Turnover of chromatin proteins in rat liver during induction of RNA synthesis by electrostimulation of the hypothalamus

It has been shown that the induction of D-RNA synthesis in rat liver nuclei by electrostimulation of hypothalamus is accompanied by a decrease in chromatin protein synthesis and an increase in phosphorylation and acetylation of chromatin proteins. The decrease of the histone synthesis is mainly due to the decrease of [14C]lysine and [14C]alanine incorporation into histones H1 and H4. The relationship between H1, H2b-H3, H2a and H4 histone fractions remains unchanged. Electrostimulation of hypothalamus increases acetylation of H2a and H4 histone fractions and phosphorylation of all histones with the exception of histone H1.     

Alterations in nicotinamide and adenine nucleotide systems during mixed-function oxidation of p-nitroanisole in perfused livers from normal and phenobarbital-treated rats

When rat liver nuclei were incubated with [adenine-3H]NAD, besides histone 1, histone 2A and especially histone 2B accepted 3H radioactivity. 3H radioactivity was also found on the non-histone proteins and on the small amounts of histones 1 and 3 released into the supernatant during incubation. [14C]Adenine uptake in vivo by liver and thymus nuclei showed radioactivity in histones 1 and 3. After digestion with Pronase and leucine aminopeptidase 14C- or 32P-labelled histone 3 released a serine phosphate-containing nucleotide, which on acid hydrolysis yielded ADP-ribose and serine phosphate. Serine phosphate was also found in the material from the nucleotide peaks from histones 2A and 2B. ADP-ribosylated histones 1 and 3 were more easily released from nuclei than their unmodified forms and showed higher [32P]Pi and [3H]lysine uptakes in vivo [Ord & Stocken (1975) FEBS Meet. Proc. 34, 113-125].     

Nonrandom assembly of chromatin during hydroxyurea inhibition of DNA synthesis

Incubation of MSB-1 chicken lymphoblastoid cells with hydroxyurea leads to a rapid 25-fold decrease in the incorporation of [3H]thymidine into DNA and a 5-fold decrease [3H]lysine into the nucleosome core histones. I have investigated whether the distortion in the normal proportion of histone-DNA synthesis results in alterations in the nucleosome assembly process and find that neither the stoichiometry of new histone synthesis nor the deposition is appreciably changed during hydroxyurea incubation. Protein cross-linking and micrococcal nuclease digestion show that the histones synthesized during hydroxyurea treatment form octamer structures and are assembled into typical nucleosome particles. Minor nucleosome subpopulations are found which exhibit altered sensitivity to nuclease digestion and which are depleted in new histones H3 and H4. When MSB-1 cells incubated in hydroxyurea are pulsed briefly with density-labeled amino acids and [3H]lysine, the radiolabeled core histone octamers formed are as dense as individual monomer histones. These results suggest that the newly synthesized histone octamers are uniformly dense and do not contain mixtures of new and old histones. Thus, histones synthesized during hydroxyurea incubation are deposited nonrandomly and do not exchange with preexisting histones.     

Effect of inhibition of DNA synthesis on histone synthesis, turnover, and deposition in the rat testis

In testicular seminiferous epithelial cells (SEC) of normal and hypophysectomized rats, 1-beta-D-arabinofuranosylcytosine and hydroxyurea (at concentrations which inhibited DNA synthesis nearly completely) inhibited histone synthesis only partially, and to a different extent for each histone fraction. In the presence of the inhibitors, the extent of synthesis relative to the corresponding control was TH1-x greater than H1 greater than TH2B-x = X2 = H2A greater than H2B = H3 greater than H4, in which synthesis of the H4 fraction was about 50% of control and that of TH1-x was 90-95% of control. The extent of inhibition of synthesis of each histone fraction was similar after hypophysectomy and, therefore, the changing of the relative populations of heterogeneous cells in the SEC did not influence the relative effects of the inhibitors of DNA synthesis on the synthesis of the various histone fractions. After [3H]leucine injection, the molar proportions of labeled histones relative to H4 decreased markedly between 1.5 h and 6-15 days; this finding indicated that there was rapid removal of histones compared to the H4 fraction during this period. When [14C]thymidine was injected 24 h prior to hydroxyurea treatment and [3H]leucine injection, the ratios of specific activities of histone H4 to DNA did not change significantly over an 11-day period. It appears that newly synthesized histone H4 and other somatic histones are associated with existing DNA in the presence of DNA inhibitors.     

