Histone Molecules Research Articles

Site-specificity of histone HL methylation by two H1-specific protein-lysine N-methyltransferases from Euglena gracilis

1. 1. The histone H1 fractions from rat spleen and liver were used as substrates for two H1-specific protein-lysine N-methyltransferases, V-A and V-B (protein methylase III) from Euglena gracilis. 2. 2. When the enzymatically [methyl- 3H]labeled H1 fractions were resolved by two-dimensional gel electrophoresis, four subtypes were found to be methylated (Hl b, Hl c, Hl d and Hl e). Both enzymes methylated Hl c and Hl b to approximately the same extent; H1 d and Hl e were methylated preferentially by enzyme V-B and V-A, respectively. 3. 3. Histone Hl c, [methyl- 3H]labeled by the methyltransferase V-A, which had been digested by arginine-specific protease (Arg C protease), showed a single radioactive peptide on HPLC, indicating methylation site specificity of the enzyme. 4. 4. Arg C protease-digestion of [methyl- 3H]labeled Hl c labeled by methyltransferase V-B indicated that this enzyme methylated two sites on the histone molecule. 5. 5. The histone Hlc methylation sites of these two enzymes did not overlap, indicating the two enzymes have different site specificity. 6. 6. In combination with the other results, this suggests that the two enzymes serve discrete purposes, possibly involving the presumed different actions of histone H1 subtypes.

12] Use of antihistone antibodies with nucleosomes

Antibodies directed against defined regions of histone molecules represent one of the most specific probes for studying the surface topography of nucleosomes and chromatin and for monitoring post-translational histone modifications. Antibodies directed against the epitopes can be used to determine the regions of histone molecules that are exposed at the surface of nucleosomes and are useful probes for analysing chromatin structure and function. Four approaches have been used to obtain antibodies suitable for these types of studies: (1) immunization with histone molecules, (2) immunization with synthetic peptide fragments of histones, (3) immunization with nucleosomes, and (4) preparation of hybridomas from nonimmunized, autoimmune mice. The method used for purifying histones and the immunization protocol, using histones complexed with RNA, influence the conformation of histone molecules and will thus affect their antigenic determinants. There is also evidence that polymerization of histones into chromatin components produces novel antigenic determinants that derive their specificity from the tertiary and quaternary structure. Changes in environmental conditions that alter the structure of chromatin or posttranslational modifications can thus be expected to modify its antigenic reactivity.

Reactivity of Autoantibodies in Systemic <i>Lupus etythematosu</i><i>s</i> with Synthetic Core Histone Peptides

The specificity of autoantibodies present in the serum of 151 patients with systemic lupus erythematosus (SLE) was investigated by ELISA using as antigen individual histones as well as 17 different core histone synthetic peptides. Many of the sera reacted with four terminal peptides (residues 1-21 and 130-135 of H3, 1-29 of H4 and 1-25 of H2B) while fewer reacted with internal peptides (residues 65-85 of H2A and 40-55 of H3). Of the 151 SLE sera, 88% reacted with one or more of the six core histone peptides whereas only 57% reacted with one or more of the complete core histone molecules. Antibodies to mononucleosomes from chicken erythrocytes were also prepared in rabbits. The rabbit antisera were tested by ELISA using as antigen chromatin subunits, native and denatured DNA, individual histones and 23 natural and synthetic peptides of histones. The antinucleosome antibodies were found to recognize the same peptide fragments as those recognized by the SLE sera.

Differences in the binding of H1 variants to DNA. Cooperativity and linker-length related distribution.

A study of the complexes formed between short linear DNA and three H1 variants, a typical somatic H1, and the extreme variants H5, from chicken erythrocytes, and spH1 from sea urchin sperm, has revealed differences between H1, H5 and spH1 that have implications for chromatin structure and folding. 1. All three histones bind cooperatively to DNA in 35 mM NaCl forming similar, but not identical, rod-like complexes. With sufficiently long DNA the complexes may be circular, circles forming more easily with H5 and spH1 than with H1. 2. The binding of H5 and spH1 to DNA is cooperative even in 5 mM NaCl, resulting in well-defined thin filaments that appear to contain two DNA molecules bridged by histone molecules. In contrast, H1 binds distributively over all the DNA molecules in 5 mM NaCl, but forms short stretches similar in appearance to the thin filaments formed with H5 and spH1. Rods appear to arise from the intertwining of regular thin filaments containing cooperatively bound histone molecules on raising the NaCl concentration to 35 mM. 3. The compositions of the rods correspond to one histone molecule for about every 47 bp (H1), 81 bp (H5) and 112 bp (spH1), suggesting average spacings of 24 bp (H1), 41 bp (H5) and 56 bp (spH1) in the component thin (double) filaments. Strikingly, these values are proportional to the linker lengths of the chromatins in which the particular H1 variant is the main or sole H1.

