The site of binding of linker histone to the nucleosome does not depend upon the amino termini of core histones.

Using nucleosomes reconstituted on a defined sequence of DNA, we have investigated the question as to whether the N-terminal tails of core histones play a role in determining the site of binding of a linker histone. Reconstitutes used histone cores of three types: intact, lacking the N-terminal H3 tails, or lacking all tails. In each case the same, single defined position for the histone core was observed, using high-resolution mapping. The affinity for binding of linker histone H1(o) was highest for the intact cores, lowest for the tailless cores. However, the location of the linker histone, as judged by micrococcal nuclease protection, was exactly the same in each case, an asymmetric site of about 17 bp to one side of the core particle DNA.     

Binding of linker histones to the core nucleosome

Binding of chicken erythrocyte linker histones H1/H5 to the core nucleosome has been studied. Histones H1/H5 bind very efficiently to the isolated core nucleosome in vitro. The binding of linker histones to the core nucleosome is associated with aggregation of the particles. Approximately one molecule of linker histone binds per core nucleosome in the aggregates, irrespective of the concentration of the linker histones and the salt used. Histone H5 shows greater binding affinity to the core nucleosome as compared to H1. The carboxyl-terminal fragment of the linker histones binds strongly to the core nucleosome while the binding of the central globular domain is weak. Each core nucleosome is capable of binding two molecules of carboxyl-terminal fragment of linker histone. The core nucleosome containing one molecule of carboxyl-terminal fragment of linker histone requires higher salt concentration for aggregation while the core nucleosome containing two molecules of carboxyl-terminal fragment of linker histone can self-associate even at lower salt concentrations. On the basis of these results we are proposing a novel mechanism for the condensation of chromatin by linker histones and other related phenomena.     

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.     

Histone hyperacetylation can induce unfolding of the nucleosome core particle.

A direct correlation exists between the level of histone H4 hyperacetylation induced by sodium butyrate and the extent to which nucleosomes lose their compact shape and become elongated (62.0% of the particles have a length/width ratio over 1.6; overall mean in the length/width ratio = 1.83 +/- 0.48) when bound to electron microscope specimen grids at low ionic strength (1mM EDTA, 10mM Tris, pH 8.0). A marked proportion of elongated core particles is also observed in the naturally occurring hyperacetylated chicken testis chromatin undergoing spermatogenesis when analyzed at low ionic strength (36.8% of the particles have a length/width ratio over 1.6). Core particles of elongated shape (length/width ratio over 1.6) generated under low ionic strength conditions are absent in the hypoacetylated chicken erythrocyte chromatin and represent only 2.3% of the untreated Hela S3 cell core particles containing a low proportion of hyperacetylated histones. The marked differences between control and hyperacetylated core particles are absent if the particles are bound to the carbon support film in the presence of 0.2 M NaCl, 6mM MgCl2 and 10mM Tris pH 8.0, conditions known to stabilize nucleosomes. A survey of the published work on histone hyperacetylation together with the present results indicate that histone hyperacetylation does not produce any marked disruption of the core particle 'per se', but that it decreases intranucleosomal stabilizing forces as judged by the lowered stability of the hyperacetylated core particle under conditions of shearing stress such as cationic competition by the carbon support film of the EM grid for DNA binding.     

DNA folding by histones: the kinetics of chromatin core particle reassembly and the interaction of nucleosomes with histones.

