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PubMed1995

Galectin-3 is a nuclear matrix protein which binds RNA.

The endogenous galectin-3 is a carbohydrate-binding protein of M(r) approximately 30,000 serving in the cytoplasm and on the cell surface as a receptor for ligands containing poly-N-acetyllactosamine sequences. In addition, galectin-3 has been demonstrated to be associated in the nucleus with ribonucleoprotein complexes and to act as a pre-mRNA splicing factor and to be involved in spliceosome assembly. However, little is known about either its nuclear localization or its ligand(s), respectively. We demonstrate directly here that galectin-3 is associated with the RNA protein skeleton of the nucleus, i.e., the nuclear matrix, and binds with single-stranded DNA (ssDNA) and with RNA. The affinity of binding was determined to be 2.3 microM. Lactose, which inhibits galectin-3 binding to glycoconjugates, failed to inhibit either galectin-3-ssDNA or galectin-3-RNA binding. Galectin-3 exhibited the highest affinity to poly(A) ribonucleotide homopolymers. The results presented here shows that galectin-3 may act as a RNA-binding protein in the nuclear matrix in a non-carbohydrate-dependent manner.

Animals↗
PubMed1998

Attachment to the nuclear matrix mediates specific alterations in chromatin structure.

The DNA in eukaryotic chromosomes is organized into a series of loops that are permanently attached at their bases to the nuclear scaffold or matrix at sequences known as scaffold-attachment or matrix-attachment regions. At present, it is not clear what effect affixation to the nuclear matrix has on chromatin architecture in important regulatory regions such as origins of replication or the promoter regions of genes. In the present study, we have investigated cell-cycle-dependent changes in the chromatin structure of a well characterized replication initiation zone in the amplified dihydrofolate reductase domain of the methotrexate-resistant Chinese hamster ovary cell line CHOC 400. Replication can initiate at any of multiple potential sites scattered throughout the 55-kilobase intergenic region in this domain, with two subregions (termed ori-beta and ori-gamma) being somewhat preferred. We show here that the chromatin in the ori-beta and ori-gamma regions undergoes dramatic alterations in micrococcal nuclease hypersensitivity as cells cross the G1/S boundary, but only in those copies of the amplicon that are affixed to the nuclear matrix. In contrast, the fine structure of chromatin in the promoter of the dihydrofolate reductase gene does not change detectably as a function of matrix attachment or cell-cycle position. We suggest that attachment of DNA to the nuclear matrix plays an important role in modulating chromatin architecture, and this could facilitate the activity of origins of replication.

Animals↗
PubMed1986

Morphometric analysis and topological organization of nuclear matrix in freeze-fractured electron microscopy.

The ultrastructural organization of nuclear matrix, purified from intact or membrane-denuded rat liver nuclei, has been analysed by means of freeze-fracturing technique. This method avoids dehydration and embedding which, in conventional thin sectioning, partly distort or mask the matrix ultrastructure. The various matrix components, and mainly the peripheral lamina and the inner network revealed complex arrangements undetectable with conventional techniques. Morphometric analyses performed with a Texture Analysis System (TAS) Leitz, allowed to obtain precise information on the matrix constituents, based on the histograms of their size distribution. These textural characteristics have been utilized in order to identify, by means of a particular computer programme, the putative matrix localization within intact freeze-fractured nuclei.

Animals↗
PubMed1987

Sub-set characteristics of DNA sequences involved in tight DNA/polypeptide complexes and their homology to nuclear matrix DNA.

Polypeptides co-isolating with DNA induce the binding of a fraction of native DNA fragments to nitrocellulose filters. Southern analysis reveals a high intensity of self-hybridization of the DNA sequences retained on nitrocellulose filters. Consistently, the DNA fraction passing the filters shows only weak hybridization when probed with DNA retained on filters. This indicates that the DNA/polypeptide complexes reside on a non-random sub-set of DNA sequences. Moreover, a high degree of homology was found between residual nuclear matrix DNA sequences and the DNA sequences retained on nitrocellulose filters. This indicates that the DNA sequences associated with tightly bound polypeptides originate from sites where the genome is salt-stably anchored in the nuclear matrix.

Animals↗
PubMed2003

Nuclear matrix association of the human beta-globin locus utilizing a novel approach to quantitative real-time PCR.

