Studies on the chromatin of barley leaves during senescence

1. The activity of soluble ribonuclease and deoxyribonuclease first declined during senescence, but later increased during advanced stages of senescence. 2. Young leaves had very low ribonuclease or deoxyribonuclease activity associated with the chromatin, but the activity of these enzymes increased progressively during senescence until the leaves died. 3. No significant changes in the composition of chromatin from first seedling leaves of barley plants during aging (from 7 to 25 days) were noted. 4. The amount of RNA synthesized by chromatin in vitro declined as the leaf aged. However, if the loss of RNA due to chromatin-associated ribonuclease was taken into account, the RNA-synthesizing activity of chromatin from senescing (15-16-day-old) leaves appeared to be somewhat higher than that of chromatin from young (7-8-day-old) leaves. In leaves at the terminal stages of senescence (23 days old) the estimates of RNA synthesis by chromatin could not be made owing to complications created by high nuclease activities. 5. It is suggested that senescence may be triggered by a decline in some hormonal factor in leaves, and that the resulting production of chromatin-associated deoxyribonuclease and ribonuclease in increasing proportions may progressively cause increased degradation of DNA and newly synthesized RNA, so that ultimately the cellular functions are impaired and the cells die.     

Poly-ADP-ribosylated histones: potent DNA suppressors

When rat liver nuclear chromatin was sonicated in buffer containing 0.35 M (NH₄)SO₄ to release the engaged RNA polymerases, a potent inhibitor was also released. This inhibitor elicited dramatic inhibition of RNA synthesis regardless of whether the free or engaged RNA polymerase was used. On further analysis, it became apparent that the site of inhibition was on the DNA template, not on the enzyme. This inhibitor could be extracted into 0.25 N HCl by the standard procedure for the isolation of histones. This acid-soluble inhibitor, showing typical histone band on gel, was RNase A and DNase I resistant, but was sensitive to both pronase and snake venom phosphodiesterase digestion, as well as to 0.1 N KOH hydrolysis. Furthermore, when [¹⁴C]adenine labeled poly-ADP-ribosylated histones were digested by snake venom phosphodiesterase, the release of radioactivity was in parallel to the loss of inhibitor activity. We conclude that the inhibitor substances are poly-ADP-ribosylated histones and propose that the poly-ADP-ribosylated histones rather than the histones are the natural suppressors of the gene.     

Histone acetyltransferase in chromatin. Extraction of transferase activity from chromatin

Previous work has attempted to localize nuclear histone acetyltransferase activity in the cromatin. Evidence was presented indicating that the transfer of ¹⁴C-acetate from ¹⁴C-acetyl CoA to histones in chromatin was an enzymatic process. We now report on the extraction of part of the histone acetyltransferase activity from rat liver chromatin, employing a procedure originally described for extraction of DNA-dependent RNA polymerase. The K_m of the extracted transferase activity for the substrate acetyl CoA was 5 × 10⁻⁷, the Q₁₀: 1.8 and the optimal pH: 7.1. Serum albumine, protamine and polylysine were poor substrates as compared to histones. Activity of extracted or heated chromatin was not restored upon incubation in the presence of extract. Also the selectivity exhibited by the transferase activity in unextracted chromatin towards arginine-rich histones, was much less pronounced in the extracts prepared from it. It is possible that the influence of steric factors contributing to this specificity in native chromatin is lost upon isolation of the enzyme from it. Alternatively, a less specific isoenzyme may have been extracted.     

Changes in enzymic activity during cultivation of human cells in vitro

The composition of chromatin, its template activity and the activity of certain chromatin-associated enzymes, including DNA polymerase (DP) and soluble RNase, DNase, DP and seryl tRNA synthetase, were examined in early and late passage of WI-38 cells and of WI-38VA13 cells.No significant changes in soluble RNase, DNase, seryl tRNA synthetase or soluble and chromatin-associated DP were found with increasing passage of WI-38 cells. The activity of seryl tRNA synthetase and DP in WI38VA13 cells was, however, significantly higher than WI-38 cells in all passages. A decline in RNA synthesizing activity of chromatin, an increase in the proportion of RNA and histone in chromatin, as well as an increase in the activities of ‘chromatin-associated enzymes’ (RNase, DNase, protease, nucleoside triphosphatase, DPN pyrophosphorylase) were noted in WI-38 cells with increasing passages. Although RNA synthesizing activity of chromatin from WI38VA13 cells was lower than that from WI-38 cells, the former also were much lower in ‘chromatin-associated enzymes’. An increase of chromatin-associated enzymes responsible for RNA, DNA and protein degradation in WI-38 cells in successive passages, and a much lower activity of these enzymes in WI-38VA13 cells (which have an indefinite doubling potential in vitro) suggests that an elevation in the activity of these enzymes, which would seriously interfere with the chromatin function, could result in ‘aging’ of WI-38 cells.     

