The diameters of frozen-hydrated chromatin fibers increase with DNA linker length: evidence in support of variable diameter models for chromatin

The diameters of chromatin fibers from Thyone briareus (sea cucumber) sperm (DNA linker length, n = 87 bp) and Necturus maculosus (mudpuppy) erythrocytes (n = 48 bp) were investigated. Soluble fibers were frozen into vitrified aqueous solutions of physiological ionic strength (124 mM), imaged by cryo-EM, and measured interactively using quantitative computer image-processing techniques. Frozen-hydrated Thyone and Necturus fibers had significantly different mean diameters of 43.5 nm (SD = 4.2 nm; SEM = 0.61 nm) and 32.0 nm (SD = 3.0 nm; SEM = 0.36 nm), respectively. Evaluation of previously published EM data shows that the diameters of chromatin from a large number of sources are proportional to linker length. In addition, the inherent variability in fiber diameter suggests a relationship between fiber structure and the heterogeneity of linker length. The cryo-EM data were in quantitative agreement with space-filling double-helical crossed-linker models of Thyone and Necturus chromatin. The data, however, do not support solenoid or twisted-ribbon models for chromatin that specify a constant 30 nm diameter. To reconcile the concept of solenoidal packing with the data, we propose a variable-diameter solid-solenoid model with a fiber diameter that increases with linker length. In principle, each of the variable diameter models for chromatin can be reconciled with local variations in linker length.     

Chromatin fibers are left-handed double helices with diameter and mass per unit length that depend on linker length

Four classes of models have been proposed for the internal structure of eukaryotic chromosome fibers--the solenoid, twisted-ribbon, crossed-linker, and superbead models. We have collected electron image and x-ray scattering data from nuclei, and isolated chromatin fibers of seven different tissues to distinguish between these models. The fiber diameters are related to the linker lengths by the equation: D(N) = 19.3 + 0.23 N, where D(N) is the external diameter (nm) and N is the linker length (base pairs). The number of nucleosomes per unit length of the fibers is also related to linker length. Detailed studies were done on the highly regular chromatin from erythrocytes of Necturus (mud puppy) and sperm of Thyone (sea cucumber). Necturus chromatin fibers (N = 48 bp) have diameters of 31 nm and have 7.5 +/- 1 nucleosomes per 10 nm along the axis. Thyone chromatin fibers (N = 87 bp) have diameters of 39 nm and have 12 +/- 2 nucleosomes per 10 nm along the axis. Fourier transforms of electron micrographs of Necturus fibers showed left-handed helical symmetry with a pitch of 25.8 +/- 0.8 nm and pitch angle of 32 +/- 3 degrees, consistent with a double helix. Comparable conclusions were drawn from the Thyone data. The data do not support the solenoid, twisted-ribbon, or supranucleosomal particle models. The data do support two crossed-linker models having left-handed double-helical symmetry and conserved nucleosome interactions.     

Small angle x-ray scattering of chromatin. Radius and mass per unit length depend on linker length.

Analyses of low angle x-ray scattering from chromatin, isolated by identical procedures but from different species, indicate that fiber diameter and number of nucleosomes per unit length increase with the amount of nucleosome linker DNA. Experiments were conducted at physiological ionic strength to obtain parameters reflecting the structure most likely present in living cells. Guinier analyses were performed on scattering from solutions of soluble chromatin from Necturus maculosus erythrocytes (linker length 48 bp), chicken erythrocytes (linker length 64 bp), and Thyone briareus sperm (linker length 87 bp). The results were extrapolated to infinite dilution to eliminate interparticle contributions to the scattering. Cross-sectional radii of gyration were found to be 10.9 +/- 0.5, 12.1 +/- 0.4, and 15.9 +/- 0.5 nm for Necturus, chicken, and Thyone chromatin, respectively, which are consistent with fiber diameters of 30.8, 34.2, and 45.0 nm. Mass per unit lengths were found to be 6.9 +/- 0.5, 8.3 +/- 0.6, and 11.8 +/- 1.4 nucleosomes per 10 nm for Necturus, chicken, and Thyone chromatin, respectively. The geometrical consequences of the experimental mass per unit lengths and radii of gyration are consistent with a conserved interaction among nucleosomes. Cross-linking agents were found to have little effect on fiber external geometry, but significant effect on internal structure. The absolute values of fiber diameter and mass per unit length, and their dependencies upon linker length agree with the predictions of the double-helical crossed-linker model. A compilation of all published x-ray scattering data from the last decade indicates that the relationship between chromatin structure and linker length is consistent with data obtained by other investigators.     

