Differential ion
Differentiation 13, 37-39 (1979)
0 Springer-Verlag 1979
Histone Conformations, Histone Modifications, and Chromatin Structure E. M. BRADBURY Portsmouth Polytechnic, Biophysics Laboratory, Portsmouth, Hampshire, UK
The past five years have witnessed major advances in our understanding of the structure and function of chromatin. These advances were initiated by Hewish and Burgoyne [ l l who showdd that nuclease digestion of chromatin in nuclei resulted in a series of discrete DNA products in which the longer DNA lengths were integral multiples of the shortest DNA length of approximately 200 base pairs (b.p.). From this finding it was proposed that chromatin was made up of a repeating subunit now called the nucleosome. In most somatic tissues the nucleosome, contains 195 f 5 b.p. of DNA, two molecules each of the histones H2A, H2B, H3, and H4 and probably one H1 histone as suggested by Kornberg [21. Chambon has shown that the DNA content of the nucleosome is not a constant and results from several l a b ratories show it to vary from 154 b.p. DNA in lower eukaryotes to 241 b.p. in sea urchin sperm 131. This variability has been associated with variability in the H 1 molecule though variability in the H2A and H2B histones cannot be excluded. Allowing the nuclease digestion process of chromatin to proceed for longer times results in a well-defined subnucleosome particle, the core particle, which appears to be a constant irrespective of the source of origin of the chromatin [4]. The core particle found by Van Holde is now known to contain 145 b.p. of DNA with two each of histones (H2A, H2B, H3, and H4). The H1 histone is thought to be associated in part with the linker DNA joining adjacent core particles and is involved in higher order chromatin structures. Nuclease digestion of chromatin has led to two major approaches in studies of chromatin structure. Firstly, digestion by staphylococcal nuclease allows the preparation of large quantities of chromatin core particles, mononucleosomw, dinucleosomes, higher oligomers of nucleosomes, and large pieces of chromatin containing several 100 nucleosomes for physical studies of
chromatin structure. Secondly, a systematic investigation of other nucleases as biochemical probes of chromatin structure has been undertaken in many laboratories. Thus No11 [Sl found that DNase I digestion of chromatin gave a very characteristic single strand DNA pattern on gels consisting of multiples of approximately 10 nucleotide lengths up to 140 nucleotides; Weintraub and Groudine (61 showed that DNase 1 attacks initially the active regions of the genome and this has been used to study the proteins associated with those regions. DNase I1 was found in Zachau’s laboratory to give a 100 b.p. DNA ladder which supported the idea that the nucleosome contained a dyad axis [71. Because of its regularity the chromatin core particle has been the subject of intense study by physical techniques. The structure of the core particle in solution has been studied extensively by the technique of neutron scatter [8,91. With neutrons it is possible to separate the scattering effects of the histone component of the core particle from those of the DNA component. Thus we have shown that the radius of gyration Rg of the DNA was about 4.7 nm while that of the histone component was about 3.3 nm. This demonstrated that the DNA was wrapped around a histone core. A full analysis of neutron scatter data allows the scatter function corresponding to the pure shape of the particle Ic to be separated from the scatter function Is resulting from internal fluctuation of scattering density within the core particle. From the shape function Ic the radial distribution function (D(r) can be derived which gives the maximum dimension of the particle 191. This was found to be 11.0 nm. Thus for the DNA Rg of 4.7 nm to be consistent with the maximum dimension of the core particle of 11.0 nm it follows that the DNA component must be located at the periphery of the particle. Beyond this point models have to be fitted to the basic scatter func0301-468 1/79/00 13/0037/6 01.00
