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PubMed2004

A-type nuclear lamins, progerias and other degenerative disorders.

Nuclear lamins were identified as core nuclear matrix constituents over 20 years ago. They have been ascribed structural roles such as maintaining nuclear integrity and assisting in nuclear envelope formation after mitosis, and have also been linked to nuclear activities including DNA replication and transcription. Recently, A-type lamin mutations have been linked to a variety of rare human diseases including muscular dystrophy, lipodystrophy, cardiomyopathy, neuropathy and progeroid syndromes (collectively termed laminopathies). Most diseases arise from dominant, missense mutations, leading to speculation as to how different mutations in the same gene can give rise to such a diverse set of diseases, some of which share little phenotypic overlap. Understanding the cellular dysfunctions that lead to laminopathies will almost certainly provide insight into specific roles of A-type lamins in nuclear organization. Here, we compare and contrast the LMNA mutations leading to laminopathies with emphasis on progerias, and discuss possible functional roles for A-type lamins in the maintenance of healthy tissues.

Aging↗
PubMed2001

Matrix attachment region-dependent function of the immunoglobulin mu enhancer involves histone acetylation at a distance without changes in enhancer occupancy.

Nuclear matrix attachment regions (MARs), which flank the immunoglobulin mu heavy-chain enhancer on either side, are required for the activation of the distal variable-region (V(H)) promoter in transgenic mice. Previously, we have shown that the MARs extend a local domain of chromatin accessibility at the mu enhancer to more distal sites. In this report, we examine the influence of MARs on the formation of a nucleoprotein complex at the enhancer and on the acetylation of histones, which have both been implicated in contributing to chromatin accessibility. By in vivo footprint analysis of transgenic mu gene constructs, we show that the occupancy of factor-binding sites at the mu enhancer is similar in transcriptionally active wild-type and transcriptionally inactive DeltaMAR genes. Chromatin immunoprecipitation experiments indicate, however, that the acetylation of histones at enhancer-distal nucleosomes is enhanced 10-fold in the presence of MARs, whereas the levels of histone acetylation at enhancer-proximal nucleosomes are similar for wild-type and DeltaMAR genes. Taken together, these data indicate that the function of MARs in mediating long-range chromatin accessibility and transcriptional activation of the V(H) promoter involves the generation of an extended domain of histone acetylation, independent of changes in the occupancy of the mu enhancer.

Acetylation↗
PubMed1999

Conserved characteristics of heterochromatin-forming DNA at the 15q11-q13 imprinting center.

Nuclear matrix binding assays (NMBAs) define certain DNA sequences as matrix attachment regions (MARs), which often have cis-acting epigenetic regulatory functions. We used NMBAs to analyze the functionally important 15q11-q13 imprinting center (IC). We find that the IC is composed of an unusually high density of MARs, located in close proximity to the germ line elements that are proposed to direct imprint switching in this region. Moreover, we find that the organization of MARs is the same at the homologous mouse locus, despite extensive divergence of DNA sequence. MARs of this size are not usually associated with genes but rather with heterochromatin-forming areas of the genome. In contrast, the 15q11-q13 region contains multiple transcribed genes and is unusual for being subject to genomic imprinting, causing the maternal chromosome to be more transcriptionally silent, methylated, and late replicating than the paternal chromosome. We suggest that the extensive MAR sequences at the IC are organized as heterochromatin during oogenesis, an organization disrupted during spermatogenesis. Consistent with this model, multicolor fluorescence in situ hybridization to halo nuclei demonstrates a strong matrix association of the maternal IC, whereas the paternal IC is more decondensed, extending into the nuclear halo. This model also provides a mechanism for spreading of the imprinting signal, because heterochromatin at the IC on the maternal chromosome may exert a suppressive position effect in cis. We propose that the germ line elements at the 15q11-q13 IC mediate their effects through the candidate heterochromatin-forming DNA identified in this study.

Amino Acid Motifs↗
PubMedDate not supplied

A new look at the cellular scaffold by embedment-free electron microscopy method.

