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PubMed1985

Properties of adenoviral DNA bound to the nuclear matrix.

The association of adenoviral DNA with the high salt (2 M NaCl) resistant nuclear fraction, termed the nuclear matrix, has been investigated in HeLa cells at different times after infection with adenovirus type 5. When nuclear matrices were prepared in the absence of exogenously added nucleases, Ad5 DNA was quantitively associated with the matrix throughout the infection period (0-24 h). Moreover, early in infection (0-10 h) Ad5 DNA was severalfold enriched in DNase I digested nuclear matrices (8-15% of total nuclear DNA) compared to the high salt soluble chromatin fraction (85-90% of total nuclear DNA). At later times after infection, progressively more Ad5 DNA appeared in the chromatin fraction until, at 24 h, the nuclear matrix was strikingly depleted in Ad5 DNA. A large proportion of the Ad5 DNA in nuclear matrices prepared early in infection, e.g., 4 h, was full length in size. At later times (12-24 h) most of the viral DNA was fragmented to a size equivalent to total matrix DNA (100-1000 base pairs). The apparent switch of the matrix-associated viral DNA from a relatively DNase I resistant to sensitive state was initiated approximately at the time when viral DNA replication began (12 h). Since no discrete portion of the Ad5 genome was significantly enriched at the sites of attachment to the nuclear matrix throughout the infection period, the switch in DNase I sensitivity is not mediated by a change in DNA sequence attachment to the matrix.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenoviruses, Human↗

PubMed1992

Further considerations on the thermal stabilization of the nuclear matrix in mouse erythroleukemia cells.

The morphology and the polypeptide composition of the nuclear matrix obtained from 37 degrees C incubated nuclei has been studied in mouse erythroleukemia cells. From a structural point of view, in the absence of heat treatment, the matrix lacked identifiable nucleolar remnants and the internal fibrogranular meshwork whereas a peripheral lamina was seen. On the contrary, the matrix obtained from heat exposed nuclei displayed very electrondense nucleolar remnants and an abundant inner network. These results were obtained irrespective of the type of extracting agent (2M NaCl or 0.2 M (NH4)2SO4) used to remove histones and other soluble proteins. The heat stabilization of the matrix could not be prevented by sulfhydryl blocking chemicals such as iodoacetamide and n-ethylmaleimide, thus suggesting that heat does not stabilize the matrix by inducing the formation of disulfide bonds. Only limited differences in the polypeptide pattern of matrix isolated under different conditions were seen using one-dimensional pore gradient polyacrylamide gels stained with both Coomassie Brilliant Blue and silver despite the fact that the matrix fraction from heat treated nuclei retained about three fold more protein in comparison with controls. The same results were obtained also by means of two-dimensional non-equilibrium gel electrophoresis.

Ammonium Sulfate↗

PubMed1990

[The intermediate filament-lamina-nuclear matrix system of BHK-21 cells].

We have employed collodial gold immuno-labelling in whole-mount cell and 2-D gel electrophoresis to demonstrate the intermediate filament (IF)-lamina-nuclear matrix (NM) system in BHK-21 (Baby Hamster Kidney) cells. Grown on grids, cells were gently extracted with salt solutions as previously described by S. Penman to preserve intact IF-lamina-NM systems. The extracted samples were fixed, postfixed, dehydrated and dried through the CO2 critical point, then examined under high voltage electron microscope (HVEM). The results revealed that the IF-lamina-NM system is a interconnecting network throughout the cell from cytoplasma to nuclear. The IF unit is 10 nm in diameter. IFs radiate away from the nuclear region into the spreading cytoplasm and the polarity of their distributing is obvious. The IF system closely connected to lamina. Immuno-gold labelling and 2-D gel proved that vimentin, a 55 KD protein (pI 5,6), is the major component of IFs in BHK-21 cells. Lamina can be precisely and specifically labelled with anti-lamin A, C proteins and as well as 2-D gel electrophoresis indicated that there are lamin A, B, C proteins in BHK-21 cells, whose molecular weights are 68 KD, 70 KD, 62 KD respectively. Its components are more complicated, but a few dots of NM proteins can be clearly distinguished in 2-D gel map, in which actin, a 45 KD protein (pI 4.5), might be involved. The nuclear matrix network was also clearly presented under HVEM. Its filaments can be labelled with anti-NM 298 KD protein precisely.

