Eur. J . Biochem. 89, 475-482 (1978)
High-Resolution Proton-Magnetic-Resonance Studies of Chromatin Core Particles Peter
L)
CARY, Tom MOSS, and E Morton BRADBURY
Biophysics Laboratories, Portsmouth Polytechnic (Received March 2. 1978)
The binding of histones in chromatin core particles and in core particles depleted of histones H2A and H2B has been studied by high-resolution proton nuclear magnetic resonance (NMR) at 270 MHz. At low ionic strengths it is shown that histones H3 and H4 are bound in the core particle. Further, whereas the apolar regions of H2A and H2B are also bound to the core particle, the basic N-terminal and C-terminal regions are more mobile and give rise to sharp resonances in the NMR spectrum of the core particle. Between 0.3 and 0.6 M NaCl there is further release of basic regions of histones H3 and H4 from the complex. The dissociation of the core particle between 0.6 and 2.0 M NaCl is accompanied by the release of the structured apolar regions of the histones as evidenced by the appearance of a complex aromatic spectrum and perturbed upfield ring-currentshifted methyl resonances. Arginine residues are implicated in the binding between histones and DNA and 69‘x of these residues are found in the apolar regions of the histones. Thc interactions between histones and DNA in the core particle thus involves H3 and H4 and the apolar regions of H2A and H2B. It is suggested that these basic regions of H2A and H2B have binding sites outside the core particle.
The concept of a repeating chromatin subunit introduced by Hewish and Burgoyne [l], Olins and Olins [2] and Woodcock [3] is now well established. This subunit, the nucleosome [4], has a variable DNA repeat depending on the tissue of origin, though for most somatic tissues the DNA repeat is 195 f 5 base pairs of DNA and in addition contains two each of the histones H2A, H2B, H3 and H4 [ 5 ] . Also associated with the nucleosome is probably one molecule of the very-lysine-rich histone H1. The structure of the nucleosome has been studied by neutron scatter techniques and found to have an apolar histone core with DNA coiled around this core. The shape which best fitted the scatter curves was an oblate spheroid with axial ratio of 0.5 [6]. Irrespective of their source of origin further digestion of nucleosomes with micrococcal nuclease results in a subnucleosomal particle, the ‘core’ particle, which contains 140 base pairs of DNA and the eight nucleosomal histones [6,7]. This ‘core’ particle is well-defined and has been studied in solution by neutron scatter techniques [7-91 and in small crystals by low-resolution X-ray crystallography [lo]. From both studies very similar low-resolution models are proposed of an oblate spheroid 11 x 11 x 5.5 nm which consist of a histone core with 1.7 k 0.2 turns of DNA of pitch 2.7- 3.0 nm. The histone core Ahhrwiution. NMR, nuclear magnetic resonance.
has been equated to a complex of the apolar regions of the histones [ 6 , 8 ] . The roles of the four histones H2A, H2B, H3 and H4 in generating chromatin structure and nucleosomes have been studied in several laboratories [I 1 - 141. Although it was initially thought that all four histones were necessary for the structure of the nucleosome [ S ] , it has now been shown by structural nuclease probes [12- 141 and X-ray diffraction [11,14] that the major structural features of the nucleosome and chromatin can be generated by histones H3 and H4 alone. Histones H2A and H2B may confer added stability to the basic structure formed by H3 and H4 and be involved in additional roles as yet undefined. High-resolution nuclear magnetic resonance (NMR) spectroscopy has been applied to the study of histone interactions and it has been demonstrated that the specific histone complexes (H32H42) [15,16] and (H2A, H2B) [17] are formed through interactions involving structured apolar regions of the histones. For H2A and H2B these structured regions lie in the centre of these molecules leaving the basic N-terminal and smaller C-terminal regions uncomplexed and mobile. For histones H3 and H4 the apolar carboxyl regions of the molecules are involved in complex formation leaving the basic N-terminal regions free. In the latter case it has been shown that residues 1-37 of H4 and 1-41 of H3 can be removed without
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affecting the ability of the apolar carboxyl regions to form complexes [I 61. These results strongly suggest that the very basic terminal regions of histones are involved in functions distinct from those of the structured apolar regions. It has been suggested that the basic N-terminal regions of histones are the major binding sites to DNA and early NMR studies of the binding of individual histones to DNA showed that the N-terminal regions were the most strongly binding to DNA [18,19]. However, in the nucleosome structure we have at least two different regions of DNA: the well defined core particles containing 140 base pairs of DNA which are joined by a linker DNA region the length of which depends on the nucleosome repeat. In addition there are higher-order chromatin structures which may require interactions outside of the nucleosome involving regions of histones H2A, H2B, H3 and H4 in addition to the interactions of H I . These interactions in higher-order chromatin structures may also require non-histone proteins. Chromatin core particles have been studied by high-resolution NMR to evaluate the extent to which the different histones are bound to the 140-base-pair DNA. EXPERIMENTAL PROCEDURE Chromatin core particles were prepared from calf thymus as described previously [20]. They contained 140 base pairs of DNA, equimolar quantities of the histones H2A, H2B, H3 and H4, and no HI histones, as demonstrated by gel electrophoresis using DNA and protein gels. The core particles were concentrated in a millipore filter to a concentration of 3- 5 mg/ml of DNA as measured by ultraviolet absorbance at 260 nm. This solution was then dialysed for 48 h in 10 mM phosphate buffer at pH 6.5-7.0 in the ratio of 1 ml of core particles to 1000 ml of buffer solution at 4 C. The dialysate was changed three times over this period of time. For the NMR studies the sample was divided into 0.5-ml aliquots which were dialysed against four changes of 'Hz0,I mM phosphate buffer/ NaCl at pH 6.5 over 48 h in the ratio 0.5 ml core particles/5 ml dialysate. Finally the core particle solution were dialysed in the ratio of 0.5 ml solution to 20 ml of 2Hz0 buffer. Core Particles Depleted of Histones H2A and H2B
To determine the contribution of histones H2A and H2B to the NMR spectrum of the core particles these histones were removed from the particles by Bio-Rad AG 50w x 2 (200-400 mesh) resin under the following conditions: 0.36 M NaCl or 0.4 M NaCI, 4 M urea, pH 7.0 at 4°C. Acrylamide gel electrophoresis showed that only histones H2A and H2B were removed from the core particles.
Magnetic-Rcsonance Studics o f Chroiiiatin Core Particles
Reconstituted Core Particles
Calf thymus histones were separated into the pairs, (H2A, H2B) and (H3, H4), by the method of van Holt and van der Westhuyzen [21]. Equiinolar mixtures of these pairs of histones in 2 M NaCl were mixed with DNA also in 2 M NaCl and dialysed down stepwise to 0 M NaCl pH 7.0. This reconstituted chromatin was then digested with micrococcal nuclease to give a 140-base-pair DNA particle as the main product. The digestion products were fractionated on a Bio-Gel A-5m (Bio-Rad) column to give 140-basepair reconstituted DNA particles purified to better than 90 7:). Nuclear Magnetic Resonance Spectroscopy
Core particle solutions were pipetted into standard 5-mm NMR tubes. NMR spectra were recorded at 270 MHz in the Fourier transform mode on a Bruker WH 270 spectrometer equipped with an Oxford lnstrument Co. superconducting magnet. Resolution enhancement was performed by the convolution difference method [22]. Signal intensities were standardized against the signal from a 2H20 solution of sodium formate doped with gadolinium nitrate and contained in an inner concentric microtube. The sodium formate signal was broadened so that its line width was independent of small variations in field homogeneity. The concentrations of the core particle solutions were monitored by the ultraviolet absorbance at 260 nm. Signal intensities were also compared with the signals from the fully dissociated histones from core particles in 2 M NaCl and 8 M urea. Under these conditions the histones are free and in the random coil conformation. Finally corrections relative to the 'H20 lock frequency were made for changes in the chemical shift of the samples in the various solution conditions.