The basic scaffold of most cells is afforded by the cytoskeleton (comprising microfilaments, intermediate filaments and the microtubules). The conventional methods of electron microscopy fail to visualize filamentous cell structure. They can show only these filaments lying at the section surface. Heavy metal staining (I), and the optical properties of the resins used for embedding are similar to those of proteins hence most proteinaceous structures remain unresolved and the cytoplasm seems to be quite homogenous (II). Aldehyde fixation could cross-link proteins and lead to the emergence of artificial structures (III). These limitations may be overcome by the use of the embedment-free electron microscopy (EF-EM). This technique present cellular scaffold as a purified, isolated, three-dimensional network with various thickness of filaments. Our study on the dynamic aspect of cellular scaffold indicate that the thickness and arrangement of filaments depend on cell type and both physiological or pathological environments. Thank also to the adaptation of immunocytochemistry to EF-EM it was possible to understand the nuclear matrix and cytomatrix structure in relation to function. Thus, combination these methods revealed findings suggesting the nuclear homing of proapoptotic proteins and their association with intermediate filaments.

Animals↗
PubMed2003

Interaction in vitro of type III intermediate filament proteins with Z-DNA and B-Z-DNA junctions.

The selection of DNA fragments containing simple d(GT)(n) and composite d(GT)(m). d(GA)(n) microsatellites during affinity binding of mouse genomic DNA to type III cytoplasmic intermediate filaments (cIFs) in vitro, and the detection of such repeats, often as parts of nuclear matrix attachment region (MAR)-like DNA, in SDS-stable DNA-vimentin crosslinkage products isolated from intact fibroblasts, prompted a detailed study of the interaction of type III cIF proteins with left-handed Z-DNA formed from d(GT)(17) and d(CG)(17) repeats under the topological tension of negatively supercoiled plasmids. Although d(GT)(n) tracts possess a distinctly lower Z-DNA-forming potential than d(CG)(n) tracts, the filament proteins produced a stronger electrophoretic mobility shift with a plasmid carrying a d(GT)(17) insert than with plasmids containing different d(CG)(n) inserts, consistent with the facts that the B-Z transition of d(GT)(n) repeats requires a higher negative superhelical density than that of d(CG)(n) repeats and the affinity of cIF proteins for plasmid DNA increases with its superhelical tension. That both types of dinucleotide repeat had indeed undergone B-Z transition was confirmed by S1 nuclease and chemical footprinting analysis of the plasmids, which also demonstrated efficient protection by cIF proteins from nucleolytic and chemical attack of the Z-DNA helices as such, as well as of the flanking B-Z junctions. The analysis also revealed sensibilization of nucleotides in the center of one of the two strands of a perfect d(CG)(17) insert toward S1 nuclease, indicating cIF protein-induced bending of the repeat. In all these assays, vimentin and glial fibrillary acidic protein (GFAP) showed comparable activities, versus desmin, which was almost inactive. In addition, vimentin and GFAP exhibited much higher affinities for the Z-DNA conformation of brominated, linear d(CG)(25) repeats than for the B-DNA configuration of the unmodified oligonucleotides. While double-stranded DNA was incapable of chasing the Z-DNA from its protein complexes, and Holliday junction and single-stranded (ss)DNA were distinguished by reasonable competitiveness, phosphatidylinositol (PI) and, particularly, phosphatidylinositol 4,5-diphosphate (PIP(2)) turned out to be extremely potent competitors. Because PIP(2) is an important member of the nuclear PI signal transduction cascade, it might exert a regulatory influence on the binding of cIF proteins to Z- and other DNA conformations. From this interaction of cIF proteins with Z- and bent DNA and their previously detected affinities for MAR-like, ss, triple helical, and four-way junction DNA, it may be concluded that the filament proteins play a general role in such nuclear matrix-associated processes as DNA replication, recombination, repair, and transcription.

