.  R e s t r i c t i o n enzyme digestion of histone deacetylase complex DNA and genomic DNA.  HeLa histone deacetylase complex DNA and HeLa genomic DNA were digested with several r e s t r i c t i o n enzymes.  The digest products were  analyzed by electrophoresis on horizontal slab gels of 0.75% agarose i n Tris-borate buffer (pH 8.3).  These were stained with 5 ug/ml of  ethidium bromide and the DNA bands were v i s u a l i z e d under UV l i g h t . histone deacetylase complex DNA.  (A)  Lane 1, TaqI digest of pBR322; 2,  H a e l l l digest; 3, EcoRI digest; 4, H i n d l l l digest of lambda DNA; 5, TaqI digest of pBR322; 6, Mspl digest; 7, H i n d l l l digest of lambda DNA; 8, TaqI digest; 9, H i n d l l l digest of lambda DNA.  (B) HeLa genomic DNA.  Lane 1, TaqI digest of pBR322; 2, H a e l l l digest; 3, EcoRI digest; 4, TaqI digest; 5, Mspl digest; 6, H i n d l l l digest of lambda DNA.  92  In view of the e a r l i e r findings that HeLa histone deacetylase complex has a high molecular weight and contains proteins and DNA, this complex was compared with the nuclear matrix.  a.  Histone Deacetylase A c t i v i t y of Nuclear Matrix  Nuclear matrix was prepared from [ H]-acetyl labelled HeLa c e l l 3  nuclei i n the presence of 50 mM sodium butyrate.  Aliquots of the labelled  nuclear matrix were taken up i n butyrate-free buffer and incubated at 22°C f o r two hours.  Released [ H]-acetate was extracted with ethyl acetate as 3  usual, and counted.  The commonly used method for preparing nuclear matrix  (164) includes an extraction with 1% T r i t o n X-100.  However, when this step  was included i n the preparation of [ H]-acetate labelled nuclear matrix, 3  no endogenous histone deacetylase a c t i v i t y was detected.  On the other  hand, i f the detergent extraction was omitted, acetate-labelled  nuclear  matrix routinely released 40% of the t o t a l counts present, while i d e n t i c a l samples boiled for 1 minute p r i o r to assaying for enzyme a c t i v i t y only 5%.  released  The protein p r o f i l e s of samples of nuclear matrix prepared either  with or without the T r i t o n X-100 wash were examined by SDS polyacrylamide g e l electrophoresis and were found to be very similar (Figure 23). The procedures for preparing nuclear matrix involve the repeated centrifugation of extracts and at each step the pellet i s recovered for the next extraction.  Ultimately, the f i n a l p e l l e t containing the nuclear  matrix i s obtained.  The p o s s i b i l i t y therefore exists that the  histone  deacetylase a c t i v i t y observed i n the nuclear matrix could have arisen by co-sedimentation of the histone deacetylase complex with large fragments of the nuclear matrix  93  b.  Protein Composition of Histone Deacetylase Complex and Nuclear Matrix  Polyacrylamide-SDS gel patterns of proteins from peak I material (deacetylase complex) and peak I I - I I I material from a Bio-Gel A-50m column are shown i n Figure 24A and B.  Since the p o s s i b i l i t y existed that the  deacetylase complex might be equivalent t o , or associated with, the nuclear matrix, corresponding patterns for the nuclear matrix from HeLa c e l l s , prepared by a standard procedure (164), are also shown.  The molecular  weights of proteins reported to belong to HeLa nuclear matrix have been published (163) and those with molecular weights of 43K, 45K, 51K, 54K, 56K and 60K were observed i n the gel patterns of both the nuclear matrix and histone deacetylase complex.  It i s seen that both the histone deacetylase  complex and polynucleosome peak contain a large variety of proteins.  non-histone  Several of the major components are found i n both the histone  deacetylase complex and nucleosome fractions as well as i n the nuclear matrix (indicated by dots i n Figure 24).  The peak I histone deacetylase  complex i s enriched i n some components, and depleted i n others r e l a t i v e to the included polynucleosomes from the column. Because of the complexity of the patterns, and the lack of complete correspondence between the proteins of the deacetylase complex and those of the nuclear matrix, i t i s not possible to establish f rom these results whether or not histone deacetylase i s associated with the nuclear matrix. This i s not s u r p r i s i n g , since there i s as yet no concensus as to which of the many components seen i n nuclear matrix preparations are s p e c i f i c these structures.  to  Furthermore, nuclear matrix i s prepared by extensive  digestion of the nuclear DNA with DNase I , whereas i n the preparation of the histone deacetylase complex, some DNA and histones must be retained,  94  Figure 23.  SDS polyacrylamide gel of HeLa nuclear matrix preparations  Nuclear matrix was prepared from HeLa nuclei by DNase I and extraction with 2 M NaCl as described i n Experimental Procedures.  The effect of  extracting nuclear matrix preparations with Triton X-100 was examined by comparing the protein p r o f i l e s of HeLa nuclear matrix prepared with and without extraction with this detergent.  SDS 15% polyacrylamide gel  electrophoresis was c a r r i e d out using the standard discontinuous system.  buffer  Lane 1, nuclear matrix prepared without exposure to T r i t o n X-100;  2, nuclear matrix extracted with 1% Triton X-100; 3, HeLa histones.  95  since t h i s i s a precondition for the detection of the i n s i t u activity.  It i s possible, indeed l i k e l y , that the  deacetylase  deacetylase-containing  material comigrates on the column with other large structures derived from the nucleus, e . g . ,  fragments of the nuclear matrix.  Thus, further work  w i l l be required to e s t a b l i s h unambiguously whether histone deacetylase  is  attached to the nuclear matrix.  IV  Effect of fi-mercaptoethanol and Neocuproine on the Histone Deacetylase Complex  Since copper chelation with 3 mM neocuproine or treatment with C-mercaptoethanol causes the d i s s o c i a t i o n of histone depleted chromosome scaffolds  (165), the effect of these reagents on the histone  deacetylase  complex was investigated.  a.  Histone Deacetylase A c t i v i t y of Treated Complex  [ H ] - a c e t y l labelled histone deacetylase complex was prepared from 3  l a b e l l e d HeLa c e l l nuclei i n the presence of 50 mM sodium butyrate by p a r t i a l micrococcal nuclease digestion and chromatography using a Bio-Gel A-50m column.The pooled fractions of histone deacetylase complex were divided into equal a l i q u o t s , treated as outlined i n Table 7, and assayed for histone deacetylase a c t i v i t y upon removal of sodium butyrate by dialysis.  A p a r a l l e l set of untreated samples served as a control to  determine the normal l e v e l of enzyme a c t i v i t y . 208,000  Each assay released  cpm, representing 48% of the t o t a l present.  The presence of  B-mercaptoethanol at either 1 mM or 10 mM caused a strong i n h i b i t i o n of the  96a  Figure 24.  SDS-polyacrylamide gel p r o f i l e s of fractions from a Bio-Gel A-50m column  The gels were run according to Laemmli (1970), and were stained with Coomassie blue.  A, 15% g e l ; B, 10% g e l .  Lanes 1, high molecular weight  markers (205K, 135K, 97.4K, 67K and 43K daltons); 2, low-molecular weight markers (97.4K, 67K, 43K, 30K, 20.IK and 14.4K daltons); 3, nuclear matrix proteins; 4, polynucleosomes from peak I material; 5, peak I I - I I I material; 6, HeLa histone markers. Figure 9. dots.  Column regions pooled are defined i n  Major bands which correspond i n lanes 3, 4 and 5 are marked by  97  endogenous h i s t o n e d e a c e t y l a s e i n the complex. whether t h i s i n h i b i t i o n was  r e v e r s i b l e , samples o f l a b e l l e d h i s t o n e  d e a c e t y l a s e complex were made 10 mM B-mercaptoethanol was butyrate.  In order to e s t a b l i s h  i n J3-mercaptoethanol  as b e f o r e , but  o m i t t e d d u r i n g t h e d i a l y s i s used t o remove sodium  I n t h i s manner, t h e h i s t o n e d e a c e t y l a s e complex was  10 mM J3-mercaptoethanol,  but none was  A l t h o u g h t h e enzyme a c t i v i t y was where B-mercaptoethanol was  exposed t o  p r e s e n t d u r i n g t h e enzyme a s s a y .  s l i g h t l y h i g h e r than i n the p r e v i o u s case  p r e s e n t d u r i n g the a s s a y , the a c t i v i t y remained  s u b s t a n t i a l l y l o w e r t h a n i n the u n t r e a t e d c o n t r o l a s s a y s , o n l y 18% o f t h e t o t a l counts b e i n g r e l e a s e d .  T h e r e f o r e i t appears from these r e s u l t s t h a t  t h e i n h i b i t i o n o f h i s t o n e d e a c e t y l a s e by J3-mercaptoethanol  i s irreversible.  Neocuproine i s a s t r o n g copper i o n c h e l a t o r (195) and i n o r d e r to observe i t s e f f e c t on h i s t o n e d e a c e t y l a s e a c t i v i t y , samples o f t h e [ H ] - a c e t y l l a b e l l e d complex were d i a l y z e d a g a i n s t b u f f e r A c o n t a i n i n g 3 3  mM neocuproine  f o r 2 hours a t 4°C and assayed.  As c o n t r o l s , e q u a l a l i q u o t s  of t h e complex were t r e a t e d i n t h e same manner, but w i t h o u t the a d d i t i o n o f neocuproine.  The  r e s u l t s i n Table 7 c l e a r l y show t h a t exposure o f the  h i s t o n e d e a c e t y l a s e complex t o 3 mM neocuproine  causes a s t r o n g i n h i b i t i o n  o f h i s t o n e d e a c e t y l a s e a c t i v i t y , a p p r o x i m a t e l y e q u a l to t h a t caused by fl-mercaptoethanol. The i n h i b i t i o n by C-mercaptoethanol c o u l d have o c c u r r e d as a r e s u l t o f i n a c t i v a t i o n o f the enyzme o r by d i s r u p t i o n o f the h i s t o n e d e a c e t y l a s e complex.  The e f f e c t o f B-mercaptoethanol on enzyme a c t i v i t y was  examined  f u r t h e r by p r e p a r i n g h i s t o n e d e a c e t y l a s e complex from u n l a b e l l e d HeLa c e l l n u c l e i and u s i n g f r e e a c e t a t e - l a b e l l e d h i s t o n e s as the s u b s t r a t e . the chromatin-bound h i s t o n e d e a c e t y l a s e r e a c t s most e f f i c i e n t l y w i t h  Although  98  Table 7  The Effect of 3-mercaptoethanol and neocuproine on histone deacetylase  complex.  Treatment of histone deacetylase complex p r i o r to d i a l y s i s to remove butyrate  Treatment of histone deacetylase complex during d i a l y s i s to remove butyrate  None  None  48  1 mM 3-mercaptoethanol  1 mM 3-mercaptoethanol  14  10 mM P-mercaptoethanol  10 mM 3-mercaptoethanol  14  10 mM 3-mercaptoethanol  None  18  Dialyzed for 2 h against buffer A containing 3 mM neocuproine  3 mM neocuproine  15  None  40  Dialyzed for 2 h against buffer A  Equal samples of  H-acetate released (%) (less background)  3  [ H]-acetyl labelled histone deacetylase complex were 3  dialyzed against buffer B (10 mM T r i s - H C l pH 7.8,  20 mM NaCl) to remove  butyrate after either being modified or dialyzed against buffer A (10 mM T r i s - H C l pH 7.4,  0.7 mM EDTA, 50 mM sodium butyrate).  