Age-related effects on the incorporation of acetate into rat liver histones.

Incorporation of sodium [3H]acetate into histones of rats was examined as a function of age. Incorporation was observed to decline with age up to 24 months, at which time a levelling occurred. Controls indicated that this decrease in histone acetylation could not be attributed to variability in isotope delivery to the liver or to alterations in intracellular 'pools' available for acetylation. Polyacrylamide gel electrophoresis established that, in all cases, acetate was incorporated primarily into histone fractions H3 and H4 and the pattern of incorporation exhibited age-dependent phenomena. H4 was predominantly labelled in 2 months animals, while in 12, 16, and 24 months animals H3 was more highly labelled; at 27 months the two fractions were labelled equally. Assessment of histone acetylase and deacetylase activities indicates that deacetylase activity increased with age.     

Histone acetylation in chicken erythrocytes. Rates of deacetylation in immature and mature red blood cells.

We analysed the rates of histone deacetylation in chicken mature and immature red blood cells. A multiplicity of deacetylation rates was observed for the histones and these rates may be subdivided into two major categories based on the extent of histone acetylation. In one set of experiments, cells were labelled with [3H]acetate in the presence of the deacetylase inhibitor n-butyrate, thereby accumulating radiolabel in the hyperacetylated forms of the histone. These hyperacetylated forms are deacetylated rapidly. [3H]Acetate-labelled tetra-acetylated H4 (H4Ac4) in mature cells was deacetylated with an initial half-life (t1/2) of approximately 5 min (time required for the removal of one-half of the labelled acetyl groups). In immature cells, all [3H]acetate-labelled H4Ac4 was deacetylated with a t1/2 of approximately 5 min. Erythrocytes were also labelled with [3H]acetate for extended periods in the absence of the deacetylase inhibitor. During this period, radiolabel accumulated predominantly in the mono- and di-acetylated forms of the histone. Using this protocol, the rate of deacetylation of H4Ac1 was observed to be approximately 145 min for mature cells, and approximately 90 min for immature cells, demonstrating that the less extensively acetylated histone is deacetylated slowly. These results are discussed in the context of the rates of histone acetylation in chicken red blood cells described in the companion paper [Zhang & Nelson (1988) Biochem. J. 250, 233-240].     

Acetylation of histones of rat testis.

When [1-¹⁴C]acetate was injected into rats intratesticularly in the presence of cycloheximide to inhibit protein synthesis, the label was incorporated into histone fractions F2a1 and F3 and into non-histone chromosomal proteins of each of the following stages of spermatogenesis: spermatogonia-preleptotene spermatocytes, leptotene-zygotene-pachytene-diplotene primary spermatocytes, and spermatids. Acetylation of histones was particularly active in the spermatid stages. There was no significant incorporation of acetate into the lysine-rich histone fractions F1 and X₁.In early periods of in vivo incorporation of [³H]amino acids into histones the acetylated histone F2a1 fractions had higher specific activities than the main band of F2a1, but with the passage of time the label moved into the principal band to the extent that specific activities in the acetylated and principal bands were approximately equal at 6 days. However, at 24–36 days the specific activities were again higher in the acetylated bands than in the principal band of F2a1. These data support the conclusions of Candido, Louie, and Dixon, from experiments with trout testis, that acetylation of histone F2a1 may be important in the process of combination of this protein with DNA in chromatin at the spermatogonia-primary spermatocyte stage and also in the subsequent removal of this histone for replacement by protamines at the spermatid stage.[³H]Amino acids were incorporated into histone fractions X₁ and F1 at approximately equal rates, and there was no evidence that one of these fractions was a precursor of the other.Chromatin of the seminiferous epithelial cells of rat testis has a firmly bound acetylase which catalyzes the in vitro acetylation of histones F3 and F2a1 by acetyl CoA.     

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.     

Alterations in histone acetylation following exposure to <Superscript>60</Superscript>Co γ-rays and their relationship with chromosome damage in human lymphoblastoid cells

Chromosome damage is related to DNA damage and erroneous repair. It can cause cell dysfunction and ultimately induce carcinogenesis. Histone acetylation is crucial for regulating chromatin structure and DNA damage repair. Ionizing radiation (IR) can alter histone acetylation. However, variations in histone acetylation in response to IR exposure and the relationship between histone acetylation and IR-induced chromosome damage remains unclear. Hence, this study investigated the variation in the total acetylation levels of H3 and H4 in human lymphocytes exposed to 0–2 Gy ⁶⁰Co γ-rays. Suberoylanilide hydroxamic acid (SAHA), a histone deacetylase (HDAC) inhibitor, was added to modify the histone acetylation state of irradiated cells. Then, the total acetylation level, enzyme activity, dicentric plus centric rings (dic?+?r) frequencies, and micronucleus (MN) frequencies of the treated cells were analyzed. Results indicated that the acetylation levels of H3 and H4 significantly decreased at 1 and 24 h, respectively, after radiation exposure. The acetylation levels of H3 and H4 in irradiated groups treated with SAHA were significantly higher than those in irradiated groups that were not treated with SAHA. SAHA treatment inhibited HDAC activity in cells exposed to 0–1 Gy ⁶⁰Co γ-rays. SAHA treatment significantly decreased dic?+?r/cell and MN/cell in cells exposed to 0.5 or 1.0 Gy ⁶⁰Co γ-rays relative to that in cells that did not receive SAHA treatment. In conclusion, histone acetylation is significantly affected by IR and is involved in chromosome damage induced by ⁶⁰Co γ-radiation.     

