Histone Fractions Research Articles

Histone H1 and chromatin interactions in human fibroblast nuclei after H1 depletion and reconstitution with H1 subfractions.

Linker histones constitute a family of lysine-rich proteins associated with nucleosome core particles and linker DNA in eukaryotic chromatin. In permeabilized cells, they can be extracted from nuclei by using salt concentration in the range of 0.3 to 0.7 M. Although other nuclear proteins are also extracted at 0.7 M salt, the remaining nucleus represents a template that is relatively intact. A cytochemical method was used to study the affinity of reconstituted linker histones for chromatin in situ in cultured human fibroblasts. We also investigated their ability to condense chromatin by using DNA-specific osmium ammine staining for electron microscopy. Permeabilized and H1-depleted fibroblast nuclei were suitable for the study of linker histone-chromatin interactions after reconstitution with purified linker histone subfractions. Our results showed that exogenous linker histones bind to chromatin with lower affinity than the native ones. We detected no significant differences between the main H1 and H1 degrees histone fractions with respect to their affinity for chromatin or in their ability to condense chromatin. Linker histone interactions with chromatin are controlled also by mechanisms independent of linker histone subtype composition.

Effects of Cadmium and Ionizing Radiation on Histones in Rat Testes

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Involvement of histone phosphorylation in apoptosis of human astrocytes after exposure to saline solution

We have previously found using inhibitors of protein phosphatase that phosphorylation of histones may be involved in thymocyte apoptosis. In this study, we examined whether histone modification occurs in astrocyte apoptosis induced by a pathological condition in the absence of drug. Incubation of cultured human astrocytes with growth medium for 24 h after exposure to saline solution for 30 min induced an increase in terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL)-positive cells and nuclear condensation, biochemical and morphological hallmarks of apoptotic cell death. Acetic acid–urea–Triton X-100 (AUT) gel electrophoresis of the nuclear histone fraction and N-terminal peptide analysis showed that the treatment with saline solution caused rapid changes in phosphorylation of H2A subfamilies, but not in histone acetylation. The phosphorylation of the two subtypes increased markedly, whereas the phosphorylation of one subtype decreased. In contrast, exposure to ACF-95, an artificial cerebrospinal fluid (CSF), was associated with little induction of apoptotic cell death and induced less changes in histone phosphorylation. These results support the previous idea that chemical modification of histones is involved in the DNA fragmentation in astrocytes undergoing apoptosis.

Effect of cadmium and ionizing radiation on histones in rat brain

Changes in constituent part of the chromatin, histones, were studied in brain of rats 1-21 days after administration of cadmium (3 mg CdCl 2 /kg i. p.) and/or whole body irradiation (3 Gy of γ-rays). In the brain hemispheres, increase in content of histones and histone/DNA ratio was found at some intervals after administration of cadmium alone or in combination with irradiation. In course of the first 2 weeks, relative proportion of individual histone fractions altered especially after combined treatment. At later intervals, in irradiated rats, relative proportion of H1 0 subfraction increased in H1 fraction. In the cerebellum, the changes of histones were more profound than in the brain hemispheres. Due to decrease in histone content, the histone/ DNA ratio decreased in the cerebellum of rats of all experimental groups. The most marked decrease in histone/DNA ratio occurred after combined treatment (Cd+lr). The changes in relative proportion of individual histone fractions were detectable from the 7th day in all groups of experimental rats, most conspicuously in the group Cd+lr. The proportion of H1 0 subtraction in the H 1 histone fraction increased mainly on the 7th day after treatments. In general, character of histone alterations induced by administration of cadmium was similar to that induced by γ-irradiation. In majority of cases, after combined treatment (cadmium administered 30 min before irradiation), the alterations of histones were more profound than after single treatment. This finding suggested that partial summation of the 2 factor effects occurred after the combined treatment.

Isolation of the chromocenter fraction from mouse liver nuclei.

A new method for isolation of the constitutive heterochromatin (chromocenters) from interphase nuclei of mouse liver has been developed. This method allows separation of chromocenters of different size. Chromocenter fractions are essentially free of nucleoli and other contaminants. In contrast to nuclei and nucleoli, the chromocenter fraction is characterized by simpler protein composition, this fraction having a reduced number of proteins (especially high molecular weight proteins). Chromocenters contain all histone fractions; however, the relative proportion of histone H1 is lower and histone H3 is higher than in the total nuclear chromatin. The amount of non-histone proteins of 51, 63, 73, and 180 kD is higher in the chromocenter fraction than in nuclei and nucleoli. The use of immunocytochemistry and immunoblotting methods revealed the presence of the specific kinetochore component, CENP A protein. This suggests tight association of some molecular kinetochore components with chromocenters in the interphase.

