Preparation and characterization of histone H1 from the sperm of the sea-urchin Sphaerechinus granularis.

The separation and purification of histone H1 from the sperm of the sea-urchin Sphaerechinus granularis is described. Physical studies were used to compare this histone H1 molecule with H1 histones from other species. C.d. and 270 MHz n.m.r. spectroscopy indicate that, despite significant compositional differences from other sea-urchin sperm H1 histones, their secondary and tertiary structures are very similar. A large difference in helicity was, however, found between S. granularis histone H1 and calf thymus histone H1, and their n.m.r. and fluorescence spectra also differ considerably. It is concluded that secondary structure and tertiary structure have not been conserved in the evolution of the H1 histone family.     

Contribution of histones H2A and H2B to the folding of nucleosomal DNA

We have studied the structural properties of nucleosomal particles deficient in histones H2A and H2B produced by modification of histone amino groups with dimethylmaleic anhydride [Jordano, J., Montero, F., & Palacián, E. (1984) Biochemistry (preceding paper in this issue)]. Digestion with DNase I of residual particles containing only 15% of the original H2A . H2B complement produces only discrete DNA fragments no longer than 70 nucleotides. As compared with the original nucleosomes, thermal denaturation of the residual particles shows a decrease from 140 to about 90 in the number of nucleotide base pairs per particle that melt at the highest temperature transition as well as a drop in the temperature of this transition. Circular dichroism spectra of the residual particles give ellipticity values around 275 nm, much higher than those corresponding to the control nucleosomes, which appears to indicate a loss in the compact DNA tertiary structure. When regeneration of the modified amino groups of the residual particles takes place in the presence of the complementary fraction containing histones H2A and H2B, but not in its absence, nucleosomal particles with the structural properties of the original nucleosomes are reconstituted. Therefore, the structural change observed in the residual particles can be assigned to the lack of histones H2A and H2B and not to the modified amino groups of the histones present in the residual particles. The results are consistent with the stabilization by histones H2A and H2B of a DNA length of 50-70 base pairs per nucleosome.     

A rapid and gentle method for the salt extraction of chromatin core histones H2A,H2B,H3 and H4 from rat liver nuclei

A complex of histones H2A, H2B, H3 and H4 has been isolated from purified rat liver nuclei by a method which is both gentle and rapid. Nuclei were homogenised in 0.25 M sucrose and the residual nuclear material obtained after centrifligation was adsorbed on calcium phosphate gel. After removing histone H1 from the adsorbed material by washing with 1M NaCl in 25 mM sodium phosphate buffer, pH 6.0, histones H2A, H2B, H3 and H4 were eluted together, with 2 M NaCl in 25 mM sodium phosphate buffer, pH 7.0. The core histones so obtained migrated as a single sharp band on polyacrylamide gel electrophoresis under non-denaturing conditions. Fractionation of the freshly prepared core histones on a Sephadex G-100 column yielded two major protein peaks. The peak having the larger elution volume contained histones H2A and H2B in equal amounts while the peak with the smaller elution volume contained all the four histones. Histones H3 and H4 were present in larger proportions in the second peak.     

Mutational analysis of the stability of the H2A and H2B histone monomers

The eukaryotic histone heterodimer H2A-H2B folds through an obligatory dimeric intermediate that forms in a nearly diffusion-limited association reaction in the stopped-flow dead time. It is unclear whether there is partial folding of the isolated monomers before association. To address the possible contributions of structure in the monomers to the rapid association, we characterized H2A and H2B monomers in the absence of their heterodimeric partner. By far-UV circular dichroism, the H2A and H2B monomers are 15% and 31% helical, respectively—significantly less than observed in X-ray crystal structures. Acrylamide quenching of the intrinsic Tyr fluorescence was indicative of tertiary structure. The H2A and H2B monomers exhibit free energies of unfolding of 2.5 and 2.9 kcal mol^− 1, respectively; at 10 μM, the sum of the stability of the monomers is ∼ 60% of the stability of the native dimer. The helical content, stability, and m values indicate that H2B has a more stable, compact structure than H2A. The monomer m values are larger than expected for the extended histone fold motif, suggesting that the monomers adopt an overly collapsed structure. Stopped-flow refolding—initiated from urea-denatured monomers or the partially folded monomers populated at low denaturant concentrations—yielded essentially identical rates, indicating that monomer folding is productive in the rapid association and folding of the heterodimer. A series of Ala and Gly mutations were introduced into H2A and H2B to probe the importance of helix propensity on the structure and stability of the monomers. The mutational studies show that the central α-helix of the histone fold, which makes extensive intermonomer contacts, is structured in H2B but only partially folded in H2A.     

