Localization of regions of DNA attachment to the nuclear skeleton within chicken alpha-globin genes in functionally active and functionally inactive nuclei

Short DNA fragments remaining associated with the nuclear skeleton after nuclease treatment and high salt extraction of chicken erythroblasts and erythrocytes nuclei were used as hybridization probes in order to find the positions of DNA attachments to a nuclear skeleton inside the domain of alpha-globin genes. It is found that in erythroblasts nuclei a rather long region including coding sequences is associated with the nuclear skeleton. This region coincides with the region preferentially digested by DNAase I in chromatin. In erythrocytes nuclei, a specific attachment point is found in the region between 4500 and 300 bp upstream to pi-gene. Several attachments in this region are detected also in erythroblast nuclei, but they are not prominent in this case. The results are discussed in terms of the existence of stable (structural) and temporal (functional) attachments of DNA to the nuclear skeleton.     

Initiated complexes of RNA polymerase II are concentrated in the nuclear skeleton associated DNA

Several preparations of nuclear matrices containing varying amounts of DNA were obtained from mouse plasmocytoma P3-X63-Ag8.653 cells and tested for the presence of RNA polymerase II activity. It has been demonstrated that about 25% of RNA polymerase II activity detected in the original nuclei can be recovered in isolated nuclear matrices. Only DNA-bound RNA polymerase II was found in the isolated matrices, while both free and DNA-bound RNA polymerase II activities were detected in the original nuclei. RNA polymerase II activity found in the isolated matrices did not depend on the portion of DNA recovered in the nuclear matrices in a large interval between 91 and 1.5% of DNA content in the original nuclei. The conclusion has been drawn that initiated RNA polymerase II molecules are non-randomly distributed along DNA loops. They are concentrated near the points of DNA attachment to the nuclear skeleton.     

Low ionic strength extraction of nuclease-treated nuclei destroys the attachment of transcriptionally active DNA to the nuclear skeleton

We have studied how the conditions in which the nuclear matrix is isolated influence the association of transcribing DNA with the nuclear matrix. Extraction of nuclease-treated nuclei with a low ionic strength solution before a high salt nuclei with a low ionic strength solution before a high salt extraction completely abolishes this association. However, RNA removal by RNAase treatment does not affect the binding of transcribing DNA to the nuclear matrix. The nature of the association of active genes with the nuclear matrix is discussed.     

Study of the structural and structure-functional subdomains in the interphase nucleus by photostabilization

The role of the nuclear matrix components in the organization of structural and functional domains of interphase nuclei was studied using irradiation with blue light in the presence of a photosensibilized agent (Ethidium bromide). Nuclear domain resistance to extractive solution (2 M NaCl) treatment served as a criterion of irradiation-induced stabilization of different nuclear domains. The following results have been obtained: 1) the structural organization of the complexes of chromatin and clusters of replication does not depend on the state of the nuclear matrix in isolated nuclei; 2) chemical stabilization of the nuclear matrix by Cu(2+)-ions is not sufficient for the organization of chromatin domains; 3) irradiation in the presence of Ethidium bromide stabilizes domains of the nuclei, but does not lead to stabilization of the nuclear matrix internal network. Hence, the irradiation prevented extraction from the nuclear domains of nonhistone proteins which were not standard matrix proteins. Based on the results obtained, a hypothesis was proposed about a coexistence of two groups of nonhistone proteins in the cell nucleus. The first group includes proteins of the nuclear matrix involved in immobilization of scafford attachment regions and active genes. The second group includes some hypothetical structural proteins participating only in compaction of DNA of condensed chromatin.     

Structural aspects of intranuclear matrix disintegration upon RNase digestion of HeLa cell nuclei

The organization of the intranuclear elements observed in histone-depleted (2 M NaCl-extracted) HeLa cell nuclei was investigated by means of electron microscopy and two-dimensional gel electrophoresis. This work was mainly aimed at verifying whether or not an intranuclear skeleton or matrix existed, which could explain the stable attachment of RNA to the residual nuclear structure after high-salt extraction, and its three-dimensional organization. We compared the ultrastructure and the polypeptide composition of RNA-containing and RNA-depleted (RNase-treated) nuclear residues, and we visualized intermediate stages of RNase action on the intranuclear material. We showed that this material was made of two types (fibrillar and granular) of salt-resistant RNP components equally sensitive to RNase when the enzyme was used prior to high-salt extraction. At least in our material and under our experimental conditions, no intranuclear matrix could be distinguished from the residual RNP material. Our results further suggest that formation of such a matrix is a path-dependent phenomenon.     

