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PubMed1997

Association of protein kinase CK2 with nuclear matrix: influence of method of preparation of nuclear matrix.

Nuclear matrix (NM) plays a role of fundamental structural and functional significance as the site of replication, transcription, and RNA processing and transport, acting as an anchor or attachment site for a variety of enzymes and other proteins involved in these activities. We have previously documented that protein kinase CK2 translocates from the cytosol to the nucleus, where it associates preferentially with chromatin and NM, in response to certain growth stimuli. Considering that characteristics of the isolated NM can depend on the procedural employed for its isolation, we compared three standard methods for NM preparation to confirm the association of intrinsic CK2 with this structure. Our data suggest that the method used for isolating the NM can qualitatively influence the measurable NM-associated CK2. However, all three methods employed yielded qualitatively similar results with respect to the stimulus-mediated modulation of NM-associated CK2, thus further supporting the notion that NM is an important site for physiologically relevant functions of CK2. In addition, core filaments and cytoskeleton that were isolated by two of the preparative methods had a small but significant level of associated CK2 activity.

Animals↗
PubMed2000

HA95 is a protein of the chromatin and nuclear matrix regulating nuclear envelope dynamics.

We report a role for HA95, a nuclear protein with high homology to the nuclear A-kinase anchoring protein AKAP95, in the regulation of nuclear envelope-chromatin interactions. Biochemical and photobleaching data indicate that HA95 is tightly associated with chromatin and the nuclear matrix/lamina network in interphase, and bound to chromatin at mitosis. HA95 resides in a complex together with lamin B receptor (LBR), lamina-associated polypeptide (LAP)2 and emerin, integral proteins of the inner nuclear membrane. Cross-linking experiments, however, illustrate a tight association of HA95 with LBR and LAP2 only. Intra-nuclear blocking of HA95 with anti-HA95 antibodies abolishes nuclear breakdown in a mitotic HeLa cell extract. The antibodies inhibit nuclear membrane breakdown and chromatin condensation - the latter independently of nuclear membranes. However, lamina disassembly is not affected, as judged by immunological analyses of A/C- and B-type lamins. In contrast, immunoblocking of HA95 bound to condensed chromosomes does not impair chromatin decondensation, nuclear membrane reassembly or lamina reformation. Our results argue for a role for HA95 in anchoring nuclear membranes and lamins to chromatin in interphase, and in releasing membranes from chromatin at mitosis. The data also suggest that HA95 is not involved in initial binding of membranes to chromatin upon nuclear reassembly. We propose that HA95 is a central platform at the chromatin/nuclear matrix interface implicated in regulating nuclear envelope-chromatin interactions during the cell cycle.

Amino Acid Sequence↗
PubMed1979

Isolation and characterization of the nuclear matrix in Friend erythroleukemia cells: chromatin and hnRNA interactions with the nuclear matrix.

Nuclear matrices from undifferentiated and differentiated Friend erythroleukemia cells have been obtained by a method which removes DNA in a physiological buffer. These matrices preserved the characteristic topographical distribution of condensed and diffuse "chromatin" regions, as do nuclei in situ or isolated nuclei. Histone H1 was released from the nuclear matrix of undifferentiated cells by 0.3 M KCl; inner core histones were released by 1 M KCl. Nuclear matrix from differentiated cells did not maintain H1, and histone cores were fully released in 0.7 M KCl. KCl removed the core histones as an octameric structure with no evidence of preferential release of any single histone. Electron microscopy of KCl-treated matrix revealed no condensed regions but rather a network of fibrils in the whole DNA-depleted nuclei. When nuclear matrices from both types of cell were exposed to conditions of very low ionic strength, inner core histones and condensed regions remained. These observations support the contention that inner core histones are bound to matrix through natural ionic bonds or saline-labile elements, and that these interactions are implicated in chromatin condensation. hnRNA remained undegraded and tenaciously associated to the matrix fibrils, and was released only by chemical means which, by breaking hydrophobic and hydrogen bonds, produced matrix lysis. Very few nonhistone proteins were released upon complete digestion of DNA from either type of nuclei. The remaining nonhistone proteins represent a large number of species of which the majority may be matrix components. The molecular architecture in both condensed and diffuse regions of interphase nuclei appears to be constructed of two distinct kinds of fibers; the thicker chromatin fibers are interwoven with the thinner matrix fibers. The latter are formed by a heteropolymer of many different proteins.

