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PubMed1998

5-Lipoxygenase expression in Langerhans cells of normal human epidermis.

We studied expression of the 5-lipoxygenase (5-LO) pathway in normal human skin. In situ hybridization revealed a 5-LO mRNA-containing epidermal cell (EC) population that was predominantly located in the midportion of the spinous layer, in outer hair root sheaths, and in the epithelial compartment of sebaceous glands. Examination of skin specimens by immunohistochemistry and of primary ECs by flow cytometry mapped the 5-LO protein exclusively to Langerhans cells (LCs). The LC 5-LO protein was largely found in the nuclear matrix, in nuclear envelopes, and perinuclear regions as indicated by in situ confocal laser scan microscopy. Reverse transcription-PCR and immunoblot analyses of purified primary EC populations further indicated that LCs are major 5-LO expressing cells. Enriched primary LCs were also found to contain 5-LO activating protein (FLAP), leukotriene (LT) C4 synthase, and LTA4 hydrolase. By contrast, 5-LO, FLAP, and LTC4 synthase were undetectable or largely reduced, but LTA4 hydrolase transcripts and protein were identified in ECs depleted of LCs. These data show that naive LCs are major, and possibly the sole, 5-LO pathway expressing cells in the normal human epidermis.

Arachidonate 5-Lipoxygenase↗

PubMed1995

The immunoglobulin heavy-chain matrix-associating regions are bound by Bright: a B cell-specific trans-activator that describes a new DNA-binding protein family.

B lymphocyte-restricted transcription of immunoglobulin heavy-chain (IgH) genes is specified by elements within the variable region (VH) promoter and the intronic enhancer (E mu). The gene encoding a protein that binds a VH promoter proximal site necessary for induced mu-heavy-chain transcription has been cloned. This B-cell specific protein, termed Bright (B cell regulator of IgH transcription), is found in both soluble and matrix insoluble nuclear fractions. Bright binds the minor groove of a restricted ATC sequence that is sufficient for nuclear matrix association. This sequence motif is present in previously described matrix-associating regions (MARs) proximal to the promoter and flanking E mu. Bright can activate E mu-driven transcription by binding these sites, but only when they occur in their natural context and in cell lines permissive for E mu activity. To bind DNA, Bright requires a novel tetramerization domain and a previously undescribed domain that shares identity with several proteins, including SWI1, a component of the SWI/SNF complex.

Amino Acid Sequence↗

PubMed1991

Differential compartmentalization of plasmid DNA microinjected into Xenopus laevis embryos relates to replication efficiency.

Circular plasmid DNA molecules and linear concatemers formed from the same plasmid exhibit strikingly different fates following microinjection into Xenopus laevis embryos. In this report, we prove quantitatively that only a minority of small, circular DNA molecules were replicated (mean = 14%) from fertilization through the blastula stage of development. At all concentrations tested, very few molecules (approximately 1%) underwent more than one round of DNA synthesis within these multiple cell cycles. In addition, unlike endogenous chromatin, the majority of circular templates became resistant to cleavage by micrococcal nuclease. The extent of nuclease resistance was similar for both replicated and unreplicated templates. Sequestration of circular molecules within a membranous compartment (pseudonucleus), rather than the formation of nucleosomes with abnormal size or spacing, apparently conferred the nuclease resistance. In contrast, most linearly concatenated DNA molecules (derived from end-to-end joining of microinjected monomeric plasmid DNA) underwent at least two rounds of DNA replication during this same period. Linear concatemers also exhibited micrococcal nuclease digestion patterns similar to those seen for endogenous chromatin yet, as judged by their failure to persist in later stages of embryogenesis, were likely to be replicated and maintained extrachromosomally. We propose, therefore, that template size and conformation determine the efficiency of replication of microinjected plasmid DNA by directing DNA to a particular compartment within the cell following injection. Template-dependent compartmentalization may result from differential localization within endogenous nuclei versus extranuclear compartments or from supramolecular assembly processes that depend on template configuration (e.g., association with nuclear matrix or nuclear envelope).

