Chromatin assembly: biochemical identities and genetic redundancy.

Investigations on chromatin assembly in vitro implicate chromatin assembly factor 1 (CAF1) as a chaperone for histones H3/H4 and nucleosome assembly protein 1 (NAP1) as a chaperone for histones H2A/H2B. Deletion analysis of CAF1 in vivo suggests multiple redundant pathways for deposition of the histones. Histone deposition requires acetylation of the amino-terminal tails and analysis of mutants suggests a specific but redundant role for acetylation of the tails in assembly. Furthermore, studies on the HAT1 acetyltransferase raise the possibility that acetylation of histones occurs following their transport into the nucleus but prior to their deposition onto DNA. Identification of the factors involved in the redundant pathways of assembly is awaited.     

Histones of Neurospora crassa.

Neurospora crassa chromatin isolated by a rapid method minimizing proteolytic degradation contains approximately one weight of acid-extractable basic protein per weight of DNA. This basic protein consists of five major polypeptide species which are similar in size to the histone proteins of higher eukaryotes and are present in approximately the same molar ratios. These five polypeptides have been purified by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Their electrophoretic mobilities in polyacrylamide gels and their amino acid compositions indicate that they are histones homologous, although not identical, to the H1, H2A, H2B, H3, and H4 histones of mammals. The first 3 residues in the amino acid sequence of Neurospora H3 histone are identical to the first 3 residues in calf and pea H3; Neurospora H1, H2A, and H4 histones have blocked NH2 termini, like their mammalian counterparts. The finding of recognizable H1, H2A, H2B, H3, and H4 histones in Neurospora extends the range of eukaryotes now shown to contain a full complement of these strongly conserved chromosomal proteins, and supports the view that histones became involved in chromosome structure at a very early point in the evolution of eukaryotes.     

Differences among subfractions of H1 histone in retention of linear and superhelical DNA on filters

Four kinds of rabbit thymus H1 histone differ among themselves in their ability to retain DNA on nitrocellulose filters. This is true for linear, or superhelical DNA, but the order of effectiveness of the different H1 histones depends on the physical conformation of the DNA. For linear DNA the binding efficiencies of the H1 histones are: RTL2 = RTL3 greater than RTL4 greater than RTL1. This order of effectiveness parallels the effectiveness of the H1 histones previously found for the condensation of linear DNA as observed by circular dichroism and viscosity. The binding efficiencies of the various histones toward superhelical DNA were: RTL4 greater than RTL3 greater than RTL1 greater than RTL2. The variation in amino acid sequence between different rabbit thymus H1 histones might thus introduce structural variations in nucleohistone fibers and perhaps in chromatin.     

Hydrolysis of histones by proteinases.

Hydrolysis of histones by proteinases from rat liver, skin and other sources was studied by using a rat thymus histone preparation as the substrate and polyacrylamide-gel electrophoresis and densitometric analysis as the methods to detect histone subtypes and their hydrolysis. The rat mast-cell proteinase I effectively hydrolysed histones except type H4. Thrombin hydrolysed effectively histones H1 and H2A, whereas plasmin hydrolysed all types of histones. Cathepsin D hydrolysed especially histone H2A. Cathepsins B and L hydrolysed all histones more slowly, and cathepsin H hydrolysed them extremely slowly. Epidermal aminoendopeptidase did not hydrolyse histones. Trypsin and chymotrypsin were used as reference enzymes, which hydrolysed all types of histones in very low concentrations. This study suggests that a variety of proteinases could play a role in histone hydrolysis. Hydrolysis of a specific subtype of histones, such as histone H2A at pH 6 by cathepsin D, may be directly involved in regulation of epidermal-cell differentiation.     

Localization of testis-variant histones in rat testis chromatin.

