higher-order structure of chromatin from resting … · characterizing the higher-order chromatin...

J. Cell Set. 62, 103-115 (1983) 103Printed in Great Britain © The Company of Biologists Limited 1983

HIGHER-ORDER STRUCTURE OF CHROMATIN

FROM RESTING CELLSII. HIGH-RESOLUTION COMPUTER ANALYSIS OF NATIVECHROMATIN FIBRES AND FREEZE-ETCHING OF NUCLEI FROMRAT LIVER CELLS

C. NICOLINI1' 6, B. CAVAZZA2' 6, V. T R E F I L E T T I 2 ' 6 ,F. PIOLI2 ' 6, F. BELTRAME3'6 , G. BRAMBILLA4'6,N. MARALDI5 AND E. PATRONE2 ' 6

1 Temple University, Philadelphia, U.SA., and Consiglio Nazionale delle Ricerche,Genova, Italy

C.N.R., Centro di Sludi Chimico Fisici di Macromolecole Sintetiche e Naturali,Genova, Italy

Istituto di Elettrotecnica, Sezione di Ingegneria Biofisica ed Elettronica, Universita diGenova, Italy

Istituto di Farmacologia, Universita di Genova, ItalyIstituto di Anatomia, Universita di Bologna, ItalyInterdisciplinary Group of Bios true lure. University of Genova, Italy

SUMMARY

Non-destructive electron microscopy of native chromatin from rat liver nuclei reveals that the30 nm fibre is formed of four 11 nm nucleofilaments, arranged in a coiled-coil (or rope-like) con-formation. At low ionic strength, native fibres show an alternating pattern of compact and unwoundregions. Freeze-etching experiments carried out on the same nuclei are compatible with the exis-tence of periodic attachments of the fibres to the nuclear envelope near the pores in a regular,drapery-like fashion.

For the first time, computer image analysis has been applied to electron micrographs of giantchromatin fibres and a few essential geometrical parameters characterizing the conformation of thehigher-order structures have been determined. No significant difference has been found betweencalf thymus and rat liver chromatin.

INTRODUCTION

The concept of regular folding of the nucleofilament into a solenoidal (Finch &Klug, 1976) or a two-order superhelical (Nicolini & Kendall, 1977) fibre,approximately 30 nm wide, has been challenged by recent electron-microscopic ob-servations on calf thymus chromatin obtained on a phospholipid monolayer (seeaccompanying paper), suggesting that the same fibre is formed of four interlinkednucleofilaments. The aim of this paper is to probe further the higher-order chromatinstructure in rat liver nuclei as revealed by a combination of 'non-destructive' electronmicroscopy resolved by a Plumbicon scanner and a computer to a level not obtainableby normal observation, and freeze-etching. The rigorous statistical analysis carried outon the computer-enhanced fibres led to the evaluation of a few relevant parameters,characterizing the higher-order chromatin organization compatible with previouslyreported observations on interphase nuclei. Furthermore, the branched multifibrillarstructure of chromatin is found to be similar in calf thymus and rat liver nuclei.

104 C. Nicolini and others

MATERIALS AND METHODS

Non-destructive electron microscopyHigh-resolution photographs of chromatin isolated from intact swollen nuclei of rat liver were

obtained by means of a recently introduced electron-microscopic technique involving aphospholipid monolayer (Cavazza et al. 1979). Nuclei were swollen in 1 mM-Tris-HCl (pH8) asdescribed (see accompanying paper). As previously pointed out, the chromatin images obtained arefree from distortion or artifacts due to fixation and/or staining, and present a limited, acceptablelevel of background noise. Rat liver nuclei were isolated using a perfusion method (Nicolini et al.1982).

Freeze-etchingIsolated rat liver nuclei (Widnell & Tata, 1964) were fixed in 2-5% glutaraldehyde in 0-1 M-

phosphate buffer (pH7-2) for 1 h and then rinsed in 015M-phosphate buffer. The nuclear pelletwas resuspended in 30% glycerol in distilled water for 30min, frozen in Freon and processed forcleaving and replication in a Balzers 360 M freeze-etch device.