Animals↗

PubMed2001

Differential subcellular distribution and transcriptional activity of sigmaE3, sigmaE4, and sigmaE3-4 isoforms of the rat estrogen receptor-alpha.

E3, E4, and E3-4 are naturally occurring estrogen receptor (ER) isoforms, generated through differential splicing of the ERalpha primary transcript and abundantly expressed in embryonic rat pituitary. Studies in COS cells transfected with full-length ERalpha or its three splice variants fused to green fluorescent protein (GFP), revealed a different subcellular localization for each isoform. In the absence of estradiol, full-length ERalpha-GFP was predominantly nuclear, and E3-GFP and E4-GFP were present both in cytoplasm and nucleus, whereas E3-4-GFP was predominantly cytoplasmic. Upon hormone treatment, a dramatic redistribution of full-length ERalpha-GFP and E3-GFP, from a diffuse to punctate pattern, occurred within the nucleus. In contrast, the distribution of E4-GFP and E3-4-GFP was unaffected. Nuclear fractionation studies showed that full-length ER-alpha and E3 displayed the same hormone-induced ability to tether to nuclear matrix, whereas nuclear E4 appeared to remain loosely associated to functional nuclear constituents. When cotransfected with an estrogen-inducible reporter plasmid (VIT-TK-CAT) in ER-negative (CHO k1) and ER-positive pituitary (GH4 C1) cells, E3-4 exhibited a very weak estrogen-dependent transactivation activity, whereas E3 had an inhibitory effect on full-length ER action. Conversely, E4 displayed estrogen-independent transcriptional activity in ER-negative cells, and in ER-positive cells, enhanced the estrogen-induced gene expression as efficiently as full-length ERalpha. In a gel mobility shift assay, phosphorylated E4 was able to form a specific complex with a consensus ERE, while E3 and E3-4 never did bind by themselves. The observed inhibitory action of E3 on estrogen-dependent transcription would rather involve protein-protein interactions such as formation of heterodimers with full-length ERalpha, as suggested by immunoprecipitation followed by Western blotting. These data suggest that E3 and E4 may play a physiologically relevant role as negative or constitutively positive modulators of transcription, in the developing rat pituitary.

Alternative Splicing↗

PubMed1993

[Urothelial tumors and the extracellular matrix].

The network of both intra- and intercellular, either physical (bioconductive connectional system) and/or chemical signals, plays a significant role in the maintenance of tissue architecture and integrity of epithelial cell layers. The basement membrane is not only a static barrier but a dynamic regulator of the urothelium. Any change in the basement membrane can lead, by the extracellular matrix-cytoskeleton-nuclear matrix interaction, to altered gene regulation of the urothelial cells. Abnormal production and deposition or proteolytic degradation of the extracellular matrix components correlate with tumour stage and progression. In some experimental models, tissue inhibitors of metalloproteinases or anti-proteinase antibodies can abrogate the proteolytic activity of those matrix metalloproteinase enzymes (collagenase IV, cathepsin, stromelysin, etc.) which promote tumour invasion. Finally, the current researches investigating the use of biologic protein (e.g., TIMP-1 e-2; agents that affect angiogenesis and spread of neoplasias) are aimed at offer new therapeutic opportunities in oncology.

Basement Membrane↗

PubMed2003

Visualization of chromatin domains created by the gypsy insulator of Drosophila.

Insulators might regulate gene expression by establishing and maintaining the organization of the chromatin fiber within the nucleus. Biochemical fractionation and in situ high salt extraction of lysed cells show that two known protein components of the gypsy insulator are present in the nuclear matrix. Using FISH with DNA probes located between two endogenous Su(Hw) binding sites, we show that the intervening DNA is arranged in a loop, with the two insulators located at the base. Mutations in insulator proteins, subjecting the cells to a brief heat shock, or destruction of the nuclear matrix lead to disruption of the loop. Insertion of an additional gypsy insulator in the center of the loop results in the formation of paired loops through the attachment of the inserted sequences to the nuclear matrix. These results suggest that the gypsy insulator might establish higher-order domains of chromatin structure and regulate nuclear organization by tethering the DNA to the nuclear matrix and creating chromatin loops.

