Regulation of nucleosomal core histone variant levels in differentiating murine erythroleukemia cells

During hexamethylenebis(acetamide)-induced terminal differentiation of murine erythroleukemia (MEL) cells in vitro, the histone variant proportions undergo changes similar to those observed in vivo in terminally differentiating cells of the young mouse. Thus, there is a rapid increase in the relative amounts of the variants H2A.1 and H2B.2 in parallel with the increase in the number of hemoglobin-producing cells and the sharp decrease in the growth rate. We show that the changes in variant proportions are not associated with slower growth per se but are most likely due to differential changes in the rates of variant synthesis as a result of commitment to terminal differentiation. In addition, we observed an inducer-specific increase in the rate of synthesis and the relative amount of the minor H2A variant 4, well before hemoglobin accumulation. We also present evidence that H2A and H2B histones are synthesized and incorporated into chromatin at a significant rate even when DNA synthesis is inhibited, suggesting turnover of these histones. H2A and H2B turnover can be detected directly even in exponentially growing cells. H2A.1 and H2B.2 have higher turnover rates than H2A.2 and H2B.1, respectively, in exponentially growing cells, a difference which is even more pronounced in induced cells. The magnitude of the differential turnover is not sufficient to account for the changes in the histone variant proportions in the short life of induced MEL cells but could explain the slow accumulation of H2A.2, H2B.1, and H3.3 in nondividing adult tissues of the mouse.     

Synthesis of nucleosomal histone variants during wheat grain development

The synthesis and distribution of histone subfractions (variants) were investigated during early grain development and in mature tissues of wheat (Tritium aestivum L.). Histones were extracted from purified chromatin and separated by two-dimensional polyacrylamide gel electrophoresis. There were no detectable differences in the patterns of histone variants from immature grain (3–16 days after fertilization), from mature embryos, from coleoptiles and roots of 4-day-old, etiolated seedlings and from leaves of 10-day-old, light-grown seedlings. Wheat H2 histones are composed of families of closely related variants. H2A consists of three major variants, and H2B consists of two major and four minor variants. The synthesis of these variants during early grain formation was determined by calculating the specific activities of the [³H]lysinelabeled proteins synthesized between 3 and 10 days after fertilization. The rate of synthesis of the nucleosomal histones closely parallels the declining rate of cell division in developing grains. Our results indicate that all the recognized wheat histone variants are present in developing wheat grains from the earliest time investigated (3 days after fertilization) and persist with no detectable changes in relative quantities throughout grain development and in several mature tissues.     

Separation of histones by reverse-phase high-performance liquid chromatography: analysis of the binding of carcinogens to histones

Reverse-phase high-performance liquid chromatography (RP-HPLC) has been examined as an approach to the rapid analysis of carcinogen-modified histones. H1 and core histone fractions were prepared by differential acid extraction of 0.35 M NaCl-extracted rat liver nuclei previously exposed to [3H]-7r,8t-dihydroxy-9t, 10t-oxy-7,8,9, 10-tetrahydrobenzo(a)pyrene [( 3H]BPDE-I). Using a sodium perchlorate-phosphate (PCP)/acetonitrile solvent system, the H1 histone fraction was eluted from an Aquapore RP-300 column in five peaks (P1-P5). The core histone fraction was resolved into eight peaks (C1-C8) using a PCP/acetonitrile-methanol solvent system. The histones of each peak were identified by sodium dodecyl sulfate and Triton/acid/urea gel electrophoresis or amino acid analysis as follows: P1, H1 degrees; P2-P5, four different H1 variant fractions; C1, H4 + A24; C2, H2B; C3, H2A X 2 + to one H2A variant; C4, H2A.1; C5, H2A.1 + two H2A variants; C6, H3.2; C7, H3.3; C8, H3.1. The bulk of radioactivity was covalently bound to histone H2A, which had higher specific activities of BPDE-I than other histones. Significant amounts of radioactivity were observed in histones H3 and H1, but not in histones H2B and H4. These RP-HPLC systems have the advantages of an analysis time within 60 min, the identification of H1, H2A, and H3 variants, and the quantitative analysis of radioactive histones. These results indicate that these RP-HPLC systems are very useful to analyze the binding of carcinogens to histones.     

