Increase in histone acetylation and transitions in histone variants during Friend cell differentiation

Histone acetylation of Murine Erythroleukemia Cells (MELC) has been re-examined. It is demonstrated that sodium butyrate causes hyperacetylation of core histones in inducible as well as non-inducible MELC strains. This indicates that histone hyperacetylation per se is not sufficient to activate genes. However, [3H]acetate incorporation into core histones of the inducible MELC line F4N increases after induction of differentiation with dimethylsulfoxide (DMSO), in contrast to the non-inducible variant F4+. Thus histone acetylation may play a role as an auxiliary mechanism for gene activation (and inactivation). In addition, the appearance of a histone H3 variant during differentiation of MELC is reported.     

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.     

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.     

Metabolism of histones and non-histone proteins in liver cell nuclei and chromatin from rats of various ages

The metabolism of various classes of histones and nonhistone proteins in intact nuclei and in liver chromatin of albino Wistar rats aged 1, 3, 12 and 24 months, was studied. It was shown that in the course of postnatal development the metabolism of nonhistone proteins extracted with 0.14 M NaCl in murine liver is increased. Later in ontogenesis, the incorporation of labeled precursors into proteins HMG 14 and HMG 17 decreases; the specific radioactivity of proteins HMG 1 + 2 is higher in 3- and 24-month-old animals. The intensity of metabolism of nonhistone proteins and histones is higher within the composition of the chromatin complex than in the intact nucleus at all stages of postnatal development. Among other histone proteins, histones H1 are characterized by the highest level specific radioactivity in rats of all age groups.     

Core histone acetylation is regulated by linker histone stoichiometry in vivo

We investigated the relationship between linker histone stoichiometry and the acetylation of core histones in vivo. Exponentially growing cell lines induced to overproduce either of two H1 variants, H1(0) or H1c, displayed significantly reduced rates of incorporation of [(3)H]acetate into all four core histones. Pulse-chase experiments indicated that the rates of histone deacetylation were similar in all cell lines. These effects were also observed in nuclei isolated from these cells upon labeling with [(3)H]acetyl-CoA. Nuclear extracts prepared from control and H1-overexpressing cell lines displayed similar levels of histone acetylation activity on chromatin templates prepared from control cells. In contrast, extracts prepared from control cells were significantly less active on chromatin templates prepared from H1-overexpressing cells than on templates prepared from control cells. Reduced levels of acetylation in H1-overproducing cell lines do not appear to depend on higher order chromatin structure, because it persists even after digestion of the chromatin with micrococcal nuclease. The results suggest that alterations in chromatin structure, resulting from changes in linker histone stoichiometry may modulate the levels or rates of core histone acetylation in vivo.     

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.     

Effect of myocardial ischemia on uridine incorporation and histone acetylation

The influence of transient myocardial ischemia on recovery uridine incorporation into RNA and histone acetylation was investigated in an isolated perfused rat heart. Hemodynamically, hearts recovered from 15 min of ischemic arrest and were stable for at least 60 min of perfusion. Uridine incorporation was reduced (P less than 0.05) in ischemic hearts by 24 and 26% after 30 and 60 min of recovery perfusion. The incorporation of uridine into RNA from purified myocytes was decreased by 50% in the ischemic muscle cells. The covalent acetylation of total nucleohistones was diminished by 37%. Histone fractionation by urea polyacrylamide gel electrophoresis clearly indicated that histones H3 and H4 preferentially incorporated less acetate during ischemic recovery. However, histone acetylation for proteins H2A + H2B was not effected. These data suggest that a brief period of ischemia disrupts nucleotide incorporation during the recovery phase, with marked decrease associated with the muscle cell. The similar change in histone acetylation indicates a possible link between nucleoproteins and chromatin function during ischemic insult to the heart.     

Histone acetylation during meiosis in Lilium microsporocytes

Acetate incorporation into histones of Lilium microsporocytes is consistently higher in early prophase of meiosis than in later stages. The pattern of acetate incorporation into histones differs from that found for the soluble nuclear proteins. Evidence is presented that acetate incorporation reflects true acetylation, not histone synthesis. e-N-acetyllysine (eNAcLys) was released from histones by enzymatic digestion. Amino acid analysis of the digests confirm the presence of significantly higher amounts of eNAcLys in early meiotic stages. These results are consistent with the hypothesis that modulation of histone charge plays a role in the binding of histones to DNA and/or chromosome condensation. In vitro levels of histone acetylating activity remain constant throughout meiosis. The results suggest that microsporocyte histone deacetylase activity increases through meiotic prophase.     