Animals↗
PubMed1983

Dynamic domains of DNA polymerase alpha in regenerating rat liver.

Intranuclear redistributions of DNA polymerase alpha activity (the presumptive replicative enzyme) are observed preceding and during in vivo replication in regenerating rat liver. The most marked induction of in vitro enzyme activity occurs coincident with the onset of in vivo replication and is seen in the high salt resistant domain of the nuclear matrix. Moreover, DNA polymerase alpha endogenous and exogenous template activities begin to redistribute prereplicatively from bulk chromatin directly to the high salt resistant matrix. These prereplicative subnuclear events are detectable prior to the induction of total cellular DNA polymerase alpha and appear to continue throughout replication. Maximal percent recovery of the total nuclear activity on the high salt resistant matrix (approximately 30%) occurs coincident with and during the time when in vivo replication rates are maximum. At the conclusion of replication, DNA polymerase alpha activities shift back into the bulk chromatin and are no longer high salt resistant. In contrast, DNA polymerase beta (presumptive repair enzyme) endogenous and exogenous template activities are randomly distributed in the nuclear subfractions through the proliferative response. We conclude that DNA polymerase alpha is dynamically assembled or activated on the nuclear matrix and that this process is initiated before the onset of in vivo replication.

Animals↗
PubMed1995

Immunocytochemical detection of phosphatidylinositol 4,5-bisphosphate localization sites within the nucleus.

Phosphatidylinositol 4,5-bisphosphate (PIP2) is a key element of signal transduction, being the preferential substrate of specific phospholipases that produce second messengers such as inositol trisphosphate (IP3) and diacylglycerol (DG). Because PIP2 has been cytochemically identified by monoclonal antibodies not only in the cytoplasmic membranes but also in the nuclear envelope and within the nucleus, we performed a study by immunoblotting and by confocal and electron microscopic immunocytochemistry to identify the nuclear sites of PIP2 localization and to exclude any cross-reactivity of the antibody with other nuclear molecules. The results confirm the specificity of the immunolabeling and indicate that PIP2 is localized at precise intranuclear sites both in in situ and in isolated nuclei. They also show that a significant amount of the phospholipid is retained by the cytoskeleton and by the inner nuclear matrix in in situ matrix preparations. Moreover the sensitivity of the immunocytochemical reaction is capable of detecting quantitative variations of PIP2 nuclear content induced by agonists that modulate the signal transduction system at the nuclear level.

3T3 Cells↗
PubMed1988

Plasmid associations with residual nuclear structures in Saccharomyces cerevisiae.

Acentric yeast plasmids are mitotically unstable, apparently because they cannot freely diffuse after replicating and therefore are not included in the daughter nucleus. This behavior could result if plasmids remain attached to structural elements of the nucleus after replicating. Since DNA replication is believed to take place on the nuclear matrix, we tested whether there was a correlation between the mitotic stability of a given plasmid and the extent to which it was found associated with residual nuclear structures. Residual nuclei were prepared from yeast nuclei by extraction with either high salt, 2 M NaCl, or low salt, 10 mM lithium diiodosalicylate (LIS). Hybridization analysis was used to estimate the fraction of plasmid molecules remaining after nuclei were extracted. We examined the extent of matrix association of three ARS1 plasmids, Trp1-RI circle (1.45 kb), YRp7 (5.7 kb) and p lambda BAT (45.1 kb) with mitotic loss rates ranging from 3-25%. In addition we examined the matrix binding of the endogenous 2 micron plasmid and the 2 micron-derived YEp13 which is relatively stable in the presence of 2 micron and less stable in cir degree strains. Among the ARS1 plasmids we observed a negative correlation between stability and matrix association, consistent with models in which binding to the nuclear matrix prevents passive segregation of ARS1 plasmid molecules. No such correlation was observed among the 2 micron plasmids. Among all plasmids examined there is a positive correlation between size and matrix association.

Cell Nucleus↗
PubMed2004

Gene positional changes relative to the nuclear substructure during carbon tetrachloride-induced hepatic fibrosis in rats.

