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PubMed1989

The effect of lead on the metabolism of a nuclear matrix protein which becomes prominent in lead-induced intranuclear inclusion bodies.

The influence of lead and calcium on the metabolism of a nuclear matrix protein has been studied in mouse neuroblastoma 2a (Nb2a) cells. This protein, p32/6.3, has an unusual distribution in that it is relatively abundant only in normal neural tissues and in intranuclear inclusion bodies induced in kidney tubule-lining cells of chronically lead-intoxicated animals (Egle, P. M., and Shelton, K. R. (1986) J. Biol. Chem. 261, 2294-2298). The mechanism(s) whereby lead increases p32/6.3 content are of interest, but its slow accumulation in intact animals over a period of weeks to months precludes studies with metabolic inhibitors. However, the enriched levels of p32/6.3 in mouse neuroblastoma 2a (Nb2a) cells permit these studies. The relative abundance of this protein was found to increase in mouse Nb2a cells after 1- and 3-day exposures to lead. This increase could be attributed to a decreased rate of degradation rather than either increased transcription or increased translation. A role for calcium in p32/6.3 regulation was also explored. Although neither increased extracellular calcium nor calcium ionophores had an effect, treatment of the cells with the calcium chelator [ethylenebis(oxyethylenenitrilo)]tetraacetic acid decreased p32/6.3 levels in a concentration-dependent manner, suggesting a role for calcium in the normal metabolism of the protein.

Animals↗
PubMed1994

The regulatory element 3' to the A gamma-globin gene binds to the nuclear matrix and interacts with special A-T-rich binding protein 1 (SATB1), an SAR/MAR-associating region DNA binding protein.

A cis-acting DNA regulatory element 3' to the A gamma-globin gene contains eight distinct regions of DNA-protein interaction distributed over 750 bp of DNA. The sequences of two foot-printed regions (sites I and IV) are A-T rich and generate a highly retarded complex on gel shift analysis with nuclear extract from human erythroleukemia (K562) cells. We have purified a 98-kD protein that reproduces this gel shift. Tryptic cleavage and peptide sequence analysis demonstrated that the 98-kD protein is identical to a recently cloned protein, special A-T-rich binding protein 1 (SATB1), that binds selectively to nuclear matrix/scaffold-associated regions of DNA (MARs/SARs). We have shown by functional analysis that the 3' A gamma regulatory element associates with the nuclear matrix. SATB1 mRNA was identified in K562 cells, and reverse transcriptase-polymerase chain reaction (RT-PCR) demonstrated its transcript in several other hematopoietic lines. Antisera to SATB1 caused ablation of the gel shift complex generated by both the crude nuclear extract and the purified 98-kD protein with the site I oligonucleotide. Furthermore, oligonucleotides that bind SATB1 inhibited formation of the site I gel shift complex when added as excess unlabeled competitor. An immunoblot analysis of the site I gel shift complex documented the presence of SATB1. Binding of SATB1 to two sites within the 3' A gamma regulatory element and its MAR/SAR activity suggests that this element may influence gene expression through interaction with the nuclear matrix.

Amino Acid Sequence↗
PubMed1986

[Isolation and fractionation of the nuclear matrix from cultured cells].

A new method for the isolation of tissue culture cell nuclei is presented which involves incubation of the nuclei in the presence of Cu2+- or Zn2+-ions. This method eliminates the danger of nuclear aggregation and permits nuclear matrix isolation and subsequent fractionation. Stabilization of the inner matrix by Cu2-ions permits analysis of the role of nucleic acids in the maintenance of the matrix structure. It is shown that solubilization of more than 95% of matrix-bound DNA and more than 90% of matrix-bound RNA did not cause any significant changes in the nuclear matrix structure.

Animals↗
PubMed1984

DNA polymerases in isolated 'nuclear matrix' of Ehrlich ascites cells.

