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PubMed1994

Subnuclear distribution of the vitamin D receptor.

The subnuclear distribution of the vitamin D receptor was investigated to begin addressing the contribution of nuclear architecture to vitamin D-responsive control of gene expression in ROS 17/2.8 rat osteosarcoma cells. The nuclear matrix is an anastomosing network of filaments that is functionally associated with DNA replication, transcription, and RNA processing. The representation of vitamin D receptor in the nuclear matrix and nonmatrix nuclear fractions was determined by the combined application of 1) sequence-specific interactions with the vitamin D receptor binding element of the rat bone-specific osteocalcin gene promoter and 2) Western blot analysis. Both methods confirmed the presence of vitamin D receptor in the nonmatrix nuclear fraction and the absence of detectable vitamin D receptors associated with the nuclear matrix. In contrast, these same nuclear matrix proteins preparations exhibited association with the general transcription factor AP-1 and a bone tissue-specific promoter binding factor NMP2. NMP-2 exhibits recognition for a promoter domain contiguous to the vitamin D-responsive element of the osteocalcin gene, although the vitamin D receptor does not appear to be a component of the nuclear matrix proteins. Interrelationships between nuclear matrix proteins and nonmatrix nuclear proteins, in mediating steroid hormone responsiveness of a vitamin D-regulated promoter, are therefore suggested.

Animals↗
PubMed1982

Nuclear matrix-bound deoxyribonucleic acid synthesis: an in vitro system.

A nuclear matrix-bound in vitro replicational system that utilizes alpha-polymerase and matrix-attached DNA has been isolated from regenerating rat liver and characterized for in vitro requirements. beta-Polymerase is a minor component of the matrix in vitro system. Evidence is presented for the native association of alpha-polymerase with the nuclear matrix and for the parallel induction of matrix-bound alpha-polymerase and in vivo DNA synthesis during the proliferative response following partial hepatectomy. Our data suggest that the attachment of alpha-polymerase to the matrix may be an important step in the assembly of functional replisomes. We also demonstrate prereplicative and late-replicative rises in nuclear and matrix-bound beta-polymerase activity and discuss these results in terms of possible genome screening.

Animals↗
PubMed1996

A nuclear matrix-specific factor that binds a specific segment of the negative regulatory element (NRE) of HIV-1 LTR and inhibits NF-kappa(B) activity.

The negative regulatory element (NRE) of human immunodeficiency virus type-1 (HIV-1) long terminal repeat (LTR) is a defined region that has been reported to downregulate LTR-directed HIV gene expression. However, information on the precise role of this region in regulating HIV gone transcription is lacking. We have investigated the possibility that these NRE sequences regulate HIV transcription by a mechanism mediated through a nuclear matrix-specific DNA-protein interaction. We find a nuclear matrix attachment region (MAR) present within the NRE of the HIV-1 LTR that recognizes a sequence-specific DNA-binding protein present in the nuclear matrix of HIV infected cells. Moreover, we also show that the purified DNA-binding nuclear matrix protein (NMP) specifically represses the DNA-binding activity of NF-kappaB. It is likely that the MAR and MAR-enriched specific DNA-binding NMP are brought into juxtaposition by the non-chromatin scaffolding of the nucleus, thus influencing NF-kappaB (and other nuclear proteins) DNA-binding activity through protein-protein and protein-DNA interactions. Our date suggest that one possible role of the NRE could be to act as a matrix attachment site in the nuclear matrix, thus, allowing interaction with a sequence-specific trans-acting factor. The negative effect on NF-kappaB activity due to this MAR-NMP-specific interaction provides a mechanism by which the NRE downregulates HIV gene expression.

Antigens, Nuclear↗
PubMed1997

Ataxin-1 with an expanded glutamine tract alters nuclear matrix-associated structures.