38
E M.Bradbury: Histone Conformations, Histone Modifications. and Chromatin Structure
tions.The shape which best fitted the Ic function was an oblate spheroid with an axial ratio of 0.5 [8,91. Thus the overall shape of the core particle was 11.0 x 11.0 x 5.5 nm3, giving a disc shaped model which contained 1.7 k 0.2 turns of DNA with pitch of 3.0 nm coiled around an apolar histone core. Neutron studies show that the DNA is highly hydrated but not the histone core. Single crystals of the core particle can be obtained and these have been studied at low resolution by electron microscopy and X-ray diffraction by Klug and coworkers (101. The low resolution model obtained from these studies is of a flat disc 11.0 x 11.0 x 5.7 nm3 with 1Y‘ turns of DNA of pitch 2.7 nm coiled on the edge of the disc. The solution and crystal structures are thus very similar at low resolution. It is quite clear that detailed structural knowledge of the core particle will soon be forthcoming from the crystal structure determinations now underway in several laboratories. The core particle described above is thought to be the fundamental structural unit of inactive chromatin. We are interested in several questions related to this particle. Firstly, what is the structural change which occurs during the transition from inactive to active chromatin? Secondly, how are the nucleosomes packed in higher order chromatin structures leading to the metaphase chromosome? Before discussing these questions we must look at the properties of the histones in relation to the structure of the core particle. Two major properties have emerged from the sequences of histones; firstly, histone sequences are highly conserved with accepted point mutation rates for H3 and H4 of 0.1, for H2A and H2B of 1.0, and for H1 of 4.0 mutations/100 residues/1O8 years. It should be noted that the most conserved histones H3‘and H4 alone are able to generate most of the structural features of chromatin [ 111. Secondly, histone sequences are highly asymmetric with very basic N-terminal regions and apolar central and Cterminal regions. Several laboratories have shown that histones form specific complexes, e.g. (H3, H4),, (H2A, H2B), (H4, H3, H2A, H2B), [41. Detailed NMR studies of histone conformations and interactions have shown that the apolar C-terminal regions become structured with increase in ionic strength and that these structured regions are the sites of interactions between histones leaving the basic N-terminal regions free and mobile [ 12, 131. More recently, we have shown that the N-terminal regions of H3 ( 1 4 1 ) and H4 (1-37) can be removed without affecting the ability of the residual C-terminal regions to form a specific complex [ 141. Now, although histone sequences are highly conserved, histones undergo two major reversible post-synthetic modifications which greatly change the nature of the modified resi-
dues; acetylation of lysines in histones H2A, H2B. H3, and H4 and phosphorylation of serines and threonines in histone HI. Acetylation of H2A, H2B, H3, and H4 has been documented in major events of DNA processing, gene activation [ 151, spermiogenesis [ 161 and DNA replication [ 171. It is significant that all sites of acetylation are located in the basic N-terminal regions of H2A, H2B, H3, and H4 [161, and acetylation is thus thought to be involved in the structural transition of the nucleosome to allow DNA to be processed. Ingram (181 has shown that the addition of Na butyrate to cell lines results in the accumulation of highly acetylated histones. With Vidali and Allfrey it has been shown that one effect of Na butyrate is to inactivate histone deacetylases and we have found that DNase I attacks preferentially the DNA associated with highly acetylated histones (191. This latter observation can be correlated with the preferential attack of DNase I on active regions