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PubMed1994

Cyclin/cdk2 complexes in the nucleus of HeLa cells.

Two different fractions of cdk2 and cdc2 have been found in the nucleus of HeLa cells. One, which can be extracted by nuclease treatment, possibly associated with DNA- or RNA-containing structures and another one, which is bound to the nuclear matrix. Nuclear cdk2 forms high molecular weight complexes which migrate at the same position as DNA polymerase alpha and proliferating cell nuclear antigen in sucrose gradient centrifugation experiments. These results suggest that nuclear cdk2 complexes could be associated with the replication factories. Immunoprecipitation experiments reveal that nuclear cdk2 complexes display histone H1-kinase activity and phosphorylate a protein of 18 kDa which is present in these complexes.

Blotting, Western↗
PubMed1997

Surface residue mutations of the PML RING finger domain alter the formation of nuclear matrix-associated PML bodies.

The human protein PML, was first identified as part of a fusion protein with retinoic acid receptor alpha as found in the chromosomal translocation which gives rise to acute promyelocytic leukaemia. PML is normally localised to large matrix-associated nuclear domains (known as ND10, Kr bodies, PODS or PML NBs) which comprise several multi-protein complexes. Within the PML protein, there are a number of identified zinc-binding domains, one of which called the RING finger is found in a large family of diverse and unrelated proteins. Here, we report the effect of site-directed mutations within the context of the whole PML protein, of amino acids found on the surface of the PML RING finger domain and PML NB formation in vivo. Mutations of a small region of the RING finger domain surface affect the size and numbers of PML NBs in a mouse fibroblast expression assay, resulting in fewer but larger exogenous PML NBs. Mutations of other surface RING residues, however, do not affect exogenous PML NB formation. Furthermore, all of the PML RING mutants co-localise to both endogenous and exogenous wild-type PML NBs. These data identify a specific region of the PML RING finger domain which is directly involved in correct PML NB formation. They also provide evidence to suggest that the PML RING finger is involved in mediating PML-PML oligomeric interactions, as part of a mechanism leading to the assembly of the PML NB complex.

3T3 Cells↗
PubMed2000

Nuclear matrix-like filaments and fibrogranular complexes form through the rearrangement of specific nuclear ribonucleoproteins.

The behavior of nuclear pre-mRNA-binding proteins after their nuclease and/or salt-induced release from RNA was investigated. After RNase digestion or salt extraction, two proteins that initially exist as tetramers (A2)(3)B1 in isolated heterogeneous nuclear ribonucleoprotein (hnRNP) complexes quantitatively reassociated to form regular helical filaments ranging in length from 100 nm to >10 microm. In highly magnified preparations prepared for scanning transmission electron microscopy, single filaments have diameters near 18 nm. In conventional negatively stained preparations viewed at low magnification, the diameters of the thinnest filaments range from 7 to 10 nm. At protein concentrations of >0.1 mg/ml, the filaments rapidly aggregated to form thicker filamentous networks that look like the fibrogranular structures termed the "nuclear matrix." Like the residual material seen in nuclear matrix preparations, the hnRNP filaments were insoluble in 2 M NaCl. Filament formation is associated with, and may be dependent on, disulfide bridge formation between the hnRNP proteins. The reducing agent 2-mercaptoethanol significantly attenuates filament assembly, and the residual material that forms is ultrastructurally distinct from the 7- to 10-nm fibers. In addition to the protein rearrangement leading to filament formation, nearly one-third of the protein present in chromatin-clarified nuclear extracts was converted to salt-insoluble material within 1 min of digestion with RNase. These observations are consistent with the possibility that the residual material termed the nuclear matrix may be enriched in, if not formed by, denatured proteins that function in pre-mRNA packaging, processing, and transport.

Actin Cytoskeleton↗
PubMed2002

The newly identified human nuclear protein NXP-2 possesses three distinct domains, the nuclear matrix-binding, RNA-binding, and coiled-coil domains.

