Structural repeating units in chromatin

Printed in Sweden Copyright 0 1976 by Academic Press. Inc. All rights of reproduction in any form resewed

Experimental Cell Research 97 (1976) 11l-l 19

STRUCTURAL

REPEATING

II. Their Isolation

UNITS

IN CHROMATIN

and Partial Characterization

C. L. F. WOODCOCK, H. E. SWEETMAN and L.-L. FRADO Department

of Zoology,

University

of Massachusetts,

Amherst,

MA 01002, USA

SUMMARY Chromatin fragments, prepared by brief digestion with DNase II or micrococcal nuclease, separated into a family of bands on sucrose gradients. The slowest migratin band with an SZRwvalue of about 11 contained spherical particles, with an average diameter of 75 1, similar to those comprising chromatin fibers. The second band (about 15S) contained mostly dimers of 75 A particles, the third trimers, etc. DNA extracted from the bands consisted of linear molecules with modal lengths of 440 A for the monomer, 1 1008, for the dimers, and 1700 8, for the trimers. These results are in accord with the rapidly accumulating evidence that chromatin is organized into discrete DNA protein units. Analysis of our data also suggests that about 640 8, of DNA is contained within each subunit, of which about 200 A is readily accessible to endonuclease attack.

In the previous paper [l], structural data were presented which suggest that the eukaryotic chromatin fiber consists of interconnected repeating units of DNA-protein. If this interpretation is correct then it should be possible to attack the DNasesensitive interconnections and liberate individual units. In this communication, we describe the results of such experiments, the ultrastructure of the isolated particles, and measurements of their DNA and histone content. While this work was in progress, a number of papers supporting a subunit structure for chromatin appeared [2-71. While these authors have all favored a basic DNA-histone repeating unit, estimates of the amount of DNA contained within each unit have varied from 400 A [ 141to 710 A [ 151.The relationship between the spherical subunits and distinct ‘connecting strands’ seen in electron micrographs is also unE-751817

clear. The data presented here indicate the probable reason for such differences in ,subunit size, and also shed some light on the nature of the ‘connecting strands’. MATERIALS

AND METHODS

Chicken erythrocyte nuclei were isolated as previously described [l] and pelleted in 0.15 M NaCl pH 8.0. For digestion with micrococcal nuclease, nuclei containing 400-600 OD,, units of chromatin were swollen bv resusoension in 1 mM CaCI, DH 7.0. and enzyme (Worthington, USA) added at fh’e rate of 10 Kunitz U per 1.0 OD,,, U of chromatin. After 30 set to 2 min at 37”C, the reaction was stopped by adding 2 mM Na,EDTA. This procedure is similar to that used by No11[6]. For DNase II digestion, the reaction mixture contained 0.005 M sodium nhosohate buffer pH 5.5 or 6.0, and was stopped by raising the pH to 8.0, and cooling rapidly. Enzyme concentration and incubation times were the same for both nucleases. To release the chromatin fragments, nuclei were broken by mild shearing (Virtis homogenizer, 20 on power scale) for 2 min. and the laraer fraements Deljeted by centrifugation at 15000 rpm for 35 min (Sorwall, RCZ-B, SS34 rotor). 3-6 ODZm units of supernatant were then layered onto 5 to 20% linear sucrose gradients adjusted to pH 7.0, and centrifuged at 35 000 Exprl Cell Res 97 (1975)

I 12

Woodcock, Sweetman and Frado

5

IO

IJ

20

a

5

I5

20

25

Fig. I. Abscissa: fraction no.; ordinate:

OD,,,. Sucrose gradient profiles of micrococcal nucleasedigested chicken erythrocyte chromatin. The top of the gradients are at left. The prominent main peak contained monomer particles. Digestion time was 2 min.

2-5 pg/ml cytochrome c. A drop of this mixture was placed on a clean wax surface for 2-10 min. to allow a surface film of cytochrome c to form and adsorb DNA [IO]. A parlodion-coated grid was touched to the surface, washed with water, and ethanol, stained with uranyl acetate freshly diluted with 90% ethanol [ 1I], and dried from isopentane. DNA length distributions were very similar with the two methods; both allowed DNA fragments down to 300 8, to be measured with confidence. Grids were examined in a Philips EM 200, and magnifications calibrated with a diffraction grating replica. For histone extraction, chromatin samples were dialysed to remove sucrose and salts (in the case of formalin-fixed material. deformvlation was netformed as described above), and then further dialyskd against 0.5 M HCI for 30 min. After centrifugation, the dialysate was lyophilized, and the residue dissolved in 0.9 M acetic acid containing 15% sucrose. Histone samples prepared in this wa; were analysed on 20% acrylamide, 2.5% urea gels [l2]. After exhaustive prerunning, using 0.9 M acetic acid in both chambers, the samples were applied, and run at 100 V for 3 h. The gels were then removed, stained with Coomassie brillJae;ttsblue, and scanned at 571 nm (Gilford Instru-