The human beta-globin locus is home to five genes that are regulated in a tissue-specific and developmental stage-specific manner. While the exact mode of expression remains somewhat enigmatic, a significant effort has been focused at the locus control region (LCR). The LCR is marked by five DNase I-hypersensitive sites (HS) approximately 15 kb upstream of the epsilon-globin gene. Nuclear matrix-associated regions (MARs) organize chromatin into functional domains and at least one of the HS appears bound to the nuclear matrix. We have employed an in vivo based PCR MAR assay to investigate the role of MAR-mediated regulation of the beta-globin locus. This was facilitated with a novel reaction efficiency based quantitative real-time PCR analysis software tool, Target Analysis Quantification. Using a log-linear regression strategy, discordances were eliminated. This allowed us to reliably estimate the relative amount of initial template associated with the nuclear matrix at 15 unique regions spanning the beta-globin locus in both non-expressing and expressing cell lines. A dynamic association dependent on expression status was revealed both at the LCR/5'HS region and within the second intron of the beta-globin gene. These results provide the first evidence that nuclear matrix association dynamically mediates the looping of the beta-globin locus to achieve transcriptional control.

Cell Line↗
PubMed1997

ATF1 and CREB trans-activate a cell cycle regulated histone H4 gene at a distal nuclear matrix associated promoter element.

Proteins of the ATF/CREB class of transcription factors stimulate gene expression of several cell growth-related genes through protein kinase A-related cAMP response elements. The promoter activity of cell cycle regulated histone H4 genes is regulated by at least four principal cis-acting elements which mediate G1/S phase control and/or enhancement of transcription during the cell cycle. Using protein-DNA interaction assays we show that the H4 promoter contains two ATF/CREB recognition motifs which interact with CREB, ATF1, and ATF2 but not with ATF4/CREB2. One ATF/CRE motif is located in the distal promoter at the nuclear matrix-associated Site IV, and the second motif is present in the proximal promoter at Site I. Both ATF/CRE motifs overlap binding sequences for the multifunctional YY1 transcription factor, which has previously been shown to be nuclear matrix associated. Subnuclear fractionation reveals that there are two ATF1 isoforms which appear to differ with respect to DNA binding activity and partition selectively between nuclear matrix and nonmatrix compartments, consistent with the role of the nuclear matrix in regulating gene expression. Site-directed mutational studies demonstrate that Site I and Site IV together support ATF1- and CREB-induced trans-activation of the H4 promoter. Thus, our data establish that ATF/CREB factors functionally modulate histone H4 gene transcription at distal and proximal promoter elements.

Activating Transcription Factor 1↗
PubMed1991

Association of topoisomerase II with the hepatoma cell nuclear matrix: the role of intermolecular disulfide bond formation.

Previous studies have resulted in conflicting data regarding the recovery of the nuclear enzymes topoisomerase (topo) II and topo I in the nuclear matrix fraction. In the present study we have assessed the effect of systematically altering a single extraction procedure on the distribution of these enzymes during the subfractionation of nuclei from HTC hepatoma tissue culture cells. When nuclear monolayers (prepared by treating attached cells in situ with the neutral detergent Nonidet-P40 at 4 degrees C) were isolated in the presence of the irreversible sulfhydryl blocking reagent iodoacetamide, subsequent treatment with DNase I and RNase A followed by 1.6 M NaCl resulted in structures which were extensively depleted of intranuclear components as assessed by phase contrast microscopy and conventional transmission electron microscopy. These structures contained 12 +/- 4% of the total protein present in the original nuclear monolayers. The lamins and polypeptides with molecular weights comparable to those of actin and vimentin were the predominant polypeptides present on SDS-polyacrylamide gels. Western blotting revealed that less than 5% of the total nuclear topo II molecules were present in these structures. In contrast, when the sulfhydryl cross-linking reagent sodium tetrathionate (NaTT) was substituted for iodoacetamide, the same extraction procedure yielded structures containing components of the nucleolus and an extensive intranuclear network. These structures contained a wide variety of nonlamin, nonhistone nuclear polypeptides including 23 +/- 4% of the total nuclear topo II. SDS-polyacrylamide gel electrophoresis performed under nonreducing conditions revealed that topo II in these nuclear matrices was present as part of a large disulfide cross-linked complex. Treatment of these structures with reducing agents in 1.6 M NaCl released the topo II. In contrast, topo I did not form disulfide cross-linked oligomers and was not detectable in any of these nuclease- and salt-resistant structures prepared at 4 degrees C. To assess the effect of in vitro heat treatment on the distribution of the topoisomerases, nuclear monolayers (isolated in the absence of iodoacetamide and NaTT) were heated to 37 degrees C for 1 h prior to treatment with nucleases and 1.6 M NaCl. The resulting structures (which retained 26 +/- 5% of the total nuclear protein) were morphologically similar to the NaTT-stabilized nuclear matrices and contained 15 +/- 4% of the total nuclear topo II. High-molecular-weight disulfide cross-linked oligomers of topo II were again demonstrated. Attempts to demonstrate these disulfide cross-linked oligomers in intact cells were unsuccessful.