The kinetics of the chromatin core particle reassembly reaction in solution were quantitatively studied under conditions such that nucleohistone aggregation did not occur. Core particles, salt-jumped rapidly by dilution from 2.5 m-NaCl (in which DNA and histones do not interact) to 0.6 m-NaCl (in which core particles are nearly intact), reassemble in two distinct time ranges. Approximately 75% of the DNA refolds into core particle-like structures “instantaneously” as measured by several physical and chemical techniques with dead times in the seconds to minutes time range. The remaining DNA refolds with relaxation times ranging from 250 minutes at 0 °C to 80 minutes at 37 °C; this slow effect cannot be attributed to sample heterogeneity. The fraction of slowly refolding DNA and the slow relaxation time are independent of the core particle concentration. Transient intermediates present during the slow phase of refolding were identified as free DNA and core particle-like structures containing excess histone. Mixing experiments with DNA, histones, and core particles showed that core particle-histone interactions are responsible for the slow kinetics of DNA refolding. Upon treatment of reassembling core particles with the protein crosslinking reagent, dimethylsuberimidate, the slow phase of the reassembly reaction was arrested and a 13 S particle containing DNA and two octamers of histone was isolated. Consistent with the nature of this kinetic intermediate, it is shown that in 0.6 m-NaCl, core particles co-operatively bind at least one additional equivalent of histones with high affinity in the form of excess octamers. Also, core particles continue to adsorb considerably more histones with a weaker association constant of the order 10⁵m^?1 (in units of octamers) to a maximum value of 12 ± 2 equivalents (octamers) per core particle. The sedimentation coefficient increases with the two-thirds power of the molecular weight of the complex, as it would in the case of clustered spheres.A reassembly mechanism consistent with the data is presented, and other simple mechanisms are excluded. In the proposed mechanism, core particles reassemble very rapidly and compete effectively with DNA for histones such that approximately one-third of the particles initially formed are complexed with an excess octamer of histones, and 25% of the total DNA remains uncomplexed. The amount of this unusual reaction intermediate decays slowly to an equilibrium value of about 10%, thereby leaving 9% of the total DNA uncomplexed. Approximate values are calculated for the free energies, rate constants, and two of the activation energies which characterize this migrating octamer mechanism. This mechanism provides a means whereby histone octamers can be temporarily stripped off DNA at a modest free energy cost, approximately 2.6 kcal per nucleosome. Also, the properties of excess histone adsorption by chromatin and octamer migration suggest an efficient mechanism, consistent with observations by others, for nucleosome assembly in vivo during replication.     

Hydrodynamic studies of the interaction between nucleosome core particles and core histones

We have studied the hydrodynamic properties of the complexes formed by interaction of nucleosome core particles with excess histone octamers containing two each of the four core histones. The results are consistent with tight binding of two to three octamers to the exterior of each core particle. The binding is dependent upon the presence of the H3/H4 histone pair: when H3/H4 alone are added to nucleosome core particles, tight binding is observed, but H2A/H2B alone are bound only weakly. We have also examined the properties of the nucleosome core in solutions containing 0·1 m to 0·7 M-NaCl. We show that in this salt range the core particle undergoes some changes in shape, reflected in a 14% increase in the frictional coefficient. Even at the highest salt concentrations used, however, the nucleosome core is still a compact, folded structure.     

Chromatin core particle unfolding induced by tryptic cleavage of histones.

Chromatin 'core particles' have been digested with trypsin to varying extents. The resulting particles are homogeneous by the criterion of ultracentrifuge boundary analysis. Sedimentation coefficients are lowered as cleavages are introduced into the histones, showing that an unfolding of the core particle occurs. This unfolding is further characterised by a lower melting temperature together with a premelting phase, higher molar ellipticity in the circular dichroism spectra at 280 nm and increased kinetics of digestion by both micrococcal nuclease and DNase I. Differences are also observed in the products of nuclease digestion. The most consistent interpretation of the data involves an unfolding process whereby free rods of DNA are released to extend from a nucleoprotein core.     

Structural dynamics of nucleosome core particle: comparison with nucleosomes containing histone variants

