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PubMed1999

Matrix attachment region binding protein MFP1 is localized in discrete domains at the nuclear envelope.

Recently, it has been suggested that nuclear processes, such as replication, transcription, and splicing, are spatially organized and associated with a nuclear framework called the nuclear matrix, a structure of unknown molecular composition. It has been shown that chromatin is attached to the nuclear matrix via specific DNA fragments called matrix attachment regions (MARs). We have begun to dissect the plant nuclear matrix by isolating a DNA binding protein with specific affinity for MARs. Here, it is shown that MAR binding filament-like protein 1 (MFP1) is associated with specklelike structures at the nuclear periphery that are part of isolated nuclei and the nuclear matrix. A predicted N-terminal transmembrane domain is necessary for the specific targeting of MFP1 to the speckles, indicating an association with the nuclear envelope-endoplasmic reticulum continuum. In addition, it is shown that a marker protein for plant microtubule organizing centers, which has been shown to be localized on the outside of the plant nuclear envelope, is also part of the nuclear matrix. These findings indicate a close and previously undescribed connection in plants between the nuclear envelope and the internal nuclear matrix, and they suggest a function for MFP1 in attaching chromatin to specific sites at the nuclear periphery.

Arabidopsis Proteins↗

PubMed1992

Nuclear matrix-associated NMN adenylyltransferase activity in human placenta.

This paper presents data about the presence of the NMN adenylyltransferase at the nuclear matrix level of human placenta nuclei. It was found that 40-45% of the activity (depending on the extraction procedure) referred to the total nuclear NMN adenylyltransferase was tightly associated with this subnuclear compartment. The matrices purified by two different procedures exhibited DNA, RNA and protein contents comparable with those described in literature. Extensive digestion of human placenta nuclei with DNase I was not able to solubilize the NMN adenylyltransferase activity. Therefore, the data we present are consistent with the conclusion that a part of the total nuclear NMN adenylyltransferase is associated with the nuclear matrix.

Cell Fractionation↗

PubMed1993

Fluorescent in situ hybridization of the telomere repeat sequence in hamster sperm nuclear structures.

The flat, hooked-shaped architecture of the hamster sperm nucleus makes this an excellent model for in situ hybridization studies of the three dimensional structure of the genome. We have examined the structure of the telomere repeat sequence (TTAGGG)n with respect to the various nuclear structures present in hamster spermatozoa, using fluorescent in situ hybridization. In fully condensed, mature sperm nuclei, the telomere sequences appeared as discrete spots of various sizes interspersed throughout the volume of the nuclei. While the pattern of these signals was non-random, it varied significantly in different nuclei. These discrete telomere foci were seen to gradually lengthen into linear, beaded signals as sperm nuclei were decondensed, in vitro, and were not associated with the nuclear annulus. We also examined the relationship of telomeres to the sperm nuclear matrix, a residual nuclear structure that retains the original size and shape of the nucleus. In these structures the DNA extends beyond the perimeter of the nucleus to form a halo around it, representing the arrangement of the chromosomal DNA into loop domains attached at their bases to the nuclear matrix. Telomere signals in these structures were also linear and equal in length to those of the decondensed nuclei, and each signal represented part of a single DNA loop domain. The telomeres were attached at one end to the nuclear matrix and extended into the halo. Sperm nuclear matrices treated with Eco RI retained the telomere signals. These data support sperm DNA packaging models in which DNA is coiled into discrete foci, rather than spread out linearly along the length of the sperm nucleus.

Animals↗

PubMed2002

RGS12TS-S localizes at nuclear matrix-associated subnuclear structures and represses transcription: structural requirements for subnuclear targeting and transcriptional repression.