Isolated nuclei from Ehrlich ascites tumor cells continue a replicative-like in vitro DNA synthesis. Polymerase alpha is the major dNTP polymerizing enzyme in nuclei. Following complete achromatinization dNTP polymerizing activities are still associated with the residual structure termed 'nuclear matrix'. In contrast to DNA synthesis in native nuclei, 'nuclear matrix' DNA synthesis is mainly due to polymerase beta-like activity.

Animals↗
PubMed1992

Localization of the nuclear matrix protein mitotin in mouse cells with a mitotic or endomitotic cell cycle.

Immunofluorescence staining was used to study the precise subcellular distribution of the nuclear matrix antigen, mitotin, in mouse cells characterized by either a mitotic or an endomitotic organization of the cell cycle. In mitotically dividing cells, mitotin showed a speckled distribution within interphase nuclei. In addition, some interphase cells exhibited a weak, focused signal adjacent to the nucleus, reflecting a possible staining of the centrosome region. Using digital contrast-enhanced immunofluorescence microscopy, a distinct association of mitotin to the centrosome, pole microtubules, and midbody could be revealed in cells at different stages of mitosis. In parallel, trophoblast giant cells characterized by an endomitotic cell cycle were derived from blastocyst outgrowths and analyzed likewise. In all giant cells examined so far, mitotin was restricted to the nuclear compartment alone, although different patterns of intranuclear staining could be detected. The present study provides further information about the precise localization of mitotin in mitotic cells, especially during mitosis. In view of the results, the staining pattern observed in endomitotic cells may allow for a better understanding of the origin and the organization of the endomitotic cell cycle.

Animals↗
PubMed2001

Small tumor virus genomes are integrated near nuclear matrix attachment regions in transformed cells.

More than 15% of human cancers have a viral etiology. In benign lesions induced by the small DNA tumor viruses, viral genomes are typically maintained extrachromosomally. Malignant progression is often associated with viral integration into host cell chromatin. To study the role of viral integration in tumorigenesis, we analyzed the positions of integrated viral genomes in tumors and tumor cell lines induced by the small oncogenic viruses, including the high-risk human papillomaviruses, hepatitis B virus, simian virus 40, and human T-cell leukemia virus type 1. We show that viral integrations in tumor cells lie near cellular sequences identified as nuclear matrix attachment regions (MARs), while integrations in nonneoplastic cells show no significant correlation with these regions. In mammalian cells, the nuclear matrix functions in gene expression and DNA replication. MARs play varied but poorly understood roles in eukaryotic gene expression. Our results suggest that integrated tumor virus genomes are subject to MAR-mediated transcriptional regulation, providing insight into mechanisms of viral carcinogenesis. Furthermore, the viral oncoproteins serve as invaluable tools for the study of mechanisms controlling cellular growth. Similarly, our demonstration that integrated viral genomes may be subject to MAR-mediated transcriptional effects should facilitate elucidation of fundamental mechanisms regulating eukaryotic gene expression.

Attachment Sites, Microbiological↗
PubMed1994

Immunocytochemical detection of the specific association of different PIC isoforms with cytoskeletal and nuclear matrix compartments in PC12 cells.

The increasing evidence of discrete roles of phosphoinositidase C (PIC) isoforms and the assessment of their localization in the cytoskeleton and in the nucleus support the involvement of particular isotypes of this enzyme in signal transduction at multiple levels. PC12 rat pheochromocytoma is one of the few cell lines expressing three immunologically distinct isoforms of PIC. We have analyzed the subcellular distribution of the PIC beta 1, gamma 1 and delta 1 isoforms using confocal and electron microscope immunocytochemistry. PIC beta 1 is mainly found in the nucleus and is associated with interchromatin domains. On the other hand, the PIC gamma 1 isoform is found in the nucleus and in the cytosol, while PIC delta 1 is exclusively cytoplasmic. Immunoblot and immunocytochemical experiments indicate that the various PIC isoforms are differently bound to structural cell compartments, such as cytoskeletal and nuclear matrix elements. In fact, PIC beta 1 and PIC gamma 1 isoforms are tightly associated with the nuclear matrix, while only about 50% of PIC gamma 1 is associated with the cytoskeleton after DNase I and high salt extractions. PIC gamma 1 is almost completely soluble under these conditions. These results further confirm the complexity of the inositide signal transduction mechanism, which involves several PIC isoforms, specifically localized in different cell compartments and support the existence of a membrane-unrelated inositol lipid-dependent signalling in the nuclear interior.