Animals↗

PubMed2005

Dynein light chain rp3 acts as a nuclear matrix-associated transcriptional modulator in a dynein-independent pathway.

Cytoplasmic dynein is a motor protein complex involved in microtubule-based cargo movement. Previous biochemical evidence suggests that dynein light chain subunits also exist outside the dynein complex. Here we show that the dynein light chain rp3 is present in both the cytoplasm and the nucleus. Nuclear rp3 binds to and assembles with the transcription factor SATB1 at nuclear matrix-associated structures. Dynein intermediate chain was also detected in the nucleus, but it was dispensable for the rp3-SATB1 interaction. SATB1 facilitates the nuclear localization of rp3, whereas rp3 and dynein motor activity are not essential for nuclear accumulation of SATB1. The nuclear rp3-SATB1 protein complex is assembled with a DNA element of the matrix attachment region of the Bcl2 gene. Finally, rp3 is involved in SATB1-mediated gene repression of Bcl2. Our data provide evidence that dynein subunit rp3 has functions independent of the dynein motor.

Cell Line↗

PubMed1996

Immunofluorescent analysis of the organization of telomeric DNA sequences and their involvement in chromosomal aberrations in hamster cells.

We have investigated the organization of telomeric TTAGGG)n repeats in the extended DNA loops of chromatin of human and hamster cells by immunofluorescent technique. In humans, telomeric repeats which are predominantly localized at the termini of all the chromosomes, have been found associated with nuclear matrix. This distribution pattern did not alter, even after the removal of 90% of the DNA from the nuclear halos by EcoRI digestion. This suggests that the telomeric sequences are tightly associated with nuclear matrix and hence cannot be solubilized by nucleases. In contrast, in Chinese hamster cells (CHO B11), a major proportion of interstitial telomeric repeats are found in the loop regions, like beads on a string, with attachments to the periphery of the nuclear matrix. Unlike in human cells, EcoRI digestion removed most of the telomeric repeats from the loop regions of Chinese hamster cells. This indicates that intrachromosomal sequences are not associated with nuclear matrix, and this finding has been further substantiated by Southern hybridization of matrix associated and loop DNA fractions of hamster cells with the (TTAGGG)n probe. The organizational differences in the telomeric repeat sequences of Chinese hamster and human cells might be due to their chromosomal location as well as their interaction with nucleoprotein complexes specific for the termini of the eukaryotic chromosomes. Furthermore, the interstitial (TTAGGG)n sequences were found to be more frequently involved in the chromosomal aberrations induced by restriction enzymes. This suggests that the intrachromosomal sites of telomeric sequences behave as hot spots for DNA damage.

Animals↗

PubMed1990

Effect of nonviable preparations from human immunodeficiency virus type 1 on nuclear matrix-associated DNA polymerase alpha and DNA topoisomerase II activities.

In a previous paper, we determined that treatment of lymphocytes with nonviable preparations of human immunodeficiency virus type 1 (HIV-1) results in an impairment of the phosphatidylinositol/protein kinase C pathway, most likely due to an inhibition of the cleavage of phosphatidylinositol bisphosphate into inositol trisphosphate and diacylglycerol, mediated by phospholipase C. Here we show that one consequence of these changes is a reduced phosphorylation of nuclear matrix-associated DNA topoisomerase II, resulting in an inhibition of the activity of this enzyme. Antibodies to the viral proteins suppressed the inhibitory effects caused by the HIV-1 preparation. Furthermore, the phytohemagglutinin A-caused augmentation of nuclear matrix-associated DNA polymerase alpha and beta activities was found to be abolished by coincubation with the HIV preparation or with the HIV-1 gp120. The phytohemagglutinin A-enhanced matrix association and processivity of DNA polymerase alpha was determined to be reduced if the lymphocytes were in contact with HIV-1 preparation. These results suggest that the reduced proliferative response of lymphocytes to phytohemagglutinin A in the presence of disrupted HIV-1 preparation is due to inhibition of at least two, perhaps separate, pathways, one involving protein kinase C resulting in a reduced phosphorylation of DNA topoisomerase II and the other changing the state of matrix association of DNA polymerase alpha and beta.

