Studies on protein organization of nucleosomes using cross-linking

When chromosomal proteins in chromatin or in mononucleosomes were extensively cross-linked with an imido ester, the H1-containing nonameric histone complex was revealed. In this complex, histone H1 is connected with the octamer of core histones. The cross-linking of H1 to the octamer is realized preferentially through H2a and H3 histones. Some HMG (high mobility group) proteins located presumably in the linker regions of a nucleosome fiber also take part in the formation of dimers, possibly with the histones of a nucleosomal core. The results suggest mutual interactions between some linker-associated proteins and intranucleosomal histones. Experiments involving extensive cross-linking of proteins in the purified mononucleosome subfractions demonstrated differences in the organization of core histones between complete nucleosomes and nucleosomes lacking H1.Abbreviations HMG proteins high mobility group proteins - DMA dimethyladipimidate dihydrochloride - DMP dimethyl-3,3-dithio-bis-propionimidate dihydrochloride     

Role of individual histone tyrosines in the formation of the nucleosome complex.

We have determined the accessibility of histone tyrosine residues to react with p-nitrobenzenesulfonyl fluoride (NBSF) in intact nuclei, salt-dissociated nucleosomes, isolated histone complexes, and individual core histones. Of the 15 core histone tyrosine residues, 13 are inaccessible in native nucleosomes; only Tyr121 near the C-terminus of H2B is fully accessible, and Tyr54 of H3 is partially accessible under near-physiological conditions. When H1 and the basic N-terminal tails of the core histones are dissociated from the DNA by treating nuclei with 0.4 and 0.8 M NaCl, the two tyrosines which are adjacent to the basic regions of H2B and H3 become accessible as well. This indicates that these tyrosine residues may be involved in histone-DNA interactions, either directly or indirectly. When the H2A-H2B dimers are dissociated from the chromatin by raising the NaCl concentration to 1.2 M, three to four tyrosines located in the structured regions of H2B and H4 are exposed, suggesting that these tyrosine residues may be located at the dimer-tetramer interface. Dissociating all the histones from the DNA at an even higher ionic strength as a mixture of dimers, tetramers, and octamers does not change the pattern of Tyr exposure, but reduces the reactivity of the tyrosines at the dimer-tetramer interface as would be expected from the reassociation of H2A-H2B dimers and H3-H4 tetramers.(ABSTRACT TRUNCATED AT 250 WORDS)     

H3 Cys-110 is in close proximity to the C-terminal regions of H2B and H4 in a nucleosome core with an altered internal arrangement of histones

A particle obtained by nuclease digestion of nucleohistone complexes prepared by direct mixing of histones with DNA in 0.15 M NaCl was indistinguishable by composition and physical properties from nucleosome cores prepared under the same conditions from nucleohistone preannealed in 0.6 M NaCl. We show here that different photo-cross-links form when these particles are prepared from H3 labeled with photoaffinity reagents on the unique histone H3 cysteine. H3-H3 histone dimers were dominant when the particles were prepared by dilution of the nucleohistone from 0.6 M NaCl while H3-H2B and H3-H4 histone dimers were prominent if the nucleohistone complex was prepared directly in 0.15 M NaCl. Peptide mapping of the novel H3-H4 and H3-H2B dimers showed that Cys-110 of histone H3 is cross-linked to the 18 amino acid C-terminal end of H4 or to the 66 amino acid C-terminal half of H2B.     

Nucleosomes from normal and regenerating rat liver

Micrococcal-nuclease digestion of rat liver nuclei selectively released mononucleosomes associated with ADP-ribosylated [Caplan, Ord & Stocken (1978) Biochem. J.174, 475-483] histone H1. Two classes of mononucleosome were detected, those that leaked out during digestion and those that were subsequently released by 5mm-sodium phosphate buffer (pH6.8)/0.2mm-NaEDTA. The former, from which histone H1 had been dissociated, contained 140-base-pair-length DNA and core histones;the latter contained core particles and mononucleosomes with histone H1 and 200-base-pair-length DNA. When normal liver nuclei were phosphorylated with [gamma-(32)P]ATP, dissociated histone H1, which could be separated from core particles with Sephadex G-200, showed (32)P uptake. (32)P uptake into histones H2A and poly(ADP-ribosyl)ated H3 was appreciable in core particles, but was less evident in nucleosomes still containing histone H1. When [(3)H]-thymidine was given to partially hepatectomized rats in S-phase, 5-10min pulses in animals of over 300g body wt. showed the presence of high-specific-radioactivity DNA in released core particles and mononucleosomes compared with DNA retained in the nuclear pellets. Mononucleosomes from rat livers in S-phase with new, [(3)H]lysine-containing histones, had higher (32)P incorporation in histones H1 and their core histones, than for di- or tri-nucleosomes. Thermal-denaturation properties of control and phosphorylated mononucleosomes and core particles were very similar; removal of histone H1 and non-histone chromosomal proteins in 0.5m-NaCl markedly increased the proportion of DNA ;melting' below 70 degrees C.     