Spinocerebellar ataxia type 1 (SCA1) is one of several neurodegenerative disorders caused by an expansion of a polyglutamine tract. It is characterized by ataxia, progressive motor deterioration, and loss of cerebellar Purkinje cells. To understand the pathogenesis of SCA1, we examined the subcellular localization of wild-type human ataxin-1 (the protein encoded by the SCA1 gene) and mutant ataxin-1 in the Purkinje cells of transgenic mice. We found that ataxin-1 localizes to the nuclei of cerebellar Purkinje cells. Normal ataxin-1 localizes to several nuclear structures approximately 0.5 microm across, whereas the expanded ataxin-1 localizes to a single approximately 2-microm structure, before the onset of ataxia. Mutant ataxin-1 localizes to a single nuclear structure in affected neurons of SCA1 patients. Similarly, COS-1 cells transfected with wild-type or mutant ataxin-1 show a similar pattern of nuclear localization; with expanded ataxin-1 occurring in larger structures that are fewer in number than those of normal ataxin-1. Colocalization studies show that mutant ataxin-1 causes a specific redistribution of the nuclear matrix-associated domain containing promyelocytic leukaemia protein. Nuclear matrix preparations demonstrate that ataxin-1 associates with the nuclear matrix in Purkinje and COS cells. We therefore propose that a critical aspect of SCA1 pathogenesis involves the disruption of a nuclear matrix-associated domain.

Animals↗
PubMed2006

Evidence of modified nuclear protein matrix in human spermatozoa with fragmented deoxyribonucleic acid.

Human spermatozoa were processed for determination of DNA fragmentation with use of an in situ diffusion assay, so that those cells containing DNA fragmentation produce extensive peripheral dissemination of DNA fragments after lysis in an agarose microgel. Quantification of specific protein staining confirmed that sperm cells without DNA fragmentation had almost complete removal of nuclear matrix proteins, whereas spermatozoa with DNA fragmentation tended to retain residual nucleoskeletal protein in a collapsed and condensed state. This result suggests that a modified nuclear protein matrix associates with fragmented sperm DNA.

Cells, Cultured↗
PubMed2003

Nuclear coactivator-62 kDa/Ski-interacting protein is a nuclear matrix-associated coactivator that may couple vitamin D receptor-mediated transcription and RNA splicing.

Nuclear coactivator-62 kDa/Ski-interacting protein (NCoA62/SKIP) is a putative vitamin D receptor (VDR) and nuclear receptor coactivator protein that is unrelated to other VDR coactivators such as those in the steroid receptor coactivator (SRC) family. The mechanism through which NCoA62/SKIP functions in VDR-activated transcription is unknown. In the present study, we identified a nuclear localization sequence in the COOH terminus of NCoA62/SKIP and showed that NCoA62/SKIP was targeted to nuclear matrix subdomains. Chromatin immunoprecipitation studies revealed that endogenous NCoA62/SKIP associated in a 1,25-dihydroxyvitamin D3-dependent manner with VDR target genes in ROS17/2.8 osteosarcoma cells. A cyclic pattern of promoter occupancy by VDR, SRC-1, and NCoA62/SKIP was observed, with NCoA62/SKIP entering these promoter complexes after SRC-1. These studies provide strong support for the proposed role of NCoA62/SKIP as a VDR transcriptional coactivator, and they indicate that key mechanistic differences probably exist between NCoA62/SKIP and SRC coactivators. To explore potential mechanisms, NCoA62/SKIP-interacting proteins were purified from HeLa cell nuclear extracts and identified by mass spectrometry. The identified proteins represent components of the spliceosome as well as other nuclear matrix-associated proteins. Here, we show that a dominant negative inhibitor of NCoA62/SKIP (dnNCoA62/SKIP) interfered with appropriate splicing of transcripts derived from 1,25-dihydroxyvitamin D3-induced expression of a growth hormone minigene cassette. Taken together, these data show that NCoA62/SKIP has properties that are consistent with those of nuclear receptor coactivators and with RNA spliceosome components, thus suggesting a potential role for NCoA62/SKIP in coupling VDR-mediated transcription to RNA splicing.

Animals↗
PubMed1987

The localization of ultraviolet-induced excision repair in the nucleus and the distribution of repair events in higher order chromatin loops in mammalian cells.

