The nucleosome is a nucleoprotein complex composed of DNA wrapped about a core of histone proteins(1, 2) . Its canonical role in the cell is the packaging of DNA, although a number of recent biochemical and genetic studies have shown that the nucleosome is also an active macromolecular complex involved in the facilitation, modulation, and repression of gene expression(3, 4, 5, 6) . Nucleosome structure has previously been investigated by a number of diverse biophysical techniques, including measurements of fluorescence properties, circular dichroism, and sedimentation coefficient(7, 8, 9, 10, 11, 12, 13) , which infer changes in conformation with changes in pH, ionic environment, and post-translational modifications. Despite this, crystallographic studies of nucleosome particles (14, 15, 16, 17) using x-ray and neutron diffraction have consistently resulted in only one structure, an oblate ellipsoid. Moreover, a high resolution structure for the nucleosome core protein octamer determined using x-ray crystallography, when wrapped with model DNA, is consistent with such an oblate form(18, 19) . In contrast, studies using electron microscopy (EM) ()and three-dimensional reconstruction techniques have indicated a different form consistent with a prolate ellipsoid(20, 21, 22, 23) . The reproducibility of the divergent results has prompted us to investigate the reason for the major differences between the structures. We have found that the structure of the nucleosome changes dramatically with ionic environment, as determined by biochemical methods, conformational characterization(21, 22, 23) , and three-dimensional reconstruction techniques based on the principle of angular reconstitution(24, 25, 26, 27, 28, 29) . Our structural characterization indicates several ionic strength-dependent conformations of the nucleosome. One of these corresponds to the three-dimensional EM structure previously determined(20) , while another corresponds to the crystallographic form of this particle(14, 15, 16, 17, 18, 19) . The study ranges from 0.0001 M to 1.75 M in ion concentration and shows the first three-dimensional reconstructions of different conformational states of the nucleosome by a single consistent technique. In total, the results not only reconcile long-standing differences observed between electron microscopic investigations of nucleosome structure (20, 21, 22) and other biophysical studies(14, 15, 16, 17, 18, 19) , but also corroborate structural changes observed at extremes of ionic strength(12, 13, 30) .

EXPERIMENTAL PROCEDURES

Purification of Nucleosomes and Electron Microscopy

Nucleosomes were prepared for conformational analysis in ionic environments of 0.1, 3.5, 10, 30, 150, 400, 750, 1200, and 1725 mM NaCl. These specific conditions were chosen as they correspond to points midway between ionic environments indicated by other studies as transition points in nucleosome structure: 0.4 mM NaCl(10) , 1 mM NaCl(7, 8, 11) , 6 mM NaCl(8, 11) , 16 mM NaCl(11) , 50 mM NaCl(11) , 250 mM NaCl(9, 11) , 550 mM NaCl(9, 12) , 950 mM NaCl(10, 12, 13) , and 1450 mM NaCl(12, 13) . Particles in the presence of <30 mM NaCl were prepared in low concentration buffer (0.15 mM triethanolamine (TEA) chloride, pH 7.4) to ensure a low effective ionic environment. At all other ionic strengths, nucleosomes were prepared in more concentrated buffer (15 mM TEA-Cl and 2 mM EDTA, pH 7.4) for additional buffering(21) . Specimens in the presence of <30 mM NaCl were also prepared in the higher concentration of buffer in order to investigate the effects of buffer on changes in nucleosome conformation. The effect of buffer was found to be similar to that of monovalent salt. Triethanolamine was chosen as a buffer in order to eliminate potential complications caused by buffers such as Tris-Cl since triethanolamine does not contain amino groups that would react with the glutaraldehyde fixative. Nucleosome particles in each ionic environment were at a concentration of 0.3 A/ml and were kept in this ionic environment for 24 h prior to fixation. Nucleosomes were then fixed in the selected ionic environments with the optimal conditions of 0.5% (w/v) glutaraldehyde and 3.0% (w/v) formaldehyde for 24 h at 4 °C as described before(21) . The effects of divalent cations (MgCl and MnCl) were also tested by including various concentrations of these salts in some of the preparations of nucleosomes.

