The core histone N-terminal tail domains function independently and additively during salt-dependent oligomerization of nucleosomal arrays

Salt-dependent oligomerization of nucleosomal arrays is related to fiber-fiber interactions and global chromosome structure. Previous studies have shown that the H2A/H2B and H3/H4 N-terminal domain (NTD) pairs are able to mediate array oligomerization. However, because of technical barriers, the function(s) of the individual core histone NTDs have not been investigated. To address this question, all possible combinations of "tailless" nucleosomal arrays were assembled from native and NTD-deleted recombinant Xenopus core histones and tandemly repeated 5 S rDNA. The recombinant arrays were characterized by differential centrifugation over the range of 0-50 mm MgCl2 to determine how each NTD affects salt-dependent oligomerization. Results indicate that all core histone NTDs participate in the oligomerization process and that the NTDs function additively and independently. These observations provide direct biochemical evidence linking all four core histone NTDs to the assembly and maintenance of global chromatin structures.     

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.     

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.     

Alteration of the nucleosomal DNA path in the crystal structure of a human nucleosome core particle

Gene expression in eukaryotes depends upon positioning, mobility and packaging of nucleosomes; thus, we need the detailed information of the human nucleosome core particle (NCP) structure, which could clarify chromatin properties. Here, we report the 2.5 Å crystal structure of a human NCP. The overall structure is similar to those of other NCPs reported previously. However, the DNA path of human NCP is remarkably different from that taken within other NCPs with an identical DNA sequence. A comparison of the structural parameters between human and Xenopus laevis DNA reveals that the DNA path of human NCP consecutively shifts by 1 bp in the regions of superhelix axis location −5.0 to −2.0 and 5.0 to 7.0. This alteration of the human DNA path is caused predominantly by tight DNA–DNA contacts within the crystal. It is also likely that the conformational change in the human H2B tail induces the local alteration of the DNA path. In human NCP, the region with the altered DNA path lacks Mn²⁺ ions and the B-factors of the DNA phosphate groups are substantially high. Therefore, in contrast to the histone octamer, the nucleosomal DNA is sufficiently flexible and mobile and can undergo drastic conformational changes, depending upon the environment.     

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.     

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.     

hSWI/SNF disrupts interactions between the H2A N-terminal tail and nucleosomal DNA.

We have employed a site-specific core histone-DNA cross-linking approach to investigate the mechanism of hSWI/SNF remodeling of a nucleosome. Remodeling results in the complete loss of canonical contacts between the N-terminal tail of H2A and DNA while new interactions are detected between this domain and DNA near the center of the original nucleosome. The data are consistent with a model in which remodeling results in the unraveling of a region of DNA from the edge of the nucleosome, leading to a repositioning of the H2A/H2B dimer to a noncanonical position near the center of the remodeled complex. Additionally, we find that prior cross-linking of the H2A N-terminal region to nucleosomal DNA does not restrict hSWI/SNF remodeling of the remainder of the nucleosome. Thus, disruption of both H2A-DNA interactions near the edge of the nucleosome is not an obligatory step in remodeling of the remainder of the complex.     

Plant and animal histones are completely interchangeable in the nucleosome core

Nucleosome core particles were reconstituted using mixtures of plant (corn or tobacco) and animal (chicken erythrocytes) histones. We show by electron microscopy and sucrose gradient sedimentation that H3 and H4 from tobacco and chicken erythrocytes can be interchanged in the nucleosome kernel. Cross-linking experiments with the protein cross-linking reagent dimethylsuberimidate reveal that, despite structural differences between the histones of the two species, H2A and H2B can be interchanged provided the homologous H2A-H2B dimers are dissociated prior to the annealing.     

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.     

Binding of mithramycin to DNA in the presence of second drugs

Comparative DNA equilibrium binding studies with mithramycin (MTR) and ethidium bromide in the presence and in the absence of second drugs were investigated by spectral titrations. Unusual curvatures (in contrast to those due to neighbor exclusion or anticooperativity) are found in the Scatchard plots of MTR-DNA titrations in the presence of netropsin, a minor-groove binder. Parallel studies with ethidium bromide indicate that although the presence of netropsin significantly reduces the binding ability of ethidium, no unusually curved Scatchard plots are obtained. The unusual curvature exhibited by the Scatchard plots of MTR titrations in the presence of netropsin indicates that the binding of netropsin greatly affects the MTR binding to DNA and can be simulated by an explicit incorporation of the second drug-DNA interaction in the binding formalism. Since netropsin is a minor-groove binder, its interference with the binding of MTR is in accord with the notion that MTR also binds at this groove. The observation of negligible effects on the DNA binding ability of MTR in the presence of either a major-groove or a phosphate group binder lends further support to this conclusion. Consistent with its guanine specificity, studies with synthetic polynucleotides suggest that MTR exhibits negligible affinity for poly(dA-dT).poly(dA-dT) or poly(dA).poly(dT).(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization and nucleosomal core localization of Achlya histones involved in stress-induced chromatin condensation

To better understand the basis for heat shock-induced chromatin condensation in Achlya, a further characterization of the histones of this organism was carried out. The nucleosomal location (i.e., core vs linker), partial peptide map, and electrophoretic behavior of each Achlya histone was determined and compared to the well-characterized histones of rabbit kidney. The results of this and previous studies suggest that in Achlya, no nucleosome linker-associated histone analogous to histone H1 of higher eucaryotes is observed and that the Achlya histone designated alpha is a novel nucleosomal core histone. These observations may reflect the existence of a mechanism of stress-induced chromatin condensation which does not involve histone H1.     

