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PubMed2003

A proteomic study of the arabidopsis nuclear matrix.

The eukaryotic nucleus has been proposed to be organized by two interdependent nucleoprotein structures, the DNA-based chromatin and the RNA-dependent nuclear matrix. The functional composition and molecular organization of the second component have not yet been resolved. Here, we describe the isolation of the nuclear matrix from the model plant Arabidopsis, its initial characterization by confocal and electron microscopy, and the identification of 36 proteins by mass spectrometry. Electron microscopy of resinless samples confirmed a structure very similar to that described for the animal nuclear matrix. Two-dimensional gel electrophoresis resolved approximately 300 protein spots. Proteins were identified in batches by ESI tandem mass spectrometry after resolution by 1D SDS-PAGE. Among the identified proteins were a number of demonstrated or predicted Arabidopsis homologs of nucleolar proteins such as IMP4, Nop56, Nop58, fibrillarins, nucleolin, as well as ribosomal components and a putative histone deacetylase. Others included homologs of eEF-1, HSP/HSC70, and DnaJ, which have also been identified in the nucleolus or nuclear matrix of human cells, as well as a number of novel proteins with unknown function. This study is the first proteomic approach towards the characterization of a higher plant nuclear matrix. It demonstrates the striking similarities both in structure and protein composition of the operationally defined nuclear matrix across kingdoms whose unicellular ancestors have separated more than one billion years ago.

Arabidopsis↗
PubMed2005

CK2 phosphorylation weakens 90 kDa MFP1 association to the nuclear matrix in Allium cepa.

MFP1 is a conserved plant coiled-coil protein located on the stroma side of the chloroplast thylakoids, as well as in the nuclear matrix. It displays species-specific variability in the number of genes, proteins, and expression. Allium cepa has two nuclear proteins antigenically related to MFP1 with different M(r), pI, distribution, and expression, but only the 90 kDa MFP1 protein is a nuclear matrix component that associates with both the nucleoskeletal filaments and a new category of nuclear bodies. The 90 kDa AcMFP1 migrates in two-dimensional blots as two sets of spots. The hypo-phosphorylated forms (pI approximately 9.5) are tightly bound to the nuclear matrix, while high ionic strength buffers release the more acidic hyper-phosphorylated ones (pI approximately 8.5), suggesting that the protein is post-translationally modified, and that these modifications control its attachment to the nuclear matrix. Dephosphorylation by exogenous alkaline phosphatase and phosphorylation by exogenous CK2, as well as specific inhibition and stimulation of endogenous CK2 with heparin and spermine and spermidine, respectively, revealed that the protein is an in vitro and in vivo substrate of this enzyme, and that CK2 phosphorylation weakens the strength of its binding to the nuclear matrix. In synchronized cells, the nuclear 90 kDa AcMFP1 phosphorylation levels vary during the cell cycle with a moderate peak in G2. These results provide the first evidence for AcMFP1 in vivo phosphorylation, and open up further research on its nuclear functions.

Casein Kinase II↗
PubMed2003

Functional association of immediate early gene c-fos with nuclear matrix.

Accumulation of c-fos gene locus DNA in the nuclear matrix of hepatocyte nuclei was observed during induction of c-fos with cycloheximide. No enhanced association with the nuclear matrix was detected for inactive immunoglobulin gene locus. The use of endogenous DNAses allows isolation of nuclear matrix preparations enriched with transcribing chromatin.

Animals↗
PubMed1987

Estrogen receptors in the nuclear matrix: direct demonstration using monoclonal antireceptor antibody.

Estradiol-binding sites, as assayed by exchange with radiolabeled steroid, become associated with the nuclear matrix of estrogen-responsive tissues after treatment with estrogen in vivo. Using monoclonal estrogen receptor antibodies, we have now obtained direct evidence that these matrix-associated estradiol-binding sites are estrogen receptor proteins similar to those found in the cytosol before estrogen treatment. Proteins of the liver nuclear matrix from untreated or ethinyl estradiol-treated female rats were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to nitrocellulose paper, and probed with the monoclonal estrogen receptor antibody H222Sp gamma. A single prominent immunoreactive 67,000 mol wt band, indicating the presence of estrogen receptors, was found in the liver nuclear matrix of estrogen-treated animals. This band was detectable, but of much lower intensity, in the liver nuclear matrix of untreated animals. Liver cytosol estrogen receptor from untreated rats also migrated as a 67,000 mol wt band. These immunoreactivity data corroborated data obtained by [3H]estradiol-binding assays. Scatchard analysis of specific high affinity [3H]estradiol-binding sites showed high levels of these sites in the liver nuclear matrix of estrogen-treated rats and low levels in untreated rats. Therefore, both direct and indirect methods of receptor identification demonstrate the specific association of estrogen receptors with the nuclear matrix after estrogen treatment in vivo.

