Current ideas on the structure of chromatin

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primary sites of interaction of the individual histones with DNA.

Current ideas on the structure of chromatin E. M. Bradbury Many studies have led to the proposal of a subunit structure for chromatin similar to a string of beads. Each bead is thought to contain 200 base pairs of DNA coiled on the outside of a multimeric histone unit comprised qf two each of the histones H2A, H2B, H3 and H4.

H2B and the arginine-rich histones H3 and H4. H 1 is about twice as large as the other histones and it is now generally accepted its function is different from that of the other four. It has been known for some time that the ratio of histone to DNA (1 .O to 1.2 ; 1.O) in multicellular organisms is relatively constant and that except for Hl the histones are present in roughly equimolar amounts. In an early model, Phillips [5] assumed that these four histones were equimolar and one of each histone could be complexed by about 100 base pairs of DNA. The sequence determination of histones [6], particularly of histone H4 by De Lange and his co-workers [2] generated a considerable amount of interest. Two properties of histones emerged from the studies. First, histone sequences are strongly conserved, particularly in those that are arginine-rich [2]. Theclassic result was that the calf thymus H4 sequence was found to be identical to that of pea except for two conservative replacements - valine and lysine in calf H4 being replaced by isoleucine and arginine in pea H4. The implication of this rigid conservation is that each and every residue is required for the function ofthe molecule and that this function is identical in all eucaryotes. The second property is an asymmetry found for the primary sequences of all histones. Well defined N-terminal segments of histones H2A, H2B, H3 and H4are very basic and contain a high proportion of residues such as proline, serine and glycine which favour extended chain structures. In contrast the carboxyl halves of H3 and H4 and the central regions of H2A and H2B contain a high proportion of apolar and other residues which favour helix formation. A general picture has emerged from studies of the conformation and interactions of individual histones in solution [7]. In aqueous solution histones are largely random coil. Increase in ionic strength induces the apolar regions to form welldefined structures which then act as sites E.M.B. is at the Biophysics Laboratory, Portsmouth interaction while the Polytechnic, Gun House, Hampshire Terrace, Ports- of histone-histone basic segments have been found to be the mouth, Hampshire, UK

In many areas of science new ideas leading to a major scientific advance often spring up in several different laboratories at the same time. These coincidences usually occur when the background knowledge in a field passes a certain threshold level and the number of research workers increase as it is perceived that an important and difficult problem is capable of solution. In this situation pivotal experiments are performed which change the direction of research and transform the field. Such is the background to the recent major advances in our understanding of the basic structure of chromatin. Cells of higher. organisms contain extraordinarily large amounts of DNA which in physical length can be of the order of metres. In current theories only a minor component of this DNA is thought to code for proteins and this poses several interrelated questions: (1) what are the mechanisms for activation and control of sets of genes in differentiated tissues? (2) why are such enormous lengths of DNA present? and (3) what are the processes for physically controlling the DNA in the nucleus through the cell cycle? We are concerned with this last question and ask whether chromatin has a basic structural unit and if so what are the possible mechanisms for packaging this unit into the higher order structures of the eucaryotic chromosomes? To find these processes we must look to the other components of the cell nucleus. In addition to DNA a second major component is a group of very basic proteins, the histones. There are five major histone fractions [ 11,each fraction exhibiting some complexity due to chemical modifications and in some cases primary sequence differences [2,3]. The following nomenclature has recently been proposed [4] to replace the nomenclature previously in use; the very lysine-rich histone is called Hl ; the intermediate histones, H2A and

Chromatin structure The basic structure ofchromatin is characterized by what appears to be a regular series of X-ray diffraction rings found at approximately 11, 5.5, 3.7,2.7 and 2.2 nm. To explain this series of rings M.H.F. Wilkins proposed a model in 1964 in which the histones coiled the DNA into a regular supercoil of diameter 10 nm and pitch 12 nm. There were two basic assumptions in this proposal [8]: (1) that there was only one structural repeat, the pitch of the helix, which gave the series of X-ray diffraction rings, and (2) the diffracted intensities came primarily from the DNA component. This model was widely accepted for almost a decade because only in the last few years has it been possible to check these assumptions by neutron diffraction. Two years ago new ideas on chromatin structure were generated in several laboratories from quite different approaches. A pivotal result was that reported by Hewish and Burgoyne [9] from studies of endonuclease digestion of chromatin in rat liver nuclei. They found that the DNA products from this digestion formed a regular series ofmolecular weights, the higher lholecular weights being integral multiples of the smallest size unit. It was later shown that this smallest size unit of DNA was approximately 200 base pairs (b.p.). They concluded that this series of DNA digestion products was ‘a reflection of regularity in the distribution of protein on the DNA’ and proposed that ‘chromatin has some simple, basic repeating substructure with a repetitive spacing of sites accessible to the endonuclease’. In parallel studies van Holde and his co-workers [lo] obtained chromatin particles called ‘P-S particles’ by micrococcal and staphylococcal nuclease digestion of calf thymus chromatin. These particles were characterized by hydrodynamic techniques and found to have the physical properties of compact globular structures. It was found that the compact structure could be unfolded by trypsin digestion showing that it was’held together by the histones. Further evidence for chromatin particles came from electron microscope studies of Olins and Olins [ 111and Woodcock [12] who reported observations of linear arrays of discrete spherical particles in gently prepared chromatin. Woodcock reported the diameter to be about 10 nm while Olins reported it to be 7 nm and called the particles ‘v-bodies’. Unfortunately, attempts made by Woodcock to publish his data in more detail were obstructed by sceptical referees. A reason why such chromatin ‘beads on a string’ were not

