Antigenic structure of histone H2B.

Antigenic determinants of histone H2B were localized using a series of 23 overlapping fragments of H2B obtained either by chemical and enzymatic cleavage of the histone or by solid-phase peptide synthesis. The ability of peptides to bind H2B antibodies was measured in an enzyme-linked immunosorbent assay, using antisera directed against calf thymus and chicken erythrocyte H2B as well as four anti H2B monoclonal antibodies obtained from autoimmune mice. Seven antigenic determinants were localized in the H2B molecule in the vicinity of residues 1-11, 6-18, 15-25, 26-35, 50-65, 94-113 and 114-125. Two of these determinants (residues 6-18 and 26-35) were revealed only through the binding properties of antibodies isolated from autoimmune mice. The usual correlation between hydrophilicity and antigenicity was found to hold for four of the epitopes, and the N- and C-termini of H2B were both antigenically active.

Comparison of different methods for localizing antigenic regions in histone H2A.

Four different variations of enzyme-linked immunosorbent assays (ELISA) were used to analyze the antigenic structure of histone H2A. Eleven natural and 10 synthetic peptides of H2A were tested for their capacity to bind antibodies raised against the complete molecule in a direct binding assay. Results were compared to those obtained in a direct test using several peptide-BSA conjugates. The capacity of peptides to inhibit the reaction between H2A antibodies and the complete H2A molecule or large fragments of it was also measured. Inhibition assays detected antigenic activity in a large number of peptides than did direct binding assays. Antisera raised against eight synthetic, unconjugated peptides all reacted with histone H2A in ELISA. Using as probes peptides of 14-21 residues, at least 11 antigenic regions could be recognized, indicating that virtually the entire H2A polypeptide chain possessed antigenic activity.

Specificity of antibodies raised against triacetylated histone H4.

Ten monoclonal antibodies (mAbs) were obtained by immunizing animals with triacetylated histone H4 from cuttle-fish. The fine specificity of these antibodies was studied using various populations of acetylated H4, (H3H4)2 tetramers and histone octamers as well as with acetylated and nonacetylated peptides of H4. None of these mAbs were found to recognize triacetylated H4. Only five of them bound to diacetylated, monoacetylated and nonacetylated H4. One antibody was specific for H4 associated in the form of histone octamers and did not bind to any nonacetylated or acetylated form of H4 monomers. Eight of the antibodies were specific for residues situated in the region 9-23 of H4. None of the mAbs was completely specific for acetylated forms of H4. In contrast, antisera raised in rabbits against triacetylated H4 reacted strongly with tri and diacetylated H4, weakly with monoacetylated H4 and barely or not at all with nonacetylated H4.

UV differential study of the histones H2A-H2B-H3-H4 octamer.

The individual calf thymus histones H2A, H2B, H3 and H4, the dimer H2A-H2B, the tetramer (H3-H4)2 and the octamer (H2A-H2B-H3-H4)2 were studied by differential UV absorption i.e. observing absorption shifts of tyrosyl residues due to thermal perturbations. The histone octamer was studied in 2 M NaCl pH 7.5 a condition under which it is stable, as demonstrated by Eickbush and Moudrianakis [18]. In addition these authors suggested that the interactions which maintain the four histones as an octamer involve the weak association of one (H3-H4)2 tetramer with two H2A-H2B dimers and might be due essentially to histidine-lysine or histidine-tyrosine hydrogen bonds. We performed the study of the octamer by UV differential absorption using the tyrosyl residues as a natural probe to follow their interaction with different residues in their neighbourhood. The main result obtained shows that the tetramer (H3-H4)2 has all its tyrosyl residues exposed to the solvent whereas the octamer has no tyrosine exposed, suggesting that with this polymer no DNA-tyrosine interactions could take place.

Successive elution by ion-exchange chromatography of H3-H4 histone complexes differing in their degree of acetylation.

On Biorex 70 ion exchanger at neutral pH the histones H3 and H4 are usually eluted by 4 M guanidinium chloride (gdm Cl). In order to protect cysteines and methionines from oxidation we systematically added 2-mercaptoethanol to the elution buffer. This resulted in the two histones being unexpectedly eluted together at around 1 M gdm Cl. The use of a shallower gradient resulted in a division in the peak of histones, with the acetylated species of H3 and H4 being eluted first and the nonacetylated species of H3 and H4 eluted last. When histone H3 or histone H4 was applied alone or when the chromatography was performed at low pH, these histones were eluted in the usual position at about 4 M gdm Cl. These events mean that the simultaneous elution of the histones H3 and H4 at about 1 M gdm Cl involves the formation of H3-H4 complexes. Therefore, the H3-H4 complex may be obtained by ion-exchange chromatography as the H2A-H2B complex was previously; furthermore, the former was fractionated according to postsynthetic modifications. This finding provides a new basis for explaining some of the previous elution profiles of chromatin extracts.