The superstructure of chromatin and its condensation mechanism

Electric dichroism and X-ray scattering measurements on solutions of uncondensed and condensed chicken erythrocyte chromatin were interpreted on the basis of model calculations. Information about the state of uncondensed fibers in the conditions of electric dichroism measurements was obtained from scattering patterns recorded as a function of pH, in the presence of spermine and at very low monovalent cation concentrations. Electric dichroism measurements on a complex of uncondensed chromatin with methylene blue were made to determine the contribution of the linker and of the nucleosomes to the total dichroism.A new approach to calculate the dichroism from realistic structural models, which also yields other structural parameters (radius of gyration, radius of gyration of the cross-section, mass per unit length) was used. Only a restricted range of structures is simultaneously compatible with all experimental results. Further, it is shown that previous interpretations of dichroism measurements on chromatin were in contradiction with X-ray scattering data and failed to take into account the distribution of orientation of the nucleosomes in the fibers. When this is done, it is found that the linker DNA in chicken erythrocyte and sea urchin chromatin must run nearly perpendicularly to the fibre axis. Taken together with the dependence of the fibre diameter on the linker length, these results provede the strongest evidence hitherto available for a model in which the linker crosses the central part of the fibre.     

Computer simulation of the 30-nanometer chromatin fiber

A new Monte Carlo model for the structure of chromatin is presented here. Based on our previous work on superhelical DNA and polynucleosomes, it reintegrates aspects of the "solenoid" and the "zig-zag" models. The DNA is modeled as a flexible elastic polymer chain, consisting of segments connected by elastic bending, torsional, and stretching springs. The electrostatic interaction between the DNA segments is described by the Debye-Hückel approximation. Nucleosome core particles are represented by oblate ellipsoids; their interaction potential has been parameterized by a comparison with data from liquid crystals of nucleosome solutions. DNA and chromatosomes are linked either at the surface of the chromatosome or through a rigid nucleosome stem. Equilibrium ensembles of 100-nucleosome chains at physiological ionic strength were generated by a Metropolis-Monte Carlo algorithm. For a DNA linked at the nucleosome stem and a nucleosome repeat of 200 bp, the simulated fiber diameter of 32 nm and the mass density of 6.1 nucleosomes per 11 nm fiber length are in excellent agreement with experimental values from the literature. The experimental value of the inclination of DNA and nucleosomes to the fiber axis could also be reproduced. Whereas the linker DNA connects chromatosomes on opposite sides of the fiber, the overall packing of the nucleosomes leads to a helical aspect of the structure. The persistence length of the simulated fibers is 265 nm. For more random fibers where the tilt angles between two nucleosomes are chosen according to a Gaussian distribution along the fiber, the persistence length decreases to 30 nm with increasing width of the distribution, whereas the other observable parameters such as the mass density remain unchanged. Polynucleosomes with repeat lengths of 212 bp also form fibers with the expected experimental properties. Systems with larger repeat length form fibers, but the mass density is significantly lower than the measured value. The theoretical characteristics of a fiber with a repeat length of 192 bp where DNA and nucleosomes are connected at the core particle are in agreement with the experimental values. Systems without a stem and a repeat length of 217 bp do not form fibers.     

Higher-order structure of long repeat chromatin.