Nucleosome core particles and oligonucleosomes were isolated by digesting rat testis nuclei with micrococcal nuclease to 20% acid-solubility, followed by fractionation of the digest on a Bio-Gel A-5m column. The core particles thus isolated were characterized on the basis of their DNA length of 151 +/- 5 base-pairs and sedimentation coefficient of 11.4S. Analysis of the acid-soluble proteins of the core particles indicated that histones TH2B and X2 are constituents of the core particles, in addition to the somatic histones H2A, H2B, H3 and H4. The acid-soluble proteins of the oligonucleosomes comprised all the histones, including both the somatic (H1, H2A, H2B, H3, H4 and X2) and the testis-specific ones (TH1 and TH2B). It was also observed that histones TH1 and H1 are absent from the core particles and were readily extracted from the chromatin by 0.6 M-NaCl, which indicated that both of them are bound to the linker DNA.     

The histones of the sperm of Spisula solidissima include a novel, cysteine-containing H-1 histone

The histones remaining at the end of the spermiogenic differentiation, which are found associated with a highly basic protamine-like component [Ausio, J. and K.E. Van Holde (1987) Eur. J. Biochem. 165, 363-371] in the mature sperm of Spisula solidissima, have been isolated and characterized for the first time. All four core histones H2A, H2B, H3, H4, and the lysine-rich histone H1 are present. The core histones are found in equal stoichiometric amounts. As has been observed in other bivalve molluscs, the amino acid compositions of the core histones of S. solidissima sperm are very close to those of their counterparts in the calf thymus somatic histones. The spermatic histone H1 exhibits an amino acid composition and structural features similar to other histones of the histone H1 family. Yet this latter histone seems to be sperm-specific, and it contains at least two cysteine residues per molecule, which makes it unique in its class.     

Isoelectric focusing and isoelectric points of bovine liver histones

A technique for isoelectric focusing of total histones in very narrow pH gradients is described. The isoelectric focusing was performed in 5% acrylamide gels at the pH range 9–11 in long quartz tubes (24 cm) in a nitrogen atmosphere. The total bovine liver histones separated into five main fractions which were identified as H1, H3, H2B, H2A, and H4 histones, and their apparent isoelectric points were determined. The main fractions were further divided into several subfractions, the maximal number of bands being 12. The isoelectric point for H1 histone in 6.25 m urea solution in the presence of a nitrogen atmosphere was 8.90, and the corresponding values for H3, H2B, H2A, and H4 histones were 9.80, 9.90, 10.10, and 10.25, respectively. The focusing technique described here has a high resolution, reproducibility, and sensitivity. The technique can be used for preparative and quantitative analysis and for studies on specificity and developmental changes of 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.     

H1° histones of normal and cancer human cells

Summary H1° histones were purified by preparative sodium dodecyl sulfate polyacrylamide gel electrophoresis from human lung carcinoma (line DMS79), human hepatoblastoma (HepG2), human adult lung and human adult and fetal liver. The purified human H1° histones were analyzed for their amino acid composition and terminal residues. The comparative analysis of the amino acid compositions of the different human H1° histones showed that: (a) all the H1° preparations have the characteristically high lysine content associated with a low arginine content, which distinguishes outer histones from core histones; (b) H1° is distinguishable from other H1 histones by the presence of methionine and histidine; (c) H1° histones from human adult, fetal and cancer cells are very similar in amino acid composition, and in cancer cells the level of the H1° histone is not inversely related with cell growth rate nor with the expression of the -fetoprotein gene.     

Core histones and linker histones are imported into the nucleus by different pathways

Histones are the major structural proteins in eukaryotic chromosomes. This group of small very basic proteins consists of the H1 linker histones and the core histones H2A, H2B, H3 and H4. Despite their small size, the nuclear import of histones occurs by an active transport mechanism and not simply by diffusion. Histones contain several nuclear localisation signals (NLS) that can be subdivided into two different types of signal structures. We have previously shown that H1 histones are transported by a heterodimeric import receptor complex consisting of importin beta and importin 7, and we now describe the receptors required for the import of the core histones. Competition experiments using the in vitro transport assay indicate that the import pathway of the core histones differs from that of the linker histones and of nuclear proteins with classical NLS. In vitro binding assays show that each of the import receptors importin beta, importin 5, importin 7 and transportin, has the capacity to bind to any of the four core histones. Reconstitution experiments with recombinant factors indicate that each of these factors can independently serve as an import receptor for each of the core histones.     

