Structural properties of barley nucleosomes

The structural properties of barley oligonucleosomes are investigated and compared to those of rat liver oligomers. Extraction of barley chromatin was performed using mild nuclease digestion of isolated nuclei leading to a low ionic strength soluble fraction. Oligonucleosomes were fractionated on sucrose gradients and characterized for DNA and histone content. Physico-chemical studies (sedimentation, circular dichroism and electric birefringence) showed that barley oligonucleosomes exhibit properties very close to those of the H1-depleted rat liver counterparts. Moreover, in situ, barley linker DNA was more sensitive to micrococcal nuclease digestion than that of rat liver. These results suggest that barley oligonucleosomes show a less compact structure than their rat liver counterparts and appear to be in contradiction with the very condensed organization of barley chromatin previously suggested.     

Nucleosomes are assembled into discrete size structures by histone H1 in vitro

The involvement of histone H1 in the formation and maintenance of higher order chromatin structures in vitro was investigated biochemically. Addition of exogenous histone H1 to isolated calf thymus mononucleosomes in low ionic strength buffer resulted in the formation of electrophoretically distinct mononucleosome assemblies (supernucleosomes). The smaller supernucleosomes were composed of about 12, 18, 24, or 30 nucleosomes and one to two molecules of histone H1 per nucleosome. It was difficult to determine accurately the size of the larger supernucleosomes, but their bands from native gels contained probably between 60 and 300 nucleosomes or more. Similar supernucleosome size classes were also obtained when oligonucleosomes instead of mononucleosomes were employed. When the assembly of mono- and oligo-nucleosomes with histone H1 took place in 0.15 M NaCl, discrete supernucleosomes containing only mono- or di-nucleosomes, but not a mixture of both, were formed. It is proposed that the small supernucleosomes containing oligomers of 6 nucleosomes may represent integral multiples of the second-order chromatin structural subunit, whereas the larger supernucleosomes containing about 60 to 300 or more nucleosomes may correspond to chromatin domains or third-order chromatin structures observed by other techniques.     

The structural organization of oligonucleosomes

We have used electric birefringence to study the structure of oligonucleosomes and to show the influence of histone H1 depletion on their conformation in solution. Measurements are made at low ionic strength on monodisperse samples containing up to 8 nucleosomes. For each oligomer, having H1 or not, the analysis of both relaxation and orientation times gives information about the particle's orientation mechanism through the ratio r of permanent over induced dipole terms. For native oligomers, the data confirm the previous finding of a discontinuity in hydrodynamic behavior between pentamer and heptamer: the rotational times are multiplied by 10 and r increases from 0.2 to 0.7 showing the appearance of a non-negligible contribution of a permanent dipole to the orientation mechanism. We suggest a model for the hexanucleosome at low ionic strength and discuss its implications for the higher-order structure of chromatin. The treatment for H1 depletion abolishes the transitions in electro-optical properties: the value of r remains constant, r = 0.15, and both rotational times increase progressively with the number of nucleosomes in the chain. That reflects an important unfolding of oligonucleosomal structure which we attributed to the unwinding of DNA tails and internucleosomal segments. The disc planes of nucleosomes become closely parallel to the nucleosomal chain axis.     

The Structural Organization of Oligonucleosomes

Abstract

We have used electric birefringence to study the structure of oligonucleosomes and to show the influence of histone H1 depletion on their conformation in solution. Measurements are made at low ionic strength on monodisperse samples containing up to 8 nucleosomes. For each oligomer, having H1 or not, the analysis of both relaxation and orientation times gives information about the particle's orientation mechanism through the ratio r of permanent over induced dipole terms. For native oligomers, the data confirm the previous finding of a discontinuity in hydrodynamic behavior between pentamer and heptamer: the rotational times are multiplied by 10 and r increases from 0.2 to 0.7 showing the appearance of a non-negligible contribution of a permanent dipole to the orientation mechanism. We suggest a model for the hexanucleosome at low ionic strength and discuss its implications for the higher-order structure of chromatin.