Actinin↗

PubMed2002

DNA binding and gene activation properties of the Nmp4 nuclear matrix transcription factors.

Splice variants of the Nmp4 gene include nuclear matrix transcription factors that regulate the type I collagen alpha1(I) polypeptide chain (COL1A1) promoter and several matrix metalloproteinase (MMP) genes. To date, these are the only Cys(2)His(2) zinc finger proteins known to bind within the minor groove of homopolymeric (dA.dT) DNA. Nmp4 isoforms contain from 5 to 8 Cys(2)His(2) zinc fingers, an SH3-binding domain that overlaps with a putative AT-hook and a polyglutamine-alanine repeat (poly(QA)). To determine the mechanistic significance of Cys(2)His(2) zinc finger association with this unusual consensus DNA binding element, we identified the Nmp4 DNA-binding and transcriptional activation domains. Zinc fingers 2, 3, and 6 mediated association with the homopolymeric (dA.dT) COL1A1/MMP DNA consensus element. The N terminus of the Nmp4 protein exhibited a strong trans-activation capacity when fused to the GAL4 DNA-binding domain, but this activity was masked within the context of the full-length Nmp4-GAL4 DNA-binding domain chimera. However, upon binding to the COL1A1/MMP homopolymeric (dA.dT) element, the native Nmp4 protein up-regulated transcription, and the poly(QA) domain acquired a significant role in trans-activation. We propose that allosteric effects induced upon zinc finger association with the homopolymeric (dA.dT) minor groove confer context-specific functionality to this unusual family of Cys(2)His(2) transcription factors.

Antigens, Nuclear↗

PubMed1999

Cell cycle-dependent nuclear location of the matricellular protein SPARC: association with the nuclear matrix.

Secreted protein acidic and rich in cysteine (SPARC) is a matricellular protein that inhibits cellular adhesion and proliferation. In this study, we report the detection of SPARC in the interphase nuclei of embryonic chicken cells in vivo. Differential partitioning of SPARC was also noted in the cytoplasm of these cells during discrete stages of M-phase: cells in metaphase and anaphase exhibited strong cytoplasmic immunoreactivity, whereas cells in telophase were devoid of labeling. Immunocytochemical analysis of embryonic chicken cells in vitro likewise showed the presence of SPARC in the nucleus. Furthermore, elution of soluble proteins and DNA from these cells indicated that SPARC might be a component of the nuclear matrix. We subsequently examined cultured bovine aortic endothelial cells, which initially appeared to express SPARC only in the cytoplasm. However, after elution of soluble proteins and chromatin, we also detected SPARC in the nuclear matrix of these cells. Embryonic chicken cells incubated with recombinant SPARC were seen to take up the protein and to translocate it to the nucleus progressively over a period of 17 h. These observations provide new information about SPARC, generally recognized as a secreted glycoprotein that mediates interactions between cells and components of the extracellular matrix. The evidence presented in this study indicates that SPARC might subserve analogous functions in the nuclear matrix.

Animals↗

PubMed2007

C-terminal domains deliver the DNA replication factor Ciz1 to the nuclear matrix.

Cip1-interacting zinc finger protein 1 (Ciz1) stimulates DNA replication in vitro and is required for mammalian cells to enter S phase. Here, we show that a significant proportion of Ciz1 is retained in nuclear foci following extraction with nuclease and high salt. This suggests that Ciz1 is normally immobilized by interaction with non-chromatin nuclear structures, consistent with the nuclear matrix. Furthermore, matrix-associated Ciz1 foci strikingly colocalize with sites of newly synthesized DNA in S phase nuclei, suggesting that Ciz1 is present in DNA replication factories. Analysis of green fluorescent protein-tagged fragments indicates that nuclear immobilization of Ciz1 is mediated by sequences in its C-terminal third, encoded within amino acids 708-830. Immobilization occurs in a cell-cycle-dependent manner, most probably during late G1 or early S phase, to coincide with its reported point of action. Although C-terminal domains are sufficient for immobilization, N-terminal domains are also required to specify focal organization. Combined with previous work, which showed that the DNA replication activity of Ciz1 is encoded by N-terminal sequences, we suggest that Ciz1 is composed of two functionally distinct domains: an N-terminal replication domain and a C-terminal nuclear matrix anchor. This could contribute to the formation or function of DNA replication factories in mammalian cells.

Animals↗

PubMed1982

Adenovirus DNA is associated with the nuclear matrix of infected cells.

