1978 Nucleic Acids Research 5 Number 5 May Volume Voue5Nme a 98NcecAisRsac

Ught scattering measurements supporting helical structures for chromatin in solution A.M. Campbell, 2* R.l. Cotter and 2J.F. Pardon

Department of Biochemistry, University of Glasgow, Glasgow G12 8QQ, 2Searle Research Laboratories, Lane End Road, High Wycombe, Bucks, HP12 4HL, UK Received 1 February 1978


Laser light scattering measurements have been made on a series of polynucleosomes containing from 50 to 150 nucleosomes. Radii of gyration have been determined as a function of polynucleosome length for different ionic strength solutions. The results suggest that at low ionic strength the chromatin adopts a loosely helical structure rather than a random coil. The helix becomes more regular on increasing the ionic strength, the dimensions resembling those proposed by Finch and Klug for their solenoid model.

INTRODUCTION The higher ordered structure of chromatin remains largely undetermined despite recent progress in determining the structure of the subunit core particle (1,2,3). In the electron microscope chromatin appears as a thread with the diameter varying depending upon the conditions used to prepare the sample, especially the ionic strength. At low ionic strength the nucleofilament (4,5,6) with diameter about lOQA almost certainly consists of a single chain of nucleosomes. It has not been established how the core particles are assem0~~~~~~ bled in the nucleofilament. Their dimensions (width x llOA, height 55A) would permit either an edge-to-edge assembly or for them to stack directly on top of each other as occurs with isolated subunits (3). When divalent ions are added to the nucleofilaments their diameter increases to 250-300A (4,5,6). Finch and Klug (6) proposed a solenoid model with diameter 300A and pitch 110 to explain both electron microscope data (4,5,6,7) and the 'llOA' reflection obtained in X-ray diffraction studies from chromatin fibres and gels (8,9). Carpenter et al. (10) have shown from neutron scattering studies both that this reflection is off meridional, as predicted for the solenoid model, and that it occurs in the absence of histone Hl at high chromatin concentrations. Another model was proposed by Renz et al. (11) who interpreted their data in terms of a superbead model with each superbead containing from six to ten Z

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Nucleic Acids Research subunits. There have been no reports that individual superbeads can be isolated intact. We have used laser light scattering to study chains of nucleosomes (polynucleosomes) containing between 50 and 150 subunits. Within this size range of chromatin fragment the technique yields both molecular weight and radius of gyration. The polynucleosomes were isolated using Staphylococcal nuclease digestion to minimise shear forces and were fractionated into discrete lengths using zonal centrifugation. Measurements were made over a range of ionic strengths where the particles remained soluble and for concentrations less than 200 ig/ml where association between individual polynucleosomes was not detected. EXPERIMENTAL METHODS

Chromatin was released from chicken erythrocyte nuclei isolated according to Shaw et al. (12) into 40mM NaCl, lOmM Tris-HCl pH 7.5 and digested with micrococcal nuclease (30 units/ml chromatin from 20 x 108 nuclei) for 20 min at 40C with shaking in the presence of 1mM CaCl2. The digest was fractionated on a 10-45% w/v sucrose gradient for 5 h at 42,000 rpm in a Til4 zonal rotor. DNA from the chromatin fractions was extracted with pronase and phenol (12) and sized on 1% agarose slab gels alongside restriction enzyme Hin III fragments of x DNA (Miles) (13) stained with ethidium bromide. Light scattering measurements were made as before (14) with minor modifications. Filtration was omitted since the chromatin binds both to cellulose acetate and cellulose nitrate. Instead, the solutions were clarified by centrifugation for 10 min in sterile tubes at 10,000 rpm using a MSE 18 centrifuge. The material from the middle of the tube was withdrawn using a sterile syringe. Data from all the samples were analysed using conventional Zimm plots (15) and angular extrapolation was from below a value of p(e) 1 of 1.3 (16). An optical density unit at 258 nm has been assumed throughout to arise from 100 1'g chromatin. RESULTS AND DISCUSSION

Figure 1 shows a Zinmn plot (15) of a typical polynucleosome preparation from which the weight average molecular weight (My) and radius of gyration are derived in the usual way. The lack of variation of Mw for different concentrations of a given polynucleosome fraction shows that there is no selfassociation between molecules (14). Each molecular weight was invariant over the ionic strength range studied. DNA extracted from polynucleosomes and run on agarose gels (figure 2) indicates the spread of molecular weights within 1572

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sin2 f + 101 xc (c in mg/ml)

