Histone-DNA binding free energy cannot be measured in dilution-driven dissociation experiments

Despite decades of study on nucleosomes, there has been no experimental determination of the free energy of association between histones and DNA. Instead, only the relative free energy of association of the histone octamer for differing DNA sequences has been available. Recently, a method was developed based on quantitative analysis of nucleosome dissociation in dilution experiments that provides a simple practical measure of nucleosome stability. Solution conditions were found in which nucleosome dissociation driven by dilution fit well to a simple model involving a noncooperative nucleosome assembly/disassembly equilibrium, suggesting that this approach might allow absolute equilibrium affinity of the histone octamer for DNA to be measured. Here, we show that the nucleosome assembly/disassembly process is not strictly reversible in these solution conditions, implying that equilibrium affinities cannot be obtained from these measurements. Increases in [NaCl] or temperature, commonly employed to suppress kinetic bottlenecks in nucleosome assembly, lead to cooperative behavior that cannot be interpreted with the simple assembly/disassembly equilibrium model. We conclude that the dilution experiments provide useful measures of kinetic but not equilibrium stability. Kinetic stability is of practical importance: it may govern nucleosome function in vivo, and it may (but need not) parallel absolute thermodynamic stability.     

Conformation of the HMG17-nucleosome complex

The nucleosome core binds more than two molecules of HMG17 at low ionic strength (8.9 mM Tris-HCl/8.9 mM boric acid/0.25 mM Na2EDTA, pH 8.3). Circular dichroism of the complexes showed only minor conformational changes of the nucleosome core DNA on binding of HMG17, with no detectable change in the histone secondary structure. The fluorescence of N-(3-pyrene) maleimide bound to -SH groups at Cys-110 of H3 histones in the core particle suggested that the structure of the histone octamer assembly changed little upon binding of HMG17 to the nucleosome. These observations support the idea that even a high level of HMG17 binding, e.g., four HMGs per nucleosome, alone, does not open up the core particle.     

Structure of histone octamers in reconstituted polynucleosomes

The salt-dependent structural changes of the histone octamer in complex with high-molecular-weight DNA have been studied by fluorescent spectroscopy. Changes in both the spectra maximum position and anisotropy of the histone tyrosine fluorescence reveal structural transitions in nucleosome within the ranges of 0.5-3 mM and 20-30 mM NaCl. Comparison of the octamer fluorescent parameters in complex with DNA as well as in a free state permits to interpret the revealed structural transitions as a change in degree of contacts stability between (H2A-H2B) dimer and (H3-H4)2 tetramer. More pronounced conformational changes in histone octamer are observed under the conditions of polynucleosome fibers interaction within the range of physiological ionic strength (100-600 mM NaCl). As far as fluorescent parameters are concerned, the aforementioned changes are connected with entire destruction of (H2A-H2B) dimer specific contacts with (H3-H4)2 tetramer. The obtained results suggest the possibility of existence of different structural states of histone octamer in the chromatin composition including those which are quite dissimilar from the octamer structure in the 2M NaCl solution.     

H3.H4 tetramer directs DNA and core histone octamer assembly in the nucleosome core particle.

The way in which histones interact with DNA during in vitro assembly of nucleohistone has been examined. Chicken erythrocyte core histones H2A, H2B, H3, and H4 and lambdaDNA in 2 M NaCl were allowed to interact by stepwise decrease in the salt concentration. Binding, although weak, was first observed at 1.4 M NaCl and was essentially completed at 0.6 M NaCl. Analysis of the DNA-bound histones revealed that each of the histones in the pairs H2A,H2B and H3,H4 was always present in equimolar amounts and that the relative proportion of each pair was constant between 1.4 and 0.8 M NaCl. Evidence is presented suggesting that binding occurred via complexes of the four histones, the nature of which is likely to reflect the equilibrium among the octamer and its products of dissociation (Ruiz-Carrillo, A., & Jorcano, J.L. (1979) Biochemistry (preceding paper in this issue)). The presence of complexes of the four core histones is, however not required for the correct assembly of the nucleosome core particle. Nucleohistones obtained by adding at progressively lower ionic strengths the dimer H2A.H2B to the H3.H4-DNA complex (split reconstitutions) had the same characteristics as those assembled with the core histone complexes.     