The higher-order structure of chromatin isolated from sea urchin sperm, which has a long nucleosomal DNA repeat length (approximately 240 bp), has been studied by electron microscopy and X-ray diffraction. Electron micrographs show that this chromatin forms 300 A filaments which are indistinguishable from those of chicken erythrocytes (approximately 212 bp repeat); X-ray diffraction patterns from partially oriented samples show that the edge-to-edge packing of nucleosomes in the direction of the 300 A filament axis, and the radial disposition of nucleosomes around it, are both similar to those of the chicken erythrocyte 300 A filament, which is described by the solenoid model. The invariance of the structure with increased linker DNA length is inconsistent with many other models proposed for the 300 A filament and, furthermore, means that the linker DNA must be bent. The low-angle X-ray scattering in the 300-400 A region both in vitro and in vivo differs from that of chicken erythrocyte chromatin. The nature of the difference suggests that 300 A filaments in sea urchin sperm in vivo are packed so tightly together that electron-density contrast between individual filaments is lost; this is consistent with electron micrographs of the chromatin in vitro.     

Higher-order structure of nucleosome oligomers from short-repeat chromatin

Sedimentation measurements and electron microscopy at a series of ionic strengths suggest that chromatin from neurons of the cerebral cortex is able to form condensed structures in vitro that are probably several turns of a solenoid with about six nucleosomes per turn. Since neuronal chromatin has a short nucleosomal repeat (approximately 165 bp) allowing virtually no linker DNA between nucleosomes, and yet forms apparently 'normal' elements of solenoid, the packing of nucleosomes in the solenoid must be highly constrained. This permits only a limited number of possible models, and enables tentative suggestions to be made about the location of the linker DNA in the typical solenoid.     

Linear dichroism of chromatin fibers

The optical anisotropy of chromatin with different length of the linker DNA isolated from a variety of sources (Friend erythroleukemia cells, calf thymus, hen erythrocytes and sea urchin sperm) has been studied in a large range of mono- and bivalent cations by the use of flow linear dichroism and electric dichroism. We have found that all chromatins studied displayed negative LD values in the range of 0.25 mM EDTA--2 mM NaCl and close positive values in the range of 2-100 mM NaCl. Mg2+ cations, in contrast to Na+ cations, induce optically isotropic chromatin fibers. All chromatin samples exhibit positive form effect amounting to 5-10% of LD amplitude observed at 260 nm. This form effect is determined by the anisotropic scattering of polarized light by single chromatin fibers. The conformational transition at 2 mM NaCl leads to the distortion of chromatin filament structure. The reversibility of this distortion depends on the length of the linker DNA--for chromatins with linker DNA 10-30 b.p. it is partially reversible, while for preparations with longer linker DNA it is irreversible. Relatively low electric fields do not have an effect on chromatin structure, while higher electric fields (more than 7 kV/cm) distort the structure of chromatin.     

A defined structure of the 30 nm chromatin fibre which accommodates different nucleosomal repeat lengths

Earlier work on the condensation of chromatins of different repeat lengths into the 30 nm fibre has been surveyed and it is shown that the external geometry of the fibre must be the same for all the chromatins. This can only be fitted by a helical coiling of nucleosomes into a solenoid with the linker DNA disposed internally. On this basis, various models were calculated and compared with published electric dichroism data. The only good fit is found with a 'reverse-loop' model, where the linker DNA forms a complete turn into the hole of the solenoid, of opposite hand to the nucleosomal DNA superhelix. This gives a topological linking number of one per nucleosome and would resolve the 'linking number paradox' if the DNA screw is the same in chromatin as in solution. The feasibility of a reverse-loop for short linkers (down to 15 base pairs) was investigated by model building and kinks of approximately 120 degrees into both DNA grooves are described, which will allow such packing. There will, however, be a 'forbidden' range for the linker DNA length, between approximately 1 and 14 bp, corresponding to nucleosomal repeats of 163 and 176 bp.     

Higher-order structures of chromatin in solution.

