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

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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 of 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 accept t h i s t h e s i s as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA September 1983 ©Colin W i l l i a m Hay, 1983 In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y available for reference and study. I further agree that permission for extensive copying of t h i s thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. I t i s understood that copying or publication of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of Biochemistry  The University of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date September 1, 1983 ABSTRACT The reversible acetylation of histones i s thought to play a role i n chromatin processing, including transcription, replication and repair. Studies on the acetyltransferases, responsible for acetylating the nucleosomal core histones, have resulted in characterization of these enzymes. However, very l i t t l e is known about the properties and distr ibution of histone deacetylase. The reversible inhibi t ion of histone deacetylase by butyrate was employed to permit studies on the chromatin-bound histone deacetylase of HeLa ce l l s using endogenous [ 3H]-acetyl labelled polynucleosomes containing the enzyme. These were prepared in the presence of 50mM butyrate and histone deacetylase was assayed upon removal of the inhibi tor . It was found that active enzyme is present only in association with a high molecular weight complex. This deacetylase-containing complex i s re lat ively resistant to digestion with micrococcal nuclease. No act iv i ty i s found on mononucleosomes or oligonucleosomes. Up to 90% of labelled acetyl groups are removed from histone deacetylase complexes incubated i n the absence of butyrate, indicating that denaturation of the histone deacetylase i s kept to a minimum using the techniques developed in this study. Free histones are a poor substrate under these conditions, but histones in mononucleosomes are deacetylated when they are incubated with histone deacetylase complex. Histone deacetylase remains bound to this complex in 1-2 M NaCl and does not dissociate from i t during i t s reaction i i i w i t h a c e t y l a t e d core hisones. Under t y p i c a l nuclease d i g e s t i o n c o n d i t i o n s , the histone deacetylase complex contains 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 of nonhistone p r o t e i n s . Comparison of the p r o t e i n composition of h i s t o n e deacetylase complexes w i t h t h a t of nuclear m a t r i x preparations shows some s i m i l a r i t i e s . Taken together, the r e s u l t s on the chromatographic behaviour, the DNA fragment s i z e s , and the p r o t e i n composition of the deacetylase complex suggest that 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 important i n maintaining i t s s t r u c t u r e and a l s o i n the bin d i n g of the deacetylase i t s e l f to the complex L a t e r research e f f o r t s were concerned w i t h c h a r a c t e r i z a t i o n of the histone deacetylase complex. The e f f e c t of J3-mercaptoethanol and neocuproine on h i s t o n e deacetylase was examined i n view of the f a c t that these reagents are known to d i s r u p t chromosome s c a f f o l d s . HeLa c e l l h i s t o n e deacetylase complex p a r t i a l l y d i s s o c i a t e s i n 10 mM B-mercaptoethanol, 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 . The presence of 10 mM J3-mercaptoethanol during the p a r t i a l m icrococcal nuclease d i g e s t i o n of HeLa c e l l n u c l e i , r e s u l t s i n a very low y i e l d of h i s t o n e deacetylase complex, w i t h a correspondingly l a r g e increase i n the production of small oligonucleosomes and mononucleosomes. Histone deacetylase a c t i v i t y on endogenous l a b e l l e d histone 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 or 10 mM J3-mercaptoethanol or 3 mM neocuproine. The l o s s of h i s t o n e deacetylase a c t i v i t i e s i s not due t o an i n a c t i v a t i o n of the enzyme, but appears to be a consequence of the d i s r u p t i o n of the s t r u c t u r e of the h i s t o n e deacetylase complex. Histone H4 i n histone deacetylase complex prepared from HeLa c e l l n u c l e i by micrococcal nuclease d i g e s t i o n was more i v h i g h l y a c e t y l a t e d than H4 i n bulk nucleosomes. R e s t r i c t i o n enzyme a n a l y s i s of the DNA a s s o c i a t e d w i t h the histone deacetylase complex revealed 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 . In view of these f i n d i n g s , h istone deacetylase appears to be a s s o c i a t e d w i t h a high molecular weight chromatin complex which may be a s i t e of r a p i d a c e t y l group turnover. V TABLE OF CONTENTS Page ABSTRACT 1 1 TABLE OF CONTENTS v LIST OF TABLES x LIST OF FIGURES x i ACKNOWLEDGEMENTS x i i i DEDICATION 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 v i 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 Ce l l Culture Conditions 31 Labelling of HeLa Cells with [ 3H]-acetate 32 Micrococcal Nuclease Digestion 32 Preparation of Histones 33 Isokinetic Sucrose Gradient Centrifugation 34 Column Chromatography 34 Precipitation 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 Si lver Staining of Polyacrylamide Gels 41 v i i Polyacrylamide Gel Scans 41 Nuclear Matrix Preparation 42 Preparation of High Mobility Group Proteins 42 Acid Hydrolysis 44 Amino Acid Analysis 44 Restriction Enzyme Digestion 44 Nucleosome Reconstitution 45 Protein Assays 46 RESULTS 4 7 PART A THE CHARACTERIZATION OF HISTONE DEACETYLASE 47 I Development of a Physiological Assay System for Histone Deacetylase 47 a. Sucrose Gradient Centrifugation 47 b. Isolation of Histone Deacetylase Act iv i ty Using a Bio-Gel A-5m Column 51 c. Substrate Preference of Chromatin-Bound Histone Deacetylase 51 II Distribution of Histone Deacetylase in Chromatin 56 III Effect of Butyrate on the Distribution of Histone Deacetylase 59 IV 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 V Effect of Nuclease Digestion on Histone Deacetylase Distribution 65 v i i i a. Reduced Nuclease Digestion 65 b. Redigestion of Histone Deacetylase Complex 67 c. Extensive Nuclease Digestion 69 VI Characteristics of Chromatin bound Histone Deacetylase.. . . 72 a. Time Course 72 b. Effect of Assay Volume on the Deacetylase Reaction 74 VII Effect of High Mobility 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 PART B CHARACTERIZATION OF HISTONE DEACETYLASE COMPLEX 83 I The Histone Content of Histone Deacetylase Complex 83 II Restriction Enzyme Analysis of Histone Deacetylase Complex DNA 88 III Comparison of Histone Deacetylase Complex and Nuclear Matrix 90 a. Histone Deacetylase Act iv i ty of Nuclear Matrix 92 b. Protein Composition of Histone Deacetylase Complex and Nuclear Matrix 93 IV Effect of fl-mercaptoethanol and Neocuproine on Histone Deacetylase Complex 95 a. Histone Deacetylase Act iv i ty of Treated Complex 95 b. Physical Properties of Treated Complex 99 ix DISCUSSION 105 I Histone Deacetylase Assay 105 II Distribution of Histone Deacetylase in Chromatin 107 III Characteristics of Chromatin Bound Histone Deacetylase.. . . I l l IV Histone Deacetylase Complex 114 BIBLIOGRAPHY 126 LIST OF TABLES Table Page 1. Histone nomenclature 3 2. Inhibition of histone deacetylase by free histone 54 3. Use of mononucleosomes as a substrate for 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 98 8. Effect of fi-mercaptoethanol on histone deacetylase 100 x i LIST OF FIGURES Figure Page 1. The amino acid sequence of trout testis 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 in nuclei 48 7. Isoskinetic sucrose gradient profi les of nucleosomes and histone deacetylase act iv i ty 50 8. Fractionation of micrococcal nuclease digest products on a Bio-Gel A5m column 52 9. Fractionation of microccocal nuclease digest products of HeLa chromatin on a Bio-Gel A-50m column 58 10. Distribution of histone deacetylase in chromatin fragments from butyrate-treated versus untreated Hela ce l l s 60 11. Effect of salt concentration on histone deacetylase distr ibution 62 12. Histone content of histone deacetylase complex prepared in the presence of 2.0 M NaCl 64 13. Effect of reduced nuclease digestion on the distr ibution of histone deacetylase 66 x i i 14. Polynucleosomes redigested with microccocal nuclease retain their endogenous histone deacetylase act iv i ty 68 15. Effect of the extent of nuclease digestion on the distribution of histone deacetylase in chromatin 70 16. Time course of chromatin-bound histone deacetylase 73 17. SDS polyacrylamide gel electrophoresis of HMG 14 and HMG 17 78 18. Effect of calf thymus HMG 14 and HMG 17 on histone deacetylase act iv i ty 81 19. Acid urea gel profi les of histone deacetylase complex and nucleosomes 86 20. Optical scans of histone H4 on acid urea polyacrylamide gels 87 21. Two-dimensional gel electrophoresis of histones 89 22. Restriction enzyme digestion of histone deacetylase complex DNA and genomic DNA 91 23. SDS polyacrylamide gel profi les of HeLa c e l l nuclear matrix preparations 94 24. SDS polyacrylamide gel profi les of fractions from a Bio-Gel A-50m column 96 25. Fractionation of B-mercaptoethanol-treated histone deacetylase complex on a Bio-Gel A-50m column 103 26. SDS polyacrylamide gel profi les of histone deacetylase complex 104 x i i i ACKNOWLEDGEMENTS I wish to express my g r a t i t u d e to my su p e r v i s o r , Dr. Peter 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 s t u d i e s . I appreciate h i s comments, encouragement and the great i n t e r e s t which he always expressed. In a d d i t i o n , I wish to thank the many people i n the Biochemistry Department who have a s s i s t e d my research e f f o r t s and career and the members of Dr. Candido's l a b o r a t o r y who helped create a pleasant atmosphere i n which to work, i n p a r t i c u l a r R. Kay f o r h i s valuable suggestions. I should a l s o l i k e to thank D. Bunyak f o r t y p i n g t h i s t h e s i s . I a l s o wish to acknowledge the f i n a n c i a l support of the Canadian i Medical Research C o u n c i l . DEDICATION to My Parents who have always given t h e i r f u l l support to my education and My Brother, Graeme 1 INTRODUCTION Histones are the major a r c h i t e c t u r a l p r o t e i n s of chromatin as they complex w i t h DNA to form nucleosomes, the fundamental repeating s t r u c t u r a l u n i t of chromatin. Two of each of the hi s t o n e s H2A, H2B, H3 and H4 combine to form a core, around which are wrapped 146 bp of DNA. The re g i o n of a DNA strand which j o i n s adjacent nucleosome core p a r t i c l e s i s termed the l i n k e r DNA. The histones can undergo a v a r i e t y of p o s t - t r a n s l a t i o n a l m o d i f i c a t i o n s i n c l u d i n g a c e t y l a t i o n , phosphorylation, methylation 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 histones at s p e c i f i c l y s y l residues i n the amino-terminal 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 of a hi 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, which reduces the i o n i c i n t e r a c t i o n between the histones and the n e g a t i v e l y charged phosphate backbone of the DNA and i s thought to a l t e r the conformation of the nucleosome, p o s s i b l y thereby i n c r e a s i n g the a c c e s s i b i l i t y of the DNA to enzymes or r e g u l a t o r y p r o t e i n s . The r a p i d a d d i t i o n and removal of histone a c e t y l groups i s brought about by the a c t i o n of s e v e r a l h istone a c e t y l t r a n s f e r a s e s and his t o n e deacetylase, r e s p e c t i v e l y . 2 I The Histones Histones were f i r s t recognized in 1884 (1) but i t was not unt i l recently that the function of this 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 in the l i terature; as a result , 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 sources of the histones are shown i n the figure legends. An examination of these sequences reveals the presence of dist inct domains. 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 sites of interaction with DNA. The carboxy-terminal portions of the nucleosomal core histones contain most of the hydrophobic and acidic amino acids, and possess a globular tert iary structure. Histone HI also contains an uneven distribution of residues and consists of three regions; however, the polarity i s the opposite to that of the core histones, the carboxy-terminal half having a ratio of basic to acidic residues of 15:1. There i s a short hydrophobic region at the re lat ive ly 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 their amino acid sequences, implying that precise binding of histones to each other and to the DNA is absolutely essential in the functioning of chromatin. Histones H3 and H4 are among the most conserved of a l l proteins with histone H4 from the cow and the pea differing by only two conservative 3 Table 1. Histone nomenclature Contemporary Alternative Molecular Amino -Nomenclature Nomenclature Weight Terminus H 1 v e r y lysine r i c h , I , F l , KAP 21,500 Acetyl-serine H 2 A lysine r i c h , I l b l , F2a2, LAK 14,004 Acetyl-serine H 2 B lysine r i c h , IIb2, F2b, KAS 13,774 Proline H 3 arginine r i c h , III , F3, ARE 15,324 Alanine H A arginine r i c h , IV, F2al, GRK 11,282 Acetyl-serine 4 AcMa-Glu-Val-Ma-Pro-Ala-Pro-Ala-Ala-Ala-Ala-Pro-Ala-Lys-Ala-1 5 10 15 Pro-Lys-Lys-Lys-Ala-Ala-Ala-Lys-Pro-Lys-Lys-Ser-Gly-Pro-Ala-20 25 30 Val-Gly-Glu-LeiJ-Ala-Gly-Lys-Ala-Val-Ala-Ala-Ser-Lys-Glu-Arg-35 40 45 Ser-Gly-Val-Ser-Leu-Ala-Ala-Leu-Lys-Lys-Ser-Leu-Ala-Ala-Gly-50 55 60 Gly-Tyr-Asp-Val-Glu-Lys-Asn-Asn-Ser-Arg-Val-Lys-Ile-Ala-Val-65 70 75 Lys-Ser-Leu-Val-Thr-Lys-Gly-Thr-l£U-Val-Glu-Thr-Lys-Gly-Thr-80 85 90 Gly-Ala-Ser-Gly-Ser-Phe-Lys-Leu-Asn-Lys-Lys-Ala-Val-Glu-Ala-95 100 105 Lys-Lys-Pro-Ala-Lys-Lys-Ala-Ala-Ala-Pro-Lys-Ala-Lys-Lys-Val-110 115 120 Ala-Ala-Lys-Lys-Pro-Ala-Ala-Ala-Lys-Lys-Pro-Lys-Lys-Val-Ala-125 130 135 Ala-Lys-Lys-Ala-Val-Ala-Ala-Lys-Lys-Ser-Pro-Lys-Lys-Ala-Lys-140 145 150 Lys-Prc>-Ma-Thr-Prci-Lys-Lys-Ma-Ma-Lys-Ser-Pro-Lys^Lys-Ala-155 160 165 Thr-Lys-Ala-Ala-Lys-Pro-Lys-Ala-Ala-Lys-Pro-Lys-Lys-Ala-Ala-170 175 180 Lys-Ser-Pro-Lys-Lys-Val-Lys-Lys-Pro-Ala-Ala-Ala-Lys-Lys-COOH 185 190 194 Figure 1. The amino a c i d sequence of trout t e s t i s histone HI (216) Ac-Ser-Gly-Arg-Gly-Lys-Gln-Gly-Gly-Lys-Ala-Arg-Ala-Lys-Ala-Lys-1 5 10 15 Thr-Arg-Ser-Ser-Arg-Ala-Gly-Leu-Gln-Phe-Pro-Val-Gly-Arg-Val-20 25 30 His-Arg-Leu-Leu-Arg-Lys-Gly-Asn-Tyr-Ala-Glu-Arg-Val-Gly-Ala-35 40 45 Gly-Ala-Pro-Val-Tyr-Leu-Ala-Ala-Val-Leu-Glu-Tyr-Leu-Thr-Ala-50 55 60 Glu-Ile-Leu-Glu-Leu-Ala-Gly-Asn-Ala-Ala-Arg-Asp-Asn-Lys-Lys-65 70 75 Thr-Arg-Ile-Ile-Pro-Arg-His-Leu-Gln-Leu-Ala-Ile-Arg-Asn-Asp-80 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-Thr-110 115 120 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-Lys-1 5 10 15 Lys-Ala-Val-Thr-Lys-Ala-Gln-Lys-Lys-Asp-Gly-Lys-Lys-Arg-Lys-20 25 30 Arg-Ser-Arg-Lys-Glu-Ser-Tyr-Ser-Val-Tyr-Val-Tyr-Lys-Val-Leu-35 40 45 Lys-Gln-Val-His-Pro-Asp-Thr-Gly-Ile-Ser-Ser-Lys-Ala-Met-Gly-50 55 60 Ile-Met-Asn-Ser-Phe-Val-Asn-Asp-Ile-Phe-Glu-Arg-Ile-Ala-Gly-65 70 75 Glu-Ala-Ser-Arg-Leu-Ala-His-Tyr-Asn-Lys-Arg-Ser-Thr-Ile-Thr-80 85 90 Ser-Arg-Glu-Ile-Gln-Thr-Ala-Val-Arg-Leu-Leu-Leu-Pro-Gly-Glu-95 100 105 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 2 N-Ala-Arg-Thr-Lys-Gln-Thr-Ala-Arg-Leu-Ser-Thr-Gly-Gly-Lys-Ala-1 5 10 15 Pro-Arg-Lys-Gln-Leu-Ala-Thr-Lys-Ala-Ala-Arg-Lys-Ser-Ala-Pro-20 25 30 Ala-Thr-Gly-Gly-Val-Lys-Lys-Pro-His-Arg-Tyr-Arg-Pro-Gly-Thr-35 40 45 Val-Ala-Leu-Arg-Glu-Ile-Arg-Arg-Tyr-Gln-Lys-Ser-Thr-Glu-Leu-50 55 60 Leu-Ile-Arg-Lys-Leu-Pro-Phe-Gln-Arg-Leu-Val-Arg-Glu-Ile-Ala-65 70 75 Gln-Asp-Phe-Lys-Thr-Asp-Leu-Arg-Phe-Gln-Ser-Ser-Ala-Val-Met-80 85 90 Ala-Leu-Gln-Glu-Ala-Cys-Glu-Ala-Tyr-Leu-Val-Gly-Leu-Phe-Glu-95 100 105 Asp-Thr-Asn-Leu-Cys-Ala-Ile-His-Ala-Lys-Arg-Val-Thr-Ile-Met-110 115 120 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-Ala-1 5 10 15 Lys-Arg-His-Arg-Lys-Val-Leu-Arg-Asp-Asn-Ile-Gln-Gly-Ile-Thr-20 25 30 Lys-Pro-Ala-Ile-Arg-Arg-Leu-Ala-Arg-Arg-Gly-Gly-Val-Lys-Arg-35 40 45 Ile-Ser-Gly-Leu-Ile-Tyr-Glu-Glu-Thr-Arg-Gly-Val-Leu-Lys-Val-50 55 60 Phe-Leu-Glu-Asn-Val-Ile-Arg-Asp-Ala-Val-Thr-Tyr-Thr-Glu-His-65 70 75 Ala-Lys-Arg-Lys-Thr-Val-Thr-Ala-Met-Asp-Val-Val-Tyr-Ala-Leu-80 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). Similarly , histone H3 from the cow and the pea dif fer 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). They can be considered as evolutionary hybrids since their histone-histone binding regions are highly conserved while other regions are more variable. 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 ver HI and trout HI (11). II Histone Modifications The histones are subject to a variety of post-translational modifications which a l ter the charge and structure of amino acid residues i n the highly basic amino-terminal portions of the core histones and in both the amino- and carboxy-terminal portions of HI. The modifications involve group substitutions such as acetylation, phosphorylation, methylation and ADP-ribosylation, and are sequence specif ic . 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 dist inct forms of enzymatic histone acetylation. The 10 f i r s t type results in 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 in the cytoplasm very shortly after synthesis and i s irrevers ible . 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 specific ly sy l residues in the basic amino-terminal portion of the nucleosomal core histones, H2A, H2B, H3 and H4. N 6 -acetylation converts a basic l y sy l residue to a neutral acetyl-lysine and thereby reduces the net positive charge of the amino-terminal region by one. Histones H2B, H3 and H4 contain 4 sites of acetylation while H2A contains a single lysy l residue which can be reversibly acetylated. The sites of acetylation in histone H4 are lysy l residues 5, 8, 12 and 16. Thus histones H2B, H3 and H4 can exist in mult i -acetylated forms with the overall positive charge in the amino-terminal region varying from +5 to +1. The acetyl groups on the lysy l residues turn over rapidly, 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 and histone deacetylase, respectively. Attempts have been made to isolate these enzymes from various tissues and their characteristics w i l l be discussed in a later section. The neutralization of the positive charges at the amino-terminal portions of the histones by acetylation i s thought to reduce the ionic interactions between the nucleosomal core histones and the negatively 11 charged DNA which is wrapped around the histones. Reduced ionic interactions between the histones and the DNA could result in an altered nucleosome conformation and increased access ibi l i ty 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 . folding of the nucleosome chain into the 30 nm fibre (91). As a consequence, histone acetylation has been considered as a possible mechanism for altering chromatin structure and regulating transcriptional act iv i ty , DNA replication and DNA repair. The possible functions of histone acetylation wi l be reviewed in a later section. b. Methylation Histones H3 and H4 can undergo methylation at the e-amino group of specific l y sy l residues after histone synthesis (14). The methylation of a lysy l residue i s irreversible and does not result i n a change in charge, but rather produces an increase in basicity and hydrophobicity. Both histones H3 and H4 are methylated i n the amino-terminal region, H4 containing one methylation site at l y sy l 20 (15,16,17). Histone H3 has four lysy l residues which are methylated d i f ferent ia l ly , l y sy l residues 9 and 27 are the two most common sites 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 in 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 substrate s p e c i f i c i t y and even ac i d - i n s o l u b l e proteins of c a l f thymus nu c l e i can be methylated i n v i t r o to form mono- and dimethyllysines (21). The modified l y s y l residues can contain one to three methyl groups and i n many cases i t i s the dimethyllysine which predominates (22). For example, histone H4 i s modified mainly as the N 6-dimethyllysine i n E h r l i c h a s c i t e s tumour c e l l s (23), c a l f thymus (24), carp t e s t i s (25) and trout t e s t i s (15). Likewise, histone H3 i s modified mainly as N 6-dimethyllysine, although a l l three forms are often present. However, methylation of the susceptible l y s y l residues need not occur as about one quarter of c a l f thymus histone H3 i s unmodified and c a l f thymus H4 contains free l y s y l residues, as well as modified forms, at 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 of histone methylation remains unresolved, as methylation does not corr e l a t e with an increase i n DNA t r a n s c r i p t i o n , DNA synthesis or with histone synthesis (23). The changes i n b a s i c i t y and hydrophobicity r e s u l t i n g from histone methylation suggest that changes i n the i n t e r a c t i o n s between histones and other chromatin components may occur, perhaps as a requirement f o r chromatin condensation and mitosis (22,26,27). c. Phosphorylation Phosphorylation of serine and threonine residues i s a major re v e r s i b l e p o s t - t r a n s l a t i o n a l modification of a l l histones. The nucleosomal core histones are phosphorylated i n the basic amino-terminal portion and histone HI can be phosphorylated i n both the amino- and carboxy-terminal regions. Histone H3 i s phosphorylated at s e r y l residues 10 and 28 while histone H2B can be phosphorylated at s e r y l residues 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+2 dependent (29). Some kinases are specific for certain histones, e.g. HeLa cel ls contain a nuclear kinase which only phosphorylates histone H3 (30) and calf thymus also contains an H3 specific 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 ives (32). The function of histone phosphorylation remains unclear. There is evidence of a relationship between phosphorylation of histones HI and H3 and chromatin condensation prior to mitosis (32,33,34,35,36). N-phosphorylation can also occur at the h i s t idy l residue of histone H4 to form N 3-phosphorylhistidine (37) and at the e-amino of a lysy l residue in HI. Two cyc l ic nucleotide independent kinases capable of N-phosphorylation have been isolated from regenerating l i ver (37) and Walker 256 carcinosarcoma cel l s (38). It has been suggested that the N-phosphorylation of histones HI and H4 i s involved with DNA replication (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 in histone HI 14 of rat l i ver (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 in length (46). The ADP-ribose polymers are formed by the enzyme poly (ADP-ribose) polymerase which has been purified to near homogeneity from rat l i ver (47), bovine thymus (48), ca l f thymus (49,50), pig thymus (51) and ascites tumour ce l l s (52). 