assayed for histone deacetylase a c t i v i t y . t r i p l i c a t e each time.  They were then  The assays were performed i n  The background release of [ H]-acetate from 3  labelled histone deacetylase complex was measured by b o i l i n g samples for 1 minute p r i o r to d i a l y s i s to remove butyrate and assaying for histone deacetylase. present.  This background was found to be 9% of the t o t a l counts  99  h i s t o n e s i n the form of nucleosomes, f r e e h i s t o n e s a r e d e a c e t y l a t e d a t a detectable rate. 1.5  ml a l i q u o t s of u n l a b e l l e d h i s t o n e d e a c e t y l a s e complex were  i n c u b a t e d w i t h 15 o r without of  10 mM  u l (30,000  cpm)  fi-mercaptoethanol,  of f r e e l a b e l l e d h i s t o n e e i t h e r as shown i n Table 8.  Although  o n l y 8% of the t o t a l counts p r e s e n t  r e l e a s e d , t h e r e was  the  radioactivity.  i n the added h i s t o n e s  was  no d i f f e r e n c e i n h i s t o n e d e a c e t y l a s e a c t i v i t y between  assays c o n t a i n i n g B-mercaptoethanol and  those f r e e of the r e d u c i n g  agent.  B-mercaptoethanol thus seems to a f f e c t the i n t e r a c t i o n between h i s t o n e  d e a c e t y l a s e and  b.  samples  h i s t o n e d e a c e t y l a s e complex were b o i l e d p r i o r to i n c u b a t i o n w i t h  l a b e l l e d h i s t o n e , to measure the background r e l e a s e of  The  Parallel  with  chromatin,  r a t h e r than the enzyme i t s e l f .  P h y s i c a l P r o p e r t i e s of T r e a t e d  Advantage was  taken of the v e r y h i g h m o l e c u l a r weight of the  d e a c e t y l a s e complex to perform p o s s i b i l i t y that  Complex  histone  a s e r i e s of simple experiments examining  fi-mercaptoethanol  might d i s r u p t i t s s t r u c t u r e .  In  the  f i r s t experiment, 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 of HeLa c e l l n u c l e i  was  a p p l i e d to a B i o - G e l A-50m column and d e a c e t y l a s e complex) was of  c o l l e c t e d and  the excluded  as shown i n F i g u r e 25A.  peak ( h i s t o n e  r e a p p l i e d to the same column.  the m a t e r i a l which absorbed a t 260 nm was  recovered  i n the excluded  However, when the experiment was  cause  repeated w i t h 10  mM  B-mercaptoethanol i n the b u f f e r d u r i n g the second column run, two were observed  All peak  R e i s o l a t i o n of the h i s t o n e d e a c e t y l a s e complex by  a second passage through a B i o - G e l A-50m column d i d not dissociation.  the  ( F i g u r e 25B).  Again,  q u a n t i t a t i v e r e c o v e r y of the A  peaks 0(;f  .-  100  Table 8  E f f e c t o f B-mercaptoethanol on h i s t o n e  A d d i t i o n s t o the Assays (mM 8-mercaptoethanol)  deacetylase.  [ H]-acetate released (cpm) 3  0  2171  1  2201  10  2145  E q u a l a l i q u o t s o f u n l a b e l l e d h i s t o n e d e a c e t y l a s e complex were i n c u b a t e d added  [ H] - a c e t y l l a b e l l e d h i s t o n e s f o r 2 hours a t 22°C. 3  with  The r e a c t i o n was  stopped by b o i l i n g f o r 1 minute and the f r e e a c e t a t e was e x t r a c t e d and counted.  101  absorbing material was obtained, but the peak corresponding to the histone deacetylase complex was greatly reduced.  The missing material was  accounted for by the second peak, which corresponded to material smaller than mononucleosomes. acetyl-labelled  When the experiment was repeated using endogenous  histone deacetylase complex, no histone deacetylase  a c t i v i t y was detected i n either peak. P a r t i a l digests of HeLa c e l l nuclei with micrococcal nuclease normally yielded a p r o f i l e such as that shown i n Figure 25C when separated on a Bio-Gel A-50m column.  A description of the protein and DNA content across  these column p r o f i l e s has been presented e a r l i e r (Figure 9 and 24) . The presence of 10 mM fi-mercaptoethanol during the preparation of the HeLa c e l l nuclei and the digestion with micrococcal nuclease, but not during the column f r a c t i o n a t i o n , resulted i n an altered column p r o f i l e as seen i n Figure 25D.  There was a pronounced decrease i n the amount of  histone deacetylase complex and a corresponding increase i n the amount of mono- and oligonucleosomes.  Thus, the extent of digestion of the material  normally eluting i n the excluded region of the column was much greater i n the presence of  fi-mercaptoethanol.  altered column p r o f i l e i s that  The most probable explanation for the  fi-mercaptoethanol  disrupts the histone  deacetylase complex, making the associated chromatin more accessible to micrococcal nuclease, which then degrades i t to mono- and oligonucleosomes more r a p i d l y . The protein compositions of HeLa c e l l nuclear matrix, histone deacetylase complex and histone deacetylase complex exposed to 10 mM fi-mercaptoethanol  were compared by SDS polyacrylamide gel  and the gels are shown i n Figure 26.  electrophoresis  The histone deacetylase complex which  was exposed to 10 mM fi-mercaptoethanol was prepared by standard micrococcal  102  n u c l e a s e d i g e s t i o n o f HeLa n u c l e i and f r a c t i o n a t e d on a B i o - G e l A-50m column w i t h o u t any B-mercaptoethanol p r e s e n t .  The r e s u l t i n g h i s t o n e  d e a c e t y l a s e complex was r e a p p l i e d t o a B i o - G e l A-50m column c o n t a i n i n g 10 mM B-mercaptoethanol and t h e f r a c t i o n s c o m p r i s i n g t h e complex were p o o l e d , c o n c e n t r a t e d and p r e c i p i t a t e d as d e s c r i b e d i n E x p e r i m e n t a l  Procedures.  A comparison o f t h e p r o t e i n s p r e s e n t i n t h e i n t a c t h i s t o n e d e a c e t y l a s e complex w i t h t h o s e i n t h e complex w h i c h i s i s o l a t e d from a B i o - G e l A-50m column c o n t a i n i n g 10 mM B-mercaptoethanol r e v e a l s an enrichment o f c o r e histones i n the l a t t e r .  The d i s s o c i a t i o n o f t h e complex by  B-mercaptoethanol t h e r e f o r e seems t o r e s u l t i n a s i g n i f i c a n t l o s s o f non-histone p r o t e i n s .  103  o  <  V o l u me ( m l )  103a  Figure 25.  Fractionation of  fi-mercaptoethanol-treated  histone deacetylase  complex on a Bio-Gel A-50m column.  (A)  Histone deacetylase complex, p u r i f i e d by Bio-Gel A-50m  chromatography, was rerun on a Bio-Gel A-50m column.  (B)  Histone  deacetylase complex was applied to the same column containing 10 mM B-mercaptoethanol.  (C)  HeLa c e l l n u c l e i were p a r t i a l l y digested with  micrococcal nuclease using standard procedures and the histone deacetylase complex and nucleosomes were isolated using a Bio-Gel A-50m column.  (D)  HeLa c e l l n u c l e i were isolated and p a r t i a l l y digested with micrococcal nuclease as i n C, except that 10 mM B-mercaptoethanol was present.  The  histone deacetylase complex and nucleosomes were analyzed on the same column, with no B-mercaptoethanol present.  104  Figure 26.  SDS-polyacrylamide gel p r o f i l e s of histone deacetylase complex.  The 15% gels were run according to Laemmli (181) and were stained with Coomassie Blue.  (A) Lane 1, HeLa histones; 2, HeLa histone deacetylase  complex; 3, HeLa nuclear matrix; 4, low molecular weight markers (97.4 K, 67 K, 43 K, 30 K, 20.1 K and 14.4 K daltons); 5, high molecular weight markers (205 K, 135 K, 97.4 K, 67 K and 43 K daltons). correspond i n lanes 2 and 3 are marked by dots.  Major bands which  (B) Lane 1, low molecular  weight markers; 2, HeLa histone deacetylase complex i s o l a t e d from a Bio-Gel A-50m column containing 10 mM C-mercaptoethanol; 3, HeLa histones.  105  DISCUSSION  The e x p e r i m e n t s d e s c r i b e d i n t h i s t h e s i s have p r o v i d e d i n f o r m a t i o n on the chromatin-bound h i s t o n e d e a c e t y l a s e o f HeLa c e l l s .  The d a t a i n t h e  f i r s t p a r t o f t h e " R e s u l t s " s e c t i o n showed t h a t t h e a c t i v e enzyme i s found o n l y i n a s s o c i a t i o n w i t h a h i g h m o l e c u l a r w e i g h t complex and f i n d i n g s d e s c r i b e d l a t e r showed t h a t t h i s h i s t o n e d e a c e t y l a s e - c o n t a i n i n g complex s h a r e s s e v e r a l o f t h e p r o p e r t i e s o f n u c l e a r m a t r i x and chromosome scaffold. chromatin  The r e l e v a n c e o f these r e s u l t s w i l l be d i s c u s s e d w i t h r e s p e c t t o p r o c e s s i n g and t h e p o s s i b l e f u n c t i o n s o f h i s t o n e a c e t y l a t i o n .  S i n c e t h e c h a r a c t e r i z a t i o n o f h i s t o n e d e a c e t y l a s e and e l u c i d a t i o n o f t h e d i s t r i b u t ion  o f t h e enzyme w i t h i n c h r o m a t i n were d e r i v e d from e x p e r i m e n t s  based upon an a s s a y system u s i n g endogenous l a b e l l e d nucleosomes, t h e h i s t o n e d e a c e t y l a s e a s s a y system w i l l be d i s c u s s e d i n some d e t a i l  I  Histone Deacetylase  The work p r e s e n t e d  first.  Assay  i n t h i s r e p o r t has shown t h a t complexes c o n t a i n i n g  l a b e l l e d h i s t o n e s t o g e t h e r w i t h h i s t o n e d e a c e t y l a s e can be i s o l a t e d i n t h e p r e s e n c e o f b u t y r a t e t o p r o v i d e a system f o r s t u d y i n g c h r o m a t i n histone deacetylase.  bound  The a d d i t i o n o f f r e e h i s t o n e s t o a c e t y l - l a b e l l e d  d e a c e t y l a s e complexes caused a c o n c e n t r a t i o n - d e p e n d e n t  reduction i n  d e a c e t y l a s e a c t i v i t y s u g g e s t i n g t h a t chromatin-bound h i s t o n e i n t e r a c t s non-productively with free histones.  deacetylase  However, when exogenous  h i s t o n e s were added i n t h e form o f mononucleosomes they were e f f i c i e n t a s substrates.  These r e s u l t s e x p l a i n t h e d i f f e r e n t h i s t o n e  deacetylase  a c t i v i t y p r o f i l e s o b t a i n e d on i s o k i n e t i c s u c r o s e g r a d i e n t s o f  chromatin  106  digests when assays are done with either added free a c e t y l - l a b e l l e d histones (Figure 7A) or on endogenous l a b e l l e d nucleosomes (Figure 7B). V i r t u a l l y a l l of the histone deacetylase a c t i v i t y was detected i n rapidly sedimenting material i n gradients containing endogenous l a b e l l e d nucleosomes, wherease gradients assayed with exogenous a c e t y l - l a b e l l e d histones showed histone deacetylase i n slowly sedimenting material, as well as some i n the rapidly sedimenting f r a c t i o n .  