Substructure of thermally unfolded chromatin subunits or nucleosomes

su

UCTURE

OF THERMALLY SUBUNITS

UNFOE

OR NUCLEOSO

N. HUNG POON and VERNER

L. SELIGY

SUMMARY Mononucleosomes containing 143+6 base pairs of duplex DNA and approximately two copies each of the histones H2n, HZ. H3 and H4 were examined durine. thermal denaturation bv high resolution electron microscopy using both bright- and dark-field (tilfed beam) modes. Co-operati;e destabilization and unfolding of the 13.2+ 1.4 nm diameter toroids occurred only after the second of the two major melting transitions. The unfolding patterns are consistent with about 1.5-1.8 turns of supercoiled DNA in intact nucleosomes. The dominant unfolded structure of samples post-fixed with glutaraldehyde is a 17.5k2.1 nm diameter open ring. Both sister DNA strands remain associated with protein. The distribution and shape of the protein patches are more irregular in unfixed, unstained samples visualized by darkfield microscopy. Image reconstruction studies on fixed and stained ring-shaped specimens indicates that there are 6-10 gIobular protein elements or patches, each about 3.9kO.5 nm in diameter, per DNA moiety.

The nucleosome is considered to be a fundamental unit of chromatin [ 1, 21. The probable pathway of the 140-145 base pair DNA component is a supercoil of about 1.7 turns [3-51. The exact arrangement and interaction domains of the four pairs of associated histones [I, 2, 6,7] are not known, but several laboratories have attempted to define them [ 1, 2, 7, 8, 91. One very fiuithas been the use of electron microscopy in conjunction with physical chemical probes to describe the organizaed nucleosomes induced by urea [IO, II] and thermal [12, 131 denaturation. As a fnrther development in this work ave found that the dominant structural of gently prepared thermally unfolded nuclessomes is an open ring and that many of these structures possess iterated subelements characteristic of globular proteins.

By application of image ~~c~~~t~~c~~~~ used in protein ~nat~r~ary techniques, structure analysis [14-16]S we determined the distribution, axial symmetry and approximate size of these iterate

folding during ther possible organization of hktones in intact and unfolded rn~~~~~c~~~§~

PreparatiQ~

Q~‘n~o~~n~~~~~~~rn~~~

Mononucleosomes were obtained from micrococcal nuclease (EC 3. i .4.7) digests of intact mature goose ervthrocvtes 112. 17, 181. DNA and nrotein were identified and calibrated according to earlier experiments [12, 181.

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Poon and Seligy

I

i

I

I

60 80 Temperature P’C)

I

I. Properties of mononucleosomes used for thermal denaturation and electron microscopy. a, DNA from whole nuclear digest; 6, DNA from nucleosomes; c, histones from mononucleosomes; d, derivative melting profile of mononucleosomes in 5 mM NaPO,, pH 7.0, and the corresponding plot of the, e, outer diameter (open symbol) of nucleosomes measured during heat denaturation. Verticle range bars indicate SD. from 504 measurements.

Fig.

Thermal denaturation microscopy

and electron

Derivative hyperchromicity-temperature profiles of thermally denatured nucleosomes were carried out in 5 mM NaPO, buffer, pH 7.0 [12]. For concomitant electron microscopic analysis a Siemens Elmiskop 1A was used in either the BF mode [ZS] or DF mode (titled beam; [18, 191). To monitor temperature fluctuations microthermocouples, linked to an X-Y plotter, were inserted into the microtransfer pipettes and on top of the individual microscope grids [12]. The methods used here for visual analysis have been reported [12, 18, 191. A detailed study comparing DF and BF images of fixed and unfixed, stained and unstained mononucleosomes has recently been done [191.