The present study provides insights on the dominant mechanisms of motions of the nucleosome core particle and the changes in its functional dynamics in response to histone variants. Comparative analysis of the global dynamics of nucleosomes with native and variant H2A histones, using normal mode analysis revealed that the dynamics of the nucleosome is highly symmetric, and its interaction with the nucleosomal DNA plays a vital role in its regulation. The collective dynamics of nucleosomes are predicted to be dominated by two types of large-scale motions: (1) a global stretching-compression of nucleosome along the dyad axis by which the nucleosome undergoes a breathing motion with a massive distortion of nucleosomal DNA, modulated by histone-DNA interactions; and (2) the flipping (or bending) of both the sides of the nucleosome in an out-of-plane fashion with respect to the dyad axis, originated by the highly dynamic N-termini of H3 and (H2A.Z-H2B) dimer in agreement with the experimentally observed perturbed dynamics of the particular N-terminus under physiological conditions. In general, the nucleosomes with variant histones exhibit higher mobilities and weaker correlations between internal motions compared to the nucleosome containing ordinary histones. The differences are more pronounced at the L1 and L2 loops of the respective monomers H2B and H2A, and at the N-termini of the monomers H3 and H4, all of which closely interact with the wrapping DNA.     

Cryo-EM structure of the nucleosome core particle containing Giardia lamblia histones

Giardia lamblia is a pathogenic unicellular eukaryotic parasite that causes giardiasis. Its genome encodes the canonical histones H2A, H2B, H3, and H4, which share low amino acid sequence identity with their human orthologues. We determined the structure of the G. lamblia nucleosome core particle (NCP) at 3.6 Å resolution by cryo-electron microscopy. G. lamblia histones form a characteristic NCP, in which the visible 125 base-pair region of the DNA is wrapped in a left-handed supercoil. The acidic patch on the G. lamblia octamer is deeper, due to an insertion extending the H2B α1 helix and L1 loop, and thus cannot bind the LANA acidic patch binding peptide. The DNA and histone regions near the DNA entry-exit sites could not be assigned, suggesting that these regions are asymmetrically flexible in the G. lamblia NCP. Characterization by thermal unfolding in solution revealed that both the H2A–H2B and DNA association with the G. lamblia H3–H4 were weaker than those for human H3–H4. These results demonstrate the uniformity of the histone octamer as the organizing platform for eukaryotic chromatin, but also illustrate the unrecognized capability for large scale sequence variations that enable the adaptability of histone octamer surfaces and confer internal stability.     

On the mechanism of nucleosome unfolding.

We have studied the relative stabilities to urea denaturation of histone-histone binding interactions as they occur both in chromatin and in histone complexes free in solution. We have used the two zero-length contact-site cross-linking agents, tetranitromethane and UV light, to measure the relative degree of H2B-H4 and H2A-H2B association under various conditions. The two interactions were disrupted coordinately when nuclei were treated with increasing concentrations of urea. In contrast, when histone complex in 2 M NaCl were treated with urea, the H2B-H4 interaction was found to be much less stable than the H2A-H2B interaction. We have shown previously that nucleosomes unfold at low ionic strengths such that the H2B-H4 but not the H2A-H2B interaction is broken in the process. We speculate that the preferential rupture of the H2B-H4 contact is of physiological significance.     

Primary organization of the nucleosome core particles sequential arrangement of histones along DNA

A high-resolution map for the arrangement of histones along DNA in the nucleosome core particles has been determined by a new sequencing procedure. The lysine groups of histones were crosslinked to partly depurinated DNA at neutral pH. One strand of DNA was split at the points of crosslinking, thus leaving the 5′-terminal DNA fragments bound to histones. The lengths of these crosslinked DNA fragments were measured to determine the position of histones on one strand of the core DNA from its 5′ end.The results demonstrate that histones are bound to regularly arranged, discrete DNA segments about six nucleotides long. These segments are separated by histone-free gaps about four nucleotides wide located at a distance of about 10n nucleotides from the 5′ end of DNA. The first 20 nucleotides from the 5′ ends of DNA seem to be free of histones. Histones appear to be arranged symmetrically and in a similar way on both DNA strands. Any one histone, being bound predominantly to discrete segments on one or other of the strands, can oscillate at the same time between the two strands across the major DNA groove. Two symmetrical models for the arrangement of two molecules of each core histone on linearized and folded DNA are proposed.     

