Identification and isolation of core histones from Schizosaccharomyces pombe

Core histones have been isolated from Schizosaccharomyces pombe and compared electrophoretically to core histones from Saccharomyces cerevisiae and rat liver. The molecular masses of all cognate histones examined were found to be very similar as determined by SDS gel electrophoresis. Histones H3, H2A and H2B from Sch. pombe migrated almost identically to their respective counterparts from S. cerevisiae as determined by acid/urea gel electrophoresis. Two-dimensional gel electrophoresis with a Triton X-100 acid/urea gel in the first dimension followed by an SDS gel in the second dimension was used to separate Sch. pombe histones from contaminating ribosomal proteins.     

A rapid and gentle method for the salt extraction of chromatin core histones H2A,H2B,H3 and H4 from rat liver nuclei

A complex of histones H2A, H2B, H3 and H4 has been isolated from purified rat liver nuclei by a method which is both gentle and rapid. Nuclei were homogenised in 0.25 M sucrose and the residual nuclear material obtained after centrifligation was adsorbed on calcium phosphate gel. After removing histone H1 from the adsorbed material by washing with 1M NaCl in 25 mM sodium phosphate buffer, pH 6.0, histones H2A, H2B, H3 and H4 were eluted together, with 2 M NaCl in 25 mM sodium phosphate buffer, pH 7.0. The core histones so obtained migrated as a single sharp band on polyacrylamide gel electrophoresis under non-denaturing conditions. Fractionation of the freshly prepared core histones on a Sephadex G-100 column yielded two major protein peaks. The peak having the larger elution volume contained histones H2A and H2B in equal amounts while the peak with the smaller elution volume contained all the four histones. Histones H3 and H4 were present in larger proportions in the second peak.     

Changes in the histones of the sea urchin Strongylocentrotus purpuratus at fertilization

Both sperm and eggs of the sea urchin Strongylocentrotus purpuratus contain specific histones in place of some of the histones found during later development. Whether these specific histones are lost upon fertilization or are retained is not known. Therefore, we have examined the histones present in the zygote nucleus to determine the fate of the gamete histones. Nuclei of zygotes which have completed DNA replication in preparation for the first mitosis were isolated by sucrose density gradient centrifugation. Histones were extracted from the isolated nuclei, and were analyzed by acid-urea and SDS polyacrylamide gel electrophoreses, and by two-dimensional electrophoresis in which both gel electrophoresis systems were combined. Electrophoretic patterns of the zygote histones were compared with those of sperm, unfertilized eggs and embryos. The results show that the zygote histone pattern is identical with the unfertilized egg histone pattern. Neither the sperm histones H1, H2A, or H2B, nor the embryonic H1, H2A, or H2B, are present in the zygote pattern. The egg and the zygote do contain a unique H2A and H2B, but not an H1. After fertilization, sperm specific histones are not present on the DNA. Egg histones become associated with both the sperm DNA and the newly replicated DNA. The association of the embryonic histones with the DNA, therefore, occurs sometime later in development.     

Reconstitution of chromatin: assembly of the nucleosome.

The order of reassociation of the four histones H2a, H2b, H3 and H4 to the DNA during the reconstitution of chromatin was determined. At each step of the reconstitution the DNA and associated histones were separated from the free histones by centrifugation in a glycerol gradient. The unbound and reassociated histones were analysed by gel electrophoresis and the histone-DNA complexes characterized by circular dichroism and electron microscopy. We show that H3 and H4 bind first to the DNA between 1.2 M NaCl and 0.85 M NaCl and impose a nucleosome like structure; in a second step histones H2a and H2b are placed around this kernel to complete the nucleosome.     

Electrophoretic separation of histones and high-mobility-group proteins on acid-urea-Triton gels

The electrophoretic mobilities of calf thymus histones and high-mobility-group (HMG) nonhistone proteins were studied on a newly modified polyacrylamide gel containing acetic acid, urea, and the nonionic detergent Triton X-100 in combination with glycine in the electrode buffer. This gel system avoids stacking gel, photopolymerization of acrylamide, and preelectrophoresis. Under extremely low Triton concentrations some H3 variant forms (H3.1) were preferentially separated by their slower migration from bulk H3. Under increasing concentrations of Triton in the gel in the presence of 3 or 6 M urea, the mobilities of H2A.1, H3.2, H2A.2, H4, and H2B were sequentially retarded. The mobilities of H1 and HMGs remained virtually unchanged under all conditions. This gel system is able to resolve charge-modified histones.     