Animals↗
PubMed1987

Distribution of nuclear matrix proteins in interphase CHO cells and rearrangements during the cell cycle. An ultrastructural study.

The nuclear matrix contains a group of residual non-histone proteins which remain structurally organized after extensive extraction of isolated nuclei with a high salt buffer, nucleases and a non-ionic detergent. Electron microscopic examination shows that the nuclear matrix is composed of a pore-complex lamina, an intranuclear network and residual nucleoli. In CHO cells biochemical analyses performed by one-dimensional SDS-PAGE show three major nuclear matrix polypeptides with molecular weights between 60 and 70 kDa. Polyclonal antibodies produced against these polypeptides were used to determine their nuclear distribution. Using immunoblotting, these proteins were found in whole nuclei, nuclear matrix, and in the intranuclear network but not in the pore-complex lamina. In order to determine the relationship between these structural proteins and the organization of the nucleus, the proteins were localized in situ. Ultrastructural detection was carried out by immunogold staining of thin sections of Lowicryl K4M-embedded cells. In interphase nuclei all condensed chromatin clumps were labelled. The nucleolus and the interchromatin granules were never immunogold-stained. During mitosis, the label was found to be associated with the chromosomes. This study shows that unlike the lamins, these 60-70 kDa nuclear matrix proteins are associated with the condensed chromatin throughout the cell cycle.

Animals↗
PubMed1982

The ovalbumin gene is associated with the nuclear matrix of chicken oviduct cells.

The DNA in a eucaryotic nucleus is arranged into a series of supercoiled loops that are anchored at their bases to the nuclear matrix. Using nuclease digestion, one can progressively cleave DNA from the loops, thereby isolating residual DNA that is progressively closer to the nuclear matrix anchorage sites. We have determined that the ovalbumin gene is preferentially associated with the nuclear matrix of chicken oviduct cells, but is not preferentially associated with the nuclear matrix of chicken liver cells. As a control, the beta-globin gene, which is not transcribed in oviduct cells, was found not to be preferentially associated with the oviduct nuclear matrix. The observation that the transcriptionally active ovalbumin gene is preferentially associated with the nuclear matrix may have significant implications for gene expression and the organization of nuclear DNA into supercoiled-loop domains.

Animals↗
PubMed1986

The nuclear matrix is the site of glucocorticoid receptor complex action in the nucleus.

Binding of highly purified glucocorticoid receptor complexes to nuclear matrix was evaluated. Extraction of purified nuclei with 2M potassium chloride and brief deoxyribonuclease digestion leaves a matrix structure containing 1% of nuclear DNA and 6-12% of nuclear proteins. The nuclear matrix retained two binding sites for receptor complexes, a high affinity, low capacity site and a low affinity, high capacity site. These sites have affinities and capacities consistent with those reported for binding of these complexes to intact nuclei. More extensive deoxyribonuclease treatment of the matrix resulted in a marked reduction of high affinity complex binding. Furthermore, the DNA binding form of the receptor complex but not the unactivated receptor complex bound to DNA fibers anchored to nuclear matrix as visualized by 18 nm gold particle receptor complexes. The data suggest that the nuclear matrix is the major site for coordinating glucocorticoid hormone action in the nucleus.

Animals↗
PubMedDate not supplied

Effect of DNA topoisomerase I inhibitor, 10-hydroxycamptothecin, on the structure and function of nuclei and nuclear matrix in bladder carcinoma MBT-2 cells.

The effects of 10-hydroxycamptothecin (HCPT), a DNA topoisomerase I inhibitor, on the structure of nuclei and nuclear matrix and new DNA replication was investigated in murine bladder carcinoma MBT-2 cells. Following 10-30 min exposure of exponentially growing cells to 10-50 microM HCPT, the rate of 2 min pulse-labeling with [3H]thymidine in whole nuclei, nonmatrix, and matrix fractions was markedly decreased in a concentration- and time-dependent manner. Reduction was highest in the nuclear matrix. After treatment of cultures with 50 microM HCPT for 10 min, inhibition of DNA synthesis was 60% in the nuclear matrix and 27% in the low-salt extract fraction tended to be stimulated after drug treatment. SDS-PAGE analysis of [35S]methionine labeled proteins indicated that 50 microM HCPT decreased protein synthesis in the matrix and other nuclear subfractions. HCPT-induced ultrastructural changes in nuclei and nuclear matrix were similar to those typically associated with lesions of DNA replication or RNA transcription.