RESULTS Properties of intact and denatured mononucleosomes The experiments shown in figs 1 and 2 and table 1 illustrate the essential properties of typical mononucleosomes. The isolated DNA is relatively discrete in size Exp CrNRes

128(1980)

(fig. la, 6); an average base pair number of 14356 was determined from reference endonuclease restriction DNA fragments [12, 201. Only four proteins, histones Hk, HZ, H3 and H4, were present (fig. 1 c) and these were in unit copy ratio with respect to each other or about two copies [2] of each per DNA molecule. Neither the protein ratio nor the relative electrophoretic mobility of these histones was altered even after heating to 97°C for 10 min. Characteristic of the melting profile of other preparations [ 1 l-13,21,22] two major melting components were distinguishable (fig. 1 d). The accompanying plot in fig. 1 e shows that gross morphological alteration does not occur until after the second melting transition is reached. The BF mode micrographs in fig. 2a, b show the dominance of the ring-shaped form. In most gently prepared BF samples the common nucleoprotein patterns were “C” and “9”. The degree of success of obtaining uniform patterns depends on sample quality, sample concentration and handling techniques. In most preparations some interstrand, multiple aggregates exist; more aggregates were seen at 80°C where complete unfolding begins to occur [12] than at 90°C or above. These aggregates generally are in the form of large, filamentous, negatively staining open or closed circles about 30.8+ 6.5 nm wide and to 87.7k30.4 nm in length. Fig. 2. Electron micrographs of thermally unfolded nucleosomes. (a) Brightfield micrograph of nucleosomes at 95°C fixed with 2% glutaraldehyde (v/v) for 1.610.3 set and stained with 1% uranylacetate (w/v) for 2.1kO.3 set before washing with glass-distilled water; (6) high magnification of select images from grids treated as in (a) to show intermittent strand separation (bars) and strand termini (arrows); (c) darkfield images of unfixed, unstained nucleosomes at 95°C. Dotted lines in the schematic indicate possible regions of strand separation. Magnification of electron image plates were double calibrated with a carbon grating of 54, 864 lines/25.4 mm and catalase crystals [12, 181.

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Table 1. Parameters

of unfolded

mononucleosomes

from

Linearspartial

microscopic

studies

BF (N=405)

YZ=73) Form Radius Inner (nm f SD.) Outer (nm + S.D.) Contour length (nm _+S.D.): Fiber width (nm) (min) (max) % B-form DNA equivalent Subelement no.” Size of subelement (nm _+S.D. dia.) Spacing of subelements (center to center) (nm + S.D.) Presumptive Strand separation % of total length (intraf terminal) (terminal)

electron

Open circle~linear

loop

46.4k4.3

4.9OkO.75 8.70f0.75 42.4163

1.3 4.1 94.6L4.0 7.451.4 3.6fO.S

1.4 4.6 86.4k4.8 7.5t1.0 3.9kO.5

4.6kO.7

5.310.8

22.7i7.8 21.5k6.7

29.414.85 18.6k5.25

a The subelements in DF mode are defined as electron-dense masses along the DNA fiber, in the BF mode these units are electron opaque. The diameters correspond to the maximum width of the unfolded nucleoprotein fibers.

Some samples also yield inherently higher grid backgrounds than others. We found that shearing or agitation of melted nucleosomes can give rise to unusual image distortion, aggregation and grid debris ([19], and unpublished). Extensive washing of the grids eliminates much of the grid debris but it also biases the types of images viewed. The selection is generally towards large multinucleoprotein filaments rather than individual nucleosomal units. The unfolded nucleosome visualized in the BF mode is largely negatively staining, whether or not briefly exposed to glutaraldehyde, which is used here as a stabilizing agent [12, 191. The negatively staining property is characteristic of a globular, hydrophobic protein moiety and not a nucleic acid [H-19, 301. As shown in fig. 2 these globular regions are often visibly iterated. Removal of histones by high salt (>2 M) or sodium dodecylsulphate (-2 %) totally destroys the negative staining and globularity of these fibres. Similar measurements and arrangements of the globular (in this Exp Cell Res 128 (1980)

Cf

z

30nm

-,

.

3. Linear tracings of darkfield visualized unfolded nucleosomes showing the distribution of globular protein elements (blocks or discs) along the DNA strands (lines). About l-50 ng samples were placed on 1.5-2.0 nm thick carbon films and photographed with tilted beam illumination. The initial magnification of images on plates was 40 K. Final enlarged micrographs were obtained microphotographically through a Leitz optical microscope (microsystem) or a Leitz projector (macrosystem) equipped with a Polaroid Land Camera. In order to retain a natural contrast, the final micrographs were taken from the contact printed plates of the originals and recorded on Polaroid Land no. 52 film. Measurements of denatured mononucleosomes were obtained directly by projecting the original images onto a screen. Fig.