Neutron scatter studies have been made on gently prepared chicken erythrocyte chromatin over a range of ionic strength. At low ionic strength the mass per unit length of the '10 nm nucleofilament corresponds to one nucleosome per 8--12 nm and a DNA packing ratio of between 6 and 9. From the contrast dependence of the cross-section radius of gyration of the nucleofilament the following parameters have been obtained; RgDNA' the cross-section radius of gyration (Rg) when DNA dominates the scatter; RgP, the cross-section Rg when protein dominates the scatter; Rc, the cross-section Rg at infinite contrast and alpha, the constant which describes the dependence of the cross-section Rg on contrast variation. From our understanding of the structure of the core particle, various arrangement of core particles in the nucleofilament have been tested. In models consistent with the above parameters the core particles are arranged edge-to-edge or with the faces of the core particles inclined to within 20 degrees to the axis of the nucleofilament. With increase of ionic strength the transition to the second-order chromatin structure has been followed. This gave the interesting result that above 20 microM NaCL or 0.4 mM MgCL2 the cross-section Rg increases abruptly to about 9 nm with a packing ratio of 0.2 nucleosome/mn and with further increase of ionic strength the Rg increases to 9.5 nm while the packing ratio increases threefold to 0.6 nucleosome/nm. This suggests a family of supercoils of nucleosomes which contract with increasing ionic strength. In its most contracted form the diameter of the hydrated supercoil has been found from the radial distribution function to be 34 nm. Models for the arrangements of core particles in the 34-nm supercoil are discussed.     

Higher order structure of chromatin: evidence from photochemically detected linear dichroism

We have used photochemically detected linear dichroism to measure the separate average angular orientations of nucleosomes and linker DNA in 30-nm chromatin fibers of varying linker size (20-80 base pairs). Our results indicate that the average tilt angles vary with linker size, but not in a monotonic manner, suggesting that the constancy of geometry of the 30-nm fiber is maintained by compensatory changes of nucleosomal tilt which accommodate packing of variable lengths of linker DNA. We discuss the compatibility of our results with the various classes of models that have been proposed for the 30-nm fiber, including the continuous solenoid model and models built from the basic unit of the zig-zag ribbon. Many models can be eliminated, and all have to be modified to fit our results for chromatins with very long linkers.     

Chromatin fiber dimensions and nucleosome orientation: a neutron scattering investigation.

Chromatin fibers were studied in solutions of mM monovalent salt by small angle neutron scattering. The variation of the cross section radius of gyration with H2O/D2O contrast shows that DNA is at much larger average radial distances from the fiber axis than histone. Consequently, the coils of DNA in a core particle must be approximately parallel to the fiber direction. The radii of gyration suggest that the maximum diameter of chromatin and nucleosomes is approximately 14 nm and that the DNA id distributed in two radial layers. The concentration dependence of the scattering maxima near 14 nm spacings furnishes independent support for a 14 nm external diameter and can be interpreted by a double DNA layer configuration.     

Optical anisotropy of chromatin. Flow linear dichroism and electric dichroism studies

The optical anisotropy of chromatin with different length of the linker DNA isolated from a variety of sources (Frend erythroleukemia cells, calf thymus, hen erythrocytes and sea urchin sperm) has been studied in a large range of mono- and bivalent cations concentrations by the use of flow linear dichroism (LD) and electric dichroism. We have found that all chromatins studied displayed negative LD values in the range of 0.25 mM EDTA - 2 mM NaCl and close positive values in the range of 2-100 mM NaCl. Mg2+ cations, in contrast to Na+ cations, induce optically isotropic chromatin fibers. All chromatin samples exhibit positive form effect amounting to 5-10% of LD amplitude observed at 260 nm. This form effect is determined by the anisotropic scattering of polarized light by single chromatin fibers. The conformational transition at 2 mM NaCl leads to the distortion of chromatin filament structure. The reversibility of this distortion depends on the length of the linker DNA - for chromatins with the linker DNA of 10-30 b.p. it is parially reversible, while for preparations with longer linker DNA it is irreversible. Relatively low electric field does not affect chromatin structure, while higher electric field (more than 7 kV/cm) distorts the structure of chromatin. Presented results explain the contradictory data obtained by electrooptical and hydrooptical methods.     