Human centromere protein B induces translational positioning of nucleosomes on alpha-satellite sequences

The human centromere proteins A (CENP-A) and B (CENP-B) are the fundamental centromere components of chromosomes. CENP-A is the centromere-specific histone H3 variant, and CENP-B specifically binds a 17-base pair sequence (the CENP-B box), which appears within every other alpha-satellite DNA repeat. In the present study, we demonstrated centromere-specific nucleosome formation in vitro with recombinant proteins, including histones H2A, H2B, H4, CENP-A, and the DNA-binding domain of CENP-B. The CENP-A nucleosome wraps 147 base pairs of the alpha-satellite sequence within its nucleosome core particle, like the canonical H3 nucleosome. Surprisingly, CENP-B binds to nucleosomal DNA when the CENP-B box is wrapped within the nucleosome core particle and induces translational positioning of the nucleosome without affecting its rotational setting. This CENP-B-induced translational positioning only occurs when the CENP-B box sequence is settled in the proper rotational setting with respect to the histone octamer surface. Therefore, CENP-B may be a determinant for translational positioning of the centromere-specific nucleosomes through its binding to the nucleosomal CENP-B box.     

H2A.Z stabilizes chromatin in a way that is dependent on core histone acetylation

The functional and structural chromatin roles of H2A.Z are still controversial. This work represents a further attempt to resolve the current functional and structural dichotomy by characterizing chromatin structures containing native H2A.Z. We have analyzed the role of this variant in mediating the stability of the histone octamer in solution using gel-filtration chromatography at different pH. It was found that decreasing the pH from neutral to acidic conditions destabilized the histone complex. Furthermore, it was shown that the H2A.Z-H2B dimer had a reduced stability. Sedimentation velocity analysis of nucleosome core particles (NCPs) reconstituted from native H2A.Z-containing octamers indicated that these particles exhibit a very similar behavior to that of native NCPs consisting of canonical H2A. Sucrose gradient fractionation of native NCPs under different ionic strengths indicated that H2A.Z had a subtle tendency to fractionate with more stabilized populations. An extensive analysis of the salt-dependent dissociation of histones from hydroxyapatite-adsorbed chromatin revealed that, whereas H2A.Z co-elutes with H3-H4, hyperacetylation of histones (by treatment of chicken MSB cells with sodium butyrate) resulted in a significant fraction of this variant eluting with the canonical H2A. These studies also showed that the late elution of this variant (correlated to enhanced binding stability) was independent of the chromatin size and of the presence or absence of linker histones.     

Site-Specific Disulfide Crosslinked Nucleosomes with Enhanced Stability

We engineered nucleosome core particles (NCPs) with two site-specific cysteine crosslinks that increase the stability of the particle. The first disulfide was introduced between the two copies of H2A via an H2A-N38C point mutation, effectively crosslinking the two H2A/H2B heterodimers together to stabilize the histone octamer against H2A/H2B dimer dissociation. The second crosslink was engineered between an R40C point mutation on the N-terminal tail of H3 and the NCP DNA ends by the introduction of a convertible nucleotide. This crosslink maintains the nucleosome DNA in a fixed translational setting relative to the histone octamer and prevents dilution-driven dissociation. The X-ray crystal structures of NCPs containing the disulfides in isolation and in combination were determined. Both disulfides stabilize the structure of the NCP without disturbing the overall structure. Nucleosomes containing these modifications will be advantageous for biochemical and structural studies as a consequence of their greater resistance to dissociation during high dilution in purification, elevated salt for crystallization and vitrification for cryogenic electron microscopy.     

