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PubMed1995

Xenopus lamin B3 has a direct role in the assembly of a replication competent nucleus: evidence from cell-free egg extracts.

Xenopus egg extracts which assemble replication competent nuclei in vitro were depleted of lamin B3 using monoclonal antibody L6 5D5 linked to paramagnetic beads. After depletion, the extracts were still capable of assembling nuclei around demembranated sperm heads. Using field emission in lens scanning electron microscopy (FEISEM) we show that most nuclei assembled in lamin B3-depleted extracts have continuous nuclear envelopes and well formed nuclear pores. However, several consistent differences were observed. Most nuclei were small and only attained diameters which were half the size of controls. In a small number of nuclei, nuclear pore baskets, normally present on the inner aspect of the nuclear envelope, appeared on its outer surface. Finally, the assembly of nuclear pores was slower in lamin B3-depleted extracts, indicating a slower overall rate of nuclear envelope assembly. The results of FEISEM were confirmed using conventional TEM thin sections, where again the majority of nuclei assembled in lamin B3-depleted extracts had well formed double unit membranes containing a high density of nuclear pores. Since nuclear envelope assembly was mostly normal but slow in these nuclei, the lamin content of 'depleted' extracts was investigated. While lamin B3 was recovered efficiently from cytosolic and membrane fractions by our procedure, a second minor lamin isoform, which has characteristics similar to those of the somatic lamin B2, remained in the extract. Thus it is likely that this lamin is necessary for nuclear envelope assembly. However, while lamin B2 did not co-precipitate with lamin B3 during immunodepletion experiments, several protein species did specifically associate with lamin B3 on paramagnetic immunobeads. The major protein species associated with lamin B3 migrated with molecular masses of 102 kDa and 57 kDa, respectively, on one-dimensional polyacrylamide gels. On two-dimensional O'Farrell gels the mobility of the 102 kDa protein was identical to the mobility of a major nuclear matrix protein, indicating a specific association between lamin B3 and other nuclear matrix proteins. Nuclei assembled in lamin B3-depleted extracts did not assemble a lamina, judged by indirect immunofluorescence, and failed to initiate semi-conservative DNA replication. However, by reinoculating depleted extracts with purified lamin B3, nuclear lamina assembly and DNA replication could both be rescued. Thus it seems likely that the inability of lamin-depleted extracts to assemble a replication competent nucleus is a direct consequence of a failure to assemble a lamina.

Animals↗
PubMed2004

Localization of the domains in Runx transcription factors required for the repression of CD4 in thymocytes.

The runt family transcription factors Runx1 and Runx3 are expressed in developing murine thymocytes. We show that enforced expression of full-length Runx1 in CD4(-)CD8(-) thymocytes results in a profound suppression of immature CD4/CD8 double-positive thymocytes and mature CD4 single-positive thymocytes compared with controls. This effect arises from Runx1- or Runx3-mediated repression of CD4 expression, and is independent of positively selecting signals. Runx1 is able to repress CD4 in CD4/CD8 double-positive thymocytes, but not in mature splenic T cells. Runx-mediated CD4 repression is independent of association with the corepressors Groucho/TLE or Sin3. Two domains are required for complete Runx-mediated CD4 repression. These are contained within Runx1 aa 212-262 and 263-360. The latter region contains the nuclear matrix targeting sequence, which is highly conserved among runt family transcription factors across species. The presence of the nuclear matrix targeting sequence is required for Runx-mediated CD4 repression, suggesting that Runx transcription factors are stabilized on the CD4 silencer via association with the nuclear matrix.

Amino Acid Sequence↗
PubMed1997

The matrix attachment region-binding protein SATB1 participates in negative regulation of tissue-specific gene expression.