In the interphase nucleus the DNA of higher eukaryotes is organized in loops anchored to a substructure known as the nuclear matrix (NM). The topological relationship between gene sequences located in the DNA loops and the NM appears to be very important for nuclear physiology because processes such as replication, transcription, and processing of primary transcripts occur at macromolecular complexes located at discrete sites upon the NM. Mammalian hepatocytes rarely divide but preserve a proliferating capacity that is displayed in vivo after specific stimulus. We have previously shown that transient changes in the relative position of specific genes to the NM occur during the process of liver regeneration after partial ablation of the liver, but also that such changes correlate with the replicating status of the cells. Moreover, since chronic exposure to carbon tetrachloride (CCl4) leads to bouts of hepatocyte damage and regeneration, and eventually to non-reversible liver fibrosis in the rat, we used this animal model in order to explore if genes that show differential activity in the liver change or modify their relative position to the NM during the process of liver fibrosis induction. We found that changes in the relative position of specific genes to the NM occur during the chronic administration of CCl4, but also that such changes correlate with the proliferating status of the hepatocytes that goes from quiescence to regeneration to replicative senescence along the course of CCl4-induced liver fibrosis, indicating that specific configurations in the higher-order DNA structure underlie the stages of progression towards liver fibrosis.

Albumins↗
PubMed1996

Construction of a chromosome specific library of human MARs and mapping of matrix attachment regions on human chromosome 19.

Using a novel procedure a representative human chromosome 19-specific library was constructed of short sequences, which bind preferentially to the nuclear matrix (matrix attachment regions, or MARs). Judging by 20 clones sequenced so far, the library contains > 50% of human inserts, about 90% of which are matrix-binding by the in vitro test. Computer analysis of sequences of eight human MARs did not reveal any significant homologies with the EMBL Nucleotide Data Base entries as well as between MARs themselves. Eight MARs were assigned to individual positions on the chromosome 19 physical map. The library constructed can serve as a good source of MAR sequences for comparative analysis and classification and for further chromosome mapping of MARs as well.

Base Sequence↗
PubMed1989

RNA metabolism in nuclei: adenovirus and heat shock alter intranuclear RNA compartmentalization.

Nuclear RNA compartmentalization has received little attention as an area where regulation of gene expression could occur. RNA transcription and processing occur in association with the nuclear matrix, a salt-insoluble proteinaceous network that fills the nuclear space and is contiguous with the peripheral lamina and pore complexes. Described here are experiments that determine the fate of nuclear RNA after it has completed these matrix-associated maturation steps. Continuous label experiments indicate that after nuclear RNA is processed it changes its state of attachment in the nucleus so that it is now removed from the nucleus in the high salt extraction step of matrix isolation. It is this salt-extractable RNA that will be transported to the cytoplasm. Late in adenovirus infection and following heat shock, when transport of cellular RNA is decreased, cellular transcripts do not make the transition from the matrix-associated to the salt-extractable nuclear pool. The implication of these data for the regulation of gene expression is discussed.

Adenoviruses, Human↗
PubMed2000

A matrix attachment region is located upstream from the high-molecular-weight glutenin gene Bx7 in wheat (Triticum aestivum L.).

A 2.2-kb nucleotide sequence rich in AT, located upstream from the Bx7 allele of the high-molecular-weight glutenin Glu-B1 locus in wheat (Triticum aestivum cv. Glenlea) was cloned following amplification by PCR. The 5' region of this sequence contains motifs typically found in matrix attachment regions (MARs) in other plants. We have shown that part of the 2.2-kb DNA binds to wheat nuclear matrix (NM) in vitro, at least as strongly as a known MAR (Adh1) from maize suggesting that there is a MAR upstream of Bx7. This MAR is approximately 800 bases in length running from -750 to -1560 bases, relative to the start codon. Although the MAR is associated with a tissue-specific gene and is beside a strong tissue-specific promoter, the MAR sequence did not lead to tissue-specific expression of the beta-glucuronidase marker gene under the control of the rice actin promoter in various tissues. Presence of the MAR was only slightly beneficial with respect to expression levels, which were not greatly altered in transient expression assays in various wheat tissues although a slight increase in the number of foci was observed in leaves, which have low transformation efficiencies.