Conformational Characterization of Nucleosomes from Their Micrographs

Three-dimensional Image Reconstruction of Nucleosomes

Reconstructions were carried out using the quaternion-assisted approach of angular reconstitution (26, 27) for reconstructing non-crystalline randomly oriented biological macromolecules from their electron micrographs. The methods utilized have been used previously by us in the reconstruction of several biological macromolecules (26, 27, 28, 36, 37) and recently in modified form by Serysheva et al.(29) . Our methods utilize sinograms and sinogram correlation functions (24, 25) and the central axis theorem (38) to determine the angular orientations of images of randomly oriented biological macromolecules with no internal symmetry. Sinograms and sinogram correlation functions were calculated as described before(26, 27, 28) . The initial orientation angles of the reconstructions were refined iteratively by a quaternion vector approach as discussed by Farrow and Ottensmeyer(26) . Resolution measurements were made as described by Czarnota et al.(28) using a phase residual approach with a conservative cutoff of 45°. Radii of gyration for reconstructions were calculated as described before(20) .

RESULTS

Nucleosome Core Particles

Electron Microscopy and Conformational Characterization of Nucleosomes

Figure 1: Images of highly purified calf thymus nucleosomes prepared in different ionic environments. Shown are nucleosomes prepared in the presence of 0.1, 3.5, 10, 30, 150, 400, 750, 1200, and 1725 mM NaCl (A-I, respectively) and dried from amyl acetate to minimize surface tension forces. Nucleosomes in A and B were prepared in low concentration buffer (0.15 mM TEA-Cl, pH 7.4) to ensure a low effective ionic environment. In C-I, particles were prepared in more concentrated buffer (15 mM TEA-Cl and 2 mM EDTA, pH 7.4). In separate experiments, specimens in the presence of <30 mM NaCl were also prepared in the higher concentration buffer, which affected nucleosome conformation similar to equivalent monovalent salt concentrations. The scale bar indicates 200 Å.

Figure 2: Conformational characterization of nucleosomes. The panels give representative results of conformational determination by principal component analysis. A-D are results of a conformational analysis by principal component analysis for nucleosomes prepared in the presence of the higher ionic strength buffer and 10, 30, 150, and 400 mM NaCl, respectively. The lower panels show corresponding distributions of length/width values as histograms. The method of conformationally characterizing nucleosomes by principal component analysis is described in detail elsewhere(21) . The longest arrow in each of the upper panels represents the best fit line through the scatter of paired length and width measurements as calculated by principal component analysis. The best fit line in A (upper panel) is consistent with a prolate ellipsoid, while the nearly vertical best fit line in C is consistent with an oblate ellipsoid. B is consistent with an intermediate form. Dimensional analysis of a perfect spheroid would generate a cluster of points resembling a half-circle about a point with equivalent length and width and a first principal component with an inclination of 45°. Tendencies toward greater or lesser slopes would indicate an oblate or prolate character, respectively. Scatters of points in each of the upper panels have been trimmed to exclude statistical extremes(21) . Dashed lines indicate mean values, and solid lines indicate mean values ± 1 S.D. The differences between data in all these panels are statistically significant to a 95% confidence level (see ``Results'' for details). Mean ratios ± 1 S.D. in the lower panels are indicated by the numbers in the upper right corner of each histogram. The number of images analyzed is given in Table 1(part A and B).

Figure 3: A compilation of primary results of the characterization of ionic strength-dependent nucleosome conformations. A shows the median observed axial length and equatorial measurements for nucleosomes in a variety of ionic environments. The bars indicate first and third quartiles. At 150 mM NaCl, the minimum dimension observed was 60 Å. An additional set of measurements exists for the lowest ionic environment shown in this panel (see Table 1, part A), but is not included on this figure for the sake of clarity. Dimensions of individually measured particles are given in Table 1(part A and B), and details are described under ``Results.'' Heights and diameters were from mean projection length and mean projection width values given in Table 1(parts A and B). If the conformation was determined to be prolate, then the mean projection length was taken as the height and the mean projection width as the diameter. If the conformation was oblate, then the projection length was taken as the particle diameter, and the mean projection width was taken as the height (see Zabal et al.(21) ). B indicates shapes and schematic representations of the various nucleosome structures deduced from electron microscopy and conformational characterization by principal component analysis. The schematic structure of the nucleosome (not to scale) is shown with increasing ionic strength from left to right. Spherical structures would correspond to the points of equal height and diameter. Dimensions given below 10 mM NaCl are for specimens in the presence of the less concentrated buffer (see ``Experimental Procedures''). For detailed discussion of conformations, see ``Results.''