This slowly sedimenting  histone deacetylase a c t i v i t y probably represents free enzyme since the peak of a c t i v i t y did not coincide with any nucleosome peak.  Deacetylase  complexes assayed with exogenous l a b e l l e d histones display much lower levels of histone deacetylase a c t i v i t y than endogenous complexes because chromatin bound histone deacetylase interacts non-productively with free histones.  In agreement with this i n t e r p r e t a t i o n , i t has been found that  histone deacetylase s o l u b i l i z e d from c a l f thymus chromatin could deacetylate added free [ H]-acetyl histones, wherease the chromatin-bound 3  histone deacetylase reacted poorly with this substrate (76). A comparison of the assay system presented i n t h i s report with previously described assays for histone deacetylase shows the advantages of using an i n vivo assembled complex of the enzyme and endogenous l a b e l l e d substrate.  Assay systems have been developed i n which enzyme preparations  were incubated with added chemically acetylated histones; however, these histones have been found to be a poor substrate for histone deacetylase because of nonspecific acetylation of amino groups within the histones (70).  Most histone deacetylase assay systems have used added free l a b e l l e d  histones as the substrate, but experiments with the p a r t i a l l y p u r i f i e d a c i d i c histone deacetylase from c a l f thymus have shown that free histones, p a r t i c u l a r l y H3 and H4, bind readily to the a c i d i c protein and maximum  107  p r e c i p i t a t i o n of the histones occurs at a histone deacetylase to histone r a t i o of approximately 1:12 (mol/mol) (74).  The binding of histones to the  chromatin-bound form of histone deacetylase may p a r t i a l l y account for i n h i b i t i o n of enzyme a c t i v i t y observed when free histones were added to samples of histone deacetylase complex (Table 2).  Other histone  deacetylase assays have u t i l i z e d p a r t i a l l y purified forms of the enzyme; however, p u r i f i c a t i o n has resulted i n alterations i n the c h a r a c t e r i s t i c s of the deacetylase.  For example i t has been shown that a crude extract of  c a l f thymus n u c l e i can deacetylate both free histones and chromatin-bound histones, wheareas the p a r t i a l l y p u r i f i e d histone deacetylase prepared by column chromatography could not deacetylate chromatin-bound histones, leading the workers to postulate that the deacetylation of nucleosomes may involve a complex process  (73).  By contrast, histone deacetylase complex contains i n vivo l a b e l l e d histones i n t h e i r physiological conformation with no free histones present and the enzyme remains i n association with chromatin during the preparation and assay of the deacetylase complex.  II  D i s t r i b u t i o n of Histone Deacetylase i n Chromatin  Digestion of HeLa nuclei with micrococcal nuclease produced mononucleosomes and polynucleosomes of up to 50 nucleosomes i n length (Figure 9).  Microccocal nuclease digestion also released a high molecular  weight complex which contained 25-55 nucleosomes worth of DNA (Figure 9). As polynucleosomes of t h i s length were normally resolved i n the included volume of a Bio-Gel A-50m column, these nucleosomes must have been associated with some other high molecular weight material.  Fractionation  108  of the nuclear digests on Bio-Gel A-50m columns containing 2 M NaCl did not a l t e r the  p r o f i l e and this suggests that very strong binding must  e x i s t between the DNA and the histone deacetylase complex.  Another example  of DNA-protein binding which i s stable i n 2 M NaCl i s the attachment of DNA loops to the nuclear matrix (168,171,172,174).  A comparison of the histone  deacetylase complex and the nuclear matrix w i l l be discussed l a t e r . Fractionations of micrococcal nuclease digests of [ H ] - a c e t y l 3  l a b e l l e d n u c l e i on Bio-Gel A-5m and A-50m columns (Figures 8 and 10) have shown that histone deacetylase a c t i v i t y i s associated with a high molecular weight complex and no a c t i v i t y was found on o l i g o - or mononucleosomes.  The  s p e c i f i c r a d i o a c t i v i t y of histones i n the histone deacetylase complex was always greater than that of the nucleosomes i n a given nuclease  digest.  Thus the nucleosomes from these p a r t i a l digests may have been derived primarily from regions of chromatin which were acetylated p r i o r to l a b e l l i n g , and which contained acetyl groups with a low turnover rate. A l s o , examination of the histones from the deacetylase complex and from nucleosomes by acid urea gel electrophoresis showed that histone H4 from deacetylase complex was acetylated to a much greater degree than H4 from nucleosomes.  The l e v e l of l a b e l l e d a c e t y l groups i n monomers and oligomers  was high enough, however, to have allowed the detection of endogenous histone deacetylase a c t i v i t y , had i t been present i n t h i s f r a c t i o n . The f r a c t i o n a t i o n of nuclease digests on Bio-Gel A-50m columns containing different concentrations of NaCl produced the surprising finding that chromatin bound histone deacetylase remains attached to large complexes i n 2 M NaCl.  Histone deacetylase must therefore interact with  chromatin i n quite a different way from acetylases (68) and most non-histone chromosomal proteins which readily dissociate from chromatin i n  109  high ionic strength solutions.  This behavior i s c o n s i s t e n t w i t h the idea  t h a t h i s t o n e d e a c e t y l a s e may be bound t o these complexes t h r o u g h protein-protein interactions. R e d i g e s t i o n of l a r g e chromatin  fragments w i t h m i c r o c o c c a l  caused t h e h i s t o n e d e a c e t y l a s e - c o n t a i n i n g c h r o m a t i n  nuclease  t o s h i f t t o a lower  s i z e d i s t r i b u t i o n , such t h a t t h e a c t i v i t y peak became c o i n c i d e n t w i t h the absorbance peak when t h e d i g e s t p r o d u c t s were s e p a r a t e d  on a B i o - G e l  A-5m  column ( F i g u r e 1 4 ) . The mononucleosomes produced d u r i n g t h e r e d i g e s t i o n o f the a c e t y l - l a b e l l e d complexes had a 3.2 f o l d h i g h e r s p e c i f i c a c t i v i t y t h a n the mononucleosomes formed d u r i n g t h e i n i t i a l d i g e s t i o n o f endogenous labelled nuclei.  Taken t o g e t h e r , these r e s u l t s suggest t h a t m i c r o c o c c a l  nuclease p r e f e r e n t i a l l y d i g e s t s chromatin  l a c k i n g histone deacetylase to  produce [ H ] - a c e t y l l a b e l e d mononucleosomes and n u c l e a s e r e s i s t a n t 3  m a t e r i a l c o n t a i n i n g t h e enzyme. These f i n d i n g s were supported  by comparisons o f p a r t i a l and e x t e n s i v e  micrococcal nuclease d i g e s t s of [ H]-acetate l a b e l l e d n u c l e i .  The  3  mononucleosomes produced d u r i n g an e x t e n s i v e n u c l e a s e d i g e s t i o n had a h i g h e r s p e c i f i c a c t i v i t y t h a n t h e mononucleosomes produced by s h o r t e r nuclease d i g e s t i o n s .  E x t e n s i v e n u c l e a s e d i g e s t i o n e v i d e n t l y caused the  r e l e a s e o f some c h r o m a t i n prepared  bound h i s t o n e d e a c e t y l a s e , s i n c e polynucleosomes  by p a r t i a l d i g e s t i o n r e l e a s e d 44.2% o f t h e i r t o t a l [ H ] - a c e t a t e  groups whereas those prepared  3  by e x t e n s i v e d i g e s t i o n r e l e a s e d o n l y 26.8%.  I f enzyme was r e l e a s e d i t d i d not r e a s s o c i a t e w i t h mononucleosomes s i n c e no d e a c e t y l a s e a c t i v i t y was d e t e c t e d i n t h i s f r a c t i o n , d e s p i t e the h i g h s p e c i f i c a c t i v i t y o f t h e h i s t o n e s i n these mononucleosomes.  Iti s  p o s s i b l e , however, t h a t the r e l e a s e d enzyme i s v e r y u n s t a b l e and becomes i n a c t i v a t e d d u r i n g the f r a c t i o n a t i o n .  When s p e c i f i c a c t i v i t y (cpm [ H ] 3  110  acetate released/A2^Q) was used to express histone deacetylase a c t i v i t y i t was found that histone deacetylase complex prepared by extensive digestion was enriched i n deacetylase a c t i v i t y by approximately 30% r e l a t i v e to those prepared by p a r t i a l nuclease digestion, again implying that micrococcal nuclease cleaves mononucleosomes from histone deacetylase complex to give a nuclease resistant core which i s enriched i n chromatin bound deacetylase.  These results suggest that histone deacetylase  is  protected from nuclease digestion which can cleave nucleosomes from the complex.  A possible binding s i t e for the deacetylase would be close to or  within the protein complex i t s e l f ,  i n association with nucleosomes.  Column fractions containing histone deacetylase complex were heterogeneous with respect to enzyme a c t i v i t y , since the proportion of acetyl groups released varied across the histone deacetylase complex peak (Figure 15).  This hetereogeneity persisted even after extensive nuclease  digestion (Figure 15). The resistance of the nucleosomes associated with the histone deacetylase complex to micrococcal nuclease digestion probably arises from physical constraints or the protein complex which l i m i t a c c e s s i b i l i t y of the nuclease.  This p o s s i b i l i t y i s supported by the observation that the  presence of B-mercaptoethanol, which has been shown to disrupt the deacetylase complex, caused greatly increased digestion of the associated polynucleosomes to y i e l d oligonucleosomes and mononucleosomes under standard nuclease digestion conditions (Figure 25). Histone deacetylase assays measured the release of [ H]-acetate from 3  histones which had been modified i n the presence of butyrate during the 2 hour l a b e l l i n g period i n v i v o .  The p o s s i b i l i t y that exposure of the c e l l s  to butyrate might have caused an a l t e r a t i o n i n the d i s t r i b u t i o n of histone  Ill  deacetylase can be discounted, as chromatin isolated from HeLa c e l l s which had been l a b e l l e d i n the absence of butyrate had exactly the same enzyme d i s t r i b u t i o n (Figure 10).  Examination of the histones from the histone  deacetylase complex by acid urea gel electrophoresis also revealed that the deacetylase complex prepared from HeLa c e l l s which had not been exposed to butyrate likewise contained hyperacetylated histones (Figure 19).  Thus  neither the incorporation of [ H]-acetyl groups into the histones of the 3  deacetylase complex over the 2 hour l a b e l l i n g period nor t h e i r subsequent release from only the high molecular weight complex were influenced by the presence of butyrate.  