Analytical image processingImages were obtained and analysed by means of the ACTA system built and installed at the

Biophysical and Electronic Engineering Section, Institute of Electrotechnics, University of Genova(Italy) (Beltrame et al. 1980). The electron microscope (EM) pictures were imaged through amacroepidiascope (final optical magnification = 24) on an European standard TV scanner target,equipped with a Plumbicon tube, which ensures a highly linear transfer function between lightintensity and electrical signal. Individual EM pictures, from chromatin on phospholipid monolayer,were obtained as an array of several thousand picture points. The final linear dimensions of eachapproximately square picture point, characteristic of the Plumbicon-equipped image analyser, weredetermined to be 0'6 or 0-9 nm, under our conditions of illumination and magnification. Individualtransmittance values for each picture point, also called a 'pixel', were acquired in a calibrated linearscale of 256 grey levels, where 0 and 256 correspond to 0 % ('black') and 100 % ('white') transmit-tance, respectively. The analogue video signal is fed through a fast A/D conversion group (8 bit,30 MHz, a monolithic integrated circuit) and each video frame can be stored in real time on amemory according to the format 512x512 pixels, 8 bit resolution per pixel. Images were transferredon a mass-memory device,-such as magnetic tape or disc, interfaced to a HP 21MX minicomputer(which controls the ACTA system); the same images were occasionally sent back from the magnetictape to the fast memory for further processing. The digital video signal originating from the memoryis sent through a look-up table system and D/A conversion to black/white and colour TV monitor.Specifically, the look-up tables, being under program control, could allow us, whenever criticallyneeded, to use pseudocolour techniques to enhance the images further. Geometric and densito-metric analysis were frequently carried out on selected regions of the fibre images, by means of aninteractive procedure involving a variable frame, program controlled for position and X—Y dimen-sions. Final images were obtained after subtraction and equalization of the background due tooptical and electronic noise, as determined in equivalent regions outside the fibre.

RESULTS

At variance with the unwinding process observed for giant chromatin fibres fromcalf thymus, which are almost invariably found in a compact form several hours afternuclear swelling (see accompanying paper), a pronounced branching of the 30nmstrand is observed at the earliest times (a few minutes) in the case of rat liverchromatin. The unwinding of the native fibre, localized in discrete regions definedby fragments of nuclear envelope material (Fig. 1A, B), leads to the subdivision of the