Binding of linker histones to the core nucleosome.

Binding of chicken erythrocyte linker histones H1/H5 to the core nucleosome has been studied. Histones H1/H5 bind very efficiently to the isolated core nucleosome in vitro. The binding of linker histones to the core nucleosome is associated with aggregation of the particles. Approximately one molecule of linker histone binds per core nucleosome in the aggregates, irrespective of the concentration of the linker histones and the salt used. Histone H5 shows greater binding affinity to the core nucleosome as compared to H1. The carboxyl-terminal fragment of the linker histones binds strongly to the core nucleosome while the binding of the central globular domain is weak. Each core nucleosome is capable of binding two molecules of carboxyl-terminal fragment of linker histone. The core nucleosome containing one molecule of carboxyl-terminal fragment of linker histone requires higher salt concentration for aggregation while the core nucleosome containing two molecules of carboxyl-terminal fragment of linker histone can self-associate even at lower salt concentrations. On the basis of these results we are proposing a novel mechanism for the condensation of chromatin by linker histones and other related phenomena.

Interaction in vitro of non-epithelial intermediate filament proteins with histones.

Non-epithelial intermediate filament (IF) subunit proteins show a high and specific affinity for core histones at physiological ionic strength. When IF proteins are titrated with a mixture of core histones and linker histone H1, in general the latter is totally excluded from complexation and in the adducts formed the moderately-arginine-rich histones H2A and H2B are progressively replaced by the very-arginine-rich histones H3 and H4. At histone saturation, 2 molecules of nonneuronal IF protein bind 1 histone H1 molecule or 8 core histone molecules, whereas due to its glutamic acid-rich, C-terminal extensions one dimer of the 68 kD neurofilament protein associates with 3 molecules of histone H1 or 24 molecules of core histones. The salt stability of the insoluble association products is dependent on the amount and arginine content of the constituent histone species. Removal of the non-alpha-helical N- and C-terminal polypeptides from IF proteins by partial chymotryptic digestion does not affect their histone-binding characteristics. Since core histones are only partially inactivated by limited tryptic digestion, they also appear to react through their alpha-helix-rich central domains; the limit peptide derived from histone H1 is completely inactive at physiological ionic strength. Affinity chromatography of rod domains of IF proteins on core histone-Sepharose 4B and of histones and their limit peptides on vimentin-Sepharose 4B has shown that the interactions involving fractions of histones H3 and H4 are extremely resistant to salt and can be dissociated only with arginine or salt under denaturing conditions. In general, the experimental results revealed close parallels between the association of histones with IF proteins and their interaction with DNA.

Antihistone antibodies detected by micro-ELISA and immunoblotting in mice with lupus-like syndrome (MRL/l, MRL/n, PN, and NZB strains)

Antihistone antibodies were searched for in autoimmune prone strains of mice: MRL/l, MRL/n, PN, and NZB by micro-enzyme-linked immunosorbent assay (micro-ELISA with total histones or H1 fraction as antigen) and immunoblotting using a solution of total histones containing H1, H2A, H2B, H3, and H4. In addition, we specified the localization of H1 fraction epitopes recognized by mouse anti-H1 autoantibodies using immunoblotting with H1 digested by α-1-chymotrypsin. All strains of autoimmune mice synthesize antihistone antibodies, principally MRL/l, then MRL/n and PN, and finally NZB. Among MRL/l mice, the histone fractions best recognized by antihistone antibodies, are, in decreasing order: H1, H3, H4, H2B, and H2A. With MRL/n and, even more strikingly with PN mice, the antihistone antibodies recognize preferentially H1 and H2B as they do in human lupus. Finally, the binding of antihistone antibodies from NZB mice is slightly stronger for H2B than for the other histone fractions. The anti-H1 autoantibodies from MRL/l, MRL/n, and PN mice are mainly directed at epitopes located on the C terminal of the histone molecule.

