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.     

Porcine follitropin. The amino-acid sequence of the beta subunit

By treatment with tRNA in the presence of 1 mM MgCl2, a chromatin preparation was obtained containing all five major histone fractions but lacking a considerable portion of non-histone proteins. This chromatin preparation as well as chromatin extracted with 0.6 M NaCl (depleted of H1 histone and some non-histone proteins) were characterized in respect of solubility and chromatin DNA accessibility. Both samples possessed practically the same solubility in the presence of 0.15 M NaCl and 1 mM MgCl2. The solubility of tRNA-treated chromatin in 5 and 10 mM MgCl2 was higher than that of salt-extracted chromation. The accessibility of the DNA of these chromatin preparations was tested with DNA-dependent RNA polymerase of Escherichia coli as a probe, using procedure that permits measurement of binding site frequency. Both tRNA-treated and salt-extracted chromatin contained as many as 33% and untreated chromatin as few as 4% of the number of binding sites found on protein-free DNA. These results demonstrate that at least in part the non-histone proteins are responsible for salt-induced insolubility and low DNA accessibility of chromatin, thus revealing the importance of non-histone proteins in the maintenance of an overall chromatin structure.     

Stimulation of V(D)J recombination by histone acetylation

V(D)J recombination assembles functional immunoglobulin and T cell receptor genes from individual gene segments [1]. A common recombination mechanism, initiated by the proteins RAG1 and RAG2 at conserved recombination signal sequences (RSSs), operates at all rearranging loci [2] [3]. It has been proposed that the key regulator of the reaction is 'accessibility' of the RSS within chromatin [4]. Recently, the packaging of RSSs into nucleosomes was shown to inhibit initiation of V(D)J recombination [5] [6]. Nevertheless, the tight tissue specificity of regulation cannot be explained by nucleosome-mediated repression alone because a significant fraction of RSSs would be predicted to lie in linker regions between nucleosomes. Therefore, some aspect of the regulation of the recombination reaction must rely on the disruption of higher-order chromatin structure. Here, we report that histone acetylation directly stimulates the recombination reaction in vivo in the correct cell- and stage-specific manner. Neither expression of RAG genes nor activity of RAG proteins was increased by acetylation. Furthermore, histone acetylation failed to overcome nucleosome-mediated repression of RSS recognition and cleavage in vitro. Our data suggest a role for histone acetylation in stimulating recombination in vivo through disruption of higher-order chromatin structures.     

Non-histone chromatin protein fractions associated with ‘active’ chromatin in embryonic chicken liver

Non-histone chromatin proteins synthesized during chicken embryonic liver development were labeled with [3H]tryptophan and [3H]methionine and characterized by electrophoresis. During embryonic development protein/DNA ratio in chromatin was low (1.30-1.62) but synthesis of non-histone protein was high. Especially one characteristic fraction K (MW 18 000), tightly bound with DNA was preferentially associated with DNAase II sensitive, active transcribed sequences. In 7-day old and adult chicken synthesis of all non-histone proteins was low, fraction K was absent or synthesized only in small amounts in association with non-active sequences, however protein/DNA ratio in chromatin was high (2.30-2.33).     

Distinct effects of histone H1 and non-histone chromosomal proteins on the structure of nucleosomes and the chromatin fibre in solution

In this study we attempt to differentiate between the effects of the non-histone chromosomal proteins and histone H1 on the structure of the nucleosomes and the chromatin fibre in solution. The properties of chromatin preparations with different histone H1 and non-histone protein compositions were compared using circular dichroism and flow linear dichroism and the following conclusions were drawn. When histone H1 is absent the non-histone proteins partially prevent the unfolding of the nucleosomes at low ionic strength. The complete blocking of this unfolding, however, is accomplished only in the presence of histone H1. The non-histone proteins do not affect the orientation of the nucleosomes along the fibre axis. Only histone H1 can maintain the positive anisotropy of the chromatin fibre.     

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.     

Liver chromatin fractions inMus andAkodon

Summary The liver chromatin fromMus musculus andAkodon molinae was separated in 8 fractions by differential centrifugation. Like fractions from both species showed approximately similar contents of DNA, equivalent ratios of histone to non-histone proteins, corresponding template activities and equal amounts of positive C-banded material. On the other hand, heavy chromatin fractions ofMus were highly enriched in satellite DNA whereas no satellite DNA was found inAkodon chromatin. Heavy chromatin fractions isolated by differential sedimentation have been usually homologued with the constitutive heterochromatin. The properties of the constitutive chromatin are discussed and the validity of the foregoing concept is challenged. It is proposed to define the constitutive heterochromatin as those chromatin regions comprising highly repeated DNA sequences clustered in restricted areas of chromosomes and not transcribed (satellite DNA).     

The composition of liver chromatin proteins from embryos, chicks and adult chickens

The chemical composition of chromatin from the livers of 12-, 15- and 19-day-old embryos, of 1-day-old chicks and of adult chickens was analysed. The process of embryonic development is accompanied by an increase in non-histone chromatin proteins and chromatin RNA, as well as in the phosphorus content of chromatin phosphoproteins. The amount of these components decreases in the livers of 1-day-old chicks and adults. Two-dimensional polyacrylamide gel electrophoresis of acid-soluble chromatin proteins showed an increase in the amount of the H1 histone in 19-day-old embryos and adult chickens. Non-histone proteins of embryo liver chromatin showed a high content of the fraction of Mr of about 40 000; this was not the case for adult chickens. The non-histone protein fraction of Mr of about 120 000, characteristic of adult chicken liver proteins, was not found in the livers of 12- and 15-day-old embryos. Non-histone chromatin proteins isolated from the livers of animals of different age exhibited also quantitative differences.     