Carcinoma, Hepatocellular↗
PubMed1999

Modulation of nuclear matrix protein phosphorylation by histones: possible involvement of NM-associated protein kinase CK2 activity.

Nuclear matrix (NM), a proteinaceous network of filaments, dictates nuclear morphology and the structure/function of DNA. Phosphorylation of NM proteins is a potential signal for regulating matrix functions. Histones also are intimately involved in DNA structure and transcription. Here, we report that various histones enhanced 32P incorporation into certain NM proteins. Modulation of NM protein phosphorylation by histones is mediated through regulation of protein kinase CK2, a messenger-independent serine/threonine kinase, which is significantly associated with the NM. The stimulatory effect of histones was mitigated by prior incubation of histones with DNA in the reaction. Phosphorylation of NM proteins was extensively reduced when an excess of the CK2-specific peptide substrate was included in the phosphorylation reaction as a competitor. Also, enhancement in the NM-associated CK2 activity by histones was blocked by inhibitors of CK2. Histone H1 effect appeared to be mediated mainly by charge effect since a stretch of polylysine induced a similar effect. Various histones also differentially affected the autophosphorylation of NM-associated CK2 subunits. This may contribute to the observed effects of histones on the NM, resulting in an enhancement and differential pattern of NM protein phosphorylation. Such a regional modification of NM protein phosphorylation might influence the nuclear functions that require histone displacement, namely, replication and transcription.

Animals↗
PubMed1991

Hepatoma-associated nuclear matrix nonhistone antigens.

Polyclonal antibodies generated against a group of high molecular weight nonhistone proteins from Morris hepatoma 7777 were used in immunological studies of hepatoma-associated nonhistone proteins in rat and hamster. We revealed the presence of cross-reactive antigens in rat Morris hepatomas 7777 and 8994, and in hamster Kirkman-Robbins hepatoma, but not in normal rat or hamster livers. These specific nonhistone proteins were found to be preferentially localized in the nuclear matrix of rat Morris hepatoma 7777 as well as hamster Kirkman-Robbins hepatoma.

Animals↗
PubMed1986

Phorbol diester-induced phosphorylation of nuclear matrix proteins in HL60 promyelocytes. Possible role in differentiation studied by cationic detergent gel electrophoresis.

Immortal HL60 promyelocytes are induced to differentiate to mortal adherent cells by a variety of agents which activate protein kinase C, including 12-O-tetradecanoylphorbol 13-acetate (TPA). In order to investigate the mechanism of this effect, we incubated HL60 cells with [32P]orthophosphate with or without TPA and extracted their proteins with the cationic detergent benzyldimethyl-n-hexadecylammonium chloride prior to electrophoresis in a discontinuous polyacrylamide gel system in the first dimension. In this system, proteins migrate toward the cathode as a function of their molecular weight, and they are separated from other radioactive components which can obscure the pattern of protein phosphorylation on sodium dodecyl sulfate (SDS) gels. SDS gel electrophoresis was used in the second dimension, resulting in the clear resolution of a large number of proteins. TPA caused many changes in the pattern of protein phosphorylation in intact cells. Two proteins which prominently increased their incorporation of 32P were investigated in particular, and they were both found to be retained in the nuclear matrix following successive extraction of cells with Triton, digestion with DNase and RNase, and extraction with 2 M NaCl. These proteins migrated with apparent molecular weights of 80,000 and 33,000 on SDS gels, and are designated NP80 and NP33, respectively. NP80 was half-maximally phosphorylated after 7 min exposure to TPA, and half-maximally phosphorylated by 10 nM TPA. NP80 co-migrated with a faint Coomassie Blue-stained protein, and NP33 co-migrated with a more prominent protein. Several proteins incorporated less 32P when the cells were exposed to TPA, including one which was extracted from nuclei with the core histones and which co-migrated with histone H2A. Further study will be needed to determine whether the differentiation of HL60 induced by TPA is mediated via phosphorylation of these nuclear matrix proteins.