of chromatin mentioned earlier. Both Allfrey’s and Dixon’s laboratories have also shown that DNaseI digestion releases a group of non-histone proteins (NHP) called HMG proteins originally identified and characterised by Johns. After s. nuclease digestion of chromatin in nuclei from Physarum polycephalum we have isolated a unit called particle A, with 140b.p. of DNA which is enriched in ribosomal RNA genes (201. Particle A is partially depleted of histones H3 and H4 but enriched in NHP which run on gels at the same positions as HMG proteins. The particle sediments at 5s which shows.that its structure is much more open than the nucleosome which sediments at 11s. Other ‘active’ chromatin particles have been proposed and the situation concerning the structure of active chromatin is far from clear. Histone HI has been implicated in maintaining and controlling higher chromatin structures. H1 is the most variable of the histones and has three well-defined structural regions; a basic N-terminal region 1-38, a globular region from 39-1 16, and a basic C-terminal region from 117-216 (211. As regards the next order of structure above the nucleosome there is limited evidence from our neutron diffraction studies of chromatin fibres [221 and from electron microscopy studies of Finch and Klug [23] to suggest that it is a flat coil of pitch 10- 11 nm and diameter 25-35 nm. Recent neutron scatter studies of solutions of gently isolated pieces of chromatin at different ionic conditions show that there are a family of coils with diameters in the above range, but with varying pitches (Suau, P., Balduin, J. P., Bradbury, E. M.: unpublished results). The most tightly coiled structure contains approximately six nucleosomes per turn with a pitch of 10-1 1 nrn. We believe that H1 stabilises this coil in vivo and is then involved in the
E. M.Bradbury: Histone Conformations, Histone Modifications. and Chromatin Structure
process of chromosome condensation leading to metaphase chromosomes. It has been proposed that control of chromosome condensation, i.e. the ‘mitotic trigger’ is through phosphorylation of serines and threonines in HI 1241. Growth associated sites of phosphorylation have been shown by Langan to be located in the basic N- and C-terminal regions of the H1. It is not known whether this phosphorylation causes condensation through H1-H1 or H1-DNA cross-linking, or whether constraints applied by H1 to decondense chromosomes are released when N and C terminal regions of H1 are phosphorylated. References 1. Hewish, D. R., Burgoyne, L. A.: Chromatin substructure. The digestion of chromatin DNA at regularly spaced sites by a nuclear deoxyribonucleax. Biochem. Biophys. Res. Commun. 52, SO4 (1973) 2. Kornberg. R. D.: Chromatin structure: a repeating unit of histones and DNA. Science 184, 868 (1974) 3. Compton, J. L., Bellard, M.,Chambon, P.: Biochemical evidence of variability in the DNA repeat length in the chromatin of higher eukaryotes. Proc. Natl. Acad. Sci. USA 73, 4382 (1976) 4. Van Holde, K. E., Iscnbcrg, I.: Histone interactions and chromatin structure. Accts. Chem. Res. 8, 327 (1975) 5. Noll, M.:Internal structure of the chromatin subunit. Nucleic Acids R a . I, 1573 (1974) 6. Wcintraub, H., Groudine, M.: Chromosomal subunits in active genes have an altered conformation. Science 193, 848 (1976) 7. Altenburga, M.,H 6 n , W., Zachau, H.: Nucleasc cleavage of chromatin at 1Wnucleotide pair intervals. Nature 264, 517 (1976) 8. Hjelm, R. P., Kneale, G. G., Suau, P., Baldwin, J. P.. Bradbury, E. M.,Ibcl, K.: Small angle neutron scattering studies of chromatin subunits in solution. Cell 10, 139 (1977) 9. Suau, P., Kneale, G. G., Braddock, G. M.,Baldwin. J. P., Bradbury, E. M.: A low resolution model for the chromatin core partick by neutron scattering. Nuclcic Acids Res. 4, 3769 (1977) 10. Finch, J. T., Lutter, L. C., Rhodes, D., Brown, R. S., Rushton, B.. L e t t , M..Klug, A.: Structure of nucleosome core particles of chromatin. Nature 269, 29 (1977)
11.