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 localized within the internucleosomal l inker regions of HeLa c e l l chromatin (53). However, i t i s not equally distributed in HeLa c e l l chromatin as i t is present only on certain 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 particular tissue i s thought to partly depend upon the act iv i ty of poly (ADP-ribose) glycohydrolase (55). Phosphodiesterase can also degrade the ADP-ribose polymer (56). Various functions have been suggested for ADP-ribosylation, including the regulation of ce l lu lar growth (57), gene expression (58), DNA synthesis (59) and DNA repair (60). ADP-ribosylation is also thought to be involved in chromosome condensation (61,62). e. Ubiquitin Attachment Histone H2A can be modified by the covalent attachment of one molecule of ubiquit in. The ubiquitin moiety i s attached through a glycylglycine bridge to lysine 119 of H2A via an isopeptide bond (196) and the modified 15 histone i s referred to as A24 or uH2A. Ubiquitin is a very highly conserved protein; human and bovine ubiquitin are identical and contain 74 amino acids (197,198). The precise function of this modification of H2A is unclear. A selective arrangement of A24 has been observed in the Drosophila genome. Approximately one in two nucleosomes of the transcribed copia and heat shock protein 70 genes in unshocked cultured cel ls contains A24 whereas less than one in 25 nucleosomes of nontranscribed sate l l i te DNA contains A24 (199). On the other hand, the replacement of H2A in chromatin with A24 does not al ter the structure of individual nucleosomes or the digestion pattern of chromatin with DNase I (200). Ubiquitin may fac i l i ta te histone degradation as i t i s identical 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 five histones contain fucose so that a minimum of 1 in 1000 nucleosomes contains a fucosylated H2A molecule (63). A l l five histones also contain mannose. If these results are duplicated in other organisms, histones w i l l have to be c lass i f ied as glycoproteins. I l l Review of Histone Acetylation The transfer of acetate from acetyl-CoA to the e-amino groups of 16 specific ly sy l residues of the core histones i s catalyzed by several histone acetyltransferases. Histone binding is a prerequisite for acetyl-CoA binding (64). The different histone acetyltransferases are c lass i f ied primarily by their subcellular local izat ion and their substrate preference with respect to histone class. Histone acetyltransferase A from rat 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 in solution (65). Acetyltransferase A from calf thymus nuclei i s released from chromatin by mild micrococcal nuclease digestion and i t acetylates histone H3 least well when the latter i s present in nucleosomes (66). Histone acetyltransferase B i s a cytoplasmic enzyme which i s specific for histone H4 (67). A third isozyme, acetyltransferase DB (DNA binding) has been prepared from bovine lymphocytes (68) and from the nuclei of African green monkey kidney ce l l s infected with SV40 (69). The enzyme has a similar histone preference as acetyltransferase A, but has a very high af f in i ty 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 distribution of this enzyme within chromatin. Histone deacetylase act iv i ty has been shown i n cal f thymus (70,71) and in rat l i ver and Novikoff hepatoma (72). Attempts to purify histone deacetylase have been largely unsuccessful as i so lat ion results in losses in substrate speci f ic i ty and enzyme act iv i ty . For example, the part ia l purif ication of histone deacetylase from calf thymus nuclei results in significant change in the ratio of the deacetylation rates of histones H3 and H4 and a complete loss of deacetylase act iv i ty against chromatin-bound histones, the physiological substrate (73). An 17 acidic protein which removes acetyl groups from free histones H4 and H3 has been part ia l ly purified from calf 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 calf thymus chromatin has been used to release histone deacetylase act iv i ty in experiments where chemically acetylated histones were used as substrate (76) . The histone deacetylase solubil ized from calf thymus chromatin could deacetylate added free acetyl-labelled histones, whereas the chromatin-bound histone deacetylase reacted poorly with this substrate. Histone deacetylase act iv i ty has been located specif ical ly in the nuclei of Physarum and sonication in high salt i s required to release the enzyme (77) . The enzyme has not been purified further. Mill imolar concentrations of butyrate reversibily inhibi t histone deacetylase by acting as a non-competitive inhibitor (78,79,80). Butyrate does not affect the rate of transfer of acetyl groups from acetyl-CoA to the histone lysy l residues by the acetyltransferases. As a consequence, butyrate causes the dynamic equilibrium between acetylation and deacetylation to shi f t , resulting in 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 in the positive charge of the acetylated core histones may result in a concomitant increase i n the access ibi l i ty of the DNA to specific nuclear enzymes or regulatory proteins. Increased histone acetylation has been reported to increase the access ibi l i ty of histone H3 to the nuclear calcium dependent H3 kinase (82). 18 High levels of histone acetylation have been associated with transcriptionally competent chromatin in fractionation experiments (83,84,85,86,87) and with nuclei that are transcriptionally very active, such as those of yeast (88) and the macronuclei of Tetrahymena (89). The acetate turnover in histone H4 during the c e l l cycle has been examined i n Physarum macroplasmodia and an increase of [ 3H]-acetate incorporation i s observed in S phase (77). Relatively high incorporations of acetate were noted i n tetraacetylated H4 in S phase and late G2 phase (M + 7 hours), correlating with high levels of tetraacetylated H4 at these times. It has also been suggested that histone acetylation may fac i l i ta te DNA replication or repair since increasing the level of histone acetylation i n human fibroblasts leads to an increase in UV-induced DNA repair (90). Recently i t has been proposed that a l l of the chromatin in 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 characteristic of chromatin structure i s the t ightly packed state of the DNA; up to a 10,000 fold contraction i s required to fold 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 structural 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 in chromatin (96) and in isolated nucleosomes (97) using zero-length crosslinkers (98) have shown that H2A-H2B and H3-H4 dimers, and (H3-H4)2 tetramers are preferentially crosslinked. Histone H2B has been found to contain separate binding sites for H2A and H4. The results are consistent with the formation of two tetrameric complexes of H2A, H2B, H3, and H4 within the nucleosome (99). Circular dichroism, laser Raman (101) and infrared spectroscopy (102) studies have shown that the core histones possess a high content of a helix with l i t t l e or no 6 sheet. The carboxy-terminal portions of the histones are involved in histone-histone interactions and are generally considered to be in a globular conformation (103). The spectral properties of a fluorescent group attached to methionine 84 in histone H4 has indicated an apolar environment for this group within nucleosomes (100). In contrast to the carboxy-terminal portions of the histone, the amino-terminal portions exist in a random c o i l conformation in 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 arginine-specific protease digestion of intact chromatin showed that one or more sites in the amino-terminal region of each core histone are accessible on the nucleosome surface (107). Digestion of nucleosomes with trypsin 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 is complexed with 146 bp of DNA (105,209) which wraps around the histones in 1 3/4 turns in a left 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 particle by digestion with DNase I (110). The sites of maximum access ibi l i ty to DNase I occur at intervals of 10.4 bp in the nucleosomes (111). This i s the same as the pitch 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 sites 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 inker 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 inker DNA varies greatly in length in different organisms. The variable length of the linker DNA results i n different nucleosomal repeat length which vary from 154 bp in 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 thin filaments of l inker 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 in the cleavage of the l inker DNA and the production of mononucleosomes and polynucleosomes containing varying numbers of nucleosome repeat units . 21 c. The Position 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 inker 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 in 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 rise to structural rearrangements indicating nucleosome sl iding (125). The pitch of the DNA helix in a nucleosome is 2.8 nm (126) and this i s very similar 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 ike ly involved in the condensation of the nucleofilament and i t s basic and hydrophilic nature suggests that i t interacts ionica l ly with the DNA (211). d. The Shape of the Nucleosome Low-angle neutron scattering studies of nucleosomes in solution have shown that the DNA is on the outside of the particle (127,128). The DNA i s 2.2 nm thick and has a pitch 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 di f fract ion 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 laser l ight scattering and NMR studies (129) suggest a 22 substructure consisting of two face to face disc-shaped heterotypic histone tetramers each surrounded by a DNA annulus. The core nucleosome particles have a sedimentation coefficient of 11 S (131). e. Higher Order Packing The 11.0 nm nucleosome represents the most extended form of chromatin and further compaction results in the formation of a 30.0 nm diameter fibre (132,133,134). Histone HI i s thought to play a role in 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 particles 30.0 nm i n diameter (135). Alternatively, nucleosomes are thought to be arranged in a solenoid to form a 25-30 nm thick chromatin f ibre (136,133,137). At a length of about 50 nucleosomes, polynucleosomes fold to form a fibre which corresponds to 8-10 turns of a solenoid with 5-6 nucleosomes per turn (138). It i s s t i l l unclear how the chromatin fibre is organized to form metaphase chromosomes. However, X-ray di f fract ion studies of chicken erythrocyte chromosomes show a 40 nm periodicity due to a structure that i s direct ly 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 structural changes in chromatin occur in vivo, in association with functional changes. The nucleosomes of transcriptionally active chromatin are more rapidly excised by micrococcal 23 nuclease (140,141) and their DNA is more rapidly degraded by DNase I (142,143,144). The sensit ivi ty of the ovalbumin gene in oviduct nuclei to micrococcal nuclease and to endogenous nuclease has been shown to para l l e l the estrogen dependent transcription of the ovalbumin gene in the immature chicken oviduct (145,146). Furthermore, nucleosomes are highly heterogeneous in 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 actively transcribed chromatin (147 - 152). Other non-histone chromatin proteins have been isolated from swine skeletal muscle, l i ver and ventricle . These proteins have molecular weights of 35-45K and appear to be important in the maturation of mRNA (153). The nucleosome repeat length of chromatin can also vary. 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 blastula stage, the repeat length has shortened considerably (212). The question of how nucleosomes are arranged relative to DNA sequences i s s t i l l unresolved. Nucleosomes could be arranged randomly or alternatively, they might be located i n unique positions with respect to DNA primary structure. The lat ter arrangement has been termed "phasing" and could theoretically be triggered by sequence specific DNA binding proteins (154). An example of precise register between nucleosomes and DNA has been reported for tRNA genes i n chicken embryos, where transcription of the tRNA genes examined depended on nucleosome position (155). An ordered nucleosome alignment has been reported in the hsp (heat shock protein) 70 24 and hsp 80 genes in Drosophila (156) and nucleosomes are precisely positioned in the non-transcribed spacer of the histone genes in Drosophila (157). Nucleosome phasing can also give rise 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 ight chain of mouse l i v e r , whereas this ordered arrangement i s lost i n the active form of the gene in a myeloma (158). VI Nuclear Skeletal Structure It i s becoming evident that chromatin in the interphase nucleus i s 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 dissociation of the histones, the DNA forms a number of independently constrained loops or domains which exhibit negative superhelicity. The lengths of the loops varies from 60 Kbp in mouse L ce l l s to 150 Kbp i n bovine l i ver 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 structural 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 ver 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 is not mediated by DNA-RNA hybrids and may involve direct protein-DNA contacts. A variety of predominant polypeptides are found in nucleosome-free nuclear matrixes (163,164,165). Several proteins have molecular weights which correspond closely to the molecular weights of the lamins, proteins localized i n the peripheral lamina of the nucleus (166). The lamina i s visualized in the electron microscope as a homogeneous structure lying between the nuclear membrane and the peripheral chromatin. Taken together, these results have led to the proposal that in interphase, chromosomal DNA is attached to the peripheral lamina of the nucleus as loops which are compacted into nucleosomes and higher order conformations which extend into the nuclear inter ior (167). The question of whether chromatin processing i s associated with nuclear skeletal structures has been investigated mainly by examining nuclear matrix preparations. Pulse-labell ing studies with thymidine indicate that DNA i s synthesized on or near the nuclear matrix i n rat l i ver and that new DNA migrates outwards (168). Similar results have been found for bovine l i v er (169,170). Studies on DNA replication have been carried out on Plasmodia of Physarum polycephalum, in which a l l of the nuclei of a single Plasmodium go through mitosis and S phase i n complete synchrony. Pulse-labelling experiments indicate that the origins of replicons are bound to the nuclear matrix during the entire c e l l cycle and that 26 replication points are bound to the matrix in S phase but are released from the binding sites after termination of replication (171). This work implies that the nuclear matrix contains specific binding sites through which chromatin moves during repl icat ion. There i s growing evidence for a non-random distribution of the unique sequences within chromatin loops, actively transcribed genes being preferentially associated with the nuclear matrix. For example, the ovalbumin gene is associated with the nuclear matrix in chicken oviduct c e l l s , but not in chicken l i ver ce l l s in which the gene i s transcriptionally inactive (172). Similarly , nuclear matrix DNA from SV40 infected 3T3 ce l l s has an enrichment of the SV40 sequences relative to the total DNA (173). The a, J3 and X globin genes in HeLa ce 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 transcription i s associated with the nuclear matrix is further supported by the observation that hnRNA is tenaciously bound to the matrix of Friend c e l l nuclei (175) and nascent hnRNA transcripts are formed in the nuclear matrix of the rat endothelium (176). The nuclear matrix has also been proposed as a site 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 enzyme act iv i t ies in these structures. 