RESULTS The NMR spectra of various constituents under different conditions were examined as follows: (a) native core particles and their dissociation into DNA and histones by salt (NaCl) and urea; (b) the histone complex formed from acid-extracted histones ; (c) core particles depleted of histones H2A and H2B; (d) reconstituted core particles and their dissociation by salt (NaCI) and urea. Native Core Particles
Fig. 1 shows the NMR spectra of core particles at different stages of salt dissociation and also the spectrum of the random coil histones from core par-
P. D. Cary, T. Moss. and E. M. Brudbul-)
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Fig. I . l+/ic‘ltl 270-MH: spcc‘tstr o/’nuiiw ‘core’ purticlc~s(140 husc p i i n ) iii I r i i M p l ~ ~ , s ~ i / i a t r , huffir ~ H ~ Ouf p H 6.5. DNA concn 3 - 5 rngrnl. (a) N o NaCI, (b)0.3 M NaCI, (c) 0.6 M NaCl including a convolulion diiTei-enceof the ring-current-shifted region, (d) 1.2 M NaCl including a convolution diference of the rinp-ciirrent-shirtecl region. (e) 2.0 M NaCl including a convolution difference of the ring-current-shifted region, Cfj 2.0 M NaCl 8 M urea
ticles dissociated in 2 M NaC1/8 M urea. The lowfield region of the spectrum of core particles, 6-10 ppm (not shown), contains resonance peaks from the aromatic residues located in the apolar regions of histones and resonances from DNA. These resonances are very broad and featurelcss, as would be expected for a slowly moving complex the size of the core particle. Only when the histones are dissociated from DNA in 2 M NaC1/8 M urea are sharp aromatic resonances observed. The upfield region of the spectrum of the core particles (Fig. 1 a) however, exhibits sharp resonances showing that some regions of the histones have mobilities additional to the overall rotation of core particlcs. A comparison of the total sum of the peak areas of this upfield region of the NMR spectrum (Fig. 1a j with that of the fully dissociated histones (Fig. 1f) shows that the residual peak area of the core particles is 17 - 20 2>of the total area of the spectrum of the free histones. In addition it can be seen that there are marked differential broadening effects which particularly affect the arginine SCH2 resonance peak at 3.25 ppm and the glycine xCH2 peak at 4.0 ppm, while the lysine E C Hresonance ~ at 3.02 ppm, although reduced to about one half of its total area, remains sharp. Also in the core particle spectrum the methyl resonances from leucine, isoleucine and valine at
about 0.9 ppin are very much reduced in area. Thus in the core particle spectrum a very substantial proportion of the upfield peak areas from the histones H2A, H2B. H3 and H4 is ‘lost’, in addition to the aromatic resonances due to broadening efl’ects from histone complex formation and binding to DNA in the particle. The effect of histone complex formation can be seen by comparing the spectrum of core particles in 2 M NaCl (Fig. 1e) when the histone complex is dissociated from DNA, with that of core particles in 2 M NaC1/8 M urea (Fig. 1 f) when the histones are fully dissociated into their random coil conformations. Again there are marked differential effects which particularly affect the peak areas of the methyl resonances at about 0.9 ppm from the apolar residues and also the arginine dCH2 resonance while the K H 2 peak from lysine residues remains sharp and well-developed. These effects, which will be described in more detail later, are attributed to complex formation between the apolar C-terminal regions of histones H3 and H4 and the apolar central regions of histones H2A and H2B. These ‘apolar’ regions contain a substantial proportion (69%) of arginine residues but only 31 ‘,!,,;, of the lysine residues. The binding of the histone complex to DNA of the core particle (cf. Fig. 1a and e) leads to a further loss of peak areas of the resonances from arginine 6CH2, glycine aCH2 and the methyl groups from the apolar residues. The resonances from aromatic residues, which are contained almost entirely in the apolar regions of the histones, are considerably broadened in both histone complexes and core particles. An estimate can be made of the chemical groups in core particles which have mobilities approaching those of the random coil conformation. This estimate, given in Table 1, is approximate because residual resonance peaks have more than one component. If it is assumed that these mobile groups are contained in continuous segments of histones then these segments are rich in lysines and alanines but contain low amounts of the apolar residues (leucines, isoleucines and valinesj, glycines, serines, threonines and very low amounts of arginines. Such regions are the N-terminal residues 1 - 30 and C-terminal 105- 125 of H2B, the N-terminal 1 - 16 and C-terminal 117- 129 of H2A and the N-terminal of H3 (18-37j. The N-terminal region of H4 contains a high proportion of glycine residues whose zCHz rcsonance is considcrably broadened in the core particle spectrum (Fig. 1 a). Individual resonances have also been identified: the observation of a sharp peak at 3.08 ppm from the cCH2 of methyl lysines suggest that some of the residual resonance comes from histone H3. It is unlikely to come from histone H4 because the glycine zCH2 peak is broad and there are eight glycines in the N-terminal region of H4. The N-terminal prolinc of histone H2B gives a sharp SCHz resonance peak at
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Magnetic-Resonance Studies of Chromatin Core Particles
Table 3 . Approximate eytimares of rhe numbers of d(fSrmr amino ucd residues contributing 10 the slzavp NMR spectrum of' native core purticles ut low ionic strength The estimated number of the residues listed was obtained from the proton N M R data of core particles in 1 mM phosphate buffer pH 6.8, 0-0.2M NaC1. (An asterisk in the list of resonant groups indicates that the estimate is difficult due to a weak resonant peak.) The numbers of these residues present in sequcnccs with high alanine and lysine contents and low arginine, glycine, apolar and aromatic contents, i.c. residues 1-30 and 105-125 of H2B, 3-36and 137-129ofH2Aand 18-37ofH3,arealsogivcnfor comparison Amino acid residue
Resonant group
Ala Arg Val Ilc Leu Lys cily
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10
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3.3 ppm while a sharp peak at 2.25 ppm shows that the N-terminal acetyl serine of histones H2A or H4 are mobile, though for reasons given above this resonance is probably from H2A. An increase in ionic strength from 0.2 M to 0.6 M NaCl causes the preferential release of regions containing the following residues : arginine, lysine, glycine, methyl-lysine, glutamic acid, glutamine and some of the apolar residues, leucine, isoleucine and valine. These residues are located in the N-terminal regions of histones H3 and H4 with possibly additional segments of H2A and H2B. The low peak areas of the apolar and aromatic residues indicate that the apolar central regions of these histones are bound in the core particle. Although the histone complex is bound to DNA in the core particles at 0.6 M NaCl, it is released by 2 M NaCl but the NMR spectra of the core particles at different stages of dissociation at 0.6, 1.2 and 2.0 M NaCl are very similar. There are small differences, for example the perturbed upfield resonances observed in the spectrum of the histone complex at 2 M NaCl are broadened and not observed in the core particle spectrum at 0.6 M NaCl. A similar change is also observed when coinparing the NMR spectrum of reconstituted histone complexes at 2 M NaCl with that of core particles as described in the
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Fig. 2. Up/idd 27O-:Ll H: :L',WR .vp'clru of narivc 'corc' parlicks (140 bnse puirs) in 1 n?M pho.sphatei0.14 M N u C ~ ; ' H Z Ohufler at p H 6.5. DNA concn 2-3 nig ml. (a) No urea, (b) 2 M urea, ( c )4 M urea, ( d ) 6 M urea