39
Moss, T., Stephens, R. M., Crane-Robinson. C., Bradbury, E. M.:A nucleosome-like structure containing DNA and the argi-
nine rich histones H, and H,. Nucleic Acid Res. 4, 2477 (1977) 12. Moss, T., Cary, P. D.. Crane-Robinson, C., Bradbury, E. M.: Physical studies on the H3/H4 histone tdramer. Biochemistry IS. 2261 (1976) 13. Moss,T., Cary, P. D., Abercrombie, B. D., Crane-Robinson, C., Bradbury, E. M.:A pHdependent interaction between histones H2A and H2B involving secondary and tertiary Folding. Eur. J. Biochem. 71, 337 (1976) 14. Bohm. L., Hayashi. H., Cary, P. D., Moss,T., Crane-Robinson, C., Bradbury, E. M.:Sites of histonehistone interaction in the H3-H4 complex. Eur. J. Biochem. 77, 487 (1977) IS. Allfrey, V. G.: Histones and nucleohistones. Phillips D.M.P. (4.). p. 264. London, New York: Plenum Press 1971 16. Louie, A. J.. Candido, E. P. H.. Dixon. G. H.: Enzymatic modifications and their possible roles in regulating the binding of basic proteins to DNA and in controlling chromosomal structure. Cold Spring Harbour Symp. Quant. Biol. 38, 803 (1974) 17. Jackson, V., Shires, A., Tanphaichitr, N., Chalkley, R.: Modifications to histones immediately aRer synthesis. J. Mol. Biol. 104, 471 (1976) 18. Riggs. M.G., Whittaker, R. G., Neumann. J. R., Ingram, V. M.: n-butyrate causes histone modification in HeLa and Friend erythroleukaemia cells. Nature 268, 462 (1977) 19. Vidali, G., Boffa, L. G., Bradbury, E. M..Allfrey, V. G.: Butyrate suppression of histone deacetylation leads to accumulation of multiacetylated forms of histones H 3 and H4 and increased DNaseI sensitivity of the associated DNA sequences. Proc. Natl. Acad. Sci. USA 75, 2239 (1978) 20. Johnson, E. M.,Allfrey, V. G., Bradbury, E. M., Matthews. H. R.: Altered nucleosome structure containing DNA sequences complementary to 19s and 26s ribosomal RNA in Physomm polycephalwn. Proc. Natl. Acad. Sci. USA 75, 1116 (1978) 21. Chapman, G. E., Hartman, P. G., Bradbury, E. M.:Studies on the role and mode of operation of the very-lysine-rich histone H I in eukaryote chromatin. Eur. J. Biochem. 61, 69 (1976) 22. Carpenter, B. G., Baldwin, J. P., Bradbury, E. M.,Ibel, K.: Organisation of subunits in chromatin. Nuclcic Acids Res. 3, 1739 (1976) 23. Finch, J. T., Klug, A.: Solenoidal model for superstructure in chromatin. Proc. Natl. Acad. Sci. USA 73, 1897 (1976) 24. Bradbury, E. M..Inglis, R. J., Matthews, H. R.: Control of cell division by very lysine rich histone (FI)phosphorylation. Nature 247, 257 (1974)
Differentiation 13, 37-39 (1979)
0 Springer-Verlag 1979
Histone Conformations, Histone Modifications, and Chromatin Structure E. M. BRADBURY Portsmouth Polytechnic, Biophysics Laboratory, Portsmouth, Hampshire, UK
The past five years have witnessed major advances in our understanding of the structure and function of chromatin. These advances were initiated by Hewish and Burgoyne [ l l who showdd that nuclease digestion of chromatin in nuclei resulted in a series of discrete DNA products in which the longer DNA lengths were integral multiples of the shortest DNA length of approximately 200 base pairs (b.p.). From this finding it was proposed that chromatin was made up of a repeating subunit now called the nucleosome. In most somatic tissues the nucleosome, contains 195 f 5 b.p. of DNA, two molecules each of the histones H2A, H2B, H3, and H4 and probably one H1 histone as suggested by Kornberg [21. Chambon has shown that the DNA content of the nucleosome is not a constant and results from several l a b ratories show it to vary from 154 b.p. DNA in lower eukaryotes to 241 b.p. in sea urchin sperm 131. This variability has been associated with variability in the H 1 molecule though variability in the H2A and H2B histones cannot be excluded. Allowing the nuclease digestion process of chromatin to proceed for longer times results in a well-defined subnucleosome particle, the core particle, which appears to be a constant irrespective of the source of origin of the chromatin [4]. The core particle found by Van Holde is now known to contain 145 b.p. of DNA with two each of histones (H2A, H2B, H3, and H4). The H1 histone is thought to be associated in part with the linker DNA joining adjacent core particles and is involved in higher order chromatin structures. Nuclease digestion of chromatin has led to two major approaches in studies of chromatin structure. Firstly, digestion by staphylococcal nuclease allows the preparation of large quantities of chromatin core particles, mononucleosomw, dinucleosomes, higher oligomers of nucleosomes, and large pieces of chromatin containing several 100 nucleosomes for physical studies of