Several lines of evidence indicate that eukaryotic DNA is arranged in highly supercoiled domains or loops, and that the repeating loops are constrained by attachment to a nuclear skeletal structure termed the nuclear matrix. Active genes are transcribed at the nuclear matrix and during replication the loops are reeled through fixed matrix-associated replication complexes. We have investigated whether the repair of DNA damage also occurs in the nuclear matrix compartment. Biochemical analysis of confluent normal human fibroblasts, ultraviolet (u.v.)-irradiated with 30 J m-2 and post-u.v. incubated in the presence of hydroxyurea, did not show any evidence for the occurrence of repair synthesis at the nuclear matrix either 30 min or 13 h after irradiation. Autoradiographic visualization of repair events in single DNA halo-matrix structures confirmed the biochemical observations. At a biologically more relevant dose of 5 J m-2 repair synthesis seems to initiate at the nuclear matrix, although only part of the total repair could be localized there. In u.v.-irradiated (30 J m-2) normal human fibroblasts post-u.v. incubated in the presence of hydroxyurea and arabinosylcytosine for 2 h, multiple single-stranded regions are generated in a DNA loop as a result of the inhibition of the excision repair process. Different biochemical approaches revealed that most of the single-stranded regions are clustered, indicating that the repair process itself is non-random or that domains in the chromatin are repaired at different rates. Preferential repair of certain domains in the chromatin was shown to occur in xeroderma pigmentosum cells of complementation group C (XP-C). In XP-C cells these domains are localized near the attachment sites of DNA loops at the nuclear matrix. In contrast, xeroderma pigmentosum cells of complementation group D as well as Syrian hamster embryonic cells with limited excision-repair capacities, revealed a random distribution of repair events in DNA loops. The preferential repair of matrix-associated DNA in XP-C cells may be related partly to repair of transcriptionally active DNA and this may account for the ability of XP-C cells, in contrast to XP-D cells, to recover u.v.-inhibited synthesis of DNA and RNA.

Animals↗
PubMed1995

Immunoglobulins anti-endonuclease 32 kDa from Cucurbita pepo var patissonina affect process of DNA synthesis.

Immunoglobulins anti-endonuclease 32 kDa inhibit DNA synthesis. We observed that low concentrations of IgGs (about 50 micrograms IgG per 1 x 10(6) cell nuclei) temporary inhibit DNA synthesis. This inhibition concerns only the synthesis of DNA bound to the nuclear matrix (associated with isolated nuclear matrix). Preincubation of cell nuclei of White bush with IgG generates longer DNA fragments than in controls. Involvement of the 32 kDa endonuclease or an endonuclease-65 kDa protein complex from the nuclear matrix in replication or structural organisation of replication is considered.

DNA, Plant↗
PubMedDate not supplied

Age-dependent increase of DNA topoisomerase II activity in quail oviduct; modulation of the nuclear matrix-associated enzyme activity by protein phosphorylation and poly(ADP-ribosyl)ation.

Nuclear DNA topoisomerase II activity in quail oviduct tissue was found to increase by about 70% with age. This age-dependent increase was observed with both the enzyme in whole nuclear extract and nuclear matrix-associated topoisomerase II. Both purified topoisomerase II and the nuclear matrix-bound enzyme were found to be modifiable by phosphorylation and poly(ADP-ribosyl)ation. Phosphorylation of the purified enzyme by isolated nuclear protein kinase NII or protein kinase C resulted in a 2- to 3-fold increase in specific activity, while poly(ADP-ribosyl)ation by soluble poly(ADP-ribose) synthetase caused a 50% inhibition of the enzyme. Using immunoprecipitation and immunoblotting procedures, phosphorylation and poly(ADP-ribosyl)ation could also be demonstrated to occur with the nuclear matrix-associated enzyme. The nuclear matrix-associated NII-like protein kinase activity, assumed to be involved in post-translational modification of topoisomerase II, displayed a 1.4- to 1.6-fold increase in old animals compared to mature ones, while the matrix-bound poly(ADP-ribose) synthetase activity decreased by about 50%. It is suggested that age-correlated enhancement of DNA topoisomerase II activity, possibly due to age-dependent changes in activities of nuclear protein kinases and poly(ADP-ribose) synthetase, may result in alterations in the topological state of DNA, possibly affecting DNA replication, transcription and repair with age.

