Features of interactions of p68 anchorosomal protein with the nuclear envelope in the cell cycle

Chromatin associated with the nuclear envelope appears in the interphase nuclei as a layer of anchorosomes, granules 20-25 nm in diameter. The fraction of chromatin directly associated with the nuclear envelope is resistant to decondensation, shows a low level of DNA methylation, and contains specific acid-soluble proteins. However, mechanisms underlying the interaction of chromatin with the nuclear envelope are not fully understood. Specifically, it is not known whether anchorosomes are permanent structures or if they undergo reversible disassembly during mitosis, when contacts between chromatin and the nuclear envelope are destroyed. We obtained immune serum recognizing a 68 kDa protein from the nuclear envelopes fraction and studied the localization of this protein in interphase and mitotic cells. We show that this protein present in the NE/anchorosomal fraction does not remain bound with chromosomes during mitosis. It dissociates from chromosomes at the beginning of the prophase and then can be identified again at the periphery of the newly forming nuclei in the telophase.     

Aspects of Interaction of Putative Anchorosomal Protein p68 with the Nuclear Envelope during the Cell Cycle

In the interphase nucleus, the chromatin associated with the nuclear envelope is represented by a layer of anchorosomes, granules with a diameter of 20–25 nm. Biochemically, the fraction of chromatin directly associated with the nuclear envelope is characterized by resistance against decondensing influences, a low level of DNA methylation, and presence of specific acid-soluble proteins. However, the mechanisms lying at the base of chromatin-nuclear envelope interaction have been insufficiently studied. Specifically, it is unknown whether anchorosomes are constant structures or subject to reversible disassembly, when the contacts between chromatin and nuclear envelope are destroyed. We obtained immune serum recognizing a 68 kDa protein from the nuclear envelopes fraction and studied the localization of this protein in interphase and mitotic cells. We show that this protein, present in the NE/anchorosomal fraction, does not remain bound with chromosomes during mitosis. It dissociates from chromosomes at the beginning of the prophase and then can be identified again at the periphery of the newly forming nuclei in the telophase.     

Structure of chromatin containing extensively acetylated H3 and H4

I have grown HeLa cells in 5 mM sodium n-butyrate leading to extensive in vivo histone acetylation, and have characterized the structure of chromatin containing the modified histones. Nuclear DNA in butyrate-treated cells is digested 5-10 fold more rapidly by DNAase I than the DNA of control cells. Staphylococcal nuclease degrades the two nuclear samples to acid-soluble material with identical rates; this nuclease, however, does excise nucleosomes with extensively acetylated histones from the nucleoprotein chain preferentially. The physical properties of unsheared chromatin and isolated core particles from control and butyrate-treated cells are closely similar, as are the rates of digestion of core particles from the two cell preparations by DNAase I. Determination of the relative susceptibilities of cleavage sites for DNAase I demonstrates that the site 60 bases from the ends of the DNA resistant in control cells, becomes susceptible to the nuclease in core particles containing acetylated histones. Similarly, the 5' terminal phosphate at the end of DNA in core prticles is removed by staphylococcal nuclease 2-3 fold faster in particles containing acetylated histones than in particles from control cells.     

Properties of mouse spleen residual condensed chromatin associated with the nuclear matrix

Properties of condensed residual chromatin of mouse spleen, a component of residual nuclear structures, were studied. Extraction of the structures with buffers of different NaCl concentrations showed that the condensed chromatin consists of condensed nucleosomal chains. On increasing the ionic strength the complexes gradually fell apart into separate nucleosomal chains. DNA of condensed chromatin was accessible to staphylococcal nuclease and DNAase I, but digestion of this DNA was not accompanied by solubilization of the residual chromatin. Besides the essentially decreased total content of nonhistone chromosomal proteins the condensed chromatin practically did not contain HMG proteins. The nucleosome repeat length of this chromatin was shorter than that of chromatin solubilized by staphylococcal nuclease.     

Spatial localization of chromosome-nuclear envelope interaction sites.