RESULTS rpm in an SW41 rotor for 12 h at 4°C. In the case of micrococcal nuclease, the gradients also contained 0.2 mM Na,EDTA. In other experiments, the digested chromatin was fixed by adding formaldehyde to 0.4% immediately before placing on the gradients. Other conditions tested were various salt (0.15, 0.50, I.0 M NaCI) concentrations in the gradient. For approximate calibration of S values, bovine serum albumen (Sigma, USA) and catalase (Sigma, USA) were used as markers. Gradient fractions were collected from the top of the tubes, and continuously monitored for absorbance at 254 nm (Isco, USA). Peak fractions were prepared for electron microscopy by negative staining with I or 2 % uranyl formate or uranyl acetate. Carbon-coated, freshly glow-discharged grids were used for all preparations. DNA was extracted from chromatin by adding SDS to 5% and sodium perchlorate to 75% w/v [8] and stirring for 10 min. The mixture was then centrifuged to separate an upper flocculent layer from the lower DNA-containing solution. The latter was carefully withdrawn, and 2 vol of 95 % ethanol added to precipitate the DNA. After standing at -20°C overnight, the DNA was collected, washed, and dried. Samples of formaldehyde-fixed chromatin were first deformylated by exhaustive dialysis at 37°C against 10m6M triethanolamine [9]. Extracted DNA was prepared for electron microscopy by two methods. In the first, the sample was dissolved in SSC (0.15 M NaCl, 0.015 M Na citrate pH 8-O), and carbon-coated grids streaked across a drop of the mixture. After drying from 95 % ethanol, the grids were shadowed with Pt in a direction perpendicular to the streak. Alternatively, the DNA was dissolved in 0.4 M ammonium acetate containing Exprl CeN Res 97 (1975)

Nuclease digestion Digestion of chicken erythrocyte chromatin with micrococcal nuclease resulted in a spectrum of fragments which could be resolved on sucrose gradients into discrete bands (fig. I). However, in contrast to similar results obtained by No11[6] with rat liver chromatin, optimum separation of the bands could not be obtained at low ionic strength. While the slowest migrating peak was well resolved under all the conditions tested, separation of the second, third, and fourth bands required either pre-fixation with formaldehyde (see Methods), or the presence of 0.5 M NaCl in the gradient (fig. 1). As discussed below, the evidence suggests that the poor resolution of unfixed chromatin fragments at low ionic strength is due to aggregation. After 2 min digestion with micrococcal nuclease, 30 % of chicken erythrocyte chromatin was converted into fragments which remained in solution after a 30 min centri-

Structural

repeating units in chromatin.

ZZ

113

80.

Sahasrabuddhe & van Holde [14], whose nuclease resistant PS particles were 12S.

70.

Analysis of gradient fractions

Further analysis of peak fractions from the gradients included electron microscopy of negatively stained preparations, electrophoresis of extracted histones, and length measurements of extracted DNA. In all cases, the 11S peak contained spherical particles with a mean diameter of 75 A (fig. 2). However, grids prepared from gradients run at low ionic strength always showed a large amount of aggregated material, even when the sample was pre-fixed with formaldehyde. Attempts to dialyse out the sucrose before making the preparations resulted in Fig. 2. Abscissa: diameter (A); ordinate: no. of particles. even more pronounced aggregation. Such Size distribution of monomer particles as measured from electron micrographs of negatively stained prep- aggregation was eliminated only in the graarations of material form the main sucrose gradient dients containing 0.5 M NaCl. Grids prepeaks. pared from these fractions showed uniform distributions of spherical particles and parfugation at 20000 g. In contrast, DNase II ticle multimers, with almost no aggregates solubilized only about 3 % of the material (figs 3-5). In some cases, the individual parunder conditions similar to those reported ticles were attached to fibers about 20 A in to liberate about 15% of rat liver chromatin diameter (fig. 3), similar to the ‘tails’ re[13]. This low value for erythrocyte chro- ported by van Holde et al. [4]. Another very matin is in agreement with the suggestion characteristic feature of the particles was a by Gottesfeld et al. [ 131that DNase II ac- central electron-opaque spot similar to the tivity is directly related to the template particles seen in chromatin fibers [l]. Maavailability of the chromatin. As with mic- terial from the 15S peak contained prerococcal nuclease, the fragments solubil- dominantly pairs of particles connected by ized by DNase II could be resolved into dis- a 20 A diameter fiber (fig. 4). The length of crete bands on sucrose gradients (data not the connecting fibers was variable, ranging up to 200 A (fig. 6). The third (about 20s) shown). Co-sedimentation of catalase (11.2 S) and peak from the gradients contained many bovine serum albumen (4.5s) with nucle- trimers, though there was a substantial conase-digested chromatin enabled approx- tamination with dimers and tetramers (fig. imate sedimentation values of 11S and 15S 5). Connecting strand length distribution of for the first and second peaks to be cal- trimers was similar to that for dimers. The distributions of DNA isolated from culated. This is in good agreement with the results of No11[6] who obtained 11.2S and the monomer, dimer and trimer peaks are 15.9s for monomer and dimer chromatin shown in fig. 7. In each case there was a particles under similar conditions, and with rather broad distribution around a distinct 60.