Lysine residues in N-terminal and C-terminal regions of human histone H2A are targets for biotinylation by biotinidase

In eukaryotic cell nuclei, DNA associates with the core histones H2A, H2B, H3 and H4 to form nucleosomal core particles. DNA binding to histones is regulated by posttranslational modifications of N-terminal tails (e.g., acetylation and methylation of histones). These modifications play important roles in the epigenetic control of chromatin structure. Recently, evidence that biotinidase and holocarboxylase synthetase (HCS) catalyze the covalent binding of biotin to histones has been provided. The primary aim of this study was to identify biotinylation sites in histone H2A and its variant H2AX. Secondary aims were to determine whether acetylation and methylation of histone H2A affect subsequent biotinylation and whether biotinidase and HCS localize to the nucleus in human cells. Biotinylation sites were identified using synthetic peptides as substrates for biotinidase. These studies provided evidence that K9 and K13 in the N-terminus of human histones H2A and H2AX are targets for biotinylation and that K125, K127 and K129 in the C-terminus of histone H2A are targets for biotinylation. Biotinylation of lysine residues was decreased by acetylation of adjacent lysines but was increased by dimethylation of adjacent arginines. The existence of biotinylated histone H2A in vivo was confirmed by using modification-specific antibodies. Antibodies to biotinidase and HCS localized primarily to the nuclear compartment, consistent with a role for these enzymes in regulating chromatin structure. Collectively, these studies have identified five novel biotinylation sites in human histones; histone H2A is unique among histones in that its biotinylation sites include amino acid residues from the C-terminus.     

pH effects on the structure of the inner histones

The structure of the inner histone complex extracted from chicken erythrocyte chromatin with 2 M NaCl has been studied as a function of pH. At pH 6, the complex dissociates to (H3–H4)₂ tetramer and H2A·H2B dimer, with little change in α-helix content (as monitored by circular dichroism at 222 nm). Although the circular dichroism of tyrosyl side chains is also largely unchanged by the dissociation, measurements of intrinsic fluorescence do suggest a change in the environment of one or more tyrosines as a result of dissociation. Below pH 4, the histones become partially unfolded, lose specific secondary and tertiary structure, and undergo nonspecific aggregation. Both the pH 6 and 4 transitions, which are largely reversible, parallel pH-induced structural changes of nucleosomes (Zama, M., Olins, D.E., Prescott, B. and Thomas, G.J. (1978) Nucleic Acids Res. 5, 3881–3897). The results are consistent with the presence of tyrosine residues at the histone subunit-subunit contacts and suggest that histone conformation within the globular regions is largely independent of histone-DNA interactions.     

Structure of polyubiquitinated histone H2A

We have recently demonstrated that trout liver histones H2A, H2B, and H2A.Z can be polyubiquitinated [Davie, J.R., Delcuve, G.P., Nickel, B.E., Moyer, R., & Bailey, G. (1987) Cancer Res. 47, 5407-5410]. In the present study we determined the arrangement of the ubiquitin molecules in polyubiquitinated histone H2A. Trout liver chromatin fragments. which had histone H1 removed, were digested with Staphylococcus aureus (V8 strain) protease which cleaves specifically on the carboxyl side of glutamic acid residues under the conditions used. The V8 protease readily degraded histone H2A and ubiquitinated (u) H2A at equivalent rates. One site in H2A and uH2A, the peptide bond between Glu 121 and Lys 122, was cleaved, yielding protein species cH2A and cuH2A, respectively. None of the other nucleosomal histones (H2B, H2A.Z, H3, and H4) including uH2B and uH2A.Z were sensitive to digestion. Trout liver histones cleaved with either V8 protease, histone H2A specific protease, or cyanogen bromide were resolved by two-dimensional gel electrophoresis and ubiquitinated peptides detected with anti-ubiquitin IgG. The results suggest that the major arrangement of ubiquitin in polyubiquitinated H2A is a chain of ubiquitin molecules joined to each other by isopeptide bonds to a ubiquitin molecule that is attached to the epsilon-amino group of lysine 119 of histone H2A.     

