Histone Synthesis Research Articles

H1 histone subtypes and subtype synthesis switches of normal and delobed embryos of Ilyanassa obsoleta

Histone H1 subtype complexity and H1 histone subtype synthesis switches were characterized during the development of normal embryos of the mud snail Ilyanassa obsoleta. The effect of the removal of the third polar lobe on the normal H1 pattern of synthesis was then investigated in the delobed embryo to determine if classical polar lobe effects are accompanied by a perturbation of these patterns. SDS-gel electrophesis and fluorography of radiolabeled 5% perchloric acid-soluble nuclear extracts resolved six H1 proteins designated bands 1–6. Bands 1–5 migrate as a cluster of individual bands with similar mobilities. Band 6 has a substantially slower mobility. The synthesis of band 6 is predominant during the first 6 hr post-trefoil. During cleavage and gastrulation bands 1 and 2 are predominant while band 3, 4, and 5 become predominant during organogenesis. In addition, it has been found that removal of the polar lobe delays the off-switch of the early bands 6, 1, and 2 and the on-switch of the late bands 3, 4, and 5. This must result in a different H1 composition in the chromatin of the two embryo types. Cell number data of normal and delobed embryos reveal that the delay in subtype synthesis switching is not caused by an overall delay of cell division in the delobed embryo. However, the data indicate that a subpopulation of cells may not divide, or may divide late, in the delayed embryo. The data also suggest that the D cell lineage may be involved in the control of histone synthesis switching in the A, B, and C cell lineages.

H1 histone synthesis and phosphorylation in mouse mammary gland in vitro

The synthesis and phosphorylation of H1 histones were studied in organ cultures of midpregnant mouse mammary glands exposed to various combinations of insulin, cortisol and prolactin over a 48-h period. The synthesis of specific H1 histone subtypes was changed only when all three hormones were present, and the effect was most pronounced during the first 24 h of culture, a period of cell replication. The 3-hormone combination also stimulated the phosphorylation of the N-terminal region of the H1 histone, and this also occurred maximally during the first 24 h of culture. The enhanced phosphorylation of the N-terminal region of the H1 histone included a site sensitive to phosphorylation by cyclic AMP-dependent protein kinase. Thus, hormones which stimulate mammary development in vitro influence the synthesis and specific phosphorylation of H1 histones during a period of cell replication preceding the expression of milk protein genes.

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.

CELL CYCLE AND ORGANELLE ENZYME CHANGES IN THE REGENERATING LIVER OF THE ALCOHOLIC RAT

Chronic ethanol effect on regenerating post-partial hepatectomy rat liver was studied from 0 to 80 hr to evaluate sequential changes. The rate of restoration in liver mass was comparable between controls (C) and ethanol (E) treated rats. Cell cycle changes included a decrease in magnitude and duration of the early peak in DNA synthesis (24 hr). However, the second peak (40 hr) was increased in magnitude so that the net change was negligible. Histone synthesis increased markedly, whereas thymidine kinase activity was moderately increased. The net effect was an accelerated accumulation of DNA (0.18 mg of DNA/g liver/hr vs 0.29 mg of DNA/g liver/hr; C vs E). The M phase of the cell cycle was initially delayed by ethanol, but once initiated, it exhibited greater numbers of mitoses reaching significance at 72 hr. It is concluded that although ethanol induces multiple changes, the overall process of restoring liver mass and cell number (DNA synthesis) is not impaired.

Histone synthesis during the cell cycle of Physarum polycephalum. Synthesis of different histone species is not under a common regulatory control.

The synthesis of histones and nonhistone nuclear proteins was studied during the naturally synchronous cell cycle of Physarum polycephalum. Contrary to the commonly accepted idea of a tight coupling of histone biosynthesis and DNA replication during the somatic cell cycle we found that 40% of total histone synthesis takes place in the G2 period in the complete absence of DNA synthesis. The core histones exhibit a maximum of synthesis during S-phase. The synthesis of histones H2A and H2B continues during the G2 period, but synthesis of H4 and H3 is restricted to the S-phase of the cell cycle. Experiments with hydroxyurea demonstrated that the synthesis of H4 and H3 is completely dependent on unperturbed DNA synthesis, whereas synthesis of H2A and H2B is independent from DNA synthesis during the entire cell cycle. This implicates significant differences between the arginine-rich histones H4 and H3 and the moderately lysine-rich histones H2A and H2B with respect to the control mechanisms of their synthesis, the metabolic stability, and the function for chromatin structure. The nonhistone nuclear proteins are synthesized throughout the cell cycle with a broad maximum in the early G2 period. The cell cycle pattern of synthesis of H1 rather resembles the pattern of the nonhistone proteins than of core histones.