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.     

Nonenzymatic glycation of histones in vitro and in vivo

Purified histones in solution, purified nuclei, or whole endothelial cells in cell culture were used to study the reactivity of histones with various sugars. The sugar incubation of purified histones produced nonenzymatic glycation and formation of histone cross-links showing disappearance of individual histone molecules and appearance of dimers and polymers in SDS-PAGE. In solution, core histones react considerably faster with sugars as compared to H1 histones. In sugar-incubated nuclei where histones are nucleosomally organized, H1 histones, which are located at the periphery of the nucleosome, and H2A-H2B dimers, which are associated with the central H3(2)-H4(2) tetramer, are more reactive as compared to H3 and H4 histones, which are most protected from the glycation reaction. Our in vivo experiments using endothelial cells show that high concentrations of ribose are able to generate protein cross-links paralleled by apoptotic cell death. High concentrations of glucose or fructose do not increase histone glycation or cell death, even after 60 days of incubation of endothelial cells. In long-time glucose- or fructose-treated cells, under nondenaturing and nonreducing SDS-PAGE conditions part of the H3 histones shifted away from their normal location. Because it is known that the mitochondrial production of reactive oxygen species (ROS) increases after hyperglycaemia, we hypothesize that ROS could be responsible for the formation of a disulphide bridge between the side chain of the cysteine residues of H3 molecules.     

Core histones H2B and H4 are mobilized during infection with herpes simplex virus 1

The infecting genomes of herpes simplex virus 1 (HSV-1) are assembled into unstable nucleosomes soon after nuclear entry. The source of the histones that bind to these genomes has yet to be addressed. However, infection inhibits histone synthesis. The histones that bind to HSV-1 genomes are therefore most likely those previously bound in cellular chromatin. In order for preexisting cellular histones to associate with HSV-1 genomes, however, they must first disassociate from cellular chromatin. Consistently, we have shown that linker histones are mobilized during HSV-1 infection. Chromatinization of HSV-1 genomes would also require the association of core histones. We therefore evaluated the mobility of the core histones H2B and H4 as measures of the mobilization of H2A-H2B dimers and the more stable H3-H4 core tetramer. H2B and H4 were mobilized during infection. Their mobilization increased the levels of H2B and H4 in the free pools and decreased the rate of H2B fast chromatin exchange. The histones in the free pools would then be available to bind to HSV-1 genomes. The mobilization of H2B occurred independently from HSV-1 protein expression or DNA replication although expression of HSV-1 immediate-early (IE) or early (E) proteins enhanced it. The mobilization of core histones H2B and H4 supports a model in which the histones that associate with HSV-1 genomes are those that were previously bound in cellular chromatin. Moreover, this mobilization is consistent with the assembly of H2A-H2B and H3-H4 dimers into unstable nucleosomes with HSV-1 genomes.     

Laser-induced crosslinking of histones to DNA in chromatin and core particles: implications in studying histone-DNA interactions.

UV laser irradiation has been used to covalently crosslink histones to DNA in nuclei, chromatin and core particles and the presence of the different histone species in the covalently linked material was detected immunochemically. When nuclei were irradiated and then trypsinized to cleave the N- and C- terminal histone tails, no histones have been found covalently linked to DNA. This finding shows that UV laser-induced crosslinking of histones to DNA is accomplished via the non-structured domains only. This unexpected way of crosslinking operated in chromatin, H1-depleted chromatin and core particles, i.e. independently of the chromatin structure. The efficiency of crosslinking, however, showed such a dependence: whilst the yield of crosslinks was similar in total and H1-depleted chromatin, in core particles the efficiency was 3-4 times lower for H2A, H2B and H4 and 10-12 times lower for H3. The decreased crosslinking efficiency, especially dramatic in the case of H3, is attributed to a reduced number of binding sites, and, respectively, is considered as a direct evidence for interaction of nonstructured tails of core histones with linker DNA.     