Relative content of cytokines in different tissues in mycosis fungoides.

The concentrations of tumor necrosis factor-alpha, interleukin-1beta, interleukin-2, interleukin-4, and the content of antibodies to H1 histone fraction in peripheral blood lymphocytes, epidermis, and serum were evaluated in patients with mycosis fungoides. It was found that as the disease progresses, cytokine synthesis is switched from Th1 to Th2 class and production of proinflammatory cytokines increases, which indirectly promotes the development of the autoimmune reaction.

Proteasomal degradation of oxidatively damaged endogenous histones in K562 human leukemic cells

A number of antitumor drugs act via the oxidation of nuclear material in the tumor cell. It is therefore important to know if tumor cells can effectively and precisely cope not only with oxidatively induced DNA damage, but also with nuclear protein oxidation. In this study, we investigated the endogenous degradation of oxidatively damaged histones in K562 human leukemic cells after oxidative challenge and demonstrated a link to the overall cellular stress response pathways by poly-ADP-ribose-polymerase (PARP). After an oxidative challenge, endogenous nuclear protein degradation, as well as histone degradation, was enhanced. Among the histone fractions, histone H1 revealed the highest degradation rate, and more than 85% of the total degraded H1 disappeared in the first 30 min after oxidative challenge. Short-term degradation of histones up to 30 min, as well as long-term degradation up to 48 h after oxidative challenge, was significantly reduced in the presence of the PARP inhibitor 3-aminobenzamide, and nearly completely abrogated by the selective proteasome inhibitor lactacystin. Immunoprecipitation experiments indicated that the proteasome specifically degraded oxidized histones. Thus, we show that the nuclear proteosome system in tumor cells is capable of preventing the accumulation of oxidized proteins in this compartment and may suggest further treatment strategies to effectively interfere with the protein “repair” and replacement strategies of tumor cells.

Prevalence and Antigen Specificity of Anti-Histone Antibodies in Patients with Polymyositis/Dermatomyositis

Anti-histone antibodies have been detected in the sera of patients with various autoimmune diseases. The existence of anti-histone antibodies in patients with polymyositis/dermatomyositis, however, has not been reported. We found anti-histone antibodies in eight (17%) of 46 sera from patients with polymyositis/dermatomyositis by an enzyme-linked immunosorbent assay. One serum was positive for both IgG anti-histone antibodies and IgM anti-histone antibodies. Six sera were positive only for IgG anti-histone antibodies. One serum was positive only for IgM anti-histone antibodies. An indirect immunofluorescence analysis using HEp-2 cells as the substrate showed that all sera positive for anti-histone antibodies produced homogeneous nuclear fluorescence. This immunofluorescence pattern disappeared after absorption of anti-histone activity with total histones. An immunoblotting analysis demonstrated that the anti-histone antibodies were predominantly directed against histone H1 in all seven sera with IgG anti-histone antibodies. Weak reactivity with H2B and H4 were also found in three sera from the patients with polymyositis/dermatomyositis. Sera from two patients with polymyositis/dermatomyositis displayed anti-H2A and H3 activity. One of the two sera showed IgM anti-histone antibodies in the enzyme-linked immunosorbent assay reacted with H1, H2A, H2B, H3, and H4, whereas the other serum reacted with no fractions of total histones. The activity of anti-histone antibodies disappeared in immunoblotting after absorption with total histones. All of the patients with anti-histone antibodies were free from lung fibrosis or internal malignancies. Thus, our data indicate that the presence of anti-histone antibodies is classified as one of the serologic abnormalities observed in polymyositis/dermatomyositis.

Effect of Ptu Treatment on Histone Acetylation Pattern in the Developing Rat Brain

The effect of hypothyroidism induced m female rats on histone acetylation pattern m the neonatal rat brain was studied. It is likely that thyroid hormone regulates the acetylation of histones and thereby influence their interaction with DNA and modulates transcription. Propylthiouracil (PTU), administered to induce hypothyroidism, resulted in a significant reduction m the thyroid and brain weight of neonatal rats. The circulating thyroxine levels were undetectable in both 14 and 21 day old pups. The hypothyroid condition was further confirmed by low levels of T4 (94.31 ng/g brain tissue vs 1811.29 ng/g in controls and 144.67 ng/g vs 1087.72 ng/g in controls at 14 and 21 days, respectively) and T3 (42.19 ng/g brain tissue vs 879.97 ng/g in controls and 60.62 ng/g vs 766.68 ng/g in controls at 14 and 21 days, respectively) in the neonatal rat brain. Histone acetylation pattern was similar in treated and control groups m the 14 day old rats. PTU treatment, however, resulted in significant (p < 0.01) reduction in acetylation in the H3 fraction at 21 days whereas no such changes were recorded in other histone fractions. Lower histone acetylation in the 21 day old pups suggest a reduction m the transcriptional activity due to fewer initiation sites for RNA polymerase. It may be concluded that thyroid hormone may stimulate transcription of specific genes by increasing the acetylation of nucleosomal histones.