Animals↗
PubMed1991

Sequence-specific binding of a c-myc nuclear-matrix-associated region shows increased nuclear matrix retention after leukemic cell (HL-60) differentiation.

HL-60 cells, a human promyelocytic leukemia cell line, contain amplified c-myc DNA sequences and mRNA transcripts. These cells can be induced to undergo macrophage differentiation by phorbol esters, which results in suppression of c-myc expression and cessation of cell proliferation. The nuclear matrix (NM), a nuclear skeleton resistant to DNase I digestion and high salt extraction, is proposed to be involved in DNA replication, gene regulation, and the correct distribution of DNA at mitosis. We have previously identified a nuclear-matrix-associated region (MAR) of the c-myc protooncogene to reside in a 1.4-kb region between Cla I and Eco RI restriction sites at the 3'-end of the gene. A 172-bp Dra I/Dra I subfragment of the 1.4-kb region was shown to be a major component of the MAR (myc-MAR), and this subfragment was demonstrated to be recognized by a nuclear protein (p25). In this report we demonstrate that phi X174 DNA, or the synthetic copolymers poly[d(G.C)] and poly[d(A.T)], are not effective suppressors of the binding of the myc-MAR to isolated NM, indicating that the binding sequence(s) are unique. We find that the addition of partially purified protein p25 increases the relative affinity of the myc-MAR for HL-60 NM in an in vitro assay system. NM isolated from HL-60 macrophages induced by phorbol esters retains significantly more myc-MAR DNA fragment in the presence of an excess amount of competitor DNA than does NM from untreated HL-60 cells. These data suggest that a change of the myc-MAR association with the NM occurs after monocytic differentiation of HL-60 cells.

Base Composition↗
PubMed1993

Death-induced changes to the nuclear matrix: the use of anti-nuclear matrix antibodies to study agents of apoptosis.

Using immunological techniques, we have shown for the first time that change in the nuclear matrix is an observable step in the cell death program. Immunometric assays detect soluble nuclear matrix proteins in the culture supernatants of cells killed by adriamycin, tumor necrosis factor and serum growth factor deprivation. Studies of cells killed by tumor necrosis factor and serum growth factor deprivation using dual immunofluorescent and 4'-6-diamidino-2-phenylindole (DAPI) staining detect many cells strongly positive for DNA that have little or no nuclear matrix protein. The staining pattern of cells killed by adriamycin is very different. There is good correspondence between the immunofluorescent and DAPI staining patterns indicating the presence of nuclear matrix proteins and DNA, although at a reduced ratio. It is concluded that the nuclear matrix changes are dependent on the agents that trigger cell death.

Apoptosis↗
PubMed1996

Histone modifications, chromatin structure, and the nuclear matrix.

The nuclear matrix has a role in the organization and function of nuclear DNA. A combination of stable and transient interactions between chromatin and the nuclear matrix is involved in organizing DNA within the nucleus. DNA sequences (matrix attachment regions) at the base of a loop bind to nuclear matrix proteins and arrange the nuclear DNA into chromatin loop domains. Multiple, transient interactions between the nuclear matrix and transcriptionally active chromatin are thought to be responsible for the insoluble feature of transcriptionally active chromatin. Current evidence suggests that histone acetyltransferase, histone deacetylase (enzymes that catalyze rapid histone acetylation and deacetylation), transcription factors, and the transcription machinery mediate the transient attachments between nuclear matrix and active chromatin. Highly acetylated core histones, which are associated with transcriptionally active DNA, are also ubiquitinated and phosphorylated. Recent studies show that specific H1 subtypes and their phosphorylated isoforms are localized in centers of RNA splicing in the nucleus. The implications of these findings and the impact of the histone modifications on the nuclear-organization of chromatin are discussed.