Histone H4 from cuttlefish testis is sequentially acetylated. Comparison with acetylation of calf thymus histone H4.

The differently acetylated subfractions of histone H4 isolated from cuttlefish testis and from calf thymus were separated by ion exchange chromatography on sulfopropyl-Sephadex, using a shallow linear gradient of guanidine hydrochloride in the presence of 6 M urea at pH 3.0. The tetra-, tri-, di-, mono-, and nonacetylated forms of cuttlefish H4 represent 2, 6.4, 18, 32.2, and 41.4% of the whole histone, respectively. The di-, mono-, and nonacetylated forms of calf H4 represent 11.7, 41.3, and 44% of the whole histone, respectively. The acetylation sites were determined in each subfraction by identification of the acetylated peptides. In each acetylated H4 subfraction, the acetylated tryptic peptides were identified by peptide mapping and amino acid analysis with reference to the peptide map of nonacetylated H4. In cuttlefish testis H4, lysine 12 is the main site of acetylation in the monoacetylated subfraction; lysines 5 and 12 are found acetylated in diacetylated H4; lysines 5, 12, and 16 are found acetylated in triacetylated H4. From these results and the stoichiometry of the different H4 subfractions, it can be concluded that lysine 5 is acetylated after lysine 12. In calf thymus, lysine 16 is the only site of acetylation in the monoacetylated subfraction. All the diacetylated forms are acetylated in lysine 16, the second site of acetylation being, in decreasing order, lysine 12, lysine 5, or lysine 8. These observations suggest that acetylation occurs in a sequential manner. Moreover, the sites of acetylation depend upon the biological event in which acetylation is involved.

Primary structure of histone H2A from nucleated erythrocyte of the marine worm Sipunculus nudus. Presence of two forms of H2A in the sipunculid chromatin.

The complete amino acid sequence (123 residues) of histone H2A from erythrocytes of the marine worm Sipunculus nudus, has been established from data provided by automated sequence analysis of large fragments generated by V8 staphylococcal protease digestion of histone H2A and by limited hydrolysis of the protein with alpha-chymotrypsin and from structural studies of tryptic peptides of the protein. By comparison with calf homologous histone, the sipunculid histone H2A shows 6 deletions and 13 substitutions. Six of the substitutions are non-conservative. Most of the evolutionary changes are mainly observed in the basic amino-terminal and carboxy-terminal regions of the molecule, which are the primary DNA-binding sites. Few conservative point changes are observed in the central region (residues 18-118) which interacts strongly with histone H2B to form the dimer H2A-H2B. 60% of the H2A molecules were found phosphorylated on the amino-terminal residue, N-acetyl-serine. The high content of phosphorylated histone H2A in the sipunculid erythrocyte chromatin could probably be related to smaller repeat length (177 +/- 5 base pairs) of nucleosomal DNA and to nuclear inactivation and chromatin condensation.

The core histones from the slime mold Physarum polycephalum. Isolation and characterization.

The histones of Physarum polycephalum have been isolated and fractionated using a combination of gel exclusion and ion-exchange chromatography or differential precipitation. The four core histones were unambiguously identified by gel electrophoresis, amino acid composition and N-terminal analysis. The molecular weight of Physarum histones H3, H2B and H4 are very close or identical to the corresponding histones from chicken erythrocytes. Physarum H2A is significantly larger than chicken erythrocyte H2A and its N-terminal residue is alanine. From the differences in amino acid compositions and in the peptide maps between Physarum and calf H4, one can expected some changes in the amino acid sequence of Physarum H4.

Antibodies from patients with drug-induced and idiopathic lupus erythematosus react with epitopes restricted to the amino and carboxyl termini of histone.