next section. This suggests that the octameric histone complex (H2A, H2B, H3, H4)2 [23] is bound to DNA at 0.6 M NaCl through the apolar regions of the four histones and the full complex is released by 2.0 M NaCI. At 2 M NaCl this histone complex is dissociated by the addition of 8 M urea. At 0.2 M NaCl, the effect of the addition of urea is shown in Fig.2. The core particle is partially dissociated by 4 M urea and except for a proportion of arginine residues is largely dissociated by 6 M urea. The 6CHz resonance from arginine is not fully developed in high urea concentration, indicating that even with 6 M urea some of the arginines are still bound to DNA. These can be released by increasing the ionic strength. The Rrconstitutcd Histone Complex
The regions of histones which are immobilised by histonc complex formation and by binding to DNA in core particles are rich in the following residues: arginine, valine, leucine, isoleucine, glycine, tyrosine and phenylalanine. Histone regions which contain these residues are the apolar central regions of H2A and H2B, the C-terminal halves of H3 and H4 and the N-terminal region of H4. The effects of histone complex formation are shown in Fig.3. The N M R spec-
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6 (PPm) Fig. 3 . 270-iMIiz N M K .spwtru o/'ocitl-c.\-tl.tic,let/ / i i . s l o i i e . ~H3, H4. H2.4, mid 112H in c.yuirnolur uinou~~t.s ut 20 nzg;'nil if7 211~0. (a) N o NaCI, pH 2.0: (b) no NaC1, pH 6.0 including a convolution dill'erencc of the ring-current-shifted upfield region; (c) 0.14 M NaCl. pH 6.0 including a convolution dillerence of the ring-current-shifted regions; (d) 2 M NaCI, pH 6.0 including a convolution diffcrence ol' the ring-currenlshifted region; (e) native 'core' particle (140 base pairs) in 0.0 M NaCI/'HzO, pH 6.0 (reference relative t o spcctrum a j
truni of the dissociated histones at pH 2 (Fig. 3 a) shows the sharp spectrum of the random coil conformation of histones with none of the perturbations characteristic of the structured histone complex. At pH 6.0 the resonances from methyl groups of leucine, isoleucine and valine are reduced in area and perturbations at 0.67, 0.44, 0.25 and 0.02 ppm are observed showing the presence of a structured histone complex. These perturbations are observed also in the (H32, H42) [15,16] and (H2A, H2B) [17] complexes. The aromatic region of the histone spectrum also shows broadened and perturbed resonances characteristic of complex behaviour (Fig. 3 b). These changes are accompanied by a reduction in peak height of the 6CH2 of arginine residues. Increasing the ionic strength to 0.14 M NaCl does not produce further marked differential changes in the NMR spectrum though by 2.0 M NaCl the spectrum is broadened presumably due to some aggregation. Even so the perturbed resonances can be observed, particularly the peak at 0.67 ppm and the complex spectrum of the aromatic residues. A model consistent with these spectral changes is a histone complex formed through interactions of the structured apolar regions, leaving the basic N-terminal regions of all four histones and the C-terminal regions of H2A and H2B mobile.
Comparing the spectrum of the histone complex in 0.14 M NaCl (Fig. 3c) with that of the core particles (Fig. 3e) shows that there are additonal differential broadening effects in the NMR spectrum of the core particle. Firstly the complex aromatic spectrum is broadened so as to be unobservable. Parallel with this the perturbed upfield resonance peaks are also broadened and cannot be observed. These two effects clearly indicate that the structured apolar regions of the histones are immobilised in the core particles. I n addition the 6CHz from the arginine residues and the aCHz of glycines are much reduced in area. This suggests that the N-terminal regions of H4 and probably H3 are also bound in the core particle at low ionic strength. I-li,stonc H 2 A and H2B Dcyletion of Core Purticks
The above NMR studies of core particles and histone complexes strongly suggest that at low ionic strength histones H3 and H4 are bound to DNA while for histones H2A and H2B the structured central regions are also bound in the core particle leaving the basic N-terminal and C-terminal regions with mobilities close to those of the random coil conformation. These mobile basic regions of H2A and H2B thus give
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Magnetic-Resonance Studies of Chromatin Core Particles
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Fig. 4. Upjieltl 27O-iMHz iVMR . s p c c ' r i u of nrrtii~c, arid pcwtitrl 'core' I niM phospliari~~ZH~O at pH 6.5. DNA concn 1 -2mg;ml. (a) Full iiativecorcparticles with no NaCl; (b) partial core particles depleted of about SO",, of H2A and H2B with no NaCl ( S O % resin extraction of H2A and H2B at 0.36 M NaCli4 M urea/'HzO at pH 7.0 plus 0.1 vol. of resin at 4 "C for 30 niin); (c) partial core particlcs containing only H3 and H4 with n o NaCl (hard extraction of H2A and H2B at 0.4 M NaC1/4 M urea pH 7.0 plus 0.1 vol. resin at 4 C for 90 min): (d) as (a) dissociated in 2.0 M NaCl; (ej as (b) dissociated in 2.0 M NaCI: (lja s ( c j disaociated in 2 M NaCI 8 M urea. Note Lhat the spcctra ( a ) and (d) are calibrated in intensity with respect to each other whilst the relations of (bj to (e) and (c) to (f) are only approximate
Fig. 5. Tlir Kt4 i4ec/rophorc,.si.s it! iiootli~c~lsulpliu".; 15 'lo nc.r:1~loniidi~ c . i l full crirrl ,ycls of hi.vtonc..c H3, H 4 , H2A (ird H 2 B ~ . ~ t r u c ~ i.fi.orii purrial 'core' purrielcs u s described in Fig.4. (a) Control H I . H3.
H4, Fi2B and H2A; (b) SO"(, removal of H2A and H2B li-om native 'core' particles; (c) full extraction of H2A and H2B leaving core particles with only H3 and H4
parlicles (140 base pairsj in
rise to the residual sharp NMR spectrum observed for core particles. Such an interpretation can be confirmed by NMR studies of core particles specifically depleted of histones H2A and H2B. Fig.4 shows the effect of specific removalof H2A and H2B on the NMR spectra of the core particles. Fig.4a gives the N M R spectrum of native core particles. Partial core particles which are depleted by about 50 ?< of histones H2A and H2B according to NM R data and gel analysis (Fig. 5 ) gives the NMR spectrum of Fig.4b. The observed area intensity of the H2A,HZB-depleted core particles is about half that of the native core particles. Dissociating the histones from native and partial core particles in 2 M NaCl gives the spectra of Fig.4d and e. The differences between these spectra can be attributed to a loss of the histones H2A and H2B. On complete depletion of the histones H2A and H2B the residual core particles containing only H3 and H4 give the NMR spectrum of F i g . 4 ~ As . can be seen, this spectrum is considerably broadened showing that at low
ionic strength histones H3 and H4 are bound to DNA in the core particles. Release of these histones at 2.0 M NaCl plus 8 M urea gives a spectrum typical of histones H3,H4 in the random coil (Fig.4f). Reconstituted Core Particles
Core particles were obtained from nuclease digestion of chromatin reconstituted from highly polymerised DNA and equimolar amounts ofthe four histones H2A, H2B, H3 and H4. The pattern of N M R behaviour is very similar to thal observed for the native core particles in Fig. 1. At low salt the same peak-broadening effects are observed and when the histones are dissociated from DNA at high salt concentration the same pattern of perturbed upfield resonances characteristic of the histone complex are observed. One resonance peak which is more prominent in the spectra of the reconstituted particle is at 3.10 ppm which is observed only in the spectrum of the H3 histone and probably comes from the cCH2 of methyl-lysine which suggests that part of the N-terminal end of H3 is mobile even at low salt concentrations.