Animals↗
PubMed1999

Reassembling proteins and chaperones in human nuclear matrix protein fractions.

To detect putative filament forming components, nuclear matrix proteins were searched for proteins extensively reassembling from urea solution. Eight proteins, ubiquitously occurring in various human cell types, but not apparent in the cytosol, were registered by means of two-dimensional gel electrophoresis. They consisted of a protein exhibiting a novel amino acid sequence; of nuclear lamin B2, RbAp46, and RbAp48; and of four as yet unknown proteins. Furthermore, partial sequencing, mass spectrometry, and immunodetection of proteins demonstrated the presence of molecular chaperones and protein folding catalysts in the nuclear matrix fractions. In addition to a TCP-1-related protein, certain members of the heat shock, PDI, and calreticulin family of proteins were detected. On the basis of the absence of several other heat shock proteins in the nuclear matrix fraction, a general contamination by cytoplasmic chaperones appears unlikely.

Amino Acid Sequence↗
PubMed1994

Parathyroid-responsive modifications in the nuclear matrix of ROS 17/2.8 rat osteosarcoma cells.

PTH is a mediator of skeletal development and remodeling that influences gene expression in osteoblastic cells. It is well established that PTH modulates the activity of membrane-associated second messenger signal transduction pathways. In these studies we have addressed the potential contribution of components of cell structure to the integration of PTH-related regulatory signals that influence the expression of bone cell genes. Chronic treatment of ROS 17/2.8 rat osteosarcoma cells with PTH is accompanied by changes in gene expression that are at least in part transcriptionally controlled. To explore the involvement of nuclear architecture in PTH-responsive modifications in gene expression, we investigated changes in the nuclear matrix after PTH treatment. Consistent with a role for the nuclear matrix in determining spatial organization and topology of chromatin as well as in the localization and targeting of transcription factors, we observed PTH-associated changes in a 200-kilodalton nuclear matrix protein in response to PTH. A significant down-regulation of synthesis was observed when nuclear matrix proteins were resolved electrophoretically in two-dimensional gels. This protein was restricted to the nuclear matrix and was not detected in the chromatin or cytoskeletal cellular fractions. These alterations in nuclear matrix proteins that occur after PTH treatment in osteosarcoma cells were phenotype related. They did not occur in UMR-106 POL or H4 hepatoma cells. Our findings support a role for the nuclear matrix in transducing PTH-mediated regulatory signals to facilitate the extent to which genes in osteoblasts are transcribed.

Animals↗
PubMed1988

Interaction of myb proteins with nuclear matrix in vitro.

Fractionation studies of isolated nuclei have shown that the proteins encoded by the retroviral oncogene v-myb and its cellular homologue c-myb are associated to a variable extent with the nuclear matrix, suggesting that the nuclear matrix might contain a cellular target for myb proteins. I have explored the possible existence of such a target by incubating soluble v-myb and c-myb protein with nuclear matrix prepared from a separate source. The results presented here suggest that nuclear matrices from various cells contain binding sites for myb proteins. Matrix-binding appears to be an intrinsic property of myb proteins. In addition to myb proteins I have demonstrated the existence of a small group of proteins possessing nuclear matrix binding activity. These findings suggest that the nuclear matrix serves as a target for a specific set of proteins, including the products of myb genes.

Animals↗
PubMed1992

The association of the human epsilon-globin gene with the nuclear matrix: a reconsideration.

The association of the human epsilon-globin gene with the nuclear matrix was studied in erythroid and non-erythroid cell lines. Using a high salt method to prepare histone depleted nuclei we studied the association of variety of fragments covering a 7.8 kb region which contains the human epsilon-globin gene. We furthermore studied the association of a set of DNA fragments covering the 13 kb human G gamma/A gamma-globin gene domain, the 16 kb psi beta/delta-globin gene domain and the 10 kb beta-globin gene domain with the nuclear matrix of K562 and Raji cells. The results show that all fragments studied are easily released from the nuclear matrix, indicating no specific association. Summarizing our results we could say that a region starting 5.7 kb 5' to the human epsilon-globin gene and ending 4 kb 3' to the human beta-globin gene seems to contain no attachment sites with the nuclear matrix of both erythroid and non-erythroid cells.