Fig. 4. Image reconstruction of select images of thermally denatured nucleosomes showing manifold symmetry of negatively staining globular subelements. Ring-shaped nucleosomes from typical grid windows, such as shown in fig. 2 were photographically enlarged to abolut 30 mm in diameter using the macrosystem [IX!. Final printing was on Kodak KC paper which is fluorescent under IJV light. For present purposes images less than 1 .O nm were optically filtered. Image enhancement or reinforcement &as obtained by photographic integration 114, 151 using a modified stroboscopic illummation system. Each photograph of a nucleosomal ring was mounted at the centre of a variable rpm platform. A single superimposed fiducial mark, at the periphery of each nucleosome image, was used to specify angle of rotation. The rpm of the platform was adjustable from 20 to 600. Stationary images were generated by synchronizing the flash

frequency of a stroboscope (Strobette, Model 964, Power Instruments Inc. 1 Ill., t&6 500 flashesiminj. placed 15 cm above the platform, to yield one flash/ revolution, 2 dn where ?t= 1 corresponds to the fiducial marker. Superposition of the negatively staining elements is achieved by advancng the strobe frequency. The value of n is obtained by direct scoring of ?he pseudo marks when ail subimages or negatively staining elements are maximally enhanced. Each trial experiment (where n = 1-12) was registered on photographic film (tvne 57, ASA 3000) using a Polaroid Eamera Model MY4 at constant shutter speed and J All experiments are carried out in a photographic dark room using the strobe lamp as illum~~at~o~~ ROWS A-C are results from the most frequent pattern observed (see table); Rows D-E are examples of aberran? and less frequently seen patterns. Vertical rows (a-f) indicate variants of n .

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Poon and Seligy

Fig. 5. Fidelity of image reconstruction technique using model structures. Simulated regular (A, 245 andB, 2~/8) or irregularly arranged subelements were constructed using white circles distributed along a

perimeter 2~. Y, where specific periodkity 27rvln and r were variable, i.e., Y=%, the average radius of a denatured nucleosome.

case electron dense) elements were obtained by DF mode microscopy in the total absence of heavy metal staining or fixative. Two typical examples of such images, are shown in fig. 2c. Appropriate measurements from other samples as well as contour measurements of unfolded nucleoprotein fibres are given in table 1. The size and electron density of the DF images are

comparable to previously visualized protein subunits of enzymes, histones and mononucleosomes [18, 191. Extensive examination of BF and DF mode micrographs at high magnification further indicated only intermittent DNA strand separation. We estimated the length of these presumptive segments to be about 3-4 nm (table 1). The evidence for a major

Exp Cell

Res 128 (1980)

