Analysis of the dynamic equilibrium of histone oligomers in a solution. The nature of forces stabilizing the (H2A-H2B-H3-H4)2 octamer structure

Dynamic equilibrium analysis of the (H2A-H2B-H3-H4)2 histone octamer with lower oligomers was performed in 2 M NaCl. Calculated data on the relative content of histone oligomers upon changing protein concentration in solution are given. The red shift of lambda max for histone tyrosine fluorescence spectra is shown to be due to hydrogen bond formation by tyrosyl OH-groups. Analysis of free energy changes of histone oligomers upon association (delta G = -17,37 +/- 0,14 kcal/mole) as well as the effect of urea on histone octamer dissociation made it possible to conclude that virtually all tyrosyls in octamer form hydrogen bonds. Intermolecular hydrogen bonds formed by tyrosyls contribute substantially to octamer stabilization. The (H2A-H2B) dimer positive cooperativity in association with the (H3-H4)2 tetramer was found. This cooperativity is caused by interaction between association sites with a two order increase in an apparent constant of dimers with tetramer association. The histone octamer was determined to be of asymmetric structure due to unequivolency of the two binding sites for the (H2A-H2B) dimers.     

Secondary structure of histones in solution

The secondary structure of histones H2B and H3 from calf thymus has been quantitatively studied in heavy water solutions in a wide range of histone concentrations, pD, and concentrations of sodium chloride by an infrared spectroscopy method. Also, the interactions between molecules of different histones in equimolar mixtures H2A-H2B, H2A-H3, H2A-H4, H2B-H3, H2B-H4, H3-H4, and H2A-H2B-H3-H4 have been investigated using the same method. For H2B and H3 conditions favourable for aggregation have been shown to induce the formation of pleated sheet structure. When the pD and concentration of NaCl are in a physiological range, the secondary structure of H2B and H3 contains about 15% of alpha-helix, 4% of parallel pleated sheet structure, 14% of antipatallel pleated sheet structure in H2B and 18% in H3. For mixtures in all cases, except H2A-H4, there is an interaction between molecules of different histones followed by a reduction of the antiparallel pleated sheet structure content. The data on the secondary structure of histones in different states (under self-association, in mixtures, in nucleosomes, and in chromatin) have been discussed and it is suggested that: 1) the secondary structure of histones in chromatin is essentially similar to that in the state of self-association; 2) in the core nucleosome particle the quantity of DNA (in nucleotide pairs), and the quantities of alpha-helix and antiparallel pleated sheet structure (in peptide groups) satisfy the relation 1 : 1 : 1.     

Fractionation of avian erythrocyte histones by hydrophobic interaction chromatography.

The interactions of H1 (H1A, H1B), H2A, H2B, H3, H4, and H5 with phenyl cross-linked agarose were studied. Procedures are described whereby all six histones can be bound, released, and fractionated by using appropriate salt concentrations or pH. The binding can be totally abolished by inclusion of hydrophobic disrupting agents. Control experiments with nonderivated cross-linked agarose ruled out a passive aggregation-disaggregation phenomenon governing the binding patterns. The absorption sequence based on the identification and quantitation of individual histones from either unfractionated (whole) histone or separate histone classes is as follows: H3 greater than or equal to H4 greater than H2B greater than or equal to H5 greater than or equal to H2A greater than H1A greater than or equal to H1B. The order differs only slightly from the reverse of the desorption sequence, H1B less than or equal to H1A less than or equal to H5 less than H2A less than or equal to H3. Preferential interaction of H2A-H2B, H3-H4, and H2A-H2B-H4 occur; these interactions can modify the original relative affinity of each individual component for the matrix. The variability in matrix affinity appears to involve simple stoichiometry of the histone components.     

Histon-histone interactions within chromatin. Preliminary location of multiple contact sites between histones 2A, 2B, and 4.