Acetylation sites in histone H3 from Physarum polycephalum

Histone H3 from Physarum polycephalum was labelled with [³H]acetate in G2 phase of the cell cycle. Only histones H3 and H4 were labelled and the H4 was removed by chromatography. Sequential Edman degradation of labelled H3 showed that acetate was incorporated into residues 9, 14, 18 and 23 which correspond to the sites of acetyl-lysine determined in histones H3 from other organisms. The results confirm the sequence conservation of H3 and support the notion that data on H3 acetylation, obtained with Physarum, can be extrapolated to higher eukaryotes.     

Failure of Long-Term Memory Formation in Juvenile Snails Is Determined by Acetylation Status of Histone H3 and Can Be Improved by NaB Treatment

Background

Animals’ capacities for different forms of learning do not mature simultaneously during ontogenesis but the molecular mechanisms behind the delayed development of specific types of memory are not fully understood. Mollusks are considered to be among the best models to study memory formation at the molecular level. Chromatin remodeling in developmental processes, as well as in long-term memory formation, was recently shown to play a major role. Histone acetylation is a key process in the chromatin remodeling and is regulated through the signaling cascades, for example MAPK/ERK. Previously, we found that MAPK/ERK is a key pathway in the formation of the food aversion reflex in Helix. Pretreatment with upstream ERK kinase inhibitor PD98059 prevented food avoidance learning in adult Helix. In contrast to adult snails, juveniles possess immature plasticity mechanisms of the avoidance reflex until the age of 2–3 months while the MAPK/ERK cascade is not activated after aversive learning. In the present study, we focused on the potential MAPK/ERK target - histone H3.

Methodology/Principal Findings

Here we found that a significant increase in histone H3 acetylation occurs in adult animals after learning, whereas no corresponding increase was observed in juveniles. The acetylation of histone H3 is regulated by ERK kinase, since the upstream ERK kinase inhibitor PD98059 prevented the increase of histone H3 acetylation upon learning. We found that the injection of histone deacetylase inhibitor sodium butyrate (NaB) prior to training led to induction in histone H3 acetylation and significantly ameliorated long-term memory formation in juvenile snails.

Conclusions/Significance

Thus, MAPK/ERK-dependent histone H3 acetylation plays an essential role in the formation of food aversion in Helix. Dysfunction of the MAPK/ERK dependent histone H3 acetylation might determine the deficiency of avoidance behavior and long-term plasticity in juvenile animals. Stimulation of histone H3 acetylation in juvenile animals by NaB promoted avoidance plasticity.     

Acetylation of histones during spermatogenesis in the rat

Acetate was actively incorporated into rat testis histones when testis cells were prepared by the trypsinization technique in the presence of [³H]acetate. The acetylation was enhanced by 10 mm sodium butyrate. Although histones H3 and H4 were the only histones which incorporated high levels of acetate, the testis-specific histones TH2B and TH3 also appeared to incorporate acetate. This was shown by electrophoresis of the histones on polyacrylamide gels containing Triton X-100. Results, obtained from analysis of histones by two-dimensional gel electrophoresis, confirmed a recent report (P. K. Trostle-Weige, M. L. Meistrich, W. A. Brock, K. Nishioka, and J. W. Bremer, (1982) J. Biol. Chem.257, 5560–5567) that TH2A was a testis-specific histone. The results also confirmed the H2A nature of a testis-enriched histone band, previously designated X2. When histones from populations of cells enriched in specific testis cell types, representing various stages of spermatogenesis, were examined, the patterns of acetylation varied dramatically. Very high levels of acetate were incorporated into multiacetylated species of histone H4 from a population of cells enriched in transition stage spermatids (steps 9–12) compared to the levels of acetate incorporated into H4 from round spermatids (steps 1–8) and earlier stages of spermatogenesis, where acetate was incorporated primarily into the monoacetylated species of H4. Thus, a striking correlation exists between the time of hyperacetylation of histone H4 and the time of removal of histones for their replacement by the basic spermatidal transition proteins designated TP, TP2, and TP4. Hyperacetylation of histone H4 may facilitate the removal of the entire histone complement during the protein transition. In any case, it must be an obligatory step in the dramatic process.     

Selective synthesis and modification of nuclear proteins during maturation of avian erythroid cells.