Isw1a does not have strict limitations on the length of extranucleosomal DNAs for mobilization of nucleosomes assembled with HeLa cell histones

The Saccharomyces cerevisiae Isw1a and Isw2 ATP-dependent chromatin-remodeling complexes have important roles in vivo in the regulation of nucleosome positioning and modulation of gene activity. We studied the ability of the Isw1a- and Isw2-remodeling enzymes to reposition nucleosomes in mono- and dinucleosomes templates with variably positioned histone octamers (in the center or at the ends of the DNA fragment). To compare the Isw1a and Isw2 nucleosome-mobilizing activities, we utilized mono- and dinucleosome templates reconstituted with purified HeLa cell histones and DNA containing one or two copies of the “601” nucleosome high-affinity sequence used to specifically position nucleosomes on the DNA. The obtained data suggest that Isw1a is able to mobilize HeLa cell histone-assembled mononucleosomes with long (more than 30?bp) extranucleosomal DNAs protruding from both sides, which contrasts to the previously reported inability of Isw1 to mobilize similar nucleosomes assembled with recombinant yeast histones. The results also suggest that Isw1a and Isw2 can mobilize nucleosomes with unfavorably short linker DNA lengths, and the presence of internucleosomal interactions promotes mobilization of nucleosomes even when the linkers are short.     

Spatial organization of histones and DNA in the nucleosome core particle: A model

We present here an attempt to build up a space-filling model of the nucleosome core particle based on the chemical crosslinking data of Mirzabekov and coworkers (23). It is shown that the models proposed earlier are inconsistent with the results of these authors. The main characteristics of our model are as follows: a) the DNA superhelix contains at least 90 base pairs (bp) per turn; b) the particle has a dyad axis of symmetry; c) the histone octamer may be regarded as consisting of two heterotypic tetramers. The possible shape and function of core histones are discussed in the light of the model.     

Nonhistone Scm3 and histones CenH3-H4 assemble the core of centromere-specific nucleosomes

The budding yeast histone H3 variant, Cse4, replaces conventional histone H3 in centromeric chromatin and, together with centromere-specific DNA-binding factors, directs assembly of the kinetochore, a multiprotein complex mediating chromosome segregation. We have identified Scm3, a nonhistone protein that colocalizes with Cse4 and is required for its centromeric association. Bacterially expressed Scm3 binds directly to and reconstitutes a stoichiometric complex with Cse4 and histone H4 but not with conventional histone H3 and H4. A conserved acidic domain of Scm3 is responsible for directing the Cse4-specific interaction. Strikingly, binding of Scm3 can replace histones H2A-H2B from preassembled Cse4-containing histone octamers. This incompatibility between Scm3 and histones H2A-H2B is correlated with diminished in vivo occupancy of histone H2B, H2A, and H2AZ at centromeres. Our findings indicate that nonhistone Scm3 serves to assemble and maintain Cse4-H4 at centromeres and may replace histone H2A-H2B dimers in a centromere-specific nucleosome core.     

X-ray scattering study of the shape of the DNA region in nucleosome core particle with synchrotron radiation.

The structure of the DNA region in rat thymus nucleosome core particle has been studied by synchrotron X-ray scattering analysis and the contrast-variation technique has been applied to determine the contribution of the DNA to the total scatterings. Small-angle contrast-matching measurements show that the entire core particle and isolated histone octamers are contrast-matched by solvents containing 64 and 54% (w/w) sucrose, respectively. At a contrast of 54% sucrose, where the scattering of the DNA dominates, the scattering data extending to higher angle of about 0.05 A-1 have been collected from relatively concentrated solutions (10 mg/ml) of core particles and interpreted on the basis of the regular helical model for the DNA region. The model calculations show that the shape of the DNA around the histone core is approximately by 1.8 turns of regular helix of 42 A radius and 28 A pitch. These values for helical parameters of our model are in good agreement with those of the structure of DNA in crystallized nucleosome cores shown by earlier diffraction studies.     