Incorporation of various amino acids into non-histone chromatin protein fractions of spleen cells of mice immunized with IgG

Summary Incorporation of three various amino acids ([³H]-tryptophan, [³H]-methionine or [³H]-leucine) into the non-histone chromatin proteins, synthesized in spleen cells of mice after immunization with IgG, is described. Two new fractions of non-histone chromatin proteins (I-mol. wt. below 3 000 and B₁-mol. wt. about 120 000), appearing during the immune reaction were labelled with [³H]-tryptophan and [³H]-methionine but not with [³H]-leucine. Synthesis of these fractions was observed only at the time of maximal RNA synthesis. A suggestion about the role of tryptophan and methionine in non-histone chromatin proteins in the regulatory processes of gene activation is discussed on the basis of their selective binding to DNA.     

Chromatin proteins associated with micrococcal nuclease-sensitive and nuclease-resistant chromatin fractions of Kirkman-Robbins hepatoma and hamster liver

Micrococcal nuclease-sensitive (SP) and nuclease-resistant (PP) chromatin fractions from Kirkman-Robbins hepatoma and hamster liver were obtained. The molecular distribution of three non-histone proteins (NHCP1, NHCP2 and NHCP3), histones, and chromatin-bound protease activity between SP and PP fractions of both tissues was compared. Differences, mainly of quantitative nature, among non-histone proteins of neoplastic and normal tissue were observed. Moreover, it was found that polypeptides with mol. wt 81 000 (NHCP1), 39 000 (NHCP2) and 21 000, 35 000, 37 000 (NHCP1), 70 000, 112 000, 141 000, 157 000 (NHCP2), 30 000–33 000 (NHCP3) were associated only with the nuclease-sensitive part of chromatin of hepatoma and normal tissue, respectively. A major difference in histone compostion of hamster hepatoma and liver concerns histones H2A and H1. Furthermore, an enrichment of high mobility group proteins as well as other soluble non-histone proteins in an acid extract of the SP fraction was observed. Apparently chromatin-bound protease activity can be found in both fractions of chromatin.     

The synthesis and decay of histone fractions and of deoxyribonucleic acid in the developing avian brain

1. The turnover of cerebral histones and DNA after injection of [4,5-(3)H]leucine or [methyl-3-(3)H]thymidine, respectively, was studied in the developing chick. 2. Chromatin was prepared from chick nuclei that had been purified by centrifugation through 1.9m-sucrose. 3. Nuclear proteins were fractionated into three major histone classes, F1 (lysine-rich), F2(b) (slightly lysine-rich) and [F3+F2(a)] (arginine-rich), and a non-histone protein residue. 4. The proportions of the histone classes remained constant throughout the period of development studied. 5. All histone fractions decayed at a similar rate, initially with a half-life of around 5 days, later with a half-life of 19 days. 6. Non-histone proteins from chromatin decayed in a heterogeneous manner with a wide range of half-lives. 7. Short-term labelling studies showed that all histone fractions were synthesized at the same rate. 8. Some non-histone proteins were very rapidly synthesized relative to histones. 9. DNA had a longer half-life than any histone fraction studied. A biphasic exponential decay curve with half-lives of 23 and 50 days was found. 10. It was concluded that the turnover of histones can occur independently of that of DNA and that different histone classes have similar rates of synthesis and decay.     

Studies on histones and non-histone proteins from rats treated with dimethylnitrosamine.

A study has been made of the histone and non-histone chromosomal proteins of rat liver after treatment in vivo with dimethylnitrosamine (DMN) (2 mg/kg). DMN was found not to affect histone turnover, as measured by 3H-labelled amino-acids incorporation. A decrease was observed in specific activity of the histones with time after injection of [14C]DMN or [14C]-formate and this was attributable to demethylation of both abnormal and normal methylation sites in these proteins. In the case of the non-histone proteins, DMN was found to increase greatly the turnover of those non-histone proteins loosely associated with chromatin DNA and RNA; turnover of those non-histone proteins tightly bound to chromatin DNA and RNA was unaffected. Demethylation of both normal and abnormal methylation sites was found to take place from both non-histone protein fractions. In the case of the loosely bound non-histone proteins a lower rate of demethylation was observed after DMN treatment.     

Two chromatin fractions with different metabolic properties of non-histone proteins and of newly synthesized RNA.

Digestion of chromatin with micrococcal nuclease under mild conditions results in the release of a minor chromatin fraction showing an increased RNA and non-histone protein content, a fast turnover of the non-histone proteins and the presence of rapidly labelled heterogeneous nuclear RNA (hnRNA) with half-life of about 20 min. Further digestion of the chromatin leads to the elimination of about 19% of the initial chromosomal DNA, thus leaving a second chromatin fraction relatively resistant to nuclease attack. This fraction has a low protein and RNA content and contains only metabolically stable non-histone proteins. No differences in the histone complement of the two fractions was found except for a 40% deficiency of H1 in the minor fraction.