chromatin structure. Secondly, a systematic investigation of other nucleases as biochemical probes of chromatin structure has been undertaken in many laboratories. Thus No11 [Sl found that DNase I digestion of chromatin gave a very characteristic single strand DNA pattern on gels consisting of multiples of approximately 10 nucleotide lengths up to 140 nucleotides; Weintraub and Groudine (61 showed that DNase 1 attacks initially the active regions of the genome and this has been used to study the proteins associated with those regions. DNase I1 was found in Zachau’s laboratory to give a 100 b.p. DNA ladder which supported the idea that the nucleosome contained a dyad axis [71. Because of its regularity the chromatin core particle has been the subject of intense study by physical techniques. The structure of the core particle in solution has been studied extensively by the technique of neutron scatter [8,91. With neutrons it is possible to separate the scattering effects of the histone component of the core particle from those of the DNA component. Thus we have shown that the radius of gyration Rg of the DNA was about 4.7 nm while that of the histone component was about 3.3 nm. This demonstrated that the DNA was wrapped around a histone core. A full analysis of neutron scatter data allows the scatter function corresponding to the pure shape of the particle Ic to be separated from the scatter function Is resulting from internal fluctuation of scattering density within the core particle. From the shape function Ic the radial distribution function (D(r) can be derived which gives the maximum dimension of the particle 191. This was found to be 11.0 nm. Thus for the DNA Rg of 4.7 nm to be consistent with the maximum dimension of the core particle of 11.0 nm it follows that the DNA component must be located at the periphery of the particle. Beyond this point models have to be fitted to the basic scatter func0301-468 1/79/00 13/0037/6 01.00
38
E M.Bradbury: Histone Conformations, Histone Modifications. and Chromatin Structure
tions.The shape which best fitted the Ic function was an oblate spheroid with an axial ratio of 0.5 [8,91. Thus the overall shape of the core particle was 11.0 x 11.0 x 5.5 nm3, giving a disc shaped model which contained 1.7 k 0.2 turns of DNA with pitch of 3.0 nm coiled around an apolar histone core. Neutron studies show that the DNA is highly hydrated but not the histone core. Single crystals of the core particle can be obtained and these have been studied at low resolution by electron microscopy and X-ray diffraction by Klug and coworkers (101. The low resolution model obtained from these studies is of a flat disc 11.0 x 11.0 x 5.7 nm3 with 1Y‘ turns of DNA of pitch 2.7 nm coiled on the edge of the disc. The solution and crystal structures are thus very similar at low resolution. It is quite clear that detailed structural knowledge of the core particle will soon be forthcoming from the crystal structure determinations now underway in several laboratories. The core particle described above is thought to be the fundamental structural unit of inactive chromatin. We are interested in several questions related to this particle. Firstly, what is the structural change which occurs during the transition from inactive to active chromatin? Secondly, how are the nucleosomes packed in higher order chromatin structures leading to the metaphase chromosome? Before discussing these questions we must look at the properties of the histones in relation to the structure of the core particle. Two major properties have emerged from the sequences of histones; firstly, histone sequences are highly conserved with accepted point mutation rates for H3 and H4 of 0.1, for H2A and H2B of 1.0, and for H1 of 4.0 mutations/100 residues/1O8 years. It should be noted that the most conserved histones H3‘and H4 alone are able to generate most of the structural features of chromatin [ 111. Secondly, histone sequences are highly asymmetric with very basic N-terminal regions and apolar central and Cterminal regions. Several laboratories have shown that histones form specific complexes, e.g. (H3, H4),, (H2A, H2B), (H4, H3, H2A, H2B), [41. Detailed NMR studies of histone conformations and interactions have shown that the apolar C-terminal regions become structured with increase in ionic strength and that these structured regions are the sites of interactions between histones leaving the basic N-terminal regions free and mobile [ 12, 131. More recently, we have shown that the N-terminal regions of H3 ( 1 4 1 ) and H4 (1-37) can be removed without affecting the ability of the residual C-terminal regions to form a specific complex [ 141. Now, although histone sequences are highly conserved, histones undergo two major reversible post-synthetic modifications which greatly change the nature of the modified resi-