RGS12TS-S, an 1,157-amino-acid RGS protein (regulator of G protein signaling), is a nuclear protein that exhibits a unique pattern of subnuclear organization into nuclear foci or dots when expressed endogenously or ectopically. We now report that RGS12TS-S is a nuclear matrix protein and identify structural determinants that target this protein to the nuclear matrix and to discrete subnuclear sites. We also determine the relationship between RGS12TS-S-decorated nuclear dots and known subnuclear domains involved in control of gene expression and provide the first evidence that RGS12TS-S is functionally involved in the regulation of transcription and cell cycle events. A novel nuclear matrix-targeting sequence was identified that is distinct from a second novel motif needed for targeting RGS12TS-S to nuclear dots. RGS12TS-S nuclear dots were distinct from Cajal bodies, SC-35 domains, promyelocytic leukemia protein nuclear bodies, Polycomb group domains, and DNA replication sites. However, RGS12TS-S inhibited S-phase DNA synthesis in various tumor cell lines independently of Rb and p53 proteins, and its prolonged expression promoted formation of multinucleated cells. Expression of RGS12TS-S dramatically reduced bromo-UTP incorporation into sites of transcription. RGS12TS-S, when tethered to a Gal4 DNA binding domain, dramatically inhibited basal transcription from a Gal4-E1b TATA promoter in a histone deacetylase-independent manner. Structural analysis revealed a role for the unique N-terminal domain of RGS12TS-S in its transcriptional repressor and cell cycle-regulating activities and showed that the RGS domain was dispensable for these functions. These results provide novel insights into the structure and function of RGS12TS-S in the nucleus and demonstrate that RGS12TS-S possesses biological activities distinct from those of other members of the RGS protein family.

Animals↗

PubMed1999

MAF1, a novel plant protein interacting with matrix attachment region binding protein MFP1, is located at the nuclear envelope.

The interaction of chromatin with the nuclear matrix via matrix attachment region (MAR) DNA is considered to be of fundamental importance for chromatin organization in all eukaryotic cells. MAR binding filament-like protein 1 (MFP1) from tomato is a novel plant protein that specifically binds to MAR DNA. Its filament protein-like structure makes it a likely candidate for a structural component of the nuclear matrix. MFP1 is located at nuclear matrix-associated, specklelike structures at the nuclear envelope. Here, we report the identification of a novel protein that specifically interacts with MFP1 in yeast two-hybrid and in vitro binding assays. MFP1 associated factor 1 (MAF1) is a small, soluble, serine/threonine-rich protein that is ubiquitously expressed and has no similarity to known proteins. MAF1, like MFP1, is located at the nuclear periphery and is a component of the nuclear matrix. These data suggest that MFP1 and MAF1 are in vivo interaction partners and that both proteins are components of a nuclear substructure, previously undescribed in plants, that connects the nuclear envelope and the internal nuclear matrix.

Amino Acid Sequence↗

PubMedDate not supplied

[Spatial organization of the chicken alpha-globin gene domain in cells of different].

We have developed a technique for mapping the sites of DNA attachment to the nuclear matrix by hybridization of nuclear matrix DNA with an oligonucleotide array. The latter was made by immobilization of 60-mer oligonucleotides distributed within the area under study with a 2 Kb step on nylon filter. Using this approach we have analyzed the mode of interaction of a 100 Kb fragment of chicken chromosome 16 including the alpha-globin gene domain with the nuclear matrix. The 40 Kb DNA loop including all alpha-globin genes was detected in erythroid cells. One of the borders of this loop colocalized with the previously mapped MAR element and CTCF-dependent enhancer-blocking element. Also, a long transcribed area was found to be preferentially associated with the nuclear matrix. The spatial organization of the area under study in lymphoid cells was drastically different from this observed in erythroid cells.

Animals↗

PubMed2003

Nuclear staining for the small heat shock protein alphaB-crystallin colocalizes with splicing factor SC35.