Alterations in amounts and covalent modifications of low-molecular-weight chromosomal proteins in Chinese hamster ovary cells during polyamine depletion

The effect of polyamine depletion on phosphorylation and ADP-ribosylation of low- M r chromosomal proteins was studied in intact, mutant Chinese hamster ovary cells (CHO-P22) devoid of ornithine decarboxylase activity. When starved of polyamines for 6 days, severe polyamine deficiency develops and the cells gradually stop growing. The rate of DNA synthesis was retarded to 16% of the control value and to 29% in density-inhibited cells. The synthesis of high-mobility-group (HMG) proteins was decreased by 65% in polyamine-depleted cells and by 40% in density-inhibited cells. The synthesis of core histones was decreased by 40% both in polyamine-depleted and density-inhibited cells. In polyamine-depleted cells the molar ratio of the higher- M r HMG proteins (HMG 1 + 2) to the lower- M r HMG proteins (HMG 14 + P) was about one-half of that found in cells grown in the presence of putrescine or in density-inhibited cells. In contrast to HMG proteins, no major differences were found in the content of core histones in these cell populations. In the perchloric acid-soluble fraction of nuclear proteins, 32P was incorporated mainly into histone H1, HMG P and a protein migrating more slowly than HMG 1 (protein P1). Specific changes in the 32P-labeling and migration of a number of protein bands, including histone H1, was observed in polyamine-depleted cells as compared to cells grown in the presence of putrescine or to density-inhibited cells. ADP-ribosylation experiments using [ 3H]adenosine showed a different pattern of label distribution; the higher- M r HMG proteins from polyamine-depleted cells contained about one-half the amount of label found in the proteins from control cells. The lower- M r HMG proteins and histone H1 were the preferentially labeled proteins in polyamine-depleted cells. Labeling of core histones with [ 32P]orthophosphate or [ 3H]adenosine did not differ markedly in the two cell populations. The results obtained using intact polyamine auxotrophic cells indicated that polyamine depletion is connected with more severe alterations in amounts and covalent modifications (phosphorylation and ADP-ribosylation) of HMG chromosomal proteins and histone H1 than core histones.

Cell-cycle-regulated translation of histone mRNA in Physarum plasmodia

In Physarum polycephalum, histone mRNA begins to accumulate 3 h before the S phase. We show that histone synthesis was limited to the S phase and that the mRNA that accumulated in the G2-phase cytoplasm could be translated in vitro. Thus, recruitment of stored mature histone mRNA did not occur until the S phase began.

Synthesis and ubiquitination of histones during myogenesis

One and two-dimensional polyacrylamide gel electrophoresis have revealed that cultures of postmitotic (G 0) chicken skeletal myotube cells synthesize significant but reduced quantities of histone proteins as compared to their proliferating myoblast precursors. In addition, modulation of variant synthesis within the histone H2A and H3 classes may accompany myotube formation. That the histone bands contain no nonhistone contaminants was shown by exclusion of [ 3H]tryptophan. It is unlikely that these results reflect synthesis of histone by contaminating replicating cells, since a single treatment with cytosine arabinoside at the time of fusion effectively removed unfused cells while suppressing synthesis of DNA in the myotube cultures. The relatively sparse incorporation of label by major variants of the H2A class in dividing myoblasts was shown to be caused by heterogeneity due to phosphorylation and extensive ubiquitination, which decline at the time of myotube formation. As determined by quantitative Western-blotting, dividing myoblasts and myotubes contain an average of 1.0 and 0.4 molecules of ubiquitinated H2A (uH2A), respectively, per 10 nucleosomes.

Coupling of histone mRNA levels to radioresistant DNA synthesis in ataxia-telangiectasia cells.