Influence of DNA Binding on the Degradation of Oxidized Histones by the 20S Proteasome

The 20S proteasome is localized in the cytosol and nuclei of mammalian cells. Previous work has shown that the cytosolic 20S proteasome is largely responsible for the selective recognition and degradation of oxidatively damaged cytosolic proteins. Since nuclear proteins are also susceptible to oxidative damage (e.g., from metabolic free radical production, ionizing radiation, xenobiotics, chemotherapy) we investigated the degradation of oxidatively damaged histones, in the presence and in the absence of DNA, by the 20S proteasome. We find that both soluble histones and DNA-bound histones are susceptible to selective proteolytic degradation by the 20S proteasome following mild oxidative damage. In contrast, more severe oxidative damage actually decreases the proteolytic susceptibility of histones. Soluble H1 showed the highest basal and maximal absolute proteolytic rates. Histone fraction H4 exhibited the greatest relative increase in proteolytic susceptibility following oxidation, almost 14-fold, and this occurred at a peroxide exposure of 5 mM. At the other end of the spectrum, histone H2A exhibited a maximal proteolytic response to H2O2of only 6-fold, and this required an H2O2exposure of 15 mM. An oxidation of reconstituted linear DNA plasmid–histone complex makes up to 95% of the histones bound to DNA susceptible to degradation, whereas undamaged protein–DNA complexes are not substrates for the proteasome. Severe oxidation by high concentrations of H2O2appears to decreases the proteolytic susceptibility of histones due to the formation of cross-linked histone–DNA aggregates which appear to inhibit the proteasome. We conclude that the degradation of nuclear proteins is highly selective and requires prior damage of the substrate protein, such as that caused by oxidation.

High-sensitivity mass spectrometry for analysis of posttranslational modifications.

Pulsed fast atom bombardment ionization (pulsed-FAB) mass spectrometry has been developed to improve the sensitivity of tandem mass spectrometry (MS/MS), allowing it to be used for the analysis of very small samples. MS/MS, when used with a magnetic four-sector instrument coupled with the pulsed-FAB system, allows significant enhancement in product ion intensity of over ten-fold in magnitude over conventional FAB. MS/MS was applied to the structural analysis of a unique nuclear protein, designated p28, which was isolated from a histone fraction obtained from starfish testes. The results clearly show that protein p28 is a heterodimer composed of testicular histones H2B and H4 which are cross-linked between Gln9 of H2B and Lys5 of H4.

Induction of renal nuclear oxalate binding activity in experimental hyperoxaluric rats.

The hyperoxaluric rat kidney nucleus exhibited a 50% increase in oxalate binding activity of control in both the residual fraction containing nuclear envelopes and the histone fraction with a concomitant increase in basal lipid peroxidation and a decrease in thiol content. However, in vitro lipid peroxidation induced by the ascorbate-ADP-Fe3+ system increased the oxalate binding activity of the residual fraction with a positive correlation but inhibited the histone oxalate binding activity with a negative correlation with depletion of thiols during peroxidation in both control and hyperoxaluric rats. A twofold increase in oxalate concentration was observed in the nucleus as well as the nuclear subfractions in hyperoxaluria. Hyperoxaluric rat kidneys showed increased H1 and oxalate binding activity, and the distribution of H1B was higher than that in the control. The present study suggests that the increased nuclear oxalate binding activity in hyperoxaluric rats was not due to lipid peroxidation but due to increased formation of histone H1.

Apoptosis: a mechanism of cell killing and lymphoid organ impairment during acute African swine fever virus infection.