Structure of chromatin from resting cells

Fig. 1A-C

106 C. Ntcolini and others

Fig. ID

Structure of chromatin from resting cells 107

30 nm strand into subfibres of smaller size down to the nucleofilament (11 nm thick), asshown in Fig. 1A, B. On a typical 60 ̂ m long chromatin fibre, emerging intact followingswelling of the nucleus (Fig. 1A, B, C) we have carried out a statistical analysis to deter-mine the size distribution of the branched (Fig. 2A) and unbranched (Fig. 2B) nativesegments of the fibre lying between the fragments of the nuclear envelope material. Thesize of these fragments has also been determined (Fig. 2c), yielding a Gaussiandistribution around a mean value of 120 nm, with two additional peaks of decreasingamplitude at 240 and 360 nm. More than 90 % of the native fibre segments, which inabout 33 % of the cases do branch, appear to have similar lengths of about 1080 and480 nm, for the branched and compact filaments, respectively; only in a few instanceshave branched filaments of up to 7000 nm been observed (Fig. 2A). Micrococcalnuclease, under mild conditions, cuts the DNA strand in regions of the chromatin fibrenot covered by the nuclear membrane fragments and prompts a local unwinding of thenucleofilament as a consequence of the coiled conformation of the native fibre; com-puter enhancement (Fig. ID) of the image reported in Fig. 4c of the accompanyingpaper reveals that, upon nuclease digestion, the bifilament strands (about 18 nm wide)display, with respect to the native 30 nm fibre, a similar change in the average inter-distance among successive regions identifiable by their increased absorbance(chromatin bodies). Fig. 3 shows the computer-enhanced image of a typical chromatinfibre from rat liver nuclei. After background subtraction a representative frequencydistribution of grey level along longitudinal scans of the chromatin fibre reveals, des-pite a great deal of random noise, discrete levels of light absorbance, presumablyreflecting discontinuous distribution of biological material not readily apparent byvisual inspection of the electron micrographs. Actually, from the computer-enhancedimage, discrete distribution of chromatin bodies ('black' regions) appears evident (Figs3, 1 D) either in the native fibre or in any of the fibres resulting from spontaneous (Fig.1A, B) or induced (Fig. ID) unwinding. The discrete distribution of black regions,equally spaced by grey regions, can be measured readily in terms of grey level intensityas a function of the distance along each individual line (1 pixel wide) crossing the fibrelongitudinally (Fig. 3). Four parallel lines yield a highly reproducible pattern withpeaks (of Gaussian distribution) and hollows regularly alternating with an average totalpitch of about 32 nm (Fig. 4). The results of a detailed analysis of the digitized pixelsignal from the top to the bottom of the fibre (i.e. from the left to the right in thefrequency grey level distribution of Fig. 3) are shown in Fig. 4.

In order to resolve the complex periodic features represented by visible spikes in

Fig. 1. A. Selected fields of the same rat liver chromatin strand with typical examples ofthe spontaneous unwinding of the native fibre into two subfibres (arrow) occurring bet-ween fragments of nuclear lamina periodically attached to the chromatin fibre (p). Bran-ching of the 30 nm fibre in up to four filaments can also be seen. Bar (A-D) , 10 nm. B. Fibreregions displaying spontaneous unwinding into successively narrower subfibres down tothe 'nucleofilament'. C. A compact portion of the several ^m long liver chromatin fibreshowing periodically attached fragments of nuclear membrane, D. Computer-enhancedimage of calf thymus nuclease-digested fibre (fig. 4c of the accompanying paper) showingthe typical 'discrete' distribution of the darkest pixels for both the heavily and lightlyplatinum-shadowed regions.


the grey level distribution, and to distinguish random noise from actual trend in thesignal, the following objective criteria were devised and utilized, (a) Pixel transmit-tance of a scan along a longitudinal line crossing the 'dark' side of the fibre ranges (Fig.3) between 35 % (grey level 90) and 70% (grey level 170); two clear peaks emergefrom the frequency distribution named I (70-110 grey level) and III (130-170), with

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Fig. 2. Statistics on a typical native fibre, about 60/xm long (Fig. 1). A. Number ofbranched segments with a given length between successive pieces of nuclear material, asa function of the length, B. AS above, but for unbranched filaments, c. Number of nuclearfragments as a function of their size measured in the direction of the fibre axis.


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Fig. 3. Left: a computer-enhanced image of a 30nm native fibre. Right: the grey (trans-mittance) level for each of the 423 pixels sequentially taken along a line crossing the samefibre longitudinally from bottom to top.

a third intermediate level (111-129) named II (Fig. 4B). (b) If we assign pixeltransmittance to either one of the three classes, the digitized signal of Fig. 3 versusdistance from the top of the fibre assumes the discontinuous form shown in Fig. 4A,where the discrete structure of the fibre can be visualized more readily and the sizeof alternating regions of type I, II and III transmittance can be determined (Fig. 4c).Considering the relatively small statistics and the 0-9 nm resolution of these measure-ments, under our operating conditions, we have plotted the dimension at 2-7 nmintervals to reduce random noise, (c) Strikingly, the size distribution of the denserbodies (type I transmittance) appears bimodal (Fig. 4c), revealing that they havedimensions equal to either 6-8nm (range 5-4—8-1; type A) or 12-1 nm (range10-8—13-5; type B). On the contrary, the light type III regions (spacer III) have aunimodal distribution around a mean value of about 8-1 nm, while the intermediatetype II transmittance regions (and those of type III connecting successive type I