The representative results in Fig. 2illustrate the differences between four different ionic strength populations of nucleosomes ranging from particles prepared in the presence of 10 mM NaCl to particles prepared in the presence of 400 mM NaCl plus 15 mM TEA and 2 mM EDTA. The differences in the inclination of the first principal component among these different populations indicate differences in nucleosome conformation between these samples. The near horizontal first principal component in Fig. 2A, which corresponds to the best fit line determined by an analysis of variance, is in agreement with a prolate conformation(21) . This conformation has mean projection length and mean projection width values consistent with those of previous studies(20, 21, 22, 23) . At 150 mM NaCl, this best fit line is nearly vertical, indicating an oblate conformation. The analysis at 30 mM NaCl indicates a best fit line that is consistent with an intermediate form between a prolate and an oblate form. Differences between the different ionic strength forms of the nucleosome are also reflected in the corresponding length/width distributions of images of single particles (Fig. 2, lower panels), although such representations do not readily permit direct structural inferences in terms of an ellipsoidal conformation.

Statistically Significant Differences between Ionic Strength-dependent Nucleosome Conformations

The differences between nucleosomes in 150 and 400 mM NaCl appeared by visual inspection to be less significant since each set had approximately equal mean projection lengths and approximately equal mean projection widths. Nevertheless, principal component analysis of the paired projection length and width measurements of each set indicates significant differences in variance, consistent with a change in shape from oblate to more spheroidal, at 150 and 400 mM NaCl, respectively (Fig. 2, C versus D; inclination of the first principal component changes from 90° to 68°, Table 1, part B). The t test also indicates statistically significant differences in width measurements between these two ionic strength-dependent populations (95% confidence level). Significant differences between the variances of these sets of width measurements were indicated by the F-test to a 95% confidence level. In general, the appropriateness of the use of these tests was confirmed by the Shapiro-Wilk test, indicating that sets of measurements could be considered to have normal distributions. The results of these tests were confirmed by the use of distribution-free non-parametric tests such as the Mann-Whitney test. This test also indicated statistically significant differences between the nucleosome populations at different ionic strengths. At the higher ionic environments (750 mM NaCl and above), where the Shapiro-Wilk test indicated that sets of measurements did not have normal distributions, the non-parametric analyses were used exclusively.

Different Nucleosome Conformations

Only one distinct conformation for the nucleosome was generally detected in each ionic environment examined by an analysis of length and width measurements and by gel electrophoretic analyses (described below). One exception was 0.1 mM NaCl, at which two forms of the nucleosome were visualized with distinctly different images, inconsistent with a single conformation. One form was significantly more elongated and slender than the other (mean length/width ratio of 8 versus 1.2), suggesting that this selected ionic environment was in a transition zone between two nucleosome conformations. This interpretation is supported by other work (30) that indicated a similar major transition in this region of ionic strength (0.1-3 mM NaCl). Additionally, gel mobility experiments of particles prepared at 0.1 mM NaCl, described below, were consistent with two populations of particles. In contrast, a more compact population with the same relative mass was not visualized in electron micrographs at other ionic environments where elongated forms were visualized (e.g. 750 mM NaCl, cf.Fig. 1G and Fig. 3and Table 1, part B). Additionally, for particles prepared at this ionic environment (750 mM NaCl), gel mobility experiments indicated only one migrating form.

Electrophoresis had previously been used to indicate a similar mobility (and by inference, shape) between unfixed and fixed nucleosomes. To assess that the morphology of fixed nucleosomes seen by electron microscopy could be corroborated with that of the same nucleosomes in solution, electrophoresis of these particles in nondenaturing gels was carried out (Fig. 4). In agreement with biophysical theories(39, 40) , slow migration of particles correlated with an elongated shape as determined via electron microscopy, and fast migration correlated with a compact shape. Consistent with this observation, the two forms of nucleosomes visualized at 0.1 mM NaCl were represented by two bands on the electrophoresis gel. At every other ionic concentration, only one band was identified in gel electrophoretic experiments, indicating by this criterion that both two-dimensional and three-dimensional analyses of nucleosome structure were carried out on a single homogeneous conformational form of the nucleosome core particle.

Figure 4: Electrophoretic analysis of nucleosomes from different ionic environments. The migration distance of nucleosomes in a 5% polyacrylamide nondenaturing gel is shown in graph format for nucleosomes optimally fixed at the salt concentrations indicated with formaldehyde and glutaraldehyde to preserve nucleosome conformation prior to analysis(21) . At 0.1 mM NaCl, a second band was present with a migration distance equivalent to that for nucleosomes in 3.5 mM NaCl (open circle; see ``Results'').

