Interaction of histone H1 molecules in a solution

Molecules of histones H1 isolated from the calf thymus, carp testicles and spermatozoa as well as trypsin-stable fragments of these proteins have been studied from the standpoint of their structure and interaction using methods of differential spectrophotometry, gel filtration and turbidimetry. The globular structure of histone H1 of the calf thymus is formed with an increase in the ionic strength of the medium and it is eluted as dimer with gel chromatography. With a considerable local increase of ionic strength (by addition of NaCl crystals) molecules of histones H1 form high-molecular aggregates from all the studied tissues. This aggregation is a result of interaction of globular trypsin-stable sites. Molecules of histone H1 from carp testicles and spermatozoa as well as their trypsin-stable fragments revealed no differences in the ability to form dimers and aggregates.     

Displacement of histones by sperm-specific proteins at different stages of spermatogenesis of squid

Changes in the composition of the chromatin basic proteins during spermatogenesis of the squid Illex argentinus were studied. The core histones of I. argentinus slightly differ from those of calf thymus in the subfractional composition of histones H2A and H2B. A similar amino acid composition is revealed in the histones H1 of the squid I. argentinus and calf thymus. Histone H1 of the squid has a lower molecular mass and a special subfractional composition as compared to those of calf thymus, grass carp and carp studied formerly [Kadura et al. (1983) Comp. Biochem. Physiol. 743, 343-350]. Neither the fractional nor subfractional composition of histones changes during spermatogenesis. The two new proteins were revealed in the chromatin composition of squid testes and spermatozoa illexines I1 and I2. Illexine I2 is composed of two subfractions I2-1 and I2-2. Illexine I2 shows a high content of arginine (75 mol/100 mol). Serine (10 mol/100 mol), histidine (3,2 mol/100 mol) and tyrosine residues (2,9 mol/100 mol) are also present. Illexine I1 shows the presence of arginine (45,6 mol/100 mol), lysine (7.6 mol/100 mol), serine (11.4 mol/100 mol), hystidine (2.3 mol/100 mol) and tyrosine residues (2.8 mol/100 mol). Molecular masses of illexines I2 and I1 are approximately 7 kDa and 9 kDa respectively. It is supposed that during spermatogenesis the histones are displaced in two-stage order: histones----I1----I2.     

Tyrosine residues in histones. Kinetics of histones F1 and F2A1 nitration by tetranitromethane]

The kinetics of nitration of tyrosine residues in histones F1 and F2a1 by tetranitromethane has been investigated. At low ionic strength and 30-fold molar excess of nitrating agent the nitration reaction results in fast modification of all tyrosine residues in both histones. At the same time the rates of modification of different tyrosine residues in histone F2a1 are not identical and markedly exceed the rate of N-Ac-OEt-Tyr nitration in a model system. The increase of reaction mixture ionic strength causes an increase of modification rates. The differential UV-absorption spectra of histone F1 obtained by temperature perturbation show an abnormal positive characteristic maximum at 286.8 nm. Analysis of the dependence of nitration rates of tyrosine residues in histones in saline solutions upon the ionic strength and of difference UV-absorption spectra of histones leads to a conclusion that there are specific interactions of definite parts of histone polypeptide chains. These interactions may arise from aggregation of histone molecules.     

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)     

The effects of daunomycin antibiotic on histone H(1): thermal denaturation and fluorescence spectroscopy studies

Using thermal denaturation and fluorescence spectroscopy, we have investigated the interaction of antitumor antibiotic, daunomycin, with calf thymus histone H(1) under several ionic strengths. The results show that daunomycin binds to histone H(1) and increases its melting temperature. Increasing ionic strength elevates this effect. Fluorescence emission data show that the interaction of daunomycin with histone H(1) decreases the emission intensity at 325 nm and induces hyperchromicity in the emission spectrum of the drug. The results suggest that histone H(1) can be considered as a new target for drug action at the chromatin level.     

Expression and purification of the full murine NPM2 and study of its interaction with protamines and histones

Mouse nucleoplasmin M.NPM2 was recombinantly expressed and the protein consisting of the complete sequence was purified and characterized. Similar to its Xenopus laevis X.NPM2 counterpart, the protein forms stable pentameric complexes and exhibits an almost undistinguishable hydrodynamic ionic strength-dependent unfolding behavior. The interaction of N.PM2 with histones and mouse P1/P2 protamines revealed that these chromosomal proteins bind preferentially to the distal part of the nucleoplasmin pentamer. Moreover, the present work highlights the critical role played by histones H2B and H4 in the association of the histone H2A-H2B dimers and histone octamer with nucleoplasmin.     

