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PubMed1986

The relationship between chromosomal origins of replication and the nuclear matrix during the cell cycle.

A cytological investigation into the dynamic behaviour of the origins of replication with respect to the nuclear matrix has been carried out on Xenopus laevis cultured cells. In order to preferentially label origins or 'non-origin' regions along DNA fibres, 5-fluoro-2'-deoxyuridine (FUdR)-treated cells were pulsed with [3H]deoxyadenosine in early or late S phase. Samples were then allowed to proceed through the cell cycle for increasing times. The DNA loops were induced in situ to completely uncoil around the nuclear matrix. The autoradiographic analysis shows that, under the experimental conditions used, 'non-origin' regions behave as expected from previous studies, i.e., they associate with the nuclear matrix only when they become part of a replication fork, whereas active origins of replication remain associated with the matrix throughout the cell cycle.

Animals↗
PubMed1998

Immunocytochemical detection of emerin within the nuclear matrix.

Emerin, the protein whose production is altered in the X-linked form of Emery-Dreifuss muscular distrophy, has been hypothesized to be associated with the nuclear matrix on the basis of biochemical studies. In addition, immunocytochemical data reported its localization at the nuclear periphery, on the nuclear lamina, in sections of several normal tissues. We investigated the association of emerin with the nuclear matrix, by using cultured cells (SaOS-2, MG63 and HeLa-S3) and their in situ extracted matrix as a model, and immunocytochemical methods, both at the light and electron microscope level. Our results show a normal presence of emerin in the cultured cells and the specific persistence of emerin on the lamina of the in situ extracted nuclear matrix. This suggests a tight binding between emerin and the nuclear lamina independently from the interactions between the C-terminal hydrophobic domain of the protein and the inner nuclear membrane.

Cell Nucleus↗
PubMedDate not supplied

[Endogenous DNases as a tool for isolation of nuclear matrix: critical parameters of nucleolysis].

Degree of nucleolysis has critical significance for isolation of nuclear matrix (NM) specifically enriched in transcribed DNA sequences as demonstrated at the example of inactive (c-fos, c-myc, and Ck) and active (p53, albumin, and 28S rRNA) genes in resting hepatocytes. Optimal degree of nucleolysis features degradation of loop domains of chromatin with preserved relatively uniform molecular weight distribution of DNA. Deviation from these parameters leads to nonspecific fragmentation of chromatin in various gene loci and isolation of NM samples nonspecifically enriched or depleted of transcribed DNA sequences. Under optimal hydrolytic conditions, the transcribed chromatin is more resistant to endogenous DNase attack, which allows selective conservation of its association with the nuclear matrix.

Albumins↗
PubMed1998

A nuclear matrix attachment region upstream of the T cell receptor beta gene enhancer binds Cux/CDP and SATB1 and modulates enhancer-dependent reporter gene expression but not endogenous gene expression.

We have previously identified a DNase I-hypersensitive site in the T cell receptor beta locus, designated HS1, that is located 400 base pairs upstream of the transcriptional enhancer Ebeta and is induced during CD4(-)CD8(-) to CD4(+)CD8(+) thymocyte differentiation. Using electrophoretic mobility shift assays, we show that HS1 induction correlates with increased binding of two nuclear factors, Cux/CDP and SATB1, to a 170-base pair DNA sequence within HS1. Furthermore, we demonstrate that HS1 is a nuclear matrix attachment region, referred to as MARbeta. These findings demonstrate that an analogous organization of cis-regulatory elements in which a nuclear matrix attachment region is in close proximity to an enhancer is conserved in the immunoglobulin and T cell receptor loci. In addition, we show that MARbeta represses Ebeta-dependent reporter gene expression in transient transfection assays. However, the targeted deletion of MARbeta from the endogenous locus does not change T cell receptor beta gene transcription in developing T cells. These contrasting results suggest a potential pitfall of functional studies of nuclear matrix attachment regions outside of their natural chromosomal context.

Animals↗
PubMed1989

Association of Tat protein and viral mRNA with nuclear matrix from HIV-1-infected H9 cells.

