Proc. Nati. Acad. Sci. USAVol. 77, No. 8, pp. 4818-4822, August 1980Cell Biology

Composition of native and reconstituted chromatin particles: Directmass determination by scanning transmission electron microscopy

(nucleosome/arginine-rich histones)

C. L. F. WOODCOCK*, L.-L. Y. FRADO*, AND J. S. WALLt*Department of Zoology, University of Massachusetts, Amherst, Massachusetts 01003; and tDepartment of Biology, Brookhaven National Laboratory,Upton, New York 11973

Communicated by Oscar L. Miller, Jr., May 23, 1980

ABSTRACT Chromatin particles reconstituted from 145-base-pair lengths of DNA and either the arginine-rich histonesH3 and H4 only or all four nucleosomal core histones have beencompared with native nucleosomes in terms of their ultra-structure and mass distribution, as determined by scanningtransmission electron microscopy (STEM). The mass of thenucleosome derived from STEM analysis was very close to thatcalculated for its DNA and histone'components. The reconsti-tuted particles showed a broader mass distribution, but it wasclear that the majority contained at least eight histone mol-ecules. This was to be expected for structures reconstituted fromall four core histones, but in the case of H3H4-DNA complexesclearly showed that an octamer rather than tetramer o thesehistones was required to fold nucleosomal DNA into a stablecompact particle. The significance of the H3H4 octamer com-plex with respect to nucleosomal structure is discussed, and theevidence that nucleosomal DNA can accept even greater num-bers of histones is considered.

One of the fundamental properties of nucleosomes (for reviewsof nucleosome structure, see refs. 1 and 2) is the ease with whichthey can be reassembled in vitro from DNA and the appro-priate histones (3-13). The reconstituted particles show manyof the features of native nucleosomes, including morphology,sedimentation velocity, and nuclease susceptibility. It has alsobeen possible to reconstitute nucleosome-like particles fromnucleosomal DNA and the two arginine-rich histones, H3 andH4 (4, 7, 11, 14-21). These H3H4 particles show many of theproperties of nucleosomes, but there has been some disagree-ment as to the stoichiometry with which these histones bind toDNA in the formation of a stable nucleosome-like particle.Bina-Stein (15) and Bina-Stein and Simpson (14) reported that140 base pairs (bp) of DNA were folded into a nucleosome-likeparticle by a single (H3H4)2 tetramer, and this conclusion wasalso reached by Oudet et al. (7), using simian virus 40 DNA. Onthe other hand, Simon et al. (18), Stockley and Thomas (21),and Thomas and Oudet (20) concluded that complexes couldbe formed from tetramers or octamers of H3 and H4 dependingon the conditions used. The question of stoichiometry is im-portant in this case because, as discussed below, it is pertinentto the question of uniqueness of the histone arrangement in thenucleosome.The problems of determining the stoichiometry of DNA-

histone complexes are threefold: first, histones, being low inaromatic amino acids, often give unreliable values with con-ventional protein assays. Second, molecular weight determi-nations based on sedimentation properties require an estimateof partial specific volume. Third, if a population of reconstituted

chromatin particles consists of more than one species withdiffering DNA-to-protein ratios, this may not be detectable withconventional assay methods. This last problem became apparentfrom the examination of electron micrographs of reconstitutedchromatin particles from a single sucrose gradient peak. Theseshowed that particles of similar sedimentation velocity haddifferent ultrastructures.A relatively new technique for mass determination that

avoids all these difficulties is the direct measurement of theelectron scattering ability of individual particles with thescanning transmission electron microscope (STEM). Such in-struments have been used to obtain both qualitative andquantitative data from chromatin and other biological materials(22-29). In this communication we describe the application ofSTEM mass measurement to native and reconstituted nucleo-somes, and we discuss the advantages and limitations of themethod.