27 VII The Present Investigation Histone acetylation has been the focus of considerable attention because of i t s possible role in chromatin processing, including transcription, replication and repair. The acetyltransferases, responsible for acetylating the nucleosomal core histones, have been f a i r l y well characterized. On the other hand, very l i t t l e is known about the properties and distribution of histone deacetylase. The reversible inhibitory effect of butyrate on histone deacetylase act iv i ty (78,79,80) has been used to fac i l i ta te investigations into the kinetics 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 in this thesis was to study histone deacetylase from HeLa c e l l nuclei (a human c e l l l ine) and to develop a physiologically meaningful assay system which would provide information on the interaction between histone deacetylase and i t s normal substrate i . e . i n vivo assembled histones in chromatin. Another major goal was to determine the distribution of the enzyme within chromatin. Advantage was taken of the non-competitive inhibit ion of histone deacetylase to develop procedures for the isolat ion of histone deacetylase i n association with endogenous [ 3H]-acetate labelled chromatin. The reversible nature of the butyrate inhibi t ion of histone deacetylase allowed enzyme act iv i ty to be assayed after removal of the sodium butyrate from the system. As mentioned ear l i er , histone deacetylase preparations commonly lose 28 the a b i l i t y to d eacetylate chromatin-bound histones and t h i s has s e v e r e l y l i m i t e d s t u d i e s on the enzyme. The i n v i v o assembled histone deacetylase-chromatin complex described i n t h i s report permitted the e l u c i d a t i o n of many of the c h a r a c t e r i s t i c s of histone deacetylase. Free histones were found to be a poor s u b s t r a t e , but histones 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 deacetylated r a p i d l y . Up to 90% of the l a b e l l e d a c e t y l groups were removed from histones i n chromatin by histone deacetylase, showing that denaturation of the enzyme had been kept to a minimum using the i s o l a t i o n techniques developed i n t h i s study. The d i s t r i b u t i o n of histone deacetylase i n chromatin was examined by d i g e s t i n g endogenous acetate 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 nuclease which p r e f e r e n t a i l l y d i g e s t s chromatin at the l i n k e r DNA to y i e l d mixtures of mononucleosomes and oligonucleosomes. Chromatin f r a c t i o n s were i s o l a t e d i n the presence of butyrate from such d i g e s t s by i s o k i n e t i c gradient 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 deacetylase a c t i v i t y a f t e r the removal of the i n h i b i t o r . A c t i v e histone deacetylase was found only i n a s s o c i a t i o n w i t h a high molecular weight, nuclease r e s i s t a n t complex and no a c t i v i t y was found on mononucleosomes or oligonucleosomes. C h a r a c t e r i z a t i o n of the high molecular weight histone deacetylase complex became the primary concern of l a t e r research 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 nuclease d i g e s t i o n c o n d i t i o n s , the histone deacetylase complex contained 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 bp, and a v a r i e t y of non-histone p r o t e i n s . 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 the maintainance of the s t r u c t u r e and the p r o t e i n composition and p h y s i c a l p r o p e r t i e s of t h i s h i s t o n e deacetylase complex were compared w i t h those of 29 nuclear m a t r i x . The s o l u b l e chromatin p r e p a r a t i o n described i n t h i s t h e s i s has provided a u s e f u l system f o r studying histone deacetylase and answering some of the questions regarding 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 counting s c i n t i l l a n t . b i s - a c r y l a m i d e : N,N'-Methylene-bis-acrylamide. bp: base p a i r BSA: Bovine serum albumin DNA: Deoxyribonucleic a c i d DNase I : Deoxyribonuclease 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 ' - t e t r a c e t l c a c i d HMG: High 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: T r i t i r a c h i u m album protease RNA: R i b o n u c l e i c a c i d RNase: Ribonuclease SDS: Sodium dodecyl sulphate TEMED: N,N,N',N'-Tetramethylethylenediamine T r i s : Tris(hydroxymethyl)aminomethane M a t e r i a l s A l l chemicals were obtained commercially and were of the highest 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; Hae l l l from BRL; TaqI from BRL; Mspl from BRL;proteinase K from Sigma; BSA for restr ict ion enzyme digests from BRL; BSA for histone deacetylase complex precipitation 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 solubi l izer from Amersham/Searle; Bio-Gel A-5m and Bio-Gel A-50m from Bio-Rad Laboratories; HeLa ce l l s from The American Type Culture Collection; feta l bovine serum from GIBCO; Eagle's minimum essential medium from GIBCO, penicill in/streptomycin from GIBCO; Amphotericin from GIBCO and [ 3H]-acetate (sodium salt) spec. act. >500 mCi/mmol from New England Nuclear. C e l l Culture Conditions HeLa ce l l s derived from the strain orig inal ly cultured by Gey et a l . (218) were grown in monolayer culture and passaged in a medium comprising 95% Eagle's minimum essential medium containing non-essential amino acids, 5% Fetal bovine serum, 100 units/ ml of penicillin-streptomycin and 0.00013% Amphotericin. Cultures of 100 ml were grown in 3.8 1 ro l l er bottles at 37°C. 32 L a b e l l i n g of HeLa C e l l s with [ 3H]-acetate HeLa c e l l s , grown to near confluence, were harvested using 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 at 700 xg f o r 7 minutes and taken up i n complete medium. Butyric acid (neutralised with NaOH) was added to a f i n a l concentration of 10 mM and the c e l l s were incubated f o r 15 minutes at 37°C with shaking to allow the butyrate to enter the c e l l s and i n h i b i t histone deacetylation. The c e l l s were then l a b e l l e d i n a 2 hour incubation with 150 uCi/ml of [ 3H]-acetate (sodium s a l t , spec. act.>500 mCi/mmol; New England Nuclear) and centrifuged at 700 xg f o r 7 minutes. The c e l l s were then taken up i n fresh medium containing 10 mM butyrate and 8 mM sodium acetate and excess l a b e l was chased out i n a 6 hour incubation. A f t e r c e n t r i f u g a t i o n at 2000 xg f o r 10 minutes, the c e l l s were frozen r a p i d l y and stored at -80°C. Micrococcal Nuclease Digestion Frozen (-80°C) c e l l p e l l e t s were homogenized gently i n 4 volumes of TMKS (0.05 M T r i s - H C l , pH 7.4, 3 mM MgCl 2, 25 mM KC1, 0.25 M sucrose, 40 mM sodium butyrate, 0.1 mM PMSF) i n a glass-Teflon hand homogenizer. 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. The r e s u l t i n g nuclear p e l l e t was suspended i n 3 volumes of TMKS made 1 mM i n CaC^, a 10 u l aliq u o t of the nuclear suspension was mixed with 0.99 ml of a s o l u t i o n containing 5 M urea and 2 M NaCl, and the absorbance at 260 nm was measured, with corrections f o r t u r b i d i t y . Micrococcal nuclease was added to the nuclear 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 25°C. Digestion was terminated by the addition of 0.1 M EGTA (pH 7.4) to a f ina l concentration of 3 mM, the nuclear suspension was chi l led on ice , centrifuged at 5,000 xg for 10 minutes and the supernatant collected. The nuclei were lysed with Buffer A (10 mM Tris-HCl pH 7.4, 0.2 mM EDTA, 50 mM sodium butyrate), containing 0.1 mM PMSF, on ice 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 ce l l s by extraction with ^SO^ (191). Frozen ( -80°C) c e l l pellets (10 s-10 s ce l l s ) were homogenized i n 2 ml TMK (10 mM T r i s - H C l , pH 8.0, 2 mM MgCl 2 , 25 mM KC1) using a glass-Teflon hand homogenizer. The sample was centrifuged at 3,000 xg for 10 minutes and the pellet was rehomogenized and centrifuged as before. The nuclear pellet was homogenized in 2 ml of 10 mM Tris-HCl (pH 7.4), layered onto 3 ml of 1 M sucrose in 10 mM Tr i s -HCl , (pH 7.4) and centrifuged at 16,000 xg for 20 minutes. The gelatinous chromatin pellet was extracted by the addition of 0.3 ml of 0.4 N ^SO^, for 15 minutes on ice . 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 -20°C and collected by centrifugation at 5,000 xg for 10 minutes. The histone pellet was dissolved in 0.3 ml of 0.1 M acetic ac id , dialyzed against the same solution for 2 hours and lyophil ized. 34 Isokinetic Sucrose Gradient Centrifugation Nucleosomes were sedimented through 10-28.3% (w/v) isokinetic sucrose gradients (184) prepared for a particle density of 1.5 g/ml (185). The gradients contained 10 mM Tr i s -HCl , (pH 7.4), 50 mM butyrate, 0.1 mM PMSF and centrifugation was carried out in 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 part ia 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 ml/h. The column contained buffer A (10 mM T r i s - H C l , pH 7.4, 0.7 mM EDTA, 50 mM sodium butyrate) and a l l procedures were performed at 4°C. Precipitation 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 in NaCl by the addition of 4.0 M NaCl. Precipitation was carried out on ice for 30 minutes and the pellet collected by centrifugation at 16,000 xg for 15 minutes. The supernatants were aspirated and the pellets 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 act iv i ty was determined by measuring the release of [ 3H]-acetate from [ 3 H]-acetyl-labelled histones. Four different procedures were employed depending upon the methods used to prepare the enzyme. For assays of whole nuclei , HeLa nuclei were isolated as described above (see Micrococcal Nuclease Digestion) except that butyrate was omitted. The nuclei were suspended in 2 volumes of Buffer C (10 mM Tr i s -HCl , pH 7.8, 3 mM MgCl 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 [ 3 H]-acetyl-labelled histones (usually 40,000 cpm) at 22°C for 4 hours, and the reaction was terminated by boil ing for 1 minute. Histone deacetylase act iv i ty i n unlabelled nucleosome fractions, obtained by isokinetic gradient centrifugation, was also measured by the release of [ 3H]-acetate from added free labelled histones. 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 isokinetic sucrose gradients which had been loaded with nucleosomes containing endogenous [ 3H]-acetyl labelled histones were assayed for histone deacetylase act iv i ty by microdialysis against Buffer B at 4°C, followed by incubation at 22°C for 4 hours without further additions. The enzyme reaction was stopped by boil ing for 1 minute. In other experiments, histone deacetylase complex with acetyl-labelled histones was precipitated with 100 mM NaCl and 36 assayed direct ly in Buffer B. The dialys is or precipitation step was required to separate nucleosomes from the sodium butyrate present in the buffers used to prepare them. Samples of [ 3H]-acetate labelled histone deacetylase complex which 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. Alternatively, equal aliquots of acetate labelled histone deacetylase complex were either untreated, boiled for 1 minute or made 1 mM or 10 mM in B-mercaptoethanol. Sodium butyrate was removed from a l l samples of histone deacetylase complex by dialysis 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 act iv i ty was measured by incubating the samples for 2 hours at 22°C and terminating the reaction by boil ing for 1 minute. The total cpm present in a sample of labelled histone deacetylase complex was determined by dissolving an aliquot i n 1.0 ml of NCS tissue solubi l izer and counting i n 10 ml ACS. Released [ 3H]-acetate was extracted and counted in the same manner for a l l of the assay systems. The incubation mixtures were acidif ied 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 collected. The extraction was repeated and the pooled ethyl acetate phases were counted i n 10 ml of ACS . 37 Precipitation 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 ted with a YM10 membrane. The concentrate was made 0.2 M in sodium acetate and 50 ug ultrapure bovine serum albumin was added as carr ier . The samples were precipitated overnight at -20°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 pellets washed in -20°C ethanol and collected by centrifugation as before. The pellets were dried in 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 ted with a YM10 membrane. The concentrated nucleosomes were made 0.2 M in sodium acetate and precipitated overnight at -20°C upon the addition of 6 volumes of ethanol. Precipitated nucleosomes were collected by centrifugation at 16,000 xg for 15 minutes. The pellet was washed i n -20°C ethanol and collected by centrifugation as before. The nucleosome pellet was dried in vacuo for 10 minutes at room temperature, taken up in 200 u l of proteinase K buffer (0.2 M Tr i s -HCl , 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 -70°C for 15 minutes. The pellet was taken up in buffer containing 50 mM Tr i s -HCl , pH 8.0, 50 mM EDTA and digested with 100 ug/ ml of pre-boiled RNase A for 45 minutes at 37°C. The RNase A digest was made 0.1 M i n NaCl by the addition of 4.0 M NaCl and predigested pronase was added to a f ina l concentration of 100 ug/ ml. Pronase digestion was carried out for 45 minutes at 37°C and the DNA was phenol extracted and precipitated in ethanol at -70°C as described above. Agarose Gel Electrophoresis Electrophoresis of DNA was carried out on horizontal slab gels of 0.9% or 0.75% agarose. The 0.9% agarose gels contained 40 mM Tris-phosphoric acid (pH 8.0), 20 mM sodium phosphate, 1 mM EDTA, 0.1% SDS and were run at 80 mA unt 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 gh t . Gels were photographed with a Polaroid camera. The 0.75% agarose gels contained 89 mM Tris-boric acid (pH 8.3), 2.5 mM EDTA, 5 ug/ml ethidium bromide and were run at 50 v unt i l the samples had entered the gel and then at 80 v u n t i l the bromophenol blue marker dye had moved approximatly 12 cm. DNA bands were visualized and photographed as above. 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 ratio of 30:0.4, w/w), 0.375 M Tr i s -HCl , pH 8.8, 0.1% SDS, 0.03% TEMED, 0.05% ammonium persulphate. The stacking gel contained 4.5% acrylamide (acrylamide:bisacrylamide ratio of 30:0.8, w/w), 0.125 M Tris -HCl pH 6.8, 0.1% SDS, 0.05% TEMED, 0.10% ammonium persulphate. Samples were boiled for 1 minute in 2 volumes of loading buffer (0.05 M Tr i s -HCl , pH 6.8, 1.0% SDS, 0.16 M B-mercaptoethanol, 7.5% sucrose) and loaded onto the gels. An aliquot of bromophenol blue in loading buffer was applied to a separate lane. The gel running buffer contained 0.05 M Tris pH ^  8.3, 0.38 M glycine, 0.1% SDS, and the gels were run at 25 v unt 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 gel . The gels were stained with 0.25% Coomassie Blue in a methanol and acetic acid system containing methanol:glacial acetic acid:water in a ratio of 2:1:5 (v/v), however, the ratio 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 acid, 0.5% TEMED, 0.125% ammonium persulphate. The separating gel was pre-electrophoresed at 200 v for 2 h at 4°C with running buffer of 0.9 M acetic acid. The stacking gel was poured after pre-electrophoresis of the separating gel and consisted of 4.5% acrylamide (acrylamide:bisacrylamide 30:0.8, w/w), 2.5 M urea, 0.375 M potassium acetate pH 4.0, 1.0% TEMED, 0.125% ammonium persulphate. Samples were taken up in loading buffer, containing 2.5 M urea, 0.9 M acetic acid, 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 as described for SDS polyacrylamide gel 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 in buffer containing 0.1 M T r i s - H C l , pH 7.4, 2.3% SDS, and 10% glycerol . The rinsed gel was cut longitudinally to y ie ld a 2 mm wide s l ice 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. Melted 1% agarose i n buffer (60 mM Tr i s -HCl , pH 6.8, 2.3% SDS, 0.002% bromophenol blue) was applied to the top of the stacking gel to seal the acid urea gel s l i c e . The gel running buffer contained 0.05 M Tris-pH ^ 8.3, 0.38M glycine, 0.1% SDS, and the gel was run at 50 v unt i l the bromophenol blue had 41 entered the stacking gel and then at 80 v unt i l the dye entered the separating ge l . Electrophoresis was performed at 120 v unt i l the bromophenol blue reached the end of the gel . The gel was stained with s i lver . Si lver Staining of Polyacrylamide Gels Si lver staining was performed on certain SDS polyacrylamide gels. The gel was washed by shaking overnight in 50% methanol and stained by shaking for 15 minutes in s i lver staining solution. The staining solution was made by dissolving 0.8 g AgN03 i n 4.0 ml H20 and adding dropwise to 20 ml of a mixture containing 95 mM NaOH and 0.52 M NH^OH. The s i lver staining solution was made up to 100 ml with 1^ 0 and used within 5 minutes. The gel was washed for 5 minutes in 1^ 0 and gently shaken in 200 ml developer (0.005% c i t r i c acid, 0.019% formaldehyde) unt i l the stained proteins were clearly v i s ib 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 dialysis membrane and dried in vacuo at 60°C. The dried gels were inserted into the gel carrier of a Beckman DU-8 spectrophotometer and the protein profiles 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 in 0.25 M sucrose and TM buffer (20 mM Tr i s -HCl , pH 7.4, 5 mM MgCl 2 , 1 mM PMSF). The digest was centrifuged at 1,000 xg for 15 minutes and the pe l le t , containing the digested nucle i , was retained and extracted with NaCl according to the standard protocol for nuclear matrix preparation (164). A l l extractions were performed on ice 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 Tr i s -HCl , pH 7.4, 0.2 mM MgCl 2 , 1 mM PMSF) three times with LM buffer containing 2.0 M NaCl and two times with LM buffer. The f i n a l pellet contained the nuclear matrix. Preparation of High Mobility Group Proteins High Mobility Group (HMG) proteins were prepared from either HeLa or cal f thymus chromatin according to the methods of Bhullar and Candido (183). Frozen ( -80°C) HeLa c e l l pellets were homogenized in 4 volumes of TMK (10 mM Tr i s -HCl , pH 8.0, 2 mM MgCl 2 , 25 mM KC1, 0.1 mM PMSF) using a glass-teflon hand homogenizer. The sample was centrifuged at 3,000 xg for 7 minutes and the pellet was rehomogenized and centrifuged as before. Calf thymus was collected and frozen within 5 minutes of slaughter. Frozen ( -80°C) calf thymus was minced with scissors and homogenized in 4 volumes 43 of PHMC (10 mM PIPES-HC1 pH 7.0, 1.0 M hexylene g lycol , 5 mM MgCl 2 > 1 mM C a C l 2 , 0.1 mM PMSF) using a motor driven teflon pestle. The sample was f i l t ered through 8 layers of cheese cloth and centrifuged at 1,000 xg for 10 minutes. The pellet was resuspended in the same buffer and rehomogenized and centrifuged twice as described above, except that hand homogenization was used. The resulting HeLa or calf thymus nuclear pellets were treated ident ical ly . The pellets were homogenized in 4 volumes of Tris buffer (10 mM Tr i s -HCl , pH 7.4, 0.1 mM PMSF), layered onto 1 M sucrose in Tris buffer and centrifuged at 17,000 xg for 20 minutes. The translucent pellet was suspended in Tris buffer in a f ina l volume of 5 ml/g i n i t i a l wet weight of the tissue, made 0.15 M in ammonium sulphate and centrifuged at 17,000 xg for 15 minutes. The supernatant was collected and 100% trichloroacetic acid was added dropwise to a f ina 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 sol id ammonium sulphate was added to give a concentration of 55%. The mixture was st irred on ice for 30 minutes and centrifuged at 17,000 xg for 30 minutes. The pel let , which contained HMG 1 and HMG 2 and the supernatant were collected and sol id ammonium sulphate was added to the supernatant to a f ina l concentration of 90%. The supernatant was st irred on ice for 30 minutes and HMG 14 and HMG 17 were collected by centrifugation at 17,000 xg for 1 hour. The HMG pellets were taken up in 0.1 M NH^CO-j, dialyzed in Spectrapor 3 dialys is tubing against the same buffer and lyophil ized. Alternatively, the HMG pellet was dialyzed against 20 mM NaCl in buffer D (15 mM Tr i s -HCl , 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 inear gradients of NaCl in 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 in buffer D. Acid Hydrolysis 50 ug samples of histones or HMG 14 and HMG 17 were dissolved in 0.4 ml of 6 N HC1 and maintained at 110°C for 20 or 36 hours in sealed glass v ia l s . The HC1 was evaporated under vacuum at 70°C. Amino Acid Analysis HMG 14 and HMG 17 proteins prepared from calf thymus were hydrolyzed in 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. Restriction Enzyme Digestion Restriction endonuclease digestion of purified DNA was carried out according to the manufacturer's (BRL) instructions. Br ie f ly , the DNA samples were taken up i n the appropriate enzyme buffer and ultrapure BSA (BRL) was added to a f ina l concentration of 100 ug/ml. Aliquots of restr ict ion enzyme were added every two hours and digestion was carried out 45 for 4 - 1 2 hours at 37°C with H a e l l l , EcoRI and Mspl, and at 65°C with Taql . The reactions were terminated by the addition of 0.5 M EDTA to a f ina l concentration of 8 mM, and heating to 68°C for 5 minutes. Nucleosome Reconstitution Acetyl- labelled core histones were prepared from labelled HeLa ce l l s by NaCl extraction in the presence of 100 mM sodium butyrate (186). 