Figure 1: Low angle Zimm plot showing light scattering from a sample containing 155 nucleosomes per polynucleosome. In 40mM NaCl, lOmM Tris HC1, 0.7mM EDTA, pH 7.5 each sample; the gels also provide a cross-check for the Mw obtained from light scattering (Table I). The agreement is less good in the larger size range because of the non-linearity and steepness of the calibration slope and the greater influence of bowing across the slab. However, the agreement between the data is sufficiently good to show that the chromatin multimers in these size and ionic strength ranges do not associate to form dimers or larger units. The errors involved in the determination of MW on our instrument have been discussed previously (17) and amount to 5%. Rg errors are of the same order but arise from slope estimation only, and are minimised by increasing the number of data points. The variation obtained for the radius of gyration of polynucleosomes as a function of ionic strength is shown in figure 3. All three sets of data are linear as would be expected for a rigid rod-like structure. These data would not support an entirely random coil type of structure for any of the ionic strength conditions studied since with such structures the radius of gyration is proportional to the square root of the molecular weight (18). Since the radi;us of gyration has been determined both for the core particle (19) and the intact nucleosome (20) it is possible to calculate plots of the variation in the radius of gyration as a function of molecular weight for different types of structure for the polynucleosome. We have used a value of 41A for the radius of gyration of the individual subunit (19) and have made use of the theorem of parallel axes to calculate distributions for a variety of models. We have made the distance between the centres of the subunits llOA. This is the approximate distance obtained from radius of gyration measurements made on small oligomers containing two, three, four (21) and six nucleosomes 1573

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110 48

32 22

12 11





Figure 2: Sizing of polynucleosome fractions on 1% agarose slab gels: (a) Calibration Hin III digest of x DNA. Numbers on left denote number of nucleosomes equivalent to DNA molecular weights taken from Murray and Murray (13). Polynucleosomes containing (b) 75, (c) 97 and (d) 110 nucleosomes.

(22). 11oA is also the value obtained from a mass per unit length measurement on more extended chromatin (27). Previous studies indicate that at low ionic strength the chromatin fibre or nucleofilament (4,5,6) has a diameter of about 110. The relationship between radius of gyration and number of nucleosomes for a linearassembly of subunits spaced 110A apart is shown in figure 3A. Such a model is more extended than the data from polynucleosomes in lOrM Tris 0.7mM EDTA pH 7.5 would indicate. The smallest distance between centres for particles stacking directly on top of each other is about 55A (1,2,3), however a linear stack of 1574

Nucleic Acids Research Sizing of Polynucleosomes


No. of Nucleosomes From light scattering


From DNA gels

24 ± 1


















4.5 7.5




1 80 ± 8




Rg )


75 0


1 00 mr

Fi gure



Nmuceosom .s/Po




(Rg)) ard size of polystrength conditionss. Data derived from

relationship between the radius-of gyration

nucleosome under different ionic light scattering measurements:* lOnM Tris, 0.7mM EDTA, pH 4citMNaCl, lOnM Tris-HCl, * 60nM NaCl, lOmM Tris-HCl,

7.5 0.7mM EDTA, pH 7.5 0.7mM EDTA, pH 7.5

(b) Calculations for linear assemblies of nucleosomes and fol helices in which nucleosomes were placed on a belix with radius 1OOA to give an outer diameter of approximately 300A. Line A represents a linear array of nucleosomes spaced 11 9apart; B represents a linear array of nucl osomes spaced 55A apart; C represents a helix with pitch 210 , six nucleosomes/turn; D represents a helix with pitch 150 , six nucleosomes/turn; E represents a helix with pitch 120 , six nucleosomes/turn. 1575

Nucleic Acids Research particles with this centre-to-centre distance is again too extended to account for the experimental data (figure 3B). At higher ionic strengths the chromatin fibre has a diameter of 250-300A (4,5,6,7). Accordingly we have investigated helical models with an outer diameter of 300A and with a spacing between nucleosomes of about llOA. Such models are able to account for the experimental data (figure 3C-E). For the highest ionic strength which we have used (6OmM NaCl, lOmM Tris-HCl, 0.7mM EDTA pH 7.5) the best agreement between the experimentally determined radii of gyration and the theoretical curve is for a helix in which the individual nucleosomes are centred on a radius of 1001 and pitch 120A. Such a helix has both outer diameter and pitch very similar to those described by Finch and Klug (6) and Carpenter et al. (10). The experimental radii of gyration obtained from solutions at lower ionic strength can also be accounted for by helices with outer diameters about 300. The lowest ionic strength studied here (l1nM Tris, 0.7mM EDTA pH 7.5) provides radii of gyration that are equivalent to a helix with radius lOOA and pitch 210A. The intermediate ionic strength (40mM NaCl, lOmM Tris-HCl 0.7mM EDTA pH 7.5) data can be explained by a helix of radius 100A and pitch 150k. These calculations therefore show that the data can be explained by a helix in which the radius remains constant but in which the pitch is reduced as the ionic strength increases. At very low ionic strength there is a significant gap between gyres whilst at higher ionic strength the helix becomes almost, if not entirely, coil-bound (i.e. adjacent gyres are in close proximity). X-ray diffraction studies of gels of chromatin show a series of diffraction maxima. Some of these maxima can be obtained from isolated subunits and are therefore attributed to the Fourier Transform of the core particle (23,24). The maximum at 11OA was originally thought to arise from a regular supercoil of DNA (25), but more recent data suggests that it is produced from the pitch of a helix formed from nucleosomes (6,24). This maximum is not observed in dilute solutions of chromatin at low ionic strength (9,26,27) but is observed either in more concentrated gels (9) or less concentrated gels (24,26) in the presenge of Mg2+ ions. Neutron scattering studies have indicated that this spacing is concentration dependent at high chromatin concentrations, becoming larger for less concentrated gels of chromatin (28). One reason why the low angle maximum has not been observed in X-ray studies of less concentrated chromatin might be that a helical structure is present but that it is less well ordered, i.e. the pitch and diameter might vary. This might lead to a broad maximum which is not resolved against the 1576

