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PubMed2000

Cell cycle-dependent phosphorylation of the ATRX protein correlates with changes in nuclear matrix and chromatin association.

Mutations in the ATRX gene are associated with an X-linked mental retardation (XLMR) syndrome most often accompanied by alpha-thalassaemia (ATR-X syndrome). The ATRX gene encodes a predicted protein of 280 kDa featuring a PHD zinc finger motif and an ATPase/helicase domain of the SWI/SNF type; the vast majority of mutations in the ATRX gene fall within these two motifs. Although these domains are suggestive of a role for ATRX in transcriptional regulation by affecting chromatin structure and/or function, the precise cellular role of the ATRX protein remains undefined. Using indirect immunofluorescence and biochemical fractionation, we demonstrate that the ATRX protein has a punctate nuclear staining pattern and that it is tightly associated with the nuclear matrix at interphase. At the onset of M phase, the ATRX protein was associated mainly with condensed chromatin. The association of the ATRX protein with chromosomes at mitosis is concomitant with phosphorylation of the protein and its association with heterochromatin protein 1alpha (HP1alpha). The phosphorylation-dependent changes in localization between the nuclear matrix and condensed chromatin are consistent with a dual role for ATRX, possibly involving gene regulation at interphase and chromosomal segregation at mitosis.

Animals↗

PubMed1995

Modification of the lipid component modulates nuclear matrix nucleolytic activity.

It was shown that lipid composition of plant nuclear matrix depends on procedure of its isolation. The matrix isolated with the use of lithium diiodosalicylate (LiS) differs in its lipid composition from the preparation isolated with the use of nonionic detergent (Triton X-100). It was also shown that the nucleolytic activity of the matrix is related to its lipid component. Matrix depleted in lipids loses half of its nucleolytic activity which is recovered after supplementation with previously extracted lipids. The extent of recovery of the nucleolytic activity is also dependent on the presence of residual DNA in matrix preparation. The recoveries of nucleolytic activities were higher in matrices not depleted in their DNA content.

Iodobenzoates↗

PubMed1990

Increase in a 55-kDa keratin-like protein in the nuclear matrix of rat liver cells during proliferative activation.

We have identified a protein (p55) with a molecular weight of 55 kDa and a pI of 6.2, which was strongly increased in the nuclear matrix of rat liver cells during proliferative activation. This protein is highly insoluble since it could not be solubilized either by detergents or by alkaline extraction. We have obtained three partial amino acid sequences which revealed that p55 has a high homology with cytokeratins. Polyclonal antibodies raised against p55 were used to carry out Western blot and immunocytochemical studies which indicated that p55 was localized only in the nuclei, specifically in the nuclear matrix. Autoradiographic experiments revealed that not all the cells presenting an increase in p55 incorporated [3H]thymidine, indicating that this protein is not related to DNA replication. Immunocytochemical studies also revealed that during mitosis p55 is localized surrounding the chromosomes and associated with the mitotic apparatus, suggesting that p55 is involved in the separation of chromosomes during cell division.

Amino Acid Sequence↗

PubMed1988

Isolation and characterization of stable nuclear matrix preparations and associated DNA from avian erythroblasts.

A novel procedure for isolation of nuclear matrices from chicken erythroblast cells was elaborated. The influence of variations in the isolation procedure on structural integrity and morphology of nuclear matrices as well as on properties of the nuclear matrix-associated DNA fractions was investigated. The incubation of isolated nuclei in the presence of Cu2+ ions provided significant stabilization of the nuclear matrix. Copper treatment of nuclei did not affect the properties of the nuclear skeleton-associated DNA fraction. In both copper-stabilized as well as unstabilized nuclei, nuclear matrix-attached DNA was digested to the same extent with nucleolytic enzymes, and could be totally removed from nuclear matrices by 2 M NaCl-2 M urea treatment. The fine morphology of the nuclear matrix did not change after extraction of nuclear skeleton-associated DNA fragments. In the presence or absence of copper ions, matrix DNA was found to be qualitatively different compared with total DNA, in particular with respect to the representation of specific repetitive sequences of the chicken beta globin gene domain.