Chromatin↗
PubMed2006

Role of NuMA in vertebrate cells: review of an intriguing multifunctional protein.

The 236 kDa large coiled-coil protein NuMA plays diverse important roles in vertebrate cells. It is an important component of the nuclear matrix in interphase cells, and is possibly involved in nuclear re-assembly after mitosis. In dividing cells, upon phosphorylation, NuMA disperses into the cytoplasm, associates with cytoplasmic dynein/dynactin to form a complex, and translocates along microtubules to the spindle poles where it organizes and tethers microtubules to spindle poles. It is thought that the stable complex of NuMA/dynein/dynactin is needed to focus microtubule minus ends to the spindle poles. But, it has also been reported that NuMA can organize microtubules in the absence of centrosomes and dynein. Another hypothesis suggests that once localized to the spindle poles, spindle-associated NuMA's exchange with cytoplasmic soluble pools and its stable crosslinking with the microtubule fibers are independent of dynein/dyactin. NuMA's function in spindle microtubule organization is regulated by RanGTP and Pins-related protein LGN. NuMA becomes dephosphorylated, loses its association with dynein/dynactin, and releases from spindle poles after anaphase onset to allow spindle disassembly and reformation of interphase daughter nuclei. The cell-cycle-dependent phosphorylation of NuMA is regulated by the balanced activities of protein kinases and phosphatases. It has been shown that phosphorylation of NuMA by cyclin B/cdc2 kinase allows NuMA to release from the nucleus and to associate with centrosomes and/or microtubules at the spindle poles, while NuMA's dephosphorylation due to the cyclin B degradation allows NuMA to dissociate from the spindle poles after anaphase onset. Overexpression of NuMA interferes with spindle-associated dynein localization and promotes multipolar spindle formation and cancer. On the other hand, NuMA is absent in many kinds of non-proliferating cells and highly differentiated cells. NuMA also functions during meiotic spindle organization in male and female germ cells. Degradation of NuMA results in the breakdown of normal nuclear structure, and has been used as a marker of cell apoptosis. The implications of NuMA protein in somatic cell animal cloning by nuclear transfer are discussed.

Active Transport, Cell Nucleus↗
PubMed2005

Functional isolation of novel nuclear proteins showing a variety of subnuclear localizations.

Nuclear proteins play key roles in the fundamental regulation of genome instability, the phases of organ development, and physiological responsiveness through gene expression. Although nuclear proteins have been shown to account for approximately one-fourth of total proteins in yeast, no efficient method to identify novel nuclear proteins has been applied to plants. In this study, a trial to isolate nuclear proteins in rice was attempted, and several novel nuclear proteins showing a variety of subnuclear localizations were identified. The nuclear transportation trap (NTT) system, which is a modified two-hybrid system, isolated many nuclear proteins from rice (Oryza sativa) NTT cDNA libraries. Nuclear localization of the isolated proteins was confirmed by transient introduction of green fluorescent protein fusion constructs for a subset of protein genes into onion (Allium cepa) cells. The majority of these proteins, including novel proteins and proteins initially categorized as cytoplasmic proteins, were revealed to be localized in the nucleus. Detailed characterization of unknown proteins revealed various subnuclear localizations, indicating their possible association with chromatin and the nuclear matrix with a foci or speckle-like distribution. Some also showed dual distribution in the nucleus and cytoplasm. In the novel protein fraction, a protein was further identified for its chromatin-associated localization in a specific organ of rice by immunostaining. Thus, a variety of novel nuclear architectural proteins with chromatin or matrix associating abilities, which are important in nuclear organization by influencing certain organ developments or cell responsiveness, can be isolated using the NTT method. Because nuclear proteins other than transcription regulators have rarely been characterized in plants, such as matrix proteins and development-specific chromatin proteins, their identification and subsequent characterization could provide important information for genome-wide regulatory mechanisms controlled by nuclear organization.

