Isolation and characterization of histones from Anopheles albimanus Weidemann

1. Histones from Anopheles albimanus adults were prepared by a combination of techniques including chromatin isolation and selective extractions. 2. The anopheline histones were identified on acid urea gels by comparing their electrophoretic profile with that of calf thymus histones and histones isolated from other tissue. 3. Excellent separation of histones was obtained after the extractions by a single electrophoretic run. 4. In addition to the five major classes of histones found in eukaryotes, a sixth class was detected and tentatively identified as histone H5. 5. This is the first report of histone H5 and its function in insects.     

Fragmentation of chromatin by endogenous nucleases, accumulation of the subnucleosomal fragments with high electrophoretic mobility]

Digestion of chromatin by endogenous nucleases to nucleosomes (140-160 base pairs of DNA) is accompanied by the accumulation of subnucleosomal DNP particles with high electrophoretic mobility (20-40 base pairs of DNA). All histones associate with the 140-160 base pairs fragment. The production of subnucleosomal DNP particles does not correlate with the degradation of histone H1 and the appearance of nucleosomes lacking histone H1. Degradation of the protein in this fragment is accompanied by the appearance of free DNA. The data obtained are in agreement with the hypothesis on the origin of subnucleosomes from the nucleosomal locus preferentially associated with the non-histone proteins and on the autonomy of these loci and of the loci associated with histone H1 in the nucleosome.     

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.     

Histones from Saccharomyces cerevisiae

Total histones and histone fractions isolated from Saccharomyces cerevisiae chromatin were analysed by polyacrylamide gel electrophoresis. The presence of the four histone fractions H2a, H2b, H3 and H4 was demonstrated. In addition, yeast chromatin contained a protein similar to histone H1 from mammals in molecular weight, charge and association properties with Triton X-100. However, it had a much lower lysine to arginine ratio, equal to about 3, as compared with H1 histones from higher eukaryotes. The order of electrophoretic mobilities of yeast histone fractions in acidic urea-polyacrylamide gels was similar to that observed for histones from plant sources, i.e. H4>H3>H2a>H2b>H1. Previously undetected protein (protein X) was extracted from yeast chromatin with 5 % HClO₄. The properties of this protein are under investigation.     

Modulation of action of wheat seedling endonucleases WEN1 and WEN2 by histones

Wheat core histones and various subfractions of histone H1 modulate differently the action of endonucleases WEN1 and WEN2 from wheat seedlings. The character of this modulation depends on the nature of the histone and the methylation status of the substrate DNA. The modulation of enzyme action occurs at different stages of processive DNA hydrolysis and is accompanied by changes in the site specificity of the enzyme action. It seems that endonuclease WEN1 prefers to bind with protein-free DNA stretches in histone H1-DNA complex. The endonuclease WEN1 does not compete with histone H1/6 for DNA binding sites, but it does compete with histone H1/1, probably for binding with methylated sites of DNA. Unlike histone H1, the core histone H2b binds with endonuclease WEN1 and significantly increases its action. This is associated with changes in the site specificity of the enzyme action that is manifested by a significant increase in the amount of low molecular weight oligonucleotides and mononucleotides produced as a result of hydrolysis of DNA fragments with 120–140-bp length. The WEN2 endonuclease binds with histone-DNA complexes only through histones. The action of WEN2 is increased or decreased depending on the nature of the histone. Histone H1/1 stimulated the exonuclease activity of WEN2. It is supposed that endonucleases WEN1 and WEN2, in addition to the catalytic domain, should have a regulatory domain that is involved in binding of histones. As histone H1 is mainly located in the linker chromatin areas, it is suggested that WEN2 should attack DNA just in the chromatin linker zones. As differentiated from WEN2, DNA hydrolysis with endonuclease WEN1 is increased in the presence of core histones and, in particular, of H2b. Endonuclease WEN1 initially attacks different DNA sites in chromatin than WEN2. Endonuclease WEN2 activity can be increased or diminished depending on presence of histone H1 subfractions. It seems that just different fractions of the histone H1 are responsible for regulation of the stepwise DNA degradation by endonuclease WEN2 during apoptosis. Modulation of the action of the endonucleases by histones can play a significant role in the epigenetic regulation of various genetic processes and functional activity of genes.     