Animals↗

PubMed1984

Induction of new proteins in the nuclear matrix of CHO cells by a heat shock: detection of a specific set in the nucleolar matrix.

An early response to an incubation of CHO cells for one hour at 43 degrees C is the appearance in the nuclear matrix of new proteins: 2 proteins (48 and 46 kDa) were associated with the extranucleolar part of the nuclear matrix, 6 other proteins (130, 100, 95, 85, 76 and 70 kDa) were present in matrices prepared from isolated nucleoli. In addition, a large increase in the amount of associated minor components is observed. It is suggested that these early modifications in composition of the matrix are related to the inhibition of rRNA and hnRNA synthesis and maturation.

Animals↗

PubMedDate not supplied

Transient storage of a nuclear matrix protein along intermediate-type filaments during mitosis: a novel function of cytoplasmic intermediate filaments.

We recently identified a nuclear matrix protein, named NMP125 for its molecular weight (M(r) 125 kDa). On the basis of immunofluorescence analysis with monoclonal anti-NMP125 antibodies of differentially extracted cells in situ, including detergents, DNase I, RNase A, and high/low ionic strength conditions, it is concluded that NMP125 is a component of a chromatin- and histone-depleted nuclear substructure, operationally defined as nuclear matrix in interphase cells. The protein revealed evolutionary conservation in man, rat, chicken, and Xenopus, at least at the level of immunological crossreactivity. The subcellular distribution of NMP125 is cell-cycle-dependent; in interphase cells NMP125 is confined to a nuclear substructure with a granular aspect, whereas after nuclear envelope breakdown, it is freed into the cytoplasm. However, most of the protein remains attached to a cytoskeletal ligand that we have identified as the intermediate-type filament vimentin. In late mitotic stages the protein forms punctuate aggregates of relatively large size, which get passively closer to the newly formed telophase nuclei together with the reorganized vimentin around the nuclei in late telophase. From the morphological point of view, although static in nature, a dynamic cell-cycle-dependent distribution of NMP125 is found, revealing dissociation and spreading throughout the cytoplasm in metaphase, binding to vimentin filaments, cytoplasmic aggregation, and transport to nuclei in telophase. The transient affinity of the nuclear protein NMP125 to vimentin filaments during mitosis together with a passive cytoplasmic dislocation of the vimentin/NMP125 conjugate toward the telophase nuclei could represent a novel and dynamic function of cytoplasmic intermediate filaments, implicating a transient repository and passive shift of nuclear proteins during mitosis.

Animals↗

PubMed1993

Adenine phosphoribosyltransferase genes in two Drosophila species: dosage compensation, a nuclear matrix attachment site, and a novel intron position.

The Aprt locus of Drosophila encodes the structural gene for the purine salvage enzyme adenine phosphoribosyltransferase. Aprt is autosomal and enzyme activity is gene-dose-dependent in Drosophila melanogaster. However, Aprt is X-linked and dosage compensated in Drosophila pseudoobscura, as shown here. The Aprt genes of both Drosophila species contain a DNA sequence associated with nuclear matrix attachment sites and these Aprt sequences specifically bind to nuclear matrix in vitro. Putative promoter sequences positioned upstream of the predicted transcriptional start site in the two Aprt genes have a similar structure of direct repeats with an overlapping dyad symmetry, but the DNA sequence of these motifs is not conserved between the two species. Biological features in mutants of Aprt as well as natural variants suggest that dosage compensation of this gene in Drosophila pseudoobscura is due to a general control mechanism on X-linked genes rather than a gene-specific mechanism.

Adenine Phosphoribosyltransferase↗

PubMed1994

Catalytic properties of DNA polymerase alpha activity associated with the heart-stabilized nuclear matrix prepared from HeLa S3 cells.