The transactivating protein from human immunodeficiency virus type 1 (HIV-1), Tat, was found to bind to the nuclear matrix from uninfected and HIV-1-infected H9 cells. Addition of the Zn2+, Cd2+ and Cu2+ chelator o-phenanthroline destroyed the matrix fibrils and the binding affinity of Tat to the matrix. A sequential treatment of the matrix, first with o-phenanthroline and then with ZnCl2, partially restored the fibrillar-like matrix structure. Infection of H9 cells with HIV-1 resulted in a displacement of cellular mRNA by viral mRNA from the nuclear matrix. Both the matrix-bound host cell and HIV-1 mRNA were found to dissociate from the matrix in the presence of o-phenanthroline. This could be prevented by coincubation with Zn2+ or Cu2+ (but not Mg2+), which stabilize the mRNA containing nuclear matrix structure.

Cell Nucleus↗
PubMed1984

Preferential repair of nuclear matrix associated DNA in xeroderma pigmentosum complementation group C.

The distribution of ultraviolet-induced DNA repair patches in the genome of xeroderma pigmentosum cells of complementation group C was investigated by determining the molecular weight distribution of repair labeled DNA and prelabeled DNA in alkaline sucrose gradients after treatment with the dimerspecific endonuclease V of bacteriophage T4. The results were consistent with the data reported by Mansbridge and Hanawalt (1983) and suggest that DNA-repair synthesis in xeroderma pigmentosum cells of complementation group C occurs in localized regions of the genome. Analysis of the spatial distribution of ultraviolet-induced repair patches in DNA loops attached to the nuclear matrix revealed that in xeroderma pigmentosum cells of complementation group C repair patches are preferentially situated near the attachment sites of DNA loops at the nuclear matrix. In normal human fibroblasts we observed no enrichment of repair-labeled DNA at the nuclear matrix and repair patches appeared to be distributed randomly along the DNA loops. The enrichment of repair-labeled DNA at the nuclear matrix in xeroderma pigmentosum cells of complementation group C may indicate that the residual DNA-repair synthesis in these cells occurs preferentially in transcribing regions of the genome.

Cell Nucleus↗
PubMed1995

Protein 4.1 is a component of the nuclear matrix of mammalian cells.

Protein 4.1 is a major component of the erythrocyte membrane skeleton that promotes the interaction of spectrin with actin and links the resulting complex network to integral membrane proteins. Here we analyse the distribution of different 4.1 proteins within the nucleus of mammalian cells. Nuclear matrices have been prepared from Madin-Darby canine kidney (MDCK) and HeLa cells and protein fractions isolated at each step of the purifications have been analysed by immunoblotting using characterized polyclonal antibodies against protein 4.1. Two 4.1 polypeptides of M(r) approximately 135,000 and 175,000 are extracted after DNase I digestion and 0.25 M ammonium sulphate treatments, suggesting that they may be associated with chromatin. Interestingly, nuclear matrices isolated after DNase I digestion and sequential treatments with increasing ionic strength contain a third 4.1 polypeptide of M(r) approximately 75,000 (4.1p75), suggesting that it is a component of the nuclear matrix. Immunoblot analyses of nuclear matrices isolated from different cell types and species indicate that 4.1p75 is a common element of the nuclear matrix of mammalian cells. Moreover, 4.1p75 distributes to typical nuclear speckles which are enriched with the spliceosome assembly factor SC35, as revealed by double-label immunofluorescence analyses. Protein 4.1p75 might be an anchoring element of the nucleoskeleton, playing a role similar to that described for the erythroid protein 4.1 in red blood cells.

3T3 Cells↗
PubMed2003

Nucleophosmin/B23 is a proliferate shuttle protein associated with nuclear matrix.