Ill  Characteristics of Chromatin Bound Histone Deacetylase  The very rapid rate and large extent of deacetylation observed i n the time course studies (Figure 16) showed that denaturation of the chromatin-bound histone deacetylase had been kept to a minimum during the i s o l a t i o n of the complexes.  A very s i m i l a r time course for histone  deacetylase has been observed using c a l f thymus homogenate and added free a c e t y l - l a b e l l e d histones as the substrate (70).  Different populations of  core histones have been shown to be modified with the metabolically active acetate at different rates (13) and, s i m i l a r l y , two d i s t i n c t populations of acetylated histones have been i d e n t i f i e d i n Hepatoma tissue culture c e l l s (180).  One population was characterized by very rapid acetylation and  deacetylation of histones  (t-^^ ^ 3 to 7 min), while the other had a  much slower rate of deacetylation.  The fact that the majority of  [ H]-acetyl groups were released i n 10 minutes shows that most of the 3  chromatin-bound histone deacetylase a c t i v i t y i s interacting with rapidly  112  t u r n i n g o v e r a c e t y l groups i n t h i s system.  However, the slow r e l e a s e o f  a c e t y l groups o v e r the n e x t 2 hours s u g g e s t s t h a t h i s t o n e d e a c e t y l a s e be r e s p o n s i b l e f o r d e a c e t y l a t i n g b o t h p o p u l a t i o n s o f h i s t o n e and d i f f e r e n c e i n r a t e s c o u l d be due preference  may  the  t o a l t e r e d a c c e s s i b i l i t y o r enzyme  f o r c e r t a i n forms o f a c e t y l a t e d h i s t o n e s .  I n these s t u d i e s , a h i g h degree of a c e t y l a t i o n has been observed i n the h i s t o n e H4 a s s o c i a t e d w i t h the h i s t o n e d e a c e t y l a s e complex f r o m b u t y r a t e t r e a t e d HeLa c e l l s ( F i g u r e 1 9 ) .  Thus i n v i v o , the  deacetylase  complex i s l i k e l y to be a s i t e o f v e r y r a p i d a c e t y l group t u r n o v e r ,  a  n o t i o n w h i c h i s c o n s i s t e n t w i t h the r a p i d k i n e t i c s o f d e a c e t y l a t i o n observed i n v i t r o w i t h t h i s m a t e r i a l . The  f i n d i n g t h a t chromatin-bound h i s t o n e d e a c e t y l a s e does not  r e v e r s i b l y d i s s o c i a t e d u r i n g the c o u r s e o f the r e a c t i o n poses the o f how  the enzyme e n c o u n t e r s i t s s u b s t r a t e .  The  two most o b v i o u s  p o s s i b i l i t i e s are that histone deacetylase i s attached t o nucleosomes i n s e c t i o n s of c h r o m a t i n o r t h a t nucleosomes a r e threaded  question  stoichiometrically  w i t h r a p i d a c e t y l group t u r n o v e r ,  p a s t the enzyme i n a manner s i m i l a r t o  t h a t o c c u r r i n g w i t h p r o c e s s i v e enzymes such as n u c l e o t i d e p o l y m e r a s e s . f i r m c h o i c e can be made a t t h i s p o i n t i n f a v o u r of e i t h e r o f  No  these  a l t e r n a t i v e s , a l t h o u g h the former mechanism would r e q u i r e r a t h e r h i g h c o n c e n t r a t i o n s o f enzyme and seems l e s s l i k e l y . h i s t o n e d e a c e t y l a s e may o f the c h r o m a t i n  The s u p p o s i t i o n t h a t the  be a t t a c h e d t o a l a r g e m a t r i x l o c a t e d a t the base  l o o p s would account f o r the o b s e r v a t i o n t h a t the enzyme  does not r e v e r s i b l y d i s s o c i a t e d u r i n g the c o u r s e of i t s r e a c t i o n .  This  would be c o n s i s t e n t w i t h the i d e a t h a t h i s t o n e d e a c e t y l a s e and nucleosomes e n c o u n t e r each o t h e r i n a p r o c e s s i v e manner. M i l l i m o l a r c o n c e n t r a t i o n s o f B-mercaptoethanol were found t o have no  113  effect on histone deacetylase a c t i v i t y when free a c e t y l - l a b e l l e d histones were used as the substrate (Table 8).  A s i m i l a r result has been reported  for c a l f thymus histone deacetylase assayed with free a c e t y l - l a b e l l e d histones  (71).  A frequently suggested function of histone acetylation i s that i t serves as a control for t r a n s c r i p t i o n , since active sections of chromatin appear to contain hyperacetylated histones (83,84,85,86,87). inactive chromatin, e.g.  Conversely,  chicken erythrocyte chromatin, appears to have low  l e v e l s of histone acetylation with a very slow turnover of acetyl groups (229).  T r a n s c r i p t i o n a l l y competent sections of chromatin have also been  shown to be enriched i n HMG 14 and HMG 17 (202).  Since these proteins can  i n h i b i t histone deacetylase i n v i t r o using nuclei and free l a b e l l e d histones (188), i t has been suggested that HMG 14 and HMG 17 could i n h i b i t histone deacetylation and produce a l o c a l i z e d enrichment i n hyperacetylated histones,  ultimately resulting i n t r a n s c r i p t i o n .  However, using high  molecular weight chromatin complexes containing histone deacetylase,  no  such effect of added HMG proteins was observed (Table 6 and Figure 17) and the removal of HMG proteins from histone deacetylase complex with 0.4 M NaCl did not affect  the rate of the enzyme reaction.  The reasons for the varying results with these two preparations are unknown.  It i s possible that nuclei contain more than one histone  deacetylase,  however, t h i s seems u n l i k e l y i n view of the strong i n h i b i t i o n  of histone deacetylase a c t i v i t y i n nuclei by millimolar concentrations of butyrate.  Rather, the micrococcal nuclease digestion of HeLa chromatin and  the i s o l a t i o n procedures which are required for the preparation of histone deacetylase complex may have resulted i n the loss of additional factors which might be necessary for the i n h i b i t o r y response to HMG proteins.  (  114  Support for this p o s s i b i l i t y comes from the observation that an i n h i b i t o r of histone deacetylase a c t i v i t y i s l o s t from the p a r t i a l l y purified enzyme from c a l f thymus a f t e r p r e c i p i t a t i o n i n acetone and chromatography on a Sepharose 6B column (74).  S i m i l a r l y , HMG 14 and HMG 17 or other proteins  may a l t e r the substrate s p e c i f i c i t y of histone deacetylase.  Histone  deacetylase appears to have a f a i r l y broad substrate s p e c i f i c i t y , as millimolar concentrations of butyrate i n h i b i t the deacetylation of each of the d i f f e r e n t l y acetylated forms of histone H4 i n rat hepatoma c e l l s  (178),  and c a l f thymus histone deacetylase has been shown to also deacetylate H3 (74) as w e l l as acetylated HMG 14 and HMG 17 (228).  Treatment of c a l f  thymus histone deacetylase with hydroxylapatite during the course of p a r t i a l p u r i f i c a t i o n has been found to result i n a s i g n i f i c a n t change i n the r a t i o of deacetylation of histones H3 and H4.  The marked a c t i v a t i o n of  H3 deacetylation a c t i v i t y suggests that an i n h i b i t o r for H3 deacetylation may have been removed (73). In view of the recent finding that HMG 14 and HMG 17 are glycosylated (230), treatment of the HMG proteins with t r i c h l o r o a c e t i c acid during t h e i r o  preparation may have resulted i n a loss of some of t h e i r b i o l o g i c a l activity.  Incubation of HMG proteins with nuclei rather than with histone  deacetylase complex may convert the proteins to t h e i r active form.  Futher  investigation w i l l be required before an unequivocal statement on the a c t i o n of HMG proteins can be made.  IV  Histone Deacetylase Complex  Several s t r i k i n g s i m i l a r i t i e s were observed between the histone deacetylase complex released from HeLa c e l l nuclei by mild micrococcal  115  nuclease digestion and the protein matrix or chromosome scaffold of nuclei.  Examination of the proteins present i n the histone deacetylase  complex by SDS polyacrylamide gel electrophoresis  (Figure 24)  revealed  several non-histone proteins which were also present i n nuclear matrix preparations.  These included the major HeLa nuclear matrix proteins c i t e d  i n the l i t e r a t u r e (163).  The lack of i d e n t i t y between the gel patterns of  the histone deacetylase complex and the nuclear matrix and uncertainty over the exact protein composition of the nuclear matrix make i t  impossible,  however, to determine conclusively whether the histone deacetylase complex and the nuclear matrix are closely  related.  When nuclear matrix was prepared from endogenous labelled HeLa c e l l n u c l e i using a reduced DNase I digestion to ensure that some histones were retained to serve as a substrate for the histone deacetylase, a s i g n i f i c a n t l e v e l of enzyme a c t i v i t y was detected.  Apart from an increase i n the  amount of histone present, the reduction i n the extent of DNase I digestion did not result i n a protein composition s i g n i f i c a n t l y different from that of conventional nuclear matrix preparations as seen by SDS polyacrylamide g e l electrophoresis.  Omission of the extraction of nuclear matrix  preparations with 1% T r i t o n X-100 did not s i g n i f i c a n t l y a l t e r the protein content of the nuclear matrix preparations (Figure 23), but was necessary for the maintenance of histone deacetylase a c t i v i t y .  Therefore,  chromatin-bound histone deacetylase must be detergent-labile.  However, i n  view of the manner i n which nuclear matrix i s i s o l a t e d , namely by successive extractions of pelleted m a t e r i a l , the p o s s i b i l i t y remains that the histone deacetylase a c t i v i t y observed i n the nuclear matrix  116  preparations was due to co-sedimentation of histone deacetylase complex with the nuclear matrix, rather than to a physiologically relevant association. Treatment of endogenous labelled histone deacetylase with 1 or 10 mM B-mercaptoethanol caused both an i r r e v e r s i b l e i n h i b i t i o n of  histone  deacetylase a c t i v i t y (Table 7) and the d i s s o c i a t i o n of the complex i t s e l f (Figure 25).  The i n h i b i t i o n of deacetylase a c t i v i t y was not due to the  reduction of t h i o l groups on the enzyme i t s e l f , B-mercaptoethanol did not affect  since 10 mM  the deacetylase a c t i v i t y of the complex  when t h i s was assayed with added histones (Table 8).  Also, neocuproine,  which should have no effect on t h i o l groups, i n h i b i t e d histone deacetylase a c t i v i t y i n the endogenously labelled histone deacetylase complex to the same extent as B-mercaptoethanol.  