Induction of programmed cell death has been described during infection with many different viruses. We have investigated the influence of African swine fever virus (ASFV) on apoptosis of different cell populations during in vitro and in vivo infection. We observed apoptosis in ASFV-infected monocyte/macrophage and peripheral blood mononuclear cell cultures. Apoptosis was demonstrated in these cells by DNA fragmentation, DNA staining and DNA-associated histone fraction detection assays. Flow cytometry analysis of infected cultures also showed morphological and functional alterations, including changes in the cell cycle and percentage of cell fractions stained with propidium iodide. After in vivo infection with three different virulent strains of ASFV, apoptosis of infected cells from the mononuclear phagocytic system and closely related elements from different tissues was observed. Additionally, infected pigs showed an intense degree of apoptosis of lymphocytes, which are not infected by the virus. In lymph nodes and other lymphoid organs, broad bands of apoptotic cells presented typical nuclear changes under light microscopy. The occurrence of DNA fragmentation was confirmed in these tissues using terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling. These findings, together with the pathological observations in infected pigs of a depletion in cell populations in lymphoid organs, suggest that virus interference with programmed cell death plays a central role in pathogenesis of this disease, being responsible for lymphoid organ impairment in acute ASFV infection.

Separation of acetylated core histones by hydrophilic-interaction liquid chromatography

Hydrophilic-interaction liquid chromatography (HILIC) has recently been introduced as a highly efficient chromatographic technique for the separation of a wide range of solutes. The present work was performed with the aim of evaluating the potential utility of HILIC for the separation of posttranslationally acetylated histones. The protein fractionations were generally achieved by using a weak cation-exchange column and an increasing sodium perchlorate gradient system in the presence of acetonitrile (70%, v/v) at pH 3.0. In combination with reversed-phase high-performance liquid chromatography (RP-HPLC) we have successfully separated various H2A variants and posttranslationally acetylated forms of H2A variants and H4 proteins in very pure form. An unambiguous assignment of the histone fractions obtained was performed using high-performance capillary and acid-urea—Triton gel electrophoresis. Our results demonstrate that for the analysis and isolation of modified core histone variants HILIC provides a new and important alternative to traditional separation techniques and will be useful in studying the biological function of histone acetylation.

Steady-state two-dimensional maps of very alkaline proteins in an immobilized pH 10–12 gradient, as exemplified by histone types

Two-dimensional steady-state patterns of histones are here reported for the first time. The first dimension run consists in nonlinear immobilized pH gradients, spanning the pH 10–12 range. The second dimension run is a standard SDS-PAGE in a constant concentration (15% T) gel slab, in presence of a 6% T stacking gel. All the histone fractions analysed (II-AS, VI-S, VII-S and VIII-S) exhibit p I values between pH 11 and 12. The M r values range from 13 to 32 kDa, with the heaviest distribution around 18 kDa. When running all different histone fractions in a single mixture, and analysing the 2-D gel by computerized image data acquisition, a total of 35 individual spots is detected.

Occurrence of histone-related oxalate binding in rat liver nucleus.

The rat liver nuclear oxalate binding protein was isolated, purified by anion and cation exchange column chromatography using Diethyl Amino Ethyl Sephadex, Carboxy Methyl Cellulose and Carboxy Methyl Sephadex C-50 ion exchangers. The purified oxalate binding protein was found to be H1B of H1 fraction of histones. Kinetic analysis of oxalate binding showed the presence of two affinity sites, one with Kd of 133.5 nM and Bmax of 40 pmoles and another with Kd of 262.5 nM and Bmax of 210 pmoles. The optimal oxalate binding was at pH 4.2 and at 28 degrees C. The oxalate binding was specific and reversible and not due to ionic charge interaction. The IC50 of other dicarboxylates was higher than that of oxalate. EGTA had no effect on oxalate binding but di- and tri-carboxylate carrier inhibitors and thiol modifying agents significantly lowered the binding activity. Oxalate binding to histones was significantly reduced in the presence of DNA or nucleotides, but RNA had no effect. ATP completely inhibited the oxalate binding activity at 1 mM concentration. Different tissues exhibited oxalate binding showing ubiquitous nature. Calf thymus H1 showed maximal binding similar to liver histones.

Mapping of B-Cell Epitopes Recognized by Antibodies to Histones in Subsets of Juvenile Chronic Arthritis

The sera of 138 patients with juvenile chronic arthritis (JCA) were tested in ELISA with the five individual histories, 34 histone peptides covering the full length of the four core histones, and two peptides corresponding to the N- and C-terminal domains of H1. The occurrence of IgG antibodies was examined regarding the different subsets of JCA (pauciarticular, polyarticular, and systemic onset) and regarding clinical features (chronic anterior uveitis, CAU) and other serological features (antinuclear antibodies, ANA). Seventy-two percent of the 138 sera reacted with at least 1 histone peptide. The peptides 204-218 of H1, 1-25 of H2B, and 111-130 of H3 were recognized by 22-28% of JCA sera, and 42% of JCA sera reacted with one or both peptides 1-25 of H2B and 111-130 of H3. The frequency of occurrence of anti-histone antibodies (AHA) regarding the type of histone fraction (H1, H2A, H2B, H3 and H4) or the regions of histones was not significantly different in the three subsets of JCA. No obvious association was found between IgG antibodies to histone peptides and uveitis. In the subset of pauciarticular JCA, 13/31 patients (41.9%) with CAU against 14/41 patients (34.1%) without CAU possessed IgG antibodies reacting with peptides of the C-terminal domain 83-135 of H3. This difference is not statistically significant. Finally, the presence of antibodies to histones and histone peptides cannot completely explain ANA reactivity found in patients' sera. Although antibodies to histone peptides occur frequently in the serum of children with JCA, the antibody profiles seem to be highly individual and do not correlate with disease subtype or activity. Identification of AHA present not only in the circulation but also in tissue deposits may provide better insight into the identification of pathogenic antibodies.