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Fig. 4. Statistics on the computer-enhanced fibre 30 nm wide, shown in Fig. 3. A. Trans-mittance signal versus distance from the top of the fibre, reduced from the row data (Fig.3) after grouping the pixels transmittance into three separate classes, as defined in B. B.Frequency (in arbitrary units) of pixels with a given transmittance value as a function oftransmittance, expressed in grey levels (see text for further explanation). C. Frequency ofbodies with given length, for bodies with grey levels I, II, and III as obtained in A and B.D. Frequency distribution of fibre pitch, as discussed in the text and apparent from A.

regions of alternating size) are only about 2-7nm long (spacer II). (d) A periodicpattern can then be identified in Fig. 4A, with repeating units consisting of spacer III- I (A or B, dark bodies) - spacer II - I (A or B) - spacer III, the total length of whichfrom centre to centre of spacers III has been determined and plotted in Fig. 4D. Withone exception, possibly due to our limited resolution or noise, these distances have aGaussian distribution around a mean value of about 32 nm (range 30-3—33*8), whichwe call the apparent pitch of the 30 nm native fibre.

This apparent pitch progressively decreases whenever the native fibre branches(Fig. 1A, B), the spacer III connecting type I dark bodies diminishing from 8-1 nm(tetrafilament or 30 nm fibre) to 6-3 nm (trifilament of 24nm fibre), to 4-5 nm (bifila-ment or 18nm fibre), to 2-7 nm (mononucleofilament or 11 nm fibre). At the sametime the size of the type I dark bodies remains constant at about 7-11 nm. A similarshrinkage (decreased pitch) occurs whenever the apparent diameter of the native fibredecreases, with the size of the dark bodies being invariant within any given fibrebefore and after its branching (see Fig. 1A, B). A rope-like structure for the native


fibre may be inferred from the spontaneous unfolding of up to four nucleofilaments(Fig. 1A, B), but also from the grey level distribution (Fig. 3) in the computer-enhanced fibre image, where grey regions, corresponding to more densely platinum-coated molecular domains (dark bodies), alternate with white regions and are out ofphase by a constant angle of 45 °, independently of fibre position, with respect to theplatinum source. Hence, this inclination probably results from the twisted path takenby the nucleofilaments.

The unfolding process occurring within the giant chromatin fragments does notpropagate along their entire length, but remains confined to narrow regions limitedby fixed points (Fig. 1A, B), often identifiable as irregular pieces of a proteinaceouslamina periodically attached to the fibre. Curiously, the size of these fragments (Fig.2c) is identical to (or a multiple of) the diameter of the pores of the nuclear membranefrom the same preparation of rat liver nuclei (Fig. 5). In the case of nuclei isolatedfrom calf thymus (see accompanying paper, Fig. 2F), these highly irregular pieces canbe fitted together to yield a continuum, suggesting that they probably come from thesame cell component (i.e. the nuclear envelope) to which chromatin fibres are linked,and which is broken during the swelling of the nucleus. Freeze-fracture micrographsgive further details of the attachment of the native fibre to the nuclear membrane (Fig.5), which seem to support the above suggestions. The freeze-etching image (Fig. 5)appears consistent with the 26-30 nm fibres being attached to the nuclear envelope,as previously apparent in the electron micrographs of thin sections of chicken erythro-cyte nuclei (Olins & Olins, 1979), and by high-resolution image analysis of Feulgen-stained tumour cells (Nicolini, 1979) and tissue-sections (Kendall et al. 1980). Thetotal length of each fibre attached to the nuclear envelope is about 450 nm, which isalso the average distance along the same fibre between the pieces of nuclear membraneas shown by the phospholipid monolayer technique (Figs 1,2). Even if Fig. 5 does

Fig. 5. Freeze-etching electron microscopy of liver nuclei. The fracture shows apparentdrapery-like chromatin fibres attached to the nuclear membrane near the pores (arrows).Bar, 200 nm.