Chromatin- and nuclei-directed ribonucleic Acid synthesis in sugar beet root

The synthesis of RNA by chromatin-bound RNA polymerase prepared from sugar beet (Beta vulgaris) root tissue is completely dependent on the presence of a divalent metal (Mg²⁺ or Mn²⁺) and the presence of four ribonucleoside triphosphates. Accumulation of labeled acid-insoluble product is inhibited by the addition of RNase and actinomycin D to the reaction. When beet root slices are washed for 25 hours, chromatin-associated RNA polymerase activity increases 7-fold over that of unwashed tissue. This enzyme activity declines with further washing. DNA template availability, as measured by saturating levels of added Escherichia coli RNA polymerase, was also found to follow a pattern similar to that for RNA polymerase. Nearest neighbor frequencies of the RNA synthesized by chromatin isolated from unwashed and washed tissue are different.     

Histone propinquity using imidoesters.

Native chromatin was reacted with bifunctional protein cross-linking reagents of varying molecular sizes. Short cross-linkers do not produce significant yields of polymeric histones whereas intermediate-sized materials such as dimethyladipimidate produce extensive interaction between histones. Dimethylsuberimidate reacts with all histone fractions but only generates polymers of F₁ and of F_2b and F_2a2.     

Initial steps in the large-scale purification of Escherichia coli deoxyribonucleic acid-dependent ribonucleic acid polymerase

Histones from exponential and stationary-phase mouse L-cells were quantitated after acrylamide gel electrophoresis in order to investigate cell cycle-dependent changes in the mode of binding of the various fractions in chromatin. By introducing various concentrations of citrate and divalent cations in the medium used for cell lysis and isolation of nuclei prior to histone extraction it was possible to demonstrate that certain histone fractions are preferentially retained in either exponential or stationary-phase nuclei. Differential retention of lysine-rich F₁ was most evident when the lysing medium contained 1 mm Mg²⁺ and Ca²⁺ and 5 mm citrate (pH 2.75). In these conditions twice as much F₁ is retained in stationary as in exponential nuclei. Differential retention of arginine-rich histones was most evident when the lysing medium contained 10 mm Mg²⁺ and Ca²⁺ and no citrate. In these conditions more F_{2a 1} is retained in exponential than in stationary nuclei while the opposite is true for F₃. However, the total amount of arginine-rich fractions (F_{2a 1} + F₃) retained was found to be the same in both cell phases. The results are discussed in relation to known structural features of the histones.     

Subcellular distribution of histone-degrading enzyme activities from rat liver.

Chromatin prepared from liver tissue contains a histone-degrading enzyme activity with a pH optimum of 7.5-8.0, whereas chromatin isolated from purified nuclei is devoid of it. The histone-degrading enzyme activity was assayed with radioactively labelled total histones from Ehrlich ascites tumor cells. Among the different subcellular fractions assayed, only lysosomes and mitochondria exhibited histone-degrading enzymes. A pH optimum around 4.0-5.0 was found for the lysosomal fraction, whereas 7.5-8.0 has been found for mitochondria. Binding studies of frozen and thawed lysosomes or mitochondria to proteinase-free chromatin demonstrate that the proteinase associated with chromatin isolated from frozen tissue originates from damaged mitochondria. The protein degradation patterns obtained after acrylamide gel electrophoresis are similar for the chromatin-associated and the mitochondrial proteinase and different from that obtained after incubation with lysosomes. The chromatin-associated proteinase as well as the mitochondrial proteinase are strongly inhibited by 1.0 mM phenylmethanesulfonyl fluoride. Weak inhibition is found for lysosomal proteinases at pH 5. Kallikrein-trypsin inhibitor, however, inhibits lysosomal proteinase activity and has no effect on either chromatin-associated or mitochondrial proteinases. The higher template activity of chromatin isolated from a total homogenate compared to chromatin prepared from nuclei may be due to the presence of this histone-degrading enzyme activity.     