Aging↗
PubMed2006

Assembly of nuclear matrix-bound protein complexes involved in non-homologous end joining is induced by inhibition of DNA topoisomerase II.

Topoisomerases maintain the DNA structure by relieving the torsional stress and alleviating other topological problems occurring in DNA during transcription and replication. Topoisomerase II appears to have a close association with the family of proteins involved in the organization of chromatin in a series of loops on the proteinaceous chromosomal matrix. Beyond its physiological functions, topoisomerase II is the target for some of the most active anticancer drugs. Inhibition of the topoisomerase II function can result in DNA double-strand breaks (DSBs) and, thus, lead to chromosomal translocations. The earliest event during DSB repair is phosphorylation of histone H2AX at S139 (so-called gammaH2AX) which is believed to serve as a focal point for the assembly of repair proteins at the DSB. In this work, we have demonstrated the formation of gammaH2AX foci in two human cell lines--K562 and HeLa--after suppression of topoisomerase II activity with etoposide. Furthermore, these foci remained visible at nuclear matrices and colocalized with the major components of non-homologous end joining (NHEJ) system of DSBs repair. Thus, inhibition of topoisomerase II activity triggers assembly of NHEJ complexes at the nuclear matrix.

Antigens, Nuclear↗
PubMed1990

Prompt heat-shock and heat-shifted proteins associated with the nuclear matrix-intermediate filament scaffold in Drosophila melanogaster cells.

Elevated temperatures induced the synthesis of several new proteins in Drosophila melanogaster cells. Besides the conventional heat shock (HS) proteins, another set of temperature-induced proteins has been found. These latter resemble the prompt HS proteins of mammalian cells. The prompt HS proteins of Drosophila differ from the well-known conventional HS proteins in the following properties: (1) synthesis of the prompt HS proteins is insensitive to the transcription inhibitor actinomycin D, which blocks the appearance of conventional HS proteins; (2) induction of the prompt HS proteins requires a significantly higher temperature than conventional HS proteins; (3) prompt HS proteins associate strictly with the nuclear matrix-intermediate filament complex (NM-IF), while the conventional HS proteins are found in all subcellular fractions; (4) prompt HS proteins of Drosophila are induced by high temperature alone while the conventional HS proteins are also produced by a variety of stress conditions. Resinless-section electron micrographs show an altered nuclear matrix morphology in heat-shocked cells. The nuclear matrix fibers are altered in spatial distribution and have much additional electron-dense material. This added material probably reflects the soluble proteins shifted into the nuclear matrix at high temperature. The prompt HS proteins can be distinguished clearly from heat-shifted proteins by several criteria. Also, the prompt HS proteins are distinct from the heat-insensitive viral proteins of a persistent virus (HPS-1).

Animals↗
PubMed1995

Targeting of adenovirus E1A and E4-ORF3 proteins to nuclear matrix-associated PML bodies.

The PML protein was first identified as part of a fusion product with the retinoic acid receptor alpha (RAR alpha), resulting from the t(15;17) chromosomal translocation associated with acute promyelocytic leukemia (APL). It has been previously demonstrated that PML, which is tightly bound to the nuclear matrix, concentrates in discrete subnuclear compartments that are disorganized in APL cells due to the expression of the PML-RAR alpha hybrid. Here we report that adenovirus infection causes a drastic redistribution of PML from spherical nuclear bodies into fibrous structures. The product encoded by adenovirus E4-ORF3 is shown to be responsible for this reorganization and to colocalize with PML into these fibers. In addition, we demonstrate that E1A oncoproteins concentrate in the PML domains, both in infected and transiently transfected cells, and that this association requires the conserved amino acid motif (D)LXCXE, common to all viral oncoproteins that bind pRB or the related p107 and p130 proteins. The SV-40 large T antigen, another member of this oncoprotein family is also found in close association with the PML nuclear bodies. Taken together, the present data indicate that the subnuclear domains containing PML represent a preferential target for DNA tumor viruses, and therefore suggest a more general involvement of the PML nuclear bodies in oncogenic processes.

