Comparison between histones FV and F2a2 of chicken erythrocyte. II. Interaction with homologous DNA.

The conformation and stability of artificial complexes between chicken erythrocyte DNA and homologous histones FV and F2a2 was studied by circular dichroism (CD) and thermal denaturation followed by both absorbance and CD measurements. The complexes are made after a stepwise potassium fluoride gradient dialysis without urea and studied at low ionic strength (10-minus 3 M). 1) No structural changes of the DNA can be detected up to r equals 0.2 with FV and r equals 0.6 for F2a2. With FV at higher values of r the CD spectrum is altered, indicating the organization of DNA and histones in some kind of aggregate. 2) The conformation of histone molecules inside the complexes is not related to the ionic strength of the medium but to an effective ionic environment close to 0.1 M. This ionic strength would also correspond to the melting temperature of histone-covered DNA. 3) From the analysis of the absorbance melting profile the length of DNA covered with an histone molecule can be estimated. A good agreement is found between the negative charge of this piece of DNA and the net positive charge of the histone. 4) Since the CD transition at 227 nm occurs before the second absorbance transition at 280 nm, the DNA is stabilized no longer by native histone but partially or fully denatured histones. The helical regions of the histone molecule are not involved in the binding process, which appears to be almost purely coulombian and most likely related to some structural fit between the pattern of negative charges in the DNA helix and that of positive charges along the peptide chain.     

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.     

Homologies in amino acid composition and structure of histone F2al isolated from human leukaemic cells

Histones F2al extracted from normal and neoplastic cells possess similar amino acid compositions. Tryptic and chymotryptic peptides of the F2al histones have identical chromato-electrophoretic R(F) values. It is concluded that histones F2al from various sources have similar overall structures. The observed differences in the ratios of in-N-monomethyl- and di-in-N-methyl-lysine in the histones from normal and neoplastic cells may be of significance with respect to gene regulation.     

Investigation of DNA complexes with histones F3 and F3+F2a2]

Characteristic viscosity, sedimentation constant and optical anisotropy were studied of the complexes formed between DNA and histone fractions F3 and F3+F2a2. The parameters mentioned continuously change with the increase of protein content within the complex. Analysis of experimental data shows that binding of a histone bads to a decrease of size and thermodynamic rigidity of the DNA molecule. On the basis of results obtained a model of F3 histone binding with DNA is suggested, amino acid sequence of this protein being taken into account. Comparison of behaviour of nucleohistones DNA+F3 and DNA+F1 studied previously testifies different way of binding of these histones to DNA.     

The evolution of histones

Summary The amino acid sequences of bovine histones H2A, H2B, H3, and H4 and the first 107 residues of rabbit thymus histone H1 were examined using newly developed procedures designed to detect and evaluate weak similarities (de Haën et al., 1976). Using the McLachlan scoring system, regions of statistically significant similarity were found between several pairs of the four smallest histones. The probability that this set of similarities could result simply from chance was estimated to be less than 10^–5. No similarity was found between the H1 sequence and the other histones. The results are interpreted to indicate that at least the C-terminal portions of the core histones evolved from a common ancestral protein.     

N-terminal acetyl-peptides from two calf thymus histones

1. The same N-terminal peptide was isolated from two major calf thymus histone fractions, F2al and F2a2. 2. The structure of the peptides is N-acetylseryl-glycyl-arginine, and the yields account for most of the N-acetyl groups previously found in these histones. 3. Some other peptides not giving a ninhydrin reaction were also investigated.     

Electrophoretic study of plant histones: Comparison with vertebrate histones

The histones of seven plant species (barley, leek, onion, pea, radish, rye, and wheat) were isolated and compared to the histones of calf thymus and rat liver using electrophoresis on polyacrylamide-urea and polyacrylamide-SDS gels. It was found that the F1 histone of plants contains more subspecies and has generally higher molecular weights than their animal histone counterparts. Histones F3 of plants and animals have identical molecular weights and similar but not identical mobilities on polyacrylamide-urea gels. No histones were found in plants which have molecular weights and mobilities on polyacrylamide-urea gels which resemble the values for histones F2a2 and F2b of animals, but instead the series of histones observed differ from any of the animal histones. These plant histones may represent either substantially modified forms of F2a2 and F2b, or else may be a different class of histone molecules unique to plants. Fractions F2al in plants and animals are identical in electrophoretic behavior, but seem to differ in degree of acetylation.     

An evolutionary comparison of plant histones.