Cloned genomic DNA for human histone H1, H3 and H4 genes has been used to determine the effects of gamma-radiation on histone mRNA levels and synthesis in ataxia-telangiectasia cells. Synthesis of histone mRNA was determined in cells synchronized with aphidicolin. Effects of irradiation on DNA synthesis and passage through S phase were also monitored. Irradiation was found to slow the passage of control cells through the cell cycle but had no effect on progression of ataxia-telangiectasia cells. H1 and core histone mRNA synthesis was inhibited by radiation in two control cell lines after release from aphidicolin block. No inhibition was observed in one ataxia-telangiectasia cell line and a small degree of inhibition in a second. An increased level of mRNA was observed in both irradiated control and ataxia-telangiectasia cells at 5-7 h post-irradiation compared to unirradiated cells. Similar results were obtained in log phase cells. These results demonstrate that histone mRNA synthesis is radioresistant in ataxia-telangiectasia cells and is coupled to radioresistant DNA synthesis in these cells.

Postsynthetic acetylation of histones during the cell cycle: a general function for the displacement of histones during chromatin rearrangements.

Postsynthetic acetylation of core histones exhibits a peak during S-phase of the Physarum cell cycle. The maximum 3H-acetate incorporation precedes the maximum of histone synthesis. Acetate is incorporated into all core histones during S-phase, but only into H2A and H2B during G2-period. Resolution of acetylated H4-subspecies reveals acetate incorporation into preexisting H4, but not into newly synthesized molecules during mitosis and early S-phase. In a protamine competition assay histones from S-phase chromatin are released at lower protamine concentrations as compared to the lower acetylated G2-chromatin. We demonstrate a preferential release of highly acetylated H4-subspecies at low protamine concentrations. Our results fit into a general model of the relationship between histone acetylation and chromatin assembly. According to this model acetylation of core histones would serve as a signal for displacement of histones from nucleosomes by modulating histone-protein or histone-DNA interactions. We propose that this mechanism operates during DNA-replication and transcription, as well as during other chromatin rearrangements.

Histones and the first cell cycle in maize germination

The timing of the onset of cell division during seed germination in maize and the role of histones for this process have been studied. Embryonic axes of maize seeds (Zea mays L. hybrid H‐30) were incubated in a sterile nutrient medium for different periods of time. For some experiments putrescine was also added. Mesocotyl, root tip and scutellar node were dissected at specific periods after incubation and the mitotic indices were determined in these tissues. Embryonic axes were incubated in the same medium either with [14C]‐lysine or [32P]‐phosphate. The incorporation of either 14C or 32P into histones was followed, both in postribosomal supernatant and in nuclei. It was found that during germination, there is specific timing for meristematic cells entering into cell division. Among the tissues tested, the mesocotyl meristem was the first to initiate this process. De novo synthesis of histones was detected as early as after 6 h of imbibition and the rate increased up to 12 h. Putrescine stimulated cell division and phosphorylation of the histones. The implications of these findings are discussed.

Changes in the nucleoprotein complexes of a baculovirus DNA during infection

The nature of the DNA-protein complexes assumed by Autographa californica nuclear polyhedrosis virus (AcNPV) DNA during infection of Spodoptera frugiperda cells was investigated by micrococcal nuclease digestion of infected nuclei. Both parental viral DNA and progeny viral DNA assumed a chromatin-like structure early in infection. By late times (24 hr) p.i., the viral DNA acquired a unique nucleoprotein structure. In addition to fragments of mononucleosome size (185 bp), two subnucleosomal bands of 120 and 90 bp were observed. The subnucleosomal bands contained exclusively viral DNA. No alteration in the nature of the host chromatin structure following AcNPV infection was observed. An examination of the basic chromatin-associated proteins revealed two major viral-induced proteins having molecular weights of 15K and 39K. The induction of the basic 15K protein between 10 and 24 hr p.i. coincided with the appearance of the altered nucleoprotein structure observed by 24 hr p.i. and the cessation of histone synthesis.

Biosynthesis and posttranslational acetylation of histones during spherulation of Physarum polycephalum.

Plasmodia of Physarum polycephalum can be induced to differentiate into dormant spherules: DNA-, RNA- and protein-synthesis cease during this process. Analysis of the histone H4 acetylation during spherulation revealed no significant changes of the relative acetate content and percentage of acetylated H4 subspecies. This result does not support a close correlation of histone acetylation and transcriptional activity. Posttranslational incorporation of 3H-acetate into core histones decreased rapidly after start of spherulation. However, acetate incorporation increased significantly at a late stage of spherulation (30 h). To elucidate the role of this elevated acetate incorporation we followed histone synthesis during spherulation. Histone synthesis decreased upon induction of differentiation and stopped after 12 h. After 38 h of spherulation histone synthesis again occurred in the absence of DNA synthesis. The peak of acetate incorporation into core histones clearly preceded this late histone synthesis, indicating acetylation of preexisting histones. We suggest, that this acetate incorporation is part of the mechanism, by which preexisting histones are replaced by newly synthesized histones. Pulse treatment with actinomycin D or cycloheximide during spherulation suggested, that the observed histone synthesis is essential for the germination of spherules. Obviously, new histones have to be synthesized for the coordinate course of the differentiation program.