-irn’L

Fig. 6. Schematic representation of mononucleosome unfolding during thermal denaturation. Only the terminal ends ofthe 13.2k1.4 nm diameter toroid can be recognized during the first melting stage (Tm’). Complete unfolding and linearization of the DNA occurs at the second melting stage (Tm2).

~~~g~e-stra~d separation at the fibre termini at the limits of resolution is practically nil. presumptive regions where intrastrand separation might have occurred are revealed by the bubble-like appearance within e ~rot~i~-~~A duplexes. Examples of this are shown by marker bars in fig. 2 and in the line tracings of DF images showing the globular regions in relation to the DNA fibre (fig. 3). Image

rotational symmetry sho at the specific angle of 2~ln=j. The intrinsic spacing of each element can be a~~r5x~mated by 21sr’!jV where i: is the average distance of each o elements from the cenlral axis of sym is s%udy 40 ring-str~~%~re~ w dividually tested over the range n = I-15 for a total of 440 operations. These resuhs are s~~~rnar~~ed in . 4 and table t. The horizontal rows A, and c show typical es of %-fold rotational sym 5%frequen% type observed. Since this holds with limited testing of strobe frequencies where h tiples of 8 are m

t less frequent

types seen.

and C as suggested fr0.m model studies ~

reconstmciion

arkham photographic integration method is suitable for enhancing image detail in electron micrographs of objects with a regularly repeating structure [14]. A modification of this procedure, which we used to produce dynamic images, is described in mg planar surface of select from prints such as in fig. 2a was arbitr y sectored over the range a smgle fiducial marker and a variable strobe lamp. A strobe frequency that yiel s a static image (n=I, one mar~~erl~mag gives a precise replica of the original stationary image. Maximum s~~e~osi~~on of reinforcement and average subelements was subsequently of iev by trial variation with higher or lower strobe frequencies than that of the static image From theory [14, 231 ring structures with inherentj-elements of j-fold T

tion [23]. As shown in Ca to Cf reinforcetranslation is only +_ one subele eter. eyond this range the intn sly affected Seque ows how image re half the repetition pattern ce more than half the elements are out of regis%er patterns dominate as shown in fig. 5 Ea to Ef. These patterns resemble those gener-

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Poon and Seligy

ated for j=5 at n>6 (AC to Af) and may explain how typical patterns in fig. 4, rows D and E, could arise from a distorted j=S structure.

The fibre width measurements per contour length from BF and DF micrographs indicate that both DNA strands are still largely associated with protein and that only relatively short stretches of singlestranded DNA occur if any. We estimate DISCUSSION the maximum length of the regions unThe main function of the histone octomer, obscured by the globular protein sections in relation to stabilization of nucleosomal is no more than 12-18 base equivalents DNA, is to lock the DNA into a relatively or about 36.8+ 12.5 bases/nucleosome. Prerigid complex [l-9, 11-13, 19-22, 24, 251. vious spectroscopic measurements indiDuring the first of the two melting transicated that histones remain associated with tions of mononucleosomes only certain re- each of the single strands of chromatin gions, about 40 base pairs [ll-13, 21, 221, DNA after thermal denaturation [13, 261. apparently gain limited mobility. This mo- Histone spacing along urea-dissociated nubility would explain the apparent reversibilcleosomes is also considered to be more ity of this melting transition and the altered or less even [lo, 271. The actual amount of melting profiles of nucleosomes treated unprotected and protected single-stranded with urea or trypsin [ll, 12, 251. We de- DNA might be critically evaluated from tected neither strand separation nor un- single-strand nuclease digests. folding at this stage. Some changes in the Many of the nucleoprotein images as surface properties of nucleosomes likely shown in fig. 26 are suggestive of a looseoccur at the end of this transition where ly, intertwined or cross-linked arrangement histone disruption takes place [13] because of sister DNA strands. Such a situation small aggregates of 2-4 nucleosomes and would arise through release of base stacking the toroid fibre ends are detectable [12]. [28] and retention of a few residual histoneOn reaching the second melting transition binding sites that are extended over one or all aggregations of this type are abolished. more gyrs of the DNA in a manner anaIn agreement with earlier work [12, 131 logous to protamine [29] or lac repressor we find that this transition coincides with [30] interactions. Glutaraldehyde-mediated disruption and release of the remaining cross-links no doubt also contribute to the majority of the DNA base pairs in stackstabilization of ring structures because they ing. The unfolding patterns shown here are not seen with the