Soluble acidic complexes containing histones H3 and H4 in nuclei of Xenopus laevis oocytes

Oocyte nuclei of Xenopus laevis contain nucleosomal-core histones in large amounts and in a soluble, non-chromatin-bound form. Supernatant fractions (100,000 X g) from isolated nuclei are enriched in complexes containing histones H3 and H4, which are of distinct size (5.6S by sucrose gradient centrifugation, approximate molecular weight of 270,000 by gel filtration) and negatively charged (isoelectric at pH 4.4). These complexes bind to DEAE-Sephacel and can be separated from nucleoplasmin. In diverse fractionation experiments, histones H3 and H4 have been found to comigrate with a pair of polypeptides of molecular weight 110,000 that represent the most acidic major protein present in these nuclei. After enrichment by gel filtration, ion exchange chromatography and electrophoresis, this pair of acidic polypeptides has been the only nonhistone protein detected in the histone-complex fraction. We suggest that in the oocyte nucleus, large proportions of the soluble histones H3 and H4 are not contained in complexes of all four nucleosomal-core histones but are differentially associated with specific, very acidic proteins into distinct 5.6S complexes.     

Timing of ubiquitin synthesis and conjugation into protein A24 during the HeLa cell cycle

The phosphorylative modification $\text{in vivo}$ of histones after shortterm (0 to 60 min) isoproterenol stimulation of confluent rat C6 glioma cell cultures has been investigated. Analysis of the phosphorylation patterns after the purification and separation of histones by SDS/polyacrylamide gel electrophoresis revealed significantly increased phosphorylation of histones H1-1 and H3 and a decrease of the phosphorylation of histones H1-3, H2A, and H2B. There was no apparent effect of isoproterenol on the net phosphorylation of histones H1-2 and H4. The data suggest an effect of isoproterenol on the phosphorylative modification of glioma cell histones via modulation of nuclear phosphorylating and dephosphorylating activities.     

Histones of the fungi, Endomyces magnusii and Neurospora crassa]

Histones of Endomyces magnusii and Neurospora crassa were found to consist of four main fractions similar to calf thymus histones in their electrophoretic mobilities, molecular sizes and chromatographic behaviour on Akrilex P-60. Two of them are homologous to the most conservative histones H3 and H4. Other two fractions correspond to the histones H2A and H2B; however, they have some pecularities. A fraction of N. crassa histones corresponding to the H2B was isolated in a homogeneous state by means of gel filtration. It appeared to be very similar to calf thymus histone H2B in its amino acid composition.     

Effects of histone acetylation on nucleosome properties as evaluated by polyacrylamide gel electrophoresis and hydroxylapatite dissociation chromatography

The release of acetylated histones from chick oviduct chromatin was analyzed by hydroxylapatite column chromatography. By raising of the NaCl concentration, acetylated histones were eluted from hydroxylapatite-bound chromatin depending on their release from nucleosomal DNA. Electrophoresis on acid-urea gel showed that hyperacetylated forms of histone H4 were eluted at a lower NaCl concentration than non-acetylated or hypoacetylated H4, suggesting that hyperacetylated H4 has decreased stability in nucleosomes. However, under milder ionic conditions which do not induce dissociation between histones and DNA, polyacrylamide gel electrophoresis of purified nucleosome cores showed no evidence for their unfolding or for increased accessibility by high mobility group protein-17.     

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.     

An Nalpha-acetyltransferase responsible for acetylation of the N-terminal residues of histones H4 and H2A

A yeast gene has been identified that encodes a novel, evolutionarily conserved Nalpha-acetyltransferase responsible for acetylation of the N-terminal residues of histones H4 and H2A. The gene has been named NAT4. Recombinant Nat4 protein acetylated a peptide corresponding to the N-terminal tail of H4, but not an H3 peptide nor the peptide adrenocorticotropin. H4 and H2A are N-terminally acetylated in all species from yeast to mammals and hence blocked from sequencing by Edman degradation. In contrast, H4 and H2A purified from a nat4 mutant were unacetylated and could be sequenced. Analysis of yeast histones by acid-urea gel electrophoresis showed that all the H4 and H2A from the mutant migrated more rapidly than the same histones from a wild type strain, consistent with the histones from the mutant having one extra positive charge due to one less acetylated amino group. A comparison of yeast proteins from wild type and a nat4 mutant by two-dimensional gel electrophoresis showed no evidence that other yeast proteins are substrates of this acetyltransferase. Thus, Nat4 may be dedicated specifically to the N-terminal acetylation of histones H4 and H2A. Surprisingly, nat4 mutants grow at a normal rate and have no readily observable phenotypes.     