DNA Polymerase II↗

PubMed1996

Association of poly(ADP-ribose) polymerase with nuclear subfractions catalyzed with sodium tetrathionate and hydrogene peroxide crosslinks.

Poly(ADP-ribose) polymerase (PARP) is a nuclear enzyme which catalyzes the transfer of ADP-ribose units from NAD+ to a variety of nuclear proteins under the stimulation of DNA strand break. To examine its role in DNA repair, we have been studying the interaction of PARP with other nuclear proteins using disulfide cross-linking, initiated by sodium tetrathionate (NaTT). Chinese Hamster Ovary (CHO) cells were extracted sequentially with Nonidet P40 (detergent), nucleases (DNase+RNase), and high salt (1.6 M NaCl) with and without the addition of a sulfhydryl reducing agent. The residual structures are referred to as the nuclear matrix, and are implicated in the organization of DNA repair and replication. Treatment of the cells with NaTT causes the crosslinking of PARP to the nuclear matrix. Activating PARP by pretreating the cells with H2O2 did not increase the cross-linking of PARP with the nuclear matrix, suggesting a lack of additional interaction of the enzyme with the nuclear matrix during DNA repair. Both NaTT and H2O2 induced crosslinks of PARP that were extractable with high salt. To shorten the procedure, these crosslinks were extracted from cells without nucleases and high salt treatment, using phosphate buffer. Using western blotting, these crosslinks appeared as a smear of high molecular weight species including a possible dimer of PARP at 230 kDa, which return to 116 kDa following reduction with beta-mercaptoethanol.

Animals↗

PubMed1994

Receptor-mediated nuclear translocation of growth hormone.

We have previously shown that the growth hormone (GH) receptor-binding protein is associated with the nucleus. We show here both by electron microscopy and nuclear isolation that GH is subject to rapid nuclear translocation. The intracellular fate of intravenously injected 125I-bovine growth hormone (bGH) was examined in the rat hepatocyte by electron microscopic autoradiography. The hormone appeared rapidly at the plasma membrane, then sequentially in lysosomal and multivesicular bodies and/or the nuclear membrane before final translocation to the nuclear matrix. Maximal translocation to the nuclear matrix occurred within 30 min of injection. Nuclear translocation of 125I-hGH was also studied by isolation of nuclei from cells stably transfected with cDNAs encoding the GH receptor, GH-binding protein, and a membrane bound but cytoplasmic domain-deficient receptor. Specific internalization and nuclear translocation of hormone only occurred in cells transfected with the full-length receptor. The translocation was rapid and became saturated within 1 h after addition of hormone to the culture media. SDS-polyacrylamide gel electrophoresis of isolated nuclei showed that GH is transported to the nucleus as the intact molecule. Pretreatment of cells with lysosomotropic agents (chloroquine, ammonium chloride, and bacitracin) decreased hormone degradation and increased nuclear translocation of GH. The nuclear translocation of GH was achieved independent of the cytoskeletal system (microtubular, microfilament, and intermediate filament networks). Thus, GH is subject to rapid receptor-dependent nuclear translocation via the endosomal pathway.

Ammonium Chloride↗

PubMed1985

Analysis of the structure and spatial distribution of ultraviolet-induced DNA repair patches in human cells made in the presence of inhibitors of replicative synthesis.