The synthesis of the nuclear proteins of duck erythroid cells at different stages of maturation has been investigated. Synthesis of histone fractions H1, H2a, H2b, H3, and H4 is restricted to the erythroblasts, while synthesis of H5 can be detected even at later stages of maturation after DNA synthesis has ceased. The synthesis of nonhistone nuclear proteins (NHNP), on the other hand, occurs in cells at all stages of maturation although their rates of synthesis decline as the cells mature. The same size classes of NHNP appear to be synthesized in erythroblasts and in early- and midpolychromatic erythrocytes. In late polychromatic erythrocytes the synthesis of a new group of NHNP of molecular weights ranging from 54,000 to 130,000 was observed. This group of proteins does not accumulate in the mature erythrocyte, indicating that their relative proportions are very small.Turnover of histone-bound phosphate was found to occur mainly at the erythroblast stage, except for histone H2a which was actively phosphorylated even at more advanced stages of maturation. Phosphorylation of most of the histones appears to be coupled to histone (and coordinate DNA) synthesis.Incorporation of radioactive acetate into histones occurs at all stages, but the rate of acetylation decreases four- to fivefold with maturation. Although the RNA synthetic activity of erythroid cells also decreases with age, experiments involving the use of RNA polymerase inhibitors suggest that the mechanisms that control RNA synthesis and histone acetylation are not tightly coupled.     

DNA methyltransferase 3b preferentially associates with condensed chromatin

In mammals, DNA methylation is catalyzed by DNA methyltransferases (DNMTs) encoded by Dnmt1, Dnmt3a and Dnmt3b. Since, the mechanisms of regulation of Dnmts are still largely unknown, the physical interaction between Dnmt3b and chromatin was investigated in vivo and in vitro. In embryonic stem cell nuclei, Dnmt3b preferentially associated with histone H1-containing heterochromatin without any significant enrichment of silent-specific histone methylation. Recombinant Dnmt3b preferentially associated with nucleosomal DNA rather than naked DNA. Incorporation of histone H1 into nucleosomal arrays promoted the association of Dnmt3b with chromatin, whereas histone acetylation reduced Dnmt3b binding in vitro. In addition, Dnmt3b associated with histone deacetylase SirT1 in the nuclease resistant chromatin. These findings suggest that Dnmt3b is preferentially recruited into hypoacetylated and condensed chromatin. We propose that Dnmt3b is a 'reader' of higher-order chromatin structure leading to gene silencing through DNA methylation.     

Droxinostat,a Histone Deacetylase Inhibitor,Induces Apoptosis in Hepatocellular Carcinoma Cell Lines via Activation of the Mitochondrial Pathway and Downregulation of FLIP

Background: The current chemotherapeutic outcomes for hepatocellular carcinoma (HCC) are not encouraging, and long-term survival of this patient group remains poor. Recent studies have demonstrated the utility of histone deacetylase inhibitors that can disrupt cell proliferation and survival in HCC management. However, the effects of droxinostat, a type of histone deacetylase inhibitor, on HCC remain to be established. Methods: The effects of droxinostat on HCC cell lines SMMC-7721 and HepG2 were investigated. Histone acetylation and apoptosis-modulating proteins were assessed via Western blot. Proliferation was examined with 3-(4, 5 dimetyl-2-thiazolyl)-2, 5-diphenyl 2H-tetrazolium bromide, cell proliferation, and real-time cell viability assays, and apoptosis with flow cytometry. Results: Droxinostat inhibited proliferation and colony formation of the HCC cell lines examined. Hepatoma cell death was induced through activation of the mitochondrial apoptotic pathway and downregulation of FLIP expression. Droxinostat suppressed histone deacetylase (HDAC) 3 expression and promoted acetylation of histones H3 and H4. Knockdown of HDAC3 induced hepatoma cell apoptosis and histone H3 and H4 acetylation. Conclusions: Droxinostat suppresses HDAC3 expression and induces histone acetylation and HCC cell death through activation of the mitochondrial apoptotic pathway and downregulation of FLIP, supporting its potential application in the treatment of HCC.     

Schizosaccharomyces pombe Hst4 functions in DNA damage response by regulating histone H3 K56 acetylation

The packaging of eukaryotic DNA into chromatin is likely to be crucial for the maintenance of genomic integrity. Histone acetylation and deacetylation, which alter chromatin accessibility, have been implicated in DNA damage tolerance. Here we show that Schizosaccharomyces pombe Hst4, a homolog of histone deacetylase Sir2, participates in S-phase-specific DNA damage tolerance. Hst4 was essential for the survival of cells exposed to the genotoxic agent methyl methanesulfonate (MMS) as well as for cells lacking components of the DNA damage checkpoint pathway. It was required for the deacetylation of histone H3 core domain residue lysine 56, since a strain with a point mutation of its catalytic domain was unable to deacetylate this residue in vivo. Hst4 regulated the acetylation of H3 K56 and was itself cell cycle regulated. We also show that MMS treatment resulted in increased acetylation of histone H3 lysine 56 in wild-type cells and hst4Delta mutants had constitutively elevated levels of histone H3 K56 acetylation. Interestingly, the level of expression of Hst4 decreased upon MMS treatment, suggesting that the cell regulates access to the site of DNA damage by changing the level of this protein. Furthermore, we find that the phenotypes of both K56Q and K56R mutants of histone H3 were similar to those of hst4Delta mutants, suggesting that proper regulation of histone acetylation is important for DNA integrity. We propose that Hst4 is a deacetylase involved in the restoration of chromatin structure following the S phase of cell cycle and DNA damage response.     

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.