Animals↗
PubMed1996

Association of transcription factors with the nuclear matrix.

The nuclear matrix is the framework scaffolding of the nucleus and has been demonstrated to be an important component in a number of nuclear processes including transcription, replication, and RNA splicing and transport. In the interphase nucleus, DNA is specifically organized in a three-dimensional fashion. An example of this fact is that actively transcribed genes have been demonstrated to associate with the nuclear matrix. In this study, nuclear matrix proteins from various rat tissues, including two androgen-regulated tissues, the seminal vesicle and ventral prostate, were examined to determine if they contained proteins that associate with consensus binding sequences for several proteins involved in the regulation of transcription. Specific interactions were identified between proteins of the nuclear matrix and these transcriptional activator binding sequences. In addition, the sizes of the complexes binding to the DNA sequences appeared to vary in some of the tissues. These data support the concept that the nuclear matrix may serve as a support structure to bring together specific DNA sequences with factors involved in the regulation of gene expression.

Animals↗
PubMed1990

Core filaments of the nuclear matrix.

The nuclear matrix is concealed by a much larger mass of chromatin, which can be removed selectively by digesting nuclei with DNase I followed by elution of chromatin with 0.25 M ammonium sulfate. This mild procedure removes chromatin almost completely and preserves nuclear matrix morphology. The complete nuclear matrix consists of a nuclear lamina with an interior matrix composed of thick, polymorphic fibers and large masses that resemble remnant nucleoli. Further extraction of the nuclear matrices of HeLa or MCF-7 cells with 2 M sodium chloride uncovered a network of core filaments. A few dark masses remained enmeshed in the filament network and may be remnants of the nuclear matrix thick fibers and nucleoli. The highly branched core filaments had diameters of 9 and 13 nm measured relative to the intermediate filaments. They may serve as the core structure around which the matrix is constructed. The core filaments retained 70% of nuclear RNA. This RNA consisted both of ribosomal RNA precursors and of very high molecular weight hnRNA with a modal size of 20 kb. Treatment with RNase A removed the core filaments. When 2 M sodium chloride was used directly to remove chromatin after DNase I digestion without a preceding 0.25 M ammonium sulfate extraction, the core filaments were not revealed. Instead, the nuclear interior was filled with amorphous masses that may cover the filaments. This reflected a requirement for a stepwise increase in ionic strength because gradual addition of sodium chloride to a final concentration of 2 M without an 0.25 M ammonium sulfate extraction uncovered core filaments.

Cell Line↗
PubMed1999

Immunocytochemical detection of structural and regulatory proteins in rat adrenal nuclear matrix.

The nuclear matrix is a specific cell structure consisting of a residual nucleoskeleton that extends from the nucleoli to the nuclear envelope. The nuclear matrix of steroidogenic cells was isolated previously from a purified nuclear fraction. We present here an in situ extraction method, modified Lutz's method, for rat glandular adrenal cell nuclear matrix. This residual organelle was characterized and studied using immunocytochemical methods. The adrenal glands were removed, the cells prepared in suspension and deposited by cytospin onto Poly-L-lysine glass slides. The nuclear matrix was extracted with Nonidet P-40, DNase I and high and low ionic strength buffers. Structural proteins, nuclear lamins, coilin and fibrillarin were detected immunocytochemically. The adrenal fasciculata cells were easily identified by this method because of their large nuclei and abundant lipid droplets in the cytoplasm. After immunocytochemical detection by antibodies against lamins A and C, a marked brown layer at the periphery of the nucleus was observed. The intensity of the staining was lower using the antibody against nuclear lamin B. Immunocytochemical detection of the protein coilin revealed punctuated stained areas, 2-6 per nucleus, that probably correspond to the coiled bodies. The protein fibrillarin was detected at the nucleolus and coiled bodies. Our technique is simple, reveals well preserved adrenal nuclear matrices, and may be a useful method for immunocytochemical analysis and in situ hybridization.