The treatment for H1 depletion abolishes the transitions in electro-optical properties: the value of r remains constant, r=0.15, and both rotational times increase progressively with the number of nucleosomes in the chain. That reflects an important unfolding of oligonucleosomal structure which we attributed to the unwinding of DNA tails and internucleosomal segments. The disc planes of nucleosomes become closely parallel to the nucleosomal chain axis.     

Different mechanism for in vitro formation of nucleosome core particles

The interaction of different histone oligomers with nucleosomes has been investigated by using nondenaturing gel electrophoresis. In the presence of 0.2 M NaCl, the addition of the pairs H2A,H2B or H3,H4 or the four core histones to nucleosome core particles produces a decrease in the intensity of the core particle band and the appearance of aggregated material at the top of the gel, indicating that all these histone oligomers are able to associate with nucleosomes. Equivalent results were obtained by using oligonucleosome core particles. Additional electrophoretic results, together with second-dimension analysis of histone composition and fluorescence and solubility studies, indicate that H2A,H2B, H3,H4, and the four core histones can migrate spontaneously from the aggregated nucleosomes containing excess histones to free core DNA. In all cases the estimated yield of histone transfer is very high. Furthermore, the results obtained from electron microscopy, solubility, and supercoiling assays demonstrate the transfer of excess histones from oligonucleosomes to free circular DNA. However, the extent of solubilization obtained in this case is lower than that observed with core DNA as histone acceptor. Our results demonstrate that nucleosome core particles can be formed in 0.2 M NaCl by the following mechanisms: (1) transfer of excess core histones from oligonucleosomes of free DNA, (2) transfer to excess H2A,H2B and H3,H4 associated separately with oligonucleosomes to free DNA, (3) transfer to excess H2A,H2B initially associated with oligonucleosomes to DNA, followed by the reaction of the resulting DNA-(H2A,H2B) complex with oligonucleosomes containing excess H3,H4, and (4) a two-step transfer reaction similar to that indicated in (3), in which excess histones H3,H4 are transferred to DNA before the reaction with oligonucleosomes containing excess H2A,H2B. The possible biological implications of these spontaneous reactions are discussed in the context of the present knowledge of the nucleosome function.     

Distribution of H1 histone in chromatin digested by micrococcal nuclease.

The relative amount of H1 histone associated with isolated nucleosomes from calf thymus was determined as a function of the extent of DNA digestion by micrococcal nuclease. Generally the amount of H1 histone associated with mononucleosomes decreases with increasing digestion until 60% of the original H1 remains associated with DNA 150 base pirs or less in size. Coincidentally, H1 histone increases relative to the other histones in aggregated material that sediments through sucrose gradients to form a pellet. However, the level of H1 histone remains at control values for oligonucleosomes (dimer to hexamer) over the 30% digestion range studied. An increase in ionic strength to 0.3 M NaCl in the density gradient reveals a different pattern of H1 binding, whereby the amount of H1 reflects the average size of the DNA fragments with which it is associated. Although there is significant binding to nucleosomes per se, it appears that the major ionic involvement of H1 is with internucleosomal spacer DNA.     

On the de novo formation of compact oligonucleosomes at high ionic strength. Evidence for nucleosomal sliding in high salt.

Histone H 1-depleted chromatin made from acid extracted, intact nuclei was exposed to various ionic strengths. NaCl concentrations above 0.3 M sufficed to generate novel oligonucleosomes formerly characterized as "compact oligomers" and "spacerless dinucleosomes". Such particles could not be identified within H 1-depleted nuclei or chromatin at low ionic strengths. Their formation, proceeding within days at 0 degrees C, was accelerated by increasing ionic strengths. The data was discussed in terms of a salt-induced motion of nucleosomal core particles along the DNA to form compact oligomers.     

Exchange of proteins during immunofractionation of chromatin

The migration and rearrangement of chromosomal proteins during immunofractionation of chromatin has been investigated. Oligonucleosomes from two different chromatins, chicken erythrocyte or rat liver, were mixed with oligonucleosomes from the other species which had been depleted of histones H1/H5 and high mobility group proteins (HMGs). The mixture was treated with buffers of various ionic strengths and immunofractionated on an anti-H1 degrees/H5 or anti-HMG-17 IgG-Sepharose column. The type of DNA, which was retained as the bound fraction on the column, was determined by slot blot analysis using nick-translated repetitive DNA probes from either chicken or rat. The results indicate that in low ionic strength buffers (i.e., below 40 mM NaCl), there is very little exchange of either histone H5 or HMG-17 among nucleosomes and therefore we suggest that it is possible to fractionate nucleosomes according to their antigenic content.     