The nuclear matrix has been implicated in several cellular processes, including DNA replication, transcription, and RNA processing. In particular, transcriptional regulation is believed to be accomplished by binding of chromatin loops to the nuclear matrix and by the concentration of specific transcription factors near these matrix attachment regions (MARs). A number of MAR-binding proteins have been identified, but few have been directly linked to tissue-specific transcription. Recently, we have identified two cellular protein complexes (NBP and UBP) that bind to a region of the mouse mammary tumor virus (MMTV) long terminal repeat (LTR) previously shown to contain at least two negative regulatory elements (NREs) termed the promoter-proximal and promoter-distal NREs. These NREs are absent from MMTV strains that cause T-cell lymphomas instead of mammary carcinomas. We show here that NBP binds to a 22-bp sequence containing an imperfect inverted repeat in the promoter-proximal NRE. Previous data showed that a mutation (p924) within the inverted repeat elevated basal transcription from the MMTV promoter and destabilized the binding of NBP, but not UBP, to the proximal NRE. By using conventional and affinity methods to purify NBP from rat thymic nuclear extracts, we obtained a single major protein of 115 kDa that was identified by protease digestion and partial sequencing analysis as the nuclear matrix-binding protein special AT-rich sequence-binding protein 1 (SATB1). Antibody ablation, distamycin inhibition of binding, renaturation and competition experiments, and tissue distribution data all confirmed that the NBP complex contained SATB1. Similar types of experiments were used to show that the UBP complex contained the homeodomain protein Cux/CDP that binds the MAR of the intronic heavy-chain immunoglobulin enhancer. By using the p924 mutation within the MMTV LTR upstream of the chloramphenicol acetyltransferase gene, we generated two strains of transgenic mice that had a dramatic elevation of reporter gene expression in lymphoid tissues compared with reporter gene expression in mice expressing wild-type LTR constructs. Thus, the 924 mutation in the SATB1-binding site dramatically elevated MMTV transcription in lymphoid tissues. These results and the ability of the proximal NRE in the MMTV LTR to bind to the nuclear matrix clearly demonstrate the role of MAR-binding proteins in tissue-specific gene regulation and in MMTV-induced oncogenesis.

Amino Acid Sequence↗
PubMed1987

Superoxide radical-induced loss of nuclear restriction of immature mRNA: a possible cause for ageing.

In eucaryotic cells, immature mRNA is normally restricted to the nucleus, where it is posttranscriptionally processed to mature mRNA. The intranuclear binding site for both the immature and mature mRNA is thought to be the nuclear matrix which serves as a platform for posttranscriptional RNA maturation and transport. The selectivity of nucleocytoplasmic transport for mature mRNA species seems to be due to the selectivity of the ATP-caused release of mature mRNA from the nuclear matrix; the attachment of immature mRNA to the matrix is not altered in the presence of this nucleotide. Here we show that in the presence of superoxide radical anions (O2-), which are very likely one of the causative factors in ageing, the selection mechanism for mature mRNA at the level of nuclear matrix attachment is disturbed. In the presence of a superoxide radical-generating system (xanthine/xanthine oxidase), both the mature ovalbumin mRNA and the immature ovalbumin mRNA precursors were found to be released from the nuclear matrix of hen oviduct cells, in the absence as well as in the presence of ATP. This result was also obtained when whole, isolated nuclei preincubated with xanthine/xanthine oxidase were used. The superoxide radical-caused effect could be partially prevented by co-addition of superoxide dismutase (SOD) which dismutates O2- to H2O2 and O2. On the other hand, in the presence of antibodies against the SOD, the effect of superoxide anions on RNA-matrix attachment was enhanced and its inhibition by SOD was abolished. Our results suggest that cellular ageing may be partially caused by superoxide radical-induced release of immature mRNA from its intranuclear binding site resulting in the appearance of immature messengers in the cytoplasm. This may cause both qualitative and quantitative changes in protein synthesis. Thus, ageing may be associated not only with the expression of genes coding for proteins not characteristic for the proper state of differentiation of a given cell (as suggested by the dysdifferentiative hypothesis of ageing) but also with impaired maturation of the primary gene transcripts due to the interference of superoxide radicals, not sufficiently eliminated by antioxidant mechanisms with age, with RNA-matrix attachment.

Adenosine Triphosphate↗
PubMed1990

Failure of differentiation of the nuclear-perinuclear skeletal complex in the round-headed human spermatozoa.