Exprl Cell Res 97 (1975)

114

Woodcock, S~veettnan und Frudo

Figs 3-5. Sucrose gradient fractions with 1% uranyl formate. Monomer, peaks respectively from gradients

negatively stained dimer and trimer containing 0.5 M

NaCI. Scales 500 A (the apparently tion of fig. 3 is caused by a greater stain).

higher amount

maynificaof residual

peak. The 450 A monomer was also re- in 0.5 M NaCl, none of this erythrocytepresented in the dimer and trimer prepara- specific histone remained (fig. 9). tions as would be expected if there was slight cross contamination. The trimer disDISCUSSION tribution showed a major peak at about 1700 A, and minor ones at about 1 100 A, Identity of the particles corresponding to contaminating dimers, In a previous paper, ultrastructural eviand at about 2300 A; corresponding to dence was presented which suggested that tetramers. Plots of the modal length from chromatin fibers are composed of interconeach distribution approximate a straight nected 75 A spherical particles [I]. The line (fig. 8), the significance of which is most crucial test of this hypothesis is the successful isolation of the particles themdiscussed below. Chromatin solubilized by micrococcal selves by destruction of the interconnecting nuclease showed an almost total loss of his- material. The experiments reported here tone f 1, and a marked decrease in histones show that micrococcal nuclease does yield f2c, f3, and f2al compared with control a population of chromatin fragments, the chromatin (fig. 9, verified by integration of smallest of which has a structure very simithe densitometer tracings). As the salt con- lar to the particles seen in whole chromatin centration in the gradients was raised, the fibers. This similarity includes size, shape, only further change in histone composition and the frequent presence of a central was a progressive loss of histone f2c, until opaque area. One of the conditions necesExprl Cell Res 97 (1975)

Structural repeating units in chromatin. II 1

7-F

100

200

300

Z?,q.6. Abscissa: length (A); ordinate: no. of dimers. Length distributio% of dimer ‘connecting strands’ measured from micrographs similar to fig. 4.

115

chromatin until at 0.5 M NaCl, this histone is absent (fig. 9). This, together with other evidence implicating f 1 type histones in chromatin condensation through histonehistone interactions [16, 171, suggests that the poor resolution of the gradients at low ionic strength is due to the same phenomenon. The increased resolution upon prefixation with formaldehyde (fig. 1) could be due to the partial loss of the histone-histone interaction sites. Further support for this comes from the microscopic examination of the gradient fractions. All attempts to pre-

50

-

40 -

n

Monomer

1 30 -

sary for visualizing the particles in whole 20chromatin is aldehyde fixation which, it could be argued, was causing the pat-tic- IOulate appearance. When isolated, however, the particles can be seen without prior fixation. Indeed, fixation of the isolated par- 16. titles does not alter their appearance in the ,2 electron microscope, or their sedimentation a_ rate. It has also been shown that the X-ray reflections of chromatin are not affected by 4 formaldehyde [15]. Thus, although formaldehyde cross-links chromatin so that histones and DNA cannot be extracted by the 40. usual methods [9], this does not seem to 32 alter the native configuration of the complex at the level of resolution of the electron 24. microscope.

L

16-

Aggregation of isola ted particles It is apparent from fig. 1 that the resolution of dimer, trimer, and tetramer particles on sucrose gradients increases as the ionic strength increases. This is accompanied by a progressive loss of histone f2c from the

a500 IOOD 1500 2000 2500 3mo Fig. 7. Abscissa: DNA length (A); ordinate: no. of fragments. Length distributions of DNA extracted from monomer, dimer and trimer sucrose gradient peaks. Exprl Cell Res 97 (1975)

Woodcock, Sweetmun and Frado

extracted DNA length (AX IO-*). Graph of modal DNA fragment length against olieomer number. The solid line is a least squares fit toihe data.points. The dashed line is a projection of the case in which oligomers are direct multiple of monomers (see text).