Thus treatment of the deacetylase  complex with reagents which disrupt chromosome scaffolds also leads to at least p a r t i a l disruption of the complex and to a loss of deacetylase activity. The digestion of HeLa nuclei with micrococcal nuclease i n the presence of 10 mM B-mercaptoethanol resulted i n a dramatic reduction i n the deacetylase-containing  peak of the Bio-Gel A-50m column, and an increase i n  the proportion of mono- and oligonucleosomes  (Figure 25).  This suggests  that the deacetylase complex i s disrupted by B-mercaptoethanol and implies that the histone deacetylase and associated nucleosomes form part of a large structure which i s sensitive to B-mercaptoethanol or neocuproine.  Such a complex, the chromosome scaffold, has been described  by Lewis and Laemmli (165).  The chromosome scaffold i s a fast-sedimenting  structure derived from metaphase chromosomes by extraction of the histones.  This protein matrix remains attached to the DNA after removal of  117  the h i s t o n e s from chromosomes w i t h SDS. d i s s o c i a t e s upon treatment  The chromosome s c a f f o l d  w i t h as l i t t l e as 1 mM  fi-mercaptoethanol  or 3  mM  n e o c u p r o i n e , and seems t o be m a i n t a i n e d by m e t a l l o p r o t e i n i n t e r a c t i o n s _l |_ j j i n v o l v i n g Cu  ions.  B i n d i n g o f the Cu  i o n s by the chromosome  s c a f f o l d r e q u i r e s the presence of f r e e s u l p h h y d r y l g r o u p s .  I t i s not c l e a r  whether the chromosome s c a f f o l d and n u c l e a r m a t r i x r e p r e s e n t a l t e r n a t i v e forms o f the same s t r u c t u r e d u r i n g d i f f e r e n t s t a g e s o f the c e l l c y c l e , o r whether they a r e d i s t i n c t s t r u c t u r e s w h i c h may The  a s s o c i a t e w i t h each o t h e r .  r e s u l t s i n t h i s report i n d i c a t e that histone deacetylase i s associated  w i t h a l a r g e s t r u c t u r e w h i c h has s e v e r a l c h a r a c t e r i s t i c s i n common w i t h chromosome s c a f f o l d s . D i s r u p t i o n of t h i s s t r u c t u r e by  fS-mercaptoethanol  o r n e o c u p r o i n e does not r e s u l t i n d i r e c t i n h i b i t i o n o f the  deacetylase  a c t i v i t y , but e v i d e n t l y a f f e c t s the a b i l i t y o f the enzyme t o i n t e r a c t w i t h the nucleosomes a s s o c i a t e d w i t h the complex. R e s t r i c t i o n enzyme d i g e s t i o n o f h i s t o n e d e a c e t y l a s e complex DNA HeLa genomic DNA  and  w i t h H a e l l l and EcoRI ( F i g u r e 22) showed t h a t s a t e l l i t e s  I I and I I I were not e n r i c h e d i n t h i s m a t e r i a l . n o t s p e c i f i c f o r s a t e l l i t e DNA,  A l t h o u g h M s p l and TaqI a r e  no d i f f e r e n c e s were observed i n the  d i g e s t i o n p a t t e r n between h i s t o n e d e a c e t y l a s e complex DNA when they were d i g e s t e d w i t h these enzymes.  and genomic  DNA  The n o t i o n t h a t h i s t o n e  d e a c e t y l a s e may  be p r e f e r e n t i a l l y a s s o c i a t e d w i t h s a t e l l i t e sequences o f  heterochromatin  and t h a t the f u n c t i o n o f the enzyme might be t o m a i n t a i n  t h e s e sequences i n an u n a c e t y l a t e d and t r a n s c r i p t i o n a l l y i n a c t i v e s t a t e can p r o b a b l y be d i s c o u n t e d .  S i m i l a r l y , r e a n n e a l i n g s t u d i e s of the s h o r t  fragments anchored t o the n u c l e a r m a t r i x o f r a t and mouse l i v e r n u c l e i and t o the metaphase s c a f f o l d o f Chinese hamster DON demonstrated t h a t t h i s DNA  DNA  interphase  cell nuclei  has the same c o m p l e x i t y as genomic DNA  and i s  118  not enriched i n either repetitive or unique sequences (203).  These results  were independent of the matrix DNA size (350-5000 bp) or of the  nuclease  used i n the preparations (micrococcal nuclease, DNase I or endogenous digestion).  Nuclear matrix, prepared from rat l i v e r nuclei under DNase I  digestion conditions which l e f t 1% of the t o t a l DNA attached to the matrix, was found to contain DNA which showed no difference i n complexity from genomic DNA (217).  Thus there are obvious s i m i l a r i t i e s i n the DNA  associated with the histone deacetylase complex and nuclear matrix.  The  p o s s i b i l i t y that the function of s a t e l l i t e DNA might be to anchor DNA loops to the nuclear matrix or other large protein complex i s u n l i k e l y i n view of the reports on nuclear matrix DNA and the r e s t r i c t i o n enzyme analysis of histone deacetylase complex DNA. The unique sequences within the DNA loops attached to the nuclear matrix appear to be distributed i n a non-random manner.  For example,  although the DNA associated with the rat l i v e r nuclear matrix described above has the same complexity as genomic DNA, i t i s enriched i n transcribed rDNA (217).  S i m i l a r l y , the transcribed ovalbumin gene of chicken oviduct  c e l l s (172) and SV40 sequences of infected 3T3 c e l l s (173) are associated with the nuclear matrix.  This i s consistent with the idea that  t r a n s c r i p t i o n i s associated with the nuclear matrix. As described e a r l i e r , acid urea gel electrophoresis  (Figure 19)  revealed that the histone deacetylase complex from butyrate-treated HeLa c e l l s contained hyperacetylated chromatin with tetraacetylated H4 as the most abundant form of t h i s histone.  S i m i l a r l y , deacetylase complex from  c e l l s which had not been treated with butyrate contained H4 which was acetylated to a greater degree than bulk histones.  The close proximity of  these histones to histone deacetylase and the rapid k i n e t i c s of  119  deacetylation suggest that histone deacetylase complex could influence chromatin processes which involve histone acetylation. In general, acetylation of histones i s thought to a l t e r the conformation of chromatin as DNase I p r e f e r e n t i a l l y digests the DNA i n chromatin containing hyperacetylated histones (85,205,232,233) and chromatin containing active genes (142).  Histone acetylation has been  assumed to result i n a weakening of the binding between the DNA and histone core to y i e l d nucleosomes with a more "open" structure.  More recently,  evidence suggests that the major effects of histone acetylation may operate at higher l e v e l s of chromatin structure, e.g. fibre  the folding of the 30 nm  (91). One of the most frequently proposed functions of histone acetylation  i s regulation of t r a n s c r i p t i o n since a c t i v e l y transcribed sequences are p r e f e r e n t i a l l y associated with hyperacetylated histones (83 - 89).  Histone  acetylation can also influence hormone binding which can ultimately lead to a change i n t r a n s c r i p t i o n .  For example, the action of the thyroid hormones  i n mammalian c e l l s i s mediated through the thyroid hormone receptor protein which associates with high molecular weight chromatin.  Butyrate-induced  hyperacetylation of histones results i n a lowered l e v e l of the chromatin associated hormone receptor; however, the chromatin associated  receptor  reappears when histone acetylation returns to normal physiological l e v e l s (231).  These results are consistent with a model i n which the a f f i n i t y  between the thyroid hormone receptor and chromatin varies with the state of histone a c e t y l a t i o n . There i s also strong evidence for other chromatin processing being influenced by the degree of chromatin a c e t y l a t i o n .  Histone acetylation may  be required for the i n i t i a t i o n of excision repair of DNA lesions or  120  re-establishment  o f t h e nucleosome s t r u c t u r e a f t e r r e p a i r s y n t h e s i s .  I n c r e a s e d h i s t o n e a c e t y l a t i o n i n human f i b r o b l a s t c e l l s has been found t o l e a d t o an i n c r e a s e i n UV-induced DNA r e p a i r ( 9 0 ) .  The i n c r e a s e i n r e p a i r  was observed i n b o t h normal and p a r t i a l l y r e p a i r d e f i c i e n t c e l l s and c o r r e l a t e d w i t h an i n c r e a s e i n t h e amount o f t h e h i g h e s t a c e t y l a t e d f o r m o f h i s t o n e H4.  Increased histone a c e t y l a t i o n l e d t o a r i s e i n the rate of  i n c i s i o n by an endonuclease a t UV-induced l e s i o n s d u r i n g t h e i n i t i a l of r e p a i r .  phase  I t i s p o s s i b l e t h a t t h e i n c r e a s e d r a t e o f DNA i n c i s i o n d u r i n g  DNA e x c i s i o n r e p a i r and enhanced DNase I d i g e s t i o n o f a c e t y l a t e d  chromatin  a r e due t o a common a l t e r a t i o n i n nucleosome s t r u c t u r e . The s i n g l e s t r a n d breaks formed i n DNA d u r i n g UV-induced r e p a i r i n human f i b r o b l a s t s have been examined and found t o remain open f o r 3-10 minutes (204).  This f i g u r e i s very s i m i l a r t o the h a l f l i f e of the r a p i d l y  d e a c e t y l a t e d h i s t o n e a c e t y l groups i n t h e h i g h m o l e c u l a r w e i g h t h i s t o n e deacetylase  complex.  I t has been proposed r e c e n t l y t h a t v i r t u a l l y a l l c h r o m a t i n c y c l e s o f a c e t y l a t i o n and d e a c e t y l a t i o n and t h a t t h i s p r o c e s s chromatin repair.  experiences  allows  t o be "opened up" f o r r e g u l a r i n s p e c t i o n o f t h e DNA and  necessary  The r e q u i r e m e n t f o r c e l l s t o c o n s t a n t l y survey t h e i r genomes may  a c c o u n t f o r t h e l a r g e amount o f energy expended by c e l l s i n r a p i d h i s t o n e a c e t y l a t i o n and d e a c e t y l a t i o n . l e a s t 2 x 10  6  F o r example, t h e s e p r o c e s s e s  e q u i v a l e n t s o f ATP/minute i n HTC c e l l s ( 9 1 ) .  consume a t The h i s t o n e  d e a c e t y l a s e complex r e p o r t e d i n t h i s t h e s i s s h o u l d be c o n s i d e r e d as a candidate f o r the s i t e of chromatin  i n s p e c t i o n and r e p a i r as i t c o n t a i n s  genomic DNA a s s o c i a t e d w i t h a c e t y l a t e d h i s t o n e s w h i c h p r o b a b l y  experience  r a p i d turnover o f t h e i r a c e t y l groups. D i f f e r e n c e s were noted i n t h e p o s i t i o n s o f s e v e r a l non-core h i s t o n e  121  p r o t e i n s i n a c i d u r e a p o l y a c r y l a m i d e g e l s between h i s t o n e d e a c e t y l a s e complex prepared  from b u t y r a t e - t r e a t e d HeLa c e l l s and h i s t o n e d e a c e t y l a s e  complex i s o l a t e d from c e l l s w h i c h had n o t e x p e r i e n c e d b u t y r a t e ( F i g u r e 19).  M i l l i m o l a r c o n c e n t r a t i o n s o f b u t y r a t e have many documented e f f e c t s on  e u k a r y o t i c c e l l s , i n c l u d i n g changes i n p r o t e i n s y n t h e s i s and a l t e r a t i o n s i n protein modification.  