same frequency in concur with a supercoiled arrangement of unfixed nucleosomes [12]. We do not know nucleoprotein in intact nucleosomes [3-5, if the globular regions observed along the 91. This pathway which is an adaptation of DNA fibres actually correspond to individthe Finch & Klug nucleosome model [5] ual histones. Linearization of the DNA is illustrated in fig. 6. Total contour length would physically partition the interacting of denatured nucleosomes is 85-92 % of the hydrophobic histone domains involved in corresponding 145 base pairs of undecore interactions [24] and rapid cooling, on natured DNA. This is comparable to esti- transfer to the electron microscope grids, mates from urea unfolded nucleosomes would lead to the formation of one or more [lo, 111 where presumably the DNA is not intrastrand globular structures from even denatured. a single renaturing protein [3 11. Mirzabekov Exp Cd

Res 128 (1980)

and co-workers have produced a highresolution map for the distribution of histones along the DNA (321. Each of the histones is apparently arranged within several adjacent or dispersed NA segments of a little less than ten nucleotides in length [33]. Our symmetry measurements indicates best of the unfolded nucleosomal ere are on average about eight proteinacious units with corresponding rotational symmetry per 145 base pairs of unfolded DNA. This constancy with respect to actual histone copy superficially points to a linear alignment of histone along DNA, at least in the denatured state. An arrangement of eight similar histone molecules about a true dyad axis is particularly appealing in view of recent critical proposals for a linear topological arrangement of histone in intact nucleosomes [4, 5, 9, 24, 331. However, while there is little question as to the fidelity of our integration method an improper rotation generated by a ring structure which is enantiomorphous [15, 231 cannot be ruled out. The Batter is also true if a major rearrangement of histone occurs along the DNA. The large target size of thermaliy unfolded nucleosomes opens up the possibility of using recently developed histone mapping and localization techniques [33, 34, 351 to determine the exact natutre and specificity of the protein globular regions. We are indebted to M. J. Dove, L. Sowden and R. Whitehead and Ii. Turner for expert technical support. This is NRCC contribution No. 18295.

EFERENCES 1. Mornberg, R 14, Ann rev biochem 46 (1977) 931. 2. Felsenfeld, G, Nature 271 (1978) 115. 3. Richards, B M, Pardon, J F, Liiley, D M J, Cotter. R I, Wooley, 9 C &Worcester, D L. Phil trans roy sot Londoc B 282 (5978) 287. 4. Finch: 9 T. Lutter. L C, Rhodes, D, Brown, R S, Rushton, B. Levitt, M & Klug, A, Nature 269 (19773 29. Printed

in Sweden

5. Finch, .I T & Klug. A, Cold Spring Harbor symp quant bio142 (1978) I. 6. Kornberg, RD. Science 184 (1974) 868. 7. van Holde, K E & Isenberg. 1. Act them res 8 (1975) 327. 8. Weintraub, H, Worcel, A & Aiberts. B. Ceii 9 (1976) 409. 9. Trifonov, E. Nucleic acids res 5 i 1978) 1371. IQ. Woodcock, C F L & Fsado, i L Y, Cord Spring Harbor symp quar,t biol 42 (1978) 43. 11. Qlins. D E. Bryan, P N, Harrington. R E; Hil!. W E & Ohs, A k. Nucleic acids I-es 4 !1977) 191 j. i2. Seligv. V L & Poon. N 61. Nucleic acids res 5 (19%) 2233. 13. Weischet. W. Tatchel!, K. van Holde, K & Klump, H. Nticleic acids res 5 (1978) !39. 14. Markham, Ii, Hit&born, J H, Hills. G J 8 Frey. S. Virology 22 (1964) 342. 15. Crowthier. R A & Klug. A, An: rev biochem 41 (1975) 161. 16. Qttensmeyer, F P, Bazett-Jones, D P & Kern. A P, Electron microscopy (ed J M Sturgess) vol. 3s p. 147. Imperial Press, Ontario, Canada (1978). 17. Finch, J T, Nell, M & Kornberg, R D. ?roc nati acad sci US 72 (1975) 3320. 18. Peon. N H & Seligy, V L, Exp cell res 1L3 (1978) 95. 19. - Ibid 125 (1980) 313. 20. Kovacic, R T & van Holde, K E, Biochemistry 16 (1977) 1490. 21. Mandel. R & Fasman. 6. Nucleic acids res 3 (1976) 1839. 22. Lewis, P N, Canj biochem 55 (1977) 736. 23. Buerger, M .I. Eiemer,tary crystallography. p. 3. Wi!ey, New York (!956). 24. Camerini-Otero. R D & Felsenfeld, 6. Nuc:eic acids res 4 (1977) i 159. 25. Lilley. D M J & Tatchell, K. Nucleic ac:ds res 4 (1977) 2039. 26. Matsuyama, A & Nagata, C. Biochim biophys acta 224 (1970) 588. 27. Jackson, V & Chalkley, R, common 67 (1975) 1391. 28. Manning, G S, @ant rev biophys Ii (1978) 179. 29. Warrant, RW & Kim. S H, Nature271 (1978) 130. 30. Zingsheim, H P, Geisler, N, Weber, K & Mayer, F. J mol bioi 115 (1977) 565. 31. Record, M T, Andersoa, C F & Lohman. ?‘ M. Ouant rev bioDhys 11 (1978) 103. 32. Mirzabekov. i e, Shick, V V, Belyavsky. A V. Karpov, V LB Bavykin, S 6, Cold Spring Harbor symp quant biol42 (1976) 149. 33. Mirzabekov. A D. Shick, V V, Belvavskv, A V & Bavykin. S ‘G, Proc nati acad scB US 75 (1978) 4184. 34. Simpson. R T, Whitlock, J P, Bina-Stein. M & Stein. A, Cold Spring Harbor symp quant biol $2 (1978) 127. 35. Bustin, M, Goldblatt. D & Sperling. R, Ce!; 7 (1976) 297.

Received December 21. 1979 Accepted February 1. 1980