Adrenal Glands↗
PubMed2005

Dynamic view of the nuclear matrix.

The nuclear matrix is an operationally defined nuclear skeletal structure that is believed to be involved in many nuclear functions including DNA replication, transcription, repair, and prem RNA processing/transport. Until relatively recently, the nuclear matrix was thought to be a rigid and static structure, but it is now thought to be dynamic. This paradigm shift was based in part on the tracking of the intranuclear movement of proteins tagged with fluorochromes. In this review, we attempt to redefine the nuclear matrix in light of recent findings and describe some useful techniques for the dynamic analysis of nuclear function.

Animals↗
PubMed2000

The nuclear matrix of Euglena gracilis (euglenophyta): a stage of nuclear matrix evolution?

Euglena gracilis cell was extracted sequentially with CSK-Triton buffer, RSB-Magik solution and DNase-As solution. DGD embedment-free electron microscopy showed that in the extracted nucleus there was a residual non-chromatin fibrous network. That it could not be removed by hot trichloroacetic acid further supported the idea that it was a non-histone, non-chromatin fibrous protein network, and should be the internal network of the nuclear matrix. After the sequential extraction, the nuclear membrane was removed, leaving behind a layer of lamina; the chromatin was digested and eluted from the dense chromosomes and residual chromosomal structures that should be chromosomal scaffold were revealed. Western blot analysis with antiserum against rat lamins showed that nuclear lamina of the cell possessed two positive polypeptides, a major one and a minor one, which had molecular masses similar to lamin B and lamin A, respectively. Comparing these data with those of the most primitive eukaryote Archezoa and of higher eukaryotes, it was suggested that the lower unicellular eukaryote E. gracilis already had the nuclear matrix structure, and its nuclear matrix (especially the lamina) might represent a stage of evolutionary history of the nuclear matrix.

Animals↗
PubMed1998

The capacity of polyomavirus enhancer binding protein 2alphaB (AML1/Cbfa2) to stimulate polyomavirus DNA replication is related to its affinity for the nuclear matrix.

The nuclear matrix is thought to play an important role in the DNA replication of eukaryotic cells, although direct evidence for such a role is still lacking. A nuclear matrix-associated transcription factor, polyomavirus (Py) enhancer binding protein 2alphaB1 (PEBP2alphaB1) (AML1/Cbfa2), was found to stimulate Py replication through its cognate binding site. The minimal replication activation domain (RAD) was identified between amino acid (aa) 302 and aa 371 by using a fusion protein containing the GAL4 DNA binding domain (GAL4-RAD). In addition, the region showed affinity for the nuclear matrix and, on the basis of competition studies, binding activity for one or more proteins involved in the initiation of Py DNA replication. A leukemogenic chimeric protein, AML1/ETO(MTG8), which does not contain this region of PEBP2alphaB1/AML1, was also localized in the nuclear matrix fraction and competed for nuclear matrix association with PEBP2alphaB1 and GAL4-RAD. Moreover, AML1/ETO inhibited Py DNA replication stimulated by PEBP2alphaB1 and GAL4-RAD. The inhibition was specific for replication mediated by PEBP2alphaB1 and GAL4-RAD, and proportional to the degree of loss of these activators from the nuclear matrix, suggesting a requirement for nuclear matrix targeting in the stimulation of Py DNA replication by RAD. These results are the first to suggest a molecular link between the initiation of DNA replication and the nuclear matrix compartment.