3 g labelled HeLa c e l l nuclei were prepared using the procedures described in "Micrococcal Nuclease Digestion". The nuclei were lysed in 90 ml buffer E (10 mM Tris-cacodylic acid pH 7.2, 0.7 mM EDTA, 100 mM sodium butyrate, 1 mM PMSF), and sol id NaCl was added to give a concentration of 0.6 M and the nuclei 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 st irred gently for 24 hours at 4°C. The mixture was centrifuged at 17,000 xg for 15 minutes and the pel let containing histone Hl-depleted chromatin, was taken up in 80 ml buffer E and st irred gently for 6 hours at 4°C and centrifuged as before. The pellet was recovered and mixed with 4 ml of buffer E containing 2 M NaCl and st irred gently overnight at 4°C. The mixture was centrifuged at 170,000 xg for 24 hours in a Beckman SW60Ti rotor and the histone fraction was collected. The histones were passed through a Sephadex G-25 column (1.0 cm x 30 cm) i n order to remove free [ 3H]-acetate and concentrated using an Amicon concentrator f i t ted 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 aliquots, containing 37 ug of DNA, were mixed with the [ 3H]-acetyl labelled core histones at a histone:DNA ratio of 0.9:1 (w/w). The histones and DNA were reconstituted by dialysis against buffer E containing successively lower ionic strength (187). Dialysis 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 ina l ly , the sodium butyrate was removed by dialys is 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 ina 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 protein. 47 RESULTS PART A. THE CHARACTERIZATION OF HISTONE DEACETYLASE Incubations of Hela nuclei with free [ 3 H]-acetyl-labelled histones at 22°C for varying periods of time resulted in the release of up to about 25% of the [ 3H]-acetyl groups after 4 hours (Figure 6). The rather slow rate of enzymatic- deacetylation of histones observed with nuclei may be due to the time required for diffusion of the free histones through the nucleus to the site 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 distribution of histone deacetylase in chromatin, Hela nuclei were part ia l ly digested with micrococcal nuclease to the extent of 12-15% solubi l izat ion of the i n i t i a l ^oC), a n < * t ^ i e resulting nucleosomes were isolated by centrifugation through an isokinetic sucrose gradient prepared for a particle density of 1.5 g/ml (185). Fractions were collected by upward displacement and sucrose was removed by 48 C O I ncubat ion Time (h) Figure 6. Time course of histone deacetylase in nuclei . HeLa nuclei were prepared as described in Experimental Procedures and incubated with added free [ 3 H]-acetyl-labelled histones (40,000 cpm) for different lengths of time. The reactions were stopped by boil ing for 1 minute and released [ 3H]-acetate was extracted and counted. 49 microdialysis. Gradient fractions were assayed for histone deacetylase act iv i ty by the addition of free acetyl-labelled histones (Figure 7A). The majority of enzyme act iv i ty was detected near the top of the gradients between free DNA and mononucleosomes, presumably representing dissociated enzyme. A second peak of enzyme act iv i ty was observed in oligonucleosomes at the bottom of the gradients. The nuclease digestion and centrifugation were repeated in the presence of butyrate with nuclei containing endogenous acetyl-labelled histones and the sucrose gradient fractions were assayed for histone deacetylase upon removal of the butyrate by dialysis (Figure 7B). In this case, a l l of the histone deacetylase was detected in high molecular weight material. In view of the finding that certain HMG proteins can inhibi t nuclear histone deacetylase act iv i ty in v i tro (188), i t was possible that the lack of detectable act iv i ty on mononucleosomes was due to the presence of such an inhibi tor . The endogenous deacetylase act iv i ty of micrococcal nuclease digests was examined on gradients run in 0.4M NaCl, to remove HMG proteins (Figure 7C). Dinucleosomes sedimented more slowly in this case due to the lack of histone HI and non-histone chromatin proteins (189,190); however, the histone deacetylase prof i le was identical to the one seen in gradients without NaCl. The above experiments indicate that histone deacetylase does not bind to mono- or dinucleosomes under the conditions typical ly used in sucrose gradient fractionations, and suggest that perhaps higher order chromatin structures are important in binding this enzyme. 50 50a Figure 7. Isokinetic sucrose gradient profi les of nucleosomes and histone deacetylase ac t iv i ty . He la nuclei were part ia l ly digested with micrococcal nuclease and nucleosomes were isolated by sedimentation through isokinetic sucrose gradients in a Beckman SW41 rotor at 25,000 rpm for 18 hr. ( - • —t - ) A260 ; [ 3H]-acetate, CPM released (XI0~ 3). (A) Free acetyl-labelled HeLa histones were added to gradient fractions which were incubated at 22°C for 4 hr and histone deacetylase act iv i ty was determined by extracting and counting the released [ 3H]-acetate. (B) Nucleosomes were prepared from acetyl-labelled HeLa nuclei using identical procedures as in A, except that a l l buffers contained 40 mM butyrate to inhibit histone deacetylase. The butyrate was removed from the gradient fractions by dialys is and the fractions were incubated for 4 hr at 22°C. t r i -acetate, released from endogenous labelled histones, was extracted and counted. (C) Endogenous acetyl-labelled nucleosomes were prepared and assayed for histone deacetylase act iv i ty as described in 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 Act iv i ty Using a Bio-Gel A-5m Column Endogenous [ 3H]-acetate-labelled HeLa c e l l nuclei were part ia l ly digested with micrococcal nuclease and the digest products were separated a Bio-Gel A-5m column, which had a molecular weight exclusion l imit of 5,000,000. Nuclease digestion and column chromatography were carried out in the presence of 50 mM butyrate and the fractions were assayed for histone deacetylase act iv i ty after dialys is as before. A profi le of the column is shown in Figure 8. The profi les of histone deacetylase act iv i ty and absorbance at 260 nm did not coincide. The histone deacetylase act iv i ty was d is t inct ly skewed to the high molecular weight side of the excluded absorbance peak. Therefore, the histone deacetylase act iv i ty 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, labelled endogenously with [ 3H]-acetate, and deacetylase were prepared by micrococcal nuclease digestion of labelled nuclei and isolated by chromatography on a Bio-Gel A-5m column in the presence of 50 mM butyrate. They were then mixed with equal amounts of either free labelled HeLa histones, free unlabelled HeLa histones or free hyper-acetylated unlabelled HeLa histones as shown i n Table 2. The added free labelled histones contained an equal number of 52 2 0 0 1 0 0 5 0 1 0 0 V o l u m e (ml ) 1 5 0 CO X o Q ? n -o ? 73 A A Q vt (D 0. X o I CJ Figure 8. Fractionation of micrococcal nuclease digest products on a Bio-Gel A-5m column. [ 3 H]-acetyl- label led HeLa nuclei were part ia l ly digested with micrococcal nuclease and the digest products were separated using a Bio-Gel A-5m column equil ibriated with 50 mM butyrate. Fractions of 1 ml were collected and the butyrate was removed by dialys is and the histone deacetylase ac t iv i ty was detected by" extracting and measuring the free [ 3H]-acetate released from the endogenous histones after incubation at 22°C for 4 hours ( ) A 2 6 Q ; ( •) [ 3H]-acetate, CPM released (x lO~ 3 ) . 53 [ 3H]-acetyl groups as were present in the endogenous labelled histones of the chromatin complexes. The reaction was ini t iated by the removal of butyrate by d ia lys i s . Each aliquot of labelled chromatin complex contained 213,000 cpm and complexes incubated alone released 88% of their [ 3H]-acetyl groups. The presence of free unlabelled histones caused 11% inhibi t ion of the deacetylase act iv i ty , while hyper-acetylated unlabelled histones caused 46% inhib i t ion . This inhibi t ion could have occurred as a result of non-productive binding between free histone and histone deacetylase, or by the preferential release of unlabelled acetyl groups from the added free histone. However, when free acetyl-labelled histones were mixed with the enzyme complexes, the release of radioactive acetate was also inhibited 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 inhibit ion of the histone deacetylase was only half as great when half as much free histones were added to the assays. Therefore, the inhibit ion of histone deacetylase by free histones appeared to be concentration dependent. To determine whether nucleosome structure is important for productive binding between histones and histone deacetylase, high molecular weight chromatin complexes labelled endogenously with [ 3H]-acetate were isolated as above and incubated either alone, with labelled mononucleosomes containing an equal number of [ 3H]-acetyl groups, or with unlabelled mononucleosomes as shown in Table 3. The mononucleosomes were prepared by part ia l micrococcal nuclease digestion of either acetyl-labelled or 54 Table 2 Inhibition 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 part ia l digestion with micrococcal nuclease and isolat ion of the polynucleosomes on a Bio-Gel A-5m column in the presence of 50 mM butyrate. HeLa histones were prepared as described in 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 dialysis and the polynucleosomes were incubated for 4 hours at 22°C. The reaction was stopped by boil ing and the released [ 3H]-acetate was extracted and counted. 55 Table 3 Use of mononcleosomes as a substrate for histone deacetylase Assay System [3 H]-Acetate, CPM Released Chromatin complex 25,539 Chromatin complex + labelled mononucleosomes 35,636 Chromatin complex + unlabelled mononucleosomes 20,400 Both unlabelled and acetyl-labelled HeLa nuclei were part ia l ly digested with micrococcal nuclease and nucleosomes were isolated using a Bio-Gel A-5m column in 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 labelled chromatin complex (containing 40,000 cpm). Butyrate was removed by dialys is and deacetylase act iv i ty was assayed by incubation at 22°C for 4 hours. The reaction was stopped by boiling and the free acetate was extracted and counted. 56 unlabelled HeLa nuclei and isolated by chromatography on identical Bio-Gel A-5m columns containing 50 mM butyrate. The addition of labelled mononucleosomes produced an increase in the amount of [ 3H]-acetate released, and the addition of unlabelled mononucleosomes (present at a lower concentration than the labelled mononucleosomes) resulted in a decrease in the amount of released [ 3H]-acetate. This shows that histones must be in their physiological conformation as nucleosome complexes in order to serve as eff icient substrates for the chromatin-bound histone deacetylase. I I . The Distribution of Histone Deacetylase in Chromatin To examine more closely the distribution of histone deacetylase act iv i ty in 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 in 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 in length, corresponding to chromatin fragment lengths of 55-25 nucleosomes (Figure 9A). In three separate preparations, the DNA from peak I polynucleosomes gave size distributions of 11-5 Kbp, 10-6 Kbp and 15.5-5 Kbp, the last 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 in length while peak III contained DNA of approximately 4.0 Kbp (20 nucleosomes) to 200 base pairs in length. Peak IV contained free nucleotides which have migrated off the end of the gel shown in Figure 9. An A-50m column 57 therefore provides good resolution of chromatin fragments over a broad size range. Peak I of the prof i l e , however, contains DNA which overlaps in size with the DNA of peak II , 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 poss ib i l i ty that peak I might consist of non-specific chromatin aggregates is rendered unlikely by the fact that the proportion of A2gQ-absorbing material in peak I is unaffected by running the columns in 1-2 M NaCl (see below). Under these conditions, histone dissociation occurs, and dissolution of such aggregates would be expected. Furthermore, the fractions corresponding to peak I exhibit no turbidity . The protein composition of the Bio-Gel A-50m chromatin fractions i s discussed in detai l below. Since polynucleosomes from both region I (excluded) and regions II-III (included) of the column contain a variety of non-histone proteins, their presence alone does not explain the difference in chromatographic properties between the two classes of polynucleosomes. The most l ike ly 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 fract ion. Micrococcal nuclease digests of [ 3 H]-acetyl-labelled chromatin were then fractionated on a Bio-Gel A-50m column in the presence of butyrate and the fractions were assayed for endogenous histone deacetylase act iv i ty as before. A prof i le of such a column is shown in Figure 10A. A l l of the enzyme act iv i ty was found in the i n i t i a l peak. No act iv i ty was detected in ol igo- or mononucleosomes (regions II and III) , confirming the results obtained with sucrose gradients (Figure 7B). The profi les of histone deacetylase act iv i ty and absorbance at 260 nm in the f i r s t peak coincided. 58 58a Figure 9. Fractionation of micrococcal nuclease digest products of HeLa chromatin on a Bio-Gel A-50m column. HeLa nuclei were part ia l ly 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 ml/hr. DNA was extracted from the column fractions and analyzed on a horizontal slab gel of 0.75% agarose in Tris-borate buffer (pH 8.3) (A) or 0.9% agarose in Tris-phosphate buffer (pH 8.0) (B). The gel was stained with 10 ug/ml of ethidium bromide and the DNA bands were visualized under UV l i gh 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) DNA from the indicated regions of the column prof i le ; (C) Absorbance profi le of the A-50m column at 260 nm. The data in (B) and (C) are from the same column run; the gel in (A) i s from a separate but identical column run. 59 III Effect of Butyrate on the Distribution of Histone Deacetylase HeLa ce l l s were normally labelled with [ 3H]-acetate for 2 hours and chased with unlabelled acetate for 6 hours in the presence of 10 mM butyrate i n order to increase the specific act iv i ty 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 in protein modification (221,223,225) and causes a reversible decrease in condensed chromatin clumps i n HeLa ce l l s (224). In order to rule out the poss ib i l i ty that the deacetylase distribution seen in the above experiments might be an art i fact of the butyrate treatment, a similar experiment was done with ce l l s labelled in the absence of butyrate. HeLa cel ls were normally labelled with 150 uCi/ml of [ 3H]-acetate. The medium used for label l ing HeLa ce l l s in the absence of butyrate contained 300 uCi/ml of [ 3H]-acetate, to compensate for the expected turnover of acetyl groups under these conditions. A label l ing time of 2 hours was used and the cel ls were frozen rapidly without an intervening cold chase. Nucleosomes were prepared from the ce 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 in vivo incorporated labe l . Fractions were then assayed for histone deacetylase as before. As seen in Figure 10B, the results were exactly the same as for ce l l s labelled in the presence of butyrate (Figure 10A), the histone deacetylase being found only in the high molecular weight material excluded by the column. This characteristic distribution of histone deacetylase therefore occurs under normal culture conditions and is not caused or influenced by exposure of the ce l l s to butyrate. This chromatin fraction 60a Figure 10. Distribution of histone deacetylase in chromatin fragments from butyrate treated versus untreated HeLa ce l l s HeLa cel ls were labelled in vivo with [ 3H]-acetate (sodium sal t , spec. act. > 500 mCi/mol) for 2 hr. Labeled nuclei were prepared from the ce l l s and part ia l ly 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 collected, dialyzed to remove the butyrate, and incubated for 4 hr at 22°C. The released [ 3H]-acetate was extracted and counted as described in Experimental Procedures. ( ) ^^Q', (••••- ) [ 3H]-acetate, CPM released (xlO 3 ) . (A) HeLa ce l l s were labelled with 150 pCi/ml of [ 3H]-acetate for 2 hr in medium containing 10 mM butyrate, and then incubated a further 6 hr i n unlabelled medium containing 8 mM sodium acetate as well as 10 mM butyrate. (B) HeLa ce l l s were labelled with 300 uCi/ml of [ 3H]-acetate for 2 hr with no butyrate present, and without a cold chase. 61 containing the deacetylase act iv i ty w i l l be referred to as the "histone deacetylase complex". IV Effect of Salt Concentration on Histone Deacetylase Distribution a. Chromatography Using a Bio-Gel A-5m Column The effect of salt concentration on the distribution of histone deacetylase was i n i t i a l l y examined by fractionating micrococcal nuclease digests of [ 3 H]-acetyl-labelled HeLa c e l l nuclei using a Bio-Gel A-5m column in the presence of sodium butyrate and varying concentrations of NaCl. Column fractions were assayed for histone deacetylase act iv i ty i n the normal manner after dialysis to remove f i r s t the NaCl, and then the butyrate. The results of fractionating nuclease digests i n the presence of 0.5 M NaCl, 1.0 M NaCl and 2.0 M NaCl are shown in Figure 11. Histone deacetylase act iv i ty was found in the high molecular weight material in a l l cases. Although 2.0 M NaCl causes the dissociation of histones from chromatin, histones were detected in 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 in the high sa l t . b. Chromatography Using a Bio-Gel A-50m Column In view of the presence of histones, and possibly other aggregates, in 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 in the case 62 62a Figure 11. Effect of salt concentration on histone deacetylase dis tr ibut ion. Endogenous labelled HeLa nuclei were part ia l ly digested with micrococcal nuclease and the resulting products were separated on a Bio-Gel A-50m column containing butyrate and different concentrations of NaCl. Fractions were assayed for histone deacetylase act iv i ty after dialys is to remove the NaCl and butyrate. Free [ 3H]-acetate was collected and counted (A) 0.5 M NaCl; (B) 1.0 M NaCl; (C) 2.0 M NaCl. 63 of 1.0 M NaCl were the same as those shown in Figure 10A, i . e . histone deacetylase was associated with the high molecular weight excluded peak. Therefore, the removal of salt dissociable non-histone chromosomal proteins at concentrations of NaCl greater than 0.35 M did not alter the prof i le . As expected, no\histone deacetylase act iv i ty was detected in the excluded fractions from the Bio-Gel A-50m column run in 2.0 M NaCl, since the polynucleosomal DNA present in the void peak did not contain histones as shown by SDS polyacrylamide gel electrophoresis (Figure 12). Since the assays of deacetylase act iv i ty in these experiments depended on the release of labelled acetate from histones present in the chromatin fractions, i t was necessary to reconstitute labelled histones with the polynucleosomal DNA in order to assay for histone deacetylase act iv i ty in the 2 M NaCl prof i l e . As the presence of histone HI during nucleosome reconstitution by salt dialysis results in precipitation of the complexes (186), [ 3 H]-acetyl-labelled core histones were prepared from labelled HeLa cel ls by NaCl extraction in the presence of 100 mM sodium butyrate. The Hl-depleted chromatin was prepared by washing labelled 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 in 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 [ 3 H]-acetyl-labelled core histones in buffer containing 2.0 M NaCl and 50 mM sodium butyrate at a histone:DNA ratio of 0.9:1 (w/w). A histone:DNA ratio of 0.9:1 (w/w) was chosen as reconstituted nucleosomes precipitate i f the ratio is greater than 1:1. The mixtures were dialyzed 64 Figure 12. The histone content of histone deacetylase complex prepared in the presence of 2.0M NaCl. He la nuclei were part ia l ly 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 equilibriated with 2.0 M NaCl; 2, HeLa histones. 