Structure and regulation of histone H2B mRNAs from Leishmania enriettii.

We have studied the structure and expression of histone H2B mRNA and genes in the parasitic protozoan Leishmania enrietti. A genomic clone containing three tandemly repeated genes has been sequenced and shown to encode three identical histone proteins and two types of closely related mRNA sequence. We have also sequenced three independent cDNA clones and demonstrated that the Leishmania H2B mRNAs are polyadenylated, similar to the basal histone mRNAs of higher eucaryotes and the histone mRNAs of yeast. In addition, the Leishmania mRNAs contain inverted repeats near the poly(A) tail which could form stem-loops similar in secondary structure, but not in sequence, to the 3' stem-loops of nonpolyadenylated replication-dependent histones of higher eucaryotes. Unlike the replication-dependent histones, the Leishmania histone H2B mRNAs do not decrease in abundance following treatment with inhibitors of DNA synthesis. The histone mRNAs are differentially expressed during the parasite life cycle and accumulate to a higher level in the extracellular promastigotes (the form which in nature lives within the gut of the insect vector) than in the intracellular amastigotes (the form that lives within the mammalian host macrophages).     

The N-terminal tails of the H2A-H2B histones affect dimer structure and stability

The histone proteins of the core nucleosome are highly basic and form heterodimers in a "handshake motif." The N-terminal tails of the histones extend beyond the canonical histone fold of the hand-shake motif and are the sites of posttranslational modifications, including lysine acetylations and serine phosphorylations, which influence chromatin structure and activity as well as alter the charge state of the tails. However, it is not well understood if these modifications are signals for recruitment of other cellular factors or if the removal of net positive charge from the N-terminal tail plays a role in the overall structure of chromatin. To elucidate the effects of the N-terminal tails on the structure and stability of histones, the highly charged N-terminal tails were truncated from the H2A and H2B histones. Three mutant dimers were studied: DeltaN-H2A/WT H2B; WT H2A/DeltaN-H2B, and DeltaN-H2A/DeltaN-H2B. The CD spectra, stabilities to urea-denaturation, and the salt-dependent stabilization of the three truncated dimers were compared with those of the wild-type dimer. The data support four conclusions regarding the effects of the N-terminal tails of H2A and H2B: (1) Removal of the N-terminal tails of H2A and H2B enhance the helical structure of the mutant heterodimers. (2) Relative to the full-length WT heterodimer, the DeltaN-H2A/WT H2B dimer is destabilized, while the WT H2A/DeltaN-H2B and DeltaN-H2A/DeltaN-H2B dimers are slightly stabilized. (3) The truncated dimers exhibit decreased m values, relative to the WT dimer, supporting the hypothesis that the N-terminal tails in the isolated dimer adopt a collapsed structure. (4) Electrostatic repulsion in the N-terminal tails decreases the stability of the H2A-H2B dimer.     

Structure of the Drosophila nucleosome core particle highlights evolutionary constraints on the H2A-H2B histone dimer

We determined the 2.45 A crystal structure of the nucleosome core particle from Drosophila melanogaster and compared it to that of Xenopus laevis bound to the identical 147 base-pair DNA fragment derived from human alpha-satellite DNA. Differences between the two structures primarily reflect 16 amino acid substitutions between species, 15 of which are in histones H2A and H2B. Four of these involve histone tail residues, resulting in subtly altered protein-DNA interactions that exemplify the structural plasticity of these tails. Of the 12 substitutions occurring within the histone core regions, five involve small, solvent-exposed residues not involved in intraparticle interactions. The remaining seven involve buried hydrophobic residues, and appear to have coevolved so as to preserve the volume of side chains within the H2A hydrophobic core and H2A-H2B dimer interface. Thus, apart from variations in the histone tails, amino acid substitutions that differentiate Drosophila from Xenopus histones occur in mutually compensatory combinations. This highlights the tight evolutionary constraints exerted on histones since the vertebrate and invertebrate lineages diverged.     

Studies on the role and mode of operation of the very-lysine-rich histones in eukaryote chromatin. The conformation of phi1 histones from marine invertebrate sperm.