MATERIALS AND METHODSPreparation of Histones and DNA. The starting material

was chicken erythrocyte nuclei prepared as described (30).Chromatin depleted of histones H1 and H5 was prepared byextensive washing with 0.65 M NaCl/0.2 mM EDTA/1 mMphenylmethylsulfonyl fluoride/10 mM Tris-HCl, pH 9.0 (10).After dissociation in 2.5 M NaCl/0.5mM phenylmethylsulfonylfluoride/50 mM sodium phosphate buffer, pH 7.0, the DNAwas absorbed onto hydroxyapatite (15), leaving four-histoneoctamers in solution. The octamers were then separated intoH3H4 and H2AH2B pairs by the method of Ruiz-Carrillo andJorcano (31). Nucleosomal DNA was extracted from mo-nonucleosomes prepared by micrococcal nuclease digestion oferythrocyte nuclei as described (12), except that the digestionbuffer contained 75 mM KCl and 5 mM 1,4-piperazinedi-ethanesulfonic acid (Pipes), pH 8.0. After purification on su-crose density gradients, the mononucleosome peaks were pooledand adjusted to 2.5 M NaCl, and the DNA was absorbed andeluted from hydroxyapatite (15).DNA was sized by electrophoresis on 3.5% polyacrylamide

gels with restriction endonuclease fragments of phage 4X174DNA as standards and quantitated in solution by using the re-lationship 1.0 A260 = 0.05 mg/ml. Histones were quantitatedby modified Lowry (32, 33) and Coomassie blue dye-binding(34) assays. In addition, samples of H3H4 pairs were subjectedto amino acid analysis (Analytical Biochemistry Labs, Columbia,MO) and various amounts of these samples were applied to 18%polyacrylamide gels (35). After staining to equilibrium with

Abbreviations: STEM, scanning transmission electron microscope(microscopy); bp, base pair(s); TMV, tobacco mosaic virus; e-, elec-tron.

4818

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Proc. Natl. Acad. Sci. USA 77 (1980) 4819

Coomassie blue, the histone bands were excised, and the dyewas eluted and measured (36).

Reconstitution. Procedures were essentially as described(13). Histones in 2.5 M NaCl/0.2 mM EDTA/10 mM Tris-HCI,pH 8.0, were added to DNA in the same solution such that thefinal volume was 1.0 ml and the final concentration of DNAwas 50 ,g/ml. The mixture was dialyzed against the startingbuffer for 4 hr, and the salt concentration then reduced con-tinuously by gradient dialysis to 25mM NaCl/0.2 mM EDTA/1mM triethanolamine-HCI buffer, pH 7.5. The dialysis was al-lowed to proceed for 16 hr at room temperature, after whichthe reconstituted products were separated on 5-20% sucrosegradients in the dialysis buffer (Beckman SW 41 rotor, 34,000rpm for 15 hr). In some cases, the reconstituted products werefixed with 0.1% glutaraldehyde for 12 hr at 00C before appli-cation to the gradients. This procedure caused no crosslinkingbetween nucleosomes.

Electron Microscopy. For conventional transmission elec-tron microscopy, peak fractions from the sucrose gradients wereadjusted to about 1 gg/ml and 50mM NaCl, applied directlyto freshly glow-dicharged thin carbon films, stained with 0.5%uranyl acetate, and finally rinsed with water. In some cases, thesamples were first fixed with glutaraldehyde as described above.Grids were examined with a Siemens Elmiskop 102, usingtilted-beam dark-field optics at 60 kV.