The repair of ultraviolet-induced damage in the presence of hydroxyurea or hydroxyurea and arabinosylcytosine was investigated in confluent human fibroblasts at the level of DNA loops attached to the nuclear matrix. Estimation of single-strand break frequencies based on the release of DNA from the DNA-nuclear matrix complex after incubation with nuclease S1 revealed the occurrence of multiple incision events per DNA loop in the presence of inhibitors. When both inhibitors were employed, over 90% of the repair-labelled DNA was not ligated within 2 h post-incubation. In the absence of ligation of repair patches, we observed a preferential release of repair-labelled DNA from the nuclear matrix by nuclease S1 compared to prelabelled DNA, regardless of the period of post-UV incubation. The results suggest that repair events are clustered to some extent in a certain area of a DNA loop. However, the position of these clusters relative to the attachment sites of DNA loops at the nuclear matrix is random. The data are discussed in terms of denaturation of a putative repair complex in the presence of hydroxyurea resulting in an excess of incisions over repaired sites.

Cell Line↗

PubMed1996

Kinetics of internalization and subcellular binding sites for T3 in mouse liver.

The intracellular fate of radiolabeled T3 taken up by mice hepatocytes in vivo was determined at specific time intervals (2-120 min) after injection by quantitative electron microscopic radioautography. Injection of a 200-fold excess of unlabeled T3 together with [125I]-T3 resulted in a more than 90% inhibition of radioactivity detected in hepatocytes. A simple grain density (GD) analysis of radioautograms revealed that a specific labeling (GD > 1) was displayed by only five cell compartments: the plasma membrane, lipid droplets, mitochondria, nuclear envelope and nuclear matrix whereas other compartments were not labeled. Labeled compartments showed distinct changes in the pattern of labeling over time: the plasma membrane was labeled only 2 min after T3 injection, whereas labeling of the nuclear envelope was high at 2 min, decreased at 15 min and progressively increased to maximal measured levels at 120 min. After a lag time of 30 min, nuclear matrix labeling increased progressively with time. Mitochondrial labeling was found to be specific at any time point studied but showed no change over time. These ultrastructural data have been confirmed in vitro by the interaction of T3 with plasma membrane, nuclear membrane, nuclear matrix and mitochondria by real-time biospecific interaction analysis in a BIAcore system. These results demonstrate that T3 binds to hepatocytes before internalization, is transported both to mitochondria and to the nuclear envelope and translocated into the nuclear matrix.

Animals↗

PubMedDate not supplied

Distribution of a novel nuclear protein in normal and regenerating liver cells.

Monoclonal antibodies to a rat liver nuclear protein (B2, 68 kda, pI: 6.5-8.2) have been established and characterized to localize the distribution of this antigen in nuclear organization. It was demonstrated that this nuclear protein B2 is associated with actively transcribed nucleosomes and the nuclear matrix as revealed by immunogold labelling. In the regenerating liver cell, the immunogold particles are predominantly in the euchromatin as compared to the resting liver cells which are mainly in nuclear matrix and heterochromatin. It was also demonstrated that the incorporation of 32P into the nuclear protein B2 is increased 6-fold in the actively transcribed nucleosomes as compared to the nuclear matrix, as analyzed by two-dimensional polyacrylamide gel electrophoresis. We hypothesize that this nuclear protein may act as an anchorage point either for actively transcribed RNA or for DNA replication. Once this protein is highly phosphorylated, it dissociates from the nuclear envelope and can then dynamically interact with active nucleosomes within the nucleus.

Animals↗

PubMed2006

Association of functional influenza viral proteins and RNAs with nuclear chromatin and sub-chromatin structure.