It has become obvious that a better understanding and potential elucidation of the nucleolar phosphoprotein B23 involving in functional interrelationship between nuclear organization and gene expression. In present study, protein B23 expression were investigated in the regenerative hepatocytes at different periods (at days 0, 1, 2, 3, 4, 7) during liver regeneration after partial hepatectomy on the rats with immunohistochemistry and Western blot analysis. Another experiment was done with immunolabeling methods and two-dimensional (2-D) gel electrophoresis for identification of B23 in the regenerating hepatocytes and HepG2 cells (hepatoblastoma cell line) after sequential extraction with detergents, nuclease, and salt. The results showed that its expression in the hepatocytes had a locative move and quantitative change during the process of liver regeneration post-operation. Its immunochemical localization in the hepatocytes during the process showed that it moved from nucleoli of the hepatocytes in the stationary stage to nucleoplasm, cytoplasm, mitotic spindles, and mitotic chromosomes of the hepatocytes in the regenerating livers. It was quantitatively increased progressively to peak level at day 3 post-operation and declined gradually to normal level at day 7. It was detected in nuclear matrix protein (NMP) composition extracted from the regenerating hepatocytes and HepG2 cells and identified with isoelectric point (pI) value of 5.1 and molecular weight of 40 kDa. These results indicated that B23 was a proliferate shuttle protein involving in cell cycle and cell proliferation associated with nuclear matrix.

Animals↗
PubMed2006

Merbarone induces activation of caspase-activated DNase and excision of chromosomal DNA loops from the nuclear matrix.

Studies were carried out to address possible cellular mechanisms by which merbarone, a catalytic inhibitor of DNA topoisomerase II, can block tumor cell growth without inducing extensive DNA cleavage. Merbarone induced the release of high molecular weight DNA fragments from the nuclear matrix of HL-60 leukemia cells, which preceded the internucleosomalsize DNA fragmentation characteristic of late-stage apoptosis. The chromatin fragments were enriched in a matrix attachment region (MAR) sequence compared with a non-MAR sequence and were similar in size to DNA loops extracted from nuclear matrices. However, merbarone did not directly induce the excision of high molecular weight DNA fragments from the nuclear matrix by promoting topoisomerase II-catalyzed DNA cleavage, because the drug inhibited topoisomerase II-mediated cleavage in isolated nuclear matrix preparations. Instead, merbarone induced rapid activation of the mitochondrial apoptosis pathway, which included the following temporal sequence of events: dissipation of the mitochondrial transmembrane potential within 30 min, release of mitochondrial cytochrome c, and activation of caspase-activated DNase (CAD) by its inhibitor ICAD. The excision of high molecular weight DNA was inhibited at least 80% in merbarone-treated cells preincubated with the pan-caspase inhibitor z-VAD-fmk [Z-Val-Ala-Asp(OMe)-fluoromethyl ketone] and in caspase-resistant Jurkat cells (ICAD/double-mutated) that express a mutant form of ICAD. These results provide evidence that merbarone can induce rapid disorganization of DNA in tumor cells that have a functional mitochondrial apoptosis pathway without inducing extensive DNA cleavage.

Catalysis↗
PubMed2005

Induction of transcription within chromosomal DNA loops flanked by MAR elements causes an association of loop DNA with the nuclear matrix.

The spatial organization of an approximately 170 kb region of human chromosome 19, including CD22 and GPR40-GPR43 genes, was studied using in situ hybridization of a set of cosmid and PAC probes with nuclear halos prepared from proliferating and differentiated HL60 cells. The whole region under study was found to be looped out into the nuclear halo in proliferating cells. It is likely that the loop observed was attached to the nuclear matrix via MAR elements present at the flanks of the area under study. Upon dimethyl sulfoxide-induced differentiation of the cells the looped fragment became associated with the nuclear matrix. This change in the spatial organization correlated with the activation of transcription of at least two (CD22 and GPR43) genes present within the loop. The data obtained are discussed in the framework of the hypothesis postulating that the spatial organization of chromosomal DNA is maintained via constitutive (basic) and facultative (transcription-related) interactions of the latter with the nuclear matrix.

Base Sequence↗
PubMed2000

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.

Ammonium Sulfate↗
PubMed2002

Characterization of the nuclear matrix proteins in a transgenic mouse model for prostate cancer.