Effect of aging and gamma radiation on acetylation of rat liver histones

We studied age-related and radiation induced changes in histone acetyltransferase activity and acetylation of histone fractions in normal and regenerating rat livers. Male Wistar rats aged 1–28 months were used through the studies. They were exposed to whole-body doses of 5.7 Gy gamma radiation from a 60Co source and partial hepatectomy was carried out 30 min after irradiation. Within nuclei isolated from normal and regenerating livers of rats, acetyltransferase activity of histones increased with age. An age-dependent decrease in histone acetyltransferase activity in normal and regenerating rat livers was also obvious at the time of maximum activity (i.e. at the 4th min of incubation). The radiation-induced drop in acetyltransferase activity was mild in normal livers. Twenty-four hours after partial hepatectomy, acetyltransferase activity in regenerating livers was almost completely inhibited by irradiation. The acetylation of histone fractions and subfractions was not equal. The highest activity among all histone fractions was found in fraction H4. In regenerating livers of non-irradiated and irradiated rats, there was a greater rate of acetylation in tetraacetylated subfractions of histone H4 compared with intact livers.

Oncogenic ras stimulates a 96-kDa histone H2b kinase activity in activated Xenopus egg extracts. Correlation with the suppression of p34cdc2 kinase.

We previously showed that purified, bacterially expressed oncogenic human rasH protein blocks or delays the progression of the embryonic cell cycle into M-phase in activated Xenopus egg extracts. This block correlates with the suppression of the activation of p34cdc2 kinase (Pan, B.-T., Chen, C.-T., and Lin, S.-M. (1994) J. Biol. Chem. 269, 5968-5975). In an attempt to identify kinases that are involved in mediating the effect of oncogenic Ras on the cell cycle, we assayed aliquots of activated Xenopus egg extracts, which were incubated at 25 degrees C for various times in the absence and presence of oncogenic Ras, for their kinase activities toward a calf thymus histone fraction (Hf1). We find that the suppression of the histone H1 kinase activity of p34cdc2 by oncogenic Ras correlates with a simultaneous stimulation of a histone H2b serine kinase activity. Using a histone H2b in-the-gel kinase assay, we further show that the stimulated histone H2b kinase activity is attributed mainly to a 96-kDa kinase and slightly to p42mapk. Although Xenopus p90rsk is also activated by oncogenic Ras, we demonstrate that activated p90rsk is not responsible for the 96-kDa histone H2b kinase activity. The identity of the 96-kDa kinase remains unclear. Our data suggests that the 96-kDa kinase may be involved in mediating the effect of oncogenic Ras on the embryonic cell cycle of Xenopus.

Histone acetyltransferase is associated with the nuclear matrix.

Only a small fraction of the adult chicken erythrocyte histones is involved in dynamic acetylation. We have reported previously that the rapidly acetylated and deacetylated H4 histones are primarily associated with the transcriptionally active DNA-enriched chromatin fragments that remain attached to the residual nuclear material following micrococcal nuclease digestion and chromatin solubilization. Furthermore, this nuclear fraction contained most of the histone deacetylase activity. In this study we show that the bulk of the nuclear histone acetyltransferase activity is located with the insoluble residual nuclear material. We demonstrate that in vitro the enzymes associated with the residual nuclear material catalyze reversible acetylation when the endogenous histones of the nuclear skeleton-bound chromatin fragments are used as substrate. Nuclear matrices isolated from adult chicken immature erythrocyte and trout liver nuclei had 60-76% of the nuclear histone acetyltransferase activity. Procedures that solubilized the internal nuclear matrix also resulted in the release of the enzyme from the nuclear matrix. Together, our observations suggest that histone acetyltransferase and deacetylase are associated with the internal nuclear matrix, and one of the functions of these enzymes may be to mediate a dynamic attachment between transcriptionally active chromatin and the nuclear matrix.