Specificity studies on anti-histone H1 antibodies obtained by different immunization methods

Antibodies were elicited against chromatographically purified histone H l subfractions or against their complexes with RNA and their specificity studied by enzyme-linked immunosorbent assay. The results show that complexing of the pure protein with RNA (i) does not lead to any significant increase in the antibody titer and (ii) results in obtaining antibodies predominantly against the common antigenic determinants present in the HI histone class. On the other hand, using pure histone fractions for immunization gives rise to antibody populations reacting mainly with the subfraction-specific determinants on the histone molecule. In view of these results the literature data should be interpreted with caution.

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.

Poly(ADP-ribose) accessibility to poly(ADP-ribose) glycohydrolase activity on poly(ADP-ribosyl)ated nucleosomal proteins.

Hydrolysis of protein-bound 32P-labelled poly(ADP-ribose) by poly(ADP-ribose) glycohydrolase shows that there is differential accessibility of poly(ADP-ribosyl)ated proteins in chromatin to poly(ADP-ribose) glycohydrolase. The rapid hydrolysis of hyper(ADP-ribosyl)ated forms of histone H1 indicates the absence of an H1 dimer complex of histone molecules. When the pattern of hydrolysis of poly(ADP-ribosyl)ated histones was analyzed it was found that poly(ADP-ribose) attached to histone H2B is more resistant than the polymer attached to histone H1 or H2A or protein A24. Polymer hydrolysis of the acceptors, which had been labelled at high substrate concentrations (greater than or equal to 10 microM), indicate that the only high molecular weight acceptor protein is poly(ADP-ribose) polymerase and that little processing of the enzyme occurs. Finally, electron microscopic evidence shows that hyper(ADP-ribosyl)ated poly(ADP-ribose) polymerase, which is dissociated from its DNA-enzyme complex, binds again to DNA after poly(ADP-ribose) glycohydrolase action.

Availability of hyperacetylated H4 histone in intact nucleosomes to specific antibodies.

Specific antibodies against the tetra-acetylated form of H4 histone have been elicited in the rabbit. They do not cross-react with the non-, mono-, and di-acetylated forms of the histone molecule but a slight cross-reactivity with the tri-acetylated form of H4 histone is observed. Our studies also show that hyperacetylated H4 histones are recognized by the antibodies in intact nucleosomes.

Chemical Alterations in Native Histone Octamer Complexes Induced by the Attak of · OH and · N3 Radicals

Abstract·OH Radicals generated by short electron‐beam pulses were allowed to attack histone octamer complexes (extracted from calf‐thymus chromatin) in N2O‐saturated dilute solution (0.5–1.3 g/l, [NaClO4] = 1 – 2M, pH 9). They induced a volume contraction due to intra‐complex cross‐linking. In this process, essentially non‐tyrosine moieties of the proteins were involved. Phenol coupling via tyrosyl radicals occurred mainly as an intramolecular reaction, i.e., it was restricted to single histone moelecules. Furthermore, it turned out that only about 55% of the tyrosine moieties were accessible to attacking ·OH and/or ·N3 radicals. When ·N3 radicals were generated via continuous irradiation of N2O‐saturated octamer solutions containing NaN3 with 60Co‐γ‐rays, dimers, trimers, and tetramers were detected by SDS gel electrophoresis, in contrast to pulse radiolysis where only dimers were found. These results were explained in terms of denaturation being induced by small chemical changes and causing partial or complete dissociation of the complexes thus permitting, in the course of the γ‐irradiation, the attack and conversion of amino‐acid moieties non‐accessible in the native octamer complexes. Removal of steric restrictions for the combination of tyrosyl radicals may also play a role. By time‐resolved absorption measurements, it was shown that, upon the attack of intact octamer complexes by ·OH radicals, tyrosyl radicals were formed which were converted to dityrosine groups according to two modes with half‐lives of several 100 m̈s and 1–2 ms, respectively. Cross‐linking of histone molecules occurred with a definitely lower rate (1st half‐life: 50 100 ms). This process was detectable both by optical absorption measurements at λ = 300 400 nm and by light‐scattering measurements.