The nuclear matrix (NM) contains a number of proteins that have been found to be associated with transformation. We have previously identified changes in the NM associated with prostate cancer. In this study, we examine the molecular changes that are associated with prostate cancer development in transgenic adenocarcinoma of mouse prostate (TRAMP) model by studying the differences in the NM proteins (NMPs). We collected prostates from the TRAMP males at six critical time points: 6 weeks (puberty), 11 and 19 weeks (development of mild hyperplasia), 25 weeks (development of severe hyperplasia), 31 and 37 weeks (development of neoplasia). The nuclear matrices from the prostates collected at these time points were then isolated and the NMPs were characterized by high-resolution two-dimensional gel electrophoresis. We found three NMPs (E1A, E1B, and E1C) that were present in the 6-week-old prostate and two NMPs (E2A and E2B) that were present in the 11-week-old prostate. These NMPs were absent in the 31- and 37-week-old prostate. We also found five NMPs (E3A-E3E) that were present in the 31-week-old prostate, but absent in the earlier time points. In addition, three NMPs (Le1, Le2, Le3) were present at higher expression in the 6-, 11-, 19-, and 25-weeks old TRAMP prostates, but they were expressed lower during the development of neoplasia at 31- and 37-weeks old. Identification of these NMPs permits the development of novel markers that can characterize various stages of prostate cancer development as well as potentially therapeutic targets.

Adenocarcinoma↗
PubMed2005

Co-localization of poly(ADPR)polymerase 1 (PARP-1) poly(ADPR)polymerase 2 (PARP-2) and related proteins in rat testis nuclear matrix defined by chemical cross-linking.

Poly(ADPR)polymerase 1 and 2 (PARP-1, PARP-2) are nuclear enzymes which function is based on specific interactions with DNA and nuclear proteins. PARPs targets include proteins involved in DNA replication, repair, and transcription and their function can be modulated either by protein-protein interaction with native PARP-1 and by non-covalent interaction with poly(ADP-ribose) (pADPR) linked to the auto-modified PARP-1. Moreover, the association of pADPR and PARP-1 with the nuclear matrix (NM) has been reported, based on the poly(ADP-ribosyl)ation of nuclear matrix proteins (NMPs). In the present article, by the use of DNA and protein cross-linking reactions, by cis-diamminedichloroplatinum II (cDDP) and sodium tetrathionate (NaTT) respectively, we present more evidences about the association of PARP-1, PARP-2, and PARPs related proteins with the NM. Our findings confirmed that NM could be seen as a fraction greatly enriched in transcription factors (i.e., C/EBP-beta) and enzymes (DNA Topo II, DNA PK) that co-localize with PARP-1 and -2 at the matrix associated regions (MARs) of chromatin. Moreover, pADPR contributes to PARP-1 localization at the NM, showing that PARP(s) activity co-operates to the functions of this nuclear fraction.

Animals↗
PubMed1986

Prediction of transcriptional activity based on gene association with the nuclear matrix.

In this report, we show that androgen-dependent genes expressed in rat ventral prostate, C1, C2 and C3, are enriched in residual DNA extracted from the nuclear matrix of ventral prostate. The enrichment is androgen-dependent and is likely to be related to their transcriptional activity. While this finding corroborates other data demonstrating that transcriptionally active genes can be protected from nuclease digestion by their association with the nuclear matrix, we have extended this analysis to show that nuclear matrix protection experiments can be used both to analyze tissue-specific expression among members of a highly related gene family and also to predict transcriptional activity of genes in particular tissues.

Androgen-Binding Protein↗
PubMed1990

A high resolution two-dimensional gel electrophoresis and silver staining protocol demonstrated with nuclear matrix proteins.

An improved two-dimensional gel electrophoresis procedure has been developed utilizing isolated nuclear matrix proteins. The proteins of the cellular nuclear matrix are tissue specific. They are an example of a protein set whose two-dimensional electrophoretic patterns afford much information of clinical significance. However, current two-dimensional gel techniques were not completely satisfactory for the small amounts of protein present in tissue samples. There was a need for a two-dimensional gel procedure which was capable of increased sensitivity and resolution and at the same time was reliable and reproducible. This has been accomplished by implementing several modifications to the current two-dimensional gel procedures. In addition, changes were introduced in the silver staining process of the gels to increase the signal to background ratio. The overall procedure affects a dramatic increase in the resolution and clarity of the proteins visualized on two-dimensional gels and is no more laborious than current techniques.

Antigens, Nuclear↗
PubMed1989

Alterations of neuronal nuclear matrix and chromatin structure after irradiation under aerobic and anoxic conditions.