Antigens, Nuclear↗
PubMed1998

Cloning and characterization of a novel zinc finger protein that associates with nuclear matrix.

We have recently reported that the nuclei of B16 melanoma cells are intensely stained with anti-rat vitronectin (Vn) antibody, which reacts with both mouse and rat Vn. In the present study, we characterized the protein immunoreactive with the antibody using NIH3T3 cells, whose nuclei were also stained with the antibody. Western blot analysis showed that a protein with an approximate molecular weight of 75 kDa (p75), which was distinct from Vn, existed in the nuclear fraction, and, more specifically, in the nuclear matrix fraction, of NIH3T3 cells. Screening of an NIH3T3 cDNA library resulted in the isolation of a nearly full-length cDNA clone encoding p75. A database search revealed that the cDNA represents a novel gene. The deduced amino acid sequence showed that the protein is 580 amino acids long and contains two C2H2-type zinc finger motifs and glutamic acid-rich domains in the C-terminal region. When a fusion protein of green fluorescence protein and p75 was expressed in NIH3T3 cells, fluorescence was preferentially observed in the nuclei, demonstrating that the protein has a nuclear localization signal. The p75 protein, termed ZAN75, exhibited DNA-binding activity in a zinc-dependent manner. Southern blot analysis demonstrated that the ZAN75 gene exists in a single copy in the mouse genome and that a closely related gene is also present in chicken, rat, and human. Northern blot analysis showed that the ZAN75 gene is ubiquitously expressed in adult mouse tissues. In the cell cycle of NIH3T3 cells, expression was low in the G0/G1 phase, increased during the G1 phase, and persisted during the S and G2/M phases, suggesting that ZAN75 plays a role in regulating cell growth.

3T3 Cells↗
PubMed1998

[Study on nuclear matrix protein in leukemia cells].

OBJECTIVE: To compare the composition of nuclear matrix proteins (NMP) between leukemia cells and normal bone marrow cells. METHODS: NMP was isolated by high-salt extration and identified by SDS-PAGE and western blotting. RESULTS: Increase in NMP was demonstrated in acute and chronic myelogenous leukemic cells as well as in the blast phase of chronic leukemia. On SDS-PAGE, NMPs with molecular weight different from what were seen in normal bone marrow cells were present in both acute and chronic myelogenous leukemias. CONCLUSION: Marked changes of NMP, not only in contents but also in compositions, exist in leukemic cells compared with normal bone marrow cells. NMP may serve as a target of chemotherapeutic drugs against leukemia.

Antigens, Nuclear↗
PubMed1995

DNA-nuclear matrix interactions analyzed by cross-linking reactions in intact nuclei from avian liver.

To detect the interactions of DNA with the nuclear matrix proteins, DNA-protein cross-linkages were induced in intact nuclei from chicken liver by the use of cis-diammine dichloroplatinum. Methods have been devised for fast purification both of the proteins and of the DNA fragments involved in the cross-linked complexes. By Southern-Western blotting a number of matrix proteins isolated from the complexes have been shown to recognize specifically DNA sequences present in the cross-linked DNA fragments. This experimental approach not only allows to identify the nuclear matrix-DNA interactions existing in the nucleus before its disruption, but also provides a preparation of matrix proteins enriched in those species which are involved in such interactions and which can therefore be detected with high sensitivity.

Animals↗
PubMed1995

The PML gene encodes a phosphoprotein associated with the nuclear matrix.

