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.     

Reconstitution of chromatin higher-order structure from histone H5 and depleted chromatin

Reconstitution of the 30 nm filament of chromatin from pure histone H5 and chromatin depleted of H1 and H5 has been studied using small-angle neutron-scattering. We find that depleted, or stripped, chromatin is saturated by H5 at the same stoichiometry as that of linker histone in native chromatin. The structure and condensation behavior of fully reconstituted chromatin is indistinguishable from that of native chromatin. Both native and reconstituted chromatin condense continuously as a function of salt concentration, to reach a limiting structure that has a mass per unit length of 6.4 nucleosomes per 11 nm. Stripped chromatin at all ionic strengths appears to be a 10 nm filament, or a random coil of nucleosomes. In contrast, both native and reconstituted chromatin have a quite different structure, showing that H5 imposes a spatial correlation between neighboring nucleosomes even at low ionic strength. Our data also suggest that five to seven contiguous nucleosomes must have H5 bound in order to be able to form a higher-order structure.     

The effect of histone H1 on the compaction of oligonucleosomes. A quasielastic light scattering study

The structural properties of H1-depleted oligonucleosomes are investigated by the use of quasielastic laser light scattering, thermal denaturation and circular dichroism and compared to those of H1-containing oligomers. To obtain information on the role of histone H1 in compaction of nucleosomes, translational diffusion coefficients (D) are determined for mono-to octanucleosomes over a range of ionic strength. The linear dependences of D on the number of nucleosomes show that the conformation of stripped oligomers is very extended and does not change drastically with increasing the ionic strength while the rigidness of the chain decreases due to the folding of linker DNA. The results prove that the salt-induced condensation is much smaller for H1-depleted than for H1-containing oligomers and that histone H1 is necessary for the formation of a supercoiled structure of oligonucleosomes, already present at low ionic strength.     

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.     

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.     

Chromatin structure. Further evidence against the existence of a beaded subunit for the 30-nm fiber

The size distribution of chromatin fragments released by micrococcal nuclease digestion of liver chromatin at various ionic strengths was examined. Below 20 mM ionic strength, gradient profiles with a peak centered at 6 nucleosomes are generated, whereas between 20 and 50 mM the peak is always centered on 12 nucleosomes, and above 50 mM ionic strength the 30-nm fiber becomes less accessible to the nuclease and there is a corresponding increase in the size distribution of fragments in the gradients. However, extensive digestions always give profiles with a peak of 12 nucleosomes as nuclease-resistant dodecamers accumulate. All of these observations are consistent with the winding of the 10-nm polynucleosome chain into a helical coil commencing at about 20 mM ionic strength. The helical turns are stabilized by histone H1 interactions between 20 and 50 mM ionic strength producing stable dodecamers. Above 50 mM ionic strength the coil condenses longitudinally and the profiles are consistent with a random attack of this fiber by the nuclease. Consequently it is not necessary to invoke the existence of a subunit bead to explain the profiles. We further define the conditions at which specific structural transitions take place and provide methodology for the preparation of chromatin at various levels of condensation.     

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.     

Effect of salt on the binding of the linker histone H1 to DNA and nucleosomes

The linker histones are involved in the salt-dependent folding of the nucleosomes into higher-order chromatin structures. To better understand the mechanism of action of these histones in chromatin, we studied the interactions of the linker histone H1 with DNA at various histone/DNA ratios and at different ionic strengths. In direct competition experiments, we have confirmed the binding of H1 to superhelical DNA in preference to linear or nicked circular DNA forms. We show that the electrophoretic mobility of the H1/supercoiled DNA complex decreases with increasing H1 concentrations and increases with ionic strengths. These results indicate that the interaction of the linker histone H1 with supercoiled DNA results in a soluble binding of H1 with DNA at low H1 or salt concentrations and aggregation at higher H1 concentrations. Moreover, we show that H1 dissociates from the DNA or nucleosomes at high salt concentrations. By the immobilized template pull-down assay, we confirm these data using the physiologically relevant nucleosome array template.     

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.     

The Role of the Central Globular Domain of Histone H5 in Chromatin Structure

Abstract

Histone H5 contains three tryosines in the central, a polar region of the molecule. All three tryosines can be spin labeled at low ionic strength. When the central globular domain is folded at high ionic strength, only one tyrosine becomes accessible to the imidazole spin label. Spin labeling the buried tyrosines prevents the folding of the globular structure, which, in turn, affects the proper binding of the H5 molecule to stripped chromatin. Chromatin complexes reconstituted from such an extensively modified H5 molecule show a weaker protection of the 168 base pair chromatosome during nuclease digestion. However, when only the surface tyrosine of the H5 molecule is labeled, such a molecule can still bind correctly to stripped chromatin, yielding a complex very similar to that of native chromatin. Our data supports the idea that not just the presence of the linker histone H5, but the presence of an intact H5 molecule with a folded, globular central domain is essential in the recognition of its specific binding sites on the nucleosomes. Our data also show that during the chromatin condensation process, the tumbling environment of the spin label attached to the surface tyrosine in the H5 molecule is not greatly hindered but remains partially mobile. This suggests that either the labeled domain of the H5 molecule is not directly involved in the condensation process or the formation of the higher-order chromatin structure does not result in a more viscous or tighter environment around the spin label. The folded globular domain of H5 molecule serves in stabilizing the nucleosome structure, as well as the higherorder chromatin structure.     