Chemical crosslinking of histone H1° to histone neighbours in nuclei and chromatin

Crosslinking of histones in mouse liver nuclei and extended chromatin with a bifunctional reagent leads to the formation of H1H1° heterodimers as well as H1°H1° homodimers. H1° can be also crosslinked to the core histones. Thus, the location of histone H1° within the basic repeating chromatin structure seems to be analogous to that of H1 histone.     

Histone dimers: a fundamental unit in histone assembly.

Histone interactions which occur, at moderate ionic strengths, when several types of purified, renatured histones are mixed at equimolar ratios have been studied. The four histones H2A,H2B,H3 and H4 complex and form dimers. Histone H1 does not interact with the other four histone types and does not form dimers. Mixing of single histone species with preformed histone pairs as well as mixing of two different types of histone pairs, leads to exchange of histones among the pairs and formation of dimers. No trimers are formed. The dimers are in equilibrium with high-molecular weight histone structures. The results indicate that histone dimers may serve as a stable intermediate in histone assembly. Because each histone type (except H1) can interact with itself as well as with each of the other three histone types we suggest that each histone type should be considered as an interchangeable subunit of a multichain protein in which the dimer species is the most stable structure.     

Histone H1 and HMG 14/17 are deposited nonrandomly in the nucleus.

We have studied the assembly of histone H1 and the high mobility group nonhistones 14/17 by isopycnic analysis after crosslinking density labeled MSB cell nuclei or chromatin. Carbodiimide crosslinking produces dense poly-H1 and hybrid density H1-H2A histone dimers, indicating that new H1 is deposited nonrandomly, albeit nonconservatively relative to new core histones. Core histone-HMG crosslinking with succinimidyl propionate yields dense HMG 14 in uniformly dense particles and new HMG 17 crosslinked to both dense and light protein, implying that HMG 14 and 17 each deposit nonrandomly; but differently with respect to new core octamers. Propionimidate crosslinking yields dense H1-HMG 17 dimers, suggesting that the interactions of new 14/17 with H1 (new HMG 14-old H1, new HMG 17-new H1) are reciprocal to their interactions with the core histones.     

Enrichment and characterization of histones by two-dimensional hydroxyapatite/reversed-phase liquid chromatography-mass spectrometry

Here we report a novel two-dimensional liquid chromatography-mass spectrometry (2D LC-MS) method that combines offline hydroxyapatite (HA) chromatography with online reversed-phase liquid chromatography-mass spectrometry (HA/RP LC-MS). The 2D LC-MS method was used to enrich and characterize histones and their posttranslational modifications. The 2D HA/RP LC-MS approach separates histones based on their relative binding affinity to DNA and relative hydrophobicity. HA/RP separations showed improvement in the number of histone isoforms detected as compared with one-dimensional RP LC-MS of acid-extracted histones. The improved histone fractionation resulted in better detection of lower abundant histone variants as well as their posttranslationally modified isoforms. Histones eluted from the HA/RP in the following order: H1, H2A/H2B heterodimers followed by H3/H4 heterotetramers, as predicted from their spatial organization in nucleosomes for binding affinity to DNA. Comparison between HA-purified and acid-extracted histones revealed similar histone profiles with the exception that the HA fractions showed a greater number of H1 isoforms. Two elution conditions were also examined: batch elution and salt gradient elution. Although both elution techniques were able to fractionate the histones sufficiently, the salt gradient approach has the most potential for purification of selected histone isoforms.     

The structure and function of Xenopus NO38-core, a histone chaperone in the nucleolus

Xenopus NO38 is an abundant nucleolar chaperone and a member of the nucleoplasmin (Np) family. Here, we report high-resolution crystal structures of the N-terminal domain of NO38, as a pentamer and a decamer. As expected, NO38 shares the Np family fold. In addition, NO38- and Np-core pentamers each use highly conserved residues and numerous waters to form their respective decamers. Further studies show that NO38 and Np each bind equal amounts of the four core histones. However, NO38 prefers the (H3-H4)(2) tetramer, while Np probably prefers H2A-H2B dimers. We also show that NO38 and Np will each bind noncognate histones when the preferred partner is absent. We suggest that these chaperones must form decamers in order to bind histones and differentiate between histone tetramers and dimers. When taken together, these data imply that NO38 may function as a histone chaperone in the nucleolus.     