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.

Interaction of tubulin with chromatin proteins. H1 and core histones.

Purified histones, H1 or core histones, induce the aggregation of tubulin. The aggregation process, studied by light scattering at 350 nm and polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, is dependent on the respective amounts of histones and tubulin; a maximum is obtained at a stoichiometry of 1 molecule of H1 or 2 molecules of core histones/tubulin dimer. At these molar ratios, all tubulin and histone molecules are found in the insoluble material which sedimented at 2000 X g. Increasing H1 or core histones, there is a progressive decrease of light scattering and a concomitant formation of soluble complexes. The minimum soluble complexes between tubulin and H1 or between tubulin and core histones have the same apparent molecular weight of 150,000-160,000; these complexes consist of one tubulin and two H1 molecules or one tubulin and four core histones. The tubulin-histone interaction is an almost instantaneous reaction which can, however, be slowed down by increasing the ionic strength of the medium. NaCl (0.5 M) decreased by 50% the formation of tubulin-H1 insoluble complexes but slightly affects the core histones-tubulin complex formation. Histones can be classified by their ability to form insoluble complexes with tubulin: H4 = H3 greater than H2B = H2A greater than H1. The reactivity of histones seems to be related to their lysine/arginine content. Electron microscopy revealed that insoluble polymers resulting from the interaction of tubulin with H1 or core histones are similar and consist of unordered aggregates of 35-40 nm ring structures.

A relationship between nuclear poly(adenosine diphosphate ribosylation) and acetylation posttranslational modifications. 1. Nucleosome studies.

The chromatin-associated enzyme poly(ADP-Rib) polymerase catalyzes the posttranslational modifications of histones. Antibody to poly(adenosine diphosphate ribose) [poly(ADP-Rib)] has been coupled to Sepharose, and the resulting immunoadsorbant was used to fractionate, specifically, oligonucleosomes derived from cells pulse labeled for the acetylation modifications of chromatin by incubation with [3H]acetate followed by treatment with sodium butyrate. Generally, about 50% of the histone H3 and H4 mass becomes acetylated under these conditions. Pulse-labeled acetylated regions of chromatin were selectively retained by the anti-poly(ADP-Rib)-Sepharose column due to the presence of endogenous poly(ADP-Rib) components. The data suggest that certain histone molecules might be mutally poly(ADP-ribosylated) and acetylated, and this phenomenon was further explored at the protein level in the accompanying paper [Wong, M., & Smulson, M. (1984) Biochemistry (following paper in this issue)].

A relationship between nuclear poly(adenosine diphosphate ribosylation) and acetylation posttranslational modifications. 2. Histone studies.

In the accompanying paper [Malik, N., & Smulson, M. (1984) Biochemistry (preceding paper in this issue)], we report that certain acetylated domains of chromatin were selectively retained by an anti-poly(ADP-Rib) antibody column. In this paper, we describe investigations of this phenomenon at the molecular level of protein interactions. We observed that the majority of endogenously hyperacetylated histones have a high affinity toward the polymer antibody column. It is speculated that these proteins were bound to the column via endogenous poly(adenosine diphosphate ribose) [poly(ADP-Rib)] since the binding was reversed upon treatment of the histones with alkali prior to immunofractionation. In order to analyze the distribution of acetate and poly(ADP-Rib) on histone proteins, [3H]acetylated nuclei were incubated in vitro with [32P]NAD. Acetate was incorporated mainly into H3 and H4 while H1 was the major acceptor protein for poly(ADP-Rib). These results suggest that a correlation may exist in vivo between the two posttranslational modification processes and that identical histone molecules may be accessible to both modifications.

Close contacts between H1 histone molecules in nuclei.

H1 histone homodimer has been purified from nuclei treated with either long (dimethylsuberimidate, dimethyl dithiobisproprionimidate) or short (ethyl dimethylaminocarbodiimide) cross-linking reagents. When such H1 dimers were cleaved with N-bromosuccinimide, chymotrypsin, or staphylococcal protease, analysis of the resulting fragments suggests that the major cross-links were formed between the COOH-terminal portions of neighboring H1 molecules, and that substantial bridging can also be made between the NH2-terminal portion of one H1 molecule and the COOH-terminal portion of another. NH2-terminal to NH2-terminal cross-links were not seen at significant levels, suggesting that the position of the NH2-terminal portion of H1 in chromatin is more restricted.