Characteristics of limited trypsinolysis of histone H5 in a solution and as a component of chromatin with different degrees of compactness

Trypsinolysis of histone H5 in solution and as a component of chromatin with different level of compactization was studied. It was demonstrated that the existence of supernucleosomal organization leads to a significant decrease of the degradation rate of histones H1 and H5 in comparison with histones H2A, H2B, H3 and H4. Analysis of trypsinolysis electrophoretic spectra of histone H5 revealed the existence of protease-resistant fragments in chromatin, but not in solution. These fragments contain not only the globular domain of histone H5 but also small-sized unstructured N- and/or C-terminal regions. The peptides were identified with the help of an immune serum specific for the globular region of histone H5. The possible role of resistant fragments in the nucleosomal organization of chromatin is discussed.     

Separation of nucleosomes containing histones H1 and H5

Hen erythrocyte chromatin was digested with staphylococcal nuclease and fractionated by electrophoresis in polyacrylamide gels. Instead of the three bands described for mouse carcinoma chromatin, four main discrete components (MN1, MN2, MN2E and MN3) were resolved in the mononucleosome fraction of erythrocyte chromatin. MN2 contained all five histones and a DNA fragment of 165–180 base pairs. MN2E comprised four nucleosomal histones plus histone H5 (but not H1) and a DNA fragment of 170–190 base pairs. The relatively nuclease resistant MN3 fraction of erythrocyte nucleosomes contained H1 but no H5 histone. A more accurate analysis of the MN2 fraction in mouse carcinoma nucleosomes revealed some additional microheterogeneity depending on the presence of two different subfractions of H1.     

Characterization of histones and chromatin of the hepatopancreas in Palaemon serratus (Crustacea Natantia)]

The chromatin of shrimp hepatopancreas has been extracted from isolated nuclei and characterized. Nuclei were prepared in the presence of Cu++ and phenyl methyl sulfonyl fluoride in order to inhibit the nuclease and protease activities throughout the different purification steps. The purified nuclei are heterogenous in size and show a density of 1,367 g/ml determined on saccharose - glucose gradients. After washing in 0,14 M NaCl and then in 10(-2) M Tris-HCL, pH = 7,6, the nuclei were disrupted in water. The solubilized chromatin was precipitated in 0,15 M.NaCl. This chromatin is characterized by a high level of RNA (RNA/DNA = 0,38) and of non histone proteins (NHP/DNA = 0,6). The denaturation curve showed only one Tm at 69 degrees in 2.10(-4) M.EDTA. When the chromatin was extracted in the presence of staphylococcal nuclease, the Tm reached 80 degrees C. The kinetics of the digestion by the staphylococcal nuclease have been studied and show that 10 per cent of hydrolysis occurs within the first minute. The repeat length of DNA as determined with the polymers of higher order is 189 +/- 5 base pairs. The existence of nucleosomes was confirmed by electron microscopy. The superstructure of chromatin was not completely destroyed after solubilisation with a Potter. The histones were studied by gel electrophoresis after differential staining. The most important feature consists in the presence of two H1, two H2A and two H4. The acetylation levels of the histones were followed after injection of 14C-acetate in vivo. The subfraction H1, 0 was acetylated. Only one H3 was present and the two H2A fractions showed the same level of acetylation. H2B migrated faster than the H2A fractions like in Echinoderms. The two H4 fractions corresponded to two differently acetylated forms. Shrimp hepatopancreas histones were fractionated by molecular sieving on Biogel P 100 and characterized according to their electrophoretic properties as well as their amino-acid content. The amino-acid compositions of the different histone fractions were nearer to Echinoderm and Sipunculid histones, than Calf thymus homologue histones. All the fractions show a weaker basicity. The H3 fraction was the only one showing a lesser variability when compared to Calf thymus H3. The non histone proteins were extracted in 10(-2) M Tris-HCL, pH = 8 and 0.1 per cent SDS. A series of 50 proteins was detected. 80 per cent of the total amount of protein was localized in a molecular weight range comprised between 40 000 and 80 000 daltons. These proteins were compared to the histones and total proteins of sonicated chromatin solubilized by SDS in order to detect proteasic effects.     