DISCUSSION Although this type of NMR study can only lead to a qualitative picture it is reasonable to conclude from the NMR data of core particles that at low ionic strengths histones H3 and H4 are bound to DNA in
P.D. Cary, T. Moss, and E. M. Bradbury
core particles. There are, however, portions of the histones which are not firmly bound to the DNA but have mobilities close to those of the random coil conformation. These portions are largely the N-terminal and C-terminal regions of histones H2A and H2B. That the arginine-rich histones H3 and H4 are bound to the DNA is demonstrated, firstly, by the observation that only a weak residual 6CH2 peak of arginine residues remains in the core particle spectrum and, secondly, on specific depletion of core particles of H2A and H2B the resulting DNA . H3,H4 particle gives only a much broadened NMR spectrum. This latter observation shows that the 24 lysine residues in histones H3 and H4 (41 % of the total number of lysines) are also broadened by the binding of these histones to DNA. Thus the simplest interpretation of the prominent &CHzlysine resonance peak in the sharp residual NMR spectrum of core particles is that these lysine residues are in histones H2A and H2B and are not firmly bound to DNA in the core particle. On the assumption that mobile continuous regions of H2A and H2B are giving rise to the sharp residual core particle spectrum then the only regions of these histones which could give this spectrum are the basic N-terminal and C-terminal regions. That histones H3 and H4 are firmly bound to DNA in the core particles at low ionic strength is not surprising in view of the findings that the important structural features of nucleosomes and chromatin are generated by H3 and H4 alone [l1 - 141. The considerable broadenings of resonances from aromatic residues and from the perturbed methyl resonances in the core particle spectrum show also that the structured apolar regions of H2A and H2B are bound in the core particles and this binding may confer additional stability to the core particle. Previous NMR studies of the specific histone complexes (H2A,H2B) [15] and (H32, H42) [17] and of the interactions of peptides of H3 and H4 [I61 clearly show that the cross-interactions of histones involve the structured apolar C-terminal regions of H3 and H4 and the central regions of H2A and H2B leaving the basic N-terminal and C-terminal regions mobile. It was suggested that these basic terminal regions of histones are structurally dissimilar from the structured apolar regions and probably have different functions. . Models for nucleosomes [6] and core particles have been obtained from neutron scatter solution studies [8,9] and from low-resolution X-ray diffraction and electron microscopy of small core particle crystals [lo]. Both studies lead to similar models for the solution and crystal states with an overall shape of a flat disc 11 x 11 x 5.5 nm. Neutron scatter studies are able to separate the scatter effects of protein and DNA and show that the chromatin particles consist of a protein core with DNA coiled around the outside. From both neutron scatter and the low-resolution X-ray study
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it has been proposed that there are 1.7 f 0.2 turns of DNA coiled with a pitch of 2.7 nm (X-ray) or 3.0 nm (neutron scatter). Features in the neutron scatter data attributed to the protein core are little affected by the state of hydration of the core particle [8] or nucleosome [6] while features attributed to DNA are much affected by hydration. Extending neutron scatter studies by the use of small molecule contrast [24] confirm that the bulk of the water of hydration is associated with the DNA. We thus have an apolar histone core with a hydrated DNA coil. This apolar histone core has been equated with a complex of the structured apolar regions of the histones. Such regions contain a high proportion of arginine residues (69 %) which are bound within the histone complex and to the DNA of the core particle. It has been proposed that the basic N-terminal regions of all the histones were associated with DNA on the outside of the apolar core. These views must now be modified. The basic N-terminal regions of histones H3 and H4 are bound to DNA at low ionic strength as shown by the NMR spectra of H2A,H2Bdepleted core particles in which all the resonance peaks are considerably broadened. With the core particles at low ionic strength the basic terminal regions of H2A and H2B are not immobilised by DNA of the core particles and these regions must require binding sites outside of the core particle. Such sites could be the linker DNA regions between core particles, non-histone proteins or an involvement in higher-order chromatin structure, e.g. the 25 - 30-nmdiameter fibre [25,26]. Increasing the ionic strength of core particle solutions to 0.6 M NaCl causes the release of the N-terminal regions of the histones H3 and H4 leaving the DNA in the core particle complexed with the apolar regions of the histones. Since four lysine residues in these N-terminal regions of histones H3 and H4 are acetylated during DNA processing it is probable that these regions would be released at lower ionic strengths after acetylation, thus changing the stability or conformation of the core particle. Studies of H1 -depleted oligomers of nucleosomes are in progress to obtain further information on the binding sites of the N-terminal regions of histones.
REFERENCES 1. Hewish, D. R. & Burgoyne, L. A . (1973) Biochrm. Bioplips. Res. Cornrnun. 52, 504-510. 2. Olins, A. L. & Olins, D. E. (1973) J . Cell Biol. 59, 252a. 3. Woodcock, C. L. F. (1973) J . Cell Biol. 59, 368a. 4. Oudet, P., Gross-Bellard, M. & Chambon, P. (1975) Cell, 4, 281 - 300. 5. Kornberg, R. D. (1974) Science IWush. D.C.) 184, 868-871. 6 . Hjelm, R. P., Kneale, G . G., Suau, P., Baldwin, J. P., Bradbury, E. M. & Ibel, K. (1977) Cell, 10, 139-151.
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P. D. Cary, T. Moss, and E. M . Bradbury: Magnetic-Resonance Studies of Chromatin Core Particles
7. Van Holde, K. E. & Isenberg, I. (1975) Arc. Chem. Res. 8, 327- 336. 8. Suau, P.,Kneale, G. G., Braddock, G. W., Baldwin, J . P. & Bradbury, E. M. (1977)Nucleic Acids Rex 4,3769-3786. 9. Richards, B. M., Pardon, J. F., Lilley, D., Cotter, R. & Wooley, 1. C. (1977)Cell B i d . Int. Rep. I , 107-116. Lutter, L. C., Rhodes, D., Brown, R. S.. Rushton, 10. Finch, J. T., B., Levitt, M. & Klug, A. (1977)Nature (Lond.) 269, 29-36. 11. Boseley, P. G., Bradbury, E. M., Butler-Brown, G. S.. Carpenter, B. G. & Stephens, R. M. (1976) Ew. J . Biochem. 62, 21-31. 12. Camerini-Otero, R. D., Sollner-Webb, B. R. & Felsenfeld, G. (1976)Cell, 8, 333-347. 13. Sollner-Webb, B. R., Camerini-Otero, R. D. & Felsenfeld, G. (1976)Cell, 9, 179-193. 14. Moss, T.,Stephens, R. M., Crane-Robinson, C. & Bradbury, E. M. (1977)Nucleic Acids Res. 4, 2477-2485. 15. Moss, T., Cary, P. D., Crane-Robinson, C. & Bradbury, E. M. (1976)Biochemi.ytry, IS, 2261 - 2267. 16. Bohm, L., Hayashi, H., Cary. P. D., Moss, T., Crane-Robinson, C. & Bradbury, E. M. (1977)Eur. J . Biocham. 77,487493.