Cell Nucleus↗
PubMed1990

Antinuclear matrix antibody. Hidden antinuclear antibody in patients with connective tissue diseases.

A total of 435 serum samples from patients with different rheumatic diseases were screened for the presence of autoantibody to nuclear matrix components by indirect immunofluorescence on 0.1 mol/L HCl extracted HEp-2 cell and WiL2 cell substrates. A total of 28 specimens were positive in this assay. Eighteen of them were from patients with systemic lupus erythematosus (18 of 250), 2 from patients with rheumatoid arthritis (2 of 115), and 8 from patients with mixed connective tissue disease (8 of 10). Antigenic material for this antibody is resistant to DNase, partially sensitive to RNase, and sensitive to trypsin. This indicates that the antigen is composed of protein and possibly RNA. In immunoblot analysis, sera positive for this antibody in indirect immunofluorescence assay recognized different peptides. This suggests that protein peptides are the major antigenic material.

Antibodies, Antinuclear↗
PubMed2003

The matrix attachment region in the Chinese hamster dihydrofolate reductase origin of replication may be required for local chromatid separation.

Centered in the Chinese hamster dihydrofolate reductase origin of replication is a prominent nuclear matrix attachment region (MAR). Indirect lines of evidence suggested that this MAR might be required for origin activation in early S phase. To test this possibility, we have deleted the MAR from a Chinese hamster ovary variant harboring a single copy of the dihydrofolate reductase locus. However, 2D gel replicon mapping shows that removal of the MAR has no significant effect either on the frequency or timing of initiation in this locus. Rather, fluorescence in situ hybridization studies on cells swollen under either neutral or alkaline conditions show that deletion of the MAR interferes with local separation of daughter chromatids. This surprising result provides direct genetic evidence that at least a subset of MARs performs an important biological function, possibly related to chromatid cohesion and separation.

Animals↗
PubMed1996

Subdomain structure of the matrix attachment region located within the mouse immunoglobulin kappa gene intron.

Using the matrix attachment region (MAR) derived from Ig kappa gene intron, we assessed the importance of internal subregions required for the specific binding to the nuclear matrix. Relative affinities of MAR subfragments were compared in an in vitro binding reaction with isolated matrix. Cleavage at the near-centric MboII site generated two subfragments retaining a significant binding affinity. Dimerization of these subfragments greatly increased the affinity. Only a partial segment (130 bp) of the 3' fragment was necessary to restore the binding. The dimerization effect was lost when the monomer units were separated by nonMAR spacers of 500 bp <. This bipartite organization of Ig kappa MAR could be a general feature of AT-rich MARs, regardless of their genomic locations.

Animals↗
PubMed1989

The adenovirus E1B 55 kd protein influences mRNA transport via an intranuclear effect on RNA metabolism.

The adenovirus type 5 early region 1B encodes a 55 kd polypeptide that functions after transcription and processing to facilitate cytoplasmic accumulation of late viral mRNAs during lytic infection. A virus, dl338, carrying a deletion within the coding region for the 55 kd product, was found to be cold-sensitive for growth. Accumulation of late viral mRNAs was more severely inhibited at 32 than at 37 degrees C in dl338-infected cells. The metabolism of late viral transcripts was analysed within the nucleus of dl338-infected cells at 32 degrees C. Late viral mRNAs failed to accumulate efficiently within a nuclear compartment defined by specific RNA extraction conditions. Normally, RNA accumulated within this operationally defined compartment after leaving the nuclear matrix and before associating with the nuclear envelope. These results indicate that the 55 kd polypeptide encoded by early region 1B facilitates an intranuclear step in the metabolism of viral mRNAs, resulting in improved transport of these RNAs to the cytoplasm.

Adenovirus Early Proteins↗