Acrosomeless round-headed spermatozoa from three men were studied under electron microscopy and indirect immunofluorescene microscopy using the anti-calicin antibody that recognizes a basic protein of the sperm perinuclear theca (Longo et al., 1987). Electron microscopy revealed the existence of anomalies of the nuclear envelope, the nuclear matrix underlying the nuclear envelope, and the perinuclear layer. The absence of sperm labeling with the anti-calicin antibody confirmed that the formation of the perinuclear theca was impaired. Data obtained from both mature spermatozoa and ejaculated spermatids suggest that i) round-headed sperm head anomalies result from a failure of differentiation of the sperm-specific skeletal complex related to the nucleus, and ii) the acrosome spreading over the nucleus, the nuclear elongation and the post-acrosomal sheath formation are dependent on such nuclear-perinuclear differentiations. In contrast, chromatin condensation, cytokinesis and some events of the acrosomal shaping appear not to depend on those nuclear-related differentiations. The possible processes allowing the maintenance of the sperm head structures and their subsequent morphogenesis are discussed.

Acrosome↗
PubMedDate not supplied

[Age-dependent changes in mRNA transport (nucleus-cytoplasm)].

Transport of mRNA from nucleus to cytoplasm is an ATP-dependent process which occurs strictly vectorially. Because the mRNA is structurally bound during transport, mRNA transport is a "solid-state" process consisting of i) mRNA release from the nuclear matrix, ii) mRNA translocation through the nuclear pore, and iii) cytoskeletal binding. We identified and purified the following components involved in the translocation step: i) the nuclear envelope (NE) nucleoside triphosphatase (NTPase) which is stimulated by the 3'poly(A) tail of mRNA, ii) the poly(A)-recognizing mRNA carrier, iii) the NE protein kinase, and iv) the NE phosphatase. In addition, we found that an RNA helicase activity is present in NE, which also may be involved in RNA transport. Our results show that, besides poly(A), also double-stranded RNA structures may modulate RNA export. The amount of mRNA released from nuclei markedly decreases with age. Evidence is presented that this age-dependent change is caused by an impairment of polyadenylation of mRNA, hnRNA processing, release of mRNA from nuclear matrix, and translocations of mRNA from nuclear to cytoplasmic compartment (decrease in activities of NE NTPase, protein kinase, and phosphatase; decrease in poly(A)-binding affinity of mRNA carrier).

Animals↗
PubMed1992

[Detection of apoprotein A-1 immunoreactivity in chromatin of rat hepatocytes].

The binding of 125I-labeled lipoproteins to subcellular structures of rat hepatocytes was studied. The protein component of HDL, LDL, and VLDL was found in large and small membranes, nuclei, mitochondria, lysosomes, microsomes, and cell cytosol. The presence in liver nuclear chromatin of proteins immunochemically related to apoA-1 was demonstrated by solid phase immunoenzymatic analysis, dot immunoanalysis, and immunoelectroblotting. The apoprotein A-1 immunoreactivity was detected in transcriptionally inactive total chromatin, transcriptionally active chromatin, and nuclear matrix. The specific apoA-1 immunoreactivity of transcriptionally active chromatin and nuclear matrix was two times as low as that of total and transcriptionally inactive chromatin. Immunoelectroblotting revealed two proteins possessing an apoA-1 immunoreactivity (M(r) 28 and 14 kDa). The 28 kDa protein was found in transcriptionally active chromatin and nuclear matrix, while the 14 kDa one--only in transcriptionally inactive chromatin. the roles of apoproteins in lipid transport to nuclei and the maintenance of chromatin in a transcriptionally active state are discussed.

Animals↗
PubMed1986

Nuclear protein matrix as a target for estramustine-induced cell death.

The effect of estramustine [estradiol 3-N-bis(2-chloroethyl)carbamate] on the human prostatic tumor cell line 1013L was investigated. Cell proliferation experiments revealed that estramustine cytotoxicity varied during the different phases of cell growth. Maximum cell killing was found in early log phase, but cell death also occurred in the stationary phase. Mitotic arrest was found at cytotoxic concentrations throughout the log phase. Subcellular distribution studies showed that the cellular uptake of estramustine increased throughout the log phase and remained steady during the stationary phase. Nuclear uptake in contrast was similar in all phases, whereas a preferential binding to the nuclear protein matrix was found to increase throughout the log phase and even during the stationary phase of growth. This implicates the nuclear protein matrix as a target for estramustine cytotoxicity.