B u t y r a t e i n d u c e s t h e s y n t h e s i s o f many new p r o t e i n s  i n F r i e n d c e l l s (222) and h i s t o n e s y n t h e s i s u l t i m a t e l y becomes independent o f DNA r e p l i c a t i o n ( 2 2 0 ) .  L i k e w i s e , b u t y r a t e causes a t r a n s f o r m e d  Syrian  hamster c e l l l i n e t o produce 2 p o l y p e p t i d e s u s u a l l y found o n l y i n untransformed  c e l l s (219).  HeLa c e l l s d i s p l a y a v a r i e d response t o  b u t y r a t e , depending upon t h e p r o t e i n f r a c t i o n a n a l y z e d ; HMG 14 and HMG 17 p h o s p h o r y l a t i o n i s enhanced by b u t y r a t e (225) whereas HeLa c e l l s a l s o e x p e r i e n c e an i n h i b i t i o n (223).  o f t h e p h o s p h o r y l a t i o n o f h i s t o n e s HI and H2A  A l t e r a t i o n s i n t h e s u l p h a t i o n o f g l y c o p r o t e i n s have a l s o been noted  i n b u t y r a t e - t r e a t e d human k i d n e y tumour c e l l s ( 2 2 1 ) .  The d i f f e r e n t  m o b i l i t i e s o f p r o t e i n s i n h i s t o n e d e a c e t y l a s e complex from b u t y r a t e t r e a t e d c e l l s may t h e r e f o r e , r e f l e c t  the a s s o c i a t i o n of m e t a b o l i c a l l y a c t i v e  p r o t e i n s w i t h t h e h i g h m o l e c u l a r complex. The n u c l e a r m a t r i x i s t h e b e s t c h a r a c t e r i z e d h i g h m o l e c u l a r w e i g h t n u c l e a r complex and appears t o be t h e s i t e o f a t l e a s t some c h r o m a t i n processing.  The c o n t e n t i o n t h a t DNA r e p l i c a t i o n o c c u r s w i t h i n t h e n u c l e a r  m a t r i x i s based upon t h e o b s e r v a t i o n s t h a t t h e s t r u c t u r e i s e n r i c h e d i n Okazaki fragments and t h a t l a b e l l e d t h y m i d i n e i s i n c o r p o r a t e d i n t o DNA i n r a t l i v e r and bovine l i v e r n u c l e a r m a t r i x (169,170).  Pulse  labelling  e x p e r i m e n t s have a l s o i n d i c a t e d t h a t t h e o r i g i n s o f r e p l i c o n s a r e bound t o the nuclear m a t r i x during the e n t i r e c e l l c y c l e (171).  Newly s y n t h e s i z e d  n u c l e o s o m a l h i s t o n e s i n r a t hepatoma c e l l s a r e more a c c e s s i b l e t o h i s t o n e  122  a c e t y l a s e i n v i v o and newly s y n t h e s i z e d c h r o m a t i n e x t e n d e d , open c o n f o r m a t i o n hyperacetylated chromatin  i s also i n a relatively  which i s t y p i c a l l y associated w i t h  (227).  When t h e r a t hepatoma c e l l s were p u l s e  l a b e l l e d w i t h a c e t a t e , i t was found t h a t newly r e p l i c a t e d c h r o m a t i n r e a c h a h i g h l y a c e t y l a t e d s t a t e u n l e s s b u t y r a t e was p r e s e n t .  d i d not  These  f i n d i n g s suggest t h a t t h e i n c r e a s e d a c c e s s i b i l i t y o f newly r e p l i c a t e d chromatin  t o h i s t o n e a c e t y l a s e s and t h e t e m p o r a r i l y i n c r e a s e d r a t e o f  a c e t y l a t i o n i s balanced  by r a p i d d e a c e t y l a t i o n and t h e r e f o r e , t h a t DNA  r e p l i c a t i o n must o c c u r i n c l o s e p r o x i m i t y t o h i s t o n e d e a c e t y l a s e .  The  h i s t o n e d e a c e t y l a s e complex s h o u l d be c o n s i d e r e d a s e i t h e r a p o s s i b l e s i t e for  DNA r e p l i c a t i o n o r as h a v i n g a r o l e i n r e p l i c a t i o n , s i n c e i t c o n t a i n s  a n a c t i v e d e a c e t y l a s e i n a s s o c i a t i o n w i t h genomic DNA. T r a n s c r i p t i o n a l a c t i v i t y has a l s o been i n f e r r e d t o o c c u r upon t h e n u c l e a r m a t r i x on t h e b a s i s o f i n d i r e c t e v i d e n c e .  The unique sequences  w i t h i n DNA l o o p s a r e a r r r a n g e d i n a non-random manner such t h a t a c t i v e genes a r e c l o s e t o t h e m a t r i x (172,173,174).  The f o r m a t i o n o f nascent  hnRNA t r a n s c r i p t s i n t h e c e l l n u c l e a r m a t r i x o f r a t e n d o t h e l i u m (176) and the s t r o n g b i n d i n g o f hnRNA t o F r i e n d c e l l n u c l e a r m a t r i x (175) add support to  t h e v i e w t h a t t r a n s c r i p t i o n o c c u r s on t h e m a t r i x .  The p o s s i b l e  f u n c t i o n s o f h i s t o n e a c e t y l a t i o n and h i s t o n e d e a c e t y l a s e complex i n t h e r e g u l a t i o n o f t r a n s c r i p t i o n have been d i s c u s s e d e a r l i e r . accumulating  However, t h e r e i s  e v i d e n c e t h a t t h e n u c l e a r m a t r i x c o n t a i n s s i t e s f o r hormone  b i n d i n g and hormone r e g u l a t e d t r a n s c r i p t i o n has been an a c c e p t e d f o r many y e a r s .  phenomenon  E s t r a d i o l b i n d i n g s i t e s have been found i n c h i c k e n  liver  n u c l e a r m a t r i x whereas t h e n u c l e a r m a t r i x from r o o s t e r l i v e r s c o n t a i n s few s i t e s f o r t h e hormone.  very  S i m i l a r l y , n u c l e a r m a t r i x p r e p a r a t i o n s from r a t  p r o s t a t e have been found t o be r i c h i n d i h y d r o t e s t o s t e r o n e b i n d i n g s i t e s  123  (177).  Nuclear pore complex proteins are always found associated with  nuclear matrix preparations containing hormone binding s i t e s . hormones are known to induce protein synthesis and i n related  The sex experiments,  nuclear matrix preparations from female rat l i v e r , endometrium and lung were examined for e s t r a d i o l binding s i t e s and hnRNA synthesis. Approximately 85% of t o t a l rapidly l a b e l l e d hnRNA was associated with the l i v e r nuclear matrix and high a f f i n i t y e s t r a d i o l binding occurred i n nuclear matrix from endometrium but not from the lung which i s not regulated by e s t r a d i o l (176).  Another study has found that the nuclear  matrix from the uterus of the female rat contains e s t r a d i o l binding s i t e s (226). Taken together, these findings are i n agreement with the proposal that the nuclear matrix and nuclear lamina form a contiguous complex and that hormones enter the nucleus through the nuclear pores and need be transported or diffuse only a very short distance to binding s i t e s on the nuclear matrix.  An ordered arrangement of the DNA loop attachment  sites,  hormone binding s i t e s and t r a n s c r i p t i o n a l apparatus on a large protein matrix would f a c i l i t a t e hormone regulated t r a n s c r i p t i o n .  One of the  prominent pore complex lamina proteins has a molecular weight of approximately 150,000 (176) and a protein with this molecular weight was observed i n the SDS polyacrylamide gel protein p r o f i l e of histone deacetylase complex (Figure 24).  Although there are i n s u f f i c i e n t  data to  conclude whether the observed protein represents a nuclear pore lamin, the p o s s i b i l i t y exists that pore-laminae are associated with the histone deacetylase complex or that fragments of the nuclear pores co-purify with the deacetylase complex.  In l i g h t of the increased levels of the thyroid  hormone chromatin associated receptor protein i n unacetylated chromatin  124  (231), the histone deacetylase complex may be involved i n hormone regulated transcription. The precise function(s) of histone acetylation and the nuclear matrix have yet to be elucidated.  Histone acetylation has been implicated i n DNA  r e p l i c a t i o n , repair and hormone binding and regulation of t r a n s c r i p t i o n . Likewise, the nuclear matrix appears to support r e p l i c a t i o n , hormone binding and t r a n s c r i p t i o n .  In general, the location of complex enzymatic  reactions within a matrix may f a c i l i t a t e chromatin processing.  Until  recently, no enzyme a c t i v i t y had been assayed d i r e c t l y i n the nuclear matrix.  However, ATP-dependent nuclear ribonucleoprotein release and  nucleoside triphosphatase a c t i v i t i e s have been detected i n rat l i v e r nuclear matrix (234).  Furthermore, antibodies generated against a nuclear  matrix glycoprotein i n h i b i t the enzyme a c t i v i t i e s  (234).  The high molecular weight histone deacetylase complex shares several properties with the nuclear matrix and chromosome scaffold as i t  contains  complex DNA, dissociates i n the presence of copper ion chelators and contains proteins found i n the nuclear matrix.  At present, no firm  conclusion can be made as to whether these different preparations represent different forms of the same structure or even whether they form an integrated complex within the nucleus. complex containing histone deacetylase  The attachment of nucleosomes to a i s consistent with the observation  that rapid acetylation and deacetylation are coupled processes that appear to occur on contiguous nucleosome arrays (205). Histone acetylation probably does not serve one specific function, but rather l i k e l y f a c i l i t a t e s  several processes which require altered  nucleosome a c c e s s i b i l i t y .  This could be accomplished by regular  acetylation and deacetylation of chromatin i n the presence of other  125  c h r o m a t i n p r o c e s s i n g enzymes.  The p r e s e n t p e r c e p t i o n o f c h r o m a t i n  may  r e q u i r e m o d i f i c a t i o n t o accommodate t h e concept t h a t c h r o m a t i n i s i n a dynamic s t a t e w i t h c o n s t a n t f l u x i n l e v e l s o f a c e t y l a t i o n .  The h i g h  m o l e c u l a r w e i g h t h i s t o n e d e a c e t y l a s e complex o u t l i n e d i n t h i s t h e s i s p r o v i d e s a u s e f u l system t o h e l p e l u c i d a t e t h e f u n c t i o n s o f h i s t o n e a c e t y l a t i o n and p o s s i b l y o f t h e n u c l e a r m a t r i x .  126  BIBLIOGRAPHY  1.  Kossel, A . (1884) Hoppe-Seyler' s Z . Physiol. Chem. 8, 511-515.  2.  DeLange, R . J . , Fambrough, D . M . , Smith, E . L . and Bonner, J . (1969) J . B i o l . Chem. 244, 319-334.  3.  Ogawa, Y . , Q u a g l i a r o t t i , G . , Jordan, J . J . , Taylor, C.W., Starbuck, W.C. and Busch, H. (1969) J . B i o l . Chem. 244, 4387-4392.  4.  Iwai, K . , Ishikawa,  K. and Hayashi, H. (1970) Nature 226, 1056-1058.  5.  Q u a g l i a r o t t i , G . , Ogawa, Y . , Taylor, C.W., Sautiere, P . , Jordan, J . , Starbuck, W.C. and Busch, H. (1969) J . B i o l . Chem. 244, 1796-1802.  6.  Bradbury, E . M . , Cary, P . D . , Chapman, G . E . , Crane-Robinson, C , Danby, S . E . and Rattle, H.W.E. (1975) Eur. J . Biochem. _52, 605-613.  7.  Chapman, G . E . , Hartman, P . G . and Bradbury, E . M . (1976) E u r . J . Biochem. _61, 69-75.  8.  DeLange, R . J . , Fambrough, D . M . , Smith, E . L . and Bonner, J . (1969) J . B i o l . Chem. 244, 5669-5679.  9.  Patthy, L . , Smith, E . L . and Johnson, J . (1973) J . B i o l . Chem. 248, 6834-6840.  10.  