Presence of non-histone proteins in nucleosomes

It has been established that nucleosomes are made of histones and DNA fragments. The purpose of this work to establish whether some non-histone proteins are also present in these chromatin subunits. We have found that nucleosome preparations contain phosphorylated non-histone proteins and protein kinases by sucrose gradient analysis. In order to establish whether these proteins are actually bound to nucleosomes or if they represent unbound or aggregated proteins, the following experiments were performed. (a) Free non-histone proteins and proteins released from chromatin by DNase overdigestion were analyzed by sucrose gradient centrifugation. No phosphoproteins but some phosvitin kinase activity was found in the part of the gradient which contained the nucleosomes. It could be assumed that part of the phosphoproteins are bound to nucleosomes. (b) A digestion of nucleosomes with DNase I suppressed the phosvitin kinase activity in the 11-S region of the gradient. (c) High ionic strength, which extracted non-histone proteins, suppressed the phosvitin kinase activity in the nucleosome region. Part of phosvitin kinase and of nuclear phosphoproteins are therefore bound to nucleosomes and are released by nuclease digestion and by high ionic strength.     

Chromatin fractionation according to the strength of its matrix bond

Extraction in low salt concentration followed by centrifugation allows rat liver nuclear chromatin to be divided into two fractions: the supernatant chromatin and matrix chromatin. The former fraction contains about 60-70% of initial DNA and about 15% of initial protein along with all five histones, and an insignificant amount of non-histone proteins. RNA synthesis in the matrix chromatin fraction is 2-3 times more intense than that in the original nuclei. The data on gradient centrifugation do not suggest the elongation of RNA molecules synthesized in the matrix fraction. The results obtained as compared with the literature data suggest that the matrix chromatin fraction is enriched with active genes.     

Ionophore A23187 raises cyclic AMP levels in macrophages by stimulating prostaglandin E formation.

A class of non-histone chromatin proteins that were bound tightly to DNA and could not be dissociated from the chromatin by high salt and urea was isolated from HeLa cell nuclei and separated from DNA by DNase digestion. These ‘tight’ proteins retained their ability to bind to single- and double-stranded DNA as assayed by nitrocellulose filter binding. Polyacrylamide gel electrophoresis showed that the most prominent proteins possessed molecular weights of about 60 000 D. In asynchronously growing HeLa cell cultures about $\text{1}\text{3}$ of the cell nuclei were immunoreactive to fluorescein-labeled antinucleoside antibodies. The immunoreactive cells were the fraction in S phase. Cycloheximide treatment of the cultures raised the fraction of immunoreactive nuclei to over $\text{sol2}\text{3}$. Exposure of the fixed cycloheximide-treated cell to tight proteins prior to staining with the antibody reduced the fraction of immunoreactive cells to the normal S phase level. Immuno-reactivity induced by X-irradiation or by the intercalating mutagen hycanthone was also suppressed by tight proteins. Cycloheximide treatment preferentially reduced the cellular content of tight proteins, suggesting that these proteins undergo a metabolic turnover with a half-life of about 5 h.     

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.     

Antibodies to 5 M urea soluble chromosomal proteins from HeLa cells

The tissue specificity of a chromosomal protein fraction, extractable from chromatin with 5 M urea at low ionic strength, has been examined in HeLa, A549 and HT 29 cells. Electrophoresis in polyacrylamide gels indicates that each cell type has a different content of 5 M urea soluble proteins which are distinguishable from the histones, from the tight DNA-binding proteins and from the high-mobility-group chromosomal proteins. Antibodies against 5 M urea soluble proteins extracted from HeLa cells were produced in mice. Although each of the mice tested prior to immunization contained a detectable amount of antibodies against both the 5 M urea soluble proteins and tight DNA-binding proteins, immunization elevated the level of the antibodies in the serum over 100-fold. The antibodies do not distinguish between the 5 M urea extracts obtained from different sources because most of the antibodies are directed against antigens shared by the cells studied. Immunofluorescence studies reveal that components which cross-react with 5 M urea soluble chromosomal proteins are also present in the cytoplasm. We conclude the following. (1) 5 M urea extracts from chromatin a group of proteins which differs among cells. (2) Mice contain detectable amounts of autoantibodies against these chromosomal proteins. (3) Immunization with the 5 M urea extractable fraction elicits antibodies against a restricted number of antigenic components which are shared among the cells studied. (4) 5 M urea extractable proteins are found both in the nucleus and cytoplasm; part of these may be cytoskeletal elements. Because the antisera do not react with histones, high-mobility-group proteins and tight DNA-binding proteins, they may be used for various functional studies on the 5 M urea extractable chromosomal protein fraction.     

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.     

Changes in chromatin structure at the replication fork. DNase I and trypsin-micrococcal nuclease effects on approximately 300- and 150-base pair nascent DNAs

DNase I, trypsin, and micrococcal nuclease are used to further probe the structure of nascent deoxyribonucleoprotein (DNP) fractions which appear after in vivo 20-s pulse labeling of sea urchin embryos with [3H]thymidine. We present evidence that the large nascent DNP which protects the approximately 300-base pair large nascent DNA consists of at least one nucleosome core. This is based on fractionation in denaturing polyacrylamide gels of DNA extracted from large nascent DNP fractions of a micrococcal nuclease + DNase I digest of nuclei. The data also suggest the existence of a DNase I-hypersensitive site(s) within the large nascent DNP; this is consistent with the hypothesis that the latter consists of closely packed dinucleosome cores. Histone H1 and non-histone proteins do not account for the previously reported unusual hyperresistance of the large nascent DNA against micrococcal nuclease. The protection offered this approximately 300-base pair nascent DNA was not eliminated by an 0.2-microgram/ml trypsin pretreatment which removes the above proteins from the chromatin. However, 5-10 micrograms/ml of trypsin, which remove a portion of the NH2 termini of the four core histones of nucleosomes, eliminate the hyperresistance of the large nascent DNA to subsequent micrococcal nuclease digestion, while nascent and bulk monomer DNAs remain unaffected. This indicates histone-histone and/or histone-DNA interactions within the large nascent DNP which differ from those of nascent and bulk mononucleosome cores.     

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.     

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.     

Tightly-bound non-histone proteins in different nucleosome-like subpopulations from pig kidney chromatin

By differential sucrose gradient centrifugation of pig kidney chromatin in the presence or absence of Na-EDTA and under varying ionic strength conditions, three nucleosome-like subpopulations with different buoyant densities can be obtained. These particles, on the basis of their histones and HMG protein pattern, of the 5-methylcytosine level of their DNA and of the RNA polymerase activity associated with them, can be considered as originating from chromatin fractions differently involved in gene expression. Two-dimensional electrophoresis of the tightly-bound non-histone proteins shows a distinct pattern for each subpopulation, such protein components being notably present in restricted numbers but in high amounts in the subpopulation which was apparently derived from condensed heterochromatin.     

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.     

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.