Equilibrium folding of the core histones: the H3-H4 tetramer is less stable than the H2A-H2B dimer

To compare the stability of structurally related dimers and to aid in understanding the thermodynamics of nucleosome assembly, the equilibrium stabilities of the recombinant wild-type H3-H4 tetramer and H2A-H2B dimer have been determined by guanidinium-induced denaturation, using fluorescence and circular dichroism spectroscopies. The unfolding of the tetramer and dimer are highly reversible. The unfolding of the H2A-H2B dimer is a two-state process, with no detected equilibrium intermediates. The H3-H4 tetramer is unstable at moderate ionic strengths (mu approximately 0.2 M). TMAO (trimethylamine-N-oxide) was used to stabilize the tetramer; the stability of the H2A-H2B dimer was determined under the same solvent conditions. The equilibrium unfolding of H3-H4 was best described by a three-state mechanism, with well-folded H3-H4 dimers as a populated intermediate. When compared to H2A-H2B, the H3-H3 tetramer interface and the H3-H4 histone fold are strikingly less stable. The free energy of unfolding, in the absence of denaturant, for the H3-H4 and H2A-H2B dimers are 12.4 and 21.0 kcal mol(-)(1), respectively, in 1 M TMAO. It is postulated that the difference in stability between the histone dimers, which contain the same fold, is the result of unfavorable tertiary interactions, most likely the partial to complete burial of three salt bridges and burial of a charged hydrogen bond. Given the conservation of these buried interactions in histones from yeast to mammals, it is speculated that the H3-H4 tetramer has evolved to be unstable, and this instability may relate to its role in nucleosome dynamics.     

The Hydrophobicity of the H3 Histone Fold differs from the Hydrophobicity of the other three Folds

The eukaryotic histone dimers, H3–H4 and H2A–H2B, are formed in the cytosol prior to being transported into the nucleus and assembled into the nucleosome. Residue side-chain distances from the interior of the histone dimers are obtained with an ellipsoidal spatial metric and structural information provided by X-ray analyses at atomic resolution of the nucleosome core particles. While the spatial hydrophobic moment profiles of the dimers are comparable with profiles obtained previously that characterize the hydrophobic core of single-chain, single-domain globular soluble proteins, correlation coefficients between the side-chain hydrophobicities and distances from the interior of the H3–H4 dimer and H2A–H2B dimer differ significantly. This difference is traced to the H3 histone fold, which segregates fewer hydrophobic residues within the protein interior than the three other folds. Examination of the correlation coefficient between residue hydrophobicity and side-chain distance from the dimer interior over local regions of the fold sequence shows that the region of reduced correlation is associated mainly with the residues at the carboxyl end of the H3 histone fold, the helical region of the fold involved in the H3–H3 binding of the (H3–H4)₂ tetramer of the nucleosome. Hydrophobic interactions apparently contribute to the binding of this fourfold helical bundle and this evolutionary requirement may trade off against the requirement for H3–H4 dimer stability. The present results provide a different view than previously proposed, albeit of similar origin, to account for the reduced stability of the H3–H4 dimer compared with the H2A–H2B dimer.Reviewing Editor: Dr. Martin Kreitman     

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.     

Yeast CAF-1 assembles histone (H3-H4)2 tetramers prior to DNA deposition

Following acetylation, newly synthesized H3-H4 is directly transferred from the histone chaperone anti-silencing factor 1 (Asf1) to chromatin assembly factor 1 (CAF-1), another histone chaperone that is critical for the deposition of H3-H4 onto replicating DNA. However, it is unknown how CAF-1 binds and delivers H3-H4 to the DNA. Here, we show that CAF-1 binds recombinant H3-H4 with 10- to 20-fold higher affinity than H2A-H2B in vitro, and H3K56Ac increases the binding affinity of CAF-1 toward H3-H4 2-fold. These results provide a quantitative thermodynamic explanation for the specific H3-H4 histone chaperone activity of CAF-1. Surprisingly, H3-H4 exists as a dimer rather than as a canonical tetramer at mid-to-low nanomolar concentrations. A single CAF-1 molecule binds a cross-linked (H3-H4)₂ tetramer, or two H3-H4 dimers that contain mutations at the (H3-H4)₂ tetramerization interface. These results suggest that CAF-1 binds to two H3-H4 dimers in a manner that promotes formation of a (H3-H4)₂ tetramer. Consistent with this idea, we confirm that CAF-1 synchronously binds two H3-H4 dimers derived from two different histone genes in vivo. Together, the data illustrate a clear mechanism for CAF-1-associated H3-H4 chaperone activity in the context of de novo nucleosome (re)assembly following DNA replication.     