Panyim, S . , B i l e k , D. and Chalkley, R. (1971) J . B i o l . Chem. 246, 4206-4215.  11.  Cole, R.D. (1977) "The Molecular Biology of the Mammalian Genetic Apparatus" Volume 1, pages 99-104. E l s e v i e r , Amsterdam.  12.  Moore, M . , Jackson, V . , Sealy, L . and Chalkley, R. (1979) Biochim. Biophys. Acta 561, 248-260.  13.  Jackson, V . , Shires, A . , Chalkley, R. and Granner, D.K. (1975) J . B i o l . Chem. 250, 4856-4863.  127  14. A l l f r e y , V . G . , Faulkner, R. and Mirsky, A . E . (1964) Proc. N a t l . Acad. S c i . U.S.A. 51, 786-794. 15. Honda, B . M . , Dixon, G.M. and Candido, E . P . M . (1975) J . ' B i o l . Chem. 250, 8681-8685. 16. Duerre, J . A . and Chakrabarty, S. (1975) J . B i o l . Chem. 250, 8457-8461. 17. Duerre, J . A . , Wallwork, J . C . , Quick, D . P . and Ford, K.M. (1977) J . B i o l . Chem. 252, 5981-5985. 18. Brandt, W . E . , S t r i c k l a n d , W.N., Morgan, M. and Van Holt, C. (1974) FEBS L e t t . 40_, 167-172. 19. Paik, W.K. and Kim, S. (1967) Biochem. Biophys. Res. Commun. _29, 14-20. 20. Greenway, P . J . and Levine, D. (1974) Biochim. Biophys. Acta 350, 374-382. 21. Wallwork, J . C . , Quick, D.P. and Duerre, J . A . (1977) J . B i o l . Chem. 5977-5980. 22. Paik, W.K. and Kim, S. (1971) Science 174, 114-119. 23. Thomas, G . , Lange, H.W. and Hempel, K. (1975) Eur. J . Biochem. 51. 609-615. 24. DeLange, R . J . , Hooper, J . A . and Smith, E . L . (1973) J . B i o l . Chem. 248, 3261-3274. 25. Hooper, J . A . , Smith, E . L . , Sommer, K.R. and Chalkley, R. (1973) J . B i o l . Chem. 248, 3275-3279. 26. T i d w e l l , T . , A l l f r e y , V . K . and Mirsky, A . E . (1968) J . B i o l . Chem. 243, 707-715. 27. Byvoet, P . , Shephard, G . R . , Hardin, J . M . and Noland, B . J . (1972) Arch. Biochem. Biophys. 148, 558-567. 28. Shoemaker, C . B . and Chalkley, R. (1978) J . B i o l . Chem. 253, 5802-5807.  128  29. Whitlock, J r . , J . P . , Augustine, R. and Schulman, M. (1980) Nature 287, 74-79. 30.  Simpson, R . T . (1978) Nucl. Acids. Res. 5_, 1109-1119.  31. Shoemaker, C . B . and Chalkley, R. (1980) J . B i o l . Chem. 255, 11048-11055. 32. Marks, D . B . , Paik, W.K. and Borum, T.W. (1973) J . B i o l . Chem. 248, 5660-5667. 33. Gurley, L . R . , Walkers, R . A . and Tobey, R . A . (1975) J . B i o l . Chem. 250, 3936-3944. 34.  Gurley, L . R . , D'Anna, J . A . , Barham, S . S . , Deaven, L . L . and Tobey, R . A . (1978) E u r . J . Biochem. 84, 1-15.  35. Hohmann, P . , Tobey, R . A . and Gurley, L . R . (1976) J . B i o l . Chem. 251, 3685-3692. 36. Lake, R . S . , G o i d l , J . A . and Salzman, N.P. (1972) Exp. C e l l Res. 73, 113-121. 37.  Smith, D . L . , Chen, C . C . , Bruegger, B . B . , Holtz, S . L . , Halper, R.M. and Smith, R.A. (1974) Biochemistry 13., 3785-3789.  38.  Smith, D . L . , Chen, C . C . , Bruegger, B . B . , Holtz, S . L . Halper, R.M. and Smith, R . A . (1974) Biochemistry 13_, 3780-3784.  39. Chen, C . C . , Bruegger, B . B . , Kern, C.W., L i n , Y . C . , Halper, R.M. and Smith, R . A . (1977) Biochemistry 16_, 4852-4855. 40. Dixon, G.H. (1976) "Life Sciences Research Reports" Volume 4, pages 197-207.  Eds: A l l f r e y , V . G . , Bautz, E . K . F . , McCarthy, B . J . , Schimke,  R . T . and T i s s i e r e s , A. 41. Riquelme, P . T . , Burzio, L . O . , and Koide, S.S. (1979) J . B i o l . Chem. 254,  3018-3028.  129  42. Ogata, N . , Ueda, K. and Hayaishi, 0. (1980) J . B i o l . Chem. 255, 7610-7615. 43. Burzio, L . O . , Riquelme, P . T . and Koide, S.S. (1979) J . B i o l . Chem. 254, 3029-3037. 44. Chambon, P . , W e i l l , J . D . , Doly, J . , Strosser, M . T . , and Mandel, P. (1966) Biochem. Biophys. Res. Commun. 25, 638-643. 45. Nishizuka, Y . , Ueda, K . , Nakazawa, K. and Hayaishi, 0. (1967). J . B i o l . Chem. 242, 3164-3171. 46. Tanaka, M . , Hayashi, K . , Sakura, H . , Miwa, M . , Matsushima, T. and Sugimura, T . (1978) Nucl. Acids Res. 5_, 3183-3194. 47. Okayama, H . , Edson, C M . , Fukushima, M . , Ueda, K. and Hayaishi, 0. (1977) J . B i o l . Chem. 252, 7000-7005. 48. Yoshihara, K . , Hashida, T . , Tanaka, Y . , Ohgushi, H . , Yoshihara, H. and Kamiya, T . (1978) J . B i o l . Chem. 253, 6459-6466. 49. Mandel, P . , Okazaki, H. and Niedergang, C. (1977) FEBS L e t t . 84, 331-336. 50.  I t o , S . , Shizuta, Y. and Hayaishi, 0. (1979) J . B i o l . Chem. 254, 3647-3651.  51. Tsopanakis,  C , McLaren, E . and S h a l l , S. (1976) Biochem. Soc. Trans.  4_, 775-777. 52. Kristensen,  T . and Holtlund, J . (1976) Eur. J . Biochem. _7£» 441-446.  53. G i r i , C P . , West, M . H . P . , Ramirez, M . L . and Smulson, M. (1978) Biochemisry _17_, 3501-3504. 54. Malik, N . , Bustin, M. and Smulson, M. (1982) Nucl. Acids Res. 10, 2939-2950. 55. Lorimer I I I , W . S . , Stone, P.R. and Kidwell, W.R. (1977) Exp. C e l l Res. 106,  261-266.  130  56. Hayaishi, 0. and Ueda, K. (1977) Annu. Rev. Biochem. 46_, 95-116. 57. F e r r i s , G.M. and Clark, J . B . (1971) Biochem. J . 2, 662-665. 58. Proctor, N.H. and Casida, J . E . (1975) Science 190, 580-582. 59. Burzio, L . and Koide, S.S. (1973) Biochem. Biophys. Res. Commun. 53, 572-579. 60. Durkacz, B.W., O m i d i j i , D . , Gray, D.A. and S h a l l , S. (1980) Nature 283, 593-596. 61. P o i r i e r , G . G . , Sevard, P . , Rajotte, D . , Morisset, J . and Lord, A. (1978) Can. J . Bioc. 56_, 784-790. 62. Wong, N.C.W., P o i r i e r , G.H..and Dixon, G.H. (1977) Eur. J . Biochem. 77_, 11-21. 63. Levy-Wilson, B. (1983) Biochemistry i n press. 64. Wiktorowicz, J . E . , Campos, K . L . and Bonner, J . (1981) Biochemistry 20, 1464-1467. 65. Garcea, R . L . and A l b e r t s , B.M. (1980) J . B i o l . Chem. 255, 11454-11463. 66. B e l i k o f f ,  E . , Wong, L . - J . and A l b e r t s , B.M. (1980) J . B i o l . Chem. 255,  11448-11453. 67. Wiegand, R . C . and Brutlag, D . L . (1981) J . B i o l . Chem. 256, 4578-4583. 68. Boehm, J . , Schlaeger, E . - J . and Knippers, R. (1980) E u r . J . Biochem. 112,  353-362.  69. Otto, B . , Boehm, J . and Knippers, R. (1980) Eur. J . Biochem. 112, 363-366. 70. Inoue, A. and Fujimoto, D. (1969) Biochem. Biophys. Res. Commun. 36, 146-150. 71. Inoue, A. and Fujimoto, D. (1970) Biochim. Biophys. Acta 220, 307-316. 72. Libby, P.R. (1970) Biochim. Biophys. Acta 213, 234-236.  131  73. K i k u c h i , H. and Fujimoto, D. (1973) FEBS L e t t . 29, 280-282. 74. V i d a l l , G . , Boffa, L . C . and A l l f r e y , V . G . (1972) J . B i o l . Chem. 247, 7365-7373. 75. Reeves, R. and Candido, E.P.M. (1979) Biochem. Biophys. Res. Commun. 89_, 571-579. 76. Libby, P.R. and Bertram, J . S . (1980) Arch. Biochem. Biophys. 201, 359-361. 77. Waterborg, J . H . , Cahal, S . S . , Muller, R.D. and Matthews, H.R. (1981) Proc. Eur. Physarum Conf. 1981 page 160. 78.  Candido, E . P . M . , Reeves, R. and Davie, J . R . (1978) C e l l 14_, 105-113.  79. Sealy, L . and Chalkley, R. (1978) C e l l 14_, 115-121. 80. V i d a l i , G. Boffa, L . C , Bradbury, E . M . and A l l f r e y , V . G . (1978) Proc. N a t l . Acad. S c i . U.S.A. 75, 2239-2243. 81. Riggs, M . G . , Whittaker, R . G . , Neumann, J . R . and Ingram, V.M. (1977) Nature 268, 462-464. 82. Whitlock, J r . , J . P . , Galaezzi, D. and Schulrnan, H. (1983) J . B i o l . Chem. 258, 1299-1304. 83. Davie, J . R . and Candido, E . P . M . (1978) Proc. N a t l . Acad. S c i . U.S.A. 75_, 3574-3577. 84. Levy-Wilson, B . , Watson, D . C and Dixon, G.H. (1979) Nucl. Acids Res. 6_, 259-274. 85. Davie, J . R . and Candido, E . P . M . (1980) FEBS L e t t . 110, 164-168. 86. Nelson, D . A . , Perry, W.M. and Chalkley, R. (1978) Biochem.  Biophys.  Res. Commun. _82, 356-363. 87. Vavra, K . J . , A l l i s , C D . and Gorovsky, M.A. (1982) J . B i o l . Chem. 257, 2591-2598.  132  88. Davie, J . R . , Saunders, C . A . , Walsh, J . M . and Weber, S . C . (1981) Nucl. Acids Res. 9_, 3205-3216. 89. Gorovsky, M . A . , Glover, C . , Johmann, C . A , Keevert, J . B . , Mathis, D . J . and Samuelson, M. (1977) Cold Spring Harbor Symp. Quant. B i o l . 42, 493-503. 90.  Smerdon, M . J . , Lan, S . Y . , Calza, R . E . and Reeves, R. (1982) J . B i o l . Chem. 257, 13441-13447.  91. Perry, M. and Chalkley, R. (1982) J . B i o l . Chem. 257, 7336-7346. 92. Hewish, D.R. and Burgoyne, L . A . (1973) Biochem. Biophys. Res. Commun. 52, 504-510. 93. Romberg, R.D. (1974) Science 184, 868-871. 94. N o l l , M. (1974) Nature 251, 249-251. 95. Thomas, J . O . and Kornberg, R.D. (1975) Proc. N a t l . Acad. S c i . U.S.A. 72, 2626-2630. 96. D'Anna, J . A . and Isenberg, I . (1974) Biochemistry 13, 4987-4992. 97. Martinson, H . G . and McCarthy, B . J . (1975) Biochemistry 14, 1073-1078. 98. Martinson, H.G. and McCarthy, B . J . (1976) Biochemistry 15, 4126-4131. 99. Campbell, A . M . and Cotter, R . I . (1976) FEBS L e t t . 70_, 209-211. 100. Lewis, P . N . (1979) E u r . J . Biochem. 99., 315-322. 101. Thomas, G . J . , Prescott, B. and O l i n s , D . E . (1977) Science 197, 385-388. 102.  Cotter, K . I . and L i l l e y , D . M . J . (1977) FEBS L e t t . 82_, 63-68.  103. Bradbury, E . M . , Moss, T . , Hayashi, H . , Hjelm, H . , Suau, P . , Stephens, R . M . , Baldwin, J . P . and Crane-Robinson, C. (1977) Cold Spring Harbor Symp. Quant. B i o l . 42_, 277-286. 104. Isenberg, I . (1979) Annu. Rev. Biochem. 4j$_, 159-192. 105. McGhee, J . D . and Felsenfeld, 1115-1156.  G. (1980) Annu. Rev. Biochem. 49,  133  106. W e i n t r a u b , J . and Van L e n t e , F. (1974) P r o c . N a t l . Acad. S c i . U.S.A. 7JL, 4249-4253.  107. R i l l , R.L. and O o s t e r h o f , D.K. (1982) J . B i o l . Chem. 257, 14875-14880. 108. W h i t l o c k , J r . , J . P . and S t e i n , A. (1978) J . B i o l . Chem. 253, 3857-3861. 109. F i n c h , J.T., L u t t e r , L.C., Rhodes, S.D., Brown, R.S., Rushton, B., L e v i t t , M. and K l u g , A. (1977) Nature 269, 29-36. 110. N o l l , M. (1974) N u c l . A c i d s Res. 1, 1573-1578. 111. L u t t e r , L.C. (1979) N u c l . A c i d s Res. 6_, 41-57. 112. T r i f o n o v , E.N. and B e t t e c k e r , T. (1979) B i o c h e m i s t r y 18, 454-456. 113. Sollner-Webb,  B., M e l c h o i r , J r . , W. and F e l s e n f e l d , G. (1978) C e l l 14,  611-627.  114. W h i t l o c k , J r . , J . P . (1977) J . B i o l . Chem. 252, 7635-7639. 115. Sollner-Webb,  B. and F e l s e n f e l d , G. (1977) C e l l 10, 537'-547.  116. W h i t l o c k , J r . , J . P . , R u s h i z k y , G.W. and Simpson, R.T. (1977) J . B i o l . Chem. 252, 3003-3006. 117. Compton, J . L . , B e l l a r d , M. and Chambon, P. (1976) P r o c . N a t l . Acad. S c i . U.S.A. 73, 4382-4386. 118. L o h r , D., Corden, J . , T a t c h e l l , K., K o v a c i c , R.T. and Van Holde, K.A. (1977) P r o c . N a t l . Acad. S c i . U.S.A. 74_, 79-83. 119. R i l l , R.L., N e l s o n , D.A., O o s t e r h o f , D.K. and H o z i e r , J.C. (1977) N u c l . A c i d s Res. 4_, 771-789. 