Adenovirus E1A Proteins↗
PubMed1991

Type IV procollagen mRNA regulation: evidence for extracellular matrix/cytoskeleton/nuclear matrix interactions in human urothelium.

The absence of basement membrane components correlates with tumor stage and progression in human bladder cancers. We have previously shown that invasive tumors possess the ability to degrade basement membrane. However, the presence of basement membrane may be affected not only by its degradation, but by its synthesis and deposition as well. Our results in the present study suggest that while the invasive human transitional carcinoma cell line EJ has an increased amount of type IV procollagen mRNA when compared to the non-invasive RT4 cell line, type IV collagen staining is absent in the invasive EJ cells and intensely present in the non-invasive RT4 cells. Moreover, when EJ cells were grown on an artificial basement membrane (Matrigel), type IV procollagen mRNA expression was down-regulated to the levels seen with the non-invasive RT4 cells. We also discovered that the invasive cells, when grown on Matrigel, appeared morphologically different from the same cells grown on plastic tissue cultures. We conclude that a deficient basement membrane in invasive cancer cells may be due not only to active proteolytic activity but also to an abnormal production and deposition of extracellular matrix components. In addition, we also demonstrated that basement membrane components may have a significant effect on epithelial cell morphology and gene regulation, and that any alterations of the extracellular matrix-cytoskeleton-nuclear matrix interactions can lead to altered gene regulations and cell function.

Basement Membrane↗
PubMed2004

The 5'-HS4 chicken beta-globin insulator is a CTCF-dependent nuclear matrix-associated element.

The protein CTCF plays an essential role in the action of a widely distributed class of vertebrate enhancer-blocking insulators, of which the first example was found in a DNA sequence element, HS4, at the 5' end of the chicken beta-globin locus. HS4 contains a binding site for CTCF that is necessary and sufficient for insulator action. Purification of CTCF has revealed that it interacts with proteins involved in subnuclear architecture, notably nucleophosmin, a 38-kDa nucleolar phosphoprotein that is concentrated in nuclear matrix preparations. In this report we show that both CTCF and the HS4 insulator element are incorporated in the matrix; HS4 incorporation depends on the presence of an intact CTCF-binding site. However the DNA sequence in the neighborhood of HS4 is not like that of canonical matrix attachment regions, and its incorporation into the matrix fraction is not sensitive to ribonuclease, suggesting that the insulator is a distinct matrix-associated element.

Animals↗
PubMed1985

Monoclonal antibodies prepared against the major Drosophila nuclear Matrix-pore complex-lamina glycoprotein bind specifically to the nuclear envelope in situ.

A high molecular weight glycoprotein found associated with a nuclear matrix-pore complex-lamina (NMPCL) preparation obtained from Drosophila melanogaster embryos has been shown by in vitro analyses to be largely confined to this subcellular fraction. In contrast with several of the NMPCL proteins, this glycoprotein remains completely insoluble after treatment with 5 M urea. It has, therefore, been possible to separate the glycoprotein from other NMPCL components by differential urea extraction. The glycoprotein in the 5 M urea-extracted pellet has been solubilized by boiling in sodium dodecyl sulfate and purified to near-homogeneity by sequential steps of chromatography on hydroxylapatite and Sephacryl S-300 (both run in the presence of 0.1% sodium dodecyl sulfate), followed by affinity chromatography on lentil lectin-Sepharose. Over 30 hybridoma cell lines producing antibodies against this glycoprotein have been obtained. Monoclonality has been established for two of these lines (designated AGP-26 and AGP-78), and the antibodies they secrete have been further characterized. Western blot analysis has shown both antibodies to be monospecific (with respect to other Drosophila embryo polypeptides) for the major NMPCL glycoprotein; in addition, antibody AGP-78 has been shown to be weakly cross-reactive with glycoproteins of similar or identical molecular weight found associated with isolated nuclear fractions obtained from Xenopus oocytes, as well as chicken, opossum, and rat livers. Finally, both antibodies AGP-26 and AGP-78 react exclusively with the Drosophila nuclear periphery (nuclear envelope) in situ as demonstrated by indirect immunofluorescence analysis of larval cryosections. Based on these results as well as upon those of biochemical studies reported previously (Berrios, M., Filson, A. J., Blobel, G, and Fisher, P. A. (1983) J. Biol. Chem. 258, 13384-13390), we conclude that the major Drosophila NMPCL glycoprotein is the specific homolog of the high molecular weight glycoprotein recently shown using immunoelectron microscopy to be a distinct component of the rat liver nuclear pore complex (Gerace, L., Ottaviano, Y., and Kondor-Koch, C. (1982) J. Cell Biol. 95, 826-837).