Circular dichroism studies of complexes of the antibiotics daunomycin, nogalamycin, chromomycin, and mithramycin with DNA

The circular dichroism spectra of complexes of the antibiotics daunomycin, nogalamycin, chromomycin, and mithramycin with calf thymus DNA have been measured over a range of drug/DNA ratios. The similarity of the CD spectra of bound chromomycin and mithramycin suggests that they have very similar binding sites, which produce strong effects on the CD spectra of the bound drugs, and remove the differences arising from local stereochemistry in the free drugs. It was found that it was not possible to predict whether the antibiotics intercalated, from studies of the CD spectra alone, even when comparisons were made with the CD spectra of aminoacridine–DNA complexes with intercalating or nonintercalating ligands.     

Structures and interactions of the core histone tail domains

The core histone tail domains are "master control switches" that help define the structural and functional characteristics of chromatin at many levels. The tails modulate DNA accessibility within the nucleosome, are essential for stable folding of oligonucleosome arrays into condensed chromatin fibers, and are important for fiber-fiber interactions involved in higher order structures. Many nuclear signaling pathways impinge upon the tail domains, resulting in posttranslational modifications that are likely to alter the charge, structure, and/or interactions of the core histone tails or to serve as targets for the binding of ancillary proteins or other enzymatic functions. However, currently we have only a marginal understanding of the molecular details of core histone tail conformations and contacts. Here we review data related to the structures and interactions of the core histone tail domains and how these domains and posttranslational modifications therein may define the structure and function of chromatin.     

Characteristics of nucleosome core DNA and their applications in predicting nucleosome positions

By analyzing dinucleotide position-frequency data of yeast nucleosome-bound DNA sequences, dinucleotide periodicities of core DNA sequences were investigated. Within frequency domains, weakly bound dinucleotides (AA, AT, and the combinations AA-TT-TA and AA-TT-TA-AT) present doublet peaks in a periodicity range of 10-11 bp, and strongly bound dinucleotides present a single peak. A time-frequency analysis, based on wavelet transformation, indicated that weakly bound dinucleotides of core DNA sequences were spaced smaller (∼10.3 bp) at the two ends, with larger (∼11.1 bp) spacing in the middle section. The finding was supported by DNA curvature and was prevalent in all core DNA sequences. Therefore, three approaches were developed to predict nucleosome positions. After analyzing a 2200-bp DNA sequence, results indicated that the predictions were feasible; areas near protein-DNA binding sites resulted in periodicity profiles with irregular signals. The effects of five dinucleotide patterns were evaluated, indicating that the AA-TT pattern exhibited better performance. A chromosome-scale prediction demonstrated that periodicity profiles perform better than previously described, with up to 59% accuracy. Based on predictions, nucleosome distributions near the beginning and end of open reading frames were analyzed. Results indicated that the majority of open reading frames’ start and end sites were occupied by nucleosomes.     

DNA stretching and extreme kinking in the nucleosome core

DNA stretching in chromatin may facilitate its compaction and influence site recognition by nuclear factors. In vivo, stretching has been estimated to occur at the equivalent of one to two base-pairs (bp) per nucleosome. We have determined the crystal structure of a nucleosome core particle containing 145 bp of DNA (NCP145). Compared to the structure with 147 bp, the NCP145 displays two incidences of stretching one to two double-helical turns from the particle dyad axis. The stretching illustrates clearly a mechanism for shifting DNA position by displacement of a single base-pair while maintaining nearly identical histone-DNA interactions. Increased DNA twist localized to a short section between adjacent histone-DNA binding sites advances the rotational setting, while a translational component involves DNA kinking at a flanking region that initiates elongation by unstacking bases. Furthermore, one stretched region of the NCP145 displays an extraordinary 55° kink into the minor groove situated 1.5 double-helical turns from the particle dyad axis, a hot spot for gene insertion by HIV-integrase, which prefers highly distorted substrate. This suggests that nucleosome position and context within chromatin could promote extreme DNA kinking that may influence genomic processes.     

DNA sequence-dependent contributions of core histone tails to nucleosome stability: differential effects of acetylation and proteolytic tail removal

Modulation of nucleosome stability in chromatin plays an important role in eukaryotic gene expression. The core histone N-terminal tail domains are believed to modulate the stability of wrapping nucleosomal DNA and the stability of the chromatin filament. We analyzed the contribution of the tail domains to the stability of nucleosomes containing selected DNA sequences that are intrinsically straight, curved, flexible, or inflexible. We find that the presence of the histone tail domains stabilizes nucleosomes containing DNA sequences that are intrinsically straight or curved. However, the tails do not significantly contribute to the free energy of nucleosome formation with flexible DNA. Interestingly, hyperacetylation of the core histone tail domains does not recapitulate the effect of tail removal by limited proteolysis with regard to nucleosome stability. We find that acetylation of the tails has the same minor effect on nucleosome stability for all the selected DNA sequences. A comparison of histone partitioning between long donor chromatin, acceptor DNA, and free histones in solution shows that the core histone tails mediate internucleosomal interactions within an H1-depleted chromatin fiber amounting to an average free energy of about 1 kcal/mol. Thus, such interactions would be significant with regard to the free energies of sequence-dependent nucleosome positioning. Last, we analyzed the contribution of the H2A/H2B dimers to nucleosome stability. We find that the intact nucleosome is stabilized by 900 cal/mol by the presence of the dimers regardless of sequence. The biological implications of these observations are discussed.