Large scale preparation of nucleosomes containing site-specifically chemically modified histones lacking the core histone tail domains

The core histone tail domains are critical regulators of chromatin structure and function and modifications such as acetylation of lysine residues within the tails are central to this regulation. Studies have shown that the removal of core histone tail domains by trypsinization in which one-half to two-thirds of each core histone tail domain are removed in gross aspects mimics the acetylation of core histone tails. In addition, removal of the tails has been useful in understanding general tail function. Thus, removal of native core histone tails by trypsinization is a widely used method. In addition, many in vitro studies now employ core histones site-specifically modified with photo activatable cross-linking probes or fluorescent probes. However, in our experience, standard methods employing trypsinized donor chromatin for reconstitution of nucleosomes containing certain chemically modified histones lacking the core histone tail domains are not uniformly applicable. Here, we describe various methods for preparing nucleosomes containing a core histone modified with a cross-linking agent, APB, and lacking the core histone tail domains.     

Interaction of non-histone proteins HMG1 and HMG2 with core histones in nucleosomes and core particles revealed by chemical cross-linking

Chemical cross-linking was used to study the interaction of the non-histone chromosomal proteins HMG1 and HMG2 with core histones in H1,H5-depleted nucleosomes or core particles. Cross-linking with a 'zero-length' cross-linker 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and with a longer (cleavable) cross-linker dimethyl-3,3'-dithiobispropionimidate revealed an interaction of HMG1 and HMG2 with (or proximity to) core histones in both types of particles. These results indicated that the presence of the 40-50-base-pairs-long segment of the 'linker' DNA in nucleosomes was not necessary for the establishment of mutual contacts of HMG1 and HMG2 proteins with core histones. Possible implications of the interaction of HMG1 and HMG2 proteins with histones for the structure and functioning of chromatin are discussed.     

The helical model of the nucleosome core.

A model of the nucleosome core is proposed based on a topologically linear array of histones attached sequentially to DNA. The linear complex folds helically forming a spring-like particle. Different variants of the particle are discussed (cylindrical springs with and without histone-histone contacts between turns of the helix, solenoidal spring). The model is consistent with known data about the nucleosome structure. Histones H3 and H4 have a special role in the model which is related also to the superstructure of chromatin.     

The roles of H1, the histone core and DNA length in the unfolding of nucleosomes at low ionic strength.

Calf thymus nucleosomes exhibit two different and independent hydrodynamic responses to diminishing salt concentration. One change is gradual over the range 40 to 0.2 mM Na+ and is accompanied by decreases in contact-site cross-linking efficiency. The other change is abrupt, being centered between 1 and 2 mM Na+. We found only one abrupt change in sedimentation rate for particles ranging in DNA content fom 144 to 230 base pairs. This response to decreasing ionic strength is similar for particles of both 169 and 230 base pairs. Core particles (144 base pairs) exhibit a somewhat diminished response. The abrupt change is blocked by formaldehyde or dimethylsuberimidate cross-linking. The blockage by dimethylsuberimidate demonstrates that the abrupt conformational change requires the participation of the core histones. H1 completely blocks the abrupt but not the gradual conformational change. Thus H1 uncouples the different responses to low ionic strength and exerts an important constraint on the conformational states available to the nucleosome core.     

Deposition of newly synthesized histones: new histones H2A and H2B do not deposit in the same nucleosome with new histones H3 and H4

We have developed procedures to study histone-histone interactions during the deposition of histones in replicating cells. Cells are labeled for 60 min with dense amino acids, and subsequently, the histones within the nucleosomes are cross-linked into an octameric complex with formaldehyde. These complexes are sedimented to equilibrium in density gradients and octamer and dioctamer complexes separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. With reversal of the cross-link, the distribution of the individual density-labeled histones in the octamer is determined. Newly synthesized H3 and H4 deposit as a tetramer and are associated with old H2A and H2B. Newly synthesized H2A and H2B deposit as a dimer associated with old H2A, H2B, H3, and H4. The significance of these results with respect to the dynamics of histone interactions in the nucleus is discussed. Control experiments are presented to test for artifactual formation of these complexes during preparative procedures. In addition, reconstitution experiments were performed to demonstrate that the composition of these octameric complexes can be determined from their distribution on density gradients.