Fig. 8. Ordinate:

pare samples of ‘low ionic strength’ monomer for the electron microscope resulted in a large number of aggregates in addition to individual 70 A particles. However, the tendency to aggregate was almost completely lost when the gradients contained 0.5 M NaCl. Length of DNA extracted from the particles If the DNA length modes (E) extracted from monomer (n=l), dimer (n=2) etc. (from fig. 7) are plotted against n, the points fall approximately on a straight line (fig. 8), which does not intercept the origin. If the nuclease treatment resulted in one doublestranded break per subunit, then, on average, the DNA lengths from different oligomers would be direct multiples of the monomer length regardless of the presence (or absence) of a discrete ‘connecting strand between subunits. In such a case, a plot of n against DNA length (E) would give a straight line through the origin (fig. 8, Exptl Cell Res 97 (1975)

dashed line). The straight line plot given by our data indicates that the oligomers are not direct multiples of the monomers, but that the series is of the type: la, 2a+h, 3u+2h . . . in which a is the length of the monomer, and b an additional length found in multimers. A least squares fit of our data to a straight line E=(u +b)n -b gives the equation: E=643n-205. Thus, while the monomer (n = I) contains 438 A of DNA, the dimer contains (2x643)-205=1081 A and so on. (A statistical treatment of the raw data, in which the regression is based on the means and variances of monomer (250600 A), dimer (700-l 200 A) and trimer (1200-2 000 A) DNA lengths gives an estimate of 195 A for the intercept, b with a projected standard error of 21 A. The ‘tetramer’ peak was excluded from this analysis.) This analysis suggests that a region of DNA, about 200 8, long, is preserved in dimers, but digested away in monomers, and an obvious site for this 200 A length is the ‘connecting strand’ seen in dimer micrographs (fig. 4). As seen from fig. 6, the length distribution of connecting strands from dimer preparations shows a cut-off at about 200 A which agrees well with the theoretical prediction. The process of nuclease digestion of chromatin can therefore be envisaged as a series of inter-particle cuts followed by digestion of the ‘free’ ends until only the 440 w of histone-protected DNA contained in the 75 A particles remains. The paucity of free ‘tails’ seen in our micrographs (40% of monomers have single tails with mean length 40 A; 2 % have double tails) suggests that once the inter-particle break has occurred, the ‘tail’ is rapidly digested away. Thus, in the formation of two monomers from a dimer, the dimer ‘tails’ would be digested away before the final inter-particle

Structural repeating units in chromatin. ZZ 117 and in our preparations of dimers and trimers in which connecting strands are visible (figs 4, 5), the length distribution is very broad (fig. 6). This suggests that DNA can be pulled from the particles during the preparation, and that it can be extended up to 200 A. A final point in favor of this model is that in dimer and trimer preparations made with a relatively thick layer of negative stain (which gives more protection against drying stresses), the 75 A subunits always remain touching. Thus, we envisage the chromatin particles to be closely packed in vivo with only a small portion of the 200 A ‘connecting strand acting as inter-subunit links.

Fig. 9. Polyacrylamide gels of histones extracted from whole chromatin, and monomer peaks run in sucrose gradients containmg no salt, 0.15 M NaCI, and 0.5 M NaCl respectively. Coomassie brilliant blue staining.

break. An alternative, but less attractive explanation of the very low frequency of double tails is that the initial break is always towards one end of the ‘connecting strand’. However, the question then arises as to the in vivo site of the 200 A segment. Does it exist as the most exposed portion of DNA on the spherical subunit, or is there really a 200 A connecting strand between sub-units? At present, the evidence seems to favor the former possibility; in preparations of whole chromatin, unstretched fibers often show no connecting strands [l],

Histone composition of isolated particle As shown in fig. 9, the monomer particles isolated in low ionic strength gradients showed substantial losses of histone. Not only was the f 1 pair absent, but also, the amountsoff2c, f3andf2al werereduced. Since this is in conflict with other determinations showing complete histone complements in DNase resistant chromatin [21], or loss of only f 1 [27], it is clear that more work must be done to rule out the possibilities of incomplete extraction, or proteolysis. Since all histone species were present in the large fragments discarded after digestion, proteolysis did not occur at this stage. Another reason for the discrepancy between our histone analyses and those of others could be the relatively small extent of digestion in our case (30 % of chromatin solubilized). If histones were liberated during digestion, then there is ample evidence [23] that they could reassociate non-specifically with the remaining chromatin. In the case of extensive digestion, this reassociation would be with already solubilized material, and the histones would be retained on the particles. After partial Exprl Cd Res 97 (1975)