Properties of soluble rat brain histone lysine methyltransferase.

Histone-lysine methyltransferase has been solubilized from rat brain chromatin by repeated extraction with distilled water. The enzyme was further purified by chromatography on DEAE-cellulose and gel filtration. With chromosomal-bound histones as substrates, the enzyme methylated only the lysyl residues in histones H3 and H4. The ratio of N epsilon-mono-: N epsilon-di-: N epsilon-trimethyllysine in histone H3 was 1.8:1.0:0.45 and the ratio of N epsilon-mono-: N epsilon-dimethyllysine in histone H4 was 0.7:1.0. The enzyme loses specificity with soluble histones as substrates; however, histones H3 and H4 were still the best methyl acceptors. The pH optima for the enzyme with soluble histones H3 and H4 as substrates were 8.2 to 8.7 and 7.2 to 8.0, respectively. S-Adenosyl-L-homocysteine, one of the products of the reaction, was a competitive inhibitor with respect to S-adenosyl-L-methionine.     

Resolution of basic cellular proteins including histone variants by two-dimensional gel electrophoresis: Evaluation of lysine to arginine ratios and phosphorylation

A procedure is described which resolves histones and other very basic cellular polypeptides from solubilized whole cells by two-dimensional gel electrophoresis. The entry of histones into the gel was apparently quantitative when salt and protamine were added to solubilization buffers containing urea and detergents. Histones and basic polypeptides in the histone region of the gels were identified and characterized by comparison with purified histones and by determining lysine to arginine ratios of individual spots. Phosphorylated derivatives of the nucleosome histones were clearly resolved from stained spots in the charge dimension. Some phosphorylated H1's comgrated with the stained spots and some were retarded in the charge dimension. Acetylated nucleosome histones were nearly coincident with stained spots. The usefulness of this technique for evaluating changes in post-translational modification of histones was illustrated by showing that one H1-like protein increased in ³²P content when C-6 cells were treated with a β-adrenergic agonist.     

The fractionation of histones isolated from Euglena gracilis.

1. The histones of Euglena gracilis were separated by gel filtration into five fractions. 2. Each fraction was characterized in terms of its electrophoretic, solubility and compositional properties. 3. Euglena gracilis clearly contains histones corresponding to vertebrate H1, H2B, H3 and H4 fractions, although they all differ in containing more lysine. 4. The remaining Euglena histone is considered to be homologous to vertebrate histone H2A, but it differs in having a much higher ratio of lysine to arginine. 5. The Euglena histone H1 appears to be lacking in aspartic acid. 6. Electrophoresis in the presence of sodium dodecyl sulphate indicates that the molecular weights of the Euglena histones are close to those of the homologous vertebrate histones.     

Poly ADP-ribosylation of histones in tumor promoter phorbol-12-myristate-13-acetate treated mouse embryo fibroblasts C3H10T1/2

The tumor promoter phorbol-12-myristate-13-acetate (PMA) increases the poly ADP-ribosylation of acid extractable (0.2N H2SO4) nuclear proteins in mouse embryo fibroblasts C3H10T1/2. Catalase suppresses the reaction by approximately 50%. Polyacrylamide gel electrophoresis reveals that the core histones H2B, A24 and H3d serve as major poly ADP-ribose acceptors. Smaller amounts of poly ADP-ribose are associated with histones H2A/H3 and H1. Poly ADP-ribosylation of histones may change the nucleosomal structure and function and play a role in PMA induced modulation of gene expression in promotion.     