In the interphase nucleus chromosomes are tightly associated with the nuclear envelope (NE) through special granular chromatin particles termed anchorosomes. It remains unclear whether anchorosomes represent constant nuclear structures, persisting throughout the cell cycle, or they appear only in the interphase during the formation of contacts between the chromosomes and NE. In other words, whether specific NE interaction sites do exist in chromosomes or any region can form anchorosome. In this work, we used micrononucleated PK cells, in which almost every micronucleus (MN) is formed by a single chromosome. The spatial distribution and quantitative characteristics of the anchorosomal layer in MN was studied using stereological analysis and three-dimensional computer reconstruction. It was shown that in cells with about 30 MN, the total surface area of NE reaches about 355 microm2, whereas in normal mononuclear cells it is 110 microm2. Hence, the NE surface increases 3-fold during MN formation. In contrast to normal cells, only 80% of the NE surface in MN is covered with anchorosomes, i.e., the total surface area of the anchorosomal layer increases by a factor of 2.5. The 3D reconstruction has demonstrated highly random distribution of anchorosome-free zones, the distribution patterns varying in individual MN. These findings are thought to be evidence for the existence of a limited number of specific chromosomal sites potentially capable of forming contacts with NE.     

Kinetics of nuclease digestion of Physarum polycephalum nuclei at different stages of the cell cycle

The kinetics of nuclease digestion of Physarum polycephalum nuclei by staphylococcal nuclease and DNase I has been studied at different stages of the cell cycle. Significant differences in the digestion behaviour of nuclei from metaphase and interphase have been detected with DNase I but not with staphylococcal nuclease. Furthermore the structure of newly replicated DNA in S phase differs from the bulk in that it is more easily degraded to acid-soluble products by either staphylococcal nuclease or by DNAase I. At least four types of chromatin structure can be distinguished by our digestion kinetics experiments.     

The anchorosome, a special chromatin granule for the anchorage of the interphase chromosome to the nuclear envelope

Peripheral chromatin granules bound to the nuclear envelope of rat liver nuclei have been further investigated. Judging by the results of Staphylococcal nuclease digestion of nuclei and electron microscopical observations, the peripheral granules have nucleosomal organization. As shown by ultraviolet radiation DNA-protein cross-linkage, the histone-like proteins present in the peripheral chromatin instead of histone H1 (Fais et al., 1982) are in close contact with DNA. The peripheral chromatin contains a DNA firmly bound to the lamina. This DNA, resistant to extraction in high salt, heparin and SDS, is protected against a DNase attack since, as shown by DNA electrophoresis data, high molecular weight molecules (up to 20 kbas) are still present in the lamina residue. However, the high molecular weight DNA disappeared if the nuclear envelope fraction was again DNase-digested after high salt treatment. Altogether, the data of the previous (Fais et al., 1982; Prusov et al., 1980: Prusov et al., 1982) and the present investigations demonstrate that the peripheral chromatin granules are endowed with properties which distinguish them from the bulk chromatin and account for the chromosome bond to the nuclear envelope during interphase. This is why we suggest the term "anchorosome" for the peripheral protein granule attached to the nuclear envelope.     

Aggregation of fragmented chromatin associated with the synthesis of products of its treatment with nuclease

Isolated cell nuclei were incubated with nucleases followed by extraction of chromatin with a low salt buffer. With an increase of nuclear chromatin degradation with DNAse I or micrococcal nuclease, solubilization of deoxyribonucleoprotein (DNP) by a low salt buffer increases, reaching a maximum upon hydrolysis with 2-4% nuclear DNA and then decreases appreciably after extensive treatment with nucleases. Soluble fragmented chromatin aggregates in the course of treatment with DNAase. I. Addition to gel chromatin preparations of exogenous products of nuclease treatment of isolated nuclei leads to its aggregation. Pretreatment of nuclear chromatin with RNAase prevents solubilization of DNP by low ionic strength solutions. Some experimental data obtained with the use of severe nuclease treatment are discussed; for a correct interpretation of these data the aggregation of fragmented chromatin by products of its nuclease degradation should be taken into consideration.     