Unravelled nucleosomes,nucleosome beads and higher order structures of chromatin: Influence of non-histone components and histone H1

Effects of non-histone components and histone H1 on the morphology of nucleosomes and chromatin were studied by electron microscopy. Soluble rat liver ehromatin was depleted of non-histone components [NH]or non-histone components and H1 [NH and H1] by dissociation and subsequent fractionation in sucrose gradients in the presence of 300 to 350 mm or 500 mm-NaCl, respectively. In reconstitution experiments the depleted samples were mixed either with [NH] or with [NH and H1] or with purified H1. The morphology of the ionic strength-dependent condensation of the samples was monitored by electron microscopy using 0 mm to 100 mm-NaCl. Based on the appearance of the different types of fibres in very low salt (0 mm up to 10 mm-NaCl), namely the zigzag-shaped, the beads-on-a-string or the DNA-like filaments, it is possible to distinguish between nucleosomes, partially unravelled nucleosomes and unravelled nucleosomes, respectively. Only those fibres which were zigzag-shaped at low ionic strength condense at increasing ionic strength into higher order structures of compact fibres. We demonstrate the dependence of the appearance of nucleosomes and chromatin upon its composition and upon the ionic strength of the solvent.[NH] have no detectable influence upon the formation of higher order chromatin structures, but they can prevent the unravelling of nucleosomes at very low ionic strength, presumably by charge shielding.For the appearance of zigzag-shaped fibres and for the condensation into compact fibres with increasing ionic strength, H1 must be present in about native amounts. Partial removal of H1 (about 10%) promotes a change from fibres into tangles. This supports the model that an H1 polymer is stabilizing the higher order chromatin structures.Reconstitution experiments with purified H1 regenerated fibres containing all the features of [NH]-depleted chromatin. Reconstitution experiments with [NH and H1] promoted fibres compatible with control chromatin. Overloading of chromatin with H1 led to additional condensation. The detailed morphology of the reconstituted fibres showed local distortions. One possibility explaining these local distortions would be competition between “main” and “additional” binding sites for histone H1.     

Histone H5 promotes the association of condensed chromatin fragments to give pseudo-higher-order structures

We describe two distinct situations in which chicken erythrocyte chromatin fragments associate in solution. The erythrocyte-specific histone H5 is implicated since chromatins that do not contain H5 do not show this behaviour. Well-defined oligomers of between approximately 6 and approximately 18 nucleosomes prepared at low ionic strength condense and associate when the ionic strength is raised to 75 mM, forming pseudo-higher-order structures. The associated forms, probably predominantly dimers, are stabilized by migration of about 10% of the H5, and of the minor lysine-rich histone H1, from the non-associated forms, probably reflecting the preference of H5 for higher-order structures observed previously [Thomas, J. O. and Rees, C. (1983) Eur. J. Biochem. 134, 109-115]. Since the final (H1 + H5) content of the aggregate at 75 mM is never higher than that of the fragment prepared at low ionic strength, migration is probably to a small proportion of sites that have inevitably become vacant due to handling losses at the higher (but not at low) ionic strength. H5 thus maximizes its interactions in the condensed state of chromatin and even maintains the association of two or more fragments without continuity of the DNA. Aggregates of oligomers larger than about 18 nucleosomes may be too long to withstand hydrodynamic shear forces in the absence of such continuity. During nuclease digestion of nuclear chromatin, H5 and, to a lesser extent, H1, are released from the ends of very short fragments and bind to larger oligomers of various sizes leading to heterogeneous aggregates that survive exposure to low ionic strength. These aggregates, in contrast to those described above, have up to 60% more H5 and 20% more H1 than chromatin prepared at low ionic strength. Whether the excess H5 and H1 bind non-specifically or to a second low-affinity binding site on each nucleosome is not known. The associated forms described above (1) are well defined and potentially useful for structural studies, whereas the other aggregates (2) seem less likely to be directly relevant to the native structure of chromatin.     