Viral DNA was found to be tightly associated with the nuclear matrix from HeLa cells lytically infected with human adenovirus type 5. The bound viral DNA, like cell DNA, was resistant to nonionic detergent and to extraction with high-salt (2 M NaCl) solution. However, whereas over 95% of the cell DNA was recovered in the matrix fraction, the amount of associated viral DNA varied during infection. Throughout the lytic cycle, the amount of matrix-associated adenovirus type 5 DNA increased until it reached a plateau level at 20 to 24 h after infection. At this stage, the matrix-bound DNA represented 87% of the total viral DNA; after this stage, additional newly synthesized viral DNA accumulated as non-matrix-associated DNA. DNase digestion studies revealed that all viral DNA sequences were equally represented in the matrix-bound DNA both early and late in infection; thus, unlike cell DNA, there seem to be no preferred attachment sites on the viral genome. An enrichment of viral DNA relative to cell DNA was found in the matrix-associated DNA after extensive DNase I digestion. This finding, together with an in situ hybridization study, suggests that the viral DNA is more intimately associated with the nuclear matrix than is cell DNA and probably does not exist in extended loops.

Adenoviruses, Human↗

PubMed1991

A region immediately adjacent to the origin of replication of bovine papilloma virus type 1 interacts in vitro with the nuclear matrix.

We have investigated the interaction of the 69% transforming fragment of the Bovine Papilloma Virus type 1 (BPV1) with the nuclear matrix from 1361.5 cells (NIH-3T3 cells transformed by a BPV chimeric construct). In vitro studies performed with end-labelled DNA fragments and nuclear matrices prepared using a high-salt extraction procedure demonstrate the binding of a 672 bp fragment adjacent to the viral origin of replication and containing the plasmid maintenance sequence (PMS-1). This fragment can be cleaved into two pieces (393 and 279 bp), both interacting equally well with the nuclear matrix. This indicates that a least two regions of the 672 bp DNA fragment are involved in the interaction.

Animals↗

PubMed1999

[Role of nuclear matrix proteins in the formation of heterochromatin].

Heterochromatin consists mainly of satellite DNAs (stDNA), the most rapidly evolving type of DNA sequences of the eucaryotic genome. On the other hand, stDNA is involved in the formation of the functionally conserved centromere structure. Centromeres and pericentromeric stDNA, are known to be in association with nuclear matrix or chromosome scaffold at all stages of the cell cycle. Several lines of evidence show that attachment of stDNA to the nuclear matrix is specific. The first defined parts of the genes found in association with nuclear matrix/scaffold, MAR/SAR, possess some common features with the stDNA. A number of different mechanisms have previously been implicated in heterochromatin formation and centromere conservation. The role of nuclear matrix proteins, which are able to recognize common structural features of MAR/SAR and stDNA, in constitutive heterochromatin organization is discussed in the current review.

Antigens, Nuclear↗

PubMed1995

Modulation of some nuclear matrix enzymatic activities by free fatty acids.

It was shown that two of main enzymatic activities of plant nucleus and nuclear matrix, namely RNA-polymerasic and DNA-nucleolytic are susceptible to modulation with free fatty acids. The effects observed were dependent to both fatty acid length and degree of unsaturation. In nuclei a stimulation of nuclease activity was observed whereas in matrices short chain fatty acids inhibited the studied activity. The effect of fatty acids on RNA-polymerase was also different in nuclei and matrices. In nuclei all fatty acids studied inhibited polymerasic activity whereas in matrices short chain fatty acids stimulated this activity by up to 80% and the long chain fatty acids inhibited by up over 70%. The overall alteration of studied activities in nuclei and matrices by unsaturated fatty acids was similar. Nucleolytic activity was stronger inhibited and polymerasic activity was stimulated when the effects of linoleic and linolenic acids were studied. The results suggest possible importance of lipid component in nuclear matrix biological function.

DNA-Directed RNA Polymerases↗

PubMed1999

Crystal structure of the nuclear matrix targeting signal of the transcription factor acute myelogenous leukemia-1/polyoma enhancer-binding protein 2alphaB/core binding factor alpha2.