We have investigated whether or not ATP or other nucleoside di- and trisphosphates (including some nonhydrolysable ATP analogues) can stimulate the activity and/or the processivity of DNA polymerase alpha associated with the nuclear matrix obtained from HeLa S3 cell nuclei that had been stabilized at 37 degrees C prior to subfractionation, as has been reported previously for DNA polymerase alpha bound to the nuclear matrix prepared from 22-h regenerating rat liver. We have found that HeLa cell matrix-associated DNA polymerase alpha activity could not be stimulated at all by ATP or other nucleotides, a behaviour which was shared also by DNA polymerase alpha activity that solubilizes from cells during the isolation of nuclei and that is thought to be a form of the enzyme not actively engaged in DNA replication. Moreover, the processivity of matrix-bound DNA polymerase alpha activity was low (< 10 nucleotides). These results were obtained with the matrix prepared with either 2 M NaCl or 0.25 M (NH4)2SO4 and led us to consider that a 37 degree incubation of isolated nuclei renders resistant to high-salt extraction a form of DNA polymerase alpha which is unlikely to be involved in DNA replication in vivo.

Adenosine Triphosphate↗

PubMed1993

Altered extracellular matrices influence cellular processes and nuclear matrix organizations of overlying human bladder urothelial cells.

A central issue in tumor biology is the understanding of the interactions between tumor cells and their environment. Using normal and ras oncogene transfected rat fibroblast cells, we now demonstrate that the transfected cells make altered extracellular matrices (ECM) and that their resulting ECM influence the proliferation and genetic regulation of human bladder cancer EJ cells. Using Western blot analyses, we observed that the ras transfected fibroblast cells lacked the ability to produce extracellular matrix component laminin whereas the normal parental fibroblast cells were able to produce intact laminin. Both transfected and nontransfected fibroblast cells were able to synthesize other extracellular matrix molecules such as type IV collagen and fibronectin. Human bladder tumor EJ cells were grown on ECM derived from normal and transfected rat fibroblast cells, and the proliferation rate and type IV collagen mRNA expression of EJ cells were determined. We observed that EJ cells, when grown on ECM derived from the ras transfected fibroblast cells, had a higher growth rate than when grown on ECM derived from the normal fibroblast cells (P < 0.037). Furthermore, EJ cells grown on ECM derived from transfected fibroblast cells showed up-regulation of type IV collagen mRNA expression when compared with EJ cells grown on ECM derived from nontransfected fibroblast cells. Finally EJ cells grown on purified laminin but not on collagen IV coated flasks showed the same level of type IV collagen mRNA expression as when grown on ECM derived from nontransfected parental fibroblast cells. Haptotactic/motility assays with EJ cells and ECM derived from ras transfected and nontransfected fibroblast cells demonstrated that ECM of ras transfected fibroblast cells, but not the parental fibroblast cells, provided a permissive or fertile soil for EJ tumor cell invasion. Finally, two-dimensional gel electrophoresis of 35S-labeled nuclear matrix proteins of EJ cells cultured on ECM derived from ras transfected fibroblast cells revealed expression of proteins in the molecular weight range of M(r) 35,000-45,000 and isoelectric focusing pH range of 5.5 to 6.0. These proteins were not present in EJ cells cultured on ECM derived from parental nontransfected fibroblast cells. We conclude that extracellular matrices derived from transformed stroma producing cells may influence the proliferation, genetic regulation, and maintenance of the overlying urothelial tumor cells. The mechanism by which the ECM may influence cellular behavior and phenotype may be in their ability to modulate the nuclear matrix proteins of the overlying cell.

Animals↗

PubMed1998

Adenovirus preterminal protein binds to the CAD enzyme at active sites of viral DNA replication on the nuclear matrix.