Histones were extracted from chromatin of the following: a moss (Polytrichum juniperinum); the primitive vascular plants Psilotum nudum and Equisetum arvense; a fern (Polypodium vulgare); the gymnosperms fir (Abies concolor), yew (Taxus canadensis) and Gingko biloba; the dicotyledonous angiosperms tobacco (Nicotiana tabacum) and maple (Acer saccharinum); and the monocotyledonous angiosperms corn (Zea mays) and lily (Lilium longiflorum). The histones were subjected to polyacrylamide gel electrophoresis and compared to standard histones of pea (Pisum sativum) and cow (Bos taurus). All species have histones of the exact electrophoretic mobility of histones F2a1 and F3 of cow and pea. All species have histones of low electrophoretic mobility assumed to be F1 histones. None of the plant histones displayed electrophoretic mobility between F3 and F2a1 while animal histone fractions F2b and F2a2 do migrate to this position. No animal histone fraction was found to migrate between F3 and F1 while a major plant fraction, designated "F2b-like" was found to migrate to this position in all plant species studied except for the moss and Psilotum. A band of similar mobility was strikingly absent from the histones of these two species.     

Comparison between histones FV and F2a2 of chicken erythrocyte. I. Structure, stability and conformation of the free proteins.

Purified chicken erythrocyte histones FV and F2a2 were studied by means of circular dichroism as a function of ionic strength and temperature. The percentage of alpha-helical regions was calculated by comparison with reference spectra obtained with four standard proteins of known tertiary structure. Maximal alpha-helical organization, reached in high ionic strength, was estimated to 14% and 23% for FV and F2a2 respectively. We have compared our experimental determinations of the secondary structure of F2a2 with predictions made from amino-acid sequence according to Fasman's rules. When instability induced by the presence of charged residues close together is taken into account, a good agreement is found between predicted and observed values. The thermal denaturation of FV is cooperative and, unlike F2a2, seems to obey a two-state transition. The classical Arrhenius plot is linear, which indicates that the heat capacity is the same in both the native and the denatured state. Such a behaviour is typical of an expanded configuration of FV even in the "native" state.     

Electrophoretic analysis of liver and testis histones of the frog Rana pipiens

Histones were extracted from frog livers and testes and analyzed by electrophoresis on long polyacrylamide gels and on sodium dodecyl sulfate (SDS)-containing polyacrylamide gels. Frog histones were found to be similar to those of calf thymus except that frog histone fraction F2A2 showed a marked dependence on the temperature at which the long gels were run, and frog histone fraction F3 could be separated from frog F2B on SDS-containing gels. Comparisons between frog liver and frog testis histones indicated that the testis contains as its major F1 component a fast migrating species not found in liver. Testis histones also showed less microheterogeneity of fractions F3 and F2A1 than liver histones. These were the only differences observed between liver and testis histones, even when testis histones were prepared from sperm suspensions that were rich in cells in the late stages of spermiogenesis. Thus it seems that, in Rana, the electrophoretic properties of the basic proteins of sperm differ from those of somatic cells only in the nature of histone F1 and in the degree of microheterogeneity of fractions F2A1 and F3.     

Reactivity of heterogeneous F1 histones with glutaraldehyde and formaldehyde

The rates of glutaraldehyde and formaldehyde fixation of F1 histones have been investigated using chromatin from rat pancreas, chicken erythrocyte, and human spleen. These chromatins differ in number, type and relative proportion of F1 species present. In all cases the rates of fixation by glutaraldehyde and formaldehyde of the F1 components is much faster than for the other histones. The rates of fixation of F1-type histones are similar in each case with the exception of one minor F1 histone from chicken which reacts slower than the rest of that F1 group. The results suggest that the interactions of all F1 type histones with DNA are similar.     

The presence of F3-F2a1 dimers and F1 oligomers in chromatin.

The oligomeric structure of histones in nuclei and chromatin has been studied by crosslinking nuclei and chromatin with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide. Crosslinked histones were detected as new high molecular weight components on SDS gels, and the protomers of the crosslinked histones were identified by their characteristic ¹²⁵I-fingerprints. The results show that a considerable portion of histones F3 and F2a1 exist in nuclei and chromatin as an F3-F2a1 dimer. Evidence is presented that histone F1 probably exists in chromatin as large oligomers.     

Dynamic equilibrium in histone assembly: self-assembly of single histones and histone pairs.