The sera of patients with systemic lupus erythematosus (SLE) and drug-induced lupus (DIL) were used to study the antigenic epitopes on nuclear histones that bind antibodies in these sera. ELISA and immunoblotting techniques showed that antibodies from both patient groups bound all classes of intact histone: H1 greater than H2B greater than H2A greater than H3 greater than H4. The different classes of histone were enzymatically or chemically cleaved to produce a series of peptide fragments which were then used to map the reactive epitopes by ELISA and immunoblotting. Ten of 11 DIL sera and 11 of 12 SLE sera bound the carboxy and amino terminal peptides. Only one sera of each group bound to the central hydrophobic polypeptide. The reactivity of DIL sera with fractionated histone polypeptides was similar to that observed with SLE sera. This observation suggests that the histone epitopes reacting with DIL sera are no less restricted than those reacting with SLE.

Site and role of the N-terminal fragment of the nucleosomal core histones in their binding to deoxyribonucleic acid as determined by vibrational spectroscopy.

The site and role of the binding of the 1-53 N-terminal part of H4 on DNA have been studied by optical spectroscopy. The structure of the 1-53 H4 fragment determined by vacuum ultraviolet circular dichroism and infrared spectroscopy is essentially aperiodic. The site of the interacion between the fragment and free DNA is localized by Raman laser spectroscopy in the small groove of the DNA, similar to the interaction site of the whole histone with DNA in nucleosomes. Infrared linear dichroism measurements show that the two 1-53 and 54-102 H4 fragments play a very important role in the histone-DNA interactions, but the roles are extremely different: the N-terminal part of the histone remains effectless on the DNA conformational flexibility and it is proposed that the structurally important interaction occurs between the globular part of the histone and the DNA. The N-terminal fragment appears to be responsible for finding the correct place on the DNA of the nucleosomal core particles.

Role of the protein alpha helixes in histone-DNA interactions studied by vibrational spectroscopy.

The localization of structurally important histone-DNA interactions has been investigated by vibrational spectroscopy. Histones H2A, H2B, and H4 and their fragments (H2A, 1-56, 1-89, 73-129; H2B, 1-59, 1-83; H4, 1-53, 1-67, 1-84, 69-84, 85-102) have been prepared, characterized, and used to reconstitute protein-DNA complexes. Evidence is given for the existence of a direct relationship between the presence of ordered alpha-helical structures in the histones and a stabilization of the DNA in a B geometry. Infrared linear and ultraviolet dichroism measurements indicate that the N-terminal fragments, rich in basic residues and mostly in a random conformation, remain without any influence on the secondary structure of the nucleic acid, leaving it free in the complexes to undergo a total B----A conformational transition. On the contrary, histone fragments that involve some alpha-helical parts of the protein partially stabilize the DNA in a B geometry. Histone fragments that contain all of the alpha helixes of the protein block the DNA in the same way as the whole corresponding histone. A model for histone-DNA interactions in the core particle is discussed.

Immunochemical detection of changes in chromatin subunits induced by histone H4 acetylation.

Native, reassociated, and reconstituted core particles from chicken erythrocytes were compared by both biophysical and immunochemical methods. No significant difference between the three types of core particles could be demonstrated by electron microscopy, circular dichroism, or immunochemical analysis with antisera to histone H2B, H2A, and H3. Core particles were also reconstituted with calf thymus non-acetylated H3, H2A, and H2B with either mono-, di-, or tri-acetylated H4 isolated from cuttle -fish testes. The hyperacetylation of H4 did not significantly alter the biophysical characteristics of core particles but it induced several changes in their immunochemical reactivity. Binding to core particles of antibodies specific for H2A, H3, and for the IRGERA (synthetic C-terminal) peptide of H3 was considerably decreased when di- or tri-acetylated H4 was used for reconstitution, whereas binding of H2B antibodies remained the same. Our results suggest that the presence of hyperacetylated H4 within core particles leads to conformational changes that alter the antigenic determinants of several of the histones present at the surface of chromatin subunits. Since histone acetylation is correlated with the open structure of active chromatin, it may become possible to monitor the activity of chromatin by immunochemical methods.

Application of electrospray and fast atom bombardment mass spectrometry to the identification of post-translational and other chemical modifications of proteins and peptides.

Mass spectrometry is a very powerful tool in the identification of chemical modifications of proteins and peptides. Often these modifications cannot be determined by conventional techniques. This report describes the combined use of electrospray ionization mass spectrometry and fast atom bombardment mass spectrometry to complete the primary structure of proteins and peptides. Examples illustrate how mass spectrometry is used to locate sites of phosphorylation, methylation and acetylation, and identify blocking groups and unexpected side reactions such as deamidation or alkylation.