Higher order structure of chromatin: orientation of nucleosomes within the 30 nm chromatin solenoid is independent of species and spacer length

We have used electric dichroism to study the arrangement of nucleosomes in 30 nm chromatin solenoidal fibers prepared from a variety of sources (CHO cells, HeLa cells, rat liver, chicken erythrocytes, and sea urchin sperm) in which the nucleosome spacer length varies from approximately 10 to approximately 80 bp. Field-free relaxation times are consistent only with structures containing 6 +/- 1 nucleosomes for every 11 nm of solenoidal length. With very few assumptions about the arrangement of the spacer DNA, our dichroism data are consistent with the same orientation of the chromatosomes for every chromatin sample examined. This orientation, which maintains the faces of the radially arranged chromatosomes inclined at an angle between 20 degrees-33 degrees to the solenoid axis, thus appears to be a general structural feature of the higher order chromatin fiber.     

A critical role for linker DNA in higher-order folding of chromatin fibers

Nucleosome-nucleosome interactions drive the folding of nucleosomal arrays into dense chromatin fibers. A better physical account of the folding of chromatin fibers is necessary to understand the role of chromatin in regulating DNA transactions. Here, we studied the unfolding pathway of regular chromatin fibers as a function of single base pair increments in linker length, using both rigid base-pair Monte Carlo simulations and single-molecule force spectroscopy. Both computational and experimental results reveal a periodic variation of the folding energies due to the limited flexibility of the linker DNA. We show that twist is more restrictive for nucleosome stacking than bend, and find the most stable stacking interactions for linker lengths of multiples of 10 bp. We analyzed nucleosomes stacking in both 1- and 2-start topologies and show that stacking preferences are determined by the length of the linker DNA. Moreover, we present evidence that the sequence of the linker DNA also modulates nucleosome stacking and that the effect of the deletion of the H4 tail depends on the linker length. Importantly, these results imply that nucleosome positioning in vivo not only affects the phasing of nucleosomes relative to DNA but also directs the higher-order structure of chromatin.     

The superstructure of chromatin and its condensation mechanism

Comparison between the internucleosomal distance found by X-ray solution scattering for chicken erythrocyte (23 nm) and sea urchin (30 nm) chromatin indicates that this distance is proportional to the linker length. The diameter of the condensed sea urchin chromatin fibers is about 45 nm which is significantly larger than in chicken erythrocyte chromatin (35 nm). Trivalent cations (Gd, Tb, Cr) and the polyamines spermine and spermidine were found to induce compaction at much lower concentrations than the divalent cations but Gd, Tb and Cr induce aggregation before full compaction of the fibers. The influence of hydrogen bonding is illustrated by comparison of the effects of NaCl, ammonium chloride and alkylammonium chlorides on condensation. Solubility experiments indicate that there is a nearly linear dependence of the Mg^-- concentration at which precipitation occures on chromatin concentration and confirm the differences between cations observed by X-ray scattering.The chicken erythrocyte chromatin samples were further characterized by their reduced electric dichroism. The values found are consistent with the model derived from X-ray scattering and are compared with those reported in the literature.     

The fine structure of chromatin in Paranosema grylli (Microsporida)

Nucleosomes were found for the first time in the nuclear chromatin of Microsporida--organisms known among the smallest eukaryotes on Earth. Chromatin of Paranosema grylli sporoplasm was studied by Miller's technique. On low ionic-strength cell spreads, this chromatin was represented by 10 nm nucleosome filaments, 20 nm filaments, and "smooth" (nucleosome-free) filaments of 3-4 nm in diameter. Nucleosome filaments display structural heterogeneity seen as irregular arrangement of nucleosome particles along the filament length. Different nucleosome filaments show 13-30 nucleosomes per 1 microm with the length of linker DNA ranging from 10 to 45 nm. The present results suggest that microsporidian chromatin is weakly condensed. Only lower-order chromatin packaging levels displayed some structural peculiarities.     