The t(15;17)(q22;q12) translocation is the cytogenetic hallmark of acute promyelocytic leukemia (APL). The PML and retinoic acid receptor-alpha (RAR alpha) transcription factor genes are involved at translocation breakpoint. To elucidate the biologic function of PML, antipeptide antibody against PML protein was raised in rabbits. This antibody was able to detect a 90-kD PML protein and a 110-kD PML-RAR alpha fusion protein by Western blotting and a nuclear speckled pattern in all cell lines by immunofluorescent staining. In K562 and NIH/3T3 cells transfected with a PML expression plasmid, we found PML to be associated with the nuclear matrix. Our results also showed that PML is a phosphorprotein. A weak signal was detected in a Western blot containing the immunoprecipitated PML protein using the phosphotyrosine-specific monoclonal antibody. Therefore, at least one of the sites was phosphorylated by a tyrosine kinase. From our analysis of the phosphoamino acids of the PML protein by complete hydrolysis and thin-layer chromatography, we concluded that both tyrosine and serine residues of PML are phosphorylated. To investigate whether expression of the PML protein is cell-cycle related, HeLa cells synchronized at various phases of the cell cycle were analyzed by immunofluorescence staining and confocal microscopy for PML expression. We found that PML was expressed at a lower level in S, G2, and M phases and at a significantly higher level in G1 phase. Our study showed that PML has many similar properties compared with the tumor suppressor, eg, Rb. These findings further support the important role of PML in APL pathogenesis.

3T3 Cells↗
PubMed1998

Differential nuclear matrix protein expression in prostate cancers: correlation with pathologic stage.

PURPOSE: Nuclear Matrix Proteins (NMP) have been shown to be tissue and cell-type specific. Several unique NMPs have been investigated in various cancerous tissues, including prostate, bladder and kidney, and some are presently utilized as tumor markers. This study was aimed at characterizing the differential NMP expression in the pathologically more aggressive prostate cancers. METHODS: High resolution two-dimensional gel electrophoresis and silver staining was used to elucidate the NMP distribution of fresh prostate cancer nuclei, obtained from 39 radical prostatectomy specimens, surgically removed from men with clinically localized prostate cancer. Based on the final pathological grading, specimens were grouped according to predicted prognosis: poor--with seminal vesicle (SV) or lymph node (LN) involvement or established capsular penetration (ECP) with gleason score >7; intermediate--organ confined (OC) or focal capsular penetration (FCP) with gleason score 7 or ECP with gleason score 6; and good--with OC or FCP and gleason score <7. RESULTS: A specific charged protein (YL-1) of molecular weight 76 kD and isoelectric range 6.0-6.6 was found to be consistently present in 19 of 19 aggressive cancers. It was present only in 1 of 10 in the group with good prognosis and weakly positive in 9 of 10 in the intermediate group. CONCLUSIONS: Within this preliminary study, the expression of YL-1 appears to be related to aggressive prostate cancer, suggesting a potential marker of poor prognosis for clinically localized prostate cancer. Further characterization of the identity and function of this NMP is needed to fully ascertain its clinical potential.

Antigens, Nuclear↗
PubMed1985

Protein composition of the chromosomal scaffold and interphase nuclear matrix.

Residual protein structures were prepared from isolated chromosomes and interphase nuclei of in vitro cultured bovine liver cells and the protein compositions were analysed. Chromosomes with minimal cytoplasmic contamination were obtained by a simple procedure using a pH 8 isolation medium containing Triton X-100 and polyamines, and residual protein-DNA complexes were prepared by extraction with 2 M NaCl. Residual protein structures were also obtained by digesting isolated chromosomes with staphylococcal nuclease. Protein compositions of both structures as obtained by SDS-polyacrylamide gel electrophoresis were essentially the same. Residual protein structures were prepared from isolated nuclei by the same procedures. The major nuclear matrix proteins, i.e., the lamins A, B, and C, were not found in the chromosomes and chromosome scaffolds. On the other hand, the residual chromosome structures contained two major polypeptides of 37 and 83 kilodalton relative molecular weights that were absent from the nuclear matrix preparations. A few polypeptides with the same or very similar electrophoretic mobilities were found in the residual structures of both the nuclei and the chromosomes.

Animals↗
PubMedDate not supplied

[Shifts in phospholipid composition of the nuclear matrix in rat liver cells exposed to insulin].

It was shown that the total content of phospholipids of rat liver nuclear matrix increased after in vivo action of insulin (2 units per 100 g of animal weight). At the same time the sphyngomyelin content was increased and the content of phosphatydilcholine and phoshpatydilinositol was decreased significantly under the hormone action. The decreasing of monophosphoinositides amount was accompanied by the increasing of triphosphoinositides content which indicates the redistribution among the phosphoinositides fractions under the hormone action and may be the result of insulin initiation of phosphoinositide pathway in nuclear membrances.

Animals↗