The human histone H3 complement anno 2011

Histones are highly basic, relatively small proteins that complex with DNA to form higher order structures that underlie chromosome topology. Of the four core histones H2A, H2B, H3 and H4, it is H3 that is most heavily modified at the post-translational level. The human genome harbours 16 annotated bona fide histone H3 genes which code for four H3 protein variants. In 2010, two novel histone H3.3 protein variants were reported, carrying over twenty amino acid substitutions. Nevertheless, they appear to be incorporated into chromatin. Interestingly, these new H3 genes are located on human chromosome 5 in a repetitive region that harbours an additional five H3 pseudogenes, but no other core histone ORFs. In addition, a human-specific novel putative histone H3.3 variant located at 12p11.21 was reported in 2011. These developments raised the question as to how many more human histone H3 ORFs there may be. Using homology searches, we detected 41 histone H3 pseudogenes in the current human genome assembly. The large majority are derived from the H3.3 gene H3F3A, and three of those may code for yet more histone H3.3 protein variants. We also identified one extra intact H3.2-type variant ORF in the vicinity of the canonical HIST2 gene cluster at chromosome 1p21.2. RNA polymerase II occupancy data revealed heterogeneity in H3 gene expression in human cell lines. None of the novel H3 genes were significantly occupied by RNA polymerase II in the data sets at hand, however. We discuss the implications of these recent developments.     

Modulation of histone H3 variant synthesis during the myoblast-myotube transition of chicken myogenesis

We have previously reported that nucleosomal histones are synthesized by cultured, postmitotic myotube cells at 9-29% of the rate in their dividing myoblast precursors (A. M. Wunsch, A. L. Haas, and J. Lough, 1987, Dev. Biol. 119, 85-93). In that study, histones were separated by two-dimensional polyacrylamide gels containing 8 M urea in the first-dimension to optimally separate variants of the H2A class. To separate and compare synthesis of variants in the H2B and H3 classes during myogenesis, 5.75 M urea has been used in the first dimension. Although no changes in the H2B variant pattern were discerned, a dramatic change in H3 variant synthesis was detected, in which a predominance of H3.2 synthesis in dividing myoblasts was almost completely replaced by a lower level of H3.3 synthesis after myotube formation. With increasing differentiation, H3.2 synthesis became undetectable, while H3.3 synthesis continued. Control experiments indicated that these results were not mediated by replicating cells in the myotube cultures, the effects of cytosine arabinoside, or contaminating non-histone proteins. These results suggest that histone H3.2 is replaced by histone H3.3 in nucleosomes during skeletal muscle maturation.     

Dynamic histone acetylation in alfalfa cells. Butyrate interference with acetate labeling

Dynamic histone acetylation of alfalfa (Medicago sativa) was studied in suspension cultures by short-term labeling with radioactive acetate. The relative labeling rates for the acetylated histones were in order of decreasing incorporation; H3.2 greater than H3.1 greater than H4 greater than H2B.1 greater than H2A.3. Histone H3 showed at least seven sites of acetylation, histone H2B.1 had six sites and histone H4 had five sites. Low numbers of acetylation sites were observed for histone H2B.2 and all histone H2A variants. The mass ratio, steady state acetylation and dynamic acetylation between major variant H3.1 and minor variant H3.2 were approx. 2:1, 1:2 and 2:5, respectively. Treatment of alfalfa cells with 50 mM n-butyrate did not lead to histone hyperacetylation, but instead interfered with histone acetylation labeling by acetate. The extent of apparent inhibition increased with time and concentration of butyrate. It is likely that the conversion of butyrate to acetylCoA results in dilution of the specific radioactivity of [3H]acetate in the acetylCoA pool thereby inhibiting the labeling reaction. This interpretation is supported by 14C-labeling of alfalfa acetylated histones by [1-14C]butyrate.     

技术与方法体外组装含有组蛋白变体H2A.Z及H3.3的核小体

核小体是构成真核生物染色质的基本结构单位,组蛋白变体H2A.Z及H3.3对染色质结构及基因转录过程发挥着重要的调控作用。体内研究核小体及染色质结构受到诸多因素限制,体外重构含有H2A.Z及H3.3的核小体结构是研究与组蛋白变体相关基因表达调控的重要方法之一。实验表达纯化了6种组蛋白,在复性的过程中装配了含有H2A.Z和H3.3的组蛋白八聚体。基于DNA序列10bp周期性及序列模体设计了3条易于形成核小体的DNA序列,通过PCR大量扩增的方法,回收了标记Cy3荧光分子的目的DNA序列。采用盐透析法体外组装了含有H2A.Z和H3.3的核小体结构,利用荧光标记、EB染色及考马斯亮蓝染色检测了含有组蛋白变体的核小体形成效率及形成过程的吉布斯自由能变化。结果发现,设计的3条DNA序列可以有效地组装形成含有组蛋白电梯的核小体结构,而且随着组蛋白八聚体与DNA比例的增加,核小体的形成效率显著提高;采用Cy3荧光标记可以灵敏且定量地计算组装过程的吉布斯自由能。该方法的建立对研究组蛋白变体相关的结构生物学及转录调控等具有一定的意义。     