dues; acetylation of lysines in histones H2A, H2B. H3, and H4 and phosphorylation of serines and threonines in histone HI. Acetylation of H2A, H2B, H3, and H4 has been documented in major events of DNA processing, gene activation [ 151, spermiogenesis [ 161 and DNA replication [ 171. It is significant that all sites of acetylation are located in the basic N-terminal regions of H2A, H2B, H3, and H4 [161, and acetylation is thus thought to be involved in the structural transition of the nucleosome to allow DNA to be processed. Ingram (181 has shown that the addition of Na butyrate to cell lines results in the accumulation of highly acetylated histones. With Vidali and Allfrey it has been shown that one effect of Na butyrate is to inactivate histone deacetylases and we have found that DNase I attacks preferentially the DNA associated with highly acetylated histones (191. This latter observation can be correlated with the preferential attack of DNase I on active regions of chromatin mentioned earlier. Both Allfrey’s and Dixon’s laboratories have also shown that DNaseI digestion releases a group of non-histone proteins (NHP) called HMG proteins originally identified and characterised by Johns. After s. nuclease digestion of chromatin in nuclei from Physarum polycephalum we have isolated a unit called particle A, with 140b.p. of DNA which is enriched in ribosomal RNA genes (201. Particle A is partially depleted of histones H3 and H4 but enriched in NHP which run on gels at the same positions as HMG proteins. The particle sediments at 5s which shows.that its structure is much more open than the nucleosome which sediments at 11s. Other ‘active’ chromatin particles have been proposed and the situation concerning the structure of active chromatin is far from clear. Histone HI has been implicated in maintaining and controlling higher chromatin structures. H1 is the most variable of the histones and has three well-defined structural regions; a basic N-terminal region 1-38, a globular region from 39-1 16, and a basic C-terminal region from 117-216 (211. As regards the next order of structure above the nucleosome there is limited evidence from our neutron diffraction studies of chromatin fibres [221 and from electron microscopy studies of Finch and Klug [23] to suggest that it is a flat coil of pitch 10- 11 nm and diameter 25-35 nm. Recent neutron scatter studies of solutions of gently isolated pieces of chromatin at different ionic conditions show that there are a family of coils with diameters in the above range, but with varying pitches (Suau, P., Balduin, J. P., Bradbury, E. M.: unpublished results). The most tightly coiled structure contains approximately six nucleosomes per turn with a pitch of 10-1 1 nrn. We believe that H1 stabilises this coil in vivo and is then involved in the
E. M.Bradbury: Histone Conformations, Histone Modifications. and Chromatin Structure
process of chromosome condensation leading to metaphase chromosomes. It has been proposed that control of chromosome condensation, i.e. the ‘mitotic trigger’ is through phosphorylation of serines and threonines in HI 1241. Growth associated sites of phosphorylation have been shown by Langan to be located in the basic N- and C-terminal regions of the H1. It is not known whether this phosphorylation causes condensation through H1-H1 or H1-DNA cross-linking, or whether constraints applied by H1 to decondense chromosomes are released when N and C terminal regions of H1 are phosphorylated. References 1. Hewish, D. R., Burgoyne, L. A.: Chromatin substructure. The digestion of chromatin DNA at regularly spaced sites by a nuclear deoxyribonucleax. Biochem. Biophys. Res. Commun. 52, SO4 (1973) 2. Kornberg. R. D.: Chromatin structure: a repeating unit of histones and DNA. Science 184, 868 (1974) 3. Compton, J. L., Bellard, M.,Chambon, P.: Biochemical evidence of variability in the DNA repeat length in the chromatin of higher eukaryotes. Proc. Natl. Acad. Sci. USA 73, 4382 (1976) 4. Van Holde, K. E., Iscnbcrg, I.: Histone interactions and chromatin structure. Accts. Chem. Res. 8, 327 (1975) 5. Noll, M.:Internal structure of the chromatin subunit. Nucleic Acids R a . I, 1573 (1974) 6. Wcintraub, H., Groudine, M.: Chromosomal subunits in active genes have an altered conformation. Science 193, 848 (1976) 7. Altenburga, M.,H 6 n , W., Zachau, H.: Nucleasc cleavage of chromatin at 1Wnucleotide pair intervals. Nature 264, 517 (1976) 8. Hjelm, R. P., Kneale, G. G., Suau, P., Baldwin, J. P.. Bradbury, E. M.,Ibcl, K.: Small angle neutron scattering studies of chromatin subunits in solution. Cell 10, 139 (1977) 9. Suau, P., Kneale, G. G., Braddock, G. M.,Baldwin. J. P., Bradbury, E. M.: A low resolution model for the chromatin core partick by neutron scattering. Nuclcic Acids Res. 4, 3769 (1977) 10. Finch, J. T., Lutter, L. C., Rhodes, D., Brown, R. S., Rushton, B.. L e t t , M..Klug, A.: Structure of nucleosome core particles of chromatin. Nature 269, 29 (1977)
11.