Three-dimensional Image Reconstruction of Nucleosomes

The three-dimensional reconstructions are given in Fig. 5. At 10 mM NaCl, the structure of the nucleosome (Fig. 5A) was a prolate form that was consistent with the previous three-dimensional EM reconstruction. At 30 mM NaCl, the structure was virtually spherical with only a slight prolate tendency (Fig. 5B), while at 150 mM NaCl, the reconstructed nucleosome was distinctly oblate, approximating the shape and size of the crystallographic conformation (Fig. 5C). The values of axial height and equatorial diameter in conjunction with radii of gyration calculated from the three-dimensional reconstructions are given in Table 2. The spatial resolution of 30 Å in the three-dimensional reconstructions, determined using a phase residual approach (28) with a cutoff of 45°, a conservative measure of resolution, was isotropic since angular orientations used for each reconstruction were in general randomly distributed. However, the preparation at 10 mM NaCl did exhibit a slight tendency toward displaying side views, while the particles prepared in the presence of 150 mM NaCl tended to display views with a circular profile. To counteract these tendencies, image classification schemes (26, 27) were used to ensure that particular views of the particle were not under-represented.

Figure 5: Three-dimensional structures for nucleosomes in three different ionic environments, determined by electron microscopy and image reconstruction techniques(26, 27, 28) . A-C are structures for the nucleosomes prepared in the presence of 10, 30, and 150 mM NaCl, respectively. These reconstructions have heights and diameters of 108 and 72 Å, 99 and 80 Å, and 80 and 110 Å, respectively. All conformations are essentially circular when viewed from the top. Structures shown correspond to the theoretical volume for the combined nucleic acid and protein components of the nucleosome. The scale bar indicates 75 Å.

DISCUSSION

The compendium of our results indicates directly that the structure of the nucleosome has a propensity to change with ionic environment and shows the existence of numerous different ionic strength-dependent conformations that exhibit statistically significant differences. Our characterization of nucleosome structure by electrophoretic analyses, two-dimensional analyses of images, and three-dimensional electron microscopic structure determination ranges over >4 orders of magnitude of ionic environment and includes the direct determination of the three-dimensional structure of three different nucleosome conformations. The different ionic environments in this study were selected as points beyond and midway between ionic environments indicated in other studies as ionic strengths at which nucleosome structure changes: 0.4 mM NaCl(10) , 1 mM NaCl(7, 8, 11) , 6 mM NaCl(8, 11) , 16 mM NaCl(11) , 50 mM NaCl(11) , 250 mM NaCl(9, 11) , 550 mM NaCl(9, 12) , 950 mM NaCl(11, 12, 13) , and 1450 mM NaCl(12, 13) . However, the corresponding structural changes in those studies were inferred or remained unaddressed.

The conformational changes detected in this study are consistent with other biophysical studies that have investigated nucleosome structure generally within narrow ranges of ionic environments (1, 2, 9) and may be similar in part to structural changes induced by physiologically important charge-modifying post-translational modifications(3, 4, 31, 32, 41, 42) . The potential of the nucleosome to undergo morphological changes is supported by early microscopic studies on polynucleosomes(43) , although most of the non-canonical forms seen were referred to as unfolded(1, 43) , and by other biophysical studies that have indicated that polynucleosomes undergo salt-dependent structural changes(44) . Although comparisons with our work are suggestive, structural changes of polynucleosomes may not correspond directly to conformational changes of mononucleosome particles as investigated here since DNA length has also been found to affect transitions of the particle(8) .

Our nucleosome structures agree with photo-footprinting results that indicate a highly elongated nucleosome structure below 0.3 mM NaCl; moreover, they support proposals of an elongated prolate nucleosome structure at this ionic environment(30) . Above this ionic strength, at 3 mM NaCl, our results indicate that the nucleosome changes to a more prolate and less extended form. This is supported by hydrodynamic and fluorescence studies (7, 8, 10, 11) that indicate a major structural change as ionic strength is increased from 0.3 to 3 mM NaCl. At 10 mM NaCl, a prolate form for the nucleosome was observed in this study by conformational characterization and by three-dimensional reconstruction. Its shape and size are consistent with previous three-dimensional (20) and two-dimensional (21, 22, 23) analyses of images. A unique form for the nucleosome at 10 mM NaCl, distinct from that at lower salt concentrations, should exist since fluorescence and sedimentation studies indicate a change in structure between 3 and 10 mM NaCl(8, 11) . The calculated radius of gyration (20) of 37.9 Å for this 10 mM NaCl reconstruction (Table 2) coincides with the experimental value of 37.6 Å determined by neutron scattering for nucleosomes in a similar ionic environment(45) .