120. Chambon, P. (1977) C o l d S p r i n g Harbor Symp. Quant. B i o l . 42, 1209-1234. 121. O l i n s , A.L. and O l i n s , D.E. (1974) S c i e n c e 183, 330-332. 122. O l i n s , A.L., S e n i o r , M.B. and O l i n s , D.E. (1976) J . B i o l . Chem. 68, 787-792.  123. Bakayev, V.V., Bakayeva, T.G. and V a r s h a v k s y , 619-629.  A . J . (1977) C e l l 11,  134  124. Thoma, F . , R o l l e r , T . L . and Klug, A. (1979) J . C e l l B i o l . 83_, 403-426. 125. Weischet, W., and Van Holde, K . E . (1980) Nucl. Acids. Res. 8, 3743-3755. 126.  Finch, J . T . , L u t t e r , L . C , Rhodes, D . , Brown, R . S . , Rushton, B . , L e v i t t , M. and Klug, A. (1977) Nature 269, 29-36.  127. Pardon, J . F . , Worcester, D . L . , Wooley, J . C , T a t c h e l l , K . , Van Holde, K . E . and Richards, B.M. (1975) Nucl. Acids Res. 2_, 2163-2176. 128. Hjelm, R . P . , Kneale, G . G . , Suan, P . , Baldwin, J . P . and Bradbury, E . M . (1977) C e l l 10, 139-151. 129. Richards, B . , Pardon, J . F . , L i l l e y , D . M . J . , Cotter, R. and Wooley, J . (1977) C e l l B i o l . I n t . Rep. 1_, 107-116. 130.  Dubochet, J . and N o l l , M. (1978) Science 202, 280-286.  131. L i l l e y , D . M . J , and T a t c h e l l , K. (1977) Nucl. Acids Res. 4_, 2039-2055. 132. Varshavsky, A . J . , Nedospasov, S . A . , Schmatchenko,  U . V . , Bakayev, V . V . ,  Chumackov, P.M. and Georgiev, C P . (1977) Nucl. Acids Res. 4_, 3303-3325. 133. Finch, J . T . and Klug, A. (1976) Proc. N a t l . Acad. S c i . U.S.A. 73, 1897-1901. 134.  Carpenter, B . C , Baldwin, J . P . , Bradbury, E . M . and I b e l , K. (1976) Nucl. Acids Res. 3_, 1739-1746.  135.  Jorcano, J . L . , Meyer, C , Day, L . A . and Renz, M (1980) Proc. N a t l . Acad. S c i . U.S.A. 77_, 6443-6447.  136. R i s , H. and Kubai, D . F . (1970) Annu. Rev. Genet. 4_, 263-294. 137. Thoma, R. , K o l l e r , T . and Klug, A . (1979) J . C e l l B i o l . 83_, 403-427. 138.  Thomas, J . 0 . and Butler, P . J . G .  (1980) J . Mol. B i o l . 144, 89-93.  139. Langmore, J . P . and Schutt, C. (1980) Nature 288, 620-622.  135  140. Belland, M . , Gannon, F . and Chambon, P. (1977) Cold Spring Harbor Symp. Quant. B i o l . 42^, 779-791. 141. Bloom, K . S . and Anderson, J . N . (1978) C e l l 15, 141-150. 142. Weintraub, H. and Groudine, M. (1976) Science 193, 848-856. 143. Garel, A. and Axel, R. (1976) Proc. N a t l . Acad. S c i . U.S.A. 73, 3966-3970. 144.  Garel, A . , Zolan M. and Axel, R. (1977) Proc. N a t l . Acad. S c i . U . S . A . 74, 4867-4871.  145.  Bloom, K . S . and Anderson, J . N . (1982) J . B i o l . Chem. 257, 13018-13027.  146. Vanderbilt, J . N . , Bloom, K . S . and Anderson, J . N . (1982) J . B i o l . Chem. 257, 13009-13017. 147.  Goodwin, G . H . , Woodhead, L . and Johns, E.W. (1977) FEBS L e t t . 73, 85-88.  148. Bakayev, V . V . , Bakayeva, T . G . , Schmatchenko, V . V . and Georgiev, G.P. (1978) Eur. J . Biochem. 91, 291-301. 149.  Levy-Wilson, B . , and Dixon, G.H. (1978) Nucl. Acids Res. 5_, 4155-4163.  150. Goodwin, G . H . , Mathew, C . G . P . , Wright, C . A . , Venkov, D . C . and Johns, E.W. (1979) Nucl. Acids Res. _7, 1815-1835. 151. Hutcheon, T . , Dixon, G.H. and Levy-Wilson, B. (1980) J . B i o l . Chem. 255, 681-685. 152.  Albanese, I . and Weintraub, H. (1980) Nucl. Acids Res. _8, 2787-2805.  153. Oku, S . , Sole, M . J . , B r i t t , B . A . and Liew, C . C . (1981) J . Neurol. S c i . 5 £ , 373-379. 154. Weissbach, A. (1977) Annu. Rev. Biochem. 46_, 25-47. 155. W i t t i g , S. and W i t t i g , B. (1982) Nature 297, 31-38. 156.  Levy A . and N o l l , M. (1980) Nucl. Acids Res. 8, 6059-6068.  136  157.  Samal, B. and Wo r e e l , A. (1981) C e l l 23_, 401-409.  158.  Pfeiffer,  W. and Zachau, H.G. (1980) Nucl. Acids Res. 8, 4621-4638.  159. Adolph, U W . (1980) J . C e l l . S c i . 42_, 291-304. 160. Adolph, L . W . , Cheung, S.M. and Laemmli, U.K. (1977) C e l l 12, 805-816. 161. Laemmli, U . K . , Cheung, S . M . , Adolph, K.W., Paulson, J . R . , Brown, J . A . and Braumbach, W.R. (1978) Cold Spring Harbor Symp. Quant. B i o l . 42, 109-118. 162. Hartwig, M. (1978) Acta. B i o l . Med. Germ. 37.. 421-432. 163. Detke, S. and K e l l e r , J . M . (1982) J . B i o l . Chem. 257, 3905-3911. 164.  Berezney, R. and Buchholtz, L . A . (1981) Biochemistry  20, 4995-5002.  165. Lewis, C D . and Laemmli, U.K. (1982) C e l l 29, 171-181. 166.  Gerace, L . , Blum, A. and Blobel, G. (1978) J . C e l l . B i o l . _79, 546-566.  167. Hancock, R. and Hughes, M . E . (1982) B i o l . C e l l 44, 201-212. 168.  Berezney, R. and Buchholtz, L . A . (1981) Exp. C e l l Res. 132, 1-13.  169. Dijkwell, P . A . , Mullenders, L . H . F . and Wanka, F . (1979) Nucl. Acids Res. 6_, 219-230. 170. Wanka, F . , Mullenders, L . H . F . , Benkers, A.G.M. Pennings, L . J . , Aelen, J . M . A . and Eygensteyn, J . (1977) Biochem. Biophys. Res. Commun. 74, 739-747. 171. Aelen, J . M . A . ,  Opstelten, R . J . G .  and Wanka, F . (1983) Nucl. Acids Res.  11, 1181-1195. 172.  Robinson, S . I . , Nelkin, B.D. and Vogelstein, B. (1982) C e l l 28^, 99-106.  173. Nelkin, B . D . , P a r d o l l , D.M. and Vogelstein, B. (1980) Nucl. Acids Res. 8, 5623-5633. 174.  Cook, P.R. and B r a z e l l , I . A . (1980) Nucl. Acids Res. 8_, 2895-2905.  175. Long, B.H. , Huang, C . - Y . and Pogo, A . 0 . (1979) C e l l 18_, 1079-1090.  137  176. Agutter, P . S . and B l r c h a l l , K. (1979) Exp. C e l l Res. 124, 453-459. 177.  Barrack, E . R . and Coffey, D.S. (1980) J . B i o l . Chem. 255, 7265-7275.  178.  Cousens, L . S . , Gallwitz, D. and A l b e r t s , B.M. (1979) J . B i o l . Chem. 254,  1716-1723.  179. Nelson, N . , Covault, J . and Chalkley, R. (1980) Nucl. Acids Res. j5, 1745-1763. 180.  Covault, J . and Chalkley, R. (1980) J . B i o l . Chem. 255, 9110-9116.  181. Laemmli, U.K. (1970) Nature 227, 680-685. 182.  Davie, J . R . (1982) Anal. Biochem. 120, 276-281.  183. Bhullar, B . S . and Candido, E . P . M .  (1981) Anal. Biochem. 118, 247-251.  184. N o l l , M. (1967) Nature 215, 360-363. 185. McCarty, J r . , K . S . Vollmer, R . T . and McCarty, K . S . (1974) Anal. Biochem. _61, 165-183. 186.  T a t c h e l l , K. and Van Holde, K . E . (1977) Biochemistry  16, 5295-5303.  187. N o l l , M . , Zimmer, S . , Engel, A. and Dubochet, J . (1980) Nucl. Acids Res. 8, 21-42. 188.  Reeves, R. and Candido, E . P . M .  (1980) Nucl. Acids Res. 8, 1947-1963.  189.  Senear, A.W. and Palmiter, R.D. (1981) J . B i o l . Chem. 256, 1191-1198.  190.  Gaubatz, J.W. and Chalkley, R. (1977) Nucl. Acids Res. 4_, 3281-3301.  191. Marushige, K. and Bonner, J . (1966) J . Mol. B i o l . 15_, 160-174. 192. Walker, J . M . , Goodwin, G.H. and John E.W. (1979) FEBS L e t t . 100, 394-398. 193. Walker, J . M . , Hastings, J . R . B . and Johns, E.W. (1977) Eur. J . Biochem. 76_, 461-468. 194. M i t c h e l l , A . R . , Beauchamp, R . S . and Bostock, C . J . (1979) J . M o l . B i o l . 135, 127-149.  138  195. Nebesar, B. (1964) Anal. Chem. 36_, 1961-1965. 196.  Goldknopf, I . L . and Busch, H. (1977) Proc. N a t l . Acad. S c i . U . S . A . 74, 864-868.  197.  Schlesinger, D.H. and Goldstein,  198.  Schlesinger, D . H . , Goldstein,  G. (1975) Nature 255, 423-424.  G. and N i a l l , H.D. (1975)  Biochemistry  14, 2214-2218. 199.  Levinger, L . and Varshavsky, A. (1982) C e l l 28, 375-385.  200. Kleinschmidt, A.M. and Martinson, H . G . (1981) Nucl. Acids Res. 9_, 2423-2431. 201. Wilkinson, K . D . and Audhya, T . K . (1981) J . B i o l . Chem. 256, 9235-9241. 202. Weisbrod, S. , Groudine, M. and Weintraub, H. (1980) C e l l 19_, 289-301. 203. Basler, J . , Hastie, N . D . , P i e t r a s , D . , Matsui, S . I . , Sandberg, A . A . and Berezney. R. (1981) Biochemistry,  20, 6921-6929.  204. Erixon, K. and Ahnstroem, G. (1979) Mut. Res. 59., 257-271. 205. Perry, M. and Chalkley, R. (1981) J . B i o l . Chem. 256, 3313-3318. 206.  Langan, T . A . (1969) Proc. N a t l . Acad. S c i . U.S.A. 64_, 1276-1283.  207. Langan, T . A . (1976) Fed. Proc. 35_, 1623. 208. Weintraub, H . , Palter, K. and Van Lente, F . (1975) C e l l 6_, 85-110. 209. L e v i t t , M. (1978) Proc. N a t l . Acad. S c i . U.S.A. 75_, 640-644. 210. Finch, J . T . , N o l l , M. and Romberg, R.D. (1975) Proc. N a t l . Acad. S c i . U.S.A. 72_, 3320-3322. 211. A l l a n , J . , Hartman, P . G . , Crane-Robinson, C. and A v i l e , F . X . (1980) Nature 288, 675-679. 212.  Savic, A . , Richman, P . , Williamson, P. and Poccia, D. (1981) Proc. N a t l . Acad. S c i . U.S.A. 78_, 3706-3710.  213. Bhullar, B . S . , Hewitt, J . and Candido, E . P . M . (1981) J . B i o l . Chem. 256, 8801-8806.  139  214.  Yoeman, L . C , Olson, M . O . J . , Sugano, N. , Jordan, J . J . , Taylor, C.W. , Starbuck,  215.  W . C and Busch, H. (1972) J . B i o l . Chem. 247, 6018-6023.  DeLange, R . J . , Hooper, J . A . and Smith, E . L . (1972) Proc. N a t l . Acad. S c i . U . S . A . 6j), 882-884.  216.  MacLeod, A . R . , Wong, N.C.W. and Dixon, G.H. (1977) Eur. J . Biochem. 78_, 281-291.  217.  P a r d o l l , D.M. and Vogelstein, B. (1980) Expt. C e l l Res. 128, 466-470.  218.  Gey, C O . , Coffman, W.D. and Kubicek, M.T. (1952) Cancer Res. 12, 264-265.  219.  Leavitt,  J . and Moyzis, T . (1978) J . B i o l . Chem. 253, 2497-2500.  220.  Zlatanova,  J . and Swetly, P. (1978) Nature 276, 276-277.  221. Heifetz, A. and Prager, M.D. (1981) J . B i o l . Chem. 256, 6529-6532. 222.  Reeves, R. and C s e r j e s i ,  P. (1979) J . B i o l . Chem. 254, 4283-4290.  223.  Boffa, L . C , Gruss, R . J . and A l l f r e y , V . G . (1981) J . B i o l . Chem. 256, 9612-9621.  224.  T r a l k a , T . S . , Rabson, A . S . , Thorgeirsson,  U . P . and Tseng, J . S . (1979)  Proc. Soc. Exp. B i o l , and Med. 161, 543-545. 225.  Levy-Wilson, B. (1981) Proc. N A t l . Acad. S c i . U . S . A . 78., 2189-2193.  226.  Barrack, E . R . , Hawkins, E . F . , A l l e n , S . L . , Hicks, L . L . and Coffey, D.S. (1977) Biochem. Biophys. Res. Commun. _7£, 829-836.  227.  Cousens, L . S . and A l b e r t s ,  B.M. (1982) J . B i o l . Chem. 257, 3945-3949.  228.  Sterner, R . , V i d a l i , G. and A l l f r e y , V . G . (1981) J . B i o l . Chem. 256, 8892-8895.  229.  Brotherton,  T.W., Covault, J . , Shines, A. and Chalkley, R. (1981)  Nucl. Acids Res. 9_, 5061-5073. 230.  Reeves, R. and Chang, D. (1983) J . B i o l . Chem. 258, 679-687.  140  231. Samuels, H . H . , Stanley, F . , Casanova, J . and Shao, T . C . (1980) J . B i o l . Chem. 255, 2499-2508. 232.  Simpson, R . T . (1978) C e l l 13_, 691-699.  233. Nelson, D . A . , Perry, M . , Sealy, L . and Chalkley, R. (1978) Biochem. Biophys. Res. Commun. 82, 1346-1353. 234. Baglia, F . A . and Maul, G.G. (1983) Proc. N a t l . Acad. S c i . U.S.A. 80, 2285-2289.  

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
http://iiif.library.ubc.ca/presentation/dsp.831.1-0095859/manifest

Comment

Related Items