Burkitt Lymphoma↗
PubMed1986

Preferential binding of DNA primase to the nuclear matrix in HeLa cells.

Studies of the spatial organization of DNA replication have provided increasing evidence of the importance of the nuclear matrix. We have previously reported a relationship between rates of DNA synthesis and the differential binding of DNA polymerase alpha to the nuclear matrix over the S-phase. We now report the detection of DNA primase bound to the HeLa nuclear matrix. Matrix-bound primase was measured both indirectly, by the incorporation of [32P]dAMP into an unprimed single-stranded template, poly(dT), and directly, by the incorporation of [3H]AMP into matrix DNA. Characteristics of this system include a requirement for ATP, inhibition by adenosine 5'-O-(thiotriphosphate), a primase inhibitor, and insensitivity to aphidicolin and alpha-amanitine, inhibitors of polymerase alpha and RNA polymerase, respectively. Subcellular quantification of primase and polymerase alpha activity revealed that while most (approximately 72%) primase activity is bound to the matrix, only a minority (approximately 32%) of polymerase alpha activity is matrix-bound. Treatment of the nuclear matrix with beta-D-octylglucoside allowed the solubilization of approximately 54% of primase activity and approximately 39% of the polymerase alpha activity. This data provides further evidence of a structural and functional role for the nuclear matrix in DNA replication. The ability to solubilize matrix-bound replicative enzymes may prove to be an important tool in the elucidation of the spatial organization of DNA replication.

Adenosine Triphosphate↗
PubMed1985

Poly(adenosine diphosphate-ribosylation) of nuclear matrix proteins in alkylating agent resistant and sensitive cell lines.

Using Walker 256 breast carcinoma cell lines either with or without acquired resistance to alkylating agents, the structural framework proteins of the nucleus, the nuclear matrix proteins, were found to be effective acceptors for poly(ADP-ribose). Incubation of isolated nuclei with nicotinamide adenine [32P] dinucleotide ([32P] NAD), followed by the isolation of the nuclear matrix, demonstrated that two polypeptides of approximate molecular weight (Mr) 105 000 and 116 000 were extensively poly(ADP-ribosylated). By an in vitro [32P] NAD assay, the nuclear matrix fraction was found to maintain approx. 15% of the total nuclear matrix activity of poly(ADP-ribose) polymerase. Confirmation that the trichloroacetic acid (TCA) precipitable material represented ADP-ribose units was achieved by enzymatic digestion of the nuclear matrix preparation with snake venom phosphodiesterase (SVP). Within 15 min, greater than 85% of the 32P label was digested by SVP and the final digestion products were found to be phosphoribosyl-AMP (PR-AMP) and adenosine 5'-monophosphate (5'-AMP) by thin layer chromatographic analysis. The average polymer chain length was estimated to be 6-7 ADP-ribose units. Because poly(ADP-ribose) polymerase has a putative role in DNA repair, a comparison of the nuclear matrix fractions from Walker resistant and sensitive tumor cell lines was made. In both cell lines, the quantitative and qualitative patterns of the nuclear matrix associated poly(ADP-ribosylation) were similar.

Adenosine Monophosphate↗
PubMed2002

Proteome analysis of nuclear matrix proteins during apoptotic chromatin condensation.

The nuclear matrix (NM) is considered a proteinaceous scaffold spatially organizing the interphase nucleus, the integrity of which is affected during apoptosis. Caspase-mediated degradation of NM proteins, such as nuclear lamins, precedes apoptotic chromatin condensation (ACC). Nevertheless, other NM proteins remain unaffected, which most likely maintain a remaining nuclear structure devoid of chromatin. We, therefore, screened various types of apoptotic cells for changes of the nuclear matrix proteome during the process of apoptotic ACC. Expectedly, we observed fundamental alterations of known chromatin-associated proteins, comprising both degradation and translocation to the cytosol. Importantly, a consistent set of abundant NM proteins, some (e.g. hNMP 200) of which displaying structural features, remained unaffected during apoptosis and might therefore represent constituents of an elementary scaffold. In addition, proteins involved in DNA replication and DNA repair were found accumulated in the NM fraction before cells became irreversibly committed to ACC, a time point characterized in detail by inhibitor studies with orthovanadate. In general, protein alterations of a consistent set of NM proteins (67 of which were identified), were reproducibly detectable in Fas-induced Jurkat cells, in UV-light treated U937 cells and also in staurosporine-treated HeLa cells. Our data indicate that substantial alterations of proteins linking chromatin to an elementary nuclear protein scaffold might play an intriguing role for the process of ACC.