Trypanosoma cruzi histones. Further characterization and comparison with higher eukaryotes

Histones extracted from T. cruzi chromatin were analyzed in three electrophoretic systems. Our results show that a basic protein with some properties similar to those of histone H1 from higher eukaryotes is present in T. cruzi. However this protein presents different electrophoretic mobilities than H1 histone from higher eukaryotes in all three electrophoretic systems tested. Considering the marked differences observed in the electrophoretic mobilities of T. cruzi histones as compared with those from higher eukaryotes, it is proposed that histones are conservative proteins primarily with regard to their function.     

Presence of histones in Aspergillus nidulans

Five major histone proteins have been extracted from chromatin isolated from purified nuclei of the fungus, Aspergillus nidulans. These proteins had chromatographic properties which were similar to reference calf thymus histones and were purified to electrophoretic homegeneity by gel chromatography of Bio-Gel P10, Bio-Gel P60, and Sephadex G-100. Electrophoresis of these proteins in three different systems (urea- starch, urea-acetic acid polyacrylamide, and discontinuous SDS polyacrylamide) showed that the A. nidulans histones H3 and H4 were nearly identical to calf thymus H3 and H4 with respect to net charge and molecular weight criteria, whereas the fungal histones H1, H2a and H2b were similar but not identical to the corresponding calf thymus histones. Amino acid analysis of A. nidulans histones H2a, H2b, and H4 showed them to be closely related to the homologous calf thymus histones. The mobility patterns of A. nidulans ribosomal basic proteins in three different electrophoretic systems were distinctly different from those of the fungal histones.     

Influence of chromatin structure, antibiotics, and endogenous histone methylation on phosphorylation of histones H1 and H3 in the presence of protein kinase A in rat liver nuclei in vitro

In vitro phosphorylation of histones H1 and H3 by cAMP-dependent protein kinase A and endogenous phosphokinases in the presence of [γ-³²P]ATP was studied in isolated rat liver nuclei with different variants of chromatin structural organization: condensed (diameter of fibrils 100–200 nm; N-1) and partly decondensed (diameter of fibrils ～30 nm; N-2). In the N-1 state histone, H1 is phosphorylated approximately twice as much than histone H3. Upon the decondensation of the chromatin in the N-2 state, 1.5-fold decrease of total phosphorylation of H1 is observed, while that of H3 does not change, although the endogenous phosphorylation of both histones is reduced by half. Changes in histone phosphorylation in the presence of low or high concentrations of distamycin and chromomycin differ for H1 and H3 in N-1 and N-2. It was found that distamycin (DM) stimulates the phosphorylation of tightly bound H1 fraction, which is not extractable by polyglutamic acid (PG), especially in N-1. Chromomycin (CM) increases the phosphorylation of both histones in PG extracts and in the nuclear pellets, particularly in N-2. At the same time, in N-1 one can detect phosphorylation of a tightly bound fraction of histones H1 whose N-termini are located on AT-rich sites that become inaccessible for protein kinase in the process of chromatin decondensation in N-2. At the same time, in N-2 the accessibility for protein kinase A of tightly bound H1 fractions, whose N-termini are located on GC-rich sites, increases dramatically. High concentrations of both CM and DM in N-1 and N-2 stimulated phosphorylation of the non-extractable by PG fraction of H1 whose N-termini are located on sites where AT ≈ GC. CM at high concentration stimulated 4–7 times the phosphorylation of a small fraction of H3, which is extracted by PG from both types of nuclei. We detected an effect of endogenous methylation of histones H1 and H3 in the nuclei on their subsequent phosphorylation depending on the chromatin structure, histone-chromatin binding strength, and concentration of DM.     

Qualitative differences in nuclear proteins correlate with neuronal terminal differentiation

1. Protein composition of neuronal nuclei was studied at two stages of brain maturation, i.e., before (embryonic day 16; E16) and after (postnatal day 10; P10) shortening of the nucleosomal repeat length. Glial nuclei were analyzed in parallel as a control. 2. Total nuclear or HCl- and 5% perchloric acid (PCA)-soluble proteins were analyzed by different electrophoretic techniques. 3. Our results show an increase in the concentration of histone H1 zero with differentiation, although the H1 class undergoes an overall decrease. 4. The chromatin of mature neurons is also enriched in the ubiquinated form of histone H2A (A24), while the high-mobility group (HMG) proteins 1 and 2 seem to decrease slightly relative to core histones. 5. Both quantitative and qualitative differences in the abundance of nonhistone proteins relative to histones accompany neuronal terminal differentiation.     