This study was undertaken to determine if structural alterations of the bulk chromatin and the amount of protein associated with the nuclear matrix in cerebellar neurons depend on radiation dose and a cell's state of oxygenation. After irradiation with 2.5 to 25.0 Gy under both aerobic and anoxic conditions, the sensitivity of the neuronal chromatin to m. nuclease digestion increase linearly with dose up to about 5 Gy, beyond which there was no further increase. The same increase in accessibility of chromatin to micrococcal nuclease digestion was observed when neuronal nuclei were irradiated at 4 degrees C. Neuronal nuclei were stained with propidium iodide (PI) for DNA and with fluorescein isothiocyanate (FITC) for protein, both before and after complete digestion with DNase I, and analyzed by flow cytometry. There was no change in either the PI (P greater than 0.4) or the FITC (P greater than 0.9) fluorescence of undigested nuclei after irradiation. For the DNase I digested nuclei, the PI fluorescence was unchanged after irradiation (P greater than 0.4), but the FITC fluorescence increased significantly (P less than 0.02). This increase in the FITC fluorescence was linear with dose up to about 5 Gy, beyond which there was no further increase. The flow cytometry results from DNase I digested nuclei were identical for neurons irradiated under aerobic or anoxic conditions, indicating that this phenomenon is oxygen independent. This increase in FITC fluorescence after irradiation was inhibited at ice-cold temperatures and probably reflects an increase in protein content at the nuclear matrix that requires metabolism. This may explain our previously observed resistance of nuclear matrix-associated DNA to digestion by DNase I. This protein increase at the nuclear matrix appears to follow "saturation" kinetics identical to that previously reported for repair of DNA strand breaks in cerebellar neurons. However, the exact molecular nature of this process and its role in DNA repair or cell survival remains to be determined.

Aerobiosis↗
PubMed1996

Tyrosine kinase-dependent release of an adenovirus preterminal protein complex from the nuclear matrix.

Adenovirus (Ad) replicative complexes form at discrete sites on the nuclear matrix (NM) through the interaction of Ad preterminal protein (pTP). The NM is a highly salt-resistant fibrillar network which is known to anchor transcription, mRNA splicing, and DNA replication complexes. Incubation of rATP with NM to which pTP was bound caused the release of pTP as a pTP-NM complex with a size of 220 to 230 kDa; incubation with 5' adenylylimidodiphosphate (rAMP-PNP) showed no significant release, indicating that rATP hydrolysis was required. With NM extracts, it was shown that a pTP-NM complex which was capable of binding Ad origin DNA could be reconstituted in vitro. A number of high-molecular-weight NM proteins ranging in size from 120 to 200 kDa were identified on Far Western blots for their ability to bind pTP. rATP-dependent release of pTP from the NM was inhibited in a dose-dependent fashion by the addition of tyrosine kinase inhibitors, such as quercetin, methyl-2,5-dihydroxycinnamate, or genistein. NM-mediated phosphorylation of a poly(Glu, Tyr) substrate was also significantly abrogated by the addition of these compounds. rATP-dependent release of Ad DNA termini bound to the NM via pTP was also blocked by the addition of these inhibitors. These results indicate that a tyrosine kinase mechanism controls the release of pTP from its binding sites on the NM. These data support the concept that phosphorylation may play a key role in the modulation of pTP binding sites on the NM.

Adenoviruses, Human↗
PubMed1990

Adenovirus terminal protein mediates both nuclear matrix association and efficient transcription of adenovirus DNA.

Adenovirus DNA is tightly bound to the nuclear matrix throughout the course of infection. Analysis of adenovirus DNA from infected HeLa cell nuclei after extraction with lithium diiodosalicylate and digestion with restriction enzymes demonstrated that the sites of tightest attachment occur in the terminal fragments of the linear viral chromosome. Analysis of viruses mutated in the precursor terminal protein coding sequence demonstrated that the terminal protein, which is covalently attached to the 5' end of each DNA strand, mediates the tight binding. Virions containing chromosomes with mutant terminal proteins were unpackaged and viral DNA accumulated in the nucleus at a normal rate and competed for the limiting component during transcription complex formation, but their early genes were transcribed at reduced efficiency by both RNA polymerases II and III. The transcriptional defects were not complemented by coinfection with a wild-type virus. We propose that the adenovirus chromosome may exist as a single chromatin domain during infection and that binding of DNA to the nuclear matrix may play a critical role in adenovirus transcription.

Adenoviruses, Human↗