AlphaB-Crystallin has for a long time been considered a specific eye lens protein. Later on it appeared that this protein belongs to the family of the small heat shock proteins and that it occurs also extra-lenticularly in many different cell types. AlphaB-Crystallin is mainly present in the cytoplasm, but there are some indications that it might have a function in the nucleus too. However, till now its presence in the nucleus is uncertain. We therefore compared the localization of alphaB-crystallin in nine cell lines cultured under normal conditions using four different antisera. All four antisera gave a diffuse staining for alphaB-crystallin in the cytoplasm, but one of the antibodies consistently showed nuclear staining in eight of the cell types, in the form of distinct speckles. These speckles are equally pronounced in the different cell types, whether or not cytoplasmic alphaB-crystallin is present. Preabsorption of the antiserum with alphaB-crystallin abolished the staining. Furthermore we demonstrate that if only minor amounts of alphaB-crystallin are present, the protein seems to be located exclusively in the nucleus. However, in case of higher amounts of protein, alphaB-crystallin is distributed between cytoplasm and nucleus. The nuclear alphaB-crystallin exists, like the cytoplasmic alphaB-crystallin, in non-phosphorylated and phosphorylated forms, is Triton-insoluble but can be extracted by 2 M NaCl. These data suggest that alphaB-crystallin might be bound to the nuclear matrix per se or to nuclear matrix proteins via other proteins. In agreement with other nuclear matrix proteins, nuclear alphaB-crystallin staining turns diffuse upon mitosis and leaves the chromosomes unstained. Double staining experiments revealed colocalization of alphaB-crystallin with the splicing factor SC35 in nuclear speckles, suggesting a role for alphaB-crystallin in splicing or protection of the splicing machinery.

3T3 Cells↗

PubMed1989

Modulation of nuclear matrix-associated 2',5'-oligoadenylate metabolism and ribonuclease L activity in H9 cells by human immunodeficiency virus.

Human T cells (H9), infected with the HTLV-IIIB strain of the human immunodeficiency virus (HIV-1), have been used to study the alteration of 2',5'-oligoadenylate [2'-5')A) metabolism in relation to virus production. The synthesis of (2'-5')A was determined to proceed in close association with the nuclear matrix. After HIV infection the (2'-5')A synthetase activity increased from 1.1 to 1.5 pmol of (2'-5')A synthesized/100 micrograms of nuclear matrix protein (during a 3-h in vitro incubation period) to 8.2 pmol at day 3 after infection. Then the activity dropped to the initial values. In non-infected H9 cells the (2'-5')A synthetase activity remained unchanged. Simultaneously with the decrease of the (2'-5')A level the cells started to release HIV. At the time of maximum synthetase levels the (2'-5')A-activated endoribonuclease (RNase L) activity strongly increased. Only one protein could be selectively cross-linked to a (2'-5')A derivative in the nuclear matrix from H9 cells; this protein is assumed to be RNase L. Experimental evidence is provided revealing that RNase L degrades HIV transcripts. A correlation could be established between high levels of (2'-5')A and RNase L and a failure of the cells to release HIV. 3'-Azido-3'-deoxythymidine was shown to cause an extension of the time period during which an RNase L-mediated degradation of viral transcripts occurred. The possibility of a novel molecular pharmacologic approach on the level of (2'-5')A metabolism is discussed.

Adenine Nucleotides↗

PubMed1994

The retinoblastoma gene product is a cell cycle-dependent, nuclear matrix-associated protein.

The retinoblastoma gene product (Rb) has been established as a tumor suppressor and cell cycle regulator, although its mechanism of action remains obscure. The observations that several Rb-binding viral oncoproteins all associate with the nuclear matrix suggest that these interactions may occur on this structure. To determine whether Rb itself is a component of the matrix, we extracted synchronized cultured cells to isolate matrix proteins while preserving nuclear architecture. Immunoblot and immunolabeling data show that a significant portion of hypophosphorylated Rb associates with the matrix only during early G1. Mutant Rb in tumor cells did not associate with the matrix, whereas Rb-reconstituted cells contained abundant matrix-bound Rb. Rb is distributed widely throughout the matrix, particularly concentrated at the nuclear periphery and in nucleolar remnants. Core filaments of the matrix contained no detectable Rb. Our screening of expression libraries for potential Rb-associated proteins has identified several that are part of the matrix. Specifically, the peripheral matrix proteins lamin A and C bound Rb in vitro. We therefore suggest that Rb interactions with the nuclear matrix may be important for its ability to regulate cell cycle progression.