Photochemical cross-linking of histones to DNA nucleosomes.

Ultraviolet (UV)-induced cross-linking was utilized in order to identify histone-DNA interacting regions in the chromatin repeating unit. Fractionated mononucleosomes which contained 185 base pairs of DNA and a full complement of the histones, including histone H1, were irradiated with light of lambda greater than 290nm in the presence of a photosensitizer. Equimolar amounts of histones H2A and H2B were found, by two independent labeling experiments, to be cross-linked to the DNA. Based on previous finding that the UV irradiation specifically cross-links residues which are in close proximity, irrespective of the nature of the amino acid side chain or the nucleotide involved, our results indicate that the four core histones are not positioned equivalently with respect to the DNA. This arrangement allows histones H2A and H2B to preferentially cross-link to the DNA. A water soluble covalent complex of DNA and histones was isolated. This complex was partially resistant to mild nuclease digestion, it exhibited a CD spectrum similar to that of chromatin, and was found to contain histone H1. These results are compatible with a model which suggests that histone H1, though anchored to the linker, is bound to the DNA at additional sites. By doing so it spans the whole length of the nucleosome and clamps together the DNA fold around the histone core.     

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.     

A Mechanism for Histone Chaperoning Activity of Nucleoplasmin: Thermodynamic and Structural Models

Nucleoplasmin (NP), a histone chaperone, acts as a reservoir for histones H2A-H2B in Xenopus laevis eggs and can displace sperm nuclear basic proteins and linker histones from the chromatin fiber of sperm and quiescent somatic nuclei. NP has been proposed to mediate the dynamic exchange of histones during the expression of certain genes and assists the assembly of nucleosomes by modulating the interaction between histones and DNA. Here, solution structural models of full-length NP and NP complexes with the functionally distinct nucleosomal core and linker histones are presented for the first time, providing a picture of the physical interactions between the nucleosomal and linker histones with NP core and tail domains. Small-angle X-ray scattering and isothermal titration calorimetry reveal that NP pentamer can accommodate five histones, either H2A-H2B dimers or H5, and that NP core and tail domains are intimately involved in the association with histones. The analysis of the binding events, employing a site-specific cooperative model, reveals a negative cooperativity-based regulatory mechanism for the linker histone/nucleosomal histone exchange. The two histone types bind with drastically different intrinsic affinity, and the strongest affinity is observed for the NP variant that mimicks the hyperphosphorylated active protein. The different “affinity windows” for H5 and H2A-H2B might allow NP to fulfill its histone chaperone role, simultaneously acting as a reservoir for the core histones and a chromatin decondensing factor. Our data are compatible with the previously proposed model where NP facilitates nucleosome assembly by removing the linker histones and depositing H2A-H2B dimers onto DNA.     

Different mechanism for in vitro formation of nucleosome core particles

The interaction of different histone oligomers with nucleosomes has been investigated by using nondenaturing gel electrophoresis. In the presence of 0.2 M NaCl, the addition of the pairs H2A,H2B or H3,H4 or the four core histones to nucleosome core particles produces a decrease in the intensity of the core particle band and the appearance of aggregated material at the top of the gel, indicating that all these histone oligomers are able to associate with nucleosomes. Equivalent results were obtained by using oligonucleosome core particles. Additional electrophoretic results, together with second-dimension analysis of histone composition and fluorescence and solubility studies, indicate that H2A,H2B, H3,H4, and the four core histones can migrate spontaneously from the aggregated nucleosomes containing excess histones to free core DNA. In all cases the estimated yield of histone transfer is very high. Furthermore, the results obtained from electron microscopy, solubility, and supercoiling assays demonstrate the transfer of excess histones from oligonucleosomes to free circular DNA. However, the extent of solubilization obtained in this case is lower than that observed with core DNA as histone acceptor. Our results demonstrate that nucleosome core particles can be formed in 0.2 M NaCl by the following mechanisms: (1) transfer of excess core histones from oligonucleosomes of free DNA, (2) transfer to excess H2A,H2B and H3,H4 associated separately with oligonucleosomes to free DNA, (3) transfer to excess H2A,H2B initially associated with oligonucleosomes to DNA, followed by the reaction of the resulting DNA-(H2A,H2B) complex with oligonucleosomes containing excess H3,H4, and (4) a two-step transfer reaction similar to that indicated in (3), in which excess histones H3,H4 are transferred to DNA before the reaction with oligonucleosomes containing excess H2A,H2B. The possible biological implications of these spontaneous reactions are discussed in the context of the present knowledge of the nucleosome function.     