Fig. 6. Computer-enhanced image of HI-depleted calf thymus chromatin (fig. 4A of theaccompanying paper) showing that the unwinding process of a 30 nm fibre into twosubfibres, each 1-6 times smaller, is symmetrical. Bar, 100 nm.

not prove definitely that the fibres are actually running from pore to pore (of constant120 nm size), visual observation seems consistent with such an interpretation. Thiswould also explain the periodic appearance along the native fibre at spacings of 120 nm


(or multiple of it) of pieces of nuclear lamina (Figs 1,2). Work is in progress to clarifythis correlation, which could be merely coincidental.

DISCUSSION

The analytical data presented here on the unwinding process of native chromatinfibres originating from rat liver nuclei seem to suggest a higher-order supercoiling, atodds with previous solenoid-like models for the structure of native chromatin (Finch6 Klug, 1976; Nicolini & Kendall, 1977). The splitting of a 30 nm fibre into subfibresreveals a strikingly positive correlation between changes in fibre width and changesin fibre pitch, with a reproducible invariance in size for the dark bodies (probablyindividual nucleosomes, or closely spaced pairs of them, resolvable by computerenhancement), periodically distributed along each fibre and separated by a distanceinversely related to fibre width. It therefore appears that the basic higher-orderchromatin structure consists of a regular folding of four nucleofilaments helicallyinterwound (rope-like) to yield the frequently reported 30 nm fibre. A repeating unit,containing nucleosome-like bodies separated by narrow spacers, appears to occuralong the fibre with a pitch of 32nm. A fibril helically interwound around the fibreaxis with 32 nm pitch and radius 15 nm would have a contour length of about 70 nm(contour length = 2 xr Vr2+(/z2/4n^), where r and h are the radius and the pitch of thefibre, respectively), thereby yielding a packing ratio of 2-2. Upon branching in twosimilar fibrils, the apparent distance among the pieces of nuclear membrane shouldincrease accordingly. Indeed, 90 % of these fibre segments are about 480 and 1080 nmlong in the folded and unfolded form, respectively. In the 11 nm fibre the size of thedark bodies is about 9-5 nm, roughly corresponding to the diameter of the nucleosomedisk (Pardon et al. 1977) oriented parallel to the nucleofilament axis, yielding aboutone nucleosome every 10nm, exactly as found by neutron scattering studies (Suau,Bradbury & Baldwin, 1978). Interestingly, in the computer-enhanced 30nm fibresfrom calf thymus the smallest size of the dark bodies is about 60 nm; this suggests, atvariance with other interpretations (McGhee, Rau, Charney & Felsenfeld, 1980), thatin going from the nucleofilament to the helical structure the orientation of the nucleo-somes changes and their flat surfaces becoming perpendicular to the fibre axis. Thistransition has been monitored by neutron scattering as a function of the ionic strength(Suau et al. 1978), the results being compatible with the crude model shown in fig.7 of the accompanying paper, and with the suggested unwinding mechanism.

Some relevant differences between rat liver and calf thymus chromatin, with res-pect to the decay mode of the 30 nm fibre to subfibres, are apparent from the presentdata. The unwinding process observed at low ionic strength is asymmetrical in thecase of calf thymus fibres (see accompanying paper), while the arc-to-chord ratio issmaller for rat liver chromatin. In this latter case the configuration of the loops is closeto that of H1 -depleted chromatin from calf thymus (Fig. 6), behaviour very probablyrelated to the presence of unwound regions within the fibres at short times afternuclear swelling. It is possible that such factors as a different degree of chemicalmodification of the histone octamer and/or the HI fraction could account for different


unwinding capabilities as well as for the variable symmetry in different cell type.Experiments on nuclease-degraded chromatin (Finch & Klug, 1976; Renz, Nehls &