Cell Cycle↗

PubMed1997

Two-dimensional electrophoresis reveals a nuclear matrix-associated nucleolin complex of basic isoelectric point.

A monoclonal antibody was raised against a salt-extractable fraction of nuclear matrix / intermediate filament scaffolds of polarized MDCK cells. The antibody recognized an approximately 100 kDa protein in total cell lysates and nuclear matrices of various human cells and tissues and stained nucleolar structures in immunofluorescence microscopy. By partial sequencing of five peptides derived from immunoprecipitated protein, the targeted antigen was found to be homologous to human nucleolin. After two-dimensional electrophoresis of total HeLa cell lysates, immunoreactive bands were detected at isoelectric point (pI) 5.5--6.1, characteristic for nucleolin, and at pI 8.5--9. Whereas the protein focusing at acidic pI was found in Triton X-100-soluble cellular fractions, the antigen focusing at basic pI was exclusively contained in the residual nuclear fraction and was solubilized upon treatment of nuclear matrices with RNAse. The component solubilized by RNAse treatment was still detected at basic pI in two-dimensional electrophoresis. However, upon immunoprecipitation of the antigen from the RNAse-released fraction in the presence of sodium dodecyl sulfate (SDS), the nuclear matrix-derived antigen was positioned at pI 5--6. The present data indicate that the nuclear matrix-bound nucleolin is associated with ribonucleoproteins and a basic component resisting dissociation under conditions of isoelectric focusing.

Animals↗

PubMedDate not supplied

Induction of nuclear matrix-estradiol receptor complex during capacitation process in human spermatozoa.

Estrogen receptor levels were determined in capacitated and noncapacitated sperm cells of normospermic men by the charcoal dextran method. The estrogen receptor of the nuclear matrix was released using hypertonic solutions. The distribution of the estrogen receptor was higher by 30% in capacitated spermatozoa as related to noncapacitated spermatozoa. The values of estrogen receptors in capacitated and noncapacitated spermatozoa were 91 +/- 21 fmol/10(8) sperm cells and 26 +/- 7 fmol/10(8) sperm cells, respectively. The association of estrogen receptor-nuclear matrix may be an important factor in the regulation of the transcription of the sperm genome of capacitated spermatozoa during the pronuclei stage.

Estradiol↗

PubMed1999

Organization of chromatin in cancer cells: role of signalling pathways.

The role of mechanical and chemical signalling pathways in the organization and function of chromatin is the subject of this review. The mechanical signalling pathway consists of the tissue matrix system that links together the three-dimensional skeletal networks, the extracellular matrix, cytoskeleton, and nuclear matrix. Intermediate filament proteins are associated with nuclear DNA, suggesting that intermediate filaments may have a role in the organization of chromatin. In human hormone-dependent breast cancer cells, the interaction between cytokeratins and chromatin is regulated by estrogens. Transcription factors, histone acetyltransferases, and histone deacetylases, which are associated with the nuclear matrix, are components of the mechanical signalling pathway. Recently, we reported that nuclear matrix-bound human and chicken histone deacetylase 1 is associated with nuclear DNA in situ, suggesting that histone deacetylase has a role in the organization of nuclear DNA. Chemical signalling pathways such as the Ras/mitogen-activated protein kinase (Ras/MAPK) pathway stimulate the activity of kinases that modify transcription factors, nonhistone chromosomal proteins, and histones. The levels of phosphorylated histones are increased in mouse fibroblasts transformed with oncogenes, the products of which stimulate the Ras/MAPK pathway. Histone phosphorylation may lead to decondensation of chromatin, resulting in aberrant gene expression.