17. Moss, T.,Cary, P. D., Abercrombie, B. D., Crane-Robinson, C. & Bradbury, E. M. (1976)Eur. J . Biochem. 71,337-350. 18. Boublik, M., Bradbury, E. M., Crane-Robinson, C. & Rattle, H. W. E. (1970)Nut. New B i d . 229, 149-150. 19. Bradbury, E. M . & Rattle, H. W. E. (1972)Eur. J . Biochrm. 27, 270- 281, 20. Moss, T.(1977)Ph.D. Thesis, Portsmouth Polytechnic. 21. Van der Westhuyzen, D. R. & Von Holt, C. (1971)FLBS Lei[. 14,333- 337. 22. Campbell, E.D., Dobson, C. M., Williams, R. J. P. & Xavier, A. V. (1973)J . M a p . Res. 11, 172-181. 23. Thomas, J . 0. (1977) in The Orgunizution and Expression of the Eukuryotic Genome (Bradbury, E. M. & Javaherian, K., eds) Academic Press, New York. 24. Baldwin. J . P., Bradbury, E. M., Carpenter, B. G.. Kneale, G. C., Simpson. J. K. & Suau, P. (1978) J. Appl. Crj.ctullogr., in the press. 25. Carpenter, B.G., Baldwin, J. P., Bradbury, E. M. & Ibel, K . (1976)Nucleic Acids Res. 3, 1739- 1746. 26. Finch, J. T. & Klug, A. (1976)Proc. Nut1 Acud. Sci. U . S . A . 73, 1897- 1901.
P. D. Cary and E. M. Bradbury, Biophysics Laboratories, Portsmouth Polytechnic, St Michael's Building, White Swan Road, Portsmouth, Great Britain, PO1 2DT T. Moss, lnstitut fur Molekularbiologie 11 der Universitlt Zurich, Winterthurerstrasse 266A,CH-8057Zurich, Switzerland
High-Resolution Proton-Magnetic-Resonance Studies of Chromatin Core Particles Peter
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CARY, Tom MOSS, and E Morton BRADBURY
Biophysics Laboratories, Portsmouth Polytechnic (Received March 2. 1978)
The binding of histones in chromatin core particles and in core particles depleted of histones H2A and H2B has been studied by high-resolution proton nuclear magnetic resonance (NMR) at 270 MHz. At low ionic strengths it is shown that histones H3 and H4 are bound in the core particle. Further, whereas the apolar regions of H2A and H2B are also bound to the core particle, the basic N-terminal and C-terminal regions are more mobile and give rise to sharp resonances in the NMR spectrum of the core particle. Between 0.3 and 0.6 M NaCl there is further release of basic regions of histones H3 and H4 from the complex. The dissociation of the core particle between 0.6 and 2.0 M NaCl is accompanied by the release of the structured apolar regions of the histones as evidenced by the appearance of a complex aromatic spectrum and perturbed upfield ring-currentshifted methyl resonances. Arginine residues are implicated in the binding between histones and DNA and 69‘x of these residues are found in the apolar regions of the histones. Thc interactions between histones and DNA in the core particle thus involves H3 and H4 and the apolar regions of H2A and H2B. It is suggested that these basic regions of H2A and H2B have binding sites outside the core particle.
The concept of a repeating chromatin subunit introduced by Hewish and Burgoyne [l], Olins and Olins [2] and Woodcock [3] is now well established. This subunit, the nucleosome [4], has a variable DNA repeat depending on the tissue of origin, though for most somatic tissues the DNA repeat is 195 f 5 base pairs of DNA and in addition contains two each of the histones H2A, H2B, H3 and H4 [ 5 ] . Also associated with the nucleosome is probably one molecule of the very-lysine-rich histone H1. The structure of the nucleosome has been studied by neutron scatter techniques and found to have an apolar histone core with DNA coiled around this core. The shape which best fitted the scatter curves was an oblate spheroid with axial ratio of 0.5 [6]. Irrespective of their source of origin further digestion of nucleosomes with micrococcal nuclease results in a subnucleosomal particle, the ‘core’ particle, which contains 140 base pairs of DNA and the eight nucleosomal histones [6,7]. This ‘core’ particle is well-defined and has been studied in solution by neutron scatter techniques [7-91 and in small crystals by low-resolution X-ray crystallography [lo]. From both studies very similar low-resolution models are proposed of an oblate spheroid 11 x 11 x 5.5 nm which consist of a histone core with 1.7 k 0.2 turns of DNA of pitch 2.7- 3.0 nm. The histone core Ahhrwiution. NMR, nuclear magnetic resonance.
has been equated to a complex of the apolar regions of the histones [ 6 , 8 ] . The roles of the four histones H2A, H2B, H3 and H4 in generating chromatin structure and nucleosomes have been studied in several laboratories [I 1 - 141. Although it was initially thought that all four histones were necessary for the structure of the nucleosome [ S ] , it has now been shown by structural nuclease probes [12- 141 and X-ray diffraction [11,14] that the major structural features of the nucleosome and chromatin can be generated by histones H3 and H4 alone. Histones H2A and H2B may confer added stability to the basic structure formed by H3 and H4 and be involved in additional roles as yet undefined. High-resolution nuclear magnetic resonance (NMR) spectroscopy has been applied to the study of histone interactions and it has been demonstrated that the specific histone complexes (H32H42) [15,16] and (H2A, H2B) [17] are formed through interactions involving structured apolar regions of the histones. For H2A and H2B these structured regions lie in the centre of these molecules leaving the basic N-terminal and smaller C-terminal regions uncomplexed and mobile. For histones H3 and H4 the apolar carboxyl regions of the molecules are involved in complex formation leaving the basic N-terminal regions free. In the latter case it has been shown that residues 1-37 of H4 and 1-41 of H3 can be removed without
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affecting the ability of the apolar carboxyl regions to form complexes [I 61. These results strongly suggest that the very basic terminal regions of histones are involved in functions distinct from those of the structured apolar regions. It has been suggested that the basic N-terminal regions of histones are the major binding sites to DNA and early NMR studies of the binding of individual histones to DNA showed that the N-terminal regions were the most strongly binding to DNA [18,19]. However, in the nucleosome structure we have at least two different regions of DNA: the well defined core particles containing 140 base pairs of DNA which are joined by a linker DNA region the length of which depends on the nucleosome repeat. In addition there are higher-order chromatin structures which may require interactions outside of the nucleosome involving regions of histones H2A, H2B, H3 and H4 in addition to the interactions of H I . These interactions in higher-order chromatin structures may also require non-histone proteins. Chromatin core particles have been studied by high-resolution NMR to evaluate the extent to which the different histones are bound to the 140-base-pair DNA. EXPERIMENTAL PROCEDURE Chromatin core particles were prepared from calf thymus as described previously [20]. They contained 140 base pairs of DNA, equimolar quantities of the histones H2A, H2B, H3 and H4, and no HI histones, as demonstrated by gel electrophoresis using DNA and protein gels. The core particles were concentrated in a millipore filter to a concentration of 3- 5 mg/ml of DNA as measured by ultraviolet absorbance at 260 nm. This solution was then dialysed for 48 h in 10 mM phosphate buffer at pH 6.5-7.0 in the ratio of 1 ml of core particles to 1000 ml of buffer solution at 4 C. The dialysate was changed three times over this period of time. For the NMR studies the sample was divided into 0.5-ml aliquots which were dialysed against four changes of 'Hz0,I mM phosphate buffer/ NaCl at pH 6.5 over 48 h in the ratio 0.5 ml core particles/5 ml dialysate. Finally the core particle solution were dialysed in the ratio of 0.5 ml solution to 20 ml of 2Hz0 buffer. Core Particles Depleted of Histones H2A and H2B
To determine the contribution of histones H2A and H2B to the NMR spectrum of the core particles these histones were removed from the particles by Bio-Rad AG 50w x 2 (200-400 mesh) resin under the following conditions: 0.36 M NaCl or 0.4 M NaCI, 4 M urea, pH 7.0 at 4°C. Acrylamide gel electrophoresis showed that only histones H2A and H2B were removed from the core particles.