Using a monoclonal antibody that recognizes a nuclear matrix protein, we selected a cDNA clone from a lambdagt11 human placenta cDNA library. This cDNA encoded a 939-amino acid protein designated nuclear matrix protein NXP-2. Northern blot analysis indicated that NXP-2 was expressed in various tissues at different levels. Forcibly expressed green fluorescent protein-tagged NXP-2 as well as endogenous NXP-2 was localized in the nucleus and distributed to the nuclear matrix. NXP-2 was released from the nuclear matrix when RNase A was included in the buffer for nuclear matrix preparation. Mapping of functional domains was carried out using green fluorescent protein-tagged truncated mutants of NXP-2. The region of amino acids 326-353 was responsible for nuclear matrix binding and contained a cluster of hydrophobic amino acids that was similar to the nuclear matrix targeting signal of acute myeloleukemia protein. The central region (amino acids 500-591) was demonstrated to be required for RNA binding by Northwestern analysis, although NXP-2 lacked a known RNA binding motif. The region of amino acid residues 682-876 was predicted to have a coiled-coil structure. The RNA-binding, nuclear matrix-binding, and coiled-coil domains are structurally separated, suggesting that NXP-2 plays important roles in diverse nuclear functions, including RNA metabolism and maintenance of nuclear architecture.

Amino Acid Sequence↗
PubMed1998

Intranuclear targeting of AML/CBFalpha regulatory factors to nuclear matrix-associated transcriptional domains.

The AML/CBFalpha runt transcription factors are key regulators of hematopoietic and bone tissue-specific gene expression. These factors contain a 31-amino acid nuclear matrix targeting signal that supports association with the nuclear matrix. We determined that the AML/CBFalpha factors must bind to the nuclear matrix to exert control of transcription. Fusing the nuclear matrix targeting signal to the GAL4 DNA binding domain transactivates a genomically integrated GAL4 responsive reporter gene. These data suggest that AML/CBFalpha must associate with the nuclear matrix to effect transcription. We used fluorescence labeling of epitope-tagged AML-1B (CBFA2) to show it colocalizes with a subset of hyperphosphorylated RNA polymerase II molecules concentrated in foci and linked to the nuclear matrix. This association of AML-1B with RNA polymerase II requires active transcription and a functional DNA binding domain. The nuclear matrix domains that contain AML-1B are distinct from SC35 RNA processing domains. Our results suggest two of the requirements for AML-dependent transcription initiation by RNA polymerase II are association of AML-1B with the nuclear matrix together with specific binding of AML to gene promoters.

Cell Compartmentation↗
PubMed1986

Localization of human histone gene transcripts predominantly in the nonmatrix nuclear fraction.

Using S1 nuclease protection assays, we have examined the representation of cell cycle-dependent H4 histone RNAs in the nuclear matrix and nonmatrix nuclear fractions of human cells. Cytoplasmic and nuclear fractions were prepared from exponentially growing HeLa S3 cells by double detergent (sodium deoxycholate and NP40) lysis. The nuclear matrix and nonmatrix nuclear fractions were then prepared by digestion of nuclei with RNase-free DNase I and subsequent high-salt [0.4 M (NH4)2SO4] extraction. Subcellular fractions were characterized by 1) DNA, RNA, and protein composition; 2) electrophoretic analysis of the proteins in each fraction; 3) the representation of 45S ribosomal RNA precursors and processed 18S and 28S ribosomal RNAs; and 4) the presence of mitochondrial RNAs. In contrast to ribosomal and messenger RNA precursors, which are largely associated with the nuclear matrix, the human H4 histone RNAs in the nucleus were found predominantly in the nonmatrix nuclear fraction. The presence of H4 histone RNA in the nonmatrix nuclear fraction appeared to be coupled to DNA replication, since inhibition of DNA synthesis by hydroxyurea resulted in a loss of histone RNA from the nucleus. Our results suggest either that the association of histone RNAs with the nuclear matrix is very transient or that posttranscriptional modifications of the rapidly processed histone gene transcripts do not involve the nuclear matrix.