For STEM, unstained samples were prepared as above, andapplied to titanium grids coated with thin (20-A) carbon on afenestrated support (37). The grids were washed with waterafter the samples had been applied, a drop of purified tobaccomosaic virus (TMV) solution was added, the excess liquid wasblotted off, and the grids were plunged into liquid nitrogen andfreeze-dried. They were then transferred under vacuum to theSTEM.Imaging and Data Analysis. Specimens were observed in

the Brookhaven STEM (38) at -1300C with a beam size of 2.5A and no pretreatment. Under these conditions, no contami-nation was observable. Essentially all electrons passing throughthe specimen were detected on either a dark field detectorsubtending 0.040-0.200 radians at the specimen or a set ofdetectors subtending 0-0.040 radians. Total transmitted currentwas measured to determine dose and to normalize the dark-fieldsignal for each picture element. This normalized dark fieldsignal was recorded digitally on a disc memory. Images fromthe disc were viewed on a display system, and, if found satis-factory, raw data (normalized) was transferred to magnetic tapefor subsequent analysm To avoid coherent scattering and obtaina signal directly proportional to mass thickness, only large-anglescattering was analyzed (39).STEM data tapes were analyzed at Brookhaven's CDC 7600

computer facility. A program was written to either: (i) locateisolated bright spots on an image, determine position and size,and plot intensity and integrated intensity (after subtractinglocal background) as a function of radius from particle center,or (ii) locate isolated linearmolecules, trace their backbone, andplot average intensity and integrated intensity as a function ofdistance from the axis.

RESULTSDigestion of chicken erythrocyte nuclei under the conditionsdescribed releases mononucleosomes with a DNA content of142-146 bp, which can be completely separated from dinu-cleosomes and subnucleosomal particles by one or two sucrosegradient centrifugations (Fig. ic). These were the source ofnative nucleosomes for STEM analysis, and the DNA extractedfrom them was used for reconstitution. The purity of the H3H4histone pair used for reconstitution is illustrated in Fig. lb.

a

a-3.I. _

1)

FIG. 1. (a) Separation of chromatin particles on 5-20% sucrosegradients. Reconstitution with all four core histones, or with only H3and H4 (using starting ratios of 1.25 histones per DNA), produces amajor class of particles with a sedimentation velocity similar to thatof native nucleosomes. A small amount of material sedimenting withfree nucleosomal DNA is also present. Material from the peak frac-tions was used for all preparations for microscopy. (b) Coomassieblue-stained gel ofthe isolated H3H4 histone pair (left track) and totalchicken histones (right track). (c) Polyacrylamide gel electrophoresisof nucleosomal DNA isolated from native nucleosomes, showing asingle band with a size of 143-145 bp. This DNA was used for the re-constitution experiments. Track at left shows OX174 DNA restrictionfragment markers, with size given in bp.

In our hands, reconstitution was most successful (as judgedby the yield of "llS" nucleosome-like particles) when per-formed by gradient dialysis from 2.5M NaCl (13) in the absenceof urea. At the end of the reconstitution, the products wereseparated on a sucrose gradient. Fig. la shows the profile fromexperiments in which the histone-to-DNA ratios were optimalfor the production of "llS" particles. In general, we found thatincreasing the DNA-to-histone ratios from the optimum causednonspecific aggregation and precipitation while reducing theratio resulted in an increase in material that sedimented withfree nucleosomal DNA and in addition, in the case of H3H4reconstitution, a variable proportion of "7S" particles that wereintermediate between free DNA and llS nucleosomes. Bina-Stein (15) and Simon et al. (18) also found that H3 and H4 wereable to fold nucleosomal DNA into a compact particle with asedimentation velocity close to that of native nucleosomes.However, Bina-Stein (15) concluded that the 9.8S H3H4 par-ticles contained a tetramer of histones, wheras Simon et al. (18)found that an octamer of histones was included in their 10.4Sparticles. In addition to the 10.4S particles, Simon et al. (18)were also able to generate 7.5S complexes at lower histone-to-DNA input ratios; these complexes contained a tetramer ofhistones. Stockley and Thomas (21), using nucleosomes depletedof histone H2A and H2B, obtained a mixture of 9.1S octamersand 6.0S tetramers.