Transcription factors of the acute myelogenous leukemia (AML)/polyoma enhancer-binding protein (PEBP2alpha)/core-binding factor alpha (CBFA) class are key transactivators of tissue-specific genes of the hematopoietic and bone lineages. AML-1/PEBP2alphaB/CBFA2 proteins participating in transcription are associated with the nuclear matrix. This association is solely dependent on a highly conserved C-terminal protein segment, designated the nuclear matrix targeting signal (NMTS). The NMTS of AML-1 is physically distinct from the nuclear localization signal, operates autonomously, and supports transactivation. Our data indicate that the related AML-3 and AML-2 proteins are also targeted to the nuclear matrix in situ by analogous C-terminal domains. Here we report the first crystal structure of an NMTS in an AML-1 segment fused to glutathione S-transferase. The model of the NMTS consists of two loops connected by a flexible U-shaped peptide chain.

Amino Acid Sequence↗

PubMed1988

Nuclear matrix associated poly(ADP-ribose) metabolism in regenerating rat liver.

We have examined a possible role for protein poly(ADP-ribosylation) during in vivo DNA replication by studying the metabolism of poly(ADP-ribose) in the nuclear matrix fraction from normal and regenerating rat liver. This fraction contains the newly replicated DNA and thus allows for the examination of the events closely associated with the replication process. It was found that 55% of the total nuclear protein-bound poly(ADP-ribose) and 15-35% of the total nuclear poly(ADP-ribose)-polymerase activity were tightly associated with this subnuclear compartment in normal liver. Surgical removal of two-thirds of the liver initiated a time-dependent decrease in nuclear matrix associated polymers of ADP-ribose and poly(ADP-ribose) polymerase activity which reached a minimum of 40% of control livers after 24 h, before returning to normal levels at 41 h post-partial hepatectomy. In contrast, the total levels of poly(ADP-ribose) in intact liver and the total polymerase activity of isolated nuclei exhibited a 2-fold increase over basal levels. These results are consistent with the conclusion that the nuclear matrix is a major poly(ADP-ribosylation) site within the nucleus and that this metabolic reaction may be closely connected with the events modulating DNA replication in this fraction.

Animals↗

PubMed1998

Involvement of a nuclear matrix association region in the regulation of the SPRR2A keratinocyte terminal differentiation marker.

The small proline-rich protein genes ( SPRRs ) code for precursors of the cornified cell envelope, and are specifically expressed during keratinocyte terminal differentiation. The single intron of SPRR2A enhanced the activity of the SPRR2A promoter in transient transfection assays. This enhancement was position dependent, and did not function in combination with a heterologous promoter, indicating that the intron does not contain a classical enhancer, and that the enhancement was not due to the splicing reaction per se. Mild DNAse-I digestion of nuclei showed the SPRR2 genes to be tightly associated with the nuclear matrix, in contrast to the other cornified envelope precursor genes mapping to the same chromosomal location (epidermal differentiation complex). In vitro binding studies indicated that both the proximal promoter and the intron of SPRR2A are required for optimal association of this gene with nuclear matrices. Neither nuclear matrix association nor the relative transcriptional enhancement by the intron changed during keratinocyte differentiation. Apparently, the association of the SPRR2A gene with the nuclear matrix results in a general, differentiation-independent enhancement of gene expression.

3T3 Cells↗

PubMed1997

Trichoderma reesei sequences that bind to the nuclear matrix enhance transformation frequency.

Three DNA fragments, trs1, 2 and 3, were isolated from the Trichoderma reesei genome on the basis of their ability to promote autonomous replication of plasmids in Saccharomyces cerevisiae. Each trs element bound specifically to the isolated T. reesei nuclear matrix in vitro, and two of them bound in vivo, indicating that they are matrix attachment regions (MARs). A similar sequence previously isolated from Aspergillus nidulans (ans1) was also shown to bind specifically to the T. reesei nuclear matrix in vitro. The T. reesei MARs are AT-rich sequences containing 70%, 86% and 73% A + T over 2.9, 0.8 and 3.7 kb, respectively for trs1, 2 and 3. They exhibited no significant sequence homology, but were shown to contain a number of sequence motifs that occur frequently in many MARs identified in other eukaryotes. However, these motifs occurred as frequently in the trs elements as in randomly generated sequences with the same A + T content. trs1 and 3 were shown to be present as single copies in the T. reesei genome. The presence of the trs elements in transforming plasmids enhanced the frequency of integrative transformation of T. reesei up to five fold over plasmids without a trs. No evidence was obtained to suggest that the trs elements promoted efficient replication of plasmids in T. reseei. A mechanism for the enhancement of transformation frequency by the trs elements is proposed.