Nucleic Acids Research strong intensity of the central maximum from the chromatin subunit. We have examined the variation of radius of gyration with polynucleosome length for a series of helical models with different radii and pitch. The experimental variation in radius of gyration at the lowest ionic strength can be accounted for by helical models with outer diameter 400A, pitch 310A, and with outer diameter 500A, pitch 4401 (figure 4A). Similarly the experimental points at the intermediate ionic strength can be accounted for by a model with outer diameter 400K and pitch 2201. As the diameter of the model increases so it becomes increasingly difficult to account for the data obtained at the higher ionic strengths. For i nstance it was not possi bl e to fi t the data at the intermediate or largest ionic strength with models having diameters greater than 400A (figure 4). We therefore conclude that for helical models the data at the higher ionic strengths can only be explained by a limited range of structures with outer diameters smaller than 400K. A good fit was obtained using parameters










so Nkwbr of




Figure 4: Calculatio s for models in which nucleosomes were placed oR a helix viwith radius 200A to give an outer diameter of approximately 500A Data points are as in figure 3. Line A represents a helix with pitch 440A 13 nucleosomes/turn of helix; Curve B represents a helix with pitch 280 , 12 beads/turn; Curve C represents a helix with pitch 240 , 12 beads/turn; Dashed lines represent linear regression fits to the data points. 1577

Nucleic Acids Research similar to those described by Finch and Klug for their solenoid model (6). For the lowest ionic strength solutions a range of models was able to account for the experimental data with the outer diameter varying from 300 to 500. For larger diameters the calculated variation becomes non linear. With the models that give good agreement the pitch varied from 2101 to 440A. Linear, non helical structures did not give good agreement. It is therefore likely that at low ionic strengths a loose helical structure forms with the limits imposed on the diameter and pitch defined above. This structure possesses a degree of flexibility not present at the higher ionic strengths where the more tightly bound helix is stabilised by forces between adjacent gyres of nucleosomes which are in close proximity. Shaw and Schmitz (29) also propose a helical superstructure or flexible coil to explain their hydrodynamic data obtained from low ionic strength chromatin solutions. Other models have been examined, especially the superbead concept described by Renz et al. (11). The radius of gyration for a superbead cona taining six nucleosomes and having an outer diameter of 300A is about llOA. The calculated variation of radius of gyration with polynucleosome length for a string of contiguous superbeads is shown in figure 5A. Clearly the calculated distribution represents a more extended structure than is present even at the lowest ionic strength. A better agreement is obtained for the lowest ionic strength data with contiguous superbeads each containing 9 nucleosomes and having an outer diameter of 300A (figure 5B) or alternatively for superbeads each containing 8 nucleosomes and with an outer diameter of 250A. However 250-300A diameter threads are not observed at such low ionic strengths and the superbead model was proposed for conditions more closely resembling those in the cell (11). Thus of the current models for higher order structure in chromatin, helical structures are most able to explain these data. We suggest that at low ionic strength the helix is relatively flexible but not random and that the chromatin adopts a loosely helical structure with pitch varying in the approximate range 210-440A and with its outer diameter in the range 300-500A. In the absence of stabilizing forces between gyres the helical nature of the structure at low ionic strength may not be entirely preserved by the procedures used in preparing materials for electron microscopy and the lOOA nucleofilament deposited on the grid from solutions at low ionic strength occurs as a result of stretching out of the loose helical structure. At the higher ionic strengths the stabilizing forces are more apparent and the 300A diameter is preserved in solution and on the electron microscope grid. Superbead 0


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100 Rg (nm)






Number of NucI.osomes/Polynucl.osome

Figure 5: Calculations for superbead models: data points are as shown in figure 3. Line A - calculated line for a string of beads diameter 300A, distance between centres 30O,each bead containing six nucleosomes; Line B - as for line A but each superbead contains 9 nucleosomes; Curve C - as for line A but each superbead contains 13 nucleosomes; Curve D - as for line A but each superbead contains 16 nucleosomes.

models would appear to be unlikely unless the structure is significantly different under more physiological conditions. ACKNOWLEDGEMENTS We wish to thank Dr. Brian Richards and Dr. David Lilley for their helpful comnents on the manuscript, and to acknowledge the technical assistance given by Inger Jonrup, John Hobbs and Ton Carr. *To whom requests for reprints should be addressed


Richards, B.M., Pardon, J.F., Lilley, D.M.J., Cotter, R.I. and Wooley, J.C. (1977) Cell Biol. Int. Reps. 1, 107-116 1579

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4. 5. 6. 7. 8. 9.