Biosynthesis of specific histones during meiotic prophase of mouse spermatogenesis

A kinetics study has demonstrated histone synthesis occurring at two distinct phases during meiotic prophase of mouse spermatogenesis. These two periods have been delineated by quantifying the synthesis of DNA and basic nuclear proteins in spermatogenic cells at discrete intervals following the intratesticular injection of [3H] thymidine and [14C] arginine, respectively. One phase of histone synthesis occurs coincident with DNA synthesis in preleptotene spermatocytes. By contrast, a second phase of histone synthesis occurs during midprophase of meiosis, independent of semiconservative DNA synthesis. The [14C] arginine incorporated into the basic nuclear proteins of pachytene spermatocytes is conserved during spermiogenesis and then subsequently discarded within the residual bodies, which are formed during late spermiogenesis. Fluorographic analyses of isotopically labeled basic nuclear proteins in pachytene spermatocytes has shown that only the somatic complement of histones are synthesized during the preleptotene period, whereas the second phase involves the synthesis of proteins H1t, H2S, and "A". In addition, several nonhistone basic nuclear proteins are synthesized concomitant with the germ cell-specific histones. Thus, the data clearly demonstrate that pachytene spermatocytes actively synthesize a number of novel chromatin-associated polypeptides.     

Synthesis and phosphorylation of histone H1 and high mobility group proteins in the regenerating rat liver after X irradiation

A rat was irradiated to the upper abdomen including the liver and then partially hepatectomized. The subsequent synthesis and phosphorylation of histone H1 and nonhistone chromosomal high mobility group (HMG) proteins were investigated. Incorporation of [3H]lysine into histone H1 was increased and reached its peak at 27 h after hepatectomy, and 14 Gy of X rays inhibited the increase. Increase in the incorporation of [3H]lysine into HMG (1 + 2), 14, and 17 which occurred around 27 h after hepatectomy was not inhibited by 14 Gy irradiation. Phosphorylation of histone H1, measured with 32Pi incorporation in vivo, was maximal between 21 and 24 h, and it was inhibited by 4.8 Gy of X rays and delayed with 1.9 Gy. Phosphorylation of HMG 14, which was the only HMG protein phosphorylated under present conditions, was not affected by X irradiation. The [3H]thymidine incorporation into nuclear DNA started increasing at 21 h and reached its maximum at 27 h after hepatectomy. X irradiation with 4.8 Gy inhibited the incorporation, and 1.9 Gy lowered it.     

Nonenzymatic acetylation of histones with acetyl phosphate and acetyl adenylate.

Nonenzymatic acetylation of calf-thymus lysine- and arginine-rich histones was demonstrated to occur when these proteins were incubated with [14C]acetyl phosphate and [14C]acetyl adenylate. The levels of acetylation depend on both pH and on reagent concentration. When acetyl [33P]phosphate and acetyl [3H]adenylate were used as reagents, we found neither histone phosphorylation nor adenylylation. Most of the radioactivity of 14C-labeled acetylated histones was recovered as Ne-acetyllysine. Furthermore, only a small amount of O-bound radioactivity was released by the 14C-labeled acetylated arginine-rich histone during treatment with hydroxylamine. Experiments on the acetylation of histones, in the presence of increasing salt concentration, gave different results for the two acetylating agents.     