In the present study, the stoichiometry of the "llS" and "7S"particles, as determined by conventional assays, suggested that,in agreement with Simon et al. (18), they contained on the orderof eight and four histones, respectively (DNA-to-histone ratiosranged from 1:0.35 to 1:0.78 for the "7S" particles, and from1:1.03 to 1:1.21 for the "llS" particles). The "tetramer" com-plex appeared in the electron microscope as largely unfolded,linear structures. Klevan et al. (4) and Stockley and Thomas (21)also concluded that the H3H4 tetramers did not form compactparticles with nucleosomal DNA. Because of the variability in

Cell Biology: Woodcock et al.

4820 Cell Biology: Woodcock et al.

formation and morphology of the "7S tetramer," most STEManalyses were performed on the "1 S" particles. Dark-fieldimages of chromatin particles recorded unstained with theSTEM, and (after light uranyl staining) with conventionaltransmission electron microscopy are shown in Fig. 2. Bothimaging methods indicate a fairly homogeneous population ofcircular profiles for the native and reconstituted 11S parti-cles.

For molecular weight determination by STEM, it was nec-essary first to measure the mass lost through electron bom-bardment during the scan needed to record the data. Mass losscurves were constructed by taking sequential measurementsof the same area after 1, 2, 4, 8, and 16 scans. Typical results areshown in Fig. 3. Nucleosomes and TMV lost mass at about thesame rate, so that after 16 scans at a dose of 12 electrons (e-)/A2per scan, from 20% to 25% of the mass was lost. However, aftera single scan, the mass loss was quite small, from 2% to 4%,depending on the method of extrapolation to zero dose. A cor-rection factor of 1.03 has been applied to the measured massvalues, but in view of the other uncertainties inherent in themethod (discussed below), the mass loss is not significant withthe scanning conditions employed.

Histograms of the mass values of native nucleosomes, ofparticles reconstituted from the four core histones and 145 bpof DNA, and of particles reconstituted from histones H3 andH4 and DNA are shown in Fig. 4. The abcissa is graduated bothin arbitrary mass units derived from the counts of electronsscattered and in daltons. In addition, the number of histonemolecule equivalents on 145 bp of DNA is indicated. The mo-lecular mass calibration is based on the scattering of definedlengths of TMV particles, using values of 3.9 X 107 daltons forthe mass and 3000 A for the length (40). Native nucleosomeshave a modal molecular mass of 215,000 and a distribution thatis somewhat assymetric, having a small secondary maximumat about 300,000 daltons. The expected molecular mass of 145bp of DNA plus two each of the histones H2A, H2B, H3, andH4 is about 202,000, close to the observed modal value. If theheavier "tail" of the distribution is omitted from the calcula-

1.0

0.9

0.8

. 0.7

°|' 0.6a 0

0

6o*° 1.0 sc

TMV

1 212 24

4 848 96

16 Scans192 e7/X2

0 1 2 4 8 16 Scans0 12 24 48 96 192 e7/X2

Electron dose

FIG. 3. Mass loss due to electron bombardment for TMV parti-cles and native nucleosomes. Selected areas of particles were scanned,using the STEM, and scattering values for individual particles werecalculated after 1, 2, 4, 8, and 16 scans at 12 e-/A2. Data are presentedas mean + 1 SD. Both types of particle lose mass at approximatelythe same rate. After one scan, less than 5% of the mass has beenlost.

tions, the mean molecular mass is 205,000, which is also closeto that expected. Langmore and Wooley (25) obtained similarvalues for nucleosome mass from a STEM analysis of chains of

20

16i

12

8'

4

0

FIG. 2. Dark-field electron micrographs of native nucleosomes(a, d) and particles reconstituted from the four core histones (b, e)and-from histones H3 and H4 only (c, f). Frames a-c were taken witha conventional transmission electron microscope after uranyl acetatestaining. Frames d-f were obtained by using the STEM. The highlyscattering object in d is a portion of aTMV particle. The STEM im-ages show considerable variation in scattering power. Scale is 1000A.

a; l (i.r, I

6

21211

8

4'

(1

4

o ~

16112'

0'i0

Calculatedmass 205kilodaltonis

1 50 200 250 300

K100 200 300 400 500 600

150 200 250 3004 t t f4 8 12 16

Mass

Native

Kilodaltons

Four-histone

Arbitrary units

Kilodaltons

iHistoneequivalents

FIG. 4. Mass distribution of native nucleosomes and of particlesreconstituted from all four core histones or from histones H3 and H4only.