Amino Acid Sequence↗

PubMed2001

PIASy, a nuclear matrix-associated SUMO E3 ligase, represses LEF1 activity by sequestration into nuclear bodies.

The Wnt-responsive transcription factor LEF1 can activate transcription in association with beta-catenin and repress transcription in association with Groucho. In search of additional regulatory mechanisms of LEF1 function, we identified the protein inhibitor of activated STAT, PIASy, as a novel interaction partner of LEF1. Coexpression of PIASy with LEF1 results in potent repression of LEF1 activity and in covalent modification of LEF1 with SUMO. PIASy markedly stimulates the sumoylation of LEF1 and multiple other proteins in vivo and functions as a SUMO E3 ligase for LEF1 in a reconstituted system in vitro. Moreover, PIASy binds to nuclear matrix-associated DNA sequences and targets LEF1 to nuclear bodies, suggesting that PIASy-mediated subnuclear sequestration accounts for the repression of LEF1 activity.

Animals↗

PubMed1992

Nuclear matrix-bound replicational sites detected in situ by 5-bromodeoxyuridine.

The nuclear matrix was prepared in situ from Swiss 3T3 cells, which were synchronized by contact inhibition and serum starvation and pulse-labelled for very short periods of time with 5-bromodeoxyuridine (5-BrdU). For the first time 5-BrdU has been employed to demonstrate the association of newly synthesized DNA with a nucleoskeleton. Immunofluorescence analysis using a monoclonal antibody to 5-BrdU revealed five different intranuclear staining patterns at different stages of the S phase. These patterns were observed also in intact cells and did not change during the matrix preparation steps which involve extraction with 2 M NaCl and DNase I digestion. Such an observation was also confirmed by spatial confocal microscopy studies. The intensity of fluorescence, which was evaluated by cytofluorometry, increased to reach a maximum during mid-S phase and then decreased. Because no significant difference was found in the time to label residual DNA of different 5-BrdU staining patterns, this strongly suggests that a different number of replicons is activated at different stages of the S phase. These results strengthen the hypothesis that eukaryotic DNA replication occurs in close association with an insoluble protein nuclear skeleton, which determines the three-dimensional spatial organization of chromosome duplication.

3T3 Cells↗

PubMed1984

Relationship between concentrations of extractable and matrix-bound nuclear androgen receptor and clinical response to endocrine therapy for prostatic adenocarcinoma.

The relationship between androgen receptor concentrations and clinical response to endocrine therapy for prostatic adenocarcinoma was investigated for 13 stage D patients. Both cytoplasmic and nuclear-androgen receptors were quantified. For nuclear androgen receptor, nuclei were isolated and treated with high ionic strength buffer (0.6 M KCl) to yield a KCl-extractable fraction; the nuclei were then treated with DNase I to yield nuclear matrices. Electron microscopy confirmed the relative nuclear purity and revealed matrix morphology. An hydroxylapatite binding assay and methyltrienolone (R1881) were used to quantify androgen receptor in cytosol, the KCl-extract and matrix preparations. Following 6 months of hormonal therapy, the clinical status of patients was re-evaluated and the patients were grouped according to disease response. The androgen receptor data obtained prior to therapy were compared for the disease response groups. The mean concentrations of cytoplasmic androgen receptor, KCl-extractable nuclear androgen receptor and nuclear matrix-bound androgen receptor, respectively, in those patients with disease progression or death (no. = 6), were 671 +/- 232, 45 +/- 17 and 119 +/- 34 fmol. per gm. of tissue +/- S.E.M., and for those with disease regression or stabilization (no. = 7), 1427 +/- 435, 193 +/- 53 and 611 +/- 92 fmol. per gm. of tissue +/- S.E.M. While cytoplasmic androgen receptor concentrations were not related to clinical status, both extractable and matrix-bound nuclear androgen receptor concentrations were significantly higher in the group which responded to hormonal therapy. These results suggest that nuclear-extractable and nonextractable androgen receptor concentrations are useful indices for the prediction of hormone-dependence of prostatic cancer.