Distinct effects of histone H1 and non-histone chromosomal proteins on the structure of nucleosomes and the chromatin fibre in solution

In this study we attempt to differentiate between the effects of the non-histone chromosomal proteins and histone H1 on the structure of the nucleosomes and the chromatin fibre in solution. The properties of chromatin preparations with different histone H1 and non-histone protein compositions were compared using circular dichroism and flow linear dichroism and the following conclusions were drawn. When histone H1 is absent the non-histone proteins partially prevent the unfolding of the nucleosomes at low ionic strength. The complete blocking of this unfolding, however, is accomplished only in the presence of histone H1. The non-histone proteins do not affect the orientation of the nucleosomes along the fibre axis. Only histone H1 can maintain the positive anisotropy of the chromatin fibre.     

Involvement of the globular domain of histone H1 in the higher order structures of chromatin

We have attacked H1-containing soluble chromatin by α-chymotrypsin under conditions where chromatin adopts different structures.Soluble rat liver chromatin fragments depleted of non-histone components were digested with α-chymotrypsin in NaCl concentrations between 0 mm and 500 mm. at pH 7, or at pH 10, or at pH 7 in the presence of 4 m-urea. α-Chymotrypsin cleaves purified rat liver histone H1 at a specific initial site (CT) located in the globular domain and produces an N-terminal half (CT-N) which contains most of the globular domain and the N-terminal tail, and a C-terminal half (CT-C) which contains the C-terminal tail and a small part of the globular domain. Since in sodium dodecyl sulfate/polyacrylamide-gel electrophoresis CT-C migrates between the core histones and H1, cleavage of chromatin-bound H1 by α-chymotrypsin can be easily monitored.The CT-C fragment was detected under conditions where chromatin fibers were unfolded or distorted: (1) under conditions of H1 dissociation at 400 mm and 500 mm-NaCl (pH 7 and 10); (2) at very low ionic strength where chromatin is unfolded into a filament with well-separated nucleosomes; (3) at pH 10 independent of the ionic strength where chromatin never assumes higher order structures; (4) in the presence of 4 m-urea (pH 7), again independent of the ionic strength. However, hardly any CT-C fragment was detected under conditions where fibers are observed in the electron microscope at pH 7 between 20 mm and 300 mm-NaCl. Under these conditions H1 is degraded by α-chymotrypsin into unstable fragments with a molecular weight higher than that of CT-C. Thus, the data show that there are at least two different modes of interaction of H1 in chromatin which correlate with the physical state of the chromatin.Since the condensation of chromatin into structurally organized fibers upon raising the ionic strength starts by internucleosomal contacts in the fiber axis (zig-zag-shaped fiber), where H1 appears to be localized, it is likely that in chromatin fibers the preferential cleavage site for α-chymotrypsin is protected because of H1-H1 contacts. The data suggest that the globular part of H1 is involved in these contacts close to the fiber axis. They appear to be hydrophobic and to be essential for the structural organization of the chromatin fibers. Based on the present and earlier observations we propose a model for H1 in which the globular domains eventually together with the N-terminal tails form a backbone in the fiber axis, and the nucleosomes are mainly attached to this polymer by the C-terminal tails.     

The role of the central globular domain of histone H5 in chromatin structure

Histone H5 contains three tyrosines in the central, apolar region of the molecule. All three tyrosines can be spin labeled at low ionic strength. When the central globular domain is folded at high ionic strength, only one tyrosine becomes accessible to the imidazole spin label. Spin labeling the buried tyrosines prevents the folding of the globular structure, which, in turn, affects the proper binding of the H5 molecule to stripped chromatin. Chromatin complexes reconstituted from such an extensively modified H5 molecule show a weaker protection of the 168 base pair chromatosome during nuclease digestion. However, when only the surface tyrosine of the H5 molecule is labeled, such a molecule can still bind correctly to stripped chromatin, yielding a complex very similar to that of native chromatin. Our data supports the idea that not just the presence of the linker histone H5, but the presence of an intact H5 molecule with a folded, globular central domain in essential in the recognition of its specific binding sites on the nucleosomes. Our data also show that during the chromatin condensation process, the tumbling environment of the spin label attached to the surface tyrosine in the H5 molecule is not greatly hindered but remains partially mobile. This suggests that either the labeled domain of the H5 molecule is not directly involved in the condensation process or the formation of the higher-order chromatin structure does not result is a more viscous or tighter environment around the spin label. The folded globular domain of H5 molecule serves in stabilizing the nucleosome structure, as well as the higher-order chromatin structure.     

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.     

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.     

The distribution of histone H1 subfractions in chromatin subunits.