Animals↗
PubMed1999

Nuclear structure, gene expression and development.

This article considers the extent to which features of nuclear structure are involved in the regulation of genome function. The recent renaissance in imaging technology has inspired a new determination to assign specific functions to nuclear domains or structures, many of which have been described as "factories" to express the idea that they coordinate nuclear processes in an efficient way. Visual data have been combined with genetic and biochemical information to support the idea that nuclear organization has functional significance. Particular DNA sequences or chromatin structures may nucleate domains that are permissive or restrictive of transcription, to which active or inactive loci could be recruited. Associations within the nucleus, as well as many nuclear structures, are transient and change dynamically during cell cycle progression and development. Despite this complexity, elucidation of the possible structural basis of epigenetic phenomena, such as the inheritance of a "cellular memory" of gene expression status, is an important goal for cell biology. Topics for discussion include the regulatory effect of chromatin structure on gene expression, putative "nuclear addresses" for genes and proteins, the functional significance of nuclear bodies, and the role of the nuclear matrix in nuclear compartmentalization.

Animals↗
PubMed1995

Nuclear matrix-intermediate filament proteins of the dental follicle/enamel epithelium and their changes during tooth eruption in dogs.

Tooth eruption activates a localized resorption and formation of alveolar bone and these activities depend upon the adjacent parts, coronal and basal, respectively, of the dental follicle-enamel epithelium. In this study the nuclear matrix-intermediate filament (NM-IF) proteins of these tissues were isolated in order to continue investigations into the molecular mechanisms underlying eruption. Dental follicles were removed from the third and fourth premolar of dogs at 13, 16 and 20 weeks (pre-, early, and mid-to-late eruption of these teeth) and NM-IF proteins were extracted from the coronal and basal halves. Most of the NM-IF protein profiles of these coronal and basal parts on one-dimensional, sodium dodecyl sulphate-polyacrylamide gel electrophoresis were remarkably constant, indicating an essentially uniform cellular composition. However, differences between these tissues were observed and some of these changed during eruption. Based on recent observations that nuclear matrix changes reflect and may even mediate cell-specific changes in gene expression, these findings suggest that changes in nuclear matrix proteins may be related to the molecular basis for some aspects of differential gene expression in the coronal and basal regions of the dental follicle and account for the ability of these tissues to activate bone resorption and formation during tooth eruption.

Alveolar Process↗
PubMed2000

The fate of the nuclear matrix-associated-region-binding protein SATB1 during apoptosis.

Special AT-rich sequence-binding protein 1 (SATB1), predominantly expressed in thymocytes, was identified as a component of the nuclear matrix protein fraction. Programmed cell death of Jurkat T-cells was induced by various stimuli in Fas-dependent and -independent fashion. During apoptosis, but not during necrosis, SATB1 was cleaved, as rapidly as was lamin B, in a caspase-dependent way yielding a stable 70 kDa fragment. The same result was obtained for apoptotic HL60-cells. We constructed various deletion constructs of SATB1, expressing protein chimeras tagged with green fluorescent protein (GFP). Transient transfection of these into Jurkat or HeLa cells followed by initiation of apoptosis allowed us to map the potential caspase-6 cleavage site VEMD to the N-terminal third of SATB1, leaving an intact DNA-binding domain in the C-terminal part of the protein. Our results suggest that apoptosis-specific breakdown of SATB1, a transcriptional activator of the CD8a gene, might be of physiological relevance during thymic clonal deletion and apoptosis of peripheral T-lymphoid cells.

Amino Acid Sequence↗