Proton magnetic resonance, circular dichroism and infrared spectroscopy are used to investigate the secondary and tertiary structure of three very lysine-rich histones from marine invertebrate sperm. At high ionic strength both Arbacia lixula and Holothuria tubulosa histone phi 1 are observed to contain 25-30% alpha-helix, no beta-structure and to form specific folded structures. Both phi 1 proton magnetic resonance spectra have perturbed methyl resonances at chemical shifts close to those observed for calf thymus H1, suggesting analogies in tertiary structure. Mytilus edulis histone phi 1 however, shows no spectroscopic evidence of secondary and tertiary structure on salt addition.     

Comprehensive structural analysis of mutant nucleosomes containing lysine to glutamine (KQ) substitutions in the H3 and H4 histone-fold domains

Post-translational modifications (PTMs) of histones play important roles in regulating the structure and function of chromatin in eukaryotes. Although histone PTMs were considered to mainly occur at the N-terminal tails of histones, recent studies have revealed that PTMs also exist in the histone-fold domains, which are commonly shared among the core histones H2A, H2B, H3, and H4. The lysine residue is a major target for histone PTM, and the lysine to glutamine (KQ) substitution is known to mimic the acetylated states of specific histone lysine residues in vivo. Human histones H3 and H4 contain 11 lysine residues in their histone-fold domains (five for H3 and six for H4), and eight of these lysine residues are known to be targets for acetylation. In the present study, we prepared 11 mutant nucleosomes, in which each of the lysine residues of the H3 and H4 histone-fold domains was replaced by glutamine: H3 K56Q, H3 K64Q, H3 K79Q, H3 K115Q, H3 K122Q, H4 K31Q, H4 K44Q, H4 K59Q, H4 K77Q, H4 K79Q, and H4 K91Q. The crystal structures of these mutant nucleosomes were determined at 2.4-3.5 ? resolutions. Some of these amino acid substitutions altered the local protein-DNA interactions and the interactions between amino acid residues within the nucleosome. Interestingly, the C-terminal region of H2A was significantly disordered in the nucleosome containing H4 K44Q. These results provide an important structural basis for understanding how histone modifications and mutations affect chromatin structure and function.     

Particular structural role of H1 in complexes with DNA and comparison with H2A- and H4-DNA complexes investigated by IR linear dichroism.

The particular role of H1 in the structure of histone–DNA associations is shown by means of ir linear dichroism. H1–, H2A–, and H4–DNA complexes are studied for different histone: DNA input ratios and various relative humidities (r.h.). The measurement of the dichroic ratios allows one to determine the secondary structure of DNA in the complexes. It is shown that the progressive addition of histone H2A or H4 to DNA inhibits the structural B → A transition and DNA remains in a B-type form at low r.h. It is found that the B → A transition is inhibited for 19 or 26 base pairs of DNA per molecule of H2A or H4. The stabilization of DNA in a B-conformation by H2A and H4 has been also observed by H2B and H3 but with a different efficiency. In contrast, histone H1, which does not belong to the core of the nucleosomes in chromatin, leaves the DNA in H1–DNA complexes free to adopt an A conformation at low r.h. for H1/DNA ratios below 0.6/1. Thus a major difference in the structural role between histone H1 and histones belonging to the nucleosomal core with respect to the conformational flexibility of DNA in the histone–DNA complexes is demonstrated.     

Deimination of histone H2A and H4 at arginine 3 in HL-60 granulocytes

Interplay of various covalent modifications of histone tails has an essential role in regulation of chromatin function. Peptidylarginine deiminase (PADI) 4 deiminates protein arginine to citrulline in a Ca(2+)-dependent manner and is present in the nucleus of granulocyte-differentiated HL-60 cells. When these cells are treated with the calcium ionophore A23187, core histone deimination occurs. To determine the deimination sites of histones, histone species were purified by reverse-phase high-performance liquid chromatography (RP-HPLC) from the cells. Immunoblotting using antimodified citrulline antibody indicated that histones H2A, H3, and H4 but not H2B were deiminated. H2A and H4 were digested with Staphylococcus aureus V8 protease, and the digests were separated by RP-HPLC. Immuno dot-blotting and mass spectrometry showed that the deiminated residues were present in H2A (1-56) and H4 (1-52) regions but not in other regions. The H2A peptide (1-56) was digested with alpha-chymotrypsin, and the deiminated peptide was separated from the corresponding nondeiminated peptide by RP-HPLC. The deiminated residue was found to be limited to residues 1-23. Similarly, digestion of the H4 peptide (1-52) with endoproteinase Asp-N and separation of the deiminated peptide from the nondeiminated peptide indicated that the deiminated residue was limited to residues 1-23. Mass spectrometry of lysylendopeptidase digests of the H2A (1-23) and H4 (1-23) peptides showed that deimination occurred at arginine 3 of the N-terminal sequence Ac-SGRGK common to H2A and H4. These results suggest that PADI4 deiminates only a restricted site of target proteins in cells. Deimination of histones is discussed in relation to chromatin structure and function.     