Animals↗
PubMed1996

The KRAB zinc finger gene ZNF74 encodes an RNA-binding protein tightly associated with the nuclear matrix.

We previously cloned ZNF74, a developmentally expressed zinc finger gene commonly deleted in DiGeorge syndrome. Here, the intron/exon organization of the human gene and the functional properties of the expressed protein are presented. This zinc finger gene from the transcription factor IIIA/Kruppel family contains three exons. A truncated Kruppel-associated box (KRAB) located at the N terminus of the predicted 64-kDa zinc finger protein is encoded by exon 2. The remainder of the protein including the zinc finger domain as well as the 3'-untranslated region (UTR) is encoded by exon 3. Both 5'-UTR (exon 1) and 3'-UTR contain repetitive Alu elements. In vitro translation of a cDNA encoding the entire ZNF74 coding region produced a 63-kDa protein as determined on sodium dodecyl sulfate-polyacrylamide gel. A bacterially expressed fusion protein shown to bind tightly to 65zinc was used to test the nucleic acid binding properties of ZNF74. By RNA binding assays, ZNF74 was found to bind specifically to poly(U) and poly(G) RNA homopolymers. The restricted binding to these homopolymers and not to poly(A) and poly(C) suggested that ZNF74 displays RNA sequence preferences. RNA binding was mediated by the zinc finger domain. Immunofluorescence studies on transfected cells revealed ZNF74 nuclear localization. The labeling pattern observed in the nuclei clearly excluded the nucleoli. The zinc finger region lacks a classical nuclear localization signal but was found to be responsible for nuclear targeting. Subcellular and in situ sequential fractionations further showed that ZNF74 is associated with the nuclear matrix. The RNA binding properties of this protein and its tight association with the nuclear matrix, a subnuclear compartment involved in DNA replication as well as RNA synthesis and processing, suggest a role for ZNF74 in RNA metabolism.

Amino Acid Sequence↗
PubMed2005

The intracellular domain of teneurin-1 interacts with MBD1 and CAP/ponsin resulting in subcellular codistribution and translocation to the nuclear matrix.

Teneurin-1 is a type II transmembrane protein expressed in neurons of the developing and adult central nervous system. To investigate the intracellular signaling of teneurin-1, we searched for proteins interacting with its intracellular domain. One of the proteins identified is the c-Cbl-associated protein CAP/ponsin, an adaptor protein containing SH3 domains. This interaction results on one hand in the recruitment of the soluble intracellular domain of teneurin-1 to the cell membrane enriched in CAP/ponsin. On the other hand, it leads to the translocation of CAP/ponsin to the nucleus, the major site of accumulation of the intracellular domain of teneurin-1. The second interacting protein identified is the methyl-CpG binding protein MBD1. In the nucleus, the intracellular domain of teneurin-1 colocalizes with this transcriptional repressor in foci associated with the nuclear matrix. We propose that these interactions are part of a specific signaling pathway. Evidence for cleavage and nuclear translocation of the intracellular domain has been obtained by the detection of endogenous teneurin-1 immunoreactivity in nuclear speckles in chick embryo fibroblasts. Furthermore, in the nuclear matrix fraction of these cells as well as in cells expressing a hormone-inducible full-length teneurin-1 protein, a teneurin-1 fragment of identical size could be detected as in cells transfected with the intracellular domain alone.

Amino Acid Sequence↗
PubMed1996

Urinary nuclear matrix protein as a marker for transitional cell carcinoma of the urinary tract.