65 against buffers of successively lower ionic strength as described i n Experimental Procedures. Butyrate was removed from the reconstituted material by dialys is and histone deacetylase act iv i ty was assayed as before. Equal aliquots of [ 3 H]-acetyl-labelled chromatin were incubated with buffer alone to serve as controls. It was found that 15% of labelled acetyl groups were released from the reconstituted histones. Thus at least some histone deacetylase remains bound to chromatin in 2 M NaCl, strongly suggesting that the major interactions involved are non-ionic i n nature. V. Effect of Nuclease Digestion on Histone Deacetylase Distribution HeLa nuclei were usually digested with micrococcal nuclease for 5 minutes as described in Experimental Procedures. In order to determine whether the degree of nuclease digestion affected the distribution of histone deacetylase in chromatin, several experiments were performed. a. Reduced Nuclease Digestion The poss ib i l i ty existed that the lack of observable histone deacetylase act iv i ty on mononucleosomes could have been due to extensive nuclease digestion or the presence of inhibitory proteins, such as HMG proteins which can inhibit nuclear histone deacetylase act iv i ty in_ vi tro (188). However, when nuclei were digested less extensively, the distribution of histone deacetylase remained the same. For example, a 30 second digest under the same conditions released only 15% as much DNA as did a 5 minute digestion and fractionation of the products of a 30 second micrococcal nuclease digestion of labelled HeLa nuclei on a Bio-Gel A-50m 66 Figure 13. Effect of reduced nuclease digestion on the distr ibution of histone deacetylase. Labelled HeLa nuclei were digested with micrococcal nuclease (2.5 units of enzyme/A2kQ of nuclei) for 30 seconds at 25°C and 15% as much DNA was released as in a normal 5 minute digestion. 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 act iv i ty upon removal of the butyrate and incubated for 4 hours at 22°C. Free [ 3H]-acetate was extracted and counted. ( ) ^60 ' (• *) [ 3H]-acetate, CPM released ( x l O - 3 ) . 67 column containing 0.4 M NaCl to dissociate any HMG proteins (Figure 13) produced a histone deacetylase profi le that was v ir tua l ly identical to that of Figure 10A. A l l of the histone deacetylase act iv i ty was present in the histone deacetylase complex in the f i r s t peak and no act iv i ty was detected in oligonucleosomes or mononucleosomes. The P r ° f i l e w a s also very similar to that seen for the standard nuclease digestion, except that the y ie ld was lower. b. Redigestion of Histone Deacetylase Complex The effect of extensive micrococcal nuclease digestion on the deacetylase distr ibution within chromatin was examined in the following experiment. Endogenous acetyl-labelled 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 in place of a Bio-Gel A-50m column for speed and convenience as greater flow rates could be achieved. It should be noted that in contrast to the Bio-Gel A-50m prof i l e , the histone deacetylase act iv i ty is d is t inct ly 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 2 , 1 mM in MgCl 2 and 2.5 mM in KC1 by the addition of lOOx solution. The pooled material was then redigested with micrococcal nuclease (10 units of enzyme/A2^Q of deacetylase fraction at 15°C for 10 minutes). The nuclease digestion was stopped by the addition of 0.1 M EGTA to a f ina l concentration of 1 mM and the redigested histone deacetylase complex was reapplied to the same column. Fractions were 68 68a Figure 14. Polynucleosomes redigested with micrococcal nuclease retain their endogenous histone deacetylase act iv i ty . A Bio-Gel A-5m column equilibrated with 50 mM butyrate was used to separate endogenous labelled chromatin fragments. Fractions of 1 ml were collected and dialyzed to remove the butyrate and the histone deacetylase act iv i ty was detected by extracting and measuring the free [ 3H]-acetate released from the endogenous histones after a 4 hr incubation at 22°C. ( ) A 2 6 Q ; (• •) [ 3H]-acetate, CPM released (xl0~ 3) . (A) Acetyl-labelled HeLa nuclei were part ia l ly digested with micrococcal nuclease and the endogenous labelled chromatin fragments were isolated by column chromatography. The polynucleosome fractions containing histone deacetylase were pooled after aliquots had been assayed. (B) The polynucleosomes pooled in A were redigested with micrococcal nuclease (10 ^260 u n : * - t s ° f enzyme/A2^Q of polynucleosomes) at 15°C for 10 min, reapplied to the column and assayed in the same way. assayed for histone deacetylase act iv i ty upon the removal of butyrate as before, and this prof i le is shown in Figure 14B. The absorbance and histone deacetylase act iv i ty present in the pooled fractions from the i n i t i a l digestion were recovered quantitatively in the fractions from the second digestion. The most obvious feature of the redigested material was the shift i n the relative positions of the and histone deacetylase peaks. The two profi les were not coincident in the i n i t i a l digestion, the histone deacetylase act iv i ty being ahead of the absorbance peak; however, the act iv i ty and the absorbance peaks did coincide after redigestion of the complex under the above conditions. The specific radioactivity of the histones in the mononucleosomes produced by redigestion of the labelled deacetylase complexes was 3.2 times higher than that of the mononucleosomes produced in the i n i t i a l digestion. In spite of this , no release of [ 3H]-acetate from mononucleosomes was detected. c. Extensive Nuclease Digestion Extensive nuclease digestions were also performed on whole nuclei in order to study their effects on histone deacetylase distr ibut ion. [ 3 H]-acetyl labelled HeLa nuclei were divided into 2 equal aliquots and one was part ia l ly digested with micrococcal nuclease (2.5 units of enzyme/A2gQ of nuclei for 5 minutes at 2 5 ° C ) . The resulting digest was fractionated on a Bio-Gel A-5m column and samples were assayed for histone deacetylase as shown in 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 in the same way as the f i r s t aliquot and the data are shown in Figure 15B. 70a Figure 15. Effect of the extent of nuclease digestion on the distr ibution Equal aliquots of HeLa nuclei containing [ 3 H]-acetyl-labelled 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 act iv i ty was determined by incubating dialyzed fractions for 4 hr at 22°C and measuring released [ 3H]-acetate. ( ) , upper curve, [ 3H]-acetate released (%); ( ), lower curve, ^60 ' ^* ~ ~ [ 3H]-acetate, CPM released (xlO 3 ) . (A) Labelled nuclei were part ia l ly digested with micrococcal nuclease (2.5 ^260 u n i t s ° f enzyme/A2^Q of nuclei) for 5 min. at 25°C. (B) Labelled nuclei were extensively digested with micrococcal nuclease (7.5 n n ^ - t s ° f enzyme/A^Q of nuclei) for 15 min at 25°C. of histone deacetylase i n chromatin. 71 The distribution of histone deacetylase act iv i ty in the digest prepared by routine conditions (Figure 15A) was the same as for previously described experiments. More extensive nuclease digestion resulted in the production of very large peaks of mononucleosomes and nucleotides but the histone deacetylase act iv i ty remained confined to the i n i t i a l peak. The ^260 a n < * e n z v m e act iv i ty profiles in 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 act iv i ty between complexes prepared by normal versus extensive digestion yielded the following results . If the percent of [ 3H]-acetate released was used for comparisons of enzyme act iv i ty , histone deacetylase complex prepared by normal digestion released 44.2% of total [ 3H]-acetyl groups whereas complexes prepared by extensive digestion released 26.8% of total [ 3H]-acetyl groups, implying that some histone deacetylase had been lost during digestion. If enzyme act iv i ty was expressed as cpm [ 3H]-acetate released/A2gQ, i t was found that deacetylase complexes prepared by normal digestion released 2.5 x 10s cpm/A^^, whereas those prepared by extensive digestion released 3.3 x 106 c p m / A „ , n , an increase of zou approximately 30%. Mononucleosomes resulting from extensive digestion had a higher specific radioactivity than mononucleosomes prepared by normal digestion, but no endogenous histone deacetylase act iv i ty was observed. It can be seen from the inserts in Figures 15A and 15B showing the percent of [ 3H]-acetate released across the deacetylase peak that the complexes were heterogeneous with respect to histone deacetylase act iv i ty after normal digestion, and remained that way after more extensive 72 digestion. A similar 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 particular assay procedures in order that enzyme characteristics could be studied. For routine assays, [ 3H]-acetyl labelled deacetylase complexes were precipitated on ice with 100 mM NaCl in the presence of 50 mM butyrate as described in detai l i n Experimental Procedures. Assays were started as soon as the complexes were redissolved in butyrate-free buffer. The precipitation 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 is shown in Figure 16. F i f ty percent of the [ 3H]-acetyl groups were released within 3 minutes. This very rapid i n i t i a l release of acetyl groups was followed by a more gradual release over the next 2-3 hours. A small percentage of chromatin bound [ 3H]-acetyl groups remained even after 15 hours of incubation. Acid hydrolysis of the acetyl-labelled histones rendered a l l of the label vo la t i l e , indicating that i t was present as acetyl groups and that none had entered the polypeptide backbone during label l ing . It i s l ike ly that the remaining [ 3H]-acetyl groups represent a-N-acetyl 73a Figure 16. Time course of chromatin-bound histone deacetylase Histone deacetylase complex containing acetyl-labelled histones was prepared from labelled HeLa nuclei by part ia 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. The reaction was stopped by boil ing and the released [ 3H]-acetate was extracted and counted. 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, this being a convenient point within the l inear 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 in the following manner. Precipitated, endogenous labelled deacetylase complexes were taken up and assayed in different volumes of buffer (Table 4). Although a 30 fold difference i n assay volumes was employed, the release of [ 3 H]-acetate over a 3 minute period was essentially unaltered in repeated experiments. Therefore, di lut ion had no significant effect on histone deacetylase ac t iv i ty , 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 inhibit ion of histone deacetylase act iv i ty when free acetyl- labelled 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 deacetylase Assay Volume (pi) [ 3H]-Acetate Released (%) 50 557*5 500 53.9 1500 56.1 Endogenously labelled histone deacetylase complex was prepared by part ia l micrococcal nuclease digestion of labelled HeLa nuclei . The histone deacetylase complex was precipitated in 100 mM NaCl containing 50 mM butyrate, taken up i n different volumes of butyrate-free buffer, and histone deacetylase act iv i ty was assayed for 3 minutes at 22°C. The reaction was stopped by boil ing 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 in histone deacetylase assays was essential , these proteins were isolated from calf thymus and hydrolyzed in 6 N HC1 as described in Experimental Procedures. The hydrolyzate was analyzed on an automatic amino acid analyzer and the amino acid content i s shown in Table 5. No corrections were made for hydrolytic losses. The preparation of cal f thymus HMG 14 and HMG 17 displayed the characteristic amino acid composition of the small HMG proteins namely, large amounts of lysine, glutamate, alanine and proline with only trace amounts of the hydrophobic amino acids and no cysteine. Comparison with the amino acid composition of calf 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 calf thymus by ammonium sulphate precipation, were present in a ratio of approximately 1:8 (mol/mol). This sample was used as a standard for comparative SDS polyacrylamide gel electrophoresis of different calf thymus and HeLa protein preparations. Preparation of HMG proteins with ammonium sulphate involves the sequential precipitation of HMG proteins with increasing concentrations of ammonium sulphate (see Experimental Procedures). It was found that a reduction in the ammonium sulphate concentration from 55% to 50% during the f i r s t precipitation to remove HMG 1 and HMG 2 resulted in an increased yie ld of HMG 14; however, large amounts of HMG 1 and HMG 2 were also present. Figure 17 shows the SDS polyacrylamide gel profi le of a typical preparation of HMG 14 and HMG 17. Amino A c i d C a l f Thymus HMG 14 + 17 Prepared by Ammonium:Sulphate P r e c i p i t a t i o n 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 A l a 17.8 14.6 18.4 17.7 Cys o 0 0 0 V a l 2.5 4.0 2.0 2.4 Met tr a c e 0.1 t r a c e t r a c e l i e u t r a c e 0.3 t r a c e t r a c e Leu 1.4 2.0 1.0 1.2 Tyr t r a c e 0.2 t r a c e t r a c e Phe trace 0.3 t r a c e t r a c e His 0.1 0.2 t r a c e t r a c e 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 isolated HMG proteins HMG 14 and HMG 17 were isolated from calf thymus by ammonium sulphate precipitation 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 calf thymus HMG 14 and HMG 17 have been published (192,193) and are included in the Table. Since the amino acid composition of the isolated calf thymus HMG proteins i s from a mixture of HMG 14 and HMG 17 in about a 1:8 mass ratio (Figure 17), approximately comparable amino acid values for this mixture have been l i s t ed . 78 Figure 17. SDS polyacrylamide gel electrophoresis of HMG 14 and HMG 17. HMG 14 and HMG 17 were prepared from calf thymus nuclei and HeLa nuclei by extraction with 2% trichloroacetic acid and precipitation with ammonium sulphate as described under Experimental Procedures. Calf thymus HMG 14 and HMG 17 were separated by column chromatography (183) and used as standards. Electrophoresis was carried out in a gel containing 15% acrylamide which was stained with Coomassie Blue. Lane 1, HeLa core histones; 2, calf thymus HMG 14; 3, calf thymus HMG 17; 4, calf 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 and HMG 17 [ ^ - A c e t a t e Released nucleosome r a t i o (mol/mol) (%) .0 46.8 •1 48.0 2 46.5 . 3 45.2 Endogenous labelled histone deacetylase complex was prepared by part ia l micrococcal nuclease digestion of labelled HeLa nucle i , and precipitated in 400 mM NaCl containing 50 mM butyrate. The HMG protein depleted histone deacetylase complex was, taken up in butyrate-free buffer containing different concentrations of HeLa HMG 14 and HMG 17, and histone deacetylase ac t iv i ty was assayed for 3 min at 22°C. The reaction was stopped by boi l ing 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 in several experiments. F i r s t , endogenous labelled complexes were precipitated i n either 100 mM NaCl, as usual, or 400 mM NaCl, in order to determine whether the release of non-histone chromatin proteins (e.g. high mobility group proteins 14 and 17) would affect histone deacetylase act iv i ty . Complexes precipitated in 100 mM NaCl released 53% of [ 3H]-acetyl groups in 3 minutes compared to 51% for those precipitated in 400 mM NaCl. Precipitation in 400 mM NaCl therefore had no significant effect on histone deacetylase ac t iv i ty . Alternatively, the effect of the addition of HeLa HMG 14 and HMG 17 to HMG-depleted HeLa histone deacetylase complex was investigated. In view of the very rapid deacetylation of histones, the histone deacetylase assays were carried out for 3 minutes at 4°C on samples of endogenous labelled histone deacetylase complex which had been collected by precipitation in 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 ratio of 1 to 3:1 (mol/mol) had no effect on histone deacetylase act iv i ty (Table 6). c. Effect of Calf Thymus HMG 14 and HMG 17 on Histone Deacetylase The poss ib i l i ty existed that only a fraction of the HMG proteins were in solution or in their correct conformation and so greatly increased concentrations of HMG proteins were used. The limited y ie ld of these 81 0 1 1 1 1 1  0 500 1000 H M G : nucleosome (mo l /mo l ) Figure' 18. Effect of calf thymus HMG 14 and HMG 17 on histone deacetylase a c t i v i t y . Endogenous labelled histone deacetylase complex was prepared by p a r t i a l digestion of HeLa nuclei with micrococcal nuclease, and equal aliquots were collected by precipitat ion in 0.4 M NaCl containing 50 mM butyrate. The HMG protein depleted histone deacetylase complex was taken up in butyrate-free buffer containing different concentrations of calf thymus HMG 14 and HMG 17, and deacetylase ac t iv i ty was assayed for 3 minutes at 4°C. The reaction was stopped by boi l ing for 1 minute and released [ 3H]-acetate was extracted and counted. Each reaction contained 80,000 cpm. 82 proteins from HeLa ce l l s necessitated the use of calf thymus nuclei as the source of HMG proteins. Endogenous labelled histone deacetylase complex was prepared from HeLa nuclei by part ia l digestion with micrococcal nuclease and aliquots were precipitated in 0.4 M NaCl containing 50 mM butyrate. The concentration of nucleosomes in samples of complex was determined by measuring the absorbance at 260 nm. The HMG-depleted deacetylase complex was taken up in butyrate-free buffer containing different concentrations of calf thymus HMG 14 and HMG 17, and histone deacetylase act iv i ty was assayed for 3 minutes at 4°C . The presence of calf thymus HMG 14 and HMG 17 at very much higher concentrations caused an increase in the release of [ 3H]-acetate (Figure 18). As the histone deacetylase assays contained a wide range of concentrations of calf thymus HMG 14 and HMG 17, the protein concentration was kept constant by the addition of BSA, which had no effect on histone deacetylase ac t iv i ty . The increase in the release of [ 3H]-acetate was only observed when the HMG:nucleosome ratio was 500:1 (mol/mol) or greater, and therefore, disruption of nucleosome structure resulting in increased access ibi l i ty of the histones i s the most l ike ly reason for an increase in [ 3H]-acetate release. 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 inhibi t ion 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 resistant, high molecular weight complex prompted investigations into the properties and characteristics of this 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 te for several forms of chromatin processing including replication (168,169,170), transcription (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 part ia l ly 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 in -20°C ethanol as described in 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 ce l l s to 10 mM sodium butyrate for 8 hours results in extensive hyperacetylation of the histones associated with the histone deacetylase complex. Although both histones H3 and H4 exist in several acetylated forms, positive identif icat ion 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 in the 84 same region of the acid urea gels. For this 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 total histone H4 being in this form. T r i - , d i - and monoacetylated H4 accounted for 27%, 23% and 16% of the total histone H4 respectively. Very l i t t l e (4%) of histone H4 was unacetylated. Comparison of this gel pattern with those of bulk histones from HeLa ce l l s exposed to butyrate (78) shows that the histones of the deacetylase complex from butyrate-treated ce l l s were acetylated to a greater degree than bulk histones from the same ce l l s . Examination of the histones from deacetylase complexes of ce l l s which had not been exposed to butyrate also showed H4 to be hyperacetylated relative to bulk H4 from the same ce l l s . The corresponding acid urea gel i s shown i n Figure 19A and the optical scan i n Figure 20B. The histones of the ol igo- and mononucleosomes produced along with histone deacetylase complex during the micrococcal nuclease digestion of HeLa c e l l nuclei were also examined and found to contain acetylated histones. The major forms of histone H4 i n the o l igo- and mononucleosomes were the unacetylated and monoacetylated derivatives. A protein which had a similar mobility to tetracetylated histone H4 (arrow, Figure 19) was observed in nucleosomes but not in 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 ce l l s ) and the second dimension was SDS 18% polyacrylamide gel electrophoresis. The protein was clearly not a histone and had an approximate molecular weight of 12K. Samples of spermine and BSA which 85 were used in 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 ce l l s in the normal manner and incubated at 22°C for 4 hours after dialys is to remove the butyrate, according to the standard procedures for assaying histone deacetylase ac t iv i ty . 