Rates of histone synthesis during early development of Rana pipiens

Newly synthesized histones have been extracted from Rana pipiens oocytes or cleaving embryos previously injected with [³H]lysine or [³H]arginine. The radioactive proteins were fractionated by cation-exchange chromatography and electrophoresis on acid/urea or SDS-polyacrylamide gels; histones were identified by coelectrophoresis with authentic markers. From percentage total incorporation in the putative histones, and absolute rates of lysine or arginine incorporation, rates of histone synthesis were estimated. Rates of histone synthesis in two-cell embryos were at least 10-fold higher than in maturing oocytes. Between the two-cell and blastula stages, the rate increased an additional threefold, from about 1200 pg hr^?1 per embryo to about 4500 pg hr^?1 per embryo. While all histone classes are synthesized during cleavage, synthesis of the various classes is not coordinated; histones are not synthesized in the same relative proportions at which they are found in blastula chromatin. The synthesis of histone H4 in particular is barely detectable during cleavage. This, and other observations, suggested the existence of cytoplasmic histone pools. In approaching the possible existence of histone pools, the amount of H4 present in oocytes was determined. Oocytes contain about 74 ng of H4, an amount sufficient to allow development to the blastula stage. These data are compared to those reported by others on histone synthesis during cleavage in Xenopus.     

Isolation, identification, and characterization of histones from plasmodia of the true slime mold Physarum polycephalum using extraction with guanidine hydrochloride

Histones from plasmodia of the true slime mold Physarum polycephalum have been prepared free of slime by an approach to histone isolation that uses extraction of nuclei with 40% guanidine hydrochloride and chromatography of the extract on Bio-Rex 70. This procedure followed by chromatography or electrophoresis has been used to obtain pure fractions of histones from Physarum microplasmodia. Physarum microplasmodia have five major histone fractions, and we show by amino acid analysis, apparent molecular weight on three gel systems containing sodium dodecyl sulfate, mobility on gels containing Triton X-100, and other characterizations that these fractions are analogous to mammalian histones H1, H2A, H2B, H3, and H4. Significant differences between Physarum and mammalian histones are noted, with histone H1 showing by far the greatest variation. Histones H1 and H4 from Physarum microplasmodia have similar, but not identical, products of partial chymotryptic digestion compared with those of calf thymus histones H1 and H4. Labeling experiments, in vivo, showed that histone H1 is the major phosphorylated histone and approximately 15 separate phosphopeptides are present in a tryptic digest of Physarum histone H1. The core histones from Physarum, histones H2A, H2B, H3, and H4, are rapidly acetylated; histone H4 shows five subfractions, analogous to the five subfractions of mammalian histone H4 (containing zero to four acetyllysine residues per molecule); histone H3 has a more complex pattern that we interpret as zero to four acetyllysine residues on each of two sequence variants of histone H3; histones H2A and H2B show less heterogeneity. Overall, the data show that Physarum microplasmodia have a set of histones that is closely analogous to mammalian histones.     

Metabolic activities of histones in rat liver and spleen.

Synthesis and turnover of histone I and II in normal rat liver and spleen were studied by Amberlite CG 50 column chromatography. Histone I was separated into three or four subfractions, each of which showed a different rate of incorporation of [3H]lysine. This was verified by a more shallow gradient chromatography developed by Kinkade and Cole [3] for very lysine-rich histone (F1), which showed tissue specific differences between liver and spleen in both the elution pattern and synthetic rates. These subfractions were distinguished from each other by dodecylsulphate electrophoresis. The turnover, or disassociation of histone I and II in chromatin was measured by double-labelling of normal rat liver with [3H] and [14C]lysine. A good correspondence was found between the synthesis and turnover patterns of individual histone I fractions, while the histone II synthesized was conserved for over a month. From consideration of the turnover in relation to the cell population of normal liver tissue, which consists of a very small fraction of growing cells and a very large fraction of resting ones, it was concluded that turnover of histone I must occur even in resting cells. When DNA synthesis in the spleen was completely inhibited by hydroxyurea, the synthesis of histone II was inhibited but that of histone I was only partially inhibited. The remaining synthesis seemed to occur in cells in the resting state. It was concluded tentatively, the continuous replacement of very lysine-rich histones of chromatin must occur even in resting cells in which DNA synthesis has ceased. The biological significance of disassociation of histones from chromatin was discussed.     