Proc. Nati. Acad. Sci. USA 77 (1980)

o0

Proc. Natl. Acad. Sci. USA 77 (1980) 4821

nucleosomes from HI-depleted chromatin fibers. Although theobserved mode and mean are in agreement with calculatevalues, the spread of the distribution (standard deviation is 12%of the mean) is large for a population of supposedly identicalparticles. In contrast, the standard deviation of mass values forthe TMV particles was only about 2.5% of the mean (see errorbars in Fig. 3). The large variability in the mass values fornucleosomes can be attributed to two major factors, both relatedto the small size of the nucleosome relative to the TMV particle.First, the smaller the mass of a particle, the fewer electrons willbe scattered; and because the scattering of an electron is arandom event, the mass recorded will vary. It can be calculated(27, 28) that for a particle of mass 2 X 105 daltons and a dose of10 e-/A2, this variability will be 9.3% of the mass, almost anorder of magnitude greater than the corresponding error forTMV particles.A second source of variability derives from the spot-searching

program that was used to detect particles in the original scansand determine their scattering intensity. For particles as smallas nucleosomes, the total scattering intensity calculated by theprogram varies with the assigned position of the center; thisproblem also causes a 5-10% error. Finally, it is calculated thatthe "noise" of the carbon substrate (27) gives a 6% error. Thecombined effects of these three major sources of variability,when added in quadrature, give an error of about 13%, whichcan account for the observed variation in nucleosome mass.The majority of the reconstituted particles (Fig. 4) have a

molecular mass equal to or greater than the mass of the nuc-leosome. This was expected for the particles reconstituted withfour histones, because these should in theory have an identicalcomposition to nucleosomes. In the case of particles reconsti-tuted with histones H3 and H4 only, it is clear that at least anoctamer of these histones becomes associated with nucleosomalDNA under our reconstitution conditions. An H3H4 tetramerwould have a molecular weight of about 146,000 but only about2% of the particles had molecular weights in this range. Theparticles reconstituted from 145-bp DNA and all four corehistones show a biphasic distribution similar to that observedfor native nucleosomes, although the main peak has a modalmolecular weight that is about 10% higher than that of thenative particles.The mass distributions of both types of reconstituted particle

have a significantly greater spread than the mass distributionof the native nucleosome. This suggests that the populations arenot homogeneous in mass, but contain more than one specieswith differing DNA-to-histone ratios. Crosslinking studies withH3H4-DNA complexes also indicate the presence of multipleDNA-histone species (21). In addition, the data suggest thatnucleosomal DNA is able to accept more than eight histones.In this respect, it is interesting to note that the second minorpeak on the heavy side of the distributions of native nucleosomeand four-histone-reconstituted particles have molecular weightsof about 300,000, which would correspond to the histoneequivalent of two octamers. These "heavy" particles appearas strongly scattering structures of the same width as the nu-cleosome and can be clearly distinguished from the lighterparticles in stained and unstained preparations. The possibilitythat some of these may correspond to the nucleosome-octamercomplex that appears as a stable species under certain recon-stitution conditions (41) is discussed below. The morphologyof these heavy particles excludes the possibility that they aresimply nucleosome dimers, and the narrow size distribution ofthe 145-bp input DNA (Fig. lc) makes it unlikely that they

