Rat Liver Chromatin Research Articles

The role of charge neutralization and cooperative binding of linker histone in. the higher-order structure of chromatin

The binding mode and stoichiometry of interaction between soluble rat liver chromatin and histone H1 (H1) were studied. H1 binding to chromatin is cooperative. Chromatin accepts 3.6 molecules of H1/nucleosome at 0 M salt, close to the required ratio for neutralization of 90% of the charges on the phosphate groups of chromatin (4.0 H1 molecules/nucleosome). The proposal is put forward that critical charge neutralization (90%) has a significant influence on the irregular appearance of chromatin.

Non-random reconstitution of HMG1 and HMG2 in chromatin. Determination of the histone contacts

We have studied how non-histone proteins HMG1 and HMG2 interact with rat liver chromatin using reconstitution and chemical cross-linking procedures. Both proteins were found to associate to chromatin only to some extent and always with a marked preference for short oligonucleosomes, mainly mono- and dinucleosomes. However, a slight reconstitution with the long polynucleosomal fraction can be observed in H1-depleted chromatin. Reconstitution is non-random and a clear preference for regions highly sensitive to staphylococcal nuclease (EC 3.1.31.1) is observed. Chemical cross-linking has allowed us to identify H1, H2A and H2B as the histones contacted by HMG1 and HMG2 upon reconstitution. Also, we present evidence that HMG2 and HMG2 interact with the nucleosomal particle without replacing H1 or any other histone.

Endogenous degradation of rat liver chromatin studied by agar gel electrophoresis of nuclei.

Direct agar gel electrophoresis of incubated rat liver nuclei revealed that most of the chromatin is rapidly converted to stable, large fragments, showing identical electrophoretic mobility. Short and long term incubation gave the same results. The analysis of deproteinized DNA under nondenaturing as well as denaturing conditions showed, however, a correlation between the DNA size pattern and the time of incubation. Our data on the persistance of large and uniform in size chromatin fragments despite the presence of cleaved DNA in them may indicate naturally footprinted regions of chromatin, implying most probably some strong ordered interactions of chromatin constituents. It seems that some substantial unknown features of higher order structure of chromatin are preserved in rat liver nuclei isolated and digested under the experimental conditions used.

Further purification and characterization of the DNA 3'-phosphatase from rat-liver chromatin which is also a polynucleotide 5'-hydroxyl kinase.

The DNA 3'-phosphatase activity of rat-liver chromatin has been purified. A DNA 5'-hydroxyl kinase activity comigrates at each step of purification. Both enzymes have the same molecular mass (79 kDa) and the same isoelectric point (8.6). It thus seems that the two activities are born by the same protein just as with the phage T4 enzyme which is, at the same time, a 5'-hydroxyl kinase and a 3'-phosphatase. An additional argument is that ATP, which does not influence the rate of the 3'-phosphatase reaction but which is a cosubstrate of the 5'-hydroxyl kinase, protects the 3'-phosphatase activity against thermal denaturation and trypsin digestion. The two active sites must, however, be largely independent within a common support: the thermal denaturation and trypsin inactivation rates are very different for the two activities; increasing the ionic strength activates the kinase and inhibits the phosphatase; polyvalent anions inhibit the phosphatase and have little effect on the kinase. The two active sites might belong to different domains of the protein; they could not however be separated by a partial trypsin digestion. The rates of 3'-dephosphorylation and 5'-phosphorylation by the chromatin enzyme are the same in native and denatured DNA. The 3'-phosphatase has no action on 3'-monodeoxynucleotide, but it hydrolyzes the 3'-phosphate in dinucleotides. The Km of the 3'-phosphatase is 0.548 microM. The Km (5'-OH) and Km (ATP) of the 5'-hydroxyl kinase are about 3.9 microM and 0.69 microM respectively. The chromatin enzyme is unable to hydrolyze 3'-phosphoglycolate ends in DNA.