Expression and purification of recombinant human histones

Nucleosomes reconstituted from bacterially expressed histones are useful for functional and structural analyses of histone variants, histone mutants, and histone post-translational modifications. In the present study, we developed a new method for the expression and purification of recombinant human histones. The human histone H2A, H2B, and H3 genes were expressed well in Escherichia coli cells, but the human histone H4 gene was poorly expressed. Therefore, we designed a new histone H4 gene with codons optimized for the E. coli expression system and constructed the H4 gene by chemically synthesized oligodeoxyribonucleotides. The recombinant human histones were expressed as hexahistidine-tagged proteins and were purified by one-step chromatography with nickel-nitrilotriacetic acid agarose in the presence of 6 M urea. The H2A/H2B dimer and the H3/H4 tetramer were refolded by dialysis against buffer without urea, and the hexahistidine-tags of the histones in the H2A/H2B dimer and the H3/H4 tetramer were removed by thrombin protease digestion. The H2A/H2B dimer and the H3/H4 tetramer obtained by this method were confirmed to be proficient in nucleosome formation by the salt dialysis method. The human CENP-A gene, the centromere-specific histone H3 variant, contains 28 minor codons for E. coli. A new CENP-A gene optimized for the E. coli expression system was also constructed, and we found that the purified recombinant CENP-A protein formed a nucleosome-like structure with histones H2A, H2B, and H4.     

Isolation and characterization of TH3, a germ cell-specific variant of histone 3 in rat testis

We have identified and purified TH3, a germ cell-specific histone. It has been characterized by amino acid analysis, tryptic peptide mapping, labeling with cystine, and by electrophoretic mobility as a variant of H3. On fully reduced Triton/acid/urea gels its mobility is retarded more than that of the somatic variants H3.2 and H3.3, but less than that of H3.1; it migrates between the H2As and H1s. Germinal cells from adult and sexually immature testes were purified by centrifugal elutriation followed by Percoll density gradient separation in order to study the distribution and synthesis of TH3. TH3 is found in significant levels in spermatogonia and in similar or slightly higher amounts in spermatocytes and round spermatids. The synthesis of TH3 takes place in the spermatogonia but not in spermatocytes, in contrast to the other testis-specific histones, TH2A, H1t, and TH2B. Therefore, TH3 may have a different role in spermatogenesis than do the other testis-specific histone variants.     

Sites of in vivo histone methylation in developing trout testis.

Specific lysyl residues of trout testis histones H3 and H4 are methylated partially during rainbow trout spermatogenesis. Histones H1, H2A, H2B, and protamine are not methylated. The single site (lysine 20) in histone H4 and the two major sites (lysines 9 and 27) in histone H3 are homologous to those determined for other organisms, but an additional minor site (lysine 4) occurs in histone H3. As described for calf thymus, both histones H3 and H4 contain epsilon-N-mono- and dimethyllysine, while histone H3 contains in addition, epsilon-N-trimethyllysine. The trout-specific histone H6, which accounts for 0.5 to 1.0% of total histone, contains a sequence for residues 3 to 5,-Arg-Lys-Ser-, which is the same as one methylated in histones H3, at lysines 9 and 27. However, histone H6 yields only trace amounts of [3H]methyl incorporation and no detectable methyllysines on amino acid analysis.     

Isolation and characterization of the histone variants in chicken erythrocytes.

Chicken erythrocyte histones 2A, 2B, and 3 can be resolved into nonallelic primary structure variants by polyacrylamide gel electrophoresis in the presence of Triton X-100. These variants were isolated and characterized by analysis of their tryptic and thermolytic peptides. The major variants of chicken H2A and H2B differ from the analogous component of calf thymus by a small number of conservative amino acid substitutions in the basic terminal regions, which interact with DNA. This moderate rate of allelic evolution of the slightly lysine-rich histones contrasts with the complete conservatism found in the arginine-rich histones. Chicken H4 and both chicken H3 variants are identical with their corresponding components in mammals. The amino acid substitutions distinguishing histone variants are located within the highly conserved hydrophobic regions, which are involved in histone--histone interactions.     