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 igo- and mononucleosomes under the same conditions fa i led to alter the degree of histone acetylation. These results confirm the conclusions from other experiments, that histone deacetylase is associated with a high molecular weight complex. It i s interesting to note the differences in the positions of several non-histone proteins in samples of histone deacetylase complex and nucleosomes from butyrate-treated HeLa ce l l s (Figure 19, lanes 1 and 4 of panel A, and panel B) and butyrate-untreated HeLa ce l l s (Figure 19A, lanes 2 and 3), in particular 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 in the net charge of the proteins and may reflect altered patterns of protein modification. This observation lends support to the view that butyrate has many effects on ce l l s (219,220,221, 223,224), although as was shown ear l i er , exposure of HeLa ce l l s to butyrate does not a l ter the distribution of histone deacetylase in chromatin. 86 Figure 19 Acid urea gel profiles of histone deacetylase complex and nucleosomes Histones were prepared from histone deacetylase complex and nucleosomes as described in 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 ce l l s ; 2, histone deacetylase complex prepared from HeLa ce l l s which had not been exposed to butyrate; 3, o l igo- and mononucleosomes prepared from HeLa ce l l s which had not been exposed to butyrate; 4, ol igo- and mononucleosomes prepared from butyrate-treated HeLa ce l l s ; 5, bovine serum albumin and spermine which were used in the preparation of the histones (see Experimental Procedures). (B) Lane 1, histone deacetylase complex prepared from butyrate-treated HeLa ce 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, ol igo- and mononucleosomes from butyrate-treated HeLa c e l l s . 87 87a Figure 20. Optical scans of histone H4 on acid urea polyacrylamide gels. The acid urea gels shown in Figure 19 were stained with Coomassie Blue and the portions containing histone H4 were scanned at 550 nm. The te tra- , t r i - , d i - , mono-, unacetylated forms of histone H4 are separated according to the presence of acetyl 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 ce l l s which had been exposed to 10 mM butyrate for 8 hours; (B) Histone H4 from histone deacetylase complex prepared from HeLa ce l l s which had not been exposed to butyrate; (C) Histone H4 from histone deacetylase complex of butyrate-treated HeLa ce l l s ; the complex was incubated at 22°C for 4 hours upon removal of the butyrate. 88 II Restriction Enzyme Analysis of Histone Deacetylase Complex DNA The DNA associated with the deacetylase complex was found to range from 5-11 Kbp, permitting restr ict ion endonuclease analysis to be carried out. The human genome has a number of sate l l i te DNA components which produce fragments of characteristic sizes when digested with particular restr ict ion endonucleases. Since the sizes of the fragments created by the digestion of human sate l l i tes II and III with Hael l l and EcoRI have been published (194), histone deacetylase complex DNA and total HeLa c e l l DNA were digested with these enzymes in order to ascertain i f these sate l l i te sequences were s ignif icantly enriched or depleted in the histone deacetylase complex. EcoRI recognizes the sequence GAATTC while Hae l l l recognizes GGCC. Two other restr ic t ion enzymes which recognize 4bp sequences were also used: Mspl which cuts at CCGG and TaqI which has the recognition s ite TCGA. DNA was prepared from both the deacetylase complex and HeLa c e l l nuclei by phenol extraction, and digested with the above restr ic t ion 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. Hae l l l digestion of either histone deacetylase complex DNA or genomic DNA resulted in 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 Hae l l l digestion of human sate l l i te III . 89 Figure 21. Two-dimensional gel electrophoresis of histones Butyrate-treated HeLa nuclei were part ia l ly 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 in the f i r s t dimension and SDS 18% polyacrylamide gel electrophoresis in the second dimension. The gel was stained with s i lver . The histones are identified and the protein with a molecular weight of approximately 12K is marked with an arrow. 90 Digestion of histone deacetylase complex DNA and total 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 sate l l i tes II and III . 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 Hael l l and EcoRI restr ict ion digests of histone deacetylase complex DNA and HeLa genomic DNA fai led to detect any significant differences in the proportion of sate l l i te DNAs. The DNA associated with the deacetylase complex i s therefore not noticeably enriched or depleted in sate l l i tes II and III, and appears to be of high complexity. I l l 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 nuclei 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 sufficient 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 ightly bound core histones which are resistant to extraction with 2 M NaCl has been described i n the l i terature (164). kbp kbp •23.7 ^9.5 ^6.7 •2.3 -2.0 1.4 1.3 0.6H 0.4-J 0.3 91a Figure 22. Restrict ion enzyme digestion of histone deacetylase complex DNA and genomic DNA. HeLa histone deacetylase complex DNA and HeLa genomic DNA were digested with several res tr ic t ion enzymes. The digest products were analyzed by electrophoresis on horizontal slab gels of 0.75% agarose in Tris-borate buffer (pH 8.3). These were stained with 5 ug/ml of ethidium bromide and the DNA bands were visualized under UV l ight . (A) histone deacetylase complex DNA. Lane 1, TaqI digest of pBR322; 2, Hae 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, Hae 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 earl ier 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 Act iv i ty of Nuclear Matrix Nuclear matrix was prepared from [ 3H]-acetyl labelled HeLa c e l l nuclei in the presence of 50 mM sodium butyrate. Aliquots of the labelled nuclear matrix were taken up in butyrate-free buffer and incubated at 22°C for two hours. Released [ 3H]-acetate was extracted with ethyl acetate as usual, and counted. The commonly used method for preparing nuclear matrix (164) includes an extraction with 1% Triton X-100. However, when this step was included in the preparation of [ 3H]-acetate labelled nuclear matrix, no endogenous histone deacetylase act iv i ty was detected. On the other hand, i f the detergent extraction was omitted, acetate-labelled nuclear matrix routinely released 40% of the total counts present, while identical samples boiled for 1 minute prior to assaying for enzyme act iv i ty released only 5%. The protein profi les of samples of nuclear matrix prepared either with or without the Triton X-100 wash were examined by SDS polyacrylamide gel 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 pellet containing the nuclear matrix i s obtained. The poss ibi l i ty therefore exists that the histone deacetylase act iv i ty observed in 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 II-III material from a Bio-Gel A-50m column are shown in Figure 24A and B. Since the poss ibi l i ty existed that the deacetylase complex might be equivalent to, or associated with, the nuclear matrix, corresponding patterns for the nuclear matrix from HeLa ce 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 in 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 non-histone proteins. Several of the major components are found in both the histone deacetylase complex and nucleosome fractions as well as in the nuclear matrix (indicated by dots in Figure 24). The peak I histone deacetylase complex i s enriched in some components, and depleted in others relative 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 surprising, since there i s as yet no concensus as to which of the many components seen in nuclear matrix preparations are specific to these structures. Furthermore, nuclear matrix i s prepared by extensive digestion of the nuclear DNA with DNase I , whereas in 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 in Experimental Procedures. The effect of extracting nuclear matrix preparations with Triton X-100 was examined by comparing the protein profi les of HeLa nuclear matrix prepared with and without extraction with this detergent. SDS 15% polyacrylamide gel electrophoresis was carried out using the standard discontinuous buffer system. Lane 1, nuclear matrix prepared without exposure to Triton X-100; 2, nuclear matrix extracted with 1% Triton X-100; 3, HeLa histones. 95 since this i s a precondition for the detection of the in s i tu deacetylase ac t iv i ty . It i s possible, indeed l i k e l y , that the 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 establish unambiguously whether histone deacetylase i s 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 dissociation of histone depleted chromosome scaffolds (165), the effect of these reagents on the histone deacetylase complex was investigated. a. Histone Deacetylase Act iv i ty of Treated Complex [ 3 H]-acetyl labelled histone deacetylase complex was prepared from labelled HeLa c e l l nuclei in the presence of 50 mM sodium butyrate by part ia l micrococcal nuclease digestion and chromatography using a Bio-Gel A-50m column.The pooled fractions of histone deacetylase complex were divided into equal aliquots, treated as outlined in Table 7, and assayed for histone deacetylase act iv i ty upon removal of sodium butyrate by d ia lys i s . A para l le l set of untreated samples served as a control to determine the normal level of enzyme act iv i ty . Each assay released 208,000 cpm, representing 48% of the total present. The presence of B-mercaptoethanol at either 1 mM or 10 mM caused a strong inhibit ion of the 96a Figure 24. SDS-polyacrylamide gel profi les 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% gel; B, 10% gel . 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 II-III material; 6, HeLa histone markers. Column regions pooled are defined i n Figure 9. Major bands which correspond in lanes 3, 4 and 5 are marked by dots. 97 endogenous histone deacetylase i n the complex. In order to e s t a b l i s h 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 of l a b e l l e d histone deacetylase complex were made 10 mM i n J3-mercaptoethanol as before, but B-mercaptoethanol was omitted during the d i a l y s i s used to remove sodium butyrate. In t h i s manner, the histone deacetylase complex was exposed t o 10 mM J3-mercaptoethanol, but none was present during the enzyme assay. Although the enzyme a c t i v i t y was s l i g h t l y higher than i n the previous case where B-mercaptoethanol was present during the assay, the a c t i v i t y remained s u b s t a n t i a l l y lower than i n the untreated c o n t r o l assays, only 18% of the t o t a l counts being r e l e a s e d . Therefore i t appears from these r e s u l t s that the i n h i b i t i o n of histone deacetylase by J3-mercaptoethanol i s i r r e v e r s i b l e . Neocuproine i s a strong copper i o n c h e l a t o r (195) and i n order to observe i t s e f f e c t on histone deacetylase a c t i v i t y , samples of the [ 3 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 against b u f f e r A c o n t a i n i n g 3 mM neocuproine f o r 2 hours at 4°C and assayed. As c o n t r o l s , equal a l i q u o t s of the complex were t r e a t e d i n the same manner, but without the a d d i t i o n of neocuproine. The r e s u l t s i n Table 7 c l e a r l y show that exposure of the histone deacetylase complex to 3 mM neocuproine causes a strong i n h i b i t i o n of histone deacetylase a c t i v i t y , approximately equal to that caused by fl-mercaptoethanol. The i n h i b i t i o n by C-mercaptoethanol could have occurred as a r e s u l t of i n a c t i v a t i o n of the enyzme or by d i s r u p t i o n of the histone deacetylase complex. The e f f e c t of B-mercaptoethanol on enzyme a c t i v i t y was examined f u r t h e r by preparing histone deacetylase complex from u n l a b e l l e d HeLa c e l l n u c l e i and using f r e e a c e t a t e - l a b e l l e d histones as the s u b s t r a t e . Although the chromatin-bound histone deacetylase r e a c t s most e f f i c i e n t l y w i t h 98 Table 7 The Effect of 3-mercaptoethanol and neocuproine on histone deacetylase complex. Treatment of histone deacetylase complex prior to dialysis to remove butyrate Treatment of histone deacetylase complex during dialys is to remove butyrate 3H-acetate released (%) (less background) 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 Dialyzed for 2 h against buffer A None 40 Equal samples of [ 3H]-acetyl labelled histone deacetylase complex were dialyzed against buffer B (10 mM Tris-HCl pH 7.8, 20 mM NaCl) to remove butyrate after either being modified or dialyzed against buffer A (10 mM Tris-HCl pH 7.4, 0.7 mM EDTA, 50 mM sodium butyrate). They were then assayed for histone deacetylase ac t iv i ty . The assays were performed i n t r ip l i ca te each time. The background release of [ 3H]-acetate from labelled histone deacetylase complex was measured by boil ing samples for 1 minute prior to dialys is to remove butyrate and assaying for histone deacetylase. This background was found to be 9% of the total counts present. 99 histones i n the form of nucleosomes, free histones are deacetylated at a detectable rate. 1.5 ml a l i q u o t s of unlabelled histone deacetylase complex were incubated with 15 u l (30,000 cpm) of free l a b e l l e d histone e i t h e r with or without 10 mM fi-mercaptoethanol, as shown i n Table 8. P a r a l l e l samples of histone deacetylase complex were boiled p r i o r to incubation with the l a b e l l e d histone, to measure the background release of r a d i o a c t i v i t y . Although only 8% of the t o t a l counts present i n the added histones was released, there was no difference i n histone deacetylase a c t i v i t y between assays containing B-mercaptoethanol and those free of the reducing agent. The B-mercaptoethanol thus seems to a f f e c t the i n t e r a c t i o n between histone deacetylase and chromatin, rather than the enzyme i t s e l f . b. Physical Properties of Treated Complex Advantage was taken of the very high molecular weight of the histone deacetylase complex to perform a s e r i e s of simple experiments examining the p o s s i b i l i t y that fi-mercaptoethanol might disrupt i t s structure. In the f i r s t experiment, a micrococcal nuclease digest of HeLa c e l l n u c l e i was applied to a Bio-Gel A-50m column and the excluded peak (histone deacetylase complex) was c o l l e c t e d and reapplied to the same column. A l l of the material which absorbed at 260 nm was recovered i n the excluded peak as shown i n Figure 25A. R e i s o l a t i o n of the histone deacetylase complex by a second passage through a Bio-Gel A-50m column did not cause d i s s o c i a t i o n . However, when the experiment was repeated with 10 mM B-mercaptoethanol i n the buffer during the second column run, two peaks were observed (Figure 25B). Again, quantitative recovery of the A0(;f.-100 Table 8 E f f e c t of B-mercaptoethanol on histone deacetylase. [ 3H]-acetate released (cpm) 2171 2201 2145 A d d i t i o n s to the Assays (mM 8-mercaptoethanol) 0 1 10 Equal a l i q u o t s of u n l a b e l l e d histone deacetylase complex were incubated w i t h added [ 3H] - a c e t y l l a b e l l e d histones f o r 2 hours at 22°C. 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 acetate 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. When the experiment was repeated using endogenous acetyl-labelled histone deacetylase complex, no histone deacetylase act iv i ty was detected i n either peak. Part ia l digests of HeLa c e l l nuclei with micrococcal nuclease normally yielded a prof i le such as that shown in Figure 25C when separated on a Bio-Gel A-50m column. A description of the protein and DNA content across these column profi les has been presented ear l ier (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 fractionation, resulted in an altered column profi le as seen in Figure 25D. There was a pronounced decrease in the amount of histone deacetylase complex and a corresponding increase in the amount of mono- and oligonucleosomes. Thus, the extent of digestion of the material normally eluting in the excluded region of the column was much greater in the presence of fi-mercaptoethanol. The most probable explanation for the altered column profi le i s that 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 rapidly. 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 electrophoresis and the gels are shown in Figure 26. The histone deacetylase complex which was exposed to 10 mM fi-mercaptoethanol was prepared by standard micrococcal 102 nuclease d i g e s t i o n of HeLa n u c l e i and f r a c t i o n a t e d on a Bio-Gel A-50m column without any B-mercaptoethanol present. The r e s u l t i n g histone deacetylase complex was r e a p p l i e d to a Bio-Gel A-50m column c o n t a i n i n g 10 mM B-mercaptoethanol and the f r a c t i o n s comprising the complex were pooled, concentrated and p r e c i p i t a t e d as described i n Experimental Procedures. A comparison of the p r o t e i n s present i n the i n t a c t h istone deacetylase complex w i t h those i n the complex which i s i s o l a t e d from a Bio-Gel 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 of core histones i n the l a t t e r . The d i s s o c i a t i o n of the complex by B-mercaptoethanol t h e r e f o r e seems to r e s u l t i n a s i g n i f i c a n t l o s s of non-histone p r o t e i n s . 103 o < V o l u me (ml) 103a Figure 25. Fractionation of fi-mercaptoethanol-treated histone deacetylase complex on a Bio-Gel A-50m column. (A) Histone deacetylase complex, purified 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 nuclei were part ia l ly 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 nuclei were isolated and part ia l ly 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 profiles 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). Major bands which correspond in lanes 2 and 3 are marked by dots. (B) Lane 1, low molecular weight markers; 2, HeLa histone deacetylase complex isolated from a Bio-Gel A-50m column containing 10 mM C-mercaptoethanol; 3, HeLa histones. 