Gibberellic Acid-mediated Protein Synthesis and Poly(ADP-ribosyl)ation in Maize Shoot Chromatin

Summary We have recently reported [Wielgat and Kleczkowski, Plant Physiol. 81, 404–407 (1986)], that purified chromatin has the ability to incorporate amino acids into acid precipitable material. In the present study the effect of gibberellic acid (GA)3) on this process as well as on poly (ADP-ribosyl)ation in maize shoot chromatin was studied. Chromatin isolated from 5-day-old etiolated maize seedlings treated with 10−5 M GA3 showed about 50% inhibition of the protein-synthesizing activity when compared to control (−GA3) plants. This effect was not observed when GA3 (10−8–10−3M) was added in vitro to the incubation mixture containing chromatin isolated from the control plants. It was also demonstrated that treatment with GA3in vivo considerably inhibited the chromatin-mediated synthesis of poly(ADP-ribose) from NAD+ . Poly (ADP-ribose) has been postulated [Umansky et al., Eur. J. Biochem. 105, 117–129 (1980)] to be a primary energy source for peptide bond formation by rat liver chromatin. We suggest, that the GA3-dependent inhibition of protein synthesis by maize chromatin results from the repression of the poly(ADP-ribose) synthetase activity.

Gene-specific differences in the supranucleosomal organization of rat liver chromatin

Rat liver chromatin was separated into a solubilized portion and insoluble nuclear material, and the solubilized portion was fractionated by sucrose gradient sedimentation. The chromatin encompassing three transcribed genes (albumin, tryptophan oxygenase, and alpha-fetoprotein), which are expressed at very different levels, partitions preferentially with insoluble nuclear material and possesses a disrupted nucleosome structure. On the contrary, the chromatin encompassing three inactive genes fractionates into the solubilized chromatin portion and exhibits a canonical nucleosome repeat structure. By sucrose gradient sedimentation, all size classes of inactive chromatin particles are found to contain internal cleavages in the linker region between nucleosomes; they are probably held together by histone H1 and mono- and divalent cations. When the chromatin encompassing two flanking sequences of the tyrosine aminotransferase gene is studied, the 0.35-kilobase upstream-located chromatin exhibits features of active genes, while the 2.55-kilobase upstream-located chromatin partitions preferentially with solubilized chromatin and sediments in internally cleaved particles.

Accessibility of the globular domain of histones H1 and H5 to antibodies upon folding of chromatin.

Antibodies to the globular domain of histones H1 and H5 were purified by affinity chromatography and used to study the accessibility of this region of H1 and H5 in folded and unfolded rat liver and hen erythrocyte chromatin respectively. The different conformations of the chromatin filament were induced by varying the ionic strength from 1 mM to 80 mM NaCl and maintained by fixation with glutaraldehyde. Treatment with glutaraldehyde at a given salt concentration affected neither the orientation of nucleosomes relative to the fiber axis nor the compactness of chromatin. Solid-phase immunoassay and inhibition experiments showed no binding of the antibody against the globular domain of H1 to chromatin at the entire range of salt concentrations, while the antibody to the whole H1 molecule reacted with chromatin at low salt. On the other hand, the antibody to the globular region of H5 reacted with hen erythrocyte chromatin independently of the extent of chromatin condensation. These results indicate that the antigenic determinants of the globular domain of H5 are accessible to the antibody both in folded and unfolded chromatin, while those of the same region of H1 are masked, probably by interaction with DNA or proteins.

The alteration in conformation of rat liver chromatin upon the administration of fluometurone and butiphose: [formula omitted], [formula omitted] Institute of Biochemistry, Tashkent, USSR

The alteration in conformation of rat liver chromatin upon the administration of fluometurone and butiphose: [formula omitted], [formula omitted] Institute of Biochemistry, Tashkent, USSR

Release of core DNA from nucleosomal core particles following (ADP-ribose)n-modification [formula omitted][formula omitted

Isolated nucleosomal core particles from rat liver chromatin were ADP-ribosylated in vitro and the consequences of this modification on intranucleosomal DNA-protein interactions were studied by retention gel analysis. A separating force of 11 fN caused the complete release of 146 bp DNA fragments from ADP-ribosylated core particles, while the DNA of unmodified core particles remained protein-associated. We conclude that posttranslational (ADP-ribose)n-modification of chromatin proteins may reduce DNA-protein interactions at the nucleosomal level of chromatin organization.