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8 observed in earlier electron microscopic studies is probably due to sample preparation although a clear description of globular chromatin units is to be found in a premature study of sea urchin spermatozoa by Afzelius [13] in 1955 in which it is stated that ‘the nucleus consists of granules often in a linear arrangement; the measured mean size is a little less than 10 nm’. Later, very strong evidence for the particle nature of chromatin came from studies of the minichromosome formed between SV40 DNA and histones in the electron microscope [ 14,151. In particular, the very beautiful study by Chambon and co-workers [ 151 showed how circular SV40 DNA containing 21 twists could form a roughly equivalent number of beads on complexing with the four histones H2A, H2B, H3 and H4. Histone complexes Although early studies were concerned with the isolation and characterization of individual histones, more recent studies on the complexes formed between histones have led to important results. Kornberg and Thomas [16] used mild methods to extract histones from chromatin and fractionated them into three groups: (1) Hl, (2) H2A and H2B and (3) H3 and H4. On cross-linking these pairs of histones with dimethyl suberimidate they found evidence for a tetramer (H3),(H4)2 and oligimers of H2A and H2B. Roark et al. [ 171 isolated histones in a similar manner and from equilibrium ultracentrifugation studies proposed a concentration dependent dimer + tetramer equilibrium. Kornberg [18] has proposed a model in which the chromatin subunit is comprised of the tetramer (H3)2(H4)2 together with two each of H2A and H2B combined with 200 base pairs of DNA [9] to give the correct histone/DNA ratio. In the model it is assumed that the tetramer is fully globular and that DNA would be on the outside of this unit. Isenberg and D’Anna [ 191have studied the interactions between pairs of isolated histones and found that the pairs (H3, H4), (H2A, H2B) and (H2B, H4) form strong complexes. It is now generally thought that all four histones form an interacting unit. Neutron diffraction How then do we reconcile the proposal of a regular supercoil from X-ray diffraction data with the above evidence from many sources in favour of a subunit structure? It occurred to us three years ago that the use of neutrons would allow a simple and direct test of the two assumptions given earlier, involving the X-ray proposal of a supercoil. This is because the neutron scattering of hydrogen is

anomalous and has a negative value while for all other atoms in proteins and DNA the values are positive. Thus, the higher proportion of hydrogens in proteins compared to DNA results in proteins having a much lower average scattering power for neutrons than DNA. Without going into detail it is possible to use neutrons to distinguish between the scatter of proteins and DNA. It was found [20] that the 11 nm spacing came largely from the protein component and was attributed to the regular distribution of multimeric histone units in chromatin, basically the inter subunit spacing. The 5.5 and 2.7 nm rings came largely from the DNA component while the 3.7 nm was more complex but had a larger scatter component from protein than from DNA. Taking into account also the properties of histones a model was proposed which consisted of an interacting core of the apolar segments of the four histones H2A, H2B, H3 and H4 with DNA complexed with the basic histone segments coiled on the outside of the apolar core. Structure in the chromatin beads The exact length of DNA in the bead and whether there is a spacer region between the beads is now the subject of some discussion. No11 [21] has reported unit lengths of 205 b.p. while SollnerWebb and Felsenfeld [22] report 185 b.p. units. Such differences are largely points of detail and will be resolved with more experiments. Of more interest is the appearance of DNA fragments smaller than the approximate 200 b.p. length with increasing nuclease digestion. Using staphylococcal nuclease No11 [21] reported a second digestion product of 170 b.p. while Sollner-Webb and Felsenfeld have reported [22] 140 b.p. which with further digestion gave a series of ten discrete DNA lengths between 140 and 52 b.p. It now appears that the 140 b.p. unit is probably the correct value and the series of discrete DNA lengths reflect in some way the arrangement of histones and DNA in the subunit. Of particular interest is Noll’s [23] DNAase I digestion of rat liver chromatin. The DNA products were found to make up an arithmetic series of 10,20,30,. . .,220 b.p. This high accessibility of DNA was given as a strong argument for placing DNA on the outside of the chromatin subunit, either coiled or kinked [24]. Control of chromosome structure An interesting paradox exists between the high degree of sequence conservation of histones and the large number and variety of post-synthetic modifications, such as phosphorylation, methylation and acetylation of histones which occur during the cell cycle [3]. Such modifications