Adenovirus (Ad) replicative complexes form at discrete sites on the nuclear matrix (NM) via an interaction mediated by the precursor of the terminal protein (pTP). The identities of cellular proteins involved in these complexes have remained obscure. We present evidence that pTP binds to a multifunctional pyrimidine biosynthesis enzyme found at replication domains on the NM. Far-Western blotting identified proteins of 150 and 240 kDa that had pTP binding activity. Amino acid sequencing of the 150-kDa band revealed sequence identity to carbamyl phosphate synthetase I (CPS I) and a high degree of homology to the related trifunctional enzyme known as CAD (for carbamyl phosphate synthetase, aspartate transcarbamylase, and dihydroorotase). Western blotting with an antibody directed against CAD detected a 240-kDa band that comigrated with that detected by pTP far-Western blotting. Binding experiments showed that a pTP-CAD complex was immunoprecipitable from cell extracts in which pTP was expressed by a vaccinia virus recombinant. Additionally, in vitro-translated epitope-tagged pTP and CAD were immunoprecipitable as a complex, indicating the occurrence of a protein-protein interaction. Confocal fluorescence microscopy of Ad-infected NM showed that pTP and CAD colocalized in nuclear foci. Both pTP and CAD were confirmed to colocalize with active sites of replication detected by bromodeoxyuridine incorporation. These data support the concept that the pTP-CAD interaction may allow anchorage of Ad replication complexes in the proximity of required cellular factors and may help to segregate replicated and unreplicated viral DNA.

Adenoviridae↗

PubMed2001

Isolation of SDS-stable complexes of the intermediate filament protein vimentin with repetitive, mobile, nuclear matrix attachment region, and mitochondrial DNA sequence elements from cultured mouse and human fibroblasts.

Crosslinkage of vimentin to DNA in mouse L929 cells by formaldehyde and isolation of SDS-stable DNA-vimentin complexes from normal L929 cells and mouse and human embryo fibroblasts indicated close spatial relations between these components in the intact cell. The adducts, obtained by immunoprecipitation with anti-vimentin antibody, contained substantial quantities, not only of repetitive and mobile sequence elements such as centromeric satellite DNA, telomere DNA, microsatellites and minisatellites, long and short interspersed nucleotide elements, and retroposons, but also of mitochondrial (mt) DNA. Because the SDS-stable complexes could be isolated with distinctly higher yields from oxidatively stressed, senescent fibroblasts and were dissociated by boiling, they possibly arose from accidental condensation reactions mediated by unsaturated and dialdehydes, products of free radical-induced lipid peroxidation. They can therefore be considered vestiges of a general interaction of vimentin with cellular DNA. The sequence patterns of their DNA fragments were similar to those of extrachromosomal circular and linear DNA, including retroviral elements, markers and enhancers of genomic instability that also occur in the cytoplasm and are able to transport vimentin into the nucleus. Many of the fragments were also remarkably similar to AT-rich nuclear matrix attachment regions (MARs) in that they contained, in addition to various mobile elements, a palette of typical MAR motifs. With its tendency to multimerize and to interact with single-stranded and supercoiled DNA, vimentin thus behaves like a nuclear matrix protein and may as such participate in a variety of nuclear matrix-associated processes such as replication, recombination, repair, and transcription of DNA. These activities seem to be extendible to the mitochondrial compartment, as vimentin was also crosslinked to mtDNA, preferentially to its D-loop and hypervariable main control region. These sites are prone to point and deletion mutations and, like nuclear MARs, are associated with the cyto-karyomatrix. Moreover, as a developmentally regulated and tissue-specific cyto-karyomatrix protein, vimentin may contribute to the organization of chromatin, including centromeric and telomeric heterochromatin at the nuclear periphery, with all its consequences for genomic activities during embryogenesis and in adulthood of vertebrates. However, because of its high affinity for hypervariable, recombinogenic DNA sequences, vimentin is proposed to play a major role in both the preservation and the evolution of the nuclear and mitochondrial genome.

Animals↗

PubMed2000

Groucho/TLE/R-esp proteins associate with the nuclear matrix and repress RUNX (CBF(alpha)/AML/PEBP2(alpha)) dependent activation of tissue-specific gene transcription.