Role of histone pairs H2A,H2B and H3,H4 in the self-assembly of nucleosome core particles

The role of the histone pairs H2A,H2B and H3,H4 in the kinetics of core particle formation was investigated by using N-(1-pyrene)maleimide-labeled histone H3. The excimer emission intensity of a DNA-core histone complex prepared by direct mixing of DNA and histones in 0.2 m-NaCl is reduced by half when H2A,H2B is omitted. Fluorescence quenching studies and lifetime measurements indicate that the emission differences are probably due to static quenching. In a correctly folded nucleosome or a DNA-(H3,H4) complex, the two pyrene rings are buried and are held very close. DNA-(H3,H4) can interact with additional copies of H3,H4, but only when two dimers of H2A,H2B are correctly bound is there a specific twofold increase in excimer emission.The kinetics of the reaction of H3,H4 with DNA in 0.2 m-NaCl were followed by measuring the increase in 460 nm fluorescence. The apparent rate constant of the dominant kinetic component is ~ 2 × 10^?1 s^?1. If histones H2A,H2B are added immediately after the preparation of the DNA-(H3,H4) complex, an increase in excimer fluorescence is observed, with an apparent rate constant of ~ 6 × 10^?3 s^?1. However, if histones H2A,H2B are added one hour after DNA-(H3,H4) complex formation, there is no increase in excimer fluorescence. These results suggest that an intermediate involving the H3,H4 tetramer is formed first in nucleosome assembly. In the presence of H2A,H2B, this intermediate evolves to the final folded nucleosome, but in the absence of H2A,H2B it rearranges to an unmaturable dead-end complex. Additional experiments show that a very fast transfer of histone pairs (probably H2A,H2B) can take place between partially reconstituted nucleosomes.     

Relaxation of chromatin structure upon removal of histones H2A and H2B

Modification of chromatin from chicken erythrocytes with dimethylmaleic anhydride is accompanied by its solubilization and the dissociation of histones H1, H5, H2A and H2B. Histone H1 is the first to dissociate and H5 the last. After regeneration of the modified amino groups, residual chromatin preparations with different histone composition were studied by circular dichroism and thermal denaturation. In addition to the effects produced by the lack of histones H1 and H5, both techniques show a substantial relaxation of chromatin structure induced by the loss of histones H2A and H2B, which appear to play an important role in the superhelical folding of DNA.     

Effect of urea on proteolysis of histone H2A in calf thymus nuclei

The 1 hour incubation of calf thymus nuclei at 37 degrees C leads to a proteolysis of the histones H1, H3 and H2B. Urea does not influence the histone degradation while 1.5 and 2.0 M NaCl lead to the proteolysis of the H2A histone. On this background, 2 M urea restrains the degradation of the H2A histone. It is assumed that hydrogen bonds are very important for the activity of the proteinases and its interaction with the H2A histone.     

Accessibility of tyrosyl residues altered by formation of the histone 2A/2B complex

The availability of tyrosyl residues to surface iodination was analyzed for histone 2A (H2A), histone 2B (H2B), and the H2A/H2B complex. When H2A is free in solution (200 mM NaCl, pH 7.4) tyrosine-39 and one or both tyrosines-50 and -57 were readily iodinated. Tyrosines-83 and -121 of H2B were iodinated, both when the histone was free in solution and when it was associated with H2A, while tyrosines-37, -40, and -42 of H2B were not iodinated under either condition. When H2A and H2B were associated or covalently cross-linked, all tyrosyl residues of H2A were unavailable for iodination. We also found that the iodination of nondenatured H2A and H2B did not inhibit formation of the H2A/H2B complex. These results indicate that the amino-terminal regions of the hydrophobic portions of H2A and H2B undergo significant conformational changes upon formation of the H2A/H2B complex. These conformational shifts occur in the same region of the H2A/H2B complex that contains a contact site between H2A and H2B in the nucleosome, thus indicating an involvement of this region in chromatin assembly.     

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.