Evidence for the existence of a nucleolar skeleton attached to the pore complex-lamina in human fibroblasts

This work deals with the types of nuclear skeletal structures obtained from human fibroblast nuclei isolated by different procedures. It is confirmed that, in somatic vertebrate cells, the pore complex-lamina is always observed, whereas the presence of internal nucleolar and extranucleolar residual structures depends upon the method of nuclear isolation used. Furthermore, the results reported here argue for the existence of a nucleolar skeleton different from the nucleolar matrix often observed in different cell types by other investigators. The conditions of nuclear isolation which allow us to visualize this nucleolar skeleton without any other internal residual structures are described. The attachment of the nucleolar skeleton to the lamina suggested by the present data is considered in relation to the in situ position of nucleoli near the nuclear envelope.     

Aging and Loss of Germination in Rye Seeds Is Accompanied by a Decreased Fragmentation of Nuclear DNA at Loop Domain Boundaries

To investigate the processes that occur in the embryo cell nuclei in the course of natural and accelerated aging of rye seeds, nuclear DNA structural organization into chromatin loop domains was studied. The loss of germination was shown to be accompanied by a decreased excision of chromatin loop domains. The study of chromatin accessibility to DNase I did not reveal any considerable changes in chromatin architecture that would explain the decreased DNA fragmentation at matrix attachment regions. A soluble nuclear protein of ca. 31 kD was found to manifest nuclease activity, which declined with the loss of germination. The study of DNA fragmentation in histone-depleted nuclei (nucleoids) disclosed a nuclease activity resistant to 2 M NaCl extraction and sensitive to the specific inhibitors of DNA topoisomerase II; the latter activity also declined with aging. The authors conclude that the changes in DNA fragmentation patterns in aging seeds were primarily caused by a decreased activity of the enzymes accounting for the excision of chromatin loop domains.     

The type of DNA attachment sites recovered from nuclear matrix depends on isolation procedure used

A large variety of DNA sequences have been described in nuclear matrix attachment regions. It could be most likely a result of the different methods used for their isolation. The idea about how different types of known DNA sequences (strongly attached to the nuclear matrix, weakly attached, or not attached) directly participate in anchoring DNA loops to the nuclear matrices isolated by different experimental procedures was tested in this study. Matrix-attached (M) and matrix-independent or loop (L) fractions as well as nuclear matrices were isolated using extractions of nuclei with 25 mM lithium 3,5-diiodosalicylate (LIS), 2 M NaCl, 0.65 M ammonium sulphate containing buffers followed by DNase I/RNase A digestion, or according to so designated conventional method. Using PCR-based and in vitro binding assays it was established that LIS and ammonium sulphate extractions gave similar results for the type of attachment of sequences investigated. The harsh extraction with 2 M NaCl or the conventional procedure led to some rearrangements in the attachment of DNA loops. As a result a big part of matrix attached sequences were found detached in the loop fractions. However, the in vitro binding abilities of the MARs to the nuclear matrices isolated by different methods did not change.     

The effect of in vitro heat exposure on the recovery of nuclear matrix-bound DNA polymerase alpha activity during the different phases of the cell cycle in synchronized HeLa S3 cells.

HeLa S3 cells were synchronized by a double thymidine block or aphidicolin treatment and the levels of nuclear matrix-bound DNA polymerase alpha activity were then measured using activated calf thymus DNA as template. The nuclear matrix was obtained by 2 M NaCl extraction and DNase I digestion of isolated nuclei incubated at 37 degrees C for 45 min prior to subfractionation. In all phases of the cell cycle 25-30% of nuclear DNA polymerase alpha activity remained matrix-bound, even when cells were in the G1 phase. No dynamic association of DNA polymerase alpha activity with the matrix was seen, at variance with previous results obtained in regenerating rat liver. The variations measured in matrix-bound activity closely followed those detected in isolated nuclei throughout the cell cycle. If nuclei were not heat-stabilized very low levels of DNA polymerase alpha activity were measured in the matrix (1-2% of total nuclear activity). Heat incubation of nuclei failed to produce any enrichment in matrix-associated newly replicated DNA, whereas the sulfhydryl cross-linking chemical sodium tetrathionate did. Therefore the results obtained after the heat stabilization procedure do not completely fit with the model that envisions the nuclear matrix as the active site where eucaryotic DNA replication takes place.     