The amino-terminal tails of the core histones and the translational position of the TATA box determine TBP/TFIIA association with nucleosomal DNA.

We establish that the TATA binding protein (TBP) in the presence of TFIIA recognizes the TATA box in nucleosomal DNA dependent on the dissociation of the amino-terminal tails of the core histones from the nucleosome and the position of the TATA box within the nucleosome. We examine TBP/TFIIA access to the TATA box with this sequence placed in four distinct rotational frames with reference to the histone surface and at three distinct translational positions at the edge, side and dyad axis of the nucleosome. Under our experimental conditions, we find that the preferential translational position at which TBP/TFIIA can bind the TATA box is within linker DNA at the edge of the nucleosome and that binding is facilitated if contacts made by the amino-terminal tails of the histones with nucleosomal DNA are eliminated. TBP/TFIIA binding to DNA at the edge of the nucleosome occurs with the TATA box in all four rotational positions. This is indicative of TBP/TFIIA association directing the dissociation of the TATA box from the surface of the histone octamer.     

Influence of histone tails and H4 tail acetylations on nucleosome-nucleosome interactions

Nucleosome–nucleosome interaction plays a fundamental role in chromatin folding and self-association. The cation-induced condensation of nucleosome core particles (NCPs) displays properties similar to those of chromatin fibers, with important contributions from the N-terminal histone tails. We study the self-association induced by addition of cations [Mg²⁺, Ca²⁺, cobalt(III)hexammine³⁺, spermidine³⁺ and spermine⁴⁺] for NCPs reconstituted with wild-type unmodified histones and with globular tailless histones and for NCPs with the H4 histone tail having lysine (K) acetylations or lysine-to-glutamine mutations at positions K5, K8, K12 and K16. In addition, the histone construct with the single H4K16 acetylation was investigated. Acetylated histones were prepared by a semisynthetic native chemical ligation method. The aggregation behavior of NCPs shows a general cation-dependent behavior similar to that of the self-association of nucleosome arrays. Unlike nucleosome array self-association, NCP aggregation is sensitive to position and nature of the H4 tail modification. The tetra-acetylation in the H4 tail significantly weakens the nucleosome–nucleosome interaction, while the H4 K → Q tetra-mutation displays a more modest effect. The single H4K16 acetylation also weakens the self-association of NCPs, which reflects the specific role of H4K16 in the nucleosome–nucleosome stacking. Tailless NCPs can aggregate in the presence of oligocations, which indicates that attraction also occurs by tail-independent nucleosome–nucleosome stacking and DNA–DNA attraction in the presence of cations. The experimental data were compared with the results of coarse-grained computer modeling for NCP solutions with explicit presence of mobile ions.     

Salt-dependent interconversion of inner histone oligomers.

The inner histone complex, extracted from chicken erythrocyte chromatin in 2 M NaCL AT pH 7.4, has been characterized by sedimentation equilibrium and sedimentation velocity. High speed sedimentation equilibrium studies indicate that in 2 M NaCl the inner histones are a weakly associating system with contributions from species ranging in molecular weight from dimer to octamer. The appearance of a single boundary (3.8S at 2 M NaCl) in sedimentation velocity studies conducted over a wide range of protein concentrations and ionic conditions indicates that the various histone oligomers present are in rapid equilibrium with one another. At higher salts the equilibrium is shifted to favor higher molecular weight species; in 4 M NaCl essentially all of the histone is octameric at protein concentrations above 0.2 mg/ml. The facile interconversion of histone oligomers suggests that small alterations in histone-histone interactions may be responsible for changes in nucleosome conformations during various biological processes.     