High-performance liquid chromatography of chromatin histones

A method is described for the rapid analysis of histones by high-performance liquid chromatography on reversed-phase μBondapak columns, containing either octadecylsilane (C18) or cyanopropylsilane (CN) bonded to silica particles packed in either steel columns or Radial-Pak cartridges. A linear gradient progressing from water—acetonitrile (80:20) to water—acetonitrile (40:60) and increasing in acetonitrile concentration at the rate of 10%/h was used to elute the histones at a flow-rate of 1 ml/min for steel columns or 2 ml/min for Radial-Pak cartridges. Two conditions were found to be necessary to achieve histone, fractionation: (1) silylation of the active silanol groups on the silica matrix, and (2) 0.1–0.3% trifluoroacetic acid in the eluting solvent. More than 95% of the total [3H]lysine-labeled protein applied to the CN column was eluted. The histone fractions were identified by their electrophoretic mobilities in both acid—urea and Triton DF-16 polyacrylamide gels. Histones were eluted from the columns in the following order: H1, H2B, (LHP)H2A, (MHP)H2A, H4, (LHP)H3, and (MHP)H3 (where LHP and MHP refer to the less-hydrophobic and more-hydrophobic histone variants). Phosphorylated and acetylated histone molecules were not separated from their unmodified parent molecules. The volatile nature of the water—acetonitrile—trifluoroacetic acid eluting solvent facilitated recovery of salt-free histones from the fractions by direct lyophilization of the column effluents. The best resolution of histone fractions was obtained with the Radial-Pak μBondapak C18 cartridge using 0.3% TFA. However, for analytical studies, the best detection was obtained by using the μBondapak CN steel column. Poorer resolution was obtained by using the non-silica based PRP-1 reversed-phase column, containing a polystyrene-divinylbenzene resin under the same conditions.

Triiodothyronine nuclear receptor: Role of histones and DNA in hormone binding

The triiodothyronine (T 3) nuclear receptor was previously shown to lose rapidly its high affinity hormone-binding property after a partial purification from the nuclear extract. It was then found that histones + DNA added to the incubation medium with labeled T 3 could restore, at least in part, the high affinity T 3 binding. We now demonstrate that DNA alone increases the high affinity T 3 binding site concentration moderately, and only at low ionic strength where it can bind to the receptor. Total histones and all histone fractions studied (total core histones, F2a, H2B, H3, H4, H1) specifically increase, at low concentrations, the level of T 3 binding; but higher concentrations of some individualized histones, particularly arginine-rich histones, have an inhibitory effect. DNA, or several other polynucleotides, in the presence of histones increase the stimulating histone effect and reverse the inhibitory effect into a true activation. Histones increase the number of T 3 binding sites but decrease the affinity for T 3; addition of DNA restores the high affinity for T 3 and stabilizes the T 3-receptor complexes. Thus, some of the histone molecules could play a role in the maintenance of the T 3 binding site, but multiple interactions between histones or with DNA seem necessary to impair the negative effect exerted by other parts of the histone molecules. Whether these positive and negative effects of histones on the T 3 binding site are of biological relevance in the regulation of T 3 binding to its receptor remains to be determined.

Assessment of estrogen receptor--histone interactions.

Several different in vitro binding assays show that the estrogen receptor from rabbit uterus interacts selectively with purified histones from calf thymus. The estrogen receptor binds strongly to histones H2B and H2A, moderately to histones H3 and H4, and poorly to histone H1. In the presence of histones H2B or H2A, the position at which the estrogen receptor focuses in an isoelectric gradient is shifted to a more basic zone. Kinetic experiments show that, if histone H2B is bound to a DNA, the estrogen receptor dissociates more slowly from that DNA. The portion of the estrogen receptor molecule required for binding to histone H2B is relatively stable to tryptic digestion; in contrast, the portion of the receptor molecule responsible for DNA binding is promptly lost during limited tryptic digestion. These in vitro findings suggest that the mechanism by which the estrogen receptor selectively alters gene expression may involve specific contacts with histone molecules.