The superstructure of chromatin and its condensation mechanism. III: Effect of monovalent and divalent cations X-ray solution scattering and hydrodynamic studies.

Solutions of rat liver and chicken erythrocyte chromatin at different ionic strengths were characterized by synchrotron X-ray solution scattering, ultracentrifugation, density and viscosity measurements. Previous observations on nuclei were extended to rat liver, calf thymus and yeast nuclei. It is shown that with monovalent cations condensation is independent of the nature of the cation whereas with divalent cations there are significant differences related to the preference of base binding over phosphate binding. The consistency of hydrodynamic and scattering results confirm the view that chromatin in solution at low ionic strength has a helix-like superstructure. A survey of X-ray and neutron scattering results in the literature shows that previous interpretations, e.g. in terms of a 10 nm filament, are incompatible with the experimental data at low resolution.

Studies on the stability of the higher-order structure of rat liver chromatin containing high-mobility-group proteins.

The stability of the higher-order structure of chromatin containing high-mobility-group (HMG) proteins has been studied in rat liver nuclei by mild micrococcal nuclease digestion at low temperature and fractionation by sucrose gradient centrifugation. Nuclei preparation and digestion, chromatin solubilization and analysis have been carried out in two ionic conditions, 140 mM and 40 mM monovalent cation concentration, avoiding drastic changes in ionic conditions and temperature during preparation and analysis. During the time course of digestion at 140 mM ionic strength a material stable at 80 S appears, whose DNA is cleaved at values around 12 nucleosomes. The distribution of HMG proteins in different chromatin fractions was analyzed by immunodot using antibodies elicited against proteins HMG-1, HMG-2, and HMG-14 and 17. It appears that these proteins have a distribution distinctly different from the bulk of chromatin. They are never found in the chromatin fragments that keep their internucleosomal interactions, indicating that these proteins tend to accumulate in points where the chromatin has a less stable structure.

Analysis of nucleosome arrangement on satellite DNA of rat liver chromatin

The arrangement of nucleosomes on the nucleotide sequence of satellite DNA of Oceanian rat (Rattus rattus) has been studied. Nucleosome cores were prepared from rat liver nuclei with micrococcal nuclease, exonucleaseIII and nuclease Sl. From the total population of core DNA fragments, the satellite-containing fragments were selected by molecular cloning and the complete nucleotide sequence of these clones was determined. The data show that nucleosomes occupy a number of preferred positions on satellite DNA. These positions are strictly defined. Thus location of nucleosomes along the satellite sequence is non-random. Such finding may have important biological significance.

Stimulation of human neuroblastoma DNA polymerase alpha and primase activities by a protein factor isolated from rat liver chromatin.

Nuclear protein factor type 1 (NPF-1) that simulates IMR-32 primase-associated DNA polymerase alpha 1 and alpha 2 activities has been purified from a high-salt extract of liver chromatin from 6-month-old rats. The final purified factor lacks DNA polymerase alpha, RNA polymerase, and DNA-unwinding or topoisomerase type I activities. The stimulatory activity is destroyed by trypsin (60 min at 37 degrees C), DNase II (60 min at 37 degrees C), and heat treatment (2 min at 68 degrees C). The 125I-labeled NPF-1 does not bind to activated calf thymus DNA or poly(dC). However, it forms a ternary complex with DNA in the presence of DNA polymerase alpha-primase complex (alpha 1 and alpha 2). The ternary complex sediments on sucrose density gradient as a heavier band (11S). The NPF-1 also stimulates (2.5-fold) primase-catalyzed incorporation of GMP and dGMP from the corresponding triphosphates on poly(dC) template even in the presence of a high concentration of alpha-amanitin (400 micrograms/ml). The labeled duplex containing the poly(dC) template, [32P]-GTP, and [3H]dGTP loses 80% of the 32P label and 70% of the 3H label after treatment with 0.3 M KOH and DNase I, respectively. The products were isolated from reaction mixtures incubated with and without NPF-1 and subjected to alkaline sucrose-density-gradient sedimentation analysis. The results suggest that the rate of synthesis of DNA short chains is increased in the presence of NPF-1 without a concomitant increase in the chain length of the newly synthesized products.