The Runt related transcription factors RUNX (AML/CBF(alpha)/PEBP2(alpha)) are key regulators of hematopoiesis and osteogenesis. Using co-transfection experiments with four natural promoters, including those of the osteocalcin (OC), multi drug resistance (MDR), Rous Sarcoma Virus long terminal repeat (LTR), and bone sialoprotein (BSP) genes, we show that each of these promoters responds differently to the forced expression of RUNX proteins. However, the three RUNX subtypes (i.e. AML1, AML2, and AML3) regulate each promoter in a similar manner. Although the OC promoter is activated in a C terminus dependent manner, the MDR, LTR and BSP promoters are repressed by three distinct mechanisms, either independent of or involving the AML C terminus, or requiring only the conserved C-terminal pentapeptide VWRPY. Using yeast two hybrid assays we find that the C terminus of AML1 interacts with a Groucho/TLE/R-esp repressor protein. Co-expression assays reveal that TLE proteins repress AML dependent activation of OC gene transcription. Western and northern blot analyses suggest that TLE expression is regulated reciprocally with the levels of OC gene expression during osteoblast differentiation. Digital immunofluorescence microscopy results show that TLE1 and TLE2 are both associated with the nuclear matrix, and that a significant subset of each colocalizes with AML transcription factors. This co-localization of TLE and AML proteins is lost upon removing the C terminus of AML family members. Our findings indicate that suppression of AML-dependent gene activation by TLE proteins involves functional interactions with the C terminus of AML at the nuclear matrix in situ. Our data are consistent with the concept that the C termini of AML proteins support activation or repression of cell-type specific genes depending on the regulatory organization of the target promoter and subnuclear localization.

Animals↗

PubMed1999

Origin and roles of nuclear matrix proteins. Specific functions of the MAR-binding protein MeCP2/ARBP.

HnRNP proteins are the major protein components of the nuclear matrix, and sites of nascent transcripts and RNA maturation are its main sources. The evidence for and the roles of functional and structural loops in interphase chromatin and metaphase chromosomes is discussed. Recent data suggest a specific role for the matrix attachment region (MAR)- and methyl-CpG-binding protein MeCP2/ARBP. This repressor protein binds to MARs and, through interaction with mSin3A, recruits a corepressor complex containing histone deacetylases. This in turn is thought to generate a localized silenced chromatin structure. Transfection experiments are presented in support of this model.

Animals↗

PubMed1987

Identification of a meiotic prophase-specific nuclear matrix protein in the rat.

We have identified a 110-kDa pI 5.6 phosphoprotein with DNA binding properties in the rat pachytene spermatocyte nuclear matrix. By immunoblotting and indirect immunofluorescence assays using polyclonal antibodies against the 110-kDa protein, we observed that it was germ cell nuclear matrix specific, more prominent in pachytene spermatocytes compared to premeiotic spermatogonia or postmeiotic round spermatids, and present in rat oocytes and in germ cells of mouse and monkey. We propose that this protein could play an important role in the meiotic process.

Animals↗

PubMed2000

A human common nuclear matrix protein homologous to eukaryotic translation initiation factor 4A.

Amino acid sequencing and mass spectrometry revealed identity of a human nuclear matrix protein, termed hNMP 265, with a predicted protein of gene KIAA0111. Two-dimensional electrophoresis and Northern hybridization showed the protein to ubiquitously occur in various human cell types. Exhibiting DEAD-box motifs characteristic for RNA helicases, hNMP 265 is highly similar to the human initiation factors eIF4A-I and -II. On the other hand, hNMP 265 greatly differs from the initiation factors by a N-terminal sequence rich in charged amino acids. Sequence searches and alignments indicate proteins related to hNMP 265 in other eukaryotes. Chimeras between hNMP 265 and green fluorescence protein or hapten appeared as speckles in extranucleolar regions in the nucleus, but not in the cytoplasm. Experiments with tagged deletion mutants indicated that the N-terminal amino acid sequence is necessary for nuclear localization. A putative role of hNMP 265 in pre-mRNA processing is discussed.