105 DISCUSSION The experiments described i n t h i s t h e s i s have provided i n f o r m a t i o n on the chromatin-bound histone deacetylase of HeLa c e l l s . The data i n the f i r s t part of the " R e s u l t s " s e c t i o n showed that the a c t i v e enzyme i s found only i n a s s o c i a t i o n w i t h a high molecular weight complex and f i n d i n g s described l a t e r showed that t h i s h i s t o n e deacetylase-containing complex shares s e v e r a l of the p r o p e r t i e s of nuclear matrix and chromosome s c a f f o l d . The relevance of these r e s u l t s w i l l be discussed w i t h respect to chromatin processing and the p o s s i b l e f u n c t i o n s of histone a c e t y l a t i o n . Since the c h a r a c t e r i z a t i o n of histone deacetylase and e l u c i d a t i o n of the d i s t r i b u t i o n of the enzyme w i t h i n chromatin were derived from experiments based upon an assay system using endogenous l a b e l l e d nucleosomes, the hi s t o n e deacetylase assay system w i l l be discussed i n some d e t a i l f i r s t . I Histone Deacetylase Assay The work presented i n t h i s report has shown that complexes c o n t a i n i n g l a b e l l e d h i s tones together w i t h histone deacetylase can be i s o l a t e d i n the presence of butyrate to provide a system f o r studying chromatin bound histone deacetylase. The a d d i t i o n of f r e e histones to a c e t y l - l a b e l l e d deacetylase complexes caused a concentration-dependent r e d u c t i o n i n deacetylase a c t i v i t y suggesting that chromatin-bound histone deacetylase i n t e r a c t s non-productively w i t h f r e e h i s t o n e s . However, when exogenous histones were added i n the form of mononucleosomes they were e f f i c i e n t as s u b s t r a t e s . These r e s u l t s e x p l a i n the d i f f e r e n t histone deacetylase a c t i v i t y p r o f i l e s obtained on i s o k i n e t i c sucrose gradients of chromatin 106 digests when assays are done with either added free acetyl-labelled histones (Figure 7A) or on endogenous labelled nucleosomes (Figure 7B). Vir tua l ly a l l of the histone deacetylase act iv i ty was detected in rapidly sedimenting material in gradients containing endogenous labelled nucleosomes, wherease gradients assayed with exogenous acetyl-labelled histones showed histone deacetylase in slowly sedimenting material, as well as some in the rapidly sedimenting fract ion. This slowly sedimenting histone deacetylase act iv i ty probably represents free enzyme since the peak of act iv i ty did not coincide with any nucleosome peak. Deacetylase complexes assayed with exogenous labelled histones display much lower levels of histone deacetylase act iv i ty than endogenous complexes because chromatin bound histone deacetylase interacts non-productively with free histones. In agreement with this interpretation, i t has been found that histone deacetylase solubilized from calf thymus chromatin could deacetylate added free [ 3H]-acetyl histones, wherease the chromatin-bound histone deacetylase reacted poorly with this substrate (76). A comparison of the assay system presented i n this report with previously described assays for histone deacetylase shows the advantages of using an in vivo assembled complex of the enzyme and endogenous labelled substrate. Assay systems have been developed in 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 labelled histones as the substrate, but experiments with the part ia l ly purified acidic histone deacetylase from cal f thymus have shown that free histones, part icularly H3 and H4, bind readily to the acidic protein and maximum 107 precipitation of the histones occurs at a histone deacetylase to histone rat io of approximately 1:12 (mol/mol) (74). The binding of histones to the chromatin-bound form of histone deacetylase may part ia l ly account for inhibi t ion of enzyme act iv i ty observed when free histones were added to samples of histone deacetylase complex (Table 2). Other histone deacetylase assays have ut i l i zed part ia l ly purified forms of the enzyme; however, purif icat ion has resulted in alterations in the characteristics of the deacetylase. For example i t has been shown that a crude extract of calf thymus nuclei can deacetylate both free histones and chromatin-bound histones, wheareas the part ia l ly purified 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 in vivo labelled histones in their physiological conformation with no free histones present and the enzyme remains in association with chromatin during the preparation and assay of the deacetylase complex. II Distribution of Histone Deacetylase in Chromatin Digestion of HeLa nuclei with micrococcal nuclease produced mononucleosomes and polynucleosomes of up to 50 nucleosomes in 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 this length were normally resolved in 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 ter the prof i le and this suggests that very strong binding must exist between the DNA and the histone deacetylase complex. Another example of DNA-protein binding which i s stable in 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 later . Fractionations of micrococcal nuclease digests of [ 3 H]-acetyl-labelled nuclei on Bio-Gel A-5m and A-50m columns (Figures 8 and 10) have shown that histone deacetylase act iv i ty i s associated with a high molecular weight complex and no act iv i ty was found on ol igo- or mononucleosomes. The specific radioactivity of histones i n the histone deacetylase complex was always greater than that of the nucleosomes in a given nuclease digest. Thus the nucleosomes from these part ia l digests may have been derived primarily from regions of chromatin which were acetylated prior to labe l l ing , and which contained acetyl groups with a low turnover rate. Also, 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 level of labelled acetyl groups in monomers and oligomers was high enough, however, to have allowed the detection of endogenous histone deacetylase act iv i ty , had i t been present i n this fract ion. The fractionation 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 in quite a different way from acetylases (68) and most non-histone chromosomal proteins which readily dissociate from chromatin in 109 high i o n i c s t r e n g t h s o l u t i o n s . This behavior i s c o n s i s t e n t w i t h the i d e a t h a t histone deacetylase may be bound to these complexes through 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 . R e d i g e s t i o n of l a r g e chromatin fragments w i t h micrococcal nuclease caused the histone d e a c e t y l a s e - c o n t a i n i n g chromatin to s h i f t to a lower s i z e d i s t r i b u t i o n , such that the a c t i v i t y peak became co i n c i d e n t w i t h the absorbance peak when the d i g e s t products were separated on a Bio-Gel A-5m column (Figure 14). The mononucleosomes produced during the r e d i g e s t i o n of the a c e t y l - l a b e l l e d complexes had a 3.2 f o l d higher s p e c i f i c a c t i v i t y than the mononucleosomes formed during the i n i t i a l d i g e s t i o n of endogenous l a b e l l e d n u c l e i . Taken together, these r e s u l t s suggest that 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 [ 3 H ] - a c e t y l l a b e l e d mononucleosomes and nuclease r e s i s t a n t m a t e r i a l c o n t a i n i n g the enzyme. These f i n d i n g s were supported by comparisons of p a r t i a l and extensive m i c r o c o c c a l nuclease d i g e s t s of [ 3H]-acetate l a b e l l e d n u c l e i . The mononucleosomes produced during an extensive nuclease d i g e s t i o n had a higher s p e c i f i c a c t i v i t y than the mononucleosomes produced by s h o r t e r nuclease d i g e s t i o n s . Extensive nuclease d i g e s t i o n e v i d e n t l y caused the re l e a s e of some chromatin bound histone deacetylase, since polynucleosomes prepared by p a r t i a l d i g e s t i o n released 44.2% of t h e i r t o t a l [ 3H]-acetate groups whereas those prepared by extensive d i g e s t i o n released only 26.8%. I f enzyme was released i t d i d not r e a s s o c i a t e w i t h mononucleosomes si n c e no deacetylase a c t i v i t y was detected i n t h i s f r a c t i o n , d e s p i t e the high s p e c i f i c a c t i v i t y of the histones i n these mononucleosomes. I t i s p o s s i b l e , however, that the released enzyme i s very unstable and becomes i n a c t i v a t e d during 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 [ 3H]-110 acetate released/A2^Q) was used to express histone deacetylase act iv i ty i t was found that histone deacetylase complex prepared by extensive digestion was enriched in deacetylase act iv i ty by approximately 30% relative to those prepared by part ia l nuclease digestion, again implying that micrococcal nuclease cleaves mononucleosomes from histone deacetylase complex to give a nuclease resistant core which is enriched in chromatin bound deacetylase. These results suggest that histone deacetylase i s protected from nuclease digestion which can cleave nucleosomes from the complex. A possible binding site for the deacetylase would be close to or within the protein complex i t s e l f , in association with nucleosomes. Column fractions containing histone deacetylase complex were heterogeneous with respect to enzyme act iv i ty , 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 imit access ibi l i ty of the nuclease. This poss ib i l i ty 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 ie ld oligonucleosomes and mononucleosomes under standard nuclease digestion conditions (Figure 25). Histone deacetylase assays measured the release of [ 3H]-acetate from histones which had been modified in the presence of butyrate during the 2 hour label l ing period in vivo. The poss ib i l i ty that exposure of the ce l l s to butyrate might have caused an alteration in the distribution of histone I l l deacetylase can be discounted, as chromatin isolated from HeLa ce l l s which had been labelled in the absence of butyrate had exactly the same enzyme distribution (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 ce l l s which had not been exposed to butyrate likewise contained hyperacetylated histones (Figure 19). Thus neither the incorporation of [ 3H]-acetyl groups into the histones of the deacetylase complex over the 2 hour label l ing period nor their subsequent release from only the high molecular weight complex were influenced by the presence of butyrate. I l l Characteristics of Chromatin Bound Histone Deacetylase The very rapid rate and large extent of deacetylation observed in the time course studies (Figure 16) showed that denaturation of the chromatin-bound histone deacetylase had been kept to a minimum during the isolat ion of the complexes. A very similar time course for histone deacetylase has been observed using calf thymus homogenate and added free acetyl-labelled 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 imi lar ly , two dist inct populations of acetylated histones have been identif ied in Hepatoma tissue culture ce 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 [ 3H]-acetyl groups were released in 10 minutes shows that most of the chromatin-bound histone deacetylase act iv i ty i s interacting with rapidly 112 t u r n i n g over a c e t y l groups i n t h i s system. However, the slow r e l e a s e of a c e t y l groups over the next 2 hours suggests that histone deacetylase may 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 both populations of histone and the d i f f e r e n c e i n r a t e s could be due to a l t e r e d a c c e s s i b i l i t y or enzyme preference f o r c e r t a i n forms of a c e t y l a t e d h i s t o n e s . In these s t u d i e s , a high degree of a c e t y l a t i o n has been observed i n the histone H4 a s s o c i a t e d w i t h the h i s t o n e deacetylase complex from butyrate t r e a t e d HeLa c e l l s (Figure 19). 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 of very r a p i d a c e t y l group turnover, a n o t i o n which 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 of 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 that chromatin-bound histone deacetylase does not r e v e r s i b l y d i s s o c i a t e during the course of the r e a c t i o n poses the question of how the enzyme encounters i t s s u b s t r a t e . The two most obvious p o s s i b i l i t i e s are that h i s t o n e deacetylase i s attached s t o i c h i o m e t r i c a l l y to nucleosomes i n s e c t i o n s of chromatin w i t h r a p i d a c e t y l group turnover, or that nucleosomes are threaded past the enzyme i n a manner s i m i l a r to that o c c u r r i n g w i t h processive enzymes such as n u c l e o t i d e polymerases. No f i r m choice can be made at t h i s p o i n t i n favour of e i t h e r of these a l t e r n a t i v e s , although the former mechanism would r e q u i r e r a t h e r high concentrations of enzyme and seems l e s s l i k e l y . The s u p p o s i t i o n that the h i s t o n e deacetylase may be attached to a l a r g e m a t r i x l o c a t e d at the base of the chromatin loops would account f o r the observation that the enzyme does not r e v e r s i b l y d i s s o c i a t e during the course 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 idea that histone deacetylase and nucleosomes encounter each other i n a processive manner. M i l l i m o l a r concentrations of B-mercaptoethanol were found to have no 113 effect on histone deacetylase act iv i ty when free acetyl-labelled histones were used as the substrate (Table 8). A similar result has been reported for calf thymus histone deacetylase assayed with free acetyl-labelled histones (71). A frequently suggested function of histone acetylation is that i t serves as a control for transcription, since active sections of chromatin appear to contain hyperacetylated histones (83,84,85,86,87). Conversely, inactive chromatin, e.g. chicken erythrocyte chromatin, appears to have low levels of histone acetylation with a very slow turnover of acetyl groups (229). Transcriptionally competent sections of chromatin have also been shown to be enriched in HMG 14 and HMG 17 (202). Since these proteins can inhibit histone deacetylase in v i tro using nuclei and free labelled histones (188), i t has been suggested that HMG 14 and HMG 17 could inhibit histone deacetylation and produce a localized enrichment in hyperacetylated histones, ultimately resulting in transcription. 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, this seems unlikely i n view of the strong inhibi t ion of histone deacetylase act iv i ty in nuclei by millimolar concentrations of butyrate. Rather, the micrococcal nuclease digestion of HeLa chromatin and the isolat ion 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 inhibitory response to HMG proteins. ( 114 Support for this poss ib i l i ty comes from the observation that an inhibitor of histone deacetylase act iv i ty i s lost from the part ia l ly purified enzyme from cal f thymus after precipitation i n acetone and chromatography on a Sepharose 6B column (74). Similarly , HMG 14 and HMG 17 or other proteins may alter the substrate specif ic i ty of histone deacetylase. Histone deacetylase appears to have a f a i r l y broad substrate spec i f ic i ty , as millimolar concentrations of butyrate inhibi t the deacetylation of each of the differently acetylated forms of histone H4 in rat hepatoma ce l l s (178), and calf thymus histone deacetylase has been shown to also deacetylate H3 (74) as well as acetylated HMG 14 and HMG 17 (228). Treatment of calf thymus histone deacetylase with hydroxylapatite during the course of par t ia l purif icat ion has been found to result in a significant change in the ratio of deacetylation of histones H3 and H4. The marked activation of H3 deacetylation act iv i ty suggests that an inhibitor 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 trichloroacetic acid during their o preparation may have resulted in a loss of some of their biological ac t iv i ty . Incubation of HMG proteins with nuclei rather than with histone deacetylase complex may convert the proteins to their active form. Futher investigation w i l l be required before an unequivocal statement on the action of HMG proteins can be made. IV Histone Deacetylase Complex Several striking s imi lar i t ies 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 nucle i . Examination of the proteins present in the histone deacetylase complex by SDS polyacrylamide gel electrophoresis (Figure 24) revealed several non-histone proteins which were also present in nuclear matrix preparations. These included the major HeLa nuclear matrix proteins cited i n the l i terature (163). The lack of identity 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 nuclei using a reduced DNase I digestion to ensure that some histones were retained to serve as a substrate for the histone deacetylase, a significant leve l of enzyme act iv i ty was detected. Apart from an increase in the amount of histone present, the reduction in the extent of DNase I digestion did not result in a protein composition signif icantly different from that of conventional nuclear matrix preparations as seen by SDS polyacrylamide gel electrophoresis. Omission of the extraction of nuclear matrix preparations with 1% Triton X-100 did not signif icantly al ter the protein content of the nuclear matrix preparations (Figure 23), but was necessary for the maintenance of histone deacetylase ac t iv i ty . Therefore, chromatin-bound histone deacetylase must be detergent-labile. However, in view of the manner in which nuclear matrix i s isolated, namely by successive extractions of pelleted material, the poss ibi l i ty remains that the histone deacetylase act iv i ty observed in 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 irreversible inhibit ion of histone deacetylase act iv i ty (Table 7) and the dissociation of the complex i t s e l f (Figure 25). The inhibit ion of deacetylase act iv i ty was not due to the reduction of th io l groups on the enzyme i t s e l f , since 10 mM B-mercaptoethanol did not affect the deacetylase act iv i ty of the complex when this was assayed with added histones (Table 8). Also, neocuproine, which should have no effect on th io l groups, inhibited histone deacetylase act iv i ty in 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 part ia l disruption of the complex and to a loss of deacetylase ac t iv i ty . The digestion of HeLa nuclei with micrococcal nuclease in the presence of 10 mM B-mercaptoethanol resulted in a dramatic reduction i n the deacetylase-containing peak of the Bio-Gel A-50m column, and an increase in the proportion of mono- and oligonucleosomes (Figure 25). This suggests that the deacetylase complex is 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 histones from chromosomes w i t h SDS. The chromosome s c a f f o l d d i s s o c i a t e s upon treatment w i t h as l i t t l e as 1 mM fi-mercaptoethanol or 3 mM neocuproine, and seems to be maintained 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 i o n s . Binding of the Cu ions 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 sulphhydryl groups. 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 represent a l t e r n a t i v e forms of the same s t r u c t u r e during d i f f e r e n t stages of the c e l l c y c l e , or whether they are d i s t i n c t s t r u c t u r e s which may a s s o c i a t e w i t h each other. The 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 a s s o c i a t e d w i t h a l a r g e s t r u c t u r e which 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 or neocuproine 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 of 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 of the enzyme to 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 of histone deacetylase complex DNA and HeLa genomic DNA w i t h H a e l l l and EcoRI (Figure 22) showed that s a t e l l i t e s I I and I I I were not enriched i n t h i s m a t e r i a l . Although Mspl and TaqI are not s p e c i f i c f o r s a t e l l i t e DNA, 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 histone deacetylase complex DNA and genomic DNA when they were digested w i t h these enzymes. The n o t i o n that histone deacetylase 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 of heterochromatin and that the f u n c t i o n of the enzyme might be to maintain these sequences i n an unacetylated 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 probably be discounted. S i m i l a r l y , reannealing s t u d i e s of the short DNA fragments anchored to the nuclear m a t r i x of r a t and mouse l i v e r interphase n u c l e i and to the metaphase s c a f f o l d of Chinese hamster DON c e l l n u c l e i demonstrated that t h i s DNA has the same complexity 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 in the preparations (micrococcal nuclease, DNase I or endogenous digestion). Nuclear matrix, prepared from rat l i ver nuclei under DNase I digestion conditions which lef t 1% of the total DNA attached to the matrix, was found to contain DNA which showed no difference in complexity from genomic DNA (217). Thus there are obvious s imi lari t ies i n the DNA associated with the histone deacetylase complex and nuclear matrix. The poss ibi l i ty that the function of sa te l l i te DNA might be to anchor DNA loops to the nuclear matrix or other large protein complex i s unlikely in view of the reports on nuclear matrix DNA and the restr ict ion 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 ver nuclear matrix described above has the same complexity as genomic DNA, i t i s enriched in transcribed rDNA (217). Similarly , the transcribed ovalbumin gene of chicken oviduct ce l l s (172) and SV40 sequences of infected 3T3 ce l l s (173) are associated with the nuclear matrix. This i s consistent with the idea that transcription i s associated with the nuclear matrix. As described ear l i er , acid urea gel electrophoresis (Figure 19) revealed that the histone deacetylase complex from butyrate-treated HeLa ce l l s contained hyperacetylated chromatin with tetraacetylated H4 as the most abundant form of this histone. Similarly, deacetylase complex from ce 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 kinetics 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 al ter the conformation of chromatin as DNase I preferentially digests the DNA in 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 ie ld nucleosomes with a more "open" structure. More recently, evidence suggests that the major effects of histone acetylation may operate at higher levels of chromatin structure, e.g. the folding of the 30 nm fibre (91). One of the most frequently proposed functions of histone acetylation i s regulation of transcription since actively transcribed sequences are preferentially associated with hyperacetylated histones (83 - 89). Histone acetylation can also influence hormone binding which can ultimately lead to a change in transcription. For example, the action of the thyroid hormones in mammalian ce 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 level of the chromatin associated hormone receptor; however, the chromatin associated receptor reappears when histone acetylation returns to normal physiological levels (231). These results are consistent with a model in which the af f in i ty between the thyroid hormone receptor and chromatin varies with the state of histone acetylation. There i s also strong evidence for other chromatin processing being influenced by the degree of chromatin acetylation. 