39
Moss, T., Stephens, R. M., Crane-Robinson. C., Bradbury, E. M.:A nucleosome-like structure containing DNA and the argi-
nine rich histones H, and H,. Nucleic Acid Res. 4, 2477 (1977) 12. Moss, T., Cary, P. D.. Crane-Robinson, C., Bradbury, E. M.: Physical studies on the H3/H4 histone tdramer. Biochemistry IS. 2261 (1976) 13. Moss,T., Cary, P. D., Abercrombie, B. D., Crane-Robinson, C., Bradbury, E. M.:A pHdependent interaction between histones H2A and H2B involving secondary and tertiary Folding. Eur. J. Biochem. 71, 337 (1976) 14. Bohm. L., Hayashi. H., Cary, P. D., Moss,T., Crane-Robinson, C., Bradbury, E. M.:Sites of histonehistone interaction in the H3-H4 complex. Eur. J. Biochem. 77, 487 (1977) IS. Allfrey, V. G.: Histones and nucleohistones. Phillips D.M.P. (4.). p. 264. London, New York: Plenum Press 1971 16. Louie, A. J.. Candido, E. P. H.. Dixon. G. H.: Enzymatic modifications and their possible roles in regulating the binding of basic proteins to DNA and in controlling chromosomal structure. Cold Spring Harbour Symp. Quant. Biol. 38, 803 (1974) 17. Jackson, V., Shires, A., Tanphaichitr, N., Chalkley, R.: Modifications to histones immediately aRer synthesis. J. Mol. Biol. 104, 471 (1976) 18. Riggs. M.G., Whittaker, R. G., Neumann. J. R., Ingram, V. M.: n-butyrate causes histone modification in HeLa and Friend erythroleukaemia cells. Nature 268, 462 (1977) 19. Vidali, G., Boffa, L. G., Bradbury, E. M..Allfrey, V. G.: Butyrate suppression of histone deacetylation leads to accumulation of multiacetylated forms of histones H 3 and H4 and increased DNaseI sensitivity of the associated DNA sequences. Proc. Natl. Acad. Sci. USA 75, 2239 (1978) 20. Johnson, E. M.,Allfrey, V. G., Bradbury, E. M., Matthews. H. R.: Altered nucleosome structure containing DNA sequences complementary to 19s and 26s ribosomal RNA in Physomm polycephalwn. Proc. Natl. Acad. Sci. USA 75, 1116 (1978) 21. Chapman, G. E., Hartman, P. G., Bradbury, E. M.:Studies on the role and mode of operation of the very-lysine-rich histone H I in eukaryote chromatin. Eur. J. Biochem. 61, 69 (1976) 22. Carpenter, B. G., Baldwin, J. P., Bradbury, E. M.,Ibel, K.: Organisation of subunits in chromatin. Nuclcic Acids Res. 3, 1739 (1976) 23. Finch, J. T., Klug, A.: Solenoidal model for superstructure in chromatin. Proc. Natl. Acad. Sci. USA 73, 1897 (1976) 24. Bradbury, E. M..Inglis, R. J., Matthews, H. R.: Control of cell division by very lysine rich histone (FI)phosphorylation. Nature 247, 257 (1974)