Magnetic-Rcsonance Studics o f Chroiiiatin Core Particles
Reconstituted Core Particles
Calf thymus histones were separated into the pairs, (H2A, H2B) and (H3, H4), by the method of van Holt and van der Westhuyzen [21]. Equiinolar mixtures of these pairs of histones in 2 M NaCl were mixed with DNA also in 2 M NaCl and dialysed down stepwise to 0 M NaCl pH 7.0. This reconstituted chromatin was then digested with micrococcal nuclease to give a 140-base-pair DNA particle as the main product. The digestion products were fractionated on a Bio-Gel A-5m (Bio-Rad) column to give 140-basepair reconstituted DNA particles purified to better than 90 7:). Nuclear Magnetic Resonance Spectroscopy
Core particle solutions were pipetted into standard 5-mm NMR tubes. NMR spectra were recorded at 270 MHz in the Fourier transform mode on a Bruker WH 270 spectrometer equipped with an Oxford lnstrument Co. superconducting magnet. Resolution enhancement was performed by the convolution difference method [22]. Signal intensities were standardized against the signal from a 2H20 solution of sodium formate doped with gadolinium nitrate and contained in an inner concentric microtube. The sodium formate signal was broadened so that its line width was independent of small variations in field homogeneity. The concentrations of the core particle solutions were monitored by the ultraviolet absorbance at 260 nm. Signal intensities were also compared with the signals from the fully dissociated histones from core particles in 2 M NaCl and 8 M urea. Under these conditions the histones are free and in the random coil conformation. Finally corrections relative to the 'H20 lock frequency were made for changes in the chemical shift of the samples in the various solution conditions.
RESULTS The NMR spectra of various constituents under different conditions were examined as follows: (a) native core particles and their dissociation into DNA and histones by salt (NaCl) and urea; (b) the histone complex formed from acid-extracted histones ; (c) core particles depleted of histones H2A and H2B; (d) reconstituted core particles and their dissociation by salt (NaCI) and urea. Native Core Particles
Fig. 1 shows the NMR spectra of core particles at different stages of salt dissociation and also the spectrum of the random coil histones from core par-
P. D. Cary, T. Moss. and E. M. Brudbul-)
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Fig. I . l+/ic‘ltl 270-MH: spcc‘tstr o/’nuiiw ‘core’ purticlc~s(140 husc p i i n ) iii I r i i M p l ~ ~ , s ~ i / i a t r , huffir ~ H ~ Ouf p H 6.5. DNA concn 3 - 5 rngrnl. (a) N o NaCI, (b)0.3 M NaCI, (c) 0.6 M NaCl including a convolulion diiTei-enceof the ring-current-shifted region, (d) 1.2 M NaCl including a convolution diference of the rinp-ciirrent-shirtecl region. (e) 2.0 M NaCl including a convolution difference of the ring-current-shifted region, Cfj 2.0 M NaCl 8 M urea
ticles dissociated in 2 M NaC1/8 M urea. The lowfield region of the spectrum of core particles, 6-10 ppm (not shown), contains resonance peaks from the aromatic residues located in the apolar regions of histones and resonances from DNA. These resonances are very broad and featurelcss, as would be expected for a slowly moving complex the size of the core particle. Only when the histones are dissociated from DNA in 2 M NaC1/8 M urea are sharp aromatic resonances observed. The upfield region of the spectrum of the core particles (Fig. 1 a) however, exhibits sharp resonances showing that some regions of the histones have mobilities additional to the overall rotation of core particlcs. A comparison of the total sum of the peak areas of this upfield region of the NMR spectrum (Fig. 1a j with that of the fully dissociated histones (Fig. 1f) shows that the residual peak area of the core particles is 17 - 20 2>of the total area of the spectrum of the free histones. In addition it can be seen that there are marked differential broadening effects which particularly affect the arginine SCH2 resonance peak at 3.25 ppm and the glycine xCH2 peak at 4.0 ppm, while the lysine E C Hresonance ~ at 3.02 ppm, although reduced to about one half of its total area, remains sharp. Also in the core particle spectrum the methyl resonances from leucine, isoleucine and valine at
about 0.9 ppin are very much reduced in area. Thus in the core particle spectrum a very substantial proportion of the upfield peak areas from the histones H2A, H2B. H3 and H4 is ‘lost’, in addition to the aromatic resonances due to broadening efl’ects from histone complex formation and binding to DNA in the particle. The effect of histone complex formation can be seen by comparing the spectrum of core particles in 2 M NaCl (Fig. 1e) when the histone complex is dissociated from DNA, with that of core particles in 2 M NaC1/8 M urea (Fig. 1 f) when the histones are fully dissociated into their random coil conformations. Again there are marked differential effects which particularly affect the peak areas of the methyl resonances at about 0.9 ppm from the apolar residues and also the arginine dCH2 resonance while the K H 2 peak from lysine residues remains sharp and well-developed. These effects, which will be described in more detail later, are attributed to complex formation between the apolar C-terminal regions of histones H3 and H4 and the apolar central regions of histones H2A and H2B. These ‘apolar’ regions contain a substantial proportion (69%) of arginine residues but only 31 ‘,!,,;, of the lysine residues. The binding of the histone complex to DNA of the core particle (cf. Fig. 1a and e) leads to a further loss of peak areas of the resonances from arginine 6CH2, glycine aCH2 and the methyl groups from the apolar residues. The resonances from aromatic residues, which are contained almost entirely in the apolar regions of the histones, are considerably broadened in both histone complexes and core particles. An estimate can be made of the chemical groups in core particles which have mobilities approaching those of the random coil conformation. This estimate, given in Table 1, is approximate because residual resonance peaks have more than one component. If it is assumed that these mobile groups are contained in continuous segments of histones then these segments are rich in lysines and alanines but contain low amounts of the apolar residues (leucines, isoleucines and valinesj, glycines, serines, threonines and very low amounts of arginines. Such regions are the N-terminal residues 1 - 30 and C-terminal 105- 125 of H2B, the N-terminal 1 - 16 and C-terminal 117- 129 of H2A and the N-terminal of H3 (18-37j. The N-terminal region of H4 contains a high proportion of glycine residues whose zCHz rcsonance is considcrably broadened in the core particle spectrum (Fig. 1 a). Individual resonances have also been identified: the observation of a sharp peak at 3.08 ppm from the cCH2 of methyl lysines suggest that some of the residual resonance comes from histone H3. It is unlikely to come from histone H4 because the glycine zCH2 peak is broad and there are eight glycines in the N-terminal region of H4. The N-terminal prolinc of histone H2B gives a sharp SCHz resonance peak at
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Magnetic-Resonance Studies of Chromatin Core Particles
Table 3 . Approximate eytimares of rhe numbers of d(fSrmr amino ucd residues contributing 10 the slzavp NMR spectrum of' native core purticles ut low ionic strength The estimated number of the residues listed was obtained from the proton N M R data of core particles in 1 mM phosphate buffer pH 6.8, 0-0.2M NaC1. (An asterisk in the list of resonant groups indicates that the estimate is difficult due to a weak resonant peak.) The numbers of these residues present in sequcnccs with high alanine and lysine contents and low arginine, glycine, apolar and aromatic contents, i.c. residues 1-30 and 105-125 of H2B, 3-36and 137-129ofH2Aand 18-37ofH3,arealsogivcnfor comparison Amino acid residue