PURPOSE: The purpose of this trial was to evaluate an immunoassay for urinary nuclear matrix protein, NMP22, as an indicator for transitional cell carcinoma of the urinary tract. MATERIALS AND METHODS: Three groups of subjects participated in this trial of NMP22: 1-175 with transitional cell carcinoma, 2-117 with benign urinary tract conditions and 3-375 healthy volunteers. Each subject provided a single (3 voids) urine sample for analysis at the time of study entry. Each sample was assayed for the level of NMP22. RESULTS: In normal healthy volunteers and in subjects with benign conditions median NMP22 levels were 2.9 and 3.3 units per ml., respectively. Median urinary NMP22 levels in patients with transitional cell carcinoma were significantly greater than in comparison subjects. Patients with active transitional cell carcinoma had significantly greater median urinary NMP22 levels than those with no evidence of disease (6.04 versus 4.11 units per ml., p = 0.027, 1-tailed Mann-Whitney U test). We noted no effect of tumor grade, extent of disease or exposure to intravesical therapy on urinary NMP22 levels. CONCLUSIONS: NMP22 is a promising urinary tumor marker for monitoring transitional cell carcinoma. Nuclear matrix proteins are a new class of tumor markers that represent the basis for the development of assays with increased efficacy for the detection and treatment of cancer.

Aged↗
PubMed1994

An unstable nuclear matrix may contribute to genetic instability.

Cancer cells proliferate and metastasize against the body's defense mechanisms due to their ability to change in response to challenges, both from the body's internal defenses as well as those from external sources, such as radiation and chemotherapy. This ability of tumor cell populations to change and grow in response to these stresses as well as to hold populations of cells with diverse abilities has been termed 'tumor cell heterogeneity'. Tumor cell heterogeneity is thought to arise in cancer cell populations as a result of genetic instability, an undefined process by which the genetic material of the cell is rendered more labile and more likely to undergo changes in structure, conformation, and function. DNA is structurally and functionally organized by the nuclear matrix, the dynamic RNA-protein skeleton of the nucleus. We provide here a proposal that provides a framework for understanding genetic instability in terms of an unstable nuclear matrix.

Animals↗
PubMed2002

Conserved region CR2 of Epstein-Barr virus nuclear antigen leader protein is a multifunctional domain that mediates self-association as well as nuclear localization and nuclear matrix association.

Self-association of viral proteins is important for many of their functions, including enzymatic, transcriptional, and transformational activities. Epstein-Barr virus (EBV) nuclear antigen leader protein (EBNA-LP) contains various numbers of W1W2 repeats and a unique carboxyl-terminal Y1Y2 domain. It was reported that EBNA-LP associates with a variety of cellular proteins and plays a critical role in EBV-induced transformation. We report here that EBNA-LP self-associates in vivo and the domain responsible for the homotypic association is a multifunctional domain mediating nuclear localization, nuclear matrix association, and EBNA-2-dependent coactivator function of the protein. Our conclusions are based on the following observations. (i) EBNA-LP interacts with itself or its derivatives in the yeast two-hybrid system. (ii) A purified chimeric protein consisting of glutathione S-transferase fused to EBNA-LP specifically formed complexes with EBNA-LP transiently expressed in COS-7 cells. (iii) When Flag epitope-tagged EBNA-LP with either one or two W1W2 repeats and EBNA-LP containing four W1W2 repeats were coexpressed in COS-7 cells, the latter was specifically coimmunoprecipitated with the former. (iv) Mutational analyses of EBNA-LP with deletion mutants revealed that the region between codons 19 and 39 (relative to the first amino acid residue of the W2 domain) is essential for self-association of the protein. The mapped region almost completely overlaps with CR2 and CR3, regions conserved among a subset of primate gamma-herpesviruses and critical for EBNA-2-dependent coactivator function. Amino acid substitutions in CR2 alone abolished the ability of the protein to self-interact. This laboratory previously reported that CR2 is also responsible for nuclear localization and nuclear matrix association (A. Yokoyama, Y. Kawaguchi, I. Kitabayashi, M. Ohki, and K. Hirai, Virology 279:401-413, 2001). (v) Sucrose gradient sedimentation showed that amino acid substitutions in CR2 reduced the ability of the protein to form protein complexes in B cells. These results suggest that self-association of EBNA-LP may be important for its various functions and interactions of the protein with multiple cellular proteins.

