Modification of rat liver chromatin by N-methyl-N'-nitro-N-nitrosoguanidine or N-ethyl-N'-nitro-N-nitrosoguanidine and template activity for RNA synthesis by Escherichia coli RNA polymerase after reconstitution.

Rat liver chromatin was fractionated into DNA, histones and non-histone chromosomal proteins and each component was modified with N-methyl-l-N'-nitro-N-nitrosoguanidine of N-ethyl-N'-nitrosoguanidine. The radioactivity of 14C-labeled alkyl or guanidino moieties of both compounds bound significantly to both histones and non-histone chromosomal proteins and the binding of N-methyl-N'-nitro-N-nitrosoguanidine was higher than N-ethyl-N'-nitro-N-nitrosoguanidine. However the binding of both compounds to DNA was very low and its significance was hard to evaluate. All of the three components, one of which was modified, were reconstituted into chromatin, then, [3H]UMP incorporation into acid insoluble material using Escherichia coli RNA polymerase (EC 2.7.7.6) was measured. Only with the reconstituted chromatin containing histones modified either by N-methyl-N'-nitro-N-nitrosoguanidine or N-ethyl-N'-nitro-N-nitrosoguanidine, the template activity increased drastically; i.e., about 10 or 5 times higher than that with the unmodified reconstituted chromatin, respectively. However, any remarkable alteration in the electrophoretic pattern of protein fraction of the reconstituted chromatin could not be found. The results obtained in this study are discussed in the context that the modified histones could give rise to change in the mutual interaction of chromosomal components during the reconstitution of chromatin accompanied with the increase of chromatine template activity.     

Integrity of proteins in reconstituted chromatin.

Rat liver chromatin reconstituted from fractionated histones, chromosomal non-histone proteins, and DNA is extensively degraded by chromatin-bound protease.     

Dissociation and reconstitution of chromatin without appreciable degradation of the proteins.

When chromatin from Novikoff hepatoma ascites cells was dissociated in 3 M NaCl – 7 M urea either at pH 6 or 8, degradation of chromosomal proteins was observed in two-dimensional gel electrophoretic patterns. This degradation was not prevented by 50 mM NaHSO₃ but was prevented by 1 mM PMSF (phenylmethylsulfonyl fluoride). Reconstitution of the chromatin components dissociated in 3 M NaCl – 7 M ure ? 0.05 M sodium acetate (pH 6.0) containing 1 mM PMSF resulted in reassociation of DNA, histones and the major nonhistone proteins (B24, B26, B33, BE, BJ, C1, C6, CG, CH, CM, C14, CP, C18, CR, CS and C25). Two-dimensional gel electrophoresis showed that although the proportion of the nonhistone proteins to histones was lower in reconstituted than in native chromatin, the template activity of the reconstituted chromatin was similar to that of native chromatin.     

Effect of cell trypsinization on nuclear proteins of WI-38 fibroblasts in culture.

When resting confluent monolayers of WI-38 fibrolasts are trypsinized and replated at a lower density they are stimulated to proliferate again with an interval of 18 hours between replating and the onset of DNA synthesis. Trypsinization of resting cells causes a 40% loss of nuclear proteins as well as of cytoplasmic proteins. The amount of nuclear proteins remains low for the first six hours after the cells have been replated and then it increases rapidly, reaching the same level of non-trypsinized resting cells by ten hours after plating. The proteins that are lost from the nucleus immediately after trypsinization are chromatin-associated proteins and most of them are non-histone chromosomal proteins, although a modest loss of histones cannot be ruled out. The loss of non-histone chromosomal proteins from cells that have been trypsinized causes changes in the structure of chromatin that can be detected by circular dichroism and by viscosity measurements. These results show that cell trypsinization causes an extensive loss of proteins from chromatin and that the loss is restored only several hours after the cells have been replated at a lower density.     

Phosphorylation of nuclear proteins

Many nuclear proteins are phosphorylated: they range from enzymes to several structural proteins such as histones, non-histone chromosomal proteins and the nuclear lamins. The pattern of phosphorylation varies through the cell cycle. Although histone H1 is phosphorylated during interphase its phosphorylation increases sharply during mitosis. Histone H3, chromosomal protein HMG 14 and lamins A, B and C all show reversible phosphorylation during mitosis. Several nuclear kinases have been characterized, including one that increases during mitosis and phosphorylates H1 in vitro. Factors have been demonstrated in maturing amphibian oocytes and mitotic mammalian cells that induce chromosome condensation and breakdown of the nuclear membrane. The possibility that they are autocatalytic protein kinases is considered. The location of histone phosphorylation sites within the nucleosome is consistent with a role for phosphorylation in modulating chromatin folding.     

Effect of non-histone proteins on thermal transition of chromatin and of DNA.