Changes in mouse and rat liver non-histone chromatin proteins during azo dye-induced hepatocarcinogenesis

Two recently identified liver-specific acid-soluble nonhistone chromatin proteins (LSNHP 1 and 2) show an increase related to ageing in Sprague-Dawley rat liver chromatin and a particularly sharp increase in spontaneous hepatocarcinomas in ageing male CBA mice (Medvedev et al., 1982, 1984). Sprague-Dawley male rats do not normally develop age-related spontaneous hepatocarcinomas, whereas in ageing male CBA and CH3 mice the incidence of

The distribution of nuclear proteins and transcriptionally-active sequences in rat liver chromatin fractions

Chromatin fractions from rat liver nuclei digested by nucleases were separated by differential solubility into several fractions. Material solubilized during digestion (predominantly monomer nucleosomes and polynucleosomes) had the highest HMG14+17 DNA ratios but were not enriched in active gene sequences (albumin and c-Ha- ras1 genes). Material soluble in a low ionic strength buffer containing 0.2 mM MgCl 2 (monomer nucleosomes and polynucleosomes) contained in addition to the histones, HMG14 and 17 plus a 41K non-histone protein. This fraction was depleted in active gene sequences and enriched in inactive sequences. The insoluble material was highly enriched in active sequences and had the lowest HMG14+17 DNA ratio. This fraction could be further fractionated into a histone-containing 2 M NaCl-soluble fraction and a 2 M NaCl-insoluble matrix-bound fraction, both of which were enriched in active sequences. The results show that the HMG proteins do not partition with active sequences during fractionation of chromatin. The 41K protein may be associated with inactive chromatin fraction.

Specific binding of calcium to soluble chromatin.

Equilibrium dialysis studies and sedimentation experiments with soluble rat liver chromatin and radioactive 45Ca support the hypothesis that there is a specific binding of Ca2+ to chromatin molecules. At low ionic strength the binding constant is about 12 l/mM indicating that more than half of the negative charges of the chromatin molecules are neutralized due to the binding of Ca2+ at appropriate concentrations. This effect possibly accounts for low concentration of Ca2+ and other divalent cations being effective in inducing compact higher order structures of chromatin. Monovalent cations are not specifically bound, but high concentrations cause unspecific shielding of the charges on the chromatin molecules and thus structural transformation. Even under these high salt conditions a specific binding of Ca2+ to chromatin occurs. The binding constant is, however, only 0.4 l/mM due to the lower effective concentration of the charged chromatin molecules which is reduced by unspecific neutralizing.

Soluble and Insoluble Rat Liver Chromatin is Different in Structure and Protein Composition

Rat liver chromatin has been fractionated by different solubility in solvents of 155 mM ionic strength in soluble S and insoluble I-chromatin. Histone H1 content is lower in S as compared to I-chromatin. The HMG1/2 nonhistone proteins are observed in S-chromatin and in the nuclear pelleted residue from the chromatin isolation procedure, but no amount can be detected in I-chromatin. Thermal denaturation profiles and CD-spectra are different for S and I-chromatin indicating distinct interactions between DNA and proteins in the chromatin molecules. Both effects, differing protein content and distinct DNA-protein interactions, can be correlated with solubility and insolubility being the result of charge-charge interactions between chromatin molecules and ionic components of the solvent.