Structure of the chromatin with long linker DNA

The method of velocity sedimentation have been used to investigate ionic-strength-induced compaction of sea urchin sperm chromatin characterized by extremely long linker DNA (100 b.p.). The dependence of sedimentation coefficients of oligonucleosomes on the number of nucleosomes in the chain have been studied in the range of ionic strength from 0.005 to 0.085. Analysis of these data indicates that such structural parameters of sea urchin sperm chromatin fibre as the diameter of the chain and the length of the chain per nucleosome are quite similar to those of chromatin with shorter linker DNA, but the DNA packing ratio is higher. The structure of sea urchin sperm oligonucleosomes agrees well with the model of three-dimensional zig-zag-shaped chain with linker DNA forming a loop. The possible role of alpha-helical regions of the C-terminal domain of sea urchin sperm histone H1 in the long linker DNA folding is discussed.     

Involvement of histone H1 in the organization of the nucleosome and of the salt-dependent superstructures of chromatin

We describe the results of a systematic study, using electron microscopy, of the effects of ionic strength on the morphology of chromatin and of H1-depleted chromatin. With increasing ionic strength, chromatin folds up progressively from a filament of nucleosomes at approximately 1 mM monovalent salt through some intermediate higher- order helical structures (Thoma, F., and T. Koller, 1977, Cell 12:101- 107) with a fairly constant pitch but increasing numbers of nucleosomes per turn, until finally at 60 mM (or else in approximately 0.3 mM Mg++) a thick fiber of 250 A diameter is formed, corresponding to a structurally well-organized but not perfectly regular superhelix or solenoid of pitch approximately 110 A as described by Finch and Klug (1976, Proc. Natl. Acad. Sci. U.S.A. 73:1897-1901). The numbers of nucleosomes per turn of the helical structures agree well with those which can be calculated from the light-scattering data of Campbell et al. (1978, Nucleic Acids Res. 5:1571-1580). H1-depleted chromatin also condenses with increasing ionic strength but not so densely as chromatin and not into a definite structure with a well-defined fiber direction. At very low ionic strengths, nucleosomes are present in chromatin but not in H1-depleted chromatin which has the form of an unravelled filament. At somewhat higher ionic strengths (greater than 5 mM triethanolamine chloride), nucleosomes are visible in both types of specimen but the fine details are different. In chromatin containing H1, the DNA enters and leaves the nucleosome on the same side but in chromatin depleted of H1 the entrance and exit points are much more random and more or less on opposite sides of the nucleosome. We conclude that H1 stabilizes the nucleosome and is located in the region of the exit and entry points of the DNA. This result is correlated with biochemical and x-ray crystallographic results on the internal structure of the nucleosome core to give a picture of a nucleosome in which H1 is bound to the unique region on a complete two-turn, 166 base pair particle (Fig. 15). In the formation of higher-order structures, these regions on neighboring nucleosomes come closer together so that an H1 polymer may be formed in the center of the superhelical structures.     

The structure of chromatin from the pigeon brain. II. Sedimentation analysis of oligonucleosome density

Compaction of pigeon brain and rat thymus chromatin differing in the length of the linker DNA has been studied by the method of velocity sedimentation. The dependence of sedimentation coefficients of oligonucleosomes on the number of nucleosomes in the chain in solution of different ionic strength (0.005-0.085) has been analyzed. The analyses of these dependences showed that the structure of oligonucleosomes of both cell types at low ionic conditions may be described by the model of a zig-zag-shaped nucleosomal chain. The process of compaction of the oligonucleosomes at higher ionic strength (0.045-0.085) proceeds similarly for brain and thymus chromatin. The formation of a superhelical structure is determined by the interaction of no less than 6 nucleosomes; the compactness of the structure is significantly increased when the number of nucleosomes in the chain exceeds 10. The ability of the brain oligonucleosomes to form a compact structure despite the short linker allow the suggestion that in brain short chromatin the DNA chain does not form two complete turns in the nucleosome. This provides necessary flexibility of brain chromatin.     

Iodination of nucleosomes at low ionic strength: conformational changes in H4 and stabilization by H1.