ADP-ribosylation regulates the phosphorylation of histones by the catalytic subunit of cyclic AMP-dependent protein kinase

Phosphorylation of whole histones from calf thymus by the catalytic subunit of cyclic AMP-dependent protein kinase was markedly reduced when the histones were ADP-ribosylated. NAD, nicotinamide or free ADP-ribose molecule did not suppress the phosphorylation. Urea gel electrophoretic analyses of the phosphorylated histones which had already been ADP-ribosylated revealed that the suppression of phosphorylation occurred in both H1 and core histones. Therefore, the possibility that ADP-ribosylation may regulate the phosphorylation of histones phosphorylation in nuclei warrants further investigation.     

Thyrotropin stimulation of the phosphorylation of serine in the N-terminal of thyroid H1 histones.

The analysis of thyroid nuclear proteins by polyacrylamide gel electrophoresis has demonstrated that thyrotropin and dibutyryl adenosine 3':5'-monophosphate stimulate specifically the phosphorylation of H1 histones in an intact cell system. This effect does not require new protein synthesis and implicates the phosphorylation of secrine residue(s) situated in the N-terminal part of H1 histones.     

Plant histone acetylation: in the beginning ..

The study of histone acetylation in plants started with protein purification and sequencing, with gel analysis and the use of radioactive tracers. In alfalfa, acid urea Triton gel electrophoresis and in vivo labeling with tritated acetate and lysine quantified dynamic acetylation of core histones and identified the replication-coupled and -independent expression patterns of the histone H3.1 and H3.2 variants. Pulse-chase analyses demonstrated protein turnover of newly synthesized histone H3.2 and thereby identified the replacement H3 histones of plants which maintain the nucleosome density of transcribed chromatin. Sequence analysis of histone H4 revealed acetylation of lysine 20, a site typically methylated in animals and yeasts. Histone deacetylase inhibitors butyrate and trichostatin A are metabolized in alfalfa, but loss of TSA is slow, allowing its use to induce transient hyperacetylation of histones H2B, H4 and H3. This article is part of a Special Issue entitled: Epigenetic Control of cellular and developmental processes in plants.     

Isolation and characterization of histones and other acid-soluble chromosomal proteins from Physarum polycephalum

Chromosomal basic proteins were isolated from amoebal and plasmodial stages of the acellular slime mold Physarum polycephalum. Polyacrylamide electrophoresis on high resolution acid-urea gels separated the five histone fractions in the sequence H1, H2A, H2B, H3, and H4. Under these electrophoretic conditions Physarum histones migrated more like plant (rye) than animal (calf) histones. Furthermore, Physarum histones H1, H2A, and H2B have higher molecular weights on sodium dodecyl sulfate (SDS) gels than the corresponding calf fractions. No differences were detected between amoebal and plasmodial histones on either acid-urea or SDS-polyacrylamide gel electrophoresis. Amoebal basic proteins were fractionated by exclusion chromatography. The five histone fractions plus another major acid-soluble chromosomal protein (AS) were isolated. The Physarum core histones had amino acid compositions more closely resembling those of the calf core histones than of rye, yeast, or Dictyostelium. Although generally similar in composition to the plant and animal H1 histones, the Physarum H1 had a lower lysine content. The AS protein was extracted with 5% perchloric acid or 0.5 M NaCl, migrated between histones H3 and H4 on acid-urea polyacrylamide gels, and had an apparent molecular weight of 15 900 on SDS gels. It may be related to a protein migrating near H1. Both somewhat resembled the high mobility group proteins in amino acid composition.     

组蛋白与转录因子在hAMFR基因启动子序列上的结合及相互作用

采用从鸡红细胞中分离纯化的组蛋白Ｈ１,核心组蛋白Ｈ２Ａ＋Ｈ２Ｂ和Ｈ３＋Ｈ４,以及从ＨｅＬａ细胞中萃取的含有ＲＮＡ聚合酶Ⅱ和多种Ⅱ类基因转录因子的可溶性ＨｅＬａ细胞核抽提物,通过凝胶迟滞电泳,对组蛋白和ＨｅＬａ细胞核抽提物中的转录因子在人自泌移动因子受体（Ｈｕｍａｎａｕｔｏｃｒｉｎｅｍｏｔｉｌｉｔｙｆａｃｔｏｒ,简称ｈＡＭＦＲ）基因上游启动子序列的相互作用关系进行了初步研究,得到以下结论,组蛋白Ｈ１