Amino Acid Sequence↗

PubMed1984

Nuclear matrix and transcriptional activity of the mouse alpha-globin gene.

The association of the mouse alpha-globin gene with the nuclear matrix was studied when the gene was permanently repressed in Ehrlich ascites cells, potentially active in uninduced Friend cells or actively transcribed in induced Friend cells. Matrix-associated DNA was obtained by two methods, differing in the order of treatment of the nuclei with high salt and DNase I. By using a cloned alpha-globin probe, no enrichment in alpha-globin sequences was found in the matrix-associated DNA after DNase I digestion of high-salt treated nuclei from Ehrlich ascites and uninduced Friend cells. In induced Friend cells, a high enrichment (up to 20 times) of alpha-globin sequences was detected in the DNA left with the nuclear matrix structures. The size of the DNA fragments obtained by this procedure indicated a random attack and did not correspond to a progressive top-to-bottom cleavage model. No enrichment in alpha-globin sequences was found in induced Friend cells if nuclear matrices were obtained by DNase I digestion of the nuclei before the treatment with high salt. Our data suggest that the enrichment in actively transcribed genes of matrices from nuclei treated with high salt does not reflect a localization of these genes close to the attachment sites of the chromatin loops but rather their artefactual association with some high salt-insoluble proteins of the transcriptional complexes.

Animals↗

PubMed1978

Expansion and apparent fluidity decrease of nuclear membranes induced by low Ca/Mg. Modulation of nuclear membrane lipid fluidity by the membrane-associated nuclear matrix proteins?

Macronuclei isolated from Tetrahymena are contracted in form (average diameter: 10.2 micron) at a final Ca/Mg (3:2)concentration of 5 mM. Lowering the ion concentration to 1 mM induces an expansion of the average nuclear diameter to 12.2 micron. Both contracted and expanded nuclei are surrounded by a largely intact nuclear envelope as revealed by thin-sectioning electron microscopy. Nuclear swelling is accompanied by an expansion of the nuclear envelope as indicated by the decrease in the frequency of nuclear pore complexes from 52.6 to 42.1 pores/micron2 determined by freeze-etch electron microscopy. Contracted nuclear membranes reveal particle-devoid areas (average size: 0.21 micron2) on 59% of their fracture faces at the optimal growth temperature of 28 degrees C. About three-fifths of the number of these smooth areas disappear upon nuclear membrane expansion. Electron spin resonance using 5-doxylstearic acid as a spin label indicates a higher lipid fluidity in contracted than in expa,ded nuclear membranes. Moreover, a thermotropic lipid clustering occurs at approximately 17 degrees C only in expanded nuclear membranes. In contrast to the nuclear membrane-bound lipids, free lipids extracted from the nuclei rigidify with increasing Ca/Mg concentrations. Our findings are compatible with the view that the peripheral layer of the fundamental nuclear protein-framework, the so-called nuclear matrix, can modulate, inter alia, the lipid distribution and fluidity, respectively, in nuclear membranes. We suggest that a contraction of the nuclear matrix's peripheral layer induces a contraction of the nuclear membranes which, in turn, leads to an isothermic lateral lipid segregation within nuclear membranes.

Animals↗

PubMed1995

Rapid and transient phosphorylation of nuclear matrix proteins upon interferon-alpha treatment in Daudi lymphoma cells.

Evidence for a rapid and transient hyperphosphorylation of a 45 kD protein in isolated nuclei from interferon-alpha-treated Daudi lymphoma cells is presented. Extraction of nuclear matrices from these nuclei has provided further evidence for the association of such a protein in the nuclear matrix structure. Because phosphorylation assays performed directly on nuclear matrices from interferon-treated cells have revealed rapid and transient increase of gamma 32P-ATP incorporation into this 45 kD band, an early involvement of the nuclear matrix in the response of the nucleus to the interferon antiproliferative message is suggested.

Antigens, Nuclear↗