Amino Acid Sequence↗
PubMed1999

Progressive changes of the nuclear matrix protein composition in diethylnitrosamine-induced rat hepatocarcinogenesis.

The nuclear matrix proteins (NMPs) consist of common and tissue-specific nuclear proteins, which can be altered by extracellular environments such as radiation, chemicals and virus infection. Thus, NMP profiles were analyzed in diethylnitrosamine (DEN)-treated rat liver. Male F344 rats (6 weeks old) were treated continuously with DEN (40 ppm) in drinking water. All animals were sacrificed at 10, 20 or 30 weeks during the experiment. The profiles of NMPs and cytoskeletal proteins (CSPs) progressively changed in their number and amount in DEN-treated rats. Four peptides increased in their relative amounts, while three decreased in the 10- and 20-week groups. Many NMPs were detected in DEN-induced hepatocellular carcinomas. These results suggest that the alteration of the NMPs may be involved in DEN-induced hepatocarcinogenesis.

Animals↗
PubMed1991

On the association of DNA polymerase alpha activity with the nuclear matrix in HeLa cells.

The association of DNA polymerase alpha activity with the nuclear matrix has been reinvestigated in HeLa cells. Isolated nuclei were extracted with 2M NaCl and then digested with Dnase I and the final structures were recovered by centrifugation through a sucrose cushion. Typically over 98% of the total DNA synthesized in the matrix fraction on either endogenous matrix-associated DNA or activated calf thymus DNA was due to DNA polymerase alpha as defined by inhibition to n-ethylmaleimide or aphidicolin. DNA polymerase beta activity was absent or recovered in only trace amounts. Matrix-bound DNA polymerase alpha activity demonstrated a remarkable degree of stability: DNA synthesis was essentially linear up to 3 hours at 37 degrees C. Overall, these results substantiate previous findings from regenerating rat liver, unlike other data obtained from tissue culture cells.

Aphidicolin↗
PubMed1983

Synaptonemal complexes are integral components of the isolated mouse spermatocyte nuclear matrix.

Synaptonemal complexes (SCs) have been isolated as integral components of the nuclear matrix from purified mouse pachytene spermatocytes. These nuclear synaptonemal complex-matrices are prepared by extracting Triton X-100-treated nuclei with low (0.2 M) and high (1.0 or 2.0 M) NaCl, DNase I, and RNase A to remove 85% of the nuclear proteins, 97% of the RNA, and 99% of the DNA. Studies with the light and electron microscopes indicate that these matrices, while lacking a distinct lamina, contain nuclear pores interconnected by a fiber network, residual nucleoli, and interchromatin fibers. In addition, the pachytene spermatocyte matrices contain residual XY heterochromatin and the principal components of the SCs, including two lateral elements, a central element, a presumptive centromere, and attachment plaques. These SCs are preserved within the matrix and retain their structural association with the pore-fiber complex, even when subjected to strong dissociating conditions. Nuclear matrices from pachytene spermatocytes and spermatids (steps 1-8), when analyzed by SDS PAGE, contain an array of polypeptides distinct from those of mouse liver nuclear matrices. Proteins of spermatogenic matrices range in Mr from 8,000 to approximately 150,000. The prominent lamina proteins (Mr approximately 60,000-70,000) of somatic nuclear matrices are either absent or represent only a minor part of the spermatogenic matrix. The polypeptide composition of the pachytene spermatocyte and spermatid matrices are similar, although minor quantitative and qualitative differences are evident. These observations suggest that the SC constituents may consist of a heterogeneous group of proteins present in low proportion relative to total matrix proteins, or they may be retained, but in a different form, within the spermatid matrix.

Animals↗
PubMed2003

Oligomerization of transcriptional intermediary factor 1 regulators and interaction with ZNF74 nuclear matrix protein revealed by bioluminescence resonance energy transfer in living cells.