10. 11.

12. 13. 14. 15. 16.

17. 18. 19. 20.

21. 22. 23. 24. 25. 26. 27. 28.



Pardon, J.F., Worcester, D.L., Wooley, J.C., Cotter, R.I., Lilley, D.M.J. and Richards, B.M. (1977) Nucleic Acids Res. 4, 3199-3214 Finch, J.T., Lutter, L.C., Rhodes, D., Brown, R.S., Rushton, B., Levitt, M. and Klug, A. (1977) Nature 269, 29-36 Ris, H. and Kubai, D.F. (1970) Ann. Rev. Genet. 4, 263-294 Pooley, A.S., Pardon, J.F. and Richards, B.M. (1974) J. Mol. Biol. 85, 533-549 Finch, J.T. and Klug, A. (1976) Proc. Natl. Acad. Sci. USA 73, 1897-1901 Davies, H.G. and Haynes, M.E. (1975) J. Cell Sci. 17, 263-285 Luzzati, V. and Nicolaieff, A. (1963) J. Mol. Biol. 7, 142-163 Pardon, J.F., Richards, B.M. and Cotter, R.I. (1974) Cold Spring Harbor Symp. Quant. Biol. 38, 75-81 Carpenter, B.G., Baldwin, J.P., Bradbury, E.M. and Ibel, K. (1976) Nucleic Acids Res. 3, 1739-1746 Renz, M., Nehls, P. and Hozier, J. (1977) Proc. Natl. Acad. Sci.74, 18791883 Shaw, B.R., Herman, T.M., Kovacic, R.T., Beaudreau, G.S. and Van Holde, K.E. (1976) Proc. Natl. Acad. Sci. USA 73, 505-509 Murray, K. and Murray, N.E. (1975) J. Mol. Biol. 98, 551-564 Campbell, A.M. and Cotter, R.I. (1977) Nucleic Acids Res. 4, 3877-3887 Campbell, A.M. (1976) Biochem. J. 155, 101-105 Schmid, C.W., Rinehart, F.P. and Hearst, J.E. (1971) Biopolymers 10, 883893 Campbell, A.M. and Cotter, R.I. (1976) FEBS Lett. 70, 209-211 Tanford, C. (1966) Physical Chemistry of Macromolecules, p. 306, J. Wiley & Sons Pardon, J.F., Worcester, D.L., Wooley, J.C., Tatchell, K., Van Holde, K.E. and Richards, B.M. (1975) Nucleic Acids Res. 2, 2163-2176 Hjelm, R.P., Kneale, G.G., Suau, P., Baldwin, J.P. and Bradbury, E.M. (1977) Cell 10, 139-151 Pardon, J.F., Cotter, R.I., Lilley, D.M.J., Worcester, D.L., Campbell, A.M., Wooley, J.C. and Richards, B.M. (1978) Cold Spring Harbor Symp. Quant. Biol. 42, In Press Cotter, R.I., Worcester, D.L., Lilley, D.M.J. and Pardon, J.F., In Preparation Richards, B.M., Cotter, R.I., Lilley, D.M.J., Pardon, J.F., Wooley, J.C. and Worcester, D.L. (1976) Current Chromosome Research, Ed. Jones, K. and Brandham, P.E., p. 7-16, Elsevier/North Holland Sperling, L. and Klug, A. (1977) J. Mol. Biol. 112, 253-263 Pardon, J.F. and Wilkins, M.H.F. (1972) J. Mol. Biol. 68, 115-124 Garrett, R.A. (1971) Biochim. Biophys. Acta 246, 553-560 Sperling, L. and Tardieu, A. (1976) FEBS Lett. 64, 89-91 Baldwin, J.P., Boseley, P.G. and Bradbury, E.M. (1975) Nature 253, 245249 Shaw, B.R. and Schmitz, K.S. (1976) Biochem. Biophys. Res. Corn. 73, 224232

Light scattering measurements supporting helical structures for chromatin in solution.

1978 Nucleic Acids Research 5 Number 5 May Volume Voue5Nme a 98NcecAisRsac Ught scattering measurements supporting helical structures for chromatin i...
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