Anchorage of the nucleolus in the pore complex-lamina by a DNA-bearing structure masked in situ in rat liver nuclei

We have developed a method by which nuclear shells containing nucleoli can be isolated from membrane-depleted rat liver nuclei. This method involves the removal of the internal chromatin. This chromatin is expelled from the nuclear shell using combinations of low and high ionic strength buffers. The expelled internal part is subsequently digested with DNase I or micrococcal nuclease. Examination by electron microscopy of the nuclear and the nucleolar structures at various steps of the isolation procedure shows that the nucleoli are anchored in the peripheral lamina by a pedicle that is continuous with an intranucleolar network. This network is masked in situ by nucleolar granules. The pedicle and the network which support the nucleolar DNA are composed mainly of non-histone proteins insoluble in 2M NaCl.     

Anchorage of the nucleolus in the pore complex-lamina by a DNA-bearing structure masked in situ in rat liver nuclei

We have developed a method by which nuclear shells containing nucleoli can be isolated from membrane-depleted rat liver nuclei. This method involves the removal of the internal chromatin. This chromatin is expelled from the nuclear shell using combinations of low and high ionic strength buffers. The expelled internal part is subsequently digested with DNase I or micrococcal nuclease. Examination by electron microscopy of the nuclear and the nucleolar structures at various steps of the isolation procedure shows that the nucleoli are anchored in the peripheral lamina by a pedicle that is continuous with an intranucleolar network. This network is masked in situ by nucleolar granules. The pedicle and the network which support the nucleolar DNA are composed mainly of non-histone proteins insoluble in 2M NaCl.     

Fractionation of nucleosomes by salt elution from micrococcal nuclease- digested nuclei

The solubilization of nucleosomes and histone H1 with increasing concentrations of NaCl has been investigated in rat liver nuclei that had been digested with micrococcal nuclease under conditions that did not substantially alter morphological properties with respect to differences in the extent of chromatin condensation. The pattern of nucleosome and H1 solubilization was gradual and noncoordinate and at least three different types of nucleosome packing interactions could be distinguished from the pattern. A class of nucleosomes containing 13-- 17% of the DNA and comprising the chromatin structures most available for micrococcal nuclease attack was eluted by 0.2 M NaCl. This fraction was solubilized with an acid-soluble protein of apparent molecular weight of 20,000 daltons and no histone H1. It differed from the nucleosomes released at higher NaCl concentrations in content of nonhistone chromosomal proteins. 40--60% of the nucleosomes were released by 0.3 M NaCl with 30% of the total nuclear histone H1 bound. The remaining nucleosomes and H1 were solublized by 0.4 M or 0.6 M NaCl. H1 was not nucleosome bound at these ionic strengths, and these fractions contained, respectively, 1.5 and 1.8 times more H1 per nucleosome than the population released by 0.3 M NaCl. These fractions contained the DNA least available for micrococcal nuclease attach. The strikingly different macromolecular composition, availability for nuclease digestion, and strength of the packing interactions of the nucleosomes released by 0.2 M NaCl suggest that this population is involved in a special function.     

Polyamine depletion is associated with altered chromatin structure in HeLa cells.

HeLa cells depleted of polyamines by treatment with alpha-difluoromethylornithine (DFMO), methylglyoxal bis(guanylhydrazone) (MGBG) or a combination of the two, were examined for sensitivity to micrococcal nuclease, DNAase I and DNAase II. The degrees of chromatin accessibility to DNAase I and II appeared enhanced somewhat in all three treatment groups, and the released digestion products differed from those in non-depleted cells. DNA released from MGBG- and DFMO/MGBG-treated cells by DNAase II digestion was enriched 4-7-fold for Mg2+-soluble species relative to controls. DNA released by micrococcal nuclease digestion from all three treatment groups was characterized as consisting of higher-order nucleosomal structure than was DNA released from untreated cells. At least some of the altered chromatin properties were abolished by a brief treatment of cells with polyamines, notably spermine. These studies provide the first demonstration in vivo of altered chromatin structure in cells treated with inhibitors of polyamine biosynthesis.     

Accessibility of some regions of chromatin of leukemic chicken cells to single strand nuclease S1

The sensibility to single strand nuclease S₁ of DNA from Avian leukemic cells infected with Avian Myeloblastosis virus (A.M.V.) has been studied. The resulting DNA fragments were analysed by electrophoresis on agarose gels. Fragments of discrete size appear after 10 min of digestion when less than 1 % of the DNA is rendered acid-soluble. These fragments appear as multiple of a monomeric unit and are similar to the fragments produced by micrococcal nuclease digestion. In addition integrated proviral AMV sequences were preferentially degraded by DNAase I but not by S₁ nuclease.     