Transcriptional intermediary factor 1 (TIF1) alpha and KAP-1/TIF1beta, two members of the TIF1 family of nuclear cofactors, are ubiquitous co-regulators of nuclear receptors and KRAB motif-containing zinc finger transcription factors, respectively. Despite the functional evidence suggesting a role for TIF1 proteins as modulators of transcription, the study of their interactions with transcriptional machineries in physiologically relevant systems has been difficult. Here, we have developed a bioluminescence resonance energy transfer (BRET) biophysical approach to study protein-protein interactions in the nuclear compartment of living mammalian cells. We report that TIF1alpha and KAP-1 form homo- and hetero-oligomers in intact mammalian cells. BRET titration experiments indicate that both homo- and hetero-oligomers occur with relatively high affinity suggesting that they could co-exist in cells. Furthermore, we demonstrate that KAP-1 but not TIF1alpha interacts with the KRAB multifinger ZNF74 in the nuclear matrix. Splice variants and point mutants of ZNF74 that lack transcriptional activity were found not to interact with KAP-1 confirming the physiological importance of this interaction in living cells. The interaction of ZNF74 with KAP-1 did not prevent KAP-1 homomerization indicating that the oligomers most likely represent the transcriptionally active species. Furthermore, the detection of ternary ZNF74.KAP-1.TIF1alpha complexes suggests the existence of cross-talk between KAP-1-interacting KRAB proteins and TIF1alpha-interacting nuclear receptors. In addition to providing new insights into the molecular interactions involved in the transcriptional activities of these proteins, this study shows that BRET can be advantageously used as a non-transcription-based oligomerization detection system to study the interaction of transcriptionally active proteins, including nuclear matrix proteins, in living cells.

Alternative Splicing↗
PubMed1988

[Interaction of various nuclear matrix proteins with DNA].

An attempt has been made to localize the previously detected strong and weak bonds of nuclear matrix proteins with DNA in some groups of proteins, using fractionation of the matrix into lamina and intranuclear fibrils, isolation of the "elementary globules", fractionation of matrix nucleoproteins on hydroxyapatite. It was shown that both weak and strong bonds are localized on the nuclear lamina and in the intranuclear fibrils. The single-stranded DNA enriched fraction of the matrix nucleoproteins contained mostly strong bonds. The strong bond is less resistant to pronase treatment. A method for isolating nuclear matrix nucleoprotein fractions carrying only strong or only weak bonds is proposed.

Animals↗
PubMed1991

Avian nuclear matrix proteins bind very tightly to cellular DNA of the beta-globin gene enhancer in a tissue-specific fashion.

We have previously shown that a cloned 480 bp DNA fragment that spans the 3'-enhancer region of the avian beta-globin gene cluster can become very tightly, perhaps covalently, bound to protein in avian nuclear matrices in vitro [Zenk et al. (1990) Biochemistry 29, 5221-5226]. This binding was not tissue-specific and was probably not mediated by topoisomerase enzymes. In the present study, we have examined avian nuclear matrices (or scaffolds) for the presence of very tight cellular DNA-protein complexes in the region of the beta-globin gene enhancer and of several other avian genes. Nuclear matrices were prepared by both high- and low-salt methods, and protein-DNA complexes were isolated by SDS/K+ precipitation after restriction enzyme digestion. In adult reticulocytes, up to 30% of the intact 3800 bp HindIII-EcoRI fragment that encompasses the beta-globin enhancer element may be very tightly bound to nuclear matrix protein. In adult avian thymus nuclei, the beta-globin enhancer is neither matrix-associated nor tightly bound to protein. In contrast, a 5.0-kb HindIII fragment of the malic enzyme gene is very tightly bound to nuclear matrix-associated protein in thymus cells, but not reticulocytes. The malic enzyme gene is active in thymus cells, and not in reticulocytes. These results suggests that certain regions of cellular DNA are very tightly, perhaps covalently, attached to nuclear matrix-associated proteins. Attachment follows a tissue-specific pattern that is associated with transcriptional activity.

Animals↗