The role of histone H2B from sea urchin sperm in the association of reconstituted minichromosomes

A comparative study of the condensation of reconstituted complexes of circular SV40 DNA with core histones from calf thymus and sea urchin sperm was performed using sedimentation and electron microscopic techniques. It is shown that in low ionic strength solutions both types of complexes are similar to native minichromosomes. In the region from 0.08 to 0.16 M NaCl the complexes of SV40 DNA with thymus histones form small compact particles. By contrast, the compaction of the SV40 DNA complexes with sperm histones results in the formation of giant intermolecular associates. The results obtained may mean that histone H2B of sea urchin sperm participates in the formation of a higher order structure in sperm chromatin.     

Thermal denaturation and fluorescence study of nucleosomes containing non-histone chromosomal protein HMG2

Interaction of calf thymus non-histone chromosomal protein HMG2 with H1,H5-depleted nucleosomes from chicken erythrocytes was studied by means of thermal denaturation and an N-(3-pyrene)maleimide fluorescence probe. Under low ionic conditions (2 mM Tris buffer plus EDTA) addition of 1-2 molecules of HMG2 per nucleosome markedly stabilized the segment of the linker DNA against thermal denaturation. Under approximately physiological ionic conditions (0.1 M NaCl) addition of two HMG2 molecules per nucleosome, labeled by N-(3-pyrene)maleimide at the sulfhydryl groups of Cys-110 of histones H3, resulted in a decrease of the pyrene excimer fluorescence corresponding to the slight movement of the sulfhydryl groups of the two histone H3 molecules apart.     

Association-dissociation of histone oligomers. A spin label study

The spin label method has been used to obtain information about conformational changes of histone oligomers taking advantage of the fact that at a low ionic strength and in the presence of other histones about 45% of cysteine residues of histone H3 react with the 3-maleimido-2,2,5,5-tetramethyl-1-pyrrolidinyloxyl spin label. For the labeled complexes H3-H4 and H nu the degree of immobilization of the spin label is a function of the ionic strength. This variation is identical for both complexes within a long range of ionic strengths, including the interval of 0.8-2 M NaCl, under which conditions interactions are known to exist between the tetramer (H3)2 (H4)2 and the dimer (H2A) (H2B). This finding suggests a negligible influence of the dimer for modifying the cysteine residue environment of histone H3 on octamer formation. GuHCl treatment at high ionic strength of the labeled complexes gives rise to a non-lineal increase in the degree of mobility of the spin label. This increase, at low GuHCl concentration (0-0.5 M GuHCl), is interpreted as showing a lowering in rigidity for the Cys residue environment, without affecting the general stability of the tetramer (H3)2 (H4)2. At higher GuHCl concentration (2-3 M GuHCl) the increase in the spin label mobility is related to a dissociation of the complexes in single histones. Our results are consistent with the view that the overall structure of the tetramer, as well as its conformational changes during complex structuration or denaturation, are not strongly affected by the presence of the dimer (H2A) (H2B).     

The binding of adenosine(5'')tetraphospho(5'')adenosine to calf thymus histones measured by non-equilibrium dialysis.

Binding of adenosine(5')tetraphospho(5')adenosine (Ap4A) to histones of calf thymus was investigated by non-equilibrium dialysis. Histone H1 interacts with the dinucleotide via two strong sites and competes with Mg2+ ions. Intrinsic dissociation constants were 1.6 +/- 0.1 microM and 11 +/- 1 microM for zero and 0.4 mm-Mg2+ concentration respectively. Binding of poly(dT) and of other nucleotides to histone H1 was measured in an [3H]Ap4A-competition assay. The tendency to form complexes among nucleotides was highest for bisnucleoside tetraphosphates and decreased in the order poly(dT) greater than or equal to Ap4A approximately Gp4G greater than Ap4 much greater than Ap3A approximately Ap5A greater than or equal to ATP, GTP and dTTP. The co-ordination complex derived from Ap4A and cis-diammine-dichloroplatinum(II) was not reactive. The other histones of calf thymus also bound Ap4A with affinities decreasing in the order H4 approximately H3 greater than H1 greater than H2b greater than H2a. Ap4A stimulated the exchange of histone H1 between nucleosomes, but this effect was referred to ionic strength. It did not bind to assembled nucleosomes. Binding of Ap4A to histone H1 was decreased by salt (NaCl). At physiological saline concentration the value of the dissociation constant is commensurable with the value of the Ap4A concentration in the nucleus and thus indicative of complex-formation in vivo.     