The effect of chromatin non-histone protein on DNA and chromatin stability is investigated by differential thermal denaturation method. 1) Chromatin (rat liver) yields a multiphasic melting profile. The major part of the melting curve of this chromatin is situated at temperatures higher than pure DNA, with a distinct contribution due to nucleosomes melting. A minor part melts at temperatures lower than DNA which may be assigned to chromatin non-histone protein-DNA complex which destabilized DNA structure. 2) Heparin which extracts histones lowers the melting profile of chromatin and one observes also a contribution with a Tm lower that of pure DNA. In contrast, extraction on non-histone proteins by urea supresses the low Tm peak. 3) Reconstitution of chromatin non-histone protein-DNA complexes confirms the existence of a fraction of chromatin non-histone protein which lowers the melting temperature when compared to pure DNA. It is concluded that chromatin non-histone proteins contain different fractions of proteins which are causing stabilizing and destabilizing effect on DNA structure.     

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.     

Interphase chromosomal deoxyribonucleoprotein isolated as a discrete structure from cultured cells

A new procedure is described for the preparation of interphase chromatin from cultured mouse cells (line P815). The primary objective of this procedure was to eliminate exchanges of histones between deoxynucleoprotein molecules; this objective is shown experimentally to have been attained. The chromatin is released from cells by the non-ionic detergent Nonidet P40 in medium of low ionic strength (0.1 mM-KNa₂PO₄), and may then be sedimented as a structure which conserves the general form and ultrastructural characteristics of chromatin within the cell. The nuclear envelope cannot be detected in these structures by electron microscopy, and their content of choline-containing phospholipids is less than 10% of that of nuclei. The maintenance of form in this structure must thus depend on properties of the chromatin itself, and possibly on the more compact peripheral chromatin.Soluble DNP² prepared by shearing these structures has the same relative contents of DNA, histones, non-histone proteins and RNA as DNP prepared by standard methods. Analyses by electrophoresis on polyacrylamide gels of the non-histone proteins reveals certain differences from the pattern of these proteins in DNP prepared by a salt precipitation method. The template activity for RNA synthesis, in the presence of Escherichia coli RNA polymerase of sheared, soluble DNP prepared by this procedure, is comparable to that of DNP prepared by other methods. However, in the absence of exogenous RNA polymerase the rate of RNA synthesis by structured (unsheared) chromatin is about ten times higher than the rate using sheared DNP.The rapid removal of the nuclear envelope in this lysis procedure allowed experimental examination of the origin of the histones and non-histone proteins of DNP. When DNP was prepared from a mixture of two populations of cells, one containing DNA distinguishable by a density label and the other containing radioactively labelled proteins, radioactive proteins were found exclusively in DNP of normal density, and not in dense DNP and vice versa. It is concluded that the proteins of DNP prepared in this way are not acquired during the preparation procedure but were already associated with DNA in vivo, and that other proteins are not bound non-specifically to DNA during the preparation of DNP. When a mixture of DNP molecules prepared, in this way is precipitated in 150 mm-NaCl and redissolved, some radioactively labelled histones migrate onto dense DNA molecules.This procedure is suitable for routine, quantitative isolation of chromosomal DNP from small numbers of cells; it is also applicable to cells of other cultured lines.     

Binding of the estradiol-receptor complex to reconstituted nucleoacidic protein from calf uterus

Non-histone protein-DNA complexes with acceptor activity for estradiol-receptor complexes were reconstituted from fractionated calf uterine chromatin. Acceptor activity had tissue specificity with target tissue binding exceeding non-target tissue binding. The binding of estradiol-receptor complexes to acceptor sites was dependent on intact non-histone protein-DNA complexes, reconstituted select non-histone proteins, and protein equivalent: DNA reconstitution ratios. [³H]Estradiol-receptor complexes were bound to reconstituted non-histone protein-DNA complexes (i.e., nucleoacidic protein) with a high affinity and with a limited number of binding sites. Fractionation of uterine chromatin non-histone proteins identified two major sets of non-histone proteins which had acceptor activity when reconstituted with DNA. Thus, it seems possible to reconstitute nucleoacidic protein fractions with specific acceptor activity for the calf uterine estrogen receptor.     

The effect of sodium butyrate on acetylation in vitro of chromosomal proteins in three classes of liver nuclei from different ages of rats

The optimum conditions of in vitro incorporation of sodium [³H]acetate into sliced rat liver were studied. The incubations with sliced liver from three different ages of rats were performed in the presence of sodium n-butyrate. It was found that butyrate decreases the incorporation of sodium [³H]acetate into the homogenate, isolated nuclei, non-histone chromosomal proteins and histones for all age groups. The acetylations of non-histone chromosomal proteins and histones increase with age upto 2-months and decrease in 4-month-old rats both in the absence and presence of butyrate. Liver nuclei were fractionated by the simple method of zonal centrifugation into three classes, namely diploid stromal, diploid parenchymal and tetraploid parenchymal nuclei. The acetylations of non-histone chromosomal proteins and histones in three classes of nuclei of three ages of rats were studied in the presence and absence of butyrate. Butyrate can decrease the overall acetylations of non-histone chromosomal proteins and histones but increase the amount of polyacetylated histone H4 in all classes of nuclei of the three ages.     