Rat liver chromatin was digested with micrococcal nuclease to various extents and fractionated into nucleosomes, di and trimers of nucleosomes on an isokinetic sucrose gradient. In conditions under which degradation of linker DNA within the particles was limited, the electrophoretic analysis of the histone content showed that the overall content of H1 histone increased from nucleosomes to higher order oligomers. Moreover, the histone H1 subfractions were found unevenly distributed among the chromatin subunits, one of them, H1--3 showing most variation. A more regular distribution of these subfractions was found in subunits obtained from a more extended digestion level of chromatin. It is suggested that the H1 subfractions differ in the protection they confer upon DNA.     

Ionic strength-dependent conformational transitions of chromatin. Circular dichroism and thermal denaturation studies

High-molecular-weight chicken erythrocyte chromatin was prepared by mild digestion of nuclei with micrococcal nuclease. Samples of chromatin containing both core (H3, H4, H2A, H2B) and lysine-rich (H1, H5) histone proteins (whole chromatin) or only core histone proteins (core chromatin) were examined by CD and thermal denaturation as a function of ionic strength between 0.75 and 7.0 × 10^?3M Na⁺. CD studies at 21°C revealed a conformational transition over this range of ionic strengths in core chromatin, which indicated a partial unfolding of a segment of the core particle DNA at the lowest ionic strength studied. This transition is prevented by the presence of the lysine-rich histones in whole chromatin. Thermal-denaturation profiles of both whole and core chromatins, recorded by hyperchromicity at 260 nm, reproducibly and systematically varied with the ionic strength of the medium. Both materials displayed three resolvable thermal transitions, which represented the total DNA hyperchromicity on denaturation. The fractions of the total DNA which melted in each of these transitions were extremely sensitive to ionic strength. These effects are considered to result from intra- and/or internucleosomal electrostatic repulsions in chromatin studied at very low ionic strengths. Comparison of the whole and core chromatin melting profiles indicated substantial stabilization of the core-particle DNA by binding sites between the H1/H5 histones and the 140-base-pair core particle.     

Exchange of histone H1 between segments of chromatin

We have asked whether histone H1 is tightly bound in chromatin at relatively low ionic strength (below physiological) or whether it can exchange between binding sites. We have studied this question in chromatin fragments generated by digestion with micrococcal nuclease, by mixing two fragments of known H1 content and different length (either a fragment radioactively labelled in all its histones with an unlabelled fragment, or two labelled fragments, only one of which contains H1) and then separating them again by centrifugation in sucrose gradients in order tom reexamine their H1 contents.At very low ionic strength (5 mm-Tris · HCl (pH 7.1), 1 mm-Na₂EDTA, 0.5 mm-phenylmethylsulphonyl fluoride) there was very little (less than 5 to 10%) exchange of H1. In contrast, the presence of 70 mm-NaCl in the buffer caused rapid and complete equilibration of H1 between sites in less (possibly much less) than about one hour. At 30 mm added NaCl, the result was intermediate between those at 0 mm and 70 mm added NaCl, partial exchange occurring with a half-time of one to two hours. The results were essentially the same whether or not both fragments contained H1. We infer from these results that at physiological ionic strength (~150 mm) there will be rapid and complete equilibration of H1 between sites in chromatin. We do not know whether the exchange of particular H1 subtypes is restricted to a particular class of binding site.     

Nucleomeric organization of chromatin

Chromatin in the nuclei fixed in tissue and in the nuclei isolated by low ionic strength solutions in the presence of Mg2+ is represented by globular (nucleomeric) fibrils, 20-25 nm in diameter. The staphylococcal or endogenous nuclear nuclease splits the chromatin fibrils resulting in fragments corresponding to nucleomers and their multimers. Upon removal of firmly bound Mg2+ the nucleomers unfold to form chains consisting of 4-6-8 nucleosomes. Mild hydrolysis of nuclear chromatin by staphylococcal nuclease results in a split-off of mono-, di- and trimers of nucleomers sedimenting in a sucrose density gradient in the presence of EDTA as particles with the sedimentation coefficients of 37, 47 and 55S, respectively. The sedimentation coefficient for the mononucleomer in a sucrose density gradient with MgCl2 is 45S. Determination of the length of DNA fragments of chromatin split-off by staphylococcal nuclease showed that the nucleomer consists of 8 nucleosomes, while the dimer and trimer of the nucleomer consists of 14-16 and 21-24 nucleosomes, respectively. The nucleomeric monomer undergoes structural transition from the compact (45S) to the "loose" state (37S) after removal of Mg2+. This transition is completely reversible, when the nucleomer contains histone H1. The removal of the latter or dialysis of the nucleomer against EDTA in low ionic strength solutions results in a complete unfolding of the nucleomer into a nucleosomal chain fragment. A model for the nucleomer fibril structure in which the helical organization of the nucleosomal chain in the nucleomer (2 turns with 4 nucleosomes in each) is alternated with the impaired helical bonds between the nucleomers is discussed. The functional significance of the nucleomeric organization of chromatin may be an additional restriction of the site-specific recognition of DNA in chromatin with the possibility of local (at the level of one nucleomer) changes in chromatin conformation excluding this restriction.