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 of the 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 . Increased histone 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 to lead to an increase i n UV-induced DNA r e p a i r (90). The increase i n r e p a i r was observed i n both 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 increase i n the amount of the highest a c e t y l a t e d form of his t o n e H4. Increased h i s t o n e a c e t y l a t i o n l e d to a r i s e i n the r a t e of i n c i s i o n by an endonuclease at UV-induced l e s i o n s during the i n i t i a l phase of r e p a i r . I t i s p o s s i b l e that the increased r a t e of DNA i n c i s i o n during 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 of a c e t y l a t e d chromatin are due to 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 strand breaks formed i n DNA during 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 to 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 to the h a l f l i f e of the r a p i d l y deacetylated h i s t o n e a c e t y l groups i n the high molecular weight histone 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 chromatin experiences c y c l e s of 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 that t h i s process a l l o w s chromatin 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 of the DNA and necessary r e p a i r . The requirement f o r c e l l s to c o n s t a n t l y survey t h e i r genomes may account f o r the l a r g e amount of energy expended by c e l l s i n r a p i d histone 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 . For example, these processes consume at l e a s t 2 x 10 6 e q u i v a l e n t s of ATP/minute i n HTC c e l l s (91). The histone deacetylase complex reported i n t h i s t h e s i s should be considered 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 contains 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 histones which probably experience r a p i d turnover of 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 the p o s i t i o n s of s e v e r a l non-core histone 121 p r o t e i n s i n a c i d urea polyacrylamide g e l s between histone deacetylase 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 histone deacetylase complex i s o l a t e d from c e l l s which had not experienced butyrate ( F i g u r e 19). M i l l i m o l a r concentrations of butyrate have many documented e f f e c t s on eukaryotic 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 p r o t e i n m o d i f i c a t i o n . Butyrate induces the synth e s i s of 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 histone s y n t h e s i s u l t i m a t e l y becomes independent of DNA r e p l i c a t i o n (220). L i k e w i s e , butyrate causes a transformed S y r i a n hamster c e l l l i n e to produce 2 polypeptides u s u a l l y found only 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 to butyrate, depending upon the p r o t e i n f r a c t i o n analyzed; HMG 14 and HMG 17 phosphorylation i s enhanced by butyrate (225) whereas HeLa c e l l s a l s o experience an i n h i b i t i o n of the phosphorylation of histones HI and H2A (223). A l t e r a t i o n s i n the s u l p h a t i o n of 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 kidney tumour c e l l s (221). The d i f f e r e n t m o b i l i t i e s of p r o t e i n s i n histone deacetylase complex from butyrate 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 the high molecular complex. The nu c l e a r m a t r i x i s the best c h a r a c t e r i z e d h i g h molecular weight nuclear complex and appears to be the s i t e of at l e a s t some chromatin processing. The cont e n t i o n that DNA r e p l i c a t i o n occurs w i t h i n the nuclear m a t r i x i s based upon the observations that the s t r u c t u r e i s enriched i n Okazaki fragments and that l a b e l l e d thymidine i s incorporated i n t o DNA i n r a t l i v e r and bovine l i v e r n uclear m a t r i x (169,170). Pulse l a b e l l i n g experiments have a l s o i n d i c a t e d that the o r i g i n s of r e p l i c o n s are bound to 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 synthesized nucleosomal h i s t o n e s i n r a t hepatoma c e l l s are more a c c e s s i b l e to histone 122 a c e t y l a s e i n v i v o and newly synthesized chromatin i s a l s o i n a r e l a t i v e l y extended, open conformation which i s t y p i c a l l y a s s o ciated w i t h hyperacetylated chromatin (227). When the r a t hepatoma c e l l s were pulse l a b e l l e d w i t h a c e t a t e , i t was found that newly r e p l i c a t e d chromatin d i d not reach a h i g h l y a c e t y l a t e d s t a t e unless butyrate was present. These f i n d i n g s suggest that the increased a c c e s s i b i l i t y of newly r e p l i c a t e d chromatin to h i s t o n e a c e t y l a s e s and the tempo r a r i l y increased r a t e of 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 , that DNA r e p l i c a t i o n must occur i n c l o s e p r o x i m i t y to histone deacetylase. The hist o n e deacetylase complex should be considered as e i t h e r a p o s s i b l e s i t e f o r DNA r e p l i c a t i o n or as having 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 contains an a c t i v e deacetylase 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 to occur upon the nuclear m a t r i x on the b a s i s of i n d i r e c t evidence. The unique sequences w i t h i n DNA loops are arrranged i n a non-random manner such that a c t i v e genes are c l o s e to the matrix (172,173,174). The formation of nascent hnRNA t r a n s c r i p t s i n the c e l l nuclear m a t r i x of r a t endothelium (176) and the strong binding of hnRNA to Fri e n d c e l l nuclear m a t r i x (175) add support to the view that t r a n s c r i p t i o n occurs on the matrix. The p o s s i b l e f u n c t i o n s of h i s t o n e a c e t y l a t i o n and histone deacetylase complex i n the r e g u l a t i o n of t r a n s c r i p t i o n have been discussed e a r l i e r . However, there i s accumulating evidence that the nuc l e a r m a t r i x contains s i t e s f o r hormone binding and hormone regulated t r a n s c r i p t i o n has been an accepted phenomenon f o r many years. 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 chicken l i v e r n u clear m a t r i x whereas the nuclear m a t r i x from r o o s t e r l i v e r s contains very few s i t e s f o r the hormone. S i m i l a r l y , nuclear m a t r i x preparations from r a t pro s t a t e have been found to be r i c h i n dihydrotestosterone binding s i t e s 123 (177). Nuclear pore complex proteins are always found associated with nuclear matrix preparations containing hormone binding s i tes . The sex hormones are known to induce protein synthesis and in related experiments, nuclear matrix preparations from female rat l i v e r , endometrium and lung were examined for estradiol binding sites and hnRNA synthesis. Approximately 85% of total rapidly labelled hnRNA was associated with the l i ver nuclear matrix and high af f in i ty estradiol binding occurred in nuclear matrix from endometrium but not from the lung which i s not regulated by estradiol (176). Another study has found that the nuclear matrix from the uterus of the female rat contains estradiol binding sites (226). Taken together, these findings are in 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 sites on the nuclear matrix. An ordered arrangement of the DNA loop attachment s i tes , hormone binding sites and transcriptional apparatus on a large protein matrix would fac i l i t a t e hormone regulated transcription. 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 profi le of histone deacetylase complex (Figure 24). Although there are insufficient data to conclude whether the observed protein represents a nuclear pore lamin, the poss ib i l i ty 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 ight 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 in hormone regulated transcription. The precise function(s) of histone acetylation and the nuclear matrix have yet to be elucidated. Histone acetylation has been implicated in DNA repl icat ion, repair and hormone binding and regulation of transcription. Likewise, the nuclear matrix appears to support repl icat ion, hormone binding and transcription. In general, the location of complex enzymatic reactions within a matrix may fac i l i ta te chromatin processing. Unt i l recently, no enzyme act iv i ty had been assayed direct ly in the nuclear matrix. However, ATP-dependent nuclear ribonucleoprotein release and nucleoside triphosphatase act iv i t i es have been detected in rat l i v e r nuclear matrix (234). Furthermore, antibodies generated against a nuclear matrix glycoprotein inhibi t the enzyme act iv i t ies (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 in the presence of copper ion chelators and contains proteins found in 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. The attachment of nucleosomes to a complex containing histone deacetylase 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 ike ly fac i l i ta tes several processes which require altered nucleosome access ib i l i ty . This could be accomplished by regular acetylation and deacetylation of chromatin in the presence of other 125 chromatin processing enzymes. The present perception of chromatin may re q u i r e m o d i f i c a t i o n to accommodate the concept that chromatin i s i n a dynamic s t a t e w i t h constant f l u x i n l e v e l s of a c e t y l a t i o n . The high molecular weight h i s t o n e deacetylase complex o u t l i n e d i n t h i s t h e s i s provides a u s e f u l system to help e l u c i d a t e the fu n c t i o n s of 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 of the nuclear 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 . , Quagliarotti , 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. Quagliarotti , 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) Eur. 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. , Bilek, 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. Elsevier, 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. Nat 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 . , Strickland, W.N., Morgan, M. and Van Holt, C. (1974) FEBS Lett . 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. Tidwell , T . , A l l f rey , 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) Eur. 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 . , Goidl , J . A . and Salzman, N.P. (1972) Exp. Ce 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 rey , V . G . , Bautz, E . K . F . , McCarthy, B . J . , Schimke, R.T. and Tissieres , 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 . , Wei 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 Lett . 84, 331-336. 50. Ito, S. , Shizuta, Y. and Hayaishi, 0. (1979) J . B i o l . Chem. 254, 3647-3651. 51. Tsopanakis, C , McLaren, E . and Shal 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 III , W.S., Stone, P.R. and Kidwell, W.R. (1977) Exp. Ce l l Res. 106, 261-266. 130 56. Hayaishi, 0. and Ueda, K. (1977) Annu. Rev. Biochem. 46_, 95-116. 57. Ferr 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., Omidiji , D . , Gray, D.A. and Shal l , S. (1980) Nature 283, 593-596. 61. Po ir ier , G . G . , Sevard, P . , Rajotte, D . , Morisset, J . and Lord, A. (1978) Can. J . Bioc. 56_, 784-790. 62. Wong, N.C.W., Poir ier , G.H..and Dixon, G.H. (1977) Eur. J . Biochem. 77_, 11-21. 63. Levy-Wilson, B. (1983) Biochemistry in press. 64. Wiktorowicz, J . E . , Campos, K . L . and Bonner, J . (1981) Biochemistry 20, 1464-1467. 65. Garcea, R . L . and Alberts, B.M. (1980) J . B i o l . Chem. 255, 11454-11463. 66. Belikoff , E . , Wong, L . - J . and Alberts , 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) Eur. 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. Kikuchi, H. and Fujimoto, D. (1973) FEBS Lett . 29, 280-282. 74. V i d a l l , G . , Boffa, L . C . and Al l f rey , 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) Ce l l 14_, 105-113. 79. Sealy, L . and Chalkley, R. (1978) Ce l l 14_, 115-121. 80. V i d a l i , G. Boffa, L . C , Bradbury, E.M. and Al l f rey , V . G . (1978) Proc. Natl . 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. Nat l . Acad. Sc 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 Lett . 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. No l l , M. (1974) Nature 251, 249-251. 95. Thomas, J . O . and Kornberg, R.D. (1975) Proc. Natl . Acad. Sc 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 Lett . 70_, 209-211. 100. Lewis, P.N. (1979) Eur. J . Biochem. 99., 315-322. 101. Thomas, G . J . , Prescott, B. and Olins, D.E. (1977) Science 197, 385-388. 102. Cotter, K . I . and L i l l e y , D.M.J . (1977) FEBS Lett . 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, G. (1980) Annu. Rev. Biochem. 49, 1115-1156. 133 106. Weintraub, J . and Van Lente, F. (1974) Proc. N a t l . Acad. S c i . U.S.A. 7JL, 4249-4253. 107. R i l l , R.L. and Oosterhof, D.K. (1982) J . B i o l . Chem. 257, 14875-14880. 108. Whitlock, 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 Klug, A. (1977) Nature 269, 29-36. 110. N o l l , M. (1974) Nucl. Acids Res. 1, 1573-1578. 111. L u t t e r , L.C. (1979) N u c l . Acids Res. 6_, 41-57. 112. T r i f o n o v , E.N. and Bettecker, T. (1979) Biochemistry 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. Whitlock, 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. Whitlock, J r . , J.P., Rushizky, 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) Proc. N a t l . Acad. S c i . U.S.A. 73, 4382-4386. 118. Lohr, D., Corden, J . , T a t c h e l l , K., Kovacic, R.T. and Van Holde, K.A. (1977) Proc. N a t l . Acad. S c i . U.S.A. 74_, 79-83. 119. R i l l , R.L., Nelson, D.A., Oosterhof, D.K. and Hozier, J.C. (1977) Nucl. Acids Res. 4_, 771-789. 120. Chambon, P. (1977) Cold Spring 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) Science 183, 330-332. 122. O l i n s , A.L., Senior, 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 Varshavksy, A.J. (1977) C e l l 11, 619-629. 134 124. Thoma, F . , Rol ler , T . L . and Klug, A. (1979) J . Ce 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 . , Lutter, L . C , Rhodes, D . , Brown, R . S . , Rushton, B . , Lev i t t , M. and Klug, A. (1977) Nature 269, 29-36. 127. Pardon, J . F . , Worcester, D . L . , Wooley, J . C , Tatchel 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) Ce 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) Ce l l B i o l . Int. Rep. 1_, 107-116. 130. Dubochet, J . and No l l , M. (1978) Science 202, 280-286. 131. L i l l e y , D.M.J , and Tatchel 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. Natl . Acad. Sc i . U.S.A. 73, 1897-1901. 134. Carpenter, B . C , Baldwin, J . P . , Bradbury, E.M. and Ibel , K. (1976) Nucl. Acids Res. 3_, 1739-1746. 135. Jorcano, J . L . , Meyer, C , Day, L . A . and Renz, M (1980) Proc. Nat l . Acad. S c i . U.S.A. 77_, 6443-6447. 136. Ris , H. and Kubai, D.F. (1970) Annu. Rev. Genet. 4_, 263-294. 137. Thoma, R. , Kol ler , 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) Ce l l 15, 141-150. 142. Weintraub, H. and Groudine, M. (1976) Science 193, 848-856. 143. Garel, A. and Axel, R. (1976) Proc. Natl . Acad. Sc i . U.S.A. 73, 3966-3970. 144. Garel, A . , Zolan M. and Axel, R. (1977) Proc. Natl . Acad. Sc 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 Lett . 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. Sc i . 5 £ , 373-379. 154. Weissbach, A. (1977) Annu. Rev. Biochem. 46_, 25-47. 155. Witt ig , S. and Witt ig , B. (1982) Nature 297, 31-38. 156. Levy A. and Nol l , M. (1980) Nucl. Acids Res. 8, 6059-6068. 136 157. Samal, B. and Wo ree l , A. (1981) Ce l l 23_, 401-409. 158. Pfeif fer , W. and Zachau, H.G. (1980) Nucl. Acids Res. 8, 4621-4638. 159. Adolph, UW. (1980) J . C e l l . S c i . 42_, 291-304. 160. Adolph, L.W., Cheung, S.M. and Laemmli, U.K. (1977) Cel 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 Kel ler , 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) Ce 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 . Ce l l 44, 201-212. 168. Berezney, R. and Buchholtz, L . A . (1981) Exp. Cel 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) Ce l l 28^ , 99-106. 173. Nelkin, B . D . , Pardol l , D.M. and Vogelstein, B. (1980) Nucl. Acids Res. 8, 5623-5633. 174. Cook, P.R. and Braze l l , I .A. (1980) Nucl. Acids Res. 8_, 2895-2905. 175. Long, B.H. , Huang, C . - Y . and Pogo, A.0 . (1979) Ce l l 18_, 1079-1090. 137 176. Agutter, P.S. and B l r c h a l l , K. (1979) Exp. Ce 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 Alberts, 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. No 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. Tatchel 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 Lett . 100, 394-398. 193. Walker, J . M . , Hastings, J . R . B . and Johns, E.W. (1977) Eur. J . Biochem. 76_, 461-468. 194. Mitche l l , A . R . , Beauchamp, R.S. and Bostock, C . J . (1979) J . Mol. 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. Natl . Acad. Sc i . U.S.A. 74, 864-868. 197. Schlesinger, D.H. and Goldstein, G. (1975) Nature 255, 423-424. 198. Schlesinger, D . H . , Goldstein, G. and N i a l l , H.D. (1975) Biochemistry 14, 2214-2218. 199. Levinger, L . and Varshavsky, A. (1982) Cel 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) Ce l l 19_, 289-301. 203. Basler, J . , Hastie, N.D. , Pietras, 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. Natl . Acad. Sc 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) Cel l 6_, 85-110. 209. Lev i t t , M. (1978) Proc. Natl . Acad. Sc i . U.S.A. 75_, 640-644. 210. Finch, J . T . , No l l , M. and Romberg, R.D. (1975) Proc. Natl . Acad. S c i . U.S.A. 72_, 3320-3322. 211. A l lan , J . , Hartman, P . G . , Crane-Robinson, C. and Avi l e , F .X . (1980) Nature 288, 675-679. 212. Savic, A . , Richman, P . , Williamson, P. and Poccia, D. (1981) Proc. Natl . 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, W.C and Busch, H. (1972) J . B i o l . Chem. 247, 6018-6023. 215. DeLange, R . J . , Hooper, J . A . and Smith, E . L . (1972) Proc. Nat 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. Pardol l , D.M. and Vogelstein, B. (1980) Expt. Ce 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 Cserjesi , P. (1979) J . B i o l . Chem. 254, 4283-4290. 223. Boffa, L . C , Gruss, R . J . and Al l f rey , V .G. (1981) J . B i o l . Chem. 256, 9612-9621. 224. Tralka, 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. NAtl . Acad. Sc i . U.S.A. 78., 2189-2193. 226. Barrack, E . R . , Hawkins, E . F . , A l l en , S . L . , Hicks, L . L . and Coffey, D.S. (1977) Biochem. Biophys. Res. Commun. _7£, 829-836. 227. Cousens, L . S . and Alberts, B.M. (1982) J . B i o l . Chem. 257, 3945-3949. 228. Sterner, R . , V i d a l i , G. and Al l f rey , 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) Ce 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. Natl . Acad. Sc i . U.S.A. 80, 2285-2289. 

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