The contact-site cross-linkers tetranitromethane, UV light, formaldehyde, and a monofunctional imido ester have been used to generate a collection of histone-histone dimers and trimers from nuclei and chromatin. Four different H2B-H4 dimers have been isolated. Preliminary CNBr peptide mapping has shown that all are cross-linked at different positions that are apparently clustered within the C-terminal regions of these histones. Similarily, two different H2A-H2B dimers and two different H2A-H2B-H4 trimers have been partially characterized. The data suggest a functional map for H2B in which the N-terminal third interacts with DNA, the middle third interacts with H2A, and the C-terminal third interacts with H4. We hope, by pursuing this type of analysis, to develop a detailed understanding of each histone-histone binding interaction through saturation cross-linking of the binding sites.     

A rapid method of preparing the (H3-H4-H2A-H2b)(2) histone octamer in large quantities]

A simple and fast method for isolation of large amounts of the histone octamer (H2A-H2B-H3-H4)2 is proposed. This method is based on chromatin adsorption by hydroxyapatite with subsequent extraction of the histone octamer with 50 mM sodium-phosphate buffer containing 4 M NaCl pH 8.0. It was shown that the properties of the histone octamer isolated by this extractive procedure are identical with those of the histone octamer obtained by elution on a Sephadex G-100 column. The histone tetramer (H3-H4)2 and dimer (H2A-H2B) were obtained after gel filtration on Sephadex G-100 in 50 mM sodium-acetate (pH 5.6).     

The nature of forces stabilizing nucleosome structure. Dissociation of histone octamers from DNA

We have used the measurements of the histone fluorescence parameters to study the influence of the ionic strength on histone-DNA and histone-histone interactions in reconstructed nucleosomes. The ionic strength increase lead to the two-stage nucleosome dissociation. The dimer H2A-H2B dissociates at the first stage and the tetramer (H3-H4)2 at the second one. The dimer H2A-H2B dissociation from nucleosome is a two-stage process also. The ionic bonds between (H2A-H2B) histone dimer and DNA break at first and then the dissociation of dimer from histone tetramer (H3-H4)2 occurs. According to the proposed model the dissociation accompanying a nucleosome "swelling" and an increase of DNA curvature radius. It was shown that the energy of electrostatic interactions between histone dimer and DNA is sufficiently less than the energy of dimer-tetramer interaction. We propose that the nucleosome DNA ends interact with the dimer and tetramer simultaneously. The calculated number (approximately 30 divided by 40) of ionic bonds between DNA and histone octamer globular part practically coincides with the number of exposed cationic groups on the surface of octamer globular head. On this basis we have assumed that the spatial distribution of these groups is precisely determined, which explains the high evolutionary conservatism of the histone primary structure.     

Nonallelic histone gene clusters of individual sea urchins (Lytechinus pictus): Polarity and gene organization

We have analyzed the histone genes from the sea urchin Lytechinus pictus. Examination of native DNA from individuals reveals four major Eco RI restriction endonuclease histone gene DNA fragments which have been labeled A (6.0 kb), B (4.1 kb), C (3.1 kb) and D (1.2 kb). The fragments A, B and C have been cloned into E. coli plasmids (pLpA, pLpB and pLpC). These histone gene fragments display length and sequence heterogeneity in different individuals. The plasmid pLpA contains the coding regions for H1, H4, H2B and H3 histones, and we determined that the DNA fragment D is tandem to A in native DNA and that it contains the H2A gene. The plasmids pLpB and pLpC contain the histone genes H2A-H1-H4 and H2B-H3, respectively, and together contain the sequences for the five major histones. Restriction analysis of native L. pictus DNA reveals that B and C are tandem to each other but not intermingled with the A-D-type repeat units, and are thus in separate clusters with a repeat length of 7.2 kb. Since the two cluster types do not segregate, they are not alleles. Hybridization of histone mRNA to exonuclease III-digested linear DNA demonstrated an identical polarity of the histone genes in the A-D- and B-C-type repeat units. This result revealed that the L. pictus histone genes have a polarity which is the same as other sea urchin histone genes examined to date—that is, 3′ H1-H4-H2B-H3-H2A 5′. Restriction endonuclease cleavage patterns of the cloned segments indicate that considerable sequence heterogeneity exists between the two types of histone gene repeat units.     