Chromosomal proteins of conifers

During imbibition and germination of jack pine seeds, the composition of the total extractable chromatin varied. Relative to DNA, the histone levels decreased as the nonhistone chromosomal proteins (NHCP) increased. New chromosomal proteins were synthesized after 2 days of imbibition as judged by recovery of ¹⁴C-amino acids from the major protein fractions. Phosphorylation of histones from ³²P-phosphoric acid was detected before the incorporation of ¹⁴C-amino acids. In the seed the synthesis and relative changes of chromatin coincided with a fall in total soluble protein and free arginine N. By contrast, adenylate energy charge, free glutamine N and in vitro template activity of chromatin increased during chromatin protein synthesis. When seeds had germinated for 4 days after the start of imbibition more radioactivity, derived from free ¹⁴C-amino acids, was recovered from the NHCP than from the histones. The percentage amino acid composition of most histone fractions remained stable, whereas the composition of NHCP changed more with time. The phosphorylation of NHCP was 8- to 41-fold greater than that of the histones. Phosphorylation of histone H4 was not detected at any stage of germination. Correlations between recovery of radioactivity (³²P and ¹⁴C) from chromosomal proteins and higher adenylate energy charge were positive.     

Inhibition of histone deacetylases by Trichostatin A leads to a HoxB4-independent increase of hematopoietic progenitor/stem cell frequencies as a result of selective survival

BackgroundDNA and chromatin modifications are critical mediators in the establishment and maintenance of cell type-specific gene expression patterns that constitute cellular identities. One type of modification, the acetylation and deacetylation of histones, occurs reversibly on lysine ε-NH₃⁺ groups of core histones via histone acetyl transferases (HAT) and histone deacetylases (HDAC). Hyperacetylated histones are associated with active chromatin domains, whereas hypoacetylated histones are enriched in non-transcribed loci.MethodsWe analyzed global histone H4 acetylation and HDAC activity levels in mature lineage marker-positive (Lin⁺) and progenitor lineage marker-negative (Lin^?) hematopoietic cells from murine bone marrow (BM). In addition, we studied the effects of HDAC inhibition on hematopoietic progenitor/stem cell (HPSC) frequencies, cell survival, differentiation and HoxB4 dependence.ResultsWe observed that Lin^? and Lin⁺ cells do not differ in global histone H4 acetylation but in HDAC activity levels. Further, we saw that augmented histone acetylation achieved by transient Trichostatin A (TSA) treatment increased the frequency of cells with HPSC immunophenotype and function in the heterogeneous pool of BM cells. Induction of histone hyperacetylation in differentiated BM cells was detrimental, as evidenced by preferential death of mature BM cells upon HDAC inhibition. Finally, TSA treatment of BM cells from HoxB4^?/? mice revealed that the HDAC inhibitor-mediated increase in HPSC frequencies was independent of HoxB4.ConclusionsOverall, these data indicate the potential of chromatin modifications for the regulation of HPSC. Chromatin-modifying agents may provide potential strategies for ex vivo expansion of HPSC.     

Sea urchin sperm DNP. I. Chemical composition and template properties of DNP

Electrophoretic mobility, amino acid composition and salt dissociation of histones isolated from sperm of sea urchin Strongylocentrotus intermedius and calf thymus cells were studied. The special arginine-rich histone fraction (I) has been observed in sea urchin sperm chromatin, this fraction being absent in calf thymus chromatin. Dissociation of lysine-containing histone fractions from sea urchin chromatin occured in the range of 0.7 to 1.0 M NaCl concentrations. H1 of calf thymus chromatin was totally extracted with 0.6 M NaCl. In the course of a further increase of salt concentrations (up to 1.5 M NaCl) a practically total extraction of histones from sperm chromatin was observed, while about 20% of proteins remained bound to DNA in thymus chromatin after extraction with 2.0 M NaCl. The template activity of non-extracted DNP preparations from urchin sperm was equal to 2-3% of that of totally deproteinized DNA. The template activity of DNP gradually increased at protein extraction from DNP preparations. The hybridization capacity of RNA transcribed on partially dehistonized DNP templates in vitro also increased.     

Effect of norepinephrine on chromatin protein phosphorylation in C-6 glioma cells

Rat C₆ glioma cultures were exposed to labelled sodium phosphate after treatment with NE with or without propanolol. Histones and non-histone proteins (NHP) were extracted from chromatin and there was no significant change in the specific activity of the total pool of histones and NHP between control and other two groups. However, after electrophoretic separation F₂a₂ histone showed a 60% increase while F₂b and F₃ histones exhibited a 40% decrease in phosphorylation in response to NE. There was no significant change in the gel pattern of NHP from different groups on SDS-PAGE. However, the 30k dalton NHP showed an increase in phosphorylation in response to NE and this increment was blocked by propanolol. The possible role of β-receptors on nuclear protein phosphorylation and genomic expression is discussed.