Folding mechanism of the (H3-H4)2 histone tetramer of the core nucleosome

To further understand oligomeric protein assembly, the folding and unfolding kinetics of the H3-H4 histone tetramer have been examined. The tetramer is the central protein component of the core nucleosome, which is the basic unit of DNA compaction into chromatin in the eukaryotic nucleus. This report provides the first kinetic folding studies of a protein containing the histone fold dimerization motif, a motif observed in several protein-DNA complexes. Previous equilibrium unfolding studies have demonstrated that, under physiological conditions, there is a dynamic equilibrium between the H3-H4 dimer and tetramer species. This equilibrium is shifted predominantly toward the tetramer in the presence of the organic osmolyte trimethylamine-N-oxide (TMAO). Stopped-flow methods, monitoring intrinsic tyrosine fluorescence and far-UV circular dichroism, have been used to measure folding and unfolding kinetics as a function of guanidinium hydrochloride (GdnHCl) and monomer concentrations, in 0 and 1 M TMAO. The assignment of the kinetic phases was aided by the study of an obligate H3-H4 dimer, using the H3 mutant, C110E, which destabilizes the H3-H3' hydrophobic four-helix bundle tetramer interface. The proposed kinetic folding mechanism of the H3-H4 system is a sequential process. Unfolded H3 and H4 monomers associate in a burst phase reaction to form a dimeric intermediate that undergoes a further, first-order folding process to form the native dimer in the rate-limiting step of the folding pathway. H3-H4 dimers then rapidly associate with a rate constant of > or =10(7) M(-1)sec(-1) to establish a dynamic equilibrium between the fully assembled tetramer and folded H3-H4 dimers.     

Dynamics of Nucleosome Assembly and Effects of DNA Methylation

The nucleosome is the fundamental packing unit of the eukaryotic genome, and CpG methylation is an epigenetic modification associated with gene repression and silencing. We investigated nucleosome assembly mediated by histone chaperone Nap1 and the effects of CpG methylation based on three-color single molecule FRET measurements, which enabled direct monitoring of histone binding in the context of DNA wrapping. According to our observation, (H3-H4)₂ tetramer incorporation must precede H2A-H2B dimer binding, which is independent of DNA termini wrapping. Upon CpG methylation, (H3-H4)₂ tetramer incorporation and DNA termini wrapping are facilitated, whereas proper incorporation of H2A-H2B dimers is inhibited. We suggest that these changes are due to rigidified DNA and increased random binding of histones to DNA. According to the results, CpG methylation expedites nucleosome assembly in the presence of abundant DNA and histones, which may help facilitate gene packaging in chromatin. The results also indicate that the slowest steps in nucleosome assembly are DNA termini wrapping and tetramer positioning, both of which are affected heavily by changes in the physical properties of DNA.     

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 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.     

Role of individual histone tyrosines in the formation of the nucleosome complex.