Animals↗
PubMed2004

The 33 kb transcript of the chicken alpha-globin gene domain is part of the nuclear matrix.

Giant nuclear transcripts, and in particular the RNAs of the globin gene domains which are much larger than their canonical pre-mRNAs, have been an enigma for many years. We show here that in avian erythroblastosis virus (AEV)-transformed chicken erythroleukaemic cells, where globin gene expression is abortive, the whole domain of alpha-globin genes is transcribed for about 33 kb in the globin direction and that this RNA is part of the nuclear matrix. Northern blot hybridisation with strand-specific riboprobes, recognising genes and intergenic sequences, and RT-PCR with downstream primers, show that the continuous full domain transcript (FDT) starts in the vicinity of a putative LCR and includes all the genes as well as known regulatory sites, the replication origin, and the DNA loop anchorage region in the upstream area. Absent in chicken fibroblasts, the globin FDT overlaps the major part of the ggPRX housekeeping gene that is transcribed in the opposite direction. RT-PCR and in situ hybridisation with genic and extra-genic globin probes demonstrated that the globin FDT is a component of the nuclear matrix. We suggest that the globin FDTs keep the domain in an active state, and the globin RNAs on the processing pathway are a component of the nuclear matrix. They may take part in the dynamic nuclear architecture when productively processed, or turn over slowly when globins are not synthesised.

Animals↗
PubMed2002

CpG-binding protein is a nuclear matrix- and euchromatin-associated protein localized to nuclear speckles containing human trithorax. Identification of nuclear matrix targeting signals.

CpG-binding protein (CGBP) binds unmethylated CpG dinucleotides and is essential for mammalian development. CGBP exhibits a punctate nuclear localization correlated with 4,6-diamidino-2-phenylindole light regions and is excluded from metaphase chromosomes. The distribution of CGBP is distinct from the heterochromatin-associated proteins MBD1, methyl-CpG-binding protein 2, and HP1alpha. Some CGBP-containing nuclear speckles co-localize with splicing factor SC-35 and actively transcribed regions of the genome, whereas most CGBP co-localizes with acetylated histones, indicating that CGBP is localized to active chromatin. CGBP contains two nuclear localization signals that are insufficient to direct punctate subnuclear distribution. Instead, localization of CGBP to nuclear speckles requires signals within the acidic, basic, and coiled-coil domains. CGBP associates with the nuclear matrix, and fragments of CGBP that fail to associate with the nuclear matrix fail to localize to nuclear speckles and exhibit reduced transcriptional activation activity. Mutated versions of CGBP that lack DNA binding activity exhibit a normal nuclear distribution, suggesting that CGBP accumulates at nuclear speckles as a result of protein/protein interactions. Importantly, the subcellular distribution of CGBP is identical to human trithorax, suggesting that these proteins may be components of a multimeric complex analogous to the histone-methylating Set1 complex of Saccharomyces cerevisiae that contains CGBP and trithorax homologues.

3T3 Cells↗
PubMed1995

Nuclear matrix acceptor binding sites for steroid hormone receptors: a candidate nuclear matrix acceptor protein.

Steroid/nuclear-hormone receptors are ligand-activated transcription factors that have been localized to the nuclear matrix. The classic model of hormone action suggests that, following activation, these receptors bind to specific "steroid response elements" on the DNA, then interact with other factors in the transcription initiation complex. However, evidence demonstrates the existence of specific chromatin proteins that act as accessory factors by facilitating the binding of the steroid receptors to the DNA. One such protein, the "receptor binding factor (RBF)-1", has been purified and shown to confer specific, high-affinity binding of the progesterone receptor to the DNA. Interestingly, the RBF-1 is localized to the nuclear matrix. Further, the RBF-1 binds specifically to a sequence of the c-myc proto-oncogene that has the appearance of a nuclear matrix attached region (MAR). These results, and other findings reviewed here, suggest that the nuclear matrix is involved intimately in steroid hormone-regulated gene expression.