Radioactive iodine has been used to probe the relative reactivities of nucleosomal H4 tyrosine residues under various conditions of subphysiological ionic strength. We observe that tyrosine 72 of H4, which is not reactive over the range 20-150 mM NaCl, becomes the predominant site of iodination within H4 when nucleosomes are subjected to conditions of very low ionic strength. Conversely, the other H4 tyrosine residues, which are reactive within nucleosomes in solutions of moderate ionic strength (20-150 mM NaCl), become nonreactive when the ionic strength is reduced. This "flip-flop" in the H4 iodination pattern is the manifestation of a reversible nucleosomal conformational change. A method is presented which enables the conformational status of H4 in nucleosomes to be determined by simply electrophoresing the histones on a Triton gel after probing nucleosomes with labeled iodine. Using this technique, we demonstrate that the presence of H1 on one side of the nucleosome stabilizes a histone core domain on the other side so that all four tyrosines of H4 are maintained in their physiological ionic strength conformation even under conditions of no added salt.     

Changes in chromatin folding in solution

The formation of higher order structures by nucleosome oligomers of graded sizes with increasing ionic strength has been studied in solution, by measuring sedimentation coefficients. Nucleosome monomers and dimers show no effect of ionic strength at the concentrations used, while trimers to pentamers show a linear dependence of the logarithm of sedimentation coefficient upon the logarithm of ionic strength between 5 and 25 mm, but no dependence above 25 mm. Between pentamer and hexamer a change occurs and the linear relationship is observed up to ionic strength 125 mm with hexamer and above.The simple power-law dependence of the sedimentation coefficient upon the ionic strength (s ∝ Iⁿ) is observed up to nucleosome 30mers, but by 60mer a jump in the sedimentation coefficient occurs between ionic strengths 45 and 55 mm, with the power-law applying both above and below the jump. Removal of histone H1 and non-histone proteins lowers the overall sedimentation rate and abolishes the jump.Cross-linking large oligomers at ionic strength 65 mm stabilizes the structure in the conformation found above the jump, leading to a simple power-law dependence throughout the range of ionic strength for cross-linked material. Cleavage of the cross-links restores the jump, presumably by allowing the conformational transition that causes it. Large oligomers are indistinguishable in sedimentation behaviour whether extracted from nuclei at low ionic strength or at 65 mm and maintained in the presence of salt.We interpret these results, together with the detailed electron microscopic studies reported by Thoma et al. (1979) under similar salt conditions, as showing the histone H1-dependent formation of superstructures of nucleosomes in solution induced by increasing ionic strength. The unit of higher order structure probably contains five or six nucleosomes, leading to the change in stability with hexamer. Although this size corresponds to the lower limit of size suggested for “superbeads” (Renz et al., 1977), we see no evidence that multiples of six nucleosomes have any special significance as might be predicted if superbeads had any structural importance. Rather, our results are compatible with a continuous pattern of condensation, such as a helix of nucleosomes (see e.g. Finch & Klug, 1976). The jump in sedimentation observed between ionic strengths 45 and 55 mm, together with the effect of cross-linking, suggests the co-operative stabilization of this structure at higher ionic strengths. A plausible hypothesis is that the turns of the solenoid are not tightly bonded in the axial direction below 45 mm, but come apart due to the hydrodynamic shearing forces in the larger particles leading to less compact structures with slower sedimentation rates. Above 55 mm the axial bonding is strong enough to give a stable structure of dimensions compatible with the 30 nm structures observed in the cell nucleus.     