Blotting, Southern↗

PubMed1989

Flow cytometric studies of the nuclear matrix.

We have devised a method to measure the protein and nucleic acid content of the nuclear matrix using flow cytometry. Nuclear matrices were prepared from nuclei by DNase I digestion followed by 3 M NaCl extraction. The resulting particles were stained with fluorescein isothiocyanate (FITC) for protein and propidium iodide (PI) for double-stranded nucleic acids, and fluorescence as well as forward angle light scatter was detected. The matrices were also subjected to additional chemical or enzymatic perturbations, and changes in the above parameters were measured. Results showed that matrices from heat-shocked cells not only retained the majority of heat-induced excess nuclear protein, but also exhibited higher PI signals than controls after RNase A digestion. This observation did not hold if RNase A digestion preceded high-salt extraction, suggesting that a salt-extractable moiety had been replaced or altered by heat so that double-stranded RNA was protected from the nucleolytic attack. The residual PI fluorescence in matrices from heated cells bore a linear relationship to the increased protein content in those matrices, indicating that the excess protein sequesters matrix-associated RNA. Polyacrylamide gel electrophoresis of matrix polypeptides revealed increased amounts of many proteins as a result of heat as well as the appearance of several new proteins, one of which comigrates with the HSP72/73 heat-shock proteins. The results of these studies show that flow cytometry can be used to study the nuclear matrix and is capable of detecting changes that result from alterations in its protein composition.

Cell Nucleus↗

PubMed1984

Attachment of origins of replication to the nuclear matrix and the chromosomal scaffold.

We have investigated the attachment of DNA to the nuclear matrix and chromosomal scaffold in synchronized bovine liver cells. Label incorporated at the onset of the S phase remained preferentially associated with the matrix during the subsequent G1 phase and with a residual protein structure from dehistonized chromosomes during mitosis. On the other hand label incorporated during mid or late S phase was about equally distributed over the DNA molecule after a chase into the G1 phase. These results suggest that DNA is attached to the nuclear matrix and chromosome scaffolds by the origins of replication.

Animals↗

PubMed1999

Biochemical and morphological characterization of the nuclear matrix from apoptotic HL-60 cells.

We have characterized the nuclear matrix-intermediate filament fraction from control and apoptotic HL-60 cells. Apoptosis was induced by exposure to the topoisomerase I inhibitor, camptothecin. By means of two-dimensional polyacrylamide gel electrophoresis, striking qualitative and quantitative differences were seen in the protein composition of the nuclear matrix-intermediate filament fraction obtained from apoptotic cells in comparison with controls. Western blotting analysis of apoptotic nuclear matrix proteins revealed degradation of some (topoisomerase IIalpha, SAF-A) but not other (SATB1 and nucleolin) components. Moreover, immunofluorescent staining for typical matrix antigens (NuMA protein, lamin B, SC-35) showed that in 35-40% of the structures prepared from apoptotic samples, marked changes in the subnuclear distribution of these proteins were present. Striking morphological differences between control and apoptotic samples were also detected at the ultrastructural level. These results demonstrate that both biochemical and morphological changes can be detected in the nuclear matrix prepared from apoptotic HL-60 cells.

Apoptosis↗

PubMedDate not supplied

[Effect of actinomycin D, cycloheximide, puromycin and mitomycin C on the biosynthesis of heat shock proteins in the nuclear matrix of Chinese hamster fibroblasts].

The thermal shock proteins with molecular mass of 86 kD and pI 5.5 as well as of 70 kD and pI 5.2-5.4 were isolated by means of two-dimensional electrophoresis in polyacrylamide gel from nuclear matrix of chinese hamster fibroblasts after heating of the animals. Incorporation of 35S-methionine into the thermal shock proteins was completely inhibited by actinomycin D (2 micrograms/ml), if the antibiotic was added into the cell incubation medium before heating, while the incorporation of methionine was decreased only slightly when the antibiotic was added after heating of the cells. Thus, biosynthesis of mRNA of the thermal shock proteins of 86 and 70 kD in nuclear matrix was induced by means of an increase in temperature during the incubation of the cells. Biosynthesis of the thermal shock proteins in nuclear matrix was distinctly inhibited by cycloheximide (1 microgram/ml) and was not practically inhibited by puromycin (60 micrograms/ml).

Animals↗