Analysis of Histones Associated with Neuronal and Glial Nuclei Exhibiting Divergent DNA Repeat Lengths

Abstract: Total cerebral hemisphere nuclei purified from adult rabbit brain were subfractionated into neuronal and glial populations. Previous studies have shown that chromatin in neuronal nuclei is organized in an unusual nucleosome conformation compared with glial or kidney nuclei, i.e., a short DNA repeat length is present. We now analyze whether this difference in chromatin organization is associated with an alteration in the histone component of nucleosomes. Total histone isolated by acid/urea-protamine extraction of purified neuronal, glial, and kidney nuclei was analyzed by electrophoresis on SDS-polyacrylamide slab gels. Histone H1 that was selectively extracted from nuclei was also examined. Differences were not observed on SDS gels in the electrophoretic mobilities of histones associated with either the nucleosome core particle (histones H2A, H2B, H3, H4) or the nucleosome linker region (histone H1). Total histone and selectively extracted histone H1 were also analyzed on acid/urea slab gels that resolve histones on the basis of both molecular weight and charge differences. When analyzed in this system, differences with respect to electrophoretic mobility were not detected when comparing either selectively extracted histone H1 or total histone from neuronal and glial nuclei. Quantitative analyses were also performed and neuronal nuclei were found to contain less histone H1 per milligram DNA compared with glial or kidney nuclei. Neuronal nuclei also demonstrated a lower ratio of histone H1/core histone. These results suggest that the pronounced difference in chromatin organization in neuronal compared with glial nuclei, which is reflected by a short DNA repeat length in neurons, appears to be associated with quantitative differences in neuronal histone H1.     

Cross-linking of histones with dimethyl 3,3''-dithiobispropionimidate. Interference by a one-end reaction modifying histones at lysine amino groups.

We have studied the HClO4-solubility of histones H1 and H5 in hen erythrocyte nuclei after treatment with the cross-linker dimethyl 3,3'-dithiobispropionimidate (DTPI). The amount of acid-soluble, non-cross-linked, H1 and H5 histones was drastically decreased, and that of acid-soluble H1/H5 histone dimers went through an optimum as the DTPI concentration was raised. Incubation of the HClO4-insoluble fraction with 2-mercaptoethanol regenerated the acid-solubility of H1/H5 histones in this fraction. When purified H1/H5 histones were treated with increasing concentrations of DTPI under non-cross-linking conditions, the amount of HClO4-soluble histones also greatly decreased, but to a much lesser extent if the DTPI treatment was followed by reduction with 2-mercaptoethanol. This decrease was inversely correlated to the proportion of amino groups modified. It is concluded that, when the cross-linker was used in large excess, the cross-linking reaction competed with a one-end reaction modifying the histones at lysine amino groups by cross-linker molecules, of which the imidoester groups that had not reacted were hydrolysed. It is suggested that this modification produced the changes in acid-solubility.     

Salt effects on histone subunit interactions as studied by fluorescence spectroscopy

The salt concentration dependence of the aggregation properties of calf thymus and chicken erythrocyte histones has been investigated by using fluorescence spectroscopy. The isolated H2A/H2B and H3/H4 subunit preparations were labeled with 5-(dimethylamino)naphthalene-1- sulfonyl (dansyl). This long-lived fluorescence probe allows for the observation of rotations due to tumbling of the particle and thus is a probe for changes in the size of macromolecular assemblies. The fluorescence polarization and lifetime were measured as a function of salt concentration for these isolated preparations. Next, each labeled preparation was reconstituted with its unlabeled complement, and the salt concentration dependence of histone core octamer interactions was investigated in the same manner. Salt-induced core particle formation was observed by monitoring the dansyl-labeled dimers for both the calf thymus and chicken erythrocyte preparations. Evidence for subunit dissociation of the isolated H2A-H2B preparations was also found, as well as aggregation of the isolated H3/H4 subunits to at least dimers of tetramers. The calf thymus H3/H4 preparation was in aggregated form under all conditions studied, whereas the chicken erythrocyte H3/H4 only formed aggregates at high protein or salt concentrations. We have found evidence that the dimer can displace the tetramer from the higher order aggregate in order to form core particles. Such competition between the subunit interfaces in the histone system suggests that they may play a regulatory role in histone-DNA interactions.