Quantitative analysis of chromatin structure by circular dichroism

Quantitative analysis of the circular dichroism of nucleohistones and protein-free DNA was carried out in order to determine the structure and the role of the linker region DNA in chromatin, in terms of the conformational change of chromatin as a function of the ionic strength. It is shown clearly that the circular dichroism of Hl-depleted chromatin isolated from calf thymus is determined only by the ratio of the core region to the linker region and demonstrated by the linear combination of the spectrum of protein-free DNA and that of the nucleosome core in 5 mm-Tris · HCl, 1 mm-EDTA (pH 7.8). The calculated spectrum for the linker region in the H1-depleted chromatin was in good agreement with that of protein-free DNA. From the difference spectra between nucleohistones and protein-free DNA, it is suggested that the chromatin has an additional winding of DNA other than 146 base-pairs of DNA around the histone core. By decreasing the ionic strength to values lower than 5 mm-Tris · HCl, 1 mm-EDTA, the ellipticity of H1-depleted chromatin increased greatly between 250 nm and 300 nm while the increase was small in the case of chromatin and the nucleosome core. Nucleosomes with linker region DNA but without histone H1 also show great increase in ellipticity in this range of wavelengths as the ionic strength is decreased. Therefore, the linker region in H1-depleted chromatin plays an important role in the conformational changes brought about by changes in the ionic strength, and the conformational changes caused in the DNA of chromatin by decreasing the ionic strength are suppressed by the presence of histone H1.     

Nuclease digestion promotes structural rearrangements in H1-depleted chromatin.

Digestion of H1-depleted chromatin with micrococcal nuclease at an ionic strength of 0.35M gives rise to structural rearrangements indicating nucleosomal sliding. The ionic strength necessary to reveal this effect is significantly lower than that required in the absence of an accompanying digestion. As an explanation, a model is presented in which the progressing terminal degradation of oligomeric nucleosomes is made responsible for promoting structural rearrangements.     

Chromatin on a membrane: accessibility of histone H5 for antibodies in the supernucleosomal structure

Histone H5 accessibility for the antibodies in chromatin was studied. Chromatin was immobilised on the nitrocellulose membrane in conditions which provide different levels of its compactization. Antiserum specific to the globular domain of histone H5 was used. It was shown, that for establishing real protection of histone H5 in the supernucleosomal structure it is necessary to use long fibers of chromatin. Their linking to the membrane must occur by a minimum quantity of points. It was established, that histone H5 is 5 times more accessive in the preparations of dispersed chromatin (low ionic strength) then in chromatin with the supernucleosomal organization (physiological ionic strength). We suppose that the small level of accessibility of histone H5 for the antibodies in the compact chromatin can be explained by some disruptions in the supernucleosomal organization. On the contrary, the long equable solenoid of nucleosomes provides complete protection of histone H5. In accordance with the results obtained, the model of ordered packaging of nucleosomes in the solenoid is discussed. In this model the point of entrance and exit of DNA on the nucleosomes, fixed by globular region of histone H5, is localized inside the solenoid.     

The mechanism of nucleosome assembly onto oligomers of the sea urchin 5 S DNA positioning sequence

We have used a model system composed of tandem repeats of Lytechinus variegatus 5 S rDNA (Simpson, R. T., Thoma, F., and Brubaker, J. M. (1985) Cell 42, 799-808) reconstituted into chromatin with chicken erythrocyte core histones to investigate the mechanism of chromatin assembly. Nucleosomes are assembled onto the DNA template by mixing histone octamers and DNA in 2 M NaCl followed by stepwise dialysis into very low ionic strength buffer over a 24-h period. By 1.0 M NaCl, a defined intermediate composed of arrays of H3.H4 tetramers has formed, as shown by analytical and preparative ultracentrifugation. Digestion with methidium propyl EDTA.Fe(II) indicates that these tetramers are spaced at 207 base pair intervals, i.e. one/repeat length of the DNA positioning sequence. In 0.8 M NaCl, some H2A.H2B has become associated with the H3.H4 tetramers and DNA. Surprisingly, under these conditions DNA is protected from methidium propyl EDTA.Fe(II) digestion almost as well as in the complete nucleosome, even though these structures are quite deficient in H2A.H2B. By 0.6 M NaCl, nucleosome assembly is complete, and the MPE digestion pattern is indistinguishable from that observed for oligonucleosomes at very low ionic strength. Below 0.6 M NaCl, the oligonucleosomes are involved in various salt-dependent conformational equilibria: at approximately 0.6 M, a 15% reduction in S20,w that mimics a conformational change observed previously with nucleosome core particles; at and above 0.1 M, folding into a more compact structure(s); at and above 0.1 M NaCl, a reaction involving varying amounts of dissociation of histone octamers from a small fraction of the DNA templates. In low ionic strength buffer (less than 1 mM NaCl), oligonucleosomes are present as fully loaded templates in the extended beads-on-a-string structure.