DISCUSSIONMass determination by STEM analysis has two major advan-tages over conventional methods. First, individual particles are

sampled, so that heterogeneity within the sample populationcan be detected, and second, the method is independent of theconformation of the material. These features are especiallyvaluable when dealing with fragile multicomponent complexes.The accuracy of STEM-derived mass values can be seen fromthe close correspondence between the known and observedmodal value for the mass of the native nucleosome. Because ofthe spread of the data for nucleosome-sized particles in theseexperiments (Fig. 4), the full potential of STEM in distin-guishing subspecies has not yet been realized, but it can be saidwith confidence that the reconstituted populations are moreheterogeneous than the native nucleosomes and must containsome DNA-histone complexes with more than eight histonemolecules per 145 bp of DNA. In the future, it should be pos-sible to increase the resolution of the STEM method by opti-mizing the dose/damage effect and improving the data pro-

cessing system.Despite the spread of data, the results are quite clear con-

cerning the main question addressed. Under the reconstitutionconditions used, at least an octamer of histones H3 and H4 be-comes bound to nucleosomal DNA, forming a compact particlethat is morphologically similar to the nucleosome. This raisessome important questions concerning the type and uniquenessof the DNA-histone and histone-histone bonds of the nucleo-some. The native nucleosome contains a tetramer of H3 and H4,and there is strong evidence that these arginine-rich histonesalone are able to organize nucleosomal DNA in terms of foldingand related properties (14-20). A particle consisting ofnucleosomal DNA plus two each of histones H3 and H4 can beenvisaged as a nucleosome lacking the H2 histones, but re-

taining the other, apparently dominant DNA-histone andhistone-histone contacts. Such particles, although relativelyunstable in terms of folding (14, 18, 21) often retain the uranylstaining properties of native nucleosomes (15). However, it isclear from the present work and other results (18, 20, 21) thatnucleosomal DNA can accept another four arginine-rich his-tones, becoming more compact and stably folded in the process.

The two likely mechanisms for octamer formation are (i) H3H4pairs occupy the positions normally taken by H2AH2B pairsor (ii) a second H3H4 tetramer is added to the particle. Of thesetwo possibilities, the first would probably require the leastperturbation of the native DNA-histone contacts, although itwould require dissociation of the (H3H4)2 solution complex (31,44). However, in either case, changes in histone-histone contactmust occur. Thus, the highly conserved histone-histone contactswith the nucleosome core (reviewed in ref. 45) seem not to berequired for purely structural purposes.

The formation of a significant proportion of DNA-histonecomplexes with a molecular weight of 300,000 or greater (Fig.4) raises some interesting questions. It seems likely that manyof these particles, which appear in micrographs as stronglyscattering nucleosome-like structures, contain 145 bp of DNAplus the equivalent of two octamers of histones. (Other possi-bilities such as one octamer plus two DNA strands are unlikelyfor a number of reasons: for example, the formation of theseparticles is favored by conditions of histone excess.) Similarly,the occurrence of a small peak at 300,000 daltons in the nativenucleosome mass distribution suggests that nucleosome-oc-tamer complexes are present in this population also. Others havealso noted the ability of nucleosomes to accept, or form an

equilibrium with, excess histones (10, 41, 46, 47), and there is

represent "spacerless" dinucleosomes (42, 43), because thesestructures contain about 240 bp of DNA.

Cell Biology: Woodcock et al.

also some evidence for variation from the expected 1:1 his-tone-to-DNA ratio in vivo (48). It should be noted that struc-

4822 Cell Biology: Woodcock et al.

tures containing extra histones appear to require relatively highsalt (>0.4 M NaCI) for stability (41, 47), and our interpretationof the heavy particles seen in the present study as nucleo-some-octamer complexes needs further corroboration. How-ever, as pointed out by Stein (41), the affinity of nucleosomesfor excess octamers could play a major role in the functionalorganization of the nucleus.

We thank Richard Sterner for assistance with the amino acid anal-ysis. This work was supported by National Science Foundation GrantPCM 76-04846 and National Institutes of Health Grant GM23505 (to(C.L.F.W.) and by the U.S. Department of Energy (grant to J.S.W.).The Brookhaven STEM resource is supported by the National Institutesof Health Biotechnology Resources Branch (RR00715).

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Proc. Natt. Acad. Sci. USA 77 (1980)