change the charged states of residues usually in the basic segments of histones and as there are only a small number of histones, moduications are clearly a possible mechanism for modulating the interaction of histones with DNA throughout the genome. Thus an attractive general hypothesis is that changes in the state of the eucaryotic chromosome are mediated by post-synthetic modifications of histones. In this respect it is now widely accepted that the Hl histone which is not involved in the globular structure of chromatin plays a role in generating higher order structures of the chromosome. Hl undergoes a specific pattern of phosphorylation through the cell cycle and of particular note is a large peak of phosphorylation coincident with the first visible signs of chromosome condensation in late G2. This was tirst observed in the naturally synchronous cycle of the slime mould Physarum pol.vcephalum [25] and it has been proposed largely from circumstantial evidence that the initiation of mitosis is in part controlled by the phosphorylation of the H 1 molecule [26]. In conclusion it is clear that histones have come of age and there is now a sound foundation for designing experiments to investigate further the role of histones in maintaining and controlling the state of the eucaryotic chromosome. References I Johns, E.W. (1971) in Histonesand Nucleohisrones (D.M.P. Phillips, ed.), p. 2, Plenum, New York 2 De Lange, R.G. and Smith, E.L. (1975) in The Structure and Function qfchromatin. CIBA Foundation Symposium No. 28. p. 59, Elsevier, Amsterdam 3 Dixon, G.H., Candido, E.P.M.. Honda, B.M., Louie, A.J., Macleod, A.R. and Sung, M.T. (1975) in The Structure and Function of Chromatin, CIBA Foundation Symposium No. 28. p. 229, Elsevier, Amsterdam 4 Bradbury, E.M. (1975) in The Structure und Function of Chromatin, CIBA Foundation S,vmposium No. 28, p. 1, Elsevier, Amsterdam 5 Phillips, D.M.P. (1971) in Histonesand Nucleohistones(D.M.Phillips, ed.), p. 2, Plenum, New York 6 Croft, L.R. (1973) Handbook of‘ Protein Sequences, Joynson & Bruvvers, Oxford 7 Bradbury, EM. (1975) in The Structure and Function of Chromatin, CIBA Foundation Sj,mposium No. 28, p. 13 1, Elsevier, Amsterdam 8 Pardon, J.F. and Wilkins, M.H.F. (1972) J. Mol. Biol. 68, 115 9 Hewish, D.R. and Burgoyne, L.A. (1973) Biothem. Biophys. Res. Commun 52, 504 10 Refs in Ramsay Shaw, B., Corden, J.L., Sahasrabuddhe, C.G. and Van Holde, K.E. (1974) Biothem. Biophys. Res. Commun. 61, 1193 11 Olins, A.L. and Olins, D.E. (1974) Science 183. 330 12 Woodcock, C.L.F. (1973) J. Cell. Biol. 59, 368a 13 Afzelius, B.A. (1955) 2. ZeDforsch. 42, I 34 14 Griffith, J. (1975) Science 187, 1202 15 Oudet, P., Gross Bellard, M. and Chambon. P. (1975) Cell 4, 28 I 16 Kornberg, R.D. and Thomas, J.O. (1974) Science 184, 865

TlBS - Januacy 19 76 17 Roark, D.E., Ceoghegan, T.E. and Keller. G.M. (1974) Biochem. Biophys. Res. Commun. 59. 542 18 Kornberg. R.D. (1974) Science 184.‘868 I9 D’Anna. J.A. and Isenberg. 1. (1974) Rioclt~rnisfr~~ 13. 4992 20 Ba1dwin.J.P.. Boseley. P.G., Bradbury, E.M. and [bet, K. (1975) Nafur~ 253, 245 ?I Nell, M. (1974) Nature 251. 249 22 Sollner-Webb. B. and Felsenfeld. G. (1975) Biochemi.vrn, 14. .?YI5

23 Noll, M. (1974) Nuc/& Acids Res. 1, 1573 24 Crick, F.M.C. and Klug, A. (1975) Nature 2.55, 530 25 Bradbury, E.M., Inglis, R.J.. Matthews, H.R. and Sarner, N. (1973) Eur. J. Biochem. 33, 131 26 Bradbury, E.M., Inglis. R.J. and Matthews, H.R. 11974) Nuturr 247,257

Poly ADP-ribose and ADP-ribosylation of proteins Osamu

Hayaishi

Covalent modification of proteins, by attachment of ADP-ribose, cell growvth,protein metabolism, DNA and RNA metabolism.