Distribution of H1 histone in chromatin digested by micrococcal nuclease.

The relative amount of H1 histone associated with isolated nucleosomes from calf thymus was determined as a function of the extent of DNA digestion by micrococcal nuclease. Generally the amount of H1 histone associated with mononucleosomes decreases with increasing digestion until 60% of the original H1 remains associated with DNA 150 base pirs or less in size. Coincidentally, H1 histone increases relative to the other histones in aggregated material that sediments through sucrose gradients to form a pellet. However, the level of H1 histone remains at control values for oligonucleosomes (dimer to hexamer) over the 30% digestion range studied. An increase in ionic strength to 0.3 M NaCl in the density gradient reveals a different pattern of H1 binding, whereby the amount of H1 reflects the average size of the DNA fragments with which it is associated. Although there is significant binding to nucleosomes per se, it appears that the major ionic involvement of H1 is with internucleosomal spacer DNA.     

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.     

Comparative electrophoretic properties of histones from trout and chicken erythrocytes and calf thymus at different concentrations of EDTA]

An introduction of EDTA into an electrophoretic system was found to cause specific changes in the histone distribution patterns. The electrophoretic mobility of histones H3, H2b and H2a from three evolutionally unrelated sources (trout and chicken erythrocytes and calf thymus) is increased and that for histones H1 and H5 is decreased with respect to histone H4. In general the decrease of electrophoretic mobility of the histones in the presence of EDTA is correlated with the content of basic amino acids in these histones. The effect observed can be used from electrophoretic analysis of histones.     

Histones from spermatozoa of the horseshoe crab

It is shown that histones are the nuclear proteins present in spermatozoa of the horseshoe crab Limmulus polyphemus, an arthropod which is considered a living fossil. They have been characterized and found to be closely related to calf thymus histones. The only difference is the presence of an additional histone in small amounts (2?3% of the whole histones) which has intermediate properties between H1 and H2b.     

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.     

A new fluorescence resonance energy transfer approach demonstrates that the histone variant H2AZ stabilizes the histone octamer within the nucleosome

Nucleosomes are highly dynamic macromolecular complexes that are assembled and disassembled in a modular fashion. One important way in which this dynamic process can be modulated is by the replacement of major histones with their variants, thereby affecting nucleosome structure and function. Here we use fluorescence resonance energy transfer between fluorophores attached to various defined locations within the nucleosome to dissect and compare the structural transitions of a H2A.Z containing and a canonical nucleosome in response to increasing ionic strength. We show that the peripheral regions of the DNA dissociate from the surface of the histone octamer at relatively low ionic strength, under conditions where the dimer-tetramer interaction remains unaffected. At around 550 mm NaCl, the (H2A-H2B) dimer dissociates from the (H3-H4)(2) tetramer-DNA complex. Significantly, this latter transition is stabilized in nucleosomes that have been reconstituted with the essential histone variant H2A.Z. Our studies firmly establish fluorescence resonance energy transfer as a valid method to study nucleosome stability, and shed new light on the biological function of H2A.Z.     

Structuring of H1 histone. Evidence of high-affinity binding sites for phosphate ions

Circular dichroism studies show that low concentrations of phosphate ions induce folding of the H1 histones. Sulfate and perchlorate anions have effects similar to phosphate indicating the presence on H1 histones of binding sites with high affinity for ions with tetrahedral geometry. In fact, the structuring efficiency of different ions, as determined by the midpoint value of the effect/concentration curve, is 0.05 M for NaCl, 0.005 M for NaClO4, 0.001 M for sodium phosphate and 0.0003 M for sodium sulfate on H1 histone from Chaetopterus variopedatus sperm chromatin. Phosphate shows similar folding efficiency also on calf thymus and on sea-urchin sperm H1 histones. The effect of phosphate ions on the H1 molecule is observed also by differential absorption spectroscopy in the region of absorption of amino acid side-chains. Binding studies by gel filtration chromatography on Sephadex columns show that phosphate binding occurs in the presence of structuring concentrations of sodium chloride. About 9 ATP molecules bind to H1 histones derived from non-active cell chromatins while only 3.5 ATP molecules bind to H1 derived from active somatic chromatins. The fluorescence of the tyrosine residues of Chaetopterus sperm H1 is enhanced by chloride ions and heavily quenched by phosphate ions in correlation with structuring of the molecule, demonstrating direct interactions between tyrosine residues and phosphate ions. The defined and limited number of phosphate groups bound per histone molecule, the high affinity of the interaction and the effect on the structure of the histone suggest the participation of phosphate groups in the binding of H1 histones to DNA.     