A thermodynamic model for Nap1-histone interactions

The yeast nucleosome assembly protein 1 (yNap1) plays a role in chromatin maintenance by facilitating histone exchange as well as nucleosome assembly and disassembly. It has been suggested that yNap1 carries out these functions by regulating the concentration of free histones. Therefore, a quantitative understanding of yNap1-histone interactions also provides information on the thermodynamics of chromatin. We have developed quantitative methods to study the affinity of yNap1 for histones. We show that yNap1 binds H2A/H2B and H3/H4 histone complexes with low nm affinity, and that each yNap1 dimer binds two histone fold dimers. The yNap1 tails contribute synergistically to histone binding while the histone tails have a slightly repressive effect on binding. The (H3/H4)(2) tetramer binds DNA with higher affinity than it binds yNap1.     

Structure of chromatin from the pigeon brain. I. Composition, electrophoretic motility and circular dichroism spectra of nucleosome particles

Composition and structural properties of pigeon brain hemisphere neuron chromatin have been studied. In these cells nucleosomal DNA repeat length is about 165 nucleotide pairs. The content of H1 and H2A histones was found to decrease by 18 and 30% respectively in comparison with the chromatin possessing the normal quantity of those histones. At the same time the content of protein uH2A (A-24), being the conjugate of H2A histone and ubiquitine, is increased. Mononucleosomes isolated from neuron chromatin was found to have relatively low electrophoretic mobility in polyacrylamide gel taking into account the size of their DNA fragment. Circular dichroism spectra of nucleosome particles show that the neuron mononucleosomes are more unfolded than the rat thymus ones. Data obtained allow to suggest that the short DNA repeat and accumulation of protein uH2A in neurons are the factors influencing the compactization of neuron chromatin.     

The sites of deposition of newly synthesized histone.

The chromosomal fragments produced by nuclease digestion of freshly replicated chromatin migrate more rapidly relative to bulk chromatin when analyzed in nucleoprotein gels. The cause of the anomalous migration has been studied and the evidence indicates that rather than reflecting a shorter nucleosomal repeat in vivo that it may be a consequence of nucleosome sliding during the digestion itself. The distinct electrophoretic characteristics of nucleosomal material containing newly replicated DNA have enabled us to examine their histone composition by two dimensional electrophoresis. We find that nucleosomes containing new DNA also contain newly synthesized histones H3 and H4. In contrast more than 50% of newly synthesized H2A and H2B, and essentially all of new H1, are deposited at sites on the bulk chromatin distinct from that material containing newly replicated DNA. In addition we show that newly synthesized histones H3 and H4 are bound unusually weakly when they first become associated with the chromatin.     

Accessibility of histone oligomers to the action of trypsin in a solution or in chromatin with different degrees of compactness

The accessibility to trypsin of "core" histones within the dimer (H2A-H2B), tetramer (H3-H4)2, octamer (H2A-H2B-H3-H4)2 and in chromatin was studied. It was shown that the hydrolysis of histones H2A and H2B within the dimer and octamer occurs in essentially the same way. The tetramer (H2-H4)2 becomes more compact with an increase in the ionic strength. Some of the tetramer (H3-H4)2 sites within the octamer are protected against trypsin. It was demonstrated that in terms of the histone accessibility to trypsin chromatin can exist in three states, i.e., tightly packed (in the presence of histone H1 and bivalent cations), intermediate (in the absence of histone H1 or bivalent cations) and folded (in the absence of histone H1 and bivalent cations). The folding of histones in neither of these chromatin states coincides with that within the octamer in 2M NaCl.     

Comparative electrophoretic properties of histones from trout and chicken erythrocytes and calf thymus at different concentrations of EDTA]

An introduction of EDTA into an electrophoretic system was found to cause specific changes in the histone distribution patterns. The electrophoretic mobility of histones H3, H2b and H2a from three evolutionally unrelated sources (trout and chicken erythrocytes and calf thymus) is increased and that for histones H1 and H5 is decreased with respect to histone H4. In general the decrease of electrophoretic mobility of the histones in the presence of EDTA is correlated with the content of basic amino acids in these histones. The effect observed can be used from electrophoretic analysis of histones.