Spectropolarimetric analysis of the core histone octamer and its subunits

The secondary structure of the calf thymus core histone octamer, (H2A-H2B-H3-H4)2, and its two physiological subunits, the H2A-H2B dimer and (H3-H4)2 tetramer, was analyzed by ORD spectropolarimetry as a function of temperature and solvent ionic strength within the ranges of these experimental parameters where assembly of the core histone octamer exhibits pronounced sensitivity. While the secondary structure of the dimer is relatively stable from 0.1 to 2.0 M NaCl, the secondary structure of the tetramer exhibits complex changes over this range of NaCl concentrations. Both complexes exhibit only modest responses to temperature changes. ORD spectra of very high and very low concentrations of stoichiometric mixtures of the core histones revealed no evidence of changes in the ordered structure of the histones as a result of the octamer assembly process at NaCl concentrations above 0.67 M, nor were time-dependent changes detected in the secondary structure of tetramer dissolved in low ionic strength solvent. The secondary structure of the chicken erythrocyte octamer dissolved in high concentrations of ammonium sulfate, including those of our crystallization conditions, was found to be essentially unchanged from that in 2 M NaCl when examined by both ORD and CD spectropolarimetry. The two well-defined cleaved products of the H2A-H2B dimer, cH2A-H2B and cH2A-cH2B, exhibited reduced amounts of ordered structure; in the case of the doubly cleaved moiety cH2A-cH2B, the reductions were so pronounced as to suggest marked structural rearrangements.     

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.     

Accessibility of histone oligomers to the action of trypsin in a solution or in chromatin with different degrees of compactness

The accessibility to trypsin of "core" histones within the dimer (H2A-H2B), tetramer (H3-H4)2, octamer (H2A-H2B-H3-H4)2 and in chromatin was studied. It was shown that the hydrolysis of histones H2A and H2B within the dimer and octamer occurs in essentially the same way. The tetramer (H2-H4)2 becomes more compact with an increase in the ionic strength. Some of the tetramer (H3-H4)2 sites within the octamer are protected against trypsin. It was demonstrated that in terms of the histone accessibility to trypsin chromatin can exist in three states, i.e., tightly packed (in the presence of histone H1 and bivalent cations), intermediate (in the absence of histone H1 or bivalent cations) and folded (in the absence of histone H1 and bivalent cations). The folding of histones in neither of these chromatin states coincides with that within the octamer in 2M NaCl.     

The intrinsically disordered distal face of nucleoplasmin recognizes distinct oligomerization states of histones

The role of Nucleoplasmin (NP) as a H2A-H2B histone chaperone has been extensively characterized. To understand its putative interaction with other histone ligands, we have characterized its ability to bind H3-H4 and histone octamers. We find that the chaperone forms distinct complexes with histones, which differ in the number of molecules that build the assembly and in their spatial distribution. When complexed with H3-H4 tetramers or histone octamers, two NP pentamers form an ellipsoidal particle with the histones located at the center of the assembly, in stark contrast with the NP/H2A-H2B complex that contains up to five histone dimers bound to one chaperone pentamer. This particular assembly relies on the ability of H3-H4 to form tetramers either in solution or as part of the octamer, and it is not observed when a variant of H3 (H3C110E), unable to form stable tetramers, is used instead of the wild-type protein. Our data also suggest that the distal face of the chaperone is involved in the interaction with distinct types of histones, as supported by electron microscopy analysis of the different NP/histone complexes. The use of the same structural region to accommodate all type of histones could favor histone exchange and nucleosome dynamics.     

Specific histone-histone contacts are ruptured when nucleosomes unfold at low ionic strength.