Resonant group
Ala Arg Val Ilc Leu Lys cily
fiCH3* 6CH2
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Number present in considered scquences
10
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1 8 2 1 1 1
3.3 ppm while a sharp peak at 2.25 ppm shows that the N-terminal acetyl serine of histones H2A or H4 are mobile, though for reasons given above this resonance is probably from H2A. An increase in ionic strength from 0.2 M to 0.6 M NaCl causes the preferential release of regions containing the following residues : arginine, lysine, glycine, methyl-lysine, glutamic acid, glutamine and some of the apolar residues, leucine, isoleucine and valine. These residues are located in the N-terminal regions of histones H3 and H4 with possibly additional segments of H2A and H2B. The low peak areas of the apolar and aromatic residues indicate that the apolar central regions of these histones are bound in the core particle. Although the histone complex is bound to DNA in the core particles at 0.6 M NaCl, it is released by 2 M NaCl but the NMR spectra of the core particles at different stages of dissociation at 0.6, 1.2 and 2.0 M NaCl are very similar. There are small differences, for example the perturbed upfield resonances observed in the spectrum of the histone complex at 2 M NaCl are broadened and not observed in the core particle spectrum at 0.6 M NaCl. A similar change is also observed when coinparing the NMR spectrum of reconstituted histone complexes at 2 M NaCl with that of core particles as described in the
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Fig. 2. Up/idd 27O-:Ll H: :L',WR .vp'clru of narivc 'corc' parlicks (140 bnse puirs) in 1 n?M pho.sphatei0.14 M N u C ~ ; ' H Z Ohufler at p H 6.5. DNA concn 2-3 nig ml. (a) No urea, (b) 2 M urea, ( c )4 M urea, ( d ) 6 M urea
next section. This suggests that the octameric histone complex (H2A, H2B, H3, H4)2 [23] is bound to DNA at 0.6 M NaCl through the apolar regions of the four histones and the full complex is released by 2.0 M NaCI. At 2 M NaCl this histone complex is dissociated by the addition of 8 M urea. At 0.2 M NaCl, the effect of the addition of urea is shown in Fig.2. The core particle is partially dissociated by 4 M urea and except for a proportion of arginine residues is largely dissociated by 6 M urea. The 6CHz resonance from arginine is not fully developed in high urea concentration, indicating that even with 6 M urea some of the arginines are still bound to DNA. These can be released by increasing the ionic strength. The Rrconstitutcd Histone Complex
The regions of histones which are immobilised by histonc complex formation and by binding to DNA in core particles are rich in the following residues: arginine, valine, leucine, isoleucine, glycine, tyrosine and phenylalanine. Histone regions which contain these residues are the apolar central regions of H2A and H2B, the C-terminal halves of H3 and H4 and the N-terminal region of H4. The effects of histone complex formation are shown in Fig.3. The N M R spec-
P. D. Cary, T. Moss, and E. M. Bradbury
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6 (PPm) Fig. 3 . 270-iMIiz N M K .spwtru o/'ocitl-c.\-tl.tic,let/ / i i . s l o i i e . ~H3, H4. H2.4, mid 112H in c.yuirnolur uinou~~t.s ut 20 nzg;'nil if7 211~0. (a) N o NaCI, pH 2.0: (b) no NaC1, pH 6.0 including a convolution dill'erencc of the ring-current-shifted upfield region; (c) 0.14 M NaCl. pH 6.0 including a convolution dillerence of the ring-current-shifted regions; (d) 2 M NaCI, pH 6.0 including a convolution diffcrence ol' the ring-currenlshifted region; (e) native 'core' particle (140 base pairs) in 0.0 M NaCI/'HzO, pH 6.0 (reference relative t o spcctrum a j
truni of the dissociated histones at pH 2 (Fig. 3 a) shows the sharp spectrum of the random coil conformation of histones with none of the perturbations characteristic of the structured histone complex. At pH 6.0 the resonances from methyl groups of leucine, isoleucine and valine are reduced in area and perturbations at 0.67, 0.44, 0.25 and 0.02 ppm are observed showing the presence of a structured histone complex. These perturbations are observed also in the (H32, H42) [15,16] and (H2A, H2B) [17] complexes. The aromatic region of the histone spectrum also shows broadened and perturbed resonances characteristic of complex behaviour (Fig. 3 b). These changes are accompanied by a reduction in peak height of the 6CH2 of arginine residues. Increasing the ionic strength to 0.14 M NaCl does not produce further marked differential changes in the NMR spectrum though by 2.0 M NaCl the spectrum is broadened presumably due to some aggregation. Even so the perturbed resonances can be observed, particularly the peak at 0.67 ppm and the complex spectrum of the aromatic residues. A model consistent with these spectral changes is a histone complex formed through interactions of the structured apolar regions, leaving the basic N-terminal regions of all four histones and the C-terminal regions of H2A and H2B mobile.
Comparing the spectrum of the histone complex in 0.14 M NaCl (Fig. 3c) with that of the core particles (Fig. 3e) shows that there are additonal differential broadening effects in the NMR spectrum of the core particle. Firstly the complex aromatic spectrum is broadened so as to be unobservable. Parallel with this the perturbed upfield resonance peaks are also broadened and cannot be observed. These two effects clearly indicate that the structured apolar regions of the histones are immobilised in the core particles. I n addition the 6CHz from the arginine residues and the aCHz of glycines are much reduced in area. This suggests that the N-terminal regions of H4 and probably H3 are also bound in the core particle at low ionic strength. I-li,stonc H 2 A and H2B Dcyletion of Core Purticks
The above NMR studies of core particles and histone complexes strongly suggest that at low ionic strength histones H3 and H4 are bound to DNA while for histones H2A and H2B the structured central regions are also bound in the core particle leaving the basic N-terminal and C-terminal regions with mobilities close to those of the random coil conformation. These mobile basic regions of H2A and H2B thus give
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Magnetic-Resonance Studies of Chromatin Core Particles
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Fig. 4. Upjieltl 27O-iMHz iVMR . s p c c ' r i u of nrrtii~c, arid pcwtitrl 'core' I niM phospliari~~ZH~O at pH 6.5. DNA concn 1 -2mg;ml. (a) Full iiativecorcparticles with no NaCl; (b) partial core particles depleted of about SO",, of H2A and H2B with no NaCl ( S O % resin extraction of H2A and H2B at 0.36 M NaCli4 M urea/'HzO at pH 7.0 plus 0.1 vol. of resin at 4 "C for 30 niin); (c) partial core particlcs containing only H3 and H4 with n o NaCl (hard extraction of H2A and H2B at 0.4 M NaC1/4 M urea pH 7.0 plus 0.1 vol. resin at 4 C for 90 min): (d) as (a) dissociated in 2.0 M NaCl; (ej as (b) dissociated in 2.0 M NaCI: (lja s ( c j disaociated in 2 M NaCI 8 M urea. Note Lhat the spcctra ( a ) and (d) are calibrated in intensity with respect to each other whilst the relations of (bj to (e) and (c) to (f) are only approximate
Fig. 5. Tlir Kt4 i4ec/rophorc,.si.s it! iiootli~c~lsulpliu".; 15 'lo nc.r:1~loniidi~ c . i l full crirrl ,ycls of hi.vtonc..c H3, H 4 , H2A (ird H 2 B ~ . ~ t r u c ~ i.fi.orii purrial 'core' purrielcs u s described in Fig.4. (a) Control H I . H3.