Maternal administration of cyclophosphamide induces chromosomal aberrations and inhibits cell number, histone synthesis, and DNA synthesis in preimplantation mouse embryos.

The effects of cyclophosphamide (CPA), administered to pregnant inbred CBA/Ca mice 60 h after copulation, on cell number, mitotic index, chromosome structure, histone synthesis, and DNA synthesis of 84-h blastocysts, and the subsequent development of these blastocysts cultured for a further 120 h in vitro are described. Cyclophosphamide 4, 20, and 40 mg/kg significantly increased the number of chromosomally aberrant cells, chromosomal aberrations, and chromosome breaks in the blastocysts. Chromosomal rearrangements were significantly increased in the CPA 20 and 40-mg/kg treated groups, and in the 40-mg/kg group the number of cells with ring chromosomes was significantly increased. Histone synthesis and DNA synthesis were significantly inhibited in the CPA 20 and 40-mg/kg treated groups. Blastocyst cell number in each of the treated groups was less than the controls. On subsequent culture in vitro, significantly fewer embryos in the CPA 20 and 40-mg/kg groups hatched, attached, developed trophoblast outgrowths, and expanded their inner cell masses. However, the differentiation of inner cell mass into ectoderm and endoderm was impaired by all three doses of the drug. These results demonstrate that CPA administered to pregnant mice 60 h after copulation has a clastogenic effect and interferes with synthesis of DNA and histones in the preimplantation embryo, and that the drug inhibits the subsequent development and differentiation of these embryos. Cytogenetic analysis of preimplantation embryos might be a useful adjunct to the existing methods in the evaluation of the embryotoxicity of drugs and chemicals.

Differential expression of histone sequences in Drosophila following heat shock.

The expression of the sequences encoding the four nucleosomal histone proteins was examined following heat shock of a variety of Drosophila cells and was found to be highly differential. In Drosophila melanogaster KC-O cells grown in suspension culture, there is a continuation of the synthesis of all four of the nucleosomal histone proteins following heat shock. Analysis of RNA from these cells confirms that histone messengers are transcribed and located on polysomes. This exact same pattern of histone protein synthesis occurs in KC-O cells grown to low density on plates. In contrast, KC-O cells grown to high density on plates exhibit a dramatic elevation of H2b protein synthesis relative to the synthesis of the other core histones. Organs from D melanogaster third instar larvae were examined to ascertain whether histone protein synthesis continues following heat shock in the organism. Different tissue types exhibited differential histone synthesis. Imaginal disks excised from heat-shocked larvae continue to synthesize nucleosomal histones in a variable fashion. In contrast, neither fat bodies, brains, nor salivary glands continues to synthesize core histone proteins at a significant level. D hydei plated cell cultures and larval tissues fail to synthesize histones at any detectable level following a heat shock. Based on these observations, we propose that there is a differential synthesis of nucleosomal proteins in Drosophila that is highly dependent on the state of the cells prior to the heat shock.

The structure of adenovirus chromatin in infected cells.

The structure of adenovirus chromatin in infected cells was studied by micrococcal nuclease digestion and hybridization with virus-specific probes. In the early phase of infection (5 h) a significant proportion of viral molecules was organized like actively transcribed cellular chromatin. As expected for a transcriptionally active population of molecules, even at high multiplicity of infection the nucleosomal repeating pattern was less distinct than in a transformed cell which contained the corresponding but less active genomic region. The observed repeating pattern in infected cells was unlikely to be due to integrated molecules since less than 0.07% of input genomes became associated with cellular DNA. After the onset of viral DNA replication, the pool of viral chromatin organized like cellular chromatin rapidly increased. In addition, newly replicated molecules also maintained the cellular chromatin-like organization as measured by [3H]thymidine incorporation after the cessation of cellular DNA synthesis. These data suggest that newly replicated viral molecules are organized by histones into cell-like chromatin throughout the infection cycle. Coincident with the peak of viral DNA and core protein synthesis, and the decline of histone synthesis, the late, core-like non-repeating viral chromatin became dominant, increasingly obscuring the underlying repeating pattern. Experiments suggest that this late chromatin is destined for encapsidation, that the early chromatin persists and that viral core proteins do not displace histones on viral DNA. A model is proposed suggesting that transcription and type I replication occur on histone-condensed templates, while type II replication products late in infection are condensed by core proteins and are destined for encapsidation.