The assembly of acid-extracted, purified F2a1, F3, F2a2, and F2b histones and their six possible pairwise combination into organized structures has been studied by: (1) sedimentation velocity, (2) sedimentation equilibrium, (3) electrophoresis in polyacrylamide gels containing sodium dodecyl sulfate after cross-linking the protein solution with dimethyl suberimidate, and (4) electron microscopy. Each of the purified histone fractions can renature and assemble into high molecular weight organized structures. This assembly is dependent on the ionic strength, protein concentration, and temperature of the solutions. The four histones studied assemble into structures of similar dimensions and shape. In each case the first structure observed is a bent rod with a diameter of 22 A. Conditions which favor assembly lead to formation of fibers with diameters of about 44 A. The conditions which lead to assembly into organized structures are similar for the arginine-rich histones, F2a1 and F3. Higher ionic strength is required for the assembly of the lysine-rich histones, F2a2 and F2b. Certain pairs of histones interact. Strong interactions among pairs of histones interfere with the self-assembly of single histones into large structures. Howver, increase in protein concentration or ionic stregth leads to formation of large molecular structures even in solutions of pairs of strongly interacting histones. These structures are similar to those obtained with single histones. The results suggest that aggregation and complexing of histones represent a reversible, ordered process of assembly. The various assembled forms are in a dynamic equilibrium. The final assembled form, which is similar in all cases, is dependent on the environmental conditions to which the histones are exposed. It is suggested that each of the assembled histone structures, regardless whether it is composed of a single histone or a pair of histones, can serve as a core around which the DNA can be wrapped.     

Tissue-specific histones in the erythrocytes of chicken and turtle

Mature erythrocytes from Leghorn chickens contain lysine-rich histone F1 and a tissue-specific histone F2c. The composition of the F1 fraction was found to be similar to the F1 histones in higher vertebrates. In the erythrocytes of a sea turtle (Chelonia mydas), only lysine-rich histones F1 could be detected. One of these fractions (F1b) differed in amino acid composition from the typical F1 histones described in the literature. The F1b histone fraction was not found in turtle liver. Chromatographic analysis of tryptic peptides of the chicken erythrocyte F1 and F2c histones and of the turtle erythrocyte F1a and F1b histones revealed considerable similarities between these four fractions, thus indicating their possible phylogenetic relationships.     

Amino acid composition of sperm histones in the house cricket Acheta domesticus.

Histones were isolated from late spermatids and spermatozoa of the house cricket Acheta domesticus, and the individual histone fractions were separated by electrophoresis on polyacrylamide-urea gels. The stained gels were cut so as to isolate the different histone fractions, and the amino acid compositions were determined using the technique of Houston (Houston, L.L.: Anal. Biochem. 44, 81-88 (1971). Five of the histones had amino acid compositions resembling those for the histones of calf thymus and were thus identified as fractions F1, F3, F2a2, F2b, and F2al. Another protein (SH) located exclusively in the late spermatids and spermatozoa was found to be basic and histone-like. It is a protein containing relatively high amounts of arginine (12.6%) and low amounts of lysine (7.6%), and, as a result, it has a low ratio of lysine-arginine (0.6). Other noteworthy features are its high contents of serine, glutamic acid, and glycine. It is arginine rich histone and in this regard resembles other such proteins, but it does contain unique features which distinguish it from all previously described histones.     

Iodination of Xenopus laevis histone F2a1 in chromatin.

The reaction of calf thymus and Xenopus laevis histones with radioactive iodine has been studied under various conditions that affect chromatin structure. All histones from both species contain at least one tyrosine residue and, in a denaturing solvent, all the the histones react with iodine. Histone F2a1 has been studied in detail. Calf thymus F2a1 is known to contain four tyrosyls and all four react with iodine. In high voltage paper electrophoresis, the tyrosine-containing peptides from calf co-migrate with those from Xenopus F2a1, suggesting that the amino acid sequence is strongly conserved between these two species. Therefore, the published calf thymus F2a1 sequence has been used to order the Xenopus F2a1 peptides within the molecules. When gently isolated native chromatin is iodinated in a low ionic strength medium 60% of the radioactivity in F2a1 is in tyrosyl 88, 30% in tyrosyl 51, and tyrosyl 72 and 98 have almost no radioactivity. Reagents which remove the protein from the DNA (2 M NaCl) or partially disrupt protein tertiary structure (5 M urea) increase the reactivity of each of the four tyrosyls five- to tenfold, suggesting that all four are protected about equally by the overall folding of the chromatin. Isolated F2a1 iodinated in the presence of 10 M urea shows uniform labeling in each of the four peptides, suggesting that tyrosyl 72 and 98 are afforded some protection solely by protein-protein interactions. The stepwise removal of histones in increasing NaCl concentrations differentially increases the availability of each F2a1 tyrosyl. The preferential exposure of tyrosyl 88 coincides with the removal of the majority of F1 histones at 0.5 M NaCl while the gradual and stepwise increase in reactivity of tyrosyl 51, 72, and 98 correlates with the gradual removal of histones other than F1. Radioactive iodination of chromatin has been shown to be a sensitive probe for detecting changes in the association state (or conformation) of histone F2a1.     