Iodination of nucleosomes at low ionic strength: conformational changes in H4 and stabilization by H1.

Radioactive iodine has been used to probe the relative reactivities of nucleosomal H4 tyrosine residues under various conditions of subphysiological ionic strength. We observe that tyrosine 72 of H4, which is not reactive over the range 20-150 mM NaCl, becomes the predominant site of iodination within H4 when nucleosomes are subjected to conditions of very low ionic strength. Conversely, the other H4 tyrosine residues, which are reactive within nucleosomes in solutions of moderate ionic strength (20-150 mM NaCl), become nonreactive when the ionic strength is reduced. This "flip-flop" in the H4 iodination pattern is the manifestation of a reversible nucleosomal conformational change. A method is presented which enables the conformational status of H4 in nucleosomes to be determined by simply electrophoresing the histones on a Triton gel after probing nucleosomes with labeled iodine. Using this technique, we demonstrate that the presence of H1 on one side of the nucleosome stabilizes a histone core domain on the other side so that all four tyrosines of H4 are maintained in their physiological ionic strength conformation even under conditions of no added salt.     

Modification of the lysine residues of histones H1 and H5: Effects on structure and on the binding to chromatin

The extensive modification of histone H1 from calf thymus with the amino-group reagent dimethylmaleic anhydride (over 35 lysine residues modified per molecule) produces no effect on its secondary structure detectable by circular dichroism (far UV). Fluorescence and circular dichroism (near-UV) of the modified histone show variations in the local environment of its sole tyrosine residue. These changes are reversed on regeneration of the modified amino groups. While histone H1 is easily dissociated with this reagent from calf thymus or chicken erythrocyte chromatin, a much stronger treatment is needed to liberate histone H5 from erythrocyte chromatin. This difference appears to be related to the higher arginine content of histone H5.     

Co-existence of two different types of soluble histone complexes in nuclei of Xenopus laevis oocytes

The nuclear pool of soluble histones in Xenopus laevis oocytes is organized into two major types of acidic histone complexes separable by sucrose density gradient centrifugation. One type of complex sediments at 5 S (Mr approximately 120,000), is isoelectric at pH 4.6, and contains histones H3 and/or H4 tightly bound to one polypeptide of a pair of very acidic polypeptides, designated N1 and N2 (Kleinschmidt, J. A., and Franke, W. W. (1982) Cell 29, 799-809). This complex can be selectively immunoprecipitated by guinea pig antibodies against purified protein N1/N2. In contrast, a larger complex of 7 S contains four histones and nucleoplasmin (the purified protein exists as a pentamer of a polypeptide of Mr approximately 30,000), is isoelectric over the pH range of 5-7, and can be immunoprecipitated by nucleoplasmin antibodies. Its relative molecular weight of 130,000-170,000, as determined by gel filtration, sucrose density gradient centrifugation, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the cross-linked complexes, excludes the association of a histone octamer with nucleoplasmin. In addition to histones H2A and H2B, two histones (designated H3 and H4) which are similar in their electrophoretic mobilities to histones H3 and H4 but have lower isoelectric pH values are enriched in immuno-precipitates obtained with nucleoplasmin antibodies. Cross-linking of complexes present in intact nuclei, using 1% formaldehyde at near-physiological ionic strength and pH, indicates the coexistence of these two soluble histone complexes in the living cell. In chromatin assembly experiments using SV 40 DNA, both histone fractions are able to transfer histones to DNA, resulting in an increase of DNA superhelicity and the formation of beaded nucleoprotein complexes of nucleosome-like morphology. The common principle governing both types of complexes, i.e. the association of one or two histone molecules with a karyophilic large acidic histone-binding protein is emphasized. We discuss the possible role of these complexes in storing histones utilized in chromatin assembly during early amphibian embryogenesis as well as the possible existence of similar complexes, albeit at lower concentrations, in somatic cells.