Structural changes of nucleosomal particles and isolated core-histone octamers induced by chemical modification of lysine residues

Treatment of nucleosomal particles and isolated core-histone octamers with dimethylmaleic anhydride, but not with acetic anhydride, is accompanied by a biphasic release of the two H2A.H2B dimers, the first dimer being more easily released than the second. With both kinds of particles, 50% of histones H2A and H2B are released for modification of approximately 35% of the histone amino groups. The similar behavior of nucleosomal particles and isolated core-histone octamers is consistent with the same structure of the histone octamer in the nucleosomal particle and in the free octamer in 2 M NaCl. The described release of H2A.H2B dimers allows the preparation of nucleosomal particles deficient in one H2A.H2B dimer and of the histone hexamers H2A.H2B.(H3.H4)2. For more extensive modifications, both reagents, acetic and dimethylmaleic anhydrides, cause the dissociation of nucleosomal particles with liberation of double-stranded DNA, which suggests that lysine amino groups are involved in the binding of histones to DNA. The modified nucleosomal particles are more sensitive to ionic strength than those untreated, and the presence of salt (NaCl) increases the extent of DNA release. The histones corresponding to the liberated DNA, except H2A and H2B released with dimethylmaleic anhydride, are apparently bound to the DNA-containing particles as extra histones.     

Differential dissociation of histone tails from core chromatin

The dissociation of the trypsin-sensitive basic tails of the core histones in core chromatin has been followed as a function of [NaCl] using proton NMR spectroscopy. The tails dissociate in a highly cooperative all or none manner over the salt concentration range 0.2-0.6 M, that is, below the salt concentration required to dissociate the complete molecule. Assuming that each basic tail dissociates independently, the total number of salt linkages involved in binding the tails to DNA is 103. This equals the number of basic side chains in the tails of an octamer. The standard free energy of dissociation, delta G degree, in 1 M NaCl at 297 K is 3.6 kcal/mol. Temperature had no effect on the extent of dissociation up to 45 degrees C. However, between 45 and 65 degrees C, where the premelting transition in the core chromatin occurs, the tails dissociated completely. Dissociation of the tails was associated with a conformational transition in the DNA consistent with loss of supercoiling. From this, and the results of a previous study, it can be shown that the structured, trypsin-resistant domain of each core histone octamer makes 100 salt linkages to DNA. Thus, in 10 mM salt, each core octamer makes a total of 203 salt linkages to DNA.     

Stability of the conservative mode of nucleosome assembly.

The conservative assembly of nucleosome histone octamer cores has been confirmed by electrophoretic analysis of density labeled histones following equilibrium buoyant density centrifugation. After normal replication, crosslinked octamers are shown not to contain a mixture of new and old core histones. Moreover, when DNA synthesis is inhibited by ara-C nucleosome cores are still assembled exclusively from nascent histone. Similarly, after release from cycloheximide inhibition newly synthesized core histone is conservatively deposited. Thus, a conservative mechanism of histone octamer assembly occurs when nascent histone is present in the normal stoichiometry to nascent DNA and when chromatin is assembled in nascent histone or nascent DNA excess.     

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.     

Model of the packing of DNA and histone octamer in the structure of the nucleosome

A model is proposed which describes the packing of polypeptide chains of histone molecules in the octamer (H3--H4--H2A--H2B)2, and interlocation of DNA and octamer in the nucleosome. DNA packing in the nucleosome is provided for by electrostatic interactions between DNA phosphates and cationic groups located on the globular part surface of histones octamer. The cationic groups of N- and C-end regions of the histone molecules (histones H3 and H4 in particular) additionally stabilize the nucleosome structure.     

Effects of histone acetylation on nucleosome properties as evaluated by polyacrylamide gel electrophoresis and hydroxylapatite dissociation chromatography

The release of acetylated histones from chick oviduct chromatin was analyzed by hydroxylapatite column chromatography. By raising of the NaCl concentration, acetylated histones were eluted from hydroxylapatite-bound chromatin depending on their release from nucleosomal DNA. Electrophoresis on acid-urea gel showed that hyperacetylated forms of histone H4 were eluted at a lower NaCl concentration than non-acetylated or hypoacetylated H4, suggesting that hyperacetylated H4 has decreased stability in nucleosomes. However, under milder ionic conditions which do not induce dissociation between histones and DNA, polyacrylamide gel electrophoresis of purified nucleosome cores showed no evidence for their unfolding or for increased accessibility by high mobility group protein-17.     

