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.     

Role of non-histone proteins in chromatin solubility

Treatment of chromatin gel with low ionic strength solution of tRNA has produced the dioxyribonucleoprotein (dnptRNA) in which only part of non-histone proteins was removed without loss of any major histone fraction. The solubility of DNP in the presence of 0.15 M NaCl and 1 to 5 mM MgCl2 was considerably higher than that of initial untreated chromatin. It has been assumed that the solubility of chromatin depended primarily on some non-histone proteins and not on H1 histone.     

Changes in composition of acid soluble proteins and DNA in chromatin of rat liver and brain bound and not bound to nuclear envelope as a function of age and under the influence of antioxidant ionol

In two-day rat pups, the histone H1 content in the brain chromatin was higher than in the liver chromatin, as compared to histone of the nucleosome core. The H1 content in the brain chromatin decreased with the age, while in the liver chromatin it increased. At the same time, in the adult brain chromatin bound to the nuclear envelope, a high level of H1 characteristic of chromatin of the newborn rats was preserved, while in a similar chromatin of the adult liver, the H1 content increased, but still remained less than in the chromatin not bound to the nuclear envelope. In both organs, the composition and quantitation of H1 subfractions were different in chromatins bound and not bound to the nuclear envelope. The chromatin from the liver and brain bound to the nuclear envelope differed also in the composition and quantitation of minor acid soluble proteins. In the presence of the antioxidant ionol, the 5-methylcytosine content in DNA of chromatin of the rat liver bound to the nuclear envelope increased while in the chromatin not bound to the nuclear envelope, it remained unchanged. Thus the chromatins bound and not bound to the nuclear envelope differ in the composition and mount of acid soluble proteins, including histone H1, the contents of these proteins in bound and not bound chromatin are different and change with the age in different ways. The antioxidant ionol affects differently the methylation of bound and not bound chromatin.     

Changes in Composition of Acid Soluble Proteins and DNA in Chromatin of Rat Liver and Brain Bound and Not Bound to Nuclear Envelope as a Function of Age and under the Influence of Antioxidant Ionol

In two-day rat pups, the histone H1 content in the brain chromatin was higher than in the liver chromatin, as compared to histone of the nucleosome core. The H1 content in the brain chromatin decreased with the age, while in the liver chromatin it increased. At the same time, in the adult brain chromatin bound to the nuclear envelope, a high level of H1 characteristic of chromatin of the newborn rats was preserved, while in a similar chromatin of the adult liver, the H1 content increased, but still remained less than in the chromatin not bound to the nuclear envelope. In both organs, the composition and quantitation of H1 subfractions were different in chromatins bound and not bound to the nuclear envelope. The chromatin from the liver and brain bound to the nuclear envelope differed also in the composition and quantitation of minor acid soluble proteins. In the presence of the antioxidant ionol, the 5-methylcytosine content in DNA of chromatin of the rat liver bound to the nuclear envelope increased while in the chromatin not bound to the nuclear envelope, it remained unchanged. Thus the chromatins bound and not bound to the nuclear envelope differ in the composition and mount of acid soluble proteins, including histone H1, the contents of these proteins in bound and not bound chromatin are different and change with the age in different ways. The antioxidant ionol affects differently the methylation of bound and not bound chromatin.     

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.     

The conformation of proteins in chromatin. A circular dichroism study below 250 nm.

This paper is an investigation of the circular dichroism (CD) spectra of DNA and protein in chromatin. The circular dichroism (CD) of chromatin below 250 nm is due to DNA and protein peptide chromophores. The spectrum in this region is resolved into contributions from salt-extractable proteins (histone and non-histone proteins extractable with sodium chloride), residual non-histone proteins (not extractable with 3 M sodium chloride), and DNA. Below 250 nm, DNA in chromatin has the same CD spectrum as DNA free in solution, in contrast to the CD of DNA above 250 nm (Hjelm, R. and Huang, R. C., (1974), Biochemistry 13, 5275). Histones and salt-extractable non-histone proteins in chromatin are seen to have an average CD like those observed for globular proteins. The average CD of the residual non-histone proteins is consistent with a population of proteins with more extended conformation. The CD of each of these components is found to be the same in chromatins isolated from tissues having different nuclear synthetic activities: chick embryo brain, pig cerebellum, myeloma K41, calf thymus, and chicken erythrocyte.     

Isolation of the Chromocenter Fraction from Mouse Liver Nuclei

A new method for isolation of the constitutive heterochromatin (chromocenters) from interphase nuclei of mouse liver has been developed. This method allows separation of chromocenters of different size. Chromocenter fractions are essentially free of nucleoli and other contaminants. In contrast to nuclei and nucleoli, the chromocenter fraction is characterized by simpler protein composition, this fraction having a reduced number of proteins (especially high molecular weight proteins). Chromocenters contain all histone fractions; however, the relative proportion of histone H1 is lower and histone H3 is higher than in the total nuclear chromatin. The amount of non-histone proteins of 51, 63, 73, and 180 kD is higher in the chromocenter fraction than in nuclei and nucleoli. The use of immunocytochemistry and immunoblotting methods revealed the presence of the specific kinetochore component, CENP A protein. This suggests tight association of some molecular kinetochore components with chromocenters in the interphase.     