Age-related decline in histone H1 fraction in human diploid fibroblast cultures

The age-related increase in cell volume and nuclear size of cultured human diploid fibroblasts reflected the accumulation of proteins in cytoplasm and nuclei of growth-retarded fibroblasts.Determination of the amount of nuclear proteins, which were fractionated into 0.15 M NaCl-soluble proteins, 0.4 N H₂SO₄-extractable proteins and residual acidic proteins, indicated that age-related increase in nuclear proteins was due mainly to the accumulation of residual acidic proteins.However, electrophoretic fractionation of histones from various passages of fibroblast cultures on acid urea polyacrylamide gel revealed that the relative amount of H1 fraction decreased with in vitro aging. This was further confirmed by mixing experiments examining the distribution of radioactivity of the histones from cell mixtures of young and senescent cultures labeled with [³H]lysine or [¹⁴C]lysine.A pulse label and chase experiment indicated that the observed decrease in the amount of histone H1 was mainly due to decrease in synthesis of histone H1 in senescent human fibroblast cultures.     

Histone synthesis in early amphibian development: histone and DNA syntheses are not co-ordinated

The synthesis of basic proteins has been studied in the oocytes, eggs and embryos of the South African clawed frog, Xenopus laevis. A group of newly synthesized proteins has been identified as histones by the following criteria: solubility properties; incorporation of [³H]lysine and [³H]arginine in the correct proportions, but lack of incorporation of [³H]tryptophan; co-cleotrophoresis with marker histones in various types of polyacrylamide gels, including a type run in two dimensions; peptide analysis of the arginine-rich fraction, F2A1. The four main histone fractions other than F1 were found to be synthesized at all stages of development. F1 histone synthesis was first detected at the late blastula stage.Rates of histone synthesis were estimated for the different stages of development and it was concluded that histone synthesis was not co-ordinated with DNA synthesis either temporally or quantitatively. Histone synthesis was unusual in the following major respects: histones were synthesized in oocytes, and yet in these cells DNA replication had not occurred for several months; histones were synthesized in activated or fertilized eggs at a rate far in excess (about 500 times) of the immediate requirements. We suggest that in order to provide enough histones for the late blastula embryo a store of histone is accumulated during the early cleavage stages and possibly during oogenesis.     

Regulation of histone synthesis during early Urechis caupo (Echiura) development

The histones present in mature oocytes and embryos of Urechis caupo and their pattern of synthesis during early development have been characterized. Acid-soluble proteins extracted from mature oocyte germinal vesicles and from embryonic nuclei were analyzed by two-dimensional polyacrylamide gel electrophoresis. Histones are accumulated in the mature oocytes in amounts sufficient to provide for the assembly of chromatin through the 32- to 64-cell stage of embryogenesis. Two H1 histones, which appear to be variants, were found. Germinal vesicles and cleavage-stage nuclei are enriched in H1M (maternal). During late cleavage a faster-migrating H1, H1E (embryonic), appears among the nuclear histones and, as embryogenesis continues, replaces H1M as the predominant H1. No new core histone variants are detected during early development. Examination of [³H]lysine-labeled histones from germinal vesicles and embryonic nuclei reveals stage-specific patterns of histone synthesis. H1M is the major H1 species synthesized in mature oocytes. After fertilization, a switch to the predominant synthesis of H1E occurs. Comparison of the [³H]lysine incorporated into H1E and core histones indicates that H1E synthesis is disproportionately high from midcleavage through the midblastula stage. By the gastrula stage, a balanced synthesis of H1E and each core histone is established. The results indicate that there is noncoordinate regulation of H1 and core histone synthesis during Urechis development.     

Histone methylation. Its occurrence in different cell types and relation to histone H4 metabolism in developing trout testis.