We have determined the accessibility of histone tyrosine residues to react with p-nitrobenzenesulfonyl fluoride (NBSF) in intact nuclei, salt-dissociated nucleosomes, isolated histone complexes, and individual core histones. Of the 15 core histone tyrosine residues, 13 are inaccessible in native nucleosomes; only Tyr121 near the C-terminus of H2B is fully accessible, and Tyr54 of H3 is partially accessible under near-physiological conditions. When H1 and the basic N-terminal tails of the core histones are dissociated from the DNA by treating nuclei with 0.4 and 0.8 M NaCl, the two tyrosines which are adjacent to the basic regions of H2B and H3 become accessible as well. This indicates that these tyrosine residues may be involved in histone-DNA interactions, either directly or indirectly. When the H2A-H2B dimers are dissociated from the chromatin by raising the NaCl concentration to 1.2 M, three to four tyrosines located in the structured regions of H2B and H4 are exposed, suggesting that these tyrosine residues may be located at the dimer-tetramer interface. Dissociating all the histones from the DNA at an even higher ionic strength as a mixture of dimers, tetramers, and octamers does not change the pattern of Tyr exposure, but reduces the reactivity of the tyrosines at the dimer-tetramer interface as would be expected from the reassociation of H2A-H2B dimers and H3-H4 tetramers.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Enhanced stability of histone octamers from plant nucleosomes: role of H2A and H2B histones.

Gel filtration and sedimentation studies have previously established that the vertebrate animal core histone octamer is in equilibrium with an (H3-H4)2 tetramer and an H2A-H2B dimer [Eickbush, T. H., & Moudrianakis, E. N. (1978) Biochemistry 17, 4955-4964; Godfrey, J. E., Eickbush, T. H., & Moudrianakis, E. N. (1980) Biochemistry 19, 1339-1346]. We have investigated the core histone octamer of wheat (Triticum aestivum L.) and have found it to be much more stable than its vertebrate animal counterpart. When vertebrate animal histone octamers are subjected to gel filtration in 2 M NaCl, a trailing peak of H2A-H2B dimer can be clearly resolved from the main octamer peak. When the plant octamer is subjected to the identical procedure, there is no trailing peak of H2A-H2B dimer, but rather a single peak containing the octamer. A sampling across the octamer peak from leading to trailing edge shows no change in the ratio of H2A-H2B to (H3-H4)2. Surprisingly, the plant octamer shows the same stability at 0.6 M NaCl, a salt concentration in which the vertebrate animal octamer dissociates into dimers and tetramers. Equilibrium sedimentation data indicate that the assembly potential of the wheat histones in 2 M NaCl is very high at all protein concentrations above 0.1 mg mL-1. In order to disrupt the forces stabilizing the plant histone octamer at high histone concentrations, the concentration of NaCl must be lowered to approximately 0.3 M.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation and physico-chemical properties of native histone complexes: dimer (H2-H2B), tetramer (H3-H4)2 and octamer (H3-H4-H2A-H2B)2

The paper is concerned with the isolation of the native histone complexes: dimer (H2A-H2B), tetramer (H3-H4)2 and octamer (H3-H4-H2A-H2B)2 from the calf thymus chromatin under soft conditions (hydroxyl apatite) fractionation with the subsequent gel filtration). Parameters of hydroxyl apatite saturation with chromatin are determined. The complexes obtained are free of DNA and nonhistone proteins. Absorption spectra parameters, quantum efficiencies and fluorescence spectra typical of the corresponding histone oligomers are established. Comparison of free tyrosine fluorescence spectra with histone tyrosyl ones revealed a long-wave shift in the latter.     

Thermodynamic studies of the core histones: stability of the octamer subunits is not altered by removal of their terminal domains.