Lipids as integral constituents of nuclear and chromatin fractions in Ehrlich ascites tumor cells

Lipid content and composition of DNA, histone and non-histone proteins of Ehrlich ascites tumor cell chromatin were investigated. All fractions contained small amounts of lipids, mostly neutral ones, in a specific distribution. According to isotopic studies with labeled lipid precursors, incorporation took place mainly in the non-histone fraction. These findings suggest that neutral lipids attached to non-histone chromosomal proteins may also contribute to the regulatory functions ascribed to phospholipids.     

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.     

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.     

Interaction of non-histone proteins with DNA and chromatin from Drosophila and mouse cells. Specificity, isolation and analysis of complexes.

The specificity of the binding of purified non-histone proteins to DNA has been investigated through two types of experiments. Using a nitrocellulose filter assay at a low protein/DNA ratio, the binding of mouse non-histone proteins to mouse DNA was twice as great as the binding of mouse non histone protein to Drosophila DNA. The reverse experiment using Drosophila non-histone protein confirmed the interpretation that some protein . DNA complexes were specific. Protein . DNA complexes isolated by gel filtration chromatography indicated that 20% or 10% of the non-histone protein was bound to homologous or heterologous DNA respectively. Purified non-histone proteins bound with lower efficiency (15%) than unpurified but with higher specificity to soluble chromatin than to naked DNA. This binding did not result from an exchange between chromatin non-histone proteins and purified non-histone proteins added in excess. DNA-bound and chromatin-bound proteins were analysed on polyacrylamide gels. Whereas no major qualitative differences were observed with DNA-bound proteins, some proteins bound to homologous mouse chromatin were different from those bound to heterologous Drosophila chromatin. These results suggest a possible role of DNA-bound non-histone proteins in the regulation of gene expression.     

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.     

The binding of [3H]chlorambucil to nuclear proteins of the Yoshida ascites sarcoma.

The binding of [3H]chlorambucil to nuclear proteins, extracted from Yoshida ascites sarcoma cells at 6 h and 24 h after administration of 3H-labelled drug to tumour-bearing animals, has been examined. Both covalent and non-covalent binding was detected. Considerably more drug was found associated with the proteins isolated from the tumour sensitive to the effects of the drug compared with similar proteins isolated from the tumour with an acquired resistance to the effects of alkylating agents. The two-fold difference in binding to total cell protein is attributed to a higher intranuclear protein binding in sensitive cells. In particular the soluble nuclear sap fraction from sensitive cells bound at least five times as much drug as the corresponding fraction from resistant cells. Low levels of binding to histones were demonstrated compared with that to the non-histone chromatin proteins. Binding to the nuclear sap and non-histone chromatin proteins was principally to high molecular weight protein species; these did not appear to represent aggregation products as scans of stained polyacrylamide gels of the extracted protein fractions were unaltered by the treatment of tumour-bearing animals with chlorambucil. Binding to the nuclear proteins from sensitive cells tended to persist over a 24-h period, whereas it was considerably reduced in resistant cells.     

Study of peripheral chromatin granules--anchorosomes

The granular particles of chromatin peripheral layer, were isolated together, with the nuclear envelope by treatment of nuclei with nuclease. These particles differ from total chromatin by a decreased content of histone H1, a specific set of minor acid-soluble proteins and a low DNA methylation level. Taking account of the fact that these particles facilitate chromatin interaction with the nuclear envelope, the latter were termed as "anchorosomes". Using UV-induced cross-linking of DNA to the proteins, it was found that all anchorosome-specific acid-soluble proteins can directly interact with anchorosomal DNA. Treatment of anchorosomes with staphylococcal nuclease and electron microscopic data showed that anchorosomes have a nucleosomal organization. Five to ten per cent of anchorosomal DNA appear to be firmly bound to nuclear lamina. This DNA cannot be separated from the lamina by treatment with 2 M NaCl, 1% SDS or heparin (1 mg/ml). The bulk of DNA in the laminal fraction after treatment with the above reagents is protected from hydrolysis with DNAase I by anchorosomal proteins and thus has a high molecular weight (10,000-30,000 base pairs). After treatment of anchorosomes with 0.6 M or 2 M NaCl, DNAase I splits this DNA, predominantly to minor fragments.     

Chromatin-associated proteins of the developing sea urchin embryo. I. Kinetics of synthesis and characterization of non-histone proteins

Chromatin-associated non-histone proteins of the sea urchin embryo are heterogeneous, and undergo qualitative as well as quantitative changes throughout early development. The rate of synthesis of these proteins is fairly constant to the pluteus stage and, in contrast to histone synthesis, does not parallel changes in the rates of synthesis of DNA. Evidence for a pool of chromatin-associated nonhistone proteins is provided by following the kinetics of entry into chromatin of labeled protein in pulse-chase experiments of prolonged duration. This pool is synthesized during cleavage and some non-histone proteins continue to associate with chromatin until gastrulation. In addition, different rates of entry of non-histone proteins into chromatin could be detected at different stages.     

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.