Hozier, 1978; Thoma, Koller & Klug, 1979) show a compaction of the poly-nucleosomal filament with increasing salt concentration, a quite general effect that iswell-interpreted by the polyelectrolyte theory (Belmont & Nicolini, 1981). Anyway,granted that this phenomenon could be interpreted in terms of a transition to asolenoidal helix (Thoma et al. 1979), native chromatin shows, even at low ionicstrength, highly packed regions alternating with branched domains, i.e. branchingoccurs only within discrete regions of the strand. Whatever the exact structure of thetetrafilament rope is (this requires further investigation) our above findings are com-patible with the existence of chromatin domains maintaining the supercoil and negativeDNAsuperhelical turns (Cook & Brazel, 1976; Nicolini & Kendall, 1977), and of a classof strongly DNA-bound proteins probably involved in intrastrand cross-linkage(Benyajati & Worcel, 1976). Despite some inherent difficulties, our findings on thefraction of total DNA present in the branched regions and on a maximum size for theunwound filaments are strikingly compatible with the reported existence of a 40 % limitto digestion by a restriction enzyme (Igo-Kemenes & Zachau, 1978) and of a maximummolecular weight of 5 X 107 for nuclease-soluble segments. While our suggestions onthe higher-order structure are compatible with the alternating pattern of looping andextended fibre segments reported for spread human chromatin (Yunis & Bahr, 1979),those on the existence of a spatial organization in terms of 30 nm fibres attached as adrapery to the nuclear envelope are consistent with previous electron micrographs(Comings, 1968; Olins & Olins, 1979) and with calorimetric, viscoelastometric andspectropolarimetric studies on the same type of liver nuclei (Nicolinie/ al. 1982,1983).

The presence along the giant (60 fw\ long, 1010Mr) chromatin fibres of laminar frag-ments, having dimensions centred around 120, 240, 360 nm and of an upper limit in thefibre length between pieces of nuclear membrane (Fig. 2), provide an indirect justifica-tion for the recently observed (Nicolinie* al. unpublished data) clustering of pores andfor an upper limit in the interdistance among pores within each cluster. Furthermore,the recurrent value (480 nm; Figs 1, 2) of the length of chromatin segments delimited byfragments of the nuclear lamina is compatible with the existence of a repeating unit ofabout 160 nm in interpore distance (Nicolini et al. unpublished data). Speculations onthe possible implications of such findings are presented elsewhere (Nicolini, 1983).

An indication of the possible generality of the findings reported here for rat livernuclei is the existence in native chromatin from calf thymus of a similar multifibrillarstructure. Further, the detailed statistical analysis of chromatin from calf thymusfollowing computer enhancement (Fig. 6 and others not shown) yield results compar-able with those from rat liver with respect to the fibre pitch, size and distribution of thedark bodies. Also, the periodicity detected along compact strands, equal to about550 nm (or multiple of this value) in the case of calf thymus, is close to 480 nm, the valuedetermined for rat liver chromatin.

This work was partially supported by a grant (81.01304.96) from Finalized Project 'Control ofTumour Growth', Consiglio Nazionale delle Ricerche, Italy.


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COMINGS, D. E. (1968). The rationale for an ordered arrangement of chromatin in interphasenucleus. Am. J. hum. Genet. 20, 440-460.

COOK, P. & BRAZEL, I. (1976). Conformational constraints in nuclear DNA. J . Cell Sci. 22,287-302.

FINCH, J. T. & KLUG, A. (1976). Solenoidal model for superstructure in chromatin. Proc. natn.Acad. Sci. U.SA. 73, 1897-1901.

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B. M. (1977). The structure of chromatin core particles in solution. Nucl. Acids Res. 4,3199-3214.

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(Received 17 August 1982-Accepted 3 February 1983)

higher-order structure of chromatin from resting … · characterizing the higher-order chromatin...

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