Cell Cycle↗
PubMed1992

Sequence-specific DNA-binding proteins are components of a nuclear matrix-attachment site.

We have identified a nuclear matrix-attachment region within an upstream element of a human H4 histone gene promoter. Nuclear matrix proteins, isolated and solubilized from HeLa S3 cells, were found to interact with sequence specificity at this matrix-attachment region. Several types of assays for protein-DNA interaction showed that the minimal sequence for the nuclear matrix protein-DNA interaction was 5'-TGACGTCCATG-3'; the underlined region corresponds to the core consensus sequence for ATF transcription factor binding. Two proteins with molecular masses of 43 and 54 kDa were identified by UV-crosslinking analysis as integral components of this protein-DNA complex. The molecular masses of these proteins and the ATF-binding site consensus sequence suggest that these proteins are members of the ATF family. Our results provide direct evidence for nuclear matrix localization of sequence-specific DNA-binding factors for an actively transcribed gene. The proximity of a strong positive transcriptional regulatory element to the matrix-attachment region of this gene suggests that the nuclear matrix may serve to localize and concentrate trans-acting factors that facilitate regulation of gene expression.

Base Sequence↗
PubMed1999

Binding of the protein disulfide isomerase isoform ERp60 to the nuclear matrix-associated regions of DNA.

Protein ERp60, previously found in the internal nuclear matrix in chicken liver nuclei, is a member of the protein disulfide isomerase family. It binds DNA and double helical polynucleotides in vitro with a preferential recognition toward the matrix-associated regions of DNA and poly(dA) x poly(dT), and its binding is inhibited by distamycin. ERp60 can be cross-linked chemically to DNA in the intact nuclei, suggesting that its association with DNA is present in vivo. As a whole, these results indicate that ERp60 is a component of the subset of nuclear matrix proteins that are responsible for the attachment of DNA to the nuclear matrix and for the formation of DNA loops. A distinctive feature of this protein, which has two thioredoxin-like sites, is that its affinity to poly(dA) x poly(dT) is strongly dependent on its redox state. Only its oxidized form, in fact, does it bind poly(dA) x poly(dT). The hypothesis can be made that through the intervention of ERp60, the redox state of the nucleus influences the formation or the stability of some selected nuclear matrix-DNA interactions.

Animals↗
PubMed1998

The DNA-binding and tau2 transactivation domains of the rat glucocorticoid receptor constitute a nuclear matrix-targeting signal.

Using an ATP-depletion paradigm to augment glucocorticoid receptor (GR) binding to the nuclear matrix, we have identified a minimal segment of the receptor that constitutes a nuclear matrix targeting signal (NMTS). While previous studies implicated a role for the receptor's DNA-binding domain in nuclear matrix targeting, we show here that this domain of rat GR is necessary, but not sufficient, for matrix targeting. A minimal NMTS can be generated by linking the rat GR DNA-binding domain to either its tau2 transactivation domain in its natural context, or a heterologous transactivation domain derived from the Herpes simplex virus VP16 protein. The transactivation and nuclear matrix-targeting activities of tau2 are separable, as transactivation mutants were identified that either inhibited or had no apparent effect on matrix targeting of tau2. A functional interaction between the NMTS of rat GR and the RNA-binding nuclear matrix protein hnRNP U was revealed in cotransfection experiments in which hnRNP U overexpression was found to interfere with the transactivation activity of GR derivatives that possess nuclear matrix-binding capacity. We have therefore ascribed a novel function to a steroid hormone transactivation domain that could be an important component of the mechanism used by steroid hormone receptors to regulate genes in their native configuration within the nucleus.

Adenosine Triphosphate↗
PubMed1988

Effects of antileukemia agents on nuclear matrix-bound DNA replication in CCRF-CEM leukemia cells.