Apoptosis↗
PubMed2005

RNA-dependent nuclear matrix contains a 33 kb globin full domain transcript as well as prosomes but no 26S proteasomes.

Previously, we have shown that in murine myoblasts prosomes are constituents of the nuclear matrix; a major part of the latter was found to be RNase sensitive. Here, we further define the RNA-dependent matrix in avian erythroblastosis virus (AEV) transformed erythroid cells in relation to its structure, presence of specific RNA, prosomes and/or proteasomes. These cells transcribe but do not express globin genes prior to induction. Electron micrographs show little difference in matrices treated with DNase alone or with both, DNase and RNase. In situ hybridization with alpha globin riboprobes shows that this matrix includes globin transcripts. Of particular interest is that, apparently, a nearly 35 kb long globin full domain transcript (FDT), including genes, intergenic regions and a large upstream domain is a part of the RNA-dependent nuclear matrix. The 23K-type of prosomes, previously shown to be co-localized with globin transcripts in the nuclear RNA processing centers, were found all over the nuclear matrix. Other types of prosomes show different distributions in the intact cell but similar distribution patterns on the matrix. Globin transcripts and at least 80% of prosomes disappear from matrices upon RNase treatment. Interestingly, the 19S proteasome modulator complex is insensitive to RNase treatment. Only 20S prosomes but not 26S proteasomes are thus part of the RNA-dependent nuclear matrix. We suggest that giant pre-mRNA and FDTs in processing, aligning prosomes and other RNA-binding proteins are involved in the organization of the dynamic nuclear matrix. It is proposed that the putative function of RNA within the nuclear matrix and, thus, the nuclear dynamic architecture, might explain the giant size and complex organization of primary transcripts and their introns.

Base Sequence↗
PubMed2000

Ultrastructural analysis of v-myb oncogene product cooperation with components of avian cell nuclear matrix.

The cooperation of the v-Myb oncoprotein with extracted nuclear matrix of avian haematopoietic cells expressing the v-myb oncogene was studied by means of immunoelectron microscopy. The nuclear matrix was extracted by a gentle method of detergent treatment at moderate ionic strength and visualized either in ultrathin LR White sections, in unembedded resin-free sections, and in addition by the aqueous spreading technique. Using anti-Myb polyclonal antibody we have shown interaction of the v-Myb protein product with extracted nuclear matrix. This oncoprotein, however, was easily released from the structure by a detergent as well as by DNAase treatment and ammonium sulphate extraction. Prefixation of structures before detergent treatment prevented this extraction. The v-Myb protein marker was distributed in clusters or associated with fibrillar structures in most cases. Single markers decorating these fibrillar or less dense structures were also detected.

Animals↗
PubMed1992

Increase of cytokeratin D during liver regeneration: association with the nuclear matrix.

An increase of a 45 kD protein (p45) in the nuclear matrix has been observed when rat liver cells were proliferatively activated in vivo by a partial hepatectomy. The maximal levels of the association of p45 with the nuclear matrix have been detected 24 hr after hepatectomy just at the time when DNA replication is also maximal. By amino acid sequence analysis, immunoblotting and immunocytochemical methods, it has been demonstrated that p45 is identical to rat cytokeratin D. Immunogold staining of nuclear matrix-intermediate filament preparations from cultured hepatocytes indicated that p45 is associated with cytoskeletal filaments that are strongly interconnected to the lamina, whereas no intranuclear localization of the protein has been detected. With an overlay assay a specific binding of labeled p45 to two nonidentified high-molecular weight proteins and also to lamin B has been observed. Northern blot analysis revealed a biphasic pattern of expression of the messenger RNA for cytokeratin D during liver regeneration. A sharp increase in the messenger RNA levels occurred in the prereplicative phase of liver regeneration a few hours before the accumulation of the protein in the nuclear matrix fraction, and a second peak occurred 48 hr after partial hepatectomy.

Amino Acid Sequence↗