The structure and properties of histone F2a comprising the heterologous group F2a1 and F2a2 studied by 13C nuclear magnetic resonance

¹³C n.m.r. spectra have been obtained for aqueous solutions of histones F2a₁ and F2a₂, for the group F2a, for the appropriate amino acid mixturesand for the corresponding hydrolysates. These, when compared with computer simulated spectra give good agreement for secondary structure with that calculated from the known primary structure of the proteins. Evidence based on the spectra obtained at various salt concentrations leads to the conclusion that F2a is not a simple mixture but an interacting heterologous group of histones F2a₁ and F2a₂.     

The self-association of chicken-erythrocyte histones.

The self-association of the separate histone fractions isolated from chicken erythrocytes has been studied in solution at a number of different pH values and ionic strengths. The apparent molecular weights of the histones were determined over a range of macromolecular concentrations using the techniques of osmotic pressure and sedimentation equilibrium. Histone F2c (H5) did not associate under any of the conditions investigated whereas the other histone fractions all appeared to undergo self-association forming dimers, dimers of dimers, etc. The degree of association increased with the pH and ionic strength of the medium. The tendency to aggregate increased in the order; histone F2c (H5) (non-aggregating), histone F2b (H2B), histone F2a2 (H2A), histone F3 (H3), histone F2a1 (H4) (highly aggregating). In the case of histone F2a2 (H2A) at pH 3.0 and ionic strength 0.1, the apparent weight-average molecular weight was determined at a number of macromolecular concentrations at five different temperatures. The self-association was analysed according to the method of Adams (published by Beckman Instruments Inc. in 1967) and shown to be a monomer-dimer-tetramer equilibrium. The association constants were evaluated at each of the temperatures studied and from their variation with temperature the values of the enthalpy and entropy of association were calculated. The intermolecular association was characterised by only a small change in enthalpy but a large, positive, change in entropy. This suggests that the association of histones at acid pH is due to hydrophobic interactions between the relatively uncharged segments of like polypeptide chains.     

An Nalpha-acetyltransferase responsible for acetylation of the N-terminal residues of histones H4 and H2A

A yeast gene has been identified that encodes a novel, evolutionarily conserved Nalpha-acetyltransferase responsible for acetylation of the N-terminal residues of histones H4 and H2A. The gene has been named NAT4. Recombinant Nat4 protein acetylated a peptide corresponding to the N-terminal tail of H4, but not an H3 peptide nor the peptide adrenocorticotropin. H4 and H2A are N-terminally acetylated in all species from yeast to mammals and hence blocked from sequencing by Edman degradation. In contrast, H4 and H2A purified from a nat4 mutant were unacetylated and could be sequenced. Analysis of yeast histones by acid-urea gel electrophoresis showed that all the H4 and H2A from the mutant migrated more rapidly than the same histones from a wild type strain, consistent with the histones from the mutant having one extra positive charge due to one less acetylated amino group. A comparison of yeast proteins from wild type and a nat4 mutant by two-dimensional gel electrophoresis showed no evidence that other yeast proteins are substrates of this acetyltransferase. Thus, Nat4 may be dedicated specifically to the N-terminal acetylation of histones H4 and H2A. Surprisingly, nat4 mutants grow at a normal rate and have no readily observable phenotypes.     

Studies on the prolactin-releasing mechanism of histones H2A and H2B

In previous studies we demonstrated that histone preparations possess multiple effects in vivo on pituitary hormone secretion. We have now studied the specificity and signal transduction pathways involved in the prolactin (PRL)-releasing activity of histones H2A and H2B on perifused and incubated rat pituitary cells. In the perifusion experiments, freshly dispersed pituitary cells were packed into short columns and were continuously perifused with serum-free medium. The substances to be tested (stimuli) were pumped through the perifusion circuit, at the end of which perifusate fractions were collected and PRL measured by specific RIA. In the incubation studies, freshly dispersed pituitary cells were incubated in a metabolic incubator with different stimuli at different doses and for varying times. Perifusion of cells with median eminence extract (1/30), histone H2A (30 microM) or histone H2B (30 microM), generated clear PRL release responses. Cells incubated with histone H2A and H2B showed a dose- and time-dependent stimulatory effect on PRL release which, for H2A, was blocked by peptide MB-35, an 86-120 amino acid synthetic fragment of histone H2A. The polycation, poly-lys was unable to mimic the action of histones. To detect the possible signal transduction pathways involved in the response of lactotrophs to histones, cells were incubated with the calcium ionophore A23187, the calcium chelator EGTA, the intracellular phosphoinositide enhancer LiCl, the intracellular cAMP enhancers caffeine, NaF and forskolin, and the protein kinase C inhibitor, trifluoperazine (TFP). Both EGTA (or EGTA plus A23187 ionophore) and TFP were able to reduce significantly the response of lactotrophs to histones. Our results confirm previous evidence that histones may act as hypophysotropic signals. The data also suggest that calcium- and diacylglycerol-associated pathways participate in these effects.