We have investigated the role of the labile terminal domains of the core histones on the stability of the subunits of the protein core of the nucleosome by studying the thermodynamic behavior of the products of limited trypsin digestion of these subunits. The thermal stabilities of the truncated H2A-H2B dimer and the truncated (H3-H4)/(H3-H4)(2) system were studied by high-sensitivity differential scanning calorimetry and circular dichroism spectroscopy. The thermal denaturation of the truncated H2A-H2B dimer at pH 6.0 and low ionic strength is centered at 47.3 degrees C. The corresponding enthalpy change is 35 kcal/mol of 11.5 kDa monomer unit, and the heat capacity change upon unfolding is 1.2 kcal/(K mol of 11.5 kDa monomer unit). At pH 4.5 and low ionic strength, the truncated (H3-H4)/(H3-H4)(2) system, like its full-length counterpart, is quantitatively dissociated into two truncated H3-H4 dimers. The thermal denaturation of the truncated H3-H4 dimer is characterized by the presence of a single calorimetric peak centered at 60 degrees C. The enthalpy change is 25 kcal/mol of 10 kDa monomer unit, and the change in heat capacity upon unfolding is 0.5 kcal/(K mol of 10 kDa monomer unit). The thermal stabilities of both types of truncated dimers exhibit salt and pH dependencies similar to those of the full-length proteins. Finally, like their full-length counterparts, both truncated core histone dimers undergo thermal denaturation as highly cooperative units, without the involvement of any significant population of melting intermediates. Therefore, removal of the histone "tails" does not generally affect the thermodynamic behavior of the subunits of the core histone complex, indicating that the more centrally located regions of the histone fold and the extra-fold structured elements are primarily responsible for their stability and responses to parameters of their chemical microenvironment.     

Hydrophobic and acidic moments of a nucleoplasmin NP-core chaperone

A recent crystal structure, at atomic resolution, of the NO38-core chaperone has revealed a decamer comprised of a dimer of pentamers, with each pentamer consisting of closely coupled eight-stranded beta-barrel monomers. This N-terminal core domain of the chaperone shares the Nucleoplasmin family fold and is presumed to assist the binding of the core histones in their assembly into nucleosomes during DNA replication and repair. The present work provides a measure of the hydrophobic residue burial about the different interfaces and centers of the NO38-core multimeric structure. While the hydrophobic "pentameric ring," comprised of the hydrophobic cores of the monomers and prevalence of non-polar residues at their interfaces is observed, a hydrophobic bias with respect to the center of the pentamer is also found, and consequently also expected to contribute to the thermostability of the multimer. Structural and chromatographic analysis had shown the NO38-core chaperone to bind (H3-H4)2 histone tetramers as well as H2A-H2B dimers. The acidic dipole, which reflects the spatial disposition of the acidic residues of the core monomer points to the lateral region of the monomers comprising the oligomers, and thereby, shows it to be the region of charge that would optimally complement the basic charge of the histones in their electrostatic binding to the chaperone. It is also pointed out that the prevalence of basic residues on the short helices of the histone cores also provides regions of charge that would complement histone binding to the chaperone.     

NF-Y associates with H3-H4 tetramers and octamers by multiple mechanisms

NF-Y is a CCAAT-binding trimer with two histonic subunits, NF-YB and NF-YC, resembling H2A-H2B. We previously showed that the short conserved domains of NF-Y efficiently bind to the major histocompatibility complex class II Ea Y box in DNA nucleosomized with purified chicken histones. Using wild-type NF-Y and recombinant histones, we find that NF-Y associates with H3-H4 early during nucleosome assembly, under conditions in which binding to naked DNA is not observed. In such assays, the NF-YB-NF-YC dimer forms complexes with H3-H4, for whose formation the CCAAT box is not required. We investigated whether they represent octamer-like structures, using DNase I, micrococcal nuclease, and exonuclease III, and found a highly positioned nucleosome on Ea, whose boundaries were mapped; addition of NF-YB-NF-YC does not lead to the formation of octameric structures, but changes in the digestion patterns are observed. NF-YA can bind to such preformed DNA complexes in a CCAAT-dependent way. In the absence of DNA, NF-YB-NF-YC subunits bind to H3-H4, but not to H2A-H2B, through the NF-YB histone fold. These results indicate that (i) the NF-Y histone fold dimer can efficiently associate DNA during nucleosome formation; (ii) it has an intrinsic affinity for H3-H4 but does not form octamers; and (iii) the interactions between NF-YA, NF-YB-NF-YC, and H3-H4 or nucleosomes are not mutually exclusive. Thus, NF-Y can intervene at different steps during nucleosome formation, and this scenario might be paradigmatic for other histone fold proteins involved in gene regulation.