Electrophoretic study of plant histones: Comparison with vertebrate histones

The histones of seven plant species (barley, leek, onion, pea, radish, rye, and wheat) were isolated and compared to the histones of calf thymus and rat liver using electrophoresis on polyacrylamide-urea and polyacrylamide-SDS gels. It was found that the F1 histone of plants contains more subspecies and has generally higher molecular weights than their animal histone counterparts. Histones F3 of plants and animals have identical molecular weights and similar but not identical mobilities on polyacrylamide-urea gels. No histones were found in plants which have molecular weights and mobilities on polyacrylamide-urea gels which resemble the values for histones F2a2 and F2b of animals, but instead the series of histones observed differ from any of the animal histones. These plant histones may represent either substantially modified forms of F2a2 and F2b, or else may be a different class of histone molecules unique to plants. Fractions F2al in plants and animals are identical in electrophoretic behavior, but seem to differ in degree of acetylation.     

Properties of the genome in normal and SV40 transformed WI38 human diploid fibroblasts. II. Metabolism and binding of histones.

Composition, metabolism and extractability of histone fractions from WI38 human diploid fibroblasts and SV40 transformed WI38 fibroblasts are compared. Two alternate procedures were used for isolation of nuclei which allow for either optimal recovery of arginine-rich histones F3 (III) and F2a1 (IV) or for optimal retention of lysine-rich F1 (I) and slightly lysine rich F2b (II b2). While the relative amount of each histone fraction was found to be similar in normal and SV40 transformed cells, substantial increases in the levels of F 3 acetylation and F1 and F2a2 phosphorylation are reported for the histones of SV40 transformed cells. Differences in extractability of arginine-rich histones with 0.25 M HCl are also reported. While F 3 is extracted more rapidly than F 2a1 from nuclei of normal WI38 fibroblasts, the reverse is true in SV40 transformed WI38 cells. These differences are discussed in relation to modification reactions, binding of histones to DNA and SV40-induced alterations in gene readout.     

Effects of a 4-aminopyridine in calcium movements and changes of membrane potential in pinched-off nerve terminals from rat cerebral cortex

Abstract: The processes of acetylation, phosphorylation, and methylation of nuclear proteins in cerebral hemispheres of 10- and 30-day-old rats were investigated. The experiments were carried out in vitro by measuring the incorporation of labeled precursors into histones and nonhistone chromosomal proteins (NHP) extracted from nuclei and separated by polyacrylamide gel electrophoresis. The results obtained indicate that there are age-specific differences in the processes of phosphorylation and methylation of chromosomal proteins, whereas the acetylation process did not change significantly between 10 and 30 days of age. Electrophoretic analysis of histones indicated that the histone H₃ was labeled to a greater degree than the other fractions and showed major changes in the processes of phosphorylation and methylation during postnatal development. The electrophoretic analysis of NHP showed considerable changes between 10 and 30 days of age. Certain components of NHP became increasingly evident as the brain developed. The methylation of an as yet unidentified protein with a molecular weight of approximately 118,000 daltons occurred at both ages.     

Histone-acetylating enzyme of brain

1. Acetylation of histones by an enzyme system derived from rat brain and liver (histone acetylase) was studied by using [1-(14)C]acetyl-CoA as the acetyl group donor. 2. The activity of this enzyme was largely confined to the nucleus. 3. Histone-acetylating activity of cerebral nuclei purified by centrifugation through 1.9m-sucrose was not altered by the presence of the cytoplasmic fraction. 4. Cerebral nuclei from adult rats exhibited greater histone-acetylating activity than did the corresponding preparation from newborn animals. 5. Nuclear acetylating activity was higher in brain than in liver of adult rats but not in newborn animals. 6. The partially purified enzyme from cerebral nuclei, prepared by ammonium sulphate fractionation of an acetone-dried powder, specifically catalysed histone acetylation. 7. Polylysine, protamine, serum albumin and gamma-globulin were not enzymically acetylated by this preparation. 8. Soluble acetylating preparations from both brain and liver nuclei were more active towards arginine-rich F3 and slightly lysine-rich F2a and F2b histone fractions than towards the lysine-rich F1 fraction. 9. Enzymic acetylation of chromatin-bound proteins was much less extensive than that of free histones. 10. The high histone acetylase activity in mature brain may reflect the importance of this process in the genetic control of cerebral function.