Primary organization of nucleosomes containing all five histories and DNA 175 and 165 base-pairs long

The sequential arrangement of histones along DNA in nucleosomes containing all five histones and DNA about 165 and 175 base-pairs in length has been determined. The data provide evidence that core histones (H2A, H2B, H3 and H4) are arranged in nucleosomes and nucleosome core particles in a largely similar way with the following differences. (1) On nucleosomal DNA about 175 basepairs long core histones are probably shifted by 20 nucleotides on one DNA strand and by 10 nucleotides on the complementary DNA strand from the 5′ end. On nucleosomal DNA 165 base-pairs long, histones appear to be shifted by 10 nucleotides from the 5′ end of DNA on both the DNA strands. (2) Histone H3 is extended beyond core DNA and is bound to the 3′ end of DNA about 175 nucleotides long. Thus, core histones span the whole length of nucleosomal DNA. (3) Histone H2A seems to be absent from the central region of nucleosomal DNA. These results indicate that during the preparation of core particles, some rearrangement of histones or some of their regions occurs.Histone H1 has been shown to be bound mainly to the ends of nucleosomal DNA and, along the whole DNA length, to the gap regions that are free of core histones.     

Recognition and classification of histones using support vector machine.

Histones are DNA-binding proteins found in the chromatin of all eukaryotic cells. They are highly conserved and can be grouped into five major classes: H1/H5, H2A, H2B, H3, and H4. Two copies of H2A, H2B, H3, and H4 bind to about 160 base pairs of DNA forming the core of the nucleosome (the repeating structure of chromatin) and H1/H5 bind to its DNA linker sequence. Overall, histones have a high arginine/lysine content that is optimal for interaction with DNA. This sequence bias can make the classification of histones difficult using standard sequence similarity approaches. Therefore, in this paper, we applied support vector machine (SVM) to recognize and classify histones on the basis of their amino acid and dipeptide composition. On evaluation through a five-fold cross-validation, the SVM-based method was able to distinguish histones from nonhistones (nuclear proteins) with an accuracy around 98%. Similarly, we obtained an overall >95% accuracy in discriminating the five classes of histones through the application of 1-versus-rest (1-v-r) SVM. Finally, we have applied this SVM-based method to the detection of histones from whole proteomes and found a comparable sensitivity to that accomplished by hidden Markov motifs (HMM) profiles.     

Chromatin from two classes of platyhelminthes display both protist H1 and higher eukaryote core histones

Histones from the parasitic platyhelminthes, Echinococcus granulosus and Fasciola hepatica, were systematically characterized. Core histones H2A, H2B, H3 and H4, which were identified on the basis of amino acid sequencing and mass spectrometry data, showed conserved electrophoretic patterns. Histones H1, identified on the basis of physicochemical properties, amino acid composition and amino acid sequencing, showed divergence, both in their number and electrophoretic mobilities, between the two species and among other organisms. According to these data, core histones but not H1 histones, would be stabilized during evolution at the level of platyhelminthes.     

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.     

In vitro exchange of nucleosomal histones H2a and H2b

We have asked whether exogenous, radiolabeled histones can exchange with nucleosomal histones in an in vitro system. Using two different electrophoretic techniques, we were able to separate the histones contained in nucleosomes from those histones which were simply bound to the surface of the chromatin. Fluorography was used to determine which of the exogenous histones exchange with the nucleosomal histones. We observed substantial exchange of histones H1, H2a, and H2b when the chromatin and exogenous histones were incubated under approximately physiological conditions. We have also observed a small amount of exchange of H2a and H2b, as well as a substantial exchange of H1, from one chromatin fragment to another. Other conditions affecting the exchange of histones H2a and H2b are also reported.     

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.     