The effects of various antileukemic agents on DNA replication associated with the nuclear matrix were investigated in CCRF-CEM leukemia cells. Residual nuclear matrices were prepared by sequential treatment of nuclei with 1.5 M NaCl, DNase I, and Triton X-100 and contained 1-5, 10, and 37% of the total nuclear DNA, protein, and phospholipid, respectively. In control cells pulse-labeled for 45 s with [3H]thymidine, the specific activity of nascent DNA was four-fold greater in the nuclear matrix fraction relative to the specific activity of the high salt-soluble (nonmatrix) DNA fraction. Pulse-labeling and reconstitution experiments indicated that this enrichment of newly replicated DNA on the nuclear matrix did not result from aggregation of nascent DNA with the matrix. A 2-h incubation of tumor cells with either 0.1 microM teniposide (VM-26), 0.2 microM VM-26, or 0.5 microM amsacrine (m-AMSA) reduced the relative specific activity of nascent DNA on the nuclear matrix by 59, 61, and 54%, respectively, compared to control cells. In contrast hydroxyurea and cytosine arabinoside, at concentrations that markedly inhibited total nuclear DNA synthesis, did not decrease the relative specific activity of newly replicated DNA on the matrix. The results provide evidence that the antiproliferative effects of the DNA topoisomerase II inhibitors, VM-26 and m-AMSA, are localized on the nuclear matrix of CCRF-CEM leukemia cells.

Amsacrine↗
PubMed1995

Alterations in cytoskeletal and nuclear matrix-associated proteins during apoptosis.

Evidence exists that apoptosis or programmed cell death plays an important role in tumor growth. Morphologically apoptosis is characterized by membrane blebbing and nuclear fragmentation in individual cells. In this study, we investigated changes in the nuclear organization in spontaneously occurring apoptotic cells in several cancer cell lines using several antibodies to nuclear matrix constituents. It appeared that nuclear matrix components remained detectable in cells undergoing spontaneous apoptosis. The same results were found when apoptosis was induced by cycloheximide in the non-small cell lung cancer cell line MR65. Using this induction method, the percentage of apoptotic cells in MR65 cells increased, allowing a more detailed and extensive examination of nuclear matrix alterations together with cytoskeletal changes. To study the expression of cytokeratins, type A- and B lamins, a nuclear matrix-associated 13 kDa U1RNP particle and the Ki67-antigen, immunocytochemistry in combination with confocal scanning laser microscopy was used. Apoptotic cells were identified based on nuclear morphology and the in situ nick translation assay. Whereas immunoreactivity against lamins and Ki67-Ag was rapidly lost during apoptosis, expression of the 13 kDa protein and, in early apoptotic stages, also cytokeratin expression, was observed to remain present. Dead cells lacked reactivity with all the antibodies tested. The persistence of nuclear matrix components is therefore a useful marker for the detection of apoptosis.

Antigens, Nuclear↗
PubMed2000

AHM1, a novel type of nuclear matrix-localized, MAR binding protein with a single AT hook and a J domain-homologous region.

Interactions between the nuclear matrix and special regions of chromosomal DNA called matrix attachment regions (MARs) have been implicated in various nuclear functions. We have identified a novel protein from wheat, AT hook-containing MAR binding protein1 (AHM1), that binds preferentially to MARs. A multidomain protein, AHM1 has the special combination of a J domain-homologous region and a Zn finger-like motif (a J-Z array) and an AT hook. For MAR binding, the AT hook at the C terminus was essential, and an internal portion containing the Zn finger-like motif was additionally required in vivo. AHM1 was found in the nuclear matrix fraction and was localized in the nucleoplasm. AHM1 fused to green fluorescent protein had a speckled distribution pattern inside the nucleus. AHM1 is most likely a nuclear matrix component that functions between intranuclear framework and MARs. J-Z arrays can be found in a group of (hypothetical) proteins in plants, which may share some functions, presumably to recruit specific Hsp70 partners as co-chaperones.