The condensation of chromatin and histone H1-depleted chromatin by spermine

At low ionic strength, spermine induces aggregation of native and H1-depleted chromatin at spermine/phosphate (Sp/P) ratios of 0.15 and 0.3, respectively. Physico-chemical methods (electric dichroism, circular dichroism and thermal denaturation) show that spermine, at Sp/P less than 0.15, does not appreciably alter the conformation of native chromatin and interacts unspecifically with all parts of chromatin DNA (linker as well as regions slightly or tightly bound to histones). In chromatin, the role of spermine could be more important in the stabilization of higher-order structure than in the condensation of the 30 nm solenoid. The addition of spermine to H1-depleted chromatin revealed two important features: (i) spermine can partially mimic the role of histone H1 in the condensation of chromatin; (ii) the core histone octamer does not appear to play any role in the aggregation process by spermine as DNA and H1-depleted chromatin aggregate at the same Sp/P ratio.     

Chromatin fibers observed in situ in frozen hydrated sections. Native fiber diameter is not correlated with nucleosome repeat length

Chromatin fibers have been observed and measured in frozen hydrated sections of three types of cell (chicken erythrocytes and sperm of Patiria miniata and Thyone briareus) representing an approximately 20- bp range of nucleosomal repeat lengths. For sperm of the starfish P. miniata, it was possible to obtain images of chromatin fibers from cells that were swimming in seawater up to the moment of cryo- immobilization, thus providing a record of the native morphology of the chromatin of these cells. Glutaraldehyde fixation produced no significant changes in the ultrastructure or diameter of chromatin fibers, and fiber diameters observed in cryosections were similar to those recorded after low temperature embedding in Lowicryl K11M. Chromatin fiber diameters measured from cryosections of the three types of nuclei were similar, a striking contrast to the situation for chromatin isolated from these cell types, where a strong positive correlation between diameter and nucleosomal repeat length has been established. The demonstration of chromatin fibers in unfixed whole cells establishes an unequivocal baseline for the study of native chromatin and chromosome architecture. The significant differences between chromatin fibers in nucleo and after isolation supports a previous observation (P. J. Giannasca, R. A. Horowitz, and C. L. Woodcock. 1993. J. Cell Sci. 105:551-561), and suggests that structural studies on isolated material should be interpreted with caution until the changes that accompany chromatin isolation are understood.     

Chromatin dynamics of unfolding and refolding controlled by the nucleosome repeat length and the linker and core histones

Chromatin is composed of genomic DNA and histones, forming a hierarchical architecture in the nucleus. The chromatin hierarchy is common among eukaryotes despite different intrinsic properties of the genome. To investigate an effect of the differences in genome organization, chromatin unfolding processes were comparatively analyzed using Schizosaccaromyces pombe, Saccharomyces cerevisiae, and chicken erythrocyte. NaCl titration showed dynamic changes of the chromatin. 400-1000 mM NaCl facilitated beads with approximately 115 nm in diameter in S. pombe chromatin. A similar transition was also observed in S. cerevisiae chromatin. This process did not involve core histone dissociation from the chromatin, and the persistence length after the transition was approximately 26 nm for S. pombe and approximately 28 nm for S. cerevisiae, indicating a salt-induced unfolding to "beads-on-a-string" fibers. Reduced salt concentration recovered the original structure, suggesting that electrostatic interaction would regulate this discrete folding-unfolding process. On the other hand, the linker histone was extracted from chicken chromatin at 400 mM NaCl, and AFM observed the "beads-on-a-string" fibers around a nucleus. Unlike yeast chromatin, therefore, this unfolding was irreversible because of linker histone dissociation. These results indicate that the chromatin unfolding and refolding depend on the presence and absence of the linker histone, and the length of the linker DNA.