Several new examples of an unusual enzymic modification of proteins have recently been reported. In these reactions, the adenosine diphosphate ribose (ADPribose) moiety of NAD is transferred and covalently attached to an acceptor protein in either a polymeric or a monomeric form (Fig. 1). Nicotinamide and proton(s) are concomitantly released. Poly ADP-ribose has so far been found in nuclei of eucaryotes, whereas mono ADP-ribosylation of proteins has been found in procaryotes as well. These protein modifications appear to participate in the regulation of various biological functions, including cell growth and syntheses of protein, DNA and RNA. Poly ADP-ribosylation Since the original discovery of NAD by von Euler in 1936, this coenzyme has been found to be widely distributed in nature and to function as an electron carrier in various dehydrogenase systems. Although the nicotinamide-riboside linkage of NAD is a so-called high energy bond with a free energy of hydrolysis of about -8.2 kcal/mol at pH 7 and 25 “C [1], the biological significance of this bond energy has not been fully appreciated until recently. About ten years ago, three groups of investigators concurrently and independently, reported that mammalian nuclei can catalyze the polymerization of the ADP-ribose moiety of NAD into a novel homopolymer composed of repeating ADP-ribose units linked by ribose to ribose (1’ --, 2’) bonds as shown in Fig. 2 [2-41. The average chain length of this polymer, which is referred to as poly ADPribose, ranges from several up to 50. SubO.H. is Professor of Medical Chemistry at the Kyoto University Faculty qf Medicine. Sakyo-ku. Kyoto 606, Japan

appears to regulate

matin of rat liver and calf thymus nuclei. It cleaves specifically the 1’,2’-glycosidic bond of poly ADP-ribose resulting in the formation of ADP-ribose as shown in Fig. 2. On the other hand, snake venom phosphodiesterase and liver phosphodiesterase cleave the pyrophosphate linkage yielding 2’-(5”-phosphoribosyl)-5’-AMP and the terminal AMP. Although the natural existence of this unique polymer in vivo has been described from several laboratories, its biological function has not yet been clearly understood. Recently there have been a number of reports indicating a close relationship between poly ADP-ribose synthesis and DNA metabolism. For example, Kidwell showed, using nuclei from synchronized mouse L-929 and human HeLa S3 cell lines, that [ ‘HJNAD incorporation into oligo ADP-ribose in vitro is the highest at the Gz phase [8], suggesting a crucial

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sequently it was shown to be covalently attached to nuclear proteins, probably histones [5,6]. The poly ADP-ribose synthetase is present in a variety of vertebrate tissues, as well as in Tetrahymena and Physarum, a slime mold, but this activity has not yet been found either in higher plants or in procaryotic organisms. The enzyme activity is exclusively localized in the nucleus and more than 90’4 of the total activity is associated with chromatin. It has been solubilized from rat liver chromatin and purified about 5000 fold [7]. As the extent of purification increased, shorter chains were synthesized suggesting the possibility that the initial attachment of poly ADPribose to the acceptor protein and the subsequent chain elongation might be catalyzed by two different enzymes. The highly purified enzyme exhibits an absolute requirement for DNA or poly dAdT for activity. Histone appears to stimulate chain elongation. The degradation of this polymer in vivo is mainly catalyzed by poly ADP-ribose glycohydrolase, which has been solubilized and partially purified from chro-

role of this polymer in the regulation of growth cycle of cultured cells. Burzio and Koide [9] reported that treatment of rat liver nuclei with NAD markedly reduced their capacity to incorporate [3H]TTP into acid insoluble material. Subsequently Yoshihara et al. reported the ADP-ribosylation of a Ca2 +, Mg* + dependent endonuclease and the concomitant inhibition of the enzyme activity [lo]. The ADP-ribose bound to the endonuclease was in the form of monomers and oligomers and not long chain polymers. It was inferred that the ADP-ribosylation of the endonuclease led to inactivation of the enzyme and its ability to generate primer sites on DNA, thereby causing significant inhibition of the template capacity of the resultant DNA nuclear protein matrix of chromatin for DNA polymerase. In HeLa cells, however, Smulson et al. demonstrated that ADP-ribosylation led to an enhancement rather than to an inhibition in the number of primer sites for DNA polymerase [ 1I]. Although the poly ADP-ribosylation of proteins was originally discovered in eucaryotic cells and poly ADP-ribose synthesis seems to be restricted to the nuclei,