118

Woodcock, Sweetmun und Frado

digestion, however, liberated histones might tend to reassociate with undigested chromatin, and hence be excluded from the gradients. (We found almost no free histone at the top of the gradients.) Apart from the loss of histones f 1 and f2c noted above, the most dramatic change was a reduction in the amounts of f3 and f2al relative to f2a2 and f2b. This relative loss of f 3 and f 2a 1 was independent of ionic strength up to and including 0.5 M NaCl. In all cases, the ratios of f3:f2al and f2a2:f2b remained approximately constant (fig. 9; verified by integration of gel density traces). It is these two pairs of histones which are linked to form tetramers and oligomers in vivo [18, 23, 241, suggesting that more extensive histone analyses may lead to further information about the disposition of histones on the chromatin subunit.

determined by No11 [6] who also used micrococcal nuclease digests. In addition, our electron micrographs, and DNA length measurements have led to the conclusion that about 200 A of the subunit DNA is relatively accessible to DNase, a hypothesis first put forward by van Holde et al. [4]. In this respect, our data differ from these of No11[6], who did not find 440 8, fragments of DNA in micrococcal nuclease digests. Associated with the monomer of 205+ 15 base pairs (697?5 1 A) was a smaller fragment 170+10 base pairs (578+34 A) which was thought to be a product of over digestion [6]. However, even this is significantly larger than our 440 A fragments derived from monomer fractions. This discrepancy is most likely due to differences in digestion time and enzyme concentrations, since the nuclease-resistant PS particles of Sahasrabuddhe & van Holde [ 141contained about 400 8, of DNA which is Relation to other models much closer to our measurements. The In the past two years, considerable ultra- largest estimate (710 A) of the amount of structural, biochemical, and biophysical DNA contained within a chromatin subunit evidence in support of a subunit structure comes from the work of Senior & Olins for chromatin has been presented [ 1-7, 15, [ 151who used sonication of fixed material 19, 201. Our data fully support the general to prepare monomers from chicken erythroprinciple of a subunit organization in which cyte chromatin. The possibility therefore, discrete portions of DNA are complexed remains that all estimates of the monomer with histones to form spherical particles. A size from nuclease digests are artificially full discussion of the relationship between low, and that nuclease attack occurs in a the superhelical model of chromatin struc- series of steps resulting first in 700 A fragture [25, 261 and a subunit organization is ments, then 575 A, and finally 4W50 A beyond the scope of this article. However, lengths. Further analysis is clearly necesas others have pointed out [2, 5, 6, 271 the sary to establish if this is really a step-wise X-ray evidence is not an unequivocal de- reaction, or a more continuous process. monstration of superhelicity; the finding wish to thank our colleagues for their help, advice, that monomer particles isolated from chro- We and generous sharing of equipment, and L. S. Johnson matin give the same X-ray reflections as for stimulating discussions, and assistance with matheanalyses. whole chromatin [3] provides clear evi- matical This work has been supported in part by the US dence on this point. NSF (GB 43598). Our estimate of the length of DNA per subunit (about 640 A) is close to the value Expt/ Cell Res 97 (1975)

Structural repeating units in chromatin. ZZ 119 Note added during preparation

While this paper was under review, the similar study by Oosterhof et al. (Proc natl acad sci US 72 (1975) 633) was brought to our attention. In agreement with our data, these authors found that DNA isolated from monomer subunits (prepared by the action of DNase II on calf th mus chromatin) had a modal length of about 400 K. However, in contrast to our results, they found that oligomer DNA lengths were exact multiples of the monomer. At present there seems no obvious explanation for this discrepancy, and the situation is further complicated by the 680 A monomer DNA proposed by No11 [l]. We have recently shown that monomer chromatin subunits differ from oligomers in their melting profiles (Woodcock, C L F & Frado, L-L Y, Biochembiophys res comm 66 (1975)403. Monomers have a monophasic melt with a T, of about 77”C, while oligomers all show a biphasic profile with one T,,, between 45°C and 55°C and another at about 77°C. We have suggested that it is the connectingstrand DNA, present only in oligomers which melts at the lower temperature. It will be interesting to observe the melting behavior of DNase II-derived chromatin fragments.

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Exptl Cell Res 97 (1975)