Animals↗
PubMed1999

Association of chromosome territories with the nuclear matrix. Disruption of human chromosome territories correlates with the release of a subset of nuclear matrix proteins.

To study the possible role of the nuclear matrix in chromosome territory organization, normal human fibroblast cells are treated in situ via classic isolation procedures for nuclear matrix in the absence of nuclease (e.g., DNase I) digestion, followed by chromosome painting. We report for the first time that chromosome territories are maintained intact on the nuclear matrix. In contrast, complete extraction of the internal nuclear matrix components with RNase treatment followed by 2 M NaCl results in the disruption of higher order chromosome territory architecture. Correlative with territorial disruption is the formation of a faint DNA halo surrounding the nuclear lamina and a dispersive effect on the characteristically discrete DNA replication sites in the nuclear interior. Identical results were obtained using eight different human chromosome paints. Based on these findings, we developed a fractionation strategy to release the bulk of nuclear matrix proteins under conditions where the chromosome territories are maintained intact. A second treatment results in disruption of the chromosome territories in conjunction with the release of a small subset of acidic proteins. These proteins are distinct from the major nuclear matrix proteins and may be involved in mediating chromosome territory organization.

Ammonium Sulfate↗
PubMed1997

Identification of human common nuclear-matrix proteins as heterogeneous nuclear ribonucleoproteins H and H' by sequencing and mass spectrometry.

Nuclear-matrix proteins were prepared from different rat and human cells and separated by two-dimensional gel electrophoresis. By computer-assisted analysis of the images, two of the proteins were identified as ubiquitously occurring (common) nuclear-matrix proteins, which appeared in tissue-dependent concentrations. The two proteins that originated from human blood mononuclear cells were analyzed further. Tryptic digests of the blotted proteins were analyzed by partial peptide sequencing and matrix-assisted laser-desorption ionization-time-of-flight mass spectrometry. The two human common nuclear-matrix proteins were identified as heterogeneous nuclear ribonucleoproteins (hnRNP) H and H' or their variants. Furthermore, mass analysis revealed details on the N terminus of hnRNP H.

Amino Acid Sequence↗
PubMed2001

The conserved domain CR2 of Epstein-Barr virus nuclear antigen leader protein is responsible not only for nuclear matrix association but also for nuclear localization.

There is a growing body of evidence for the importance of the nuclear matrix in various nuclear events including gene expression and DNA replication. Epstein-Barr virus (EBV) nuclear antigen leader protein (EBNA-LP) is a nuclear matrix-associated protein that has been suggested to play an important role in EBV-induced transformation. To define the biological significance of the association of EBNA-LP with the nuclear matrix, we mapped the domain of EBNA-LP responsible for nuclear matrix association and investigated the functions of the EBNA-LP mutant mutagenized by substitution of alanines for the cluster of arginine residues in the mapped region. The results of the present study were as follows. (i) Transiently expressed EBNA-LP in COS-7 or BOSC23 cells was associated with the nuclear matrix, similarly to that in EBV-infected B cells. (ii) Mutational analysis of EBNA-LP revealed that a 10-amino acid segment of EBNA-LP is critical for nuclear matrix association of the protein. Interestingly, the identified region overlapped with the region CR2 of EBNA-LP conserved among a subset of primate gammaherpesviruses. The identified segment is referred to as EBNA-LP NMTS (nuclear matrix targeting signal). (iii) The EBNA-LP mutant with the arginine to alanine substitutions in NMTS was no longer localized not only to the nuclear matrix but also to the nucleus. (iv) The EBNA-LP mutant lacked its ability to coactivate EBNA-2-dependent transactivation. These results indicated that EBNA-LP needs to be localized in the nucleus and/or associated with the nuclear matrix through CR2 to elicit its function such as the coactivation of the EBNA-2-dependent transcriptional activation.

Alanine↗