Conformations of the core nucleosome: effects of ionic strength and high mobility group protein 14 and 17 binding on the fluorescence emission and polarization of dansylated methionine-84 of histone H4

Chicken histone H4 labeled at Met-84 with the fluor N-[(acetylamino)ethyl]-8-naphthyl-amine-1-sulfonic acid has been incorporated into a nucleosome which has physical characteristics virtually identical with those of native core nucleosomes. The fluorescence emission and polarization properties of the labeled nucleosome were measured as a function of ionic strength and the binding of high mobility group (HMG) proteins 14 and 17. Also, the accessibility of the fluor to the quenching agent acrylamide was determined. It was found that the fluorescence emission changes in the range 0.1-1000 mM NaCl are rather small and indicate that no major unfolding of the octamer structure occurs around Met-84 on H4 at least. Five or perhaps six discrete states were found in that ionic strength range. Each has a different accessibility to the quenching agent. The range of accessibilities varied from 9 X 10(-7) to 32 X 10(-7) mol-1 s-1 for 0.1-1000 mM NaCl, respectively. Polarization measurements showed that there was little change in the rotational relaxation lifetime of the fluor at ionic strengths less than 50 mM NaCl. Above this value, the rotational relaxation lifetimes decreased from 107 to 25 ns at 600 mM NaCl, indicating a moderately increased rotational freedom for the fluor. It is suggested that the histone octamer changes its degree of compaction in the range 0.1-600 mM NaCl but that no major protein unfolding occurs.(ABSTRACT TRUNCATED AT 250 WORDS)     

Histone H2A ubiquitination does not preclude histone H1 binding, but it facilitates its association with the nucleosome

Histone H2A ubiquitination is a bulky posttranslational modification that occurs at the vicinity of the binding site for linker histones in the nucleosome. Therefore, we took several experimental approaches to investigate the role of ubiquitinated H2A (uH2A) in the binding of linker histones. Our results showed that uH2A was present in situ in histone H1-containing nucleosomes. Notably in vitro experiments using nucleosomes reconstituted onto 167-bp random sequence and 208-bp (5 S rRNA gene) DNA fragments showed that ubiquitination of H2A did not prevent binding of histone H1 but it rather enhanced the binding of this histone to the nucleosome. We also showed that ubiquitination of H2A did not affect the positioning of the histone octamer in the nucleosome in either the absence or the presence of linker histones.     

Internal architecture of the core nucleosome: fluorescence energy transfer studies at methionine-84 of histone H4

Chicken histone H4, labeled separately at Met-84 with N-[[(iodoacetyl)amino]ethyl]-5-naphthylamine-1-sulfonic acid and 5-(iodoacetamido)fluorescein, was reassociated with unlabeled histones H2A, H2B, and H3 and 146 base pairs of DNA to produce fluorescently labeled nucleosomes having physical characteristics virtually the same as those of native core particles. Four types of particles were prepared containing respectively unlabeled H4, dansylated H4, fluoresceinated H4, and a mixture of the two labeled H4 molecules. Quantitative singlet-singlet energy-transfer measurements were carried out to determine changes in the distance between the two Met-84 H4 sites within the same nucleosome following conformational transitions which we have reported earlier. In the ionic strength range 0.1-100 mM NaCl, the distance between these sites is less than 2 nm except at 1 mM. Between 100 and 600 mM monovalent salt the distance separating the donor and acceptor fluors at Met-84 H4 increases to 3.8 nm. The conformational change centered around 200 mM NaCl is cooperative. Our results and those of others indicate that there is little unfolding of the histone octamer, at least around Met-84 H4, in the entire ionic strength range studied. A mechanism involving the rotation of the globular portion of H4 is proposed to account for this transition which occurs at physiological ionic strengths.