Cell Line↗
PubMed1987

Calmodulin and calmodulin-binding proteins in liver cell nuclei.

Three nuclear subfractions were prepared from isolated hepatocytes nuclei. The calmodulin content in whole nuclei was 79 ng/mg of protein. The soluble fraction obtained after digestion of the nuclei with DNase I and RNase A (S1 fraction) contained 252 ng of calmodulin/mg of protein. The pellet obtained after the digestion with nucleases was treated with 1.6 M NaCl, and the soluble fraction and the residual structures obtained after the treatment were called S2 fraction and nuclear matrix, respectively. The calmodulin contents of the S2 fraction and of the nuclear matrix were 68 and 190 ng/mg of protein, respectively. If nuclei were digested only with DNase I, the calmodulin content in the soluble fraction increased to 703 ng/mg of protein, indicating that part of the nuclear calmodulin is associated with active DNA. Five nuclear calmodulin-binding proteins were identified. Two, having apparent molecular masses of 240 and 150 kDa were only found in the nuclear matrix, whereas the other three, having molecular masses of 120, 65, and 40 kDa were found in different proportions in all nuclear subfractions. A calmodulin-dependent inhibition of protein phosphorylation in the S1 fraction was discovered. Purification attempts on the calmodulin-binding proteins of the S1 subfraction by calmodulin affinity chromatography yielded four major polypeptides with apparent molecular masses of about 41, 46, and 120 (two products) kDa. These polypeptides retained the ability to inhibit protein phosphorylation but not the sensitivity to calmodulin.

Animals↗
PubMed2000

Matrix attachment region regulates basal beta-lactoglobulin transgene expression.

Nuclear matrix attachment regions (MAR) have been implicated in the regulation of gene expression. We have identified a region within the proximal 3'-flanking sequences of the ovine beta-lactoglobulin (betalg) gene that interacts with the nuclear matrix in vitro. No equivalent region was detected in the 5' flanking region. We have investigated the role of this element in regulating betalg expression in vitro and in vivo. Removal of the MAR did not affect the frequency of betalg transgene expression at the mRNA level, but betalg transgenes that lacked the MAR were expressed at a lower level than wild-type betalg transgenes. In neither in-vitro HC11 transfection experiments nor transgenic mice was hormonal induction of betalg expression significantly affected by MAR removal. Nuclear run-on analysis demonstrated that the impaired basal expression of betalg transgene loci lacking the MAR was due to a reduced transcription rate. Thus, the single MAR enhances the basal transcriptional potential of the betalg gene.

Animals↗
PubMed1994

Binding of matrix attachment regions to lamin polymers involves single-stranded regions and the minor groove.

Chromatin in eukaryotic nuclei is thought to be partitioned into functional loop domains that are generated by the binding of defined DNA sequences, named MARs (matrix attachment regions), to the nuclear matrix. We have previously identified B-type lamins as MAR-binding matrix components (M. E. E. Ludérus, A. de Graaf, E. Mattia, J. L. den Blaauwen, M. A. Grande, L. de Jong, and R. van Driel, Cell 70:949-959, 1992). Here we show that A-type lamins and the structurally related proteins desmin and NuMA also specifically bind MARs in vitro. We studied the interaction between MARs and lamin polymers in molecular detail and found that the interaction is saturable, of high affinity, and evolutionarily conserved. Competition studies revealed the existence of two different types of interaction related to different structural features of MARs: one involving the minor groove of double-stranded MAR DNA and one involving single-stranded regions. We obtained similar results for the interaction of MARs with intact nuclear matrices from rat liver. A model in which the interaction of nuclear matrix proteins with single-stranded MAR regions serves to stabilize the transcriptionally active state of chromatin is discussed.

Animals↗
PubMed1996

INMP, a novel intranuclear matrix protein related to the family of intermediate filament-like proteins: molecular cloning and sequence analysis.