Histone methylation in developing trout testis has been observed in the diploid stem cells and primary spermatocytes, which actively synthesize DNA and histones. In spermatids, histone methylation is minimal and so probably plays no role in the replacement of histones by protamine which is characteristic of this cell type. No turnover of histone methyl groups could be detected over several hours, so that unlike acetylation or phosphorylation of histones, methylation in this tissue appears to be a stable, irreversible modification. When histone H4, labeled with [14C]methyl groups, is separated on starch gels into acetylated and phosphorylated derivatives, [14C]methyl label does not appear in positions characteristic of newly synthesized histone H4, i.e. the highly acetylated (di-, tri-, and tetra-acetylated), unphosphorylated species. [14C]Methyl label appears rather in the unphosphorylated, and unacetylated or monoacetylated species, shifting with time to the monophosphorylated form of histone H4. These data suggest a temporal sequence of events for histone H4: synthesis, then acetylation and deacetylation, followed by methylation and phosphorylation. Occurring late after histone synthesis and assembly into chromatin, histone methylation might then be necessary for histone interactions with other molecules (e.g. histone phosphokinase) prior to mitosis.     

Studies on the Growth in Culture of Plant Cells: XXIII ISOLATION OF NUCLEI AND HISTONES FROM CULTURED CELLS OF ACER PSEUDOPLATANUS L.

A technique is described for the isolation of purified nucleifrom suspension culture cells of Acer pseudoplatanus. This involvesa grinding medium containing 70% (v/v) glycerol, 1 mM Mg²⁺,2 mM Ca²⁺, and Tris buffer at pH 7.8, prestorage and disruptionof the cells at –20 °C in a Potter-Elvehjem homogenizer,and purification by filtration and centrifugation in the presenceof Triton X-100. The nuclear yield is c. 25% as assessed bynuclear count or DNA estimation and the nuclei are active inthe RNA synthesizing system of Tautvydas (1971). When the histones of these nuclei are extracted in H₂SO₄ andprecipitated by ethanol, 113 µg histone is obtained perµg nuclear DNA and the histone fraction contains 22% basicamino acids and has a lysine: arginine ratio of 2.6. Acid-ureagel electrophoresis shows the presence of five major histones(H1, H2A, H2B, H3, and H4 in sequence from anode to cathode)having respectively molecular weights of 24 500, 13 500, 13300, 12 800, and 11 000. There is very good correspondence betweencalf thymus histones H3 (reduced form) and H4 and two of theseAcer histones. The other Acer histones differ from the calfthymus histones H1, H2A, H2B in molecular weight but can beprovisionally equated with these by a newly developed differentialstaining reaction. Calf thymus histone H2A appears to be lessrich in lysine than the corresponding Acer histone. Evidence from a pulse-chase experiment with (¹⁴C)lysine and[³H]tryptophan is in favour of the cytoplasmic synthesis ofthe histones.     

Uncoordinate synthesis of histone H1 in cells arrested in the G1 phase

It has been known for several years that DNA replication and histone synthesis occur concomitantly in cultured mammalian cells. Normally all five classes of histones are synthesized coordinately. However, mouse myeloma cells, synchronized by starvation for isoleucine, synthesize increased amounts of histone H1 relative to the four nucleosomal core histones. This unscheduled synthesis of histone H1 is reduced within 1 h after refeeding isoleucine, and is not a normal component of G1. The synthesis of H1 increases coordinately again with other histones during the S phase. The DNA synthesis inhibitors, cytosine arabinoside and hydroxyurea, block all histone synthesis in S-phase cells. The levels of histone H1 mRNA, relative to the other histone mRNAs, is increased in isoeleucine-starved cells and decreases rapidly after refeeding isoleucine. The increased incorporation of histone H1 is at least partially due to the low isoleucine content of histone H1. Starvation of cells for lysine resulted in a decrease in H1 synthesis relative to core histones. Again the ratio was altered on refeeding the amino acid. 3T3 cells starved for serum also incorporated only H1 histones into chromatin. The ratio of different H1 proteins also changed. The synthesis of the H1⁰ protein was predominant in G₀ cells, and reduced in S-phase cells. These data indicate the metabolism of H1 is independent of the other histones when cell growth is arrested.     

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.