Degradation of sperm histones in vitro by cytoplasm of the sea urchin egg

Sperm-specific histone variants in the sea urchin Paracentrotus lividus are replaced early after fertilization with a specific embryonic set of histone variants. A possible in vitro model for the involvement of a degradation mechanism in the replacement of sperm-specific histones is presented. Soluble sperm histones are shown to be degraded quickly by egg cytoplasm. The proteolytic activity is maximal at pH 3.0; H1 and H2A histones are the most sensitive while H3 and H4 are the most resistant. H2B histones have an intermediate sensitivity. Histone degradation by egg cytoplasm or by purified fractions of it can be inhibited by chymostatin and leupeptin and, to a lesser degree, by pepstatin.     

Constitutive expression, not a particular primary sequence, is the important feature of the H3 replacement variant hv2 in Tetrahymena thermophila.

Although quantitatively minor replication-independent (replacement) histone variants have been found in a wide variety of organisms, their functions remain unknown. Like the H3.3 replacement variants of vertebrates, hv2, an H3 variant in the ciliated protozoan Tetrahymena thermophila, is synthesized and deposited in nuclei of nongrowing cells. Although hv2 is clearly an H3.3-like replacement variant by its expression, sequence analysis indicates that it evolved independently of the H3.3 variants of multicellular eukaryotes. This suggested that it is the constitutive synthesis, not the particular protein sequence, of these variants that is important in the function of H3 replacement variants. Here, we demonstrate that the gene (HHT3) encoding hv2 or either gene (HHT1 or HHT2) encoding the abundant major H3 can be completely knocked out in Tetrahymena. Surprisingly, when cells lacking hv2 are starved, a major histone H3 mRNA transcribed by the HHT2 gene, which is synthesized little, if at all, in wild-type nongrowing cells, is easily detectable. Both HHT2 and HHT3 knockout strains show no obvious defect during vegetative growth. In addition, a mutant with the double knockout of HHT1 and HHT3 is viable while the HHT2 HHT3 double-knockout mutant is not. These results argue strongly that cells require a constitutively expressed H3 gene but that the particular sequence being expressed is not critical.     

Assembly and disassembly of nucleosome core particles containing histone variants by human nucleosome assembly protein I

Histone variants play important roles in the maintenance and regulation of the chromatin structure. In order to characterize the biochemical properties of the chromatin structure containing histone variants, we investigated the dynamic status of nucleosome core particles (NCPs) that were assembled with recombinant histones. We found that in the presence of nucleosome assembly protein I (NAP-I), a histone chaperone, H2A-Barr body deficient (H2A.Bbd) confers the most flexible nucleosome structure among the mammalian histone H2A variants known thus far. NAP-I mediated the efficient assembly and disassembly of the H2A.Bbd-H2B dimers from NCPs. This reaction was accomplished more efficiently when the NCPs contained H3.3, a histone H3 variant known to be localized in the active chromatin, than when the NCPs contained the canonical H3. These observations indicate that the histone variants H2A.Bbd and H3.3 are involved in the formation and maintenance of the active chromatin structure. We also observed that acidic histone binding proteins, TAF-I/SET and B23.1, demonstrated dimer assembly and disassembly activity, but the efficiency of their activity was considerably lower than that of NAP-I. Thus, both the acidic nature of NAP-I and its other functional structure(s) may be essential to mediate the assembly and disassembly of the dimers in NCPs.     

Dramatic replacement of histone variants during genome remodeling in nuclear-transferred embryos

The genome of differentiated somatic nuclei is remodeled to a totipotent state when they are transplanted into enucleated oocytes. To clarify the mechanism of this genome remodeling, we analyzed changes in the composition of core histone variants in nuclear-transferred embryos, since recent evidence has revealed that chromatin structure can be remodeled as a result of variant histone replacement. We found that the donor cell-derived histone H3 variants H3.1, H3.2, and H3.3, as well as H2A and H2A.Z, were rapidly eliminated from the chromatin of nuclei transplanted into enucleated oocytes. Accompanying this removal, oocyte-stored histone H3 variants and H2A.X were incorporated into the transplanted nuclei, while the incorporation of H2A and H2A.Z was minimal or not detected. The incorporation of these variant histones was DNA replication-independent. These results suggest that most core histone H2A and H3 components are dynamically exchanged between donor nuclei and recipient cytoplasm, which further suggests that replacement of donor cell histones with oocyte-stored histones may play a key role in genome remodeling in nuclear-transferred embryos. In addition, the incorporation patterns of all of the histone variants in the nuclear-transferred embryos were virtually the same as in the fertilized embryos. Only the incorporation pattern of H3.1 differed; it was incorporated into the transplanted donor nuclei, but not in the pronuclei of fertilized embryos. This result suggests that the incorporation of H3.1 has a detrimental effect on the process of genome remodeling and contributes to the low success rate of somatic nuclear cloning.