Amino Acid Sequence↗
PubMed2002

Survivin exists in immunochemically distinct subcellular pools and is involved in spindle microtubule function.

Survivin is a member of the inhibitor of apoptosis gene family that has been implicated in both apoptosis inhibition and regulation of mitosis. However, the subcellular distribution of survivin has been controversial and variously described as a microtubule-associated protein or chromosomal passenger protein. Here, we show that antibodies directed to the survivin sequence Ala(3)-Ile(19) exclusively recognized a nuclear pool of survivin that segregated with nucleoplasmic proteins, but not with outer nuclear matrix or nuclear matrix proteins. By immunofluorescence, nuclear survivin localized to kinetochores of metaphase chromosomes, and to the central spindle midzone at anaphase. However, antibodies to Cys(57)-Trp(67) identified a cytosolic pool of survivin, which associated with interphase microtubules, centrosomes, spindle poles and mitotic spindle microtubules at metaphase and anaphase. Polyclonal antibodies recognizing survivin epitopes Ala(3)-Ile(19), Met(38)-Thr(48), Pro(47)-Phe(58) and Cys(57)-Trp(67) identified both survivin pools within the same mitotic cell. A ratio of approximately 1:6 for nuclear versus cytosolic survivin was obtained by quantitative subcellular fractionation. In synchronized cultures, cytosolic survivin abruptly increased at mitosis, physically associated with p34(cdc2), and was phosphorylated by p34(cdc2) on Thr(34), in vivo. By contrast, nuclear survivin began to accumulate in S phase, was not complexed with p34(cdc2) and was not phosphorylated on Thr(34). Intracellular loading of a polyclonal antibody to survivin caused microtubule defects and resulted in formation of multipolar mitotic spindles, but did not interfere with cytokinesis. These data demonstrate that although both reported localizations of survivin exist in mitotic cells, the preponderant survivin pool is associated with microtubules and participates in the assembly of a bipolar mitotic spindle.

Animals↗
PubMed2005

Initiation of DNA replication at a nuclear matrix-attached chromatin fraction.

It is still unclear what nuclear components support initiation of DNA replication. To address this issue, we developed a cell-free replication system in which the nuclear matrix along with the residual matrix-attached chromatin was used as a substrate for DNA replication. We found out that initiation occurred at late G1 residual chromatin but not at early G1 chromatin and depended on cytosolic and nuclear factors present in S phase cells but not in G1 cells. Initiation of DNA replication occurred at discrete replication foci in a pattern typical for early S phase. To prove that the observed initiation takes place at legitimate DNA replication origins, the in vitro synthesized nascent DNA strands were isolated and analyzed. It was shown that they were enriched in sequences from the core origin region of the early firing, dihydrofolate reductase origin of replication ori-beta and not in distal to the origin sequences. A conclusion is drawn that initiation of DNA replication occurs at discrete sub-chromosomal structures attached to the nuclear matrix.

Animals↗
PubMedDate not supplied

Fine structural observation of a nucleolar-nuclear matrix-lamina-intermediate filament system in transformed cells.

The nuclear matrix is a nonchromatin structure of the nucleus normally concealed by a much larger mass of chromatin. Several methods have been developed to remove chromatin from nucleus while preserving the underlying matrix architecture to some degree. The present study showed that after extraction of PtK2 cells and cervical carcinoma cells (CC3) with Triton X-100, ammonium sulfate and DNase, the nucleolar-nuclear matrix-intermediate filament (Nu-Nm-L-IF) network remained. The nucleolus was oval in shape and appeared as a fibrillar mass with an accentric dense fibrillar centre. This nucleolar skeleton was in direct connection with the nuclear matrix which in turn is connected with cytoplasmic intermediate filaments by lamins. It is concluded from these observations that the Nu-NM-L-IF system forms a continuous system which plays an important role in the maintenance of the nucleolar, nuclear and cytoplasmic integrity and cellular function.

Animals↗
PubMed1996

Binding of matrix attachment regions to nuclear lamin is mediated by the rod domain and depends on the lamin polymerization state.