Transcription and replication of the influenza virus genome occur in the nucleus. However, the intra-nuclear localization of viral RNP complexes and the function of nuclear domains involved in viral transcription and replication, if any, are not well known. In the present study, we determined the intra-nuclear localization of viral proteins and viral RNAs and the in vitro RNA synthesis activity of viral RNP complexes associated with distinct nuclear fractions prepared from infected nuclei. A majority of viral RNA polymerases and M1 were recovered in DNase-sensitive fractions, whereas some portion of RNA polymerases and approximately 25% of NP were tightly associated with so-called nuclear matrix fractions. The amount of vRNA associated with the nuclear matrix was significantly more than that of cRNA. The in vitro viral RNA synthesis activity was detected in DNase-insensitive fractions, including the nuclear matrix. In contrast, newly synthesized viral RNAs were recovered in the DNase-sensitive fraction. These observations suggest that vRNP complexes are, at least partially, associated with densely packed chromatin, where viral transcription and replication occur, and the newly synthesized vRNP complexes to be transported into the cytoplasm are released into the nucleoplasm.

Animals↗

PubMed1998

Interrelationships of nuclear structure and transcriptional control: functional consequences of being in the right place at the right time.

Functional interrelationships between components of nuclear architecture and control of gene expression are becoming increasingly evident. In this article we focus on the concept that association of genes and cognate transcription factors with the nuclear matrix may support the formation and/or activities of nuclear domains that facilitate transcriptional regulation. Several lines of evidence are consistent with the concept that association of transcription factors with the nuclear matrix may be obligatory for fidelity of gene expression and maximal transcriptional activity. The identification of specific regions of transcription factors that are responsible for intranuclear trafficking to nuclear matrix-associated sites that support transcription, reinforces the linkage of nuclear structure to regulation of genes. CBFA2/AML-1 and CBFA1/AML-3 provide paradigms for directing gene regulatory factors to RNA polymerase II containing foci within the nucleus. The implications of modifications in the intranuclear trafficking of transcription factors for developmental and tissue-specific control, as well as for aberrations in gene expression that are associated with cancer and neurological disorders, are addressed.

Amino Acid Sequence↗

PubMedDate not supplied

[Illegitimate recombination as a possible mechanism of DNA-topoisomerase II induced chromosomal rearrangements].

Using semi-quantitative PCR-based approach, we have shown that the breakpoint cluster region of the AML1 gene was associated with the nuclear matrix. We have demonstrated that inhibition of topoisomerase II by etoposide stimulates the appearance of histone gammaH2AX foci, an indicator for the presence of DNA double-strand breaks. Furthermore, the major part of these foci was associated with the nuclear matrix. We also visualized nuclear matrix--associated multiprotein complexes involved in topoisomerase II--induced DNA double-strand break repair. Colocalization studies have demonstrated that these complexes included the principal components of the non-homologous end joining repair system (Ku80, DNA-PKcs and DNA ligase IV). Thus, it is reasonable to suggest that the non-homologous DNA end joining is a possible mechanism of topoisomerase II--induced chromosomal rearrangements.

Antigens, Nuclear↗

PubMed2000

Further evidence that sperm nuclear proteins are necessary for embryogenesis.

We have recently presented evidence that the structural integrity of the mouse sperm nuclear matrix may be necessary for the proper unpackaging of sperm DNA for participation in embryogenesis. It is likely that the sperm nuclear matrix contributes to the organisation of the sperm DNA and its disturbance can seriously damage the paternal genome or its expression. In this work, we confirm our previous data and further suggest that even very subtle changes in the sperm nuclear structure may have a significant impact on embryo development. As reported previously, dithiothreitol (DTT) in the presence of an ionic detergent, ATAB, destabilized the nuclear matrix as measured by the halo assay, and oocytes injected with these nuclei failed to develop. We also discovered that omitting the protease inhibitor PMSF from the buffers used to extract spermatozoa prevented sperm injected into oocytes from participating in development. The organization of DNA into loop domains by the nuclear matrix in these nuclei appeared normal, as measured by the halo assay. Oocytes injected with sperm nuclei that had been washed with ATAB in the presence of phenylmethylsulphonyl fluoride (PMSF) but in the absence of DTT resulted in live births. Neither DTT treatment nor the absence of PMSF would be expected to disrupt the integrity of the paternal DNA. The data therefore suggest that even very subtle alterations in the structural proteins of the nucleus are enough to deprive sperm DNA of the ability to contribute to embryonic development.

Animals↗