Improved techniques for the fractionation of non-histone proteins of chromatin on hydroxyapatite.

The effect of the dissociation medium on the fractionation of chromatin on hydroxyapatite has been studied. Optimal separations of the histones and non-histone protein are only achieved when columns are run in buffers containing high concentrations of sodium ions. We have modified our previously published method such that the chromosomal proteins can be recovered in virtually quantitative yields. Each of the hydroxyapatite fractions has been analysed with respect to nucleic acid content and the proteins have been analysed by two-dimensional gel electrophoresis.     

Proteins from different classes of liver nuclei in normal and thioacetamide-treated rats

1. In normal rats the amounts of each of the main types of nuclear protein, i.e. soluble proteins, histones, non-histone chromosomal proteins and residual proteins, vary within the different classes of rat liver nuclei fractionated by zonal centrifugation. 2. Heterogeneity is observed in the non-histone chromosomal proteins prepared from different classes of liver nuclei. These differences were observed by analysis of the proteins both by sodium dodecyl sulphate-polyacrylamide-gel electrophoresis and electrofocusing electrophoresis. They are most evident between the non-histone chromosomal proteins obtained from stromal and parenchymal nuclei. However, some differences are also found for the parenchymal nuclei, between the diploid parenchymal and the tetraploid parenchymal, and between them and the nuclei involved in the synthesis of DNA respectively. 3. Drastic alterations in the nuclear proteins are found after the administration of thioacetamide. The changes observed are complex and not uniform. They vary with the age of the animal and the type of nucleus. In general an increase in the soluble proteins and non-histone chromosomal proteins and a decrease in the residual proteins is observed. There is a decrease in the specific radioactivity of soluble and residual proteins. 4. Electrophoretic analysis of the non-histone chromosomal proteins showed that specific changes occurred after administration of thioacetamide, which are different in adolescent and young adult rats.     

Protein kinase associated with chromatin and changes in substrate-specificity during germination of wheat seeds

Protein kinase activity was associated with chromatin in wheat ( Triticum aestivum L. cv. Mukakomugi) embryos. The kinase activity did not change significantly during germination, whereas the activity of poly (ADP-ribose) synthetase decreased significantly. The protein kinase activity in chromatin was inhibited by NAD, NADH, and ADP-ribose, and was enhanced by treatment of the chromatin with snake venom phosphodiesterase or soybean trypsin inhibitor. The activity in chromatin was not stimulated by cyclic AMP. Different subfractions of the histones, H1 and H2, were mainly phosphorylated in germ and 3 day-germinated seedling chromatins. The histones, H3 and H4, seemed unable to accept phosphate from ATP in the in vitro reaction system. Different acidic non-histone chromosomal proteins were phosphorylated in germ and 3-day-germinated seedling chromations, and germ-specific and seedling-specific acidic non-histone chromosomal proteins seemed unable to accept phosphate from ATP.     

Interaction of the non-histone protein PS1 with some chromatin components

The binding of non-histone protein from mouse spleen chromatin located in the sites highly sensitive to micrococcal nuclease and DNA-ase I, to DNA and histones was studied. The binding of the DNA-protein complexes to nitrocellulose filters demonstrated the absence of protein binding to DNA. A highly selective binding of protein PS1 to histones H1 and H2A and to one of the non-histone proteins (presumably HMG 14) was revealed. It is concluded that protein PS1 is incorporated into chromatin by the protein-protein interactions.     

Fractionation of chick oviduct chromatin. Nuclease-resistant deoxyribonucleic acid.

Chromatin isolated from several chick tissues was treated with micrococcal nuclease. A limited degree of tissue specificity of chromatin DNA resistance to nuclease digestion was observed. No difference in the extent of nuclease resistance of chromatin DNA was detected during oestrogen-induced oviduct differentiation. This suggested that the amount of non-histone chromosomal protein does not play an important role in the sensitivity of chromatin DNA to nuclease digestion. Studies of nuclease resistance of chromatin DNA after dissociation and reconstitution of chromatin proteins and ethanol extraction of chromatin indicate that the histones protect the DNA from nuclease attack. Slow thermal denaturation of nuclease-resistant DNA suggests that the protected DNA sequences may be (A+T)-rich, and the (G+C)-rich satellites present in total chick DNA are sensitive to nuclease.