H4, Fi2B and H2A; (b) SO"(, removal of H2A and H2B li-om native 'core' particles; (c) full extraction of H2A and H2B leaving core particles with only H3 and H4
parlicles (140 base pairsj in
rise to the residual sharp NMR spectrum observed for core particles. Such an interpretation can be confirmed by NMR studies of core particles specifically depleted of histones H2A and H2B. Fig.4 shows the effect of specific removalof H2A and H2B on the NMR spectra of the core particles. Fig.4a gives the N M R spectrum of native core particles. Partial core particles which are depleted by about 50 ?< of histones H2A and H2B according to NM R data and gel analysis (Fig. 5 ) gives the NMR spectrum of Fig.4b. The observed area intensity of the H2A,HZB-depleted core particles is about half that of the native core particles. Dissociating the histones from native and partial core particles in 2 M NaCl gives the spectra of Fig.4d and e. The differences between these spectra can be attributed to a loss of the histones H2A and H2B. On complete depletion of the histones H2A and H2B the residual core particles containing only H3 and H4 give the NMR spectrum of F i g . 4 ~ As . can be seen, this spectrum is considerably broadened showing that at low
ionic strength histones H3 and H4 are bound to DNA in the core particles. Release of these histones at 2.0 M NaCl plus 8 M urea gives a spectrum typical of histones H3,H4 in the random coil (Fig.4f). Reconstituted Core Particles
Core particles were obtained from nuclease digestion of chromatin reconstituted from highly polymerised DNA and equimolar amounts ofthe four histones H2A, H2B, H3 and H4. The pattern of N M R behaviour is very similar to thal observed for the native core particles in Fig. 1. At low salt the same peak-broadening effects are observed and when the histones are dissociated from DNA at high salt concentration the same pattern of perturbed upfield resonances characteristic of the histone complex are observed. One resonance peak which is more prominent in the spectra of the reconstituted particle is at 3.10 ppm which is observed only in the spectrum of the H3 histone and probably comes from the cCH2 of methyl-lysine which suggests that part of the N-terminal end of H3 is mobile even at low salt concentrations.
DISCUSSION Although this type of NMR study can only lead to a qualitative picture it is reasonable to conclude from the NMR data of core particles that at low ionic strengths histones H3 and H4 are bound to DNA in
P.D. Cary, T. Moss, and E. M. Bradbury
core particles. There are, however, portions of the histones which are not firmly bound to the DNA but have mobilities close to those of the random coil conformation. These portions are largely the N-terminal and C-terminal regions of histones H2A and H2B. That the arginine-rich histones H3 and H4 are bound to the DNA is demonstrated, firstly, by the observation that only a weak residual 6CH2 peak of arginine residues remains in the core particle spectrum and, secondly, on specific depletion of core particles of H2A and H2B the resulting DNA . H3,H4 particle gives only a much broadened NMR spectrum. This latter observation shows that the 24 lysine residues in histones H3 and H4 (41 % of the total number of lysines) are also broadened by the binding of these histones to DNA. Thus the simplest interpretation of the prominent &CHzlysine resonance peak in the sharp residual NMR spectrum of core particles is that these lysine residues are in histones H2A and H2B and are not firmly bound to DNA in the core particle. On the assumption that mobile continuous regions of H2A and H2B are giving rise to the sharp residual core particle spectrum then the only regions of these histones which could give this spectrum are the basic N-terminal and C-terminal regions. That histones H3 and H4 are firmly bound to DNA in the core particles at low ionic strength is not surprising in view of the findings that the important structural features of nucleosomes and chromatin are generated by H3 and H4 alone [l1 - 141. The considerable broadenings of resonances from aromatic residues and from the perturbed methyl resonances in the core particle spectrum show also that the structured apolar regions of H2A and H2B are bound in the core particles and this binding may confer additional stability to the core particle. Previous NMR studies of the specific histone complexes (H2A,H2B) [15] and (H32, H42) [17] and of the interactions of peptides of H3 and H4 [I61 clearly show that the cross-interactions of histones involve the structured apolar C-terminal regions of H3 and H4 and the central regions of H2A and H2B leaving the basic N-terminal and C-terminal regions mobile. It was suggested that these basic terminal regions of histones are structurally dissimilar from the structured apolar regions and probably have different functions. . Models for nucleosomes [6] and core particles have been obtained from neutron scatter solution studies [8,9] and from low-resolution X-ray diffraction and electron microscopy of small core particle crystals [lo]. Both studies lead to similar models for the solution and crystal states with an overall shape of a flat disc 11 x 11 x 5.5 nm. Neutron scatter studies are able to separate the scatter effects of protein and DNA and show that the chromatin particles consist of a protein core with DNA coiled around the outside. From both neutron scatter and the low-resolution X-ray study
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it has been proposed that there are 1.7 f 0.2 turns of DNA coiled with a pitch of 2.7 nm (X-ray) or 3.0 nm (neutron scatter). Features in the neutron scatter data attributed to the protein core are little affected by the state of hydration of the core particle [8] or nucleosome [6] while features attributed to DNA are much affected by hydration. Extending neutron scatter studies by the use of small molecule contrast [24] confirm that the bulk of the water of hydration is associated with the DNA. We thus have an apolar histone core with a hydrated DNA coil. This apolar histone core has been equated with a complex of the structured apolar regions of the histones. Such regions contain a high proportion of arginine residues (69 %) which are bound within the histone complex and to the DNA of the core particle. It has been proposed that the basic N-terminal regions of all the histones were associated with DNA on the outside of the apolar core. These views must now be modified. The basic N-terminal regions of histones H3 and H4 are bound to DNA at low ionic strength as shown by the NMR spectra of H2A,H2Bdepleted core particles in which all the resonance peaks are considerably broadened. With the core particles at low ionic strength the basic terminal regions of H2A and H2B are not immobilised by DNA of the core particles and these regions must require binding sites outside of the core particle. Such sites could be the linker DNA regions between core particles, non-histone proteins or an involvement in higher-order chromatin structure, e.g. the 25 - 30-nmdiameter fibre [25,26]. Increasing the ionic strength of core particle solutions to 0.6 M NaCl causes the release of the N-terminal regions of the histones H3 and H4 leaving the DNA in the core particle complexed with the apolar regions of the histones. Since four lysine residues in these N-terminal regions of histones H3 and H4 are acetylated during DNA processing it is probable that these regions would be released at lower ionic strengths after acetylation, thus changing the stability or conformation of the core particle. Studies of H1 -depleted oligomers of nucleosomes are in progress to obtain further information on the binding sites of the N-terminal regions of histones.
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P. D. Cary and E. M. Bradbury, Biophysics Laboratories, Portsmouth Polytechnic, St Michael's Building, White Swan Road, Portsmouth, Great Britain, PO1 2DT T. Moss, lnstitut fur Molekularbiologie 11 der Universitlt Zurich, Winterthurerstrasse 266A,CH-8057Zurich, Switzerland