The nuclear matrix maintains specific interactions with genomic DNA at sites known as matrix attachment regions (M/SARs). M/SARs bind in vitro to lamin polymers. We show that the polymerized alpha-helical rod domain of lamin Dm0 provides by itself the specific binding to the ftz M/SAR. In contrast, unpolymerized rod domain does not bind specifically to this M/SAR. Non-specific binding to DNA is also observed with Dm0 containing a point mutation that impairs its ability to polymerize or with the isolated tail domain. These data suggest that the specific binding of lamins to M/SARs requires the rod domain and depends on the lamin polymerization state.

Amino Acid Sequence↗
PubMed1998

Prereplicative increase of nuclear matrix-bound DNA polymerase-alpha and primase activities in HeLa S3 cells following dilution of long-term cultures.

We investigated the association of DNA polymerase and DNA primase activity with the nuclear matrix in HeLa S3 cells diluted with fresh medium after having been cultured without any medium change for 7 days. Flow cytometric analysis demonstrated that just before dilution about 85% of the cells were in the G1 phase of the cycle, whereas 8% were in the S phase. After dilution with fresh medium, 18-22 h were required for the cell population to attain a stable distribution with respect to the cell cycle. At that time, about 38% of the cells were in the S phase. DNA polymerase and DNA primase activity associated with the nuclear matrix prepared from cells just before dilution represented about 10% of nuclear activity. As judged by [3H]-thymidine incorporation and flow cytometric analysis, an increase in the number of S-phase cells was evident at least 6 h after dilution. However, as early as 2 h after dilution into fresh medium, a striking prereplicative increase of the two activities was seen in the nuclear matrix fraction but not in cytosol or isolated nuclei. Both DNA polymerase and primase activities bound to the matrix were about 60% of nuclear activity. Overall, the nuclear matrix was the cell fraction where the highest induction (about 10-fold) of both enzymatic activities was seen at 30 h after dilution, whereas in cytosol and isolated nuclei the increase was about two- and fourfold, respectively. Typical immunofluorescent patterns given by an antibody to 5-bromodeoxyuridine were seen after dilution. These findings, which are at variance with our own previous results obtained with cell cultures synchronized by either a double thymidine block or aphidicolin exposure, strengthen the contention that DNA replication is associated with an underlying nuclear structure and demonstrate the artifacts that may be generated by procedures commonly used to synchronize cell cultures.

Bromodeoxyuridine↗
PubMed2006

[Topology of chromosomes in somatic cells. Part 1].

The interphase cell nucleus is a highly compartmentalized structure in which chromosomes are located in separate, defined places called chromosome territories (CTs). Chromosome territories with interchromatin compartments (ICs) and the nuclear matrix determine the nuclear architecture. There is a connection between nuclear architecture and genome function. The topology of the chromosomes in the nuclei of somatic cells is summarized here. It is known that the size and location of chromosome territories are tissue specific and depend on the cell cycle and the size of the chromosomes and the density of the genes which are actively transcribed. The correlation between transcriptional activity, the level of chromatin structure, and the location of the chromatin domains is outlined. The tendency of the heterochromatin regions and the telomeres to associate and the influence of this on the nuclear architecture is highlighted. Some studies are focused on the indirect role of the elements of the nuclear matrix and the inner-nuclear membrane in maintaining the correct locations of chromosome territories. The role of interchromatin granule clusters (IGCs), which are located in the nuclear matrix and which remain active in nuclear processes connected with chromosome topology, is further described. The influence of cell differentiation on chromosome location is pointed out. The topology of chromosomes in evolutionarily distinct species is also mentioned in this review. The reciprocal location of the chromosome territories is probably one of the important epigenetical factors influencing correct genome function. The high level of the organization of chromatin and chromatin modifications create the unique epigenetic pattern of a particular cell type. This seems to indicate a critical role of the spatial genomic organization in regulating gene expression.

Animals↗