Nucleoids, a subnuclear system capable of chain elongation

Nucleoids, prepared by salt extraction of non-DNase-digested nuclei, have properties similar, but not identical, to those of nuclear matrices which are prepared by salt extraction of DNase-digested nuclei. Nuclear matrices retained less pulse-labelled DNA, slightly less bound DNA polymerase alpha and DNA primase, but had greater in vitro DNA synthesis and in vitro priming. Nucleoids contained larger (110 S) DNA chains than nuclear matrices (30 S). Each type of residual nuclear structure could synthesize 4.5 S Okazaki fragments. When extracted with increasing concentrations of salt, DNase-digested nucleo lost the ability for further elongation of the 4.5 S DNA intermediate after 0.1-0.2 M NaCl, whereas undigested nuclei retained this ability up to 0.9 M NaCl. Chain elongation to 28 S DNA chains could be restored to nucleoids, but not to nuclear matrices, by the addition of nuclear extracts.     

Cytoplasmic intermediate filaments are stably associated with nuclear matrices and potentially modulate their DNA-binding function

The tight association of cytoplasmic intermediate filaments (cIFs) with the nucleus and the isolation of crosslinkage products of vimentin with genomic DNA fragments, including nuclear matrix attachment regions (MARs) from proliferating fibroblasts, point to a participation of cIFs in nuclear activities. To test the possibility that cIFs are complementary nuclear matrix elements, the nuclei of a series of cultured cells were subjected to the Li-diiodosalicylate (LIS) extraction protocol developed for the preparation of nuclear matrices and analyzed by immunofluorescence microscopy and immunoblotting with antibodies directed against lamin B and cIF proteins. When nuclei released from hypotonically swollen L929 suspension cells in the presence of digitonin or Triton X-100 were exposed to such strong shearing forces that a considerable number were totally disrupted, a thin, discontinuous layer of vimentin IFs remained tenaciously adhering to still intact nuclei, in apparent coalignment with the nuclear lamina. Even in broken nuclei, the distribution of vimentin followed that of lamin B in areas where the lamina still appeared intact. The same retention of vimentin together with desmin and glial IFs was observed on the nuclei isolated from differentiating C2C12 myoblast and U333 glioma cells, respectively. Nuclei from epithelial cells shed their residual perinuclear IF layers as coherent cytoskeletal ghosts, except for small fractions of vimentin and cytokeratin IFs, which remained in a dot-to cap-like arrangement on the nuclear surface, in apparent codistribution with lamin B. LIS extraction did not bring about a reduction in the cIF protein contents of such nuclei upon their transformation into nuclear matrices. Moreover, in whole mount preparations of mouse embryo fibroblasts, DNA/chromatin emerging from nuclei during LIS extraction mechanically and chemically cleaned the nuclear surface and perinuclear area from loosely anchored cytoplasmic material with the production of broad, IF-free annular spaces, but left substantial fractions of the vimentin IFs in tight association with the nuclear surface. Accordingly, double-immunogold electron microscopy of fixed and permeabilized fibroblasts disclosed a close neighborhood of vimentin IFs and lamin B, with a minimal distance between the nanogold particles of ca. 30 nm. These data indicate an extremely solid interconnection of cIFs with structural elements of the nuclear matrix, and make them, together with their susceptibility to crosslinkage to MARs and other genomic DNA sequences under native conditions, complementary or even integral constituents of the karyoskeleton.     

Simian virus 40 associates with nuclear superstructures at early times of infection.

The association of infecting simian virus 40 with insoluble nuclear structures was assayed by disrupting infected nuclei and assaying insoluble fractions for virus. Three methods were used which lyse nuclei but maintain the insolubility of residual nuclear structures: sonication, high-salt-Triton-EDTA extraction, and low-salt-lithium diiodosalicylate extraction. After each type of nuclear extraction, infecting simian virus 40 remained associated with the residual nuclear structures. This association depended strictly on natural viral infections and on the use of buffers containing moderate amounts of salt and Mg2+ for the isolation of infected nuclei. These viral interactions exhibited behavior similar to host cell DNA interactions studied by analogous assays. Both viral DNA and coat proteins were found associated with the host cell nuclear superstructure. We concluding that at early times after infection the viral templates mimic the state of the host cell chromatin by attaching to the cellular nuclear matrix.     

Magnesium and Calcium in Isolated Cell Nuclei

The calcium and magnesium contents of thymus nuclei have been determined and the nuclear sites of attachment of these two elements have been studied. The nuclei used for these purposes were isolated in non-aqueous media and in sucrose solutions. Non-aqueous nuclei contain 0.024 per cent calcium and 0.115 per cent magnesium. Calcium and magnesium are held at different sites. The greater part of the magnesium is bound to DNA, probably to its phosphate groups. Evidence is presented that the magnesium atoms combined with the phosphate groups of DNA are also attached to mononucleotides. There is reason to believe that those DNA-phosphate groups to which magnesium is bound, less than 1/10th of the total, are metabolically active, while those to which histones are attached seem to be inactive.