Differential condensation of mouse DNA families in chromatin: accessibility to nuclease probes

A number of minor, previously unidentified GC-rich mouse DNA families have been observed in CsCl gradients. Since these DNA families are found in the DNA of mouse nuclei which is most resistant to both micrococcal nuclease and DNAase I, they must occur in highly condensed chromatin. Fractionation of high molecular weight nuclease digested DNA by sequential polyethylene glycol (PEG) precipitation demonstrates differential enrichment of these DNA families implying a differential condensation of these DNA fractions in chromatin.     

Non-histone proteins in fractionated chromatin before and after DNAse II treatment

Chromatin from spleen cells of normal, non-immunized mice and from mice 3 days after immunization with human immunoglobulin G was fractionated at increasing salt concentrations into three fractions: 0.35 M NaCl-soluble, 2 M NaCl-soluble and a residual fraction, dissociated in 2 M NaCl/5 M urea. The residual fraction of chromatin, homogeneous by ultracentrifugation and containing only 25% of the total chromatin DNA, was associated with proteins strongly labeled with [3H]tryptophan, [3H]methionine and [3H]leucine. This fraction was more sensitive to DNAase II treatment than was native, non-fractionated chromatin and it contained approx. 40% Mg2+-soluble DNA sequences. The template activity of the residual fraction was 6--7-times higher than that of non-fractionated chromatin. Fraction A, characteristic for non-immunized spleen cells, was present in three chromatin fractions and after DNAase II treatment it remained only in the residual fraction, which suggests that this fraction is associated with genes non-transcribed in non-immunized mice. Fractions I and B1 were found mainly in the residual fraction, and only in smaller amounts in the 0.35 M NaCl-soluble fraction. After DNAase II treatment, fractions I and B1 in chromatin from immunized mice disappeared, which suggests that these fractions may be associated with active transcribed sequences during the immune reaction.     

Selective release of chromosomal proteins during limited DNAase 1 digestion of avian erythrocyte chromatin

Duck erythrocyte chromatin has been treated with DNAase 1 under conditions that are known to digest selectively the structural genes coding for globin mRNAs. This limited digestion releases specific sets of nonhistone chromosomal proteins that are not preferentially released during limited digestion with micrococcal nuclease, which does not selectively attack the globin sequences. Analysis of nucleosome monomer and multimer peaks separated on sucrose gradients after limited digestion with micrococcal nuclease shows that the proteins which are released by DNAase 1 digestion remain associated with the chromatin subunits and can be removed by extraction in 0.5 M NaCl. These proteins are tentatively identified as members of the high mobility group (HMG) proteins (originally described by Goodwin, Sanders and Johns, 1973) in terms of their extractability, electrophoretic characteristics and amino acid composition.     

Analysis of cellular nucleoproteins by the nucleoprotein-celite chromatography method. III. Two types of DNA-nuclear matrix interaction

There are two types of DNA-nuclear matrix interactions in animal cells as revealed by the release of DNA from isolated nuclei by three successive gradients: NaCl, LiCl-urea and temperature. Nuclei were treated with dissociating agents while being adsorbed on the Celite columns. "Weak" DNA-matrix interactions which dissociate in 1.5 M LiCl-3 M urea at 2 degrees appear to be sensitive to ethidium bromide and resistant to exogeneous nucleases (DNAase I, DNAase II and micrococcal nuclease), to DNA-damaging agents, including alkylators and gamma-irradiation, and also to psoralen-induced cross-links. "Strong" DNA-matrix interactions proved to be very different. They dissociate in 4 M LiCl-8 M urea at approximately 90 degrees, are very sensitive to DNAase I and other nucleases, slightly sensitive to chemicals and irradiation at doses stimulating single-stranded DNA breaks, but resistant to ethidium bromide. DNA strand separation seems to be necessary prerequisite for DNA release from its "strong" complex with nuclear matrix. A model for the topological link between DNA and the nuclear matrix involved in DNA replication complex is discussed.