Another, more spherical form for the nucleosome is indicated by this study at 30 mM NaCl. This is supported by observations of a conformational change between 10 and 30 mM NaCl (11) and by neutron scattering experiments that indicate a spherical conformation in a virtually equivalent ionic environment(46) . The radius of gyration for this reconstruction (Table 2) was determined to be 38.5 Å, an increase from the value at 10 mM NaCl. The value determined using neutron scattering is larger as well, 39.4 Å (46) .

Upon further increases in ionic strength to 150 mM NaCl, a major change in nucleosome structure occurs in which its shape becomes oblate. The existence of a modification in conformation upon increasing ionic strength from 30 to 150 mM NaCl is also supported by the work of others(11) . The oblate form detected in this study at an ionic environment resembling physiological conditions (in terms of monovalent cation concentration) approaches the crystallographic nucleosome structure(14, 15, 16, 17) . The three-dimensional reconstruction exhibits the same 110-Å diameter as the crystallographic conformation (15) and also approximates the high resolution core histone octamer structure modeled with DNA(18, 19) . However, each hemisphere of the nucleosome is 10 Å greater in height than that in the crystallographic conformation. This is not necessarily surprising given that the ionic conditions used are not the same as the crystallographic conditions that typically include divalent cations, polyamines, and detergents (14, 15, 16, 17, 18, 19) , resulting in a chemical environment with an overall higher effective ionic strength. In addition, it is possible that the exact edge-on view showing the minimum particle height was under-represented in the electron micrographs and therefore underweighted in the three-dimensional reconstruction. The calculated radius of gyration of 39.1 Å for this reconstruction (Table 2) is close to calculated values that range from 39.2 to 40.9 Å for the particles that have conformations consistent with the canonical crystallographic structure (47, 48, 49) .

A further conformational change to a less oblate form observed in this study by microscopy of particles prepared in the presence of 400 mM NaCl is supported by findings of a transition between 150 and 400 mM NaCl by fluorescence studies(9, 11) . This change is consistent with the overall structural change of the nucleosome characterized by Dong et al.(50) in the range of 100-600 mM NaCl. Using detailed physicochemical analyses in that study, it was concluded that both DNA and histones exhibit changes as salt is increased in this range, consistent with a trend to a more relaxed secondary structure(50) . At still higher ionic strengths, the nucleosome structure undergoes a major elongation and disruption as reflected in our micrographs of the particle and supported by a variety of studies(12, 13, 51) . At 750 mM NaCl and higher ionic strengths, the particle appeared in our study as a highly extended bent rod. Above 950 mM NaCl, H2A-H2B dimer disassociation has been reported using circular dichroism and fluorescence spectroscopy(12, 13) . This is consistent with our micrographs of the particle prepared in the presence of 1200 mM NaCl and evident as a thinning of the nucleosome particle (Fig. 1G and Fig. 3and Table 1, part B), a decrease in mass, as well as the appearance of smaller particles consistent with the relative mass of a H2A-H2B dimer (data not shown). At a still higher ionic strength, 1725 mM NaCl, the structure appears even thinner in micrographs (Fig. 1H and Fig. 3and Table 1, part B) and has a decreased mass, consistent with observations that (H3-H4) tetramer dissociation occurs above 1450 mM NaCl(12, 13) . However, as characterized by Yager and van Holde(52) , the observed nucleosome disassociation is expected to be more acute at the nucleosome concentration of 0.3 A/ml used here than at higher nucleosome concentrations.

In summary, the results presented here indicate that nucleosome structure changes with ionic environment, indicating the effects of pervasive ionic interactions and charge effects(18, 19, 53) . This report provides a structural basis for the study of conformational changes elicited by divalent ions and by specific charge modifications as the result of physiological requirements during the cell cycle or differentiation such as the acetylation, phosphorylation, and poly(ADP-ribosyl)ation of nucleosomes, which result in altered structures(32, 33, 54, 55) . It also explains directly the different structures obtained previously by three-dimensional reconstruction and by crystallography. Moreover, the results establish a consistency between changes in nucleosome conformation seen in structural studies and alterations of the nucleosome observed by genetic and biochemical approaches, which also indicate a dynamic nature for this biochemically active macromolecular complex.

FOOTNOTES

*

This work was supported by the MRC Canada, NCIC, and OCTRF. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§

Recipient of a Steve Fonyo Fellowship from the NCIC. To whom correspondence should be addressed: Div. of Molecular and Structural Biology, Ontario Cancer Inst., 610 University Ave., Toronto, Ontario M5G 2M9, Canada. Tel.: 416-946-2000 (ext. 4929) Fax: 416-946-6529; :gczarnot{at}oci.utoronto.ca.

()

The abbreviations used are: EM, electron microscopy; TEA, triethanolamine.

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