Histone synthesis and deposition in the G1 and S phases of hepatoma tissue culture cells.

Hepatoma tissue culture cells were synchronized in G1 and in S phase in order to examine the level of synthesis of different histone types and to determine the rate, timing, and location of their deposition onto DNA. We observe a basal level of synthesis in G1 (5% of that seen in S phase) for H2A.1, H2A.2, H3.2, H2B, and H4. The minor histone variants X and Z are synthesized at 30% of the rate observed in S cells. The rate of synthesis of the ubiquinated histones uH2A.1,2 is not as depressed in G1 cells as seen for H2A.1 and H2A.2. Histones synthesized in G1 are not deposited on the DNA of these cells at equivalent rates. Thus, histones H3.2 and H4 are not deposited significantly until S phase begins, at which time deposition occurs selectively on newly synthesized DNA. The deposition of H2A.1, H2A.2, H2B, X, and Z proceeds in G1; however, it occurs to a 2-4-fold lower extent than seen for the deposition of H1, HMG 14, and HMG 17. The deposition of all histones synthesized in S phase occurs rapidly, but there are variations in the sites of deposition. Thus, newly synthesized H3.1, H3.2, and H4 deposit primarily on newly replicated DNA whereas H2A.1, H2A.2, uH2A.1, 2, and H2B deposit only partially on new DNA (30%) and mostly on old. H1, HMG 14, and HMG 17 are deposited in an apparently fully random manner over the chromatin. To interpret these observations, we propose a model which includes a measure of histone exchange on the chromatin fiber. The model emphasizes the dynamics of histone-histone and histone-DNA interactions in regions of active genes and at replication forks.

Histone segregation on replicating chromatin.

We have reinvestigated the mode of segregation of preexisting histones onto replicating chromosomes. Since our previous data have indicated that only histones H3 and H4 do not appear to move from their association with the DNA strand with which they are bound until the next round of replication, we have concentrated our attention on these two histones. The strategy we have employed involved density labeling of DNA and radiolabeling of the histones of interest. Subsequently, we followed the association of histones and DNA during further rounds of DNA replication. One can make predictions concerning the nature of the association between specific histones and particular DNA strands depending on the mode of deposition. The results have confirmed our previous findings that histones segregate randomly. The possibility that such a result is a consequence of turnover of radiolabel in non-histone proteins and subsequent reutilization for histone synthesis has been tested directly. This process appears to be occurring to only a very limited extent. The implications of these conclusions for chromatin structure and gene control are discussed.

Changes in histone acetylation during the development of rabbit bone marrow erythroid cells

The acetylation of histones in rabbit bone marrow erythroid cells was investigated by measuring the incorporation of labelled acetate into erythroblasts which were separated by velocity sedimentation at unit gravity into five fractions corresponding to different stages of development. Histone acetylation decreased during erythroid development in concert with a decline in DNA and histone synthesis. Some acetylation persisted after condensation of the nucleus and cessation of DNA synthesis in late orthochromatic cells. This residual acetylation may be related to the low level of transcription which is still present at this stage. Sodium butyrate increased the acetylation of histones 2- to 7-fold, with the greatest stimulation occurring in the most immature cells. The general decline in acetylation of histones during erythroid cell development was similar in the presence and absence of butyrate.

Interrelationships of protein and DNA syntheses during replication of mammalian cells.

During the replication of chromatin, the syntheses of the histone protein and DNA components are closely coordinated but not totally linked. The interrelationships of total protein synthesis, histone protein synthesis, DNA synthesis, and mRNA levels have been investigated in Chinese hamster ovary cells subjected to several different types of inhibitors in several different temporal combinations. The results from these studies and results reported elsewhere can be brought together into a consistent framework which combines the idea of autoregulation of histone biosynthesis as originally proposed by W. B. Butler and G. C. Mueller (Biochim. Biophys. Acta 294:481-496, 1973] with the presence of basal histone synthesis and the effects of protein synthesis on DNA synthesis. The proposed framework obviates the difficulties of Butler and Mueller's model and may have wider application in understanding the control of cell growth.