Immunofractionation of chromatin regions associated with histone H1°

Two monoclonal antibodies, which were elicited against histone H5, bind to purified rat liver chromatin and to rat liver H1o but not to rat liver H1. The monoclonal antibodies were immobilized on CNBr-Sepharose and the resulting immunoaffinity column was used to fractionate rat liver oligonucleosomes. Enzyme-linked immunoabsorbant assay (ELISA) and immunoblotting experiments indicate that the nucleosomes bound to the column were tenfold enriched in their content of H1o. Oligonucleosomes, prepared from the livers of either untreated or 3-methylcholanthrene-treated adult rats, were fractionated on the anti-H1o affinity column. The DNA purified from the unfractionated nucleosomes, from the unbound nucleosomes and from the nucleosomes which were bound to the column was examined with various 32P-labeled probes. A slight enrichment in H1o was detected in the coding region of the rat albumin gene. In contrast DNA which was bound to the column was significantly depleted in sequences hybridizing with total cellular RNA (which contains mostly ribosomal RNA) and with sequences hybridizing to the 3'-terminal region of a cytochrome P-450 gene, which is inducible by the chemical carcinogen 3-methylcholanthrene, regardless of whether isolated from control or from carcinogen-treated rat livers. Our experiments clearly demonstrate that chromatin can be efficiently immunofractionated. The results suggest that the H1o content of chromatin regions containing genes which are constitutively transcribed is not necessarily different from that of regions containing non-transcribed genes and that highly inducible genes may be segregated into chromatin regions which are depleted of H1o.

Detection and partial characterization of the chromatin-associated proteases of yeast Saccharomyces cerevisiae.

The chromatin fraction was prepared from yeast Saccharomyces cerevisiae free from cytoplasmic contamination except for a trace of mitochondria. When the yeast chromatin was incubated with histones as a substrate it showed three peaks of proteolytic activity as approximately pH 4, pH 7 and pH 11. These activities were separated from each other by differential extractions from chromatin and successive gel filtration through Sephadex G-100. Proteases were partially characterized by affinity labeling with [3H]diisopropylfluorophosphate (iPr2P-F) and by various protease inhibitors. The neutral and the alkaline proteases were serine proteases with a molecular mass of 35 kDa and 25 kDa respectively. The acidic protease showed a molecular size larger than 100 kDa on the gel filtration, and was probably an aspartyl protease because it was most strongly inhibited by pepstatin. A iPr2P-F-binding protein with a molecular mass of 66 kDa, found in chromatin, was likely to be converted to the alkaline protease of 25 kDa when chromatin was incubated at pH 10 or in 6 M urea/0.1 M phosphoric acid at the extraction. The distribution of proteolytic activities and iPr2P-F-binding proteins were compared among chromatins from different strains and from cells in different growth phases and it was found that these three proteases were present in all of them but with different proportions. Considering that rat liver chromatin contains equivalents to these proteases [Tsurugi, K. and Ogata, K. (1982) J. Biochem. (Tokyo) 92, 1369-1381], the results suggested that they play some important roles in the function of eukaryotic chromatin.

A chromatin core particle obtained by selective cleavage of histones by clostripain.

Rat liver chromatin core particles digested with clostripain yield a structurally well-defined nucleoprotein particle with an octameric core made up of fragmented histone species (designated H'2A, H'2B, H'3 and H'4, respectively) after selective loss of a sequence segment located in the N-terminal region of each core histone. Sequential Edman degradation and carboxypeptidase digestion unambiguously establish that histones H2A, H2B, H3 and H4 are selectively cleaved at the carboxyl side of Arg 11, Lys 20, Arg 26 and Arg 19 respectively and that the C-terminal sequences remain unaffected. Despite the loss of the highly basic N-terminal regions, including approximately 17% of the total amino acids, the characteristic structural organization of the nucleosome core particle appears to be fully retained in the proteolyzed core particle, as judged by physicochemical and biochemical evidence. Binding of spermidine to native and proteolyzed core particles shows that DNA accessibility differs markedly in both structures. As expected the proteolyzed particle, which has lost all the in vivo acetylation sites, is not enzymatically acetylated, in contrast to the native particle. However, proteolyzed histones act as substrates of the acetyltransferase in the absence of DNA, as a consequence of the occurrence of potential acetylation sites in the core histones thus rendered accessible. The possible role of the histone N-terminal regions on chromatin structure and function is discussed in the light of the present observations with the new core particle obtained by clostripain proteolysis.