The ordered unfolding of the nucleosome core within chromatin at low ionic strengths has been studied. The results show that, when nuclei are lysed gently in solutions of very low ionic strength, their constituent nucleosomes rupture at a major H2B-H4 binding site but remain unperturbed at the site of the H2A-H2B interaction. These conclusions are based on data which show that at least four separate but closely spaced H2B-H4 contacts, identifiable by contact-site cross-linking in intact nuclei, are broken when nuclei are suspended in very dilute buffers. Appropriate controls on purified nucleosomes monomers demonstrate that the H2B-H4 contacts being broken are indeed intranucleosomal. Sedimentation of nucleosomes in the ultracentrifuge at various salt concentrations reveals that a significant conformational transition occurs in the range of ionic strength over which the H2B-H4 binding site ruptures.     

Characterization of a cloned histone gene cluster of the newt Notophthalamus viridescens.

We report the cloning and characterization of a histone gene cluster of the newt Notophthalamus viridescens. Fragments containing newt histone genes were identified in whole genome Southern blots; these fragments were cloned into a bacteriophage lambda cloning vector constructed for this purpose. The positions of most of the histone genes were determined by hybridizing subcloned sea urchin histone genes to digests of the cloned newt gene cluster. The position of each gene was verified, and its polarity determined by sequencing a portion of each. The order of the genes in the cloned segment is H1-H3-H2B-H2A-H4, with each of the genes but H2B being transcribed in the same direction. Subcloned segments of the histone gene repeat were used to determine the size of each newt oocyte histone mRNA.     

Chromosomal organization of chicken histone genes: preferred associations and inverted duplications.

We present a detailed picture of the disposition of core and H1 histone genes in the chicken genome. Forty-two genes were located within four nonoverlapping regions totalling approximately 175 kilobases and covered by three cosmid clones and a number of lambda clones. The genes for the tissue-specific H5 histone and other variant histones were not found in these regions. The longest continuous region mapped was 67 kilobases and contained 21 histone genes in five dissimilar clusters. No long-range repeat was evident, but there were preferred associations, such as H1 genes with paired, divergently transcribed H2A-H2B genes and H3-H4 associations. However, there were exceptions, and even when associations such as H1-H2A-H2B we maintained, the order of those genes within a cluster may not have been. Another feature was the presence of three (unrelated) clusters in which genes were symmetrically ordered around central H3 genes; in one such cluster, the boundaries of a duplicated H2A-H4 gene pair contained related repeat sequences. Despite the dispersed nature of chicken histone genes, the number of each type was approximately equal, being represented as follows: 6 H1, 10 H2A, 8 H2B, 10 H3, and 8 H4.     

Spatial organization of the (H3-H4-H2A-H2B)2 histone octamer

Structure of the (H2A-H2B-H3-H4)2 histone octamer isolated from calf thymus chromatin at ionic strength 0.1 to 4.0 M NaCl, pH 7.6, was studied spectrofluorometrically. Sensitivity of lambda max tyrosine fluorescence position to structural changes of histone oligomers and to the processes of their association was shown. It were detect two ranges of cooperative changes in histone optical parameters at 0.6-1.4 M NaCl (transition I) and at 2.4-3.4 M NaCl (transition II): Transition I corresponds to the formation of equilibrium system (hexamer) + (dimer) in equilibrium octamer. Transition II corresponds to the structural changes of the histone octamer. Thus, fluorescence anisotropy increases, lambda max for fluorescence spectrum is shifted to the longer wavelengths, contributions of two components to fluorescence decay change, a fraction of fluorescence accessible to the quenching by I- decreases. Histone octamer formation is characterized by making specific contacts between the (H2A-H2B) dimer and (H3-H4)2 tetramer. These contacts are realized at gradual changing of ionic strengths (by dialysis). In the case of abrupt local changes of the environment the process is irreversibly shifted to formation of unspecific high molecular aggregates. The important function role for energetically degenerated states of histone oligomers, energy barriers between which can be overcome by changing total conditions of histone microenvironment in chromatin is discussed.