Prothymosin alpha interacts with free core histones in the nucleus of dividing cells

The acidic protein prothymosin alpha (ProTalpha), with a broad presence in mammalian cells, has been widely considered to have a role in cell division, through an unrevealed mechanism in which histones may be involved in view of their ability to interact with ProTalpha in vitro. Results of co-immunoprecipitation experiments presented here demonstrate that ProTalpha interacts in vivo with core histones in proliferating B-lymphocytes (NC-37 cells). This interaction occurs with histones H3, H2A, H2B and H4 located free in the nucleoplasm, whereas no interaction was detected with histone H1, mono-nucleosome particles or chromatin. Moreover, the core histones form part of a nuclear multiprotein complex of about 700 kDa separated by ProTalpha-Sepharose affinity, with components including H3 and H4 acetyltranferases, H3 methyltransferases, hnRNP isotypes A3, A2/B1 and R, ATP-dependent and independent DNA helicases II, beta-actin and vimentin, all co-purifying by gel filtration. This indicates that the interaction of ProTalpha with core histones in the nucleus may be related to the structural modification of histones H3 and H4, and hence to chromatin activity, raising the possibility that the other proteins in the nuclear complex may play a role in this process.     

The ubiquitinated histone species are enriched in histone H1-depleted chromatin regions

Bovine thymus and trout testis chromatin were fractionated into regions which differed in their micrococcal nuclease accessibility and solubility properties, and the distribution of the ubiquitinated histone species among these chromatin regions was elucidated. Ubiquitinated (u) species of histones H2A and H2B were enriched in the nuclease-sensitive, low-ionic-strength, soluble fraction of both chromatins. These results indicate that the presence of ubiquitinated histones may alter nucleosome-nucleosome interactions and destabilize higher-order chromatin structures. Bovine thymus chromatin was separated into aggregation-resistant, salt-soluble and aggregation-prone, salt-insoluble chromatin fractions. The aggregation-resistant chromatin fraction depleted in H1 histones was enriched in uH2A and uH2B, with uH2B showing the greater enrichment. The chromatin fragments were also stripped and reconstituted with the H1 histones prior to fractionation. The results were the same as above: uH2A and uH2B were preferentially localized in the aggregation-resistant. H1-depleted chromatin fraction, suggesting that chromatin regions enriched in ubiquitinated histone species have a reduced affinity for the H1 histones. Thus, ubiquitinated histone species may be one of the contributing factors in the differential assembly of various parts of the genome.     

Nucleosome assembly in vitro: separate histone transfer and synergistic interaction of native histone complexes purified from nuclei of Xenopus laevis oocytes.

High speed supernatants of Xenopus laevis oocyte nuclei efficiently assemble DNA into nucleosomes in vitro under physiological salt conditions. The assembly activity cofractionates with two histone complexes composed of the acidic protein N1/N2 in complex with histones H3 and H4, and nucleoplasmin in complex with histones H2B and H2A. Both histone complexes have been purified and their nucleosome assembly activities have been analysed separately and in combination. While the histones from the N1/N2 complexes are efficiently transferred to DNA and induce supercoils into relaxed circular plasmid DNA, the nucleoplasmin complexes show no supercoil induction, but can also transfer their histones to DNA. In combination, the complexes act synergistically in supercoil induction thereby increasing the velocity and the number of supercoils induced. Electron microscopic analysis of the reaction products shows fully packaged nucleoprotein structures with the typical nucleosomal appearance resulting in a compaction ratio of 2.8 under low ionic strength conditions. The high mobility group protein HMG-1, which is also present in the soluble nuclear homogenate from X. laevis oocytes, is not required for nucleosome core assembly. Fractionation experiments show that the synergistic effect in the supercoiling reaction can be exerted by histones H3 and H4 bound to DNA and the nucleoplasmin complexes alone. This indicates that it is not the synchronous action of both complexes which is required for nucleosome assembly, but that their cooperative action can be resolved into two steps: deposition of H3 and H4 from the N1/N2 complexes onto the DNA and completion of nucleosome core formation by addition of H2B and H2A from the nucleoplasmin complexes.