Adenocarcinoma↗

PubMed1996

[Nuclear matrix-lamina-intermediate filament system in PtK 2 cells].

Selective extraction, whole mount cell preparation and DGD embeddment-free section were involved in visualizing the nuclear matrix-lamina-intermediate filament system in PtK 2 cells. After extraction the anaphase chromosome residues adjoined to the cytoplasmic intermediate filament in some areas. Immunofluorescent staining showed us that the intermediate filament reacted with AE 1 and AE 3; McAb 223 could be localized specifically on the lamina while McAb C 23 could crossreact with cytoplasmic intermediate filament beside the lamina location, monoclonal antibody against lamin A (C) could also bind to chromosome residues. Antibody to 280 kD nuclear matrix protein which were positive stained in HeLa cells could not react with the nuclear matrix components of PtK 2 cells. 2-D electrophoresis demonstrated that there were some differences in the composition of the nuclear matrix-lamina-intermediate filament system of HeLa and PtK 2 cells. TdR treatment could lead to alteration of nuclear matrix proteins.

Animals↗

PubMed2001

The role of ATM in telomere structure and function.

Ataxia telangiectasia (AT) is a rare human autosomal recessive disorder with a wide variety of phenotypic manifestations. AT patients are cancer prone and hypersensitive to ionizing radiation. Cells derived from AT patients require higher levels of serum factors, exhibit cytoskeletal defects, and undergo premature senescence in culture. The gene responsible for AT is ATM (ataxia-telangiectasia mutated), and its product has been implicated in mitogenic signal transduction, chromosome condensation, meiotic recombination, and cell cycle control. Because of the homology of the human ATM gene to the TEL1 and rad3 genes of yeast, it has been suggested that mutations in ATM could lead to defective telomere maintenance. The ATM gene product influences chromosome end associations, telomere length, and telomere clustering. The defective telomere metabolism in AT cells could be due to altered interactions between the telomeres and the nuclear matrix. These interactions were studied in nuclear matrix halos before and after irradiation. Altered telomere-nuclear matrix interactions were observed in cells derived from individuals with AT. AT cells also had different nucleosomal periodicity in their telomeres from normal cells. Both telomere-nuclear matrix interactions and nucleosomal periodicity were altered by treatment of primary AT fibroblasts with ionizing radiation. This effect was not observed in cells derived from normal individuals. A link was also found between altered telomere-nuclear matrix interactions, aberrant telomere clustering, and gonadal atrophy. The telomere defect was not corrected by the ectopic expression of the catalytic subunit of telomerase (TERT). Since alteration of the yeast telomere chromatin structure is known to influence gene expression, we compared expressed sequence tags (ESTs) of Atm-null mouse cells and normal mouse cells. Several ESTs were found to be aberrantly expressed in Atm-null mouse cells. This paper summarizes our recent publications and presents some new data on the influence of ATM on telomere metabolism.

Ataxia Telangiectasia Mutated Proteins↗

PubMed1998

Nuclear dreams: the malignant alteration of nuclear architecture.

Cancer is diagnosed by examining the architectural alterations to cells and tissues. Changes in nuclear structure are among the most universal of these and include increases in nuclear size, deformities in nuclear shape, and changes in the internal organization of the nucleus. These may all reflect changes in the nuclear matrix, a non-chromatin nuclear scaffolding determining nuclear form, higher order chromatin folding, and the spatial organization of nucleic acid metabolism. Malignancy-induced changes in this structure may have profound effects on chromatin folding, on the fidelity of genome replication, and on gene expression. Elucidating the mechanisms and the biological consequences of nuclear changes will require the identification of the major structural molecules of the internal nuclear matrix and an understanding of their assembly into structural elements. If biochemical correlates to malignant alterations in nuclear structure can be identified then nuclear matrix proteins and, perhaps nuclear matrix-associated structural RNAs, may be an attractive set of diagnostic markers and therapeutic targets.

Animals↗