An important step in understanding nuclear structure and its function in replication and gene regulation is the cloning and characterisation of nuclear matrix proteins. A full-length cDNA-clone, encoding a novel nuclear matrix protein, was isolated from a (lambda gt11 cDNA library derived from murine macrophages. The antibody used for the screen was raised against a single polypeptide isolated from two-dimensional gel electrophoresis of nuclear matrix preparations. The cDNA encodes a protein of 446 amino acids named INMP for intranuclear matrix protein. INMP displays several salient features, a coiled-coil domain, a leucine zipper, a number of phosphorylation sites and a putative nuclear localisation signal. Sequence homology comparisons indicate that INMP is a unique protein which is evolutionary related to the gene family of intermediate filament-like proteins, especially the nuclear lamins.

Amino Acid Sequence↗
PubMed2003

Menin associates with FANCD2, a protein involved in repair of DNA damage.

Multiple endocrine neoplasia type I (MEN1) is an inherited tumor syndrome characterized by tumors in multiple endocrine organs including the parathyroids, pancreatic islets, and the pituitary. The gene mutated in MEN1 patients, Men1, encodes a protein of 610 amino acid residues, menin, and mutations in the Men1 gene lead to the MEN1 syndrome. Although the chromosomal instability in the peripheral lymphocytes from the MEN1 patients has been reported previously, it is not clear whether menin is involved in repair of DNA damage. Here we show that menin specifically interacts with FANCD2, a protein encoded by a gene involved in DNA repair and mutated in patients with an inherited cancer-prone syndrome, Fanconi anemia. The interaction between menin and FANCD2 is enhanced by gamma-irradiation. Moreover, loss of menin expression in mouse embryonic fibroblasts leads to increased sensitivity to DNA damage. Furthermore, menin is localized to chromatin and nuclear matrix, and the association with nuclear matrix is enhanced by gamma-irradiation. Together, these results suggest that menin plays a critical role in repair of DNA damage in concert with FANCD2.

3T3 Cells↗
PubMed1996

MFP1, a novel plant filament-like protein with affinity for matrix attachment region DNA.

The interaction of chromatin with the nuclear matrix via matrix attachment regions (MARs) on the DNA is considered to be of fundamental importance for higher order chromatin organization and regulation of gene expression. Here, we report a novel nuclear matrix-localized MAR DNA binding protein, designated MAR binding filament-like protein 1 (MFP1), from tomato. In contrast to the few animal MAR DNA binding proteins thus far identified, MFP1 contains a predicted N-terminal transmembrane domain and a long filament-like alpha-helical domain that is similar to diverse nuclear and cytoplasmic filament proteins from animals and yeast. DNA binding assays established that MFP1 can discriminate between animal and plant MAR DNAs and non-MAR DNA fragments of similar size and AT content. Deletion mutants of MFP1 revealed a novel, discrete DNA binding domain near the C terminus of the protein. MFP1 is an in vitro substrate for casein kinase II, a nuclear matrix-associated protein kinase. Its structure, MAR DNA binding activity, and nuclear matrix localization suggest that MFP1 is likely to participate in nuclear architecture by connecting chromatin with the nuclear matrix and potentially with the nuclear envelope.

Amino Acid Sequence↗
PubMed1992

Nuclear distribution pattern of tumour-associated nonhistone protein of mol. wt 48,000.

1. As a further step toward characterizing nonhistone protein of mol. wt 48,000 which was found to be much more abundant in animal tumour cells than in normal ones [Krajewska W.M., Lipínska A., Marszatek M., Kiliańska Z., Wojtkowiak Z. and Kłyszejko-Stefanowicz L. Cell. Biochem. Funct. 8, 79-89 (1990)] its intranuclear localization in hamster liver and Kirkman-Robbins hepatoma was studied. The protein was identified by immunoblotting technique in the presence of antibodies against polypeptide of mol. wt about 48,000 from Kirkman-Robbins hepatoma. 2. Distribution of antigen with mol. wt of 48,000 in nuclear fractions representing different levels of nuclear material organization, i.e. in nucleoli, nuclease-sensitive and nuclease-resistant fractions, and extensive nuclease digestion products separated by size on Bio-Gel A-50m; implied the structural role of this component. 3. Fractionation of endogenously digested nuclei into low salt extract, high salt extract and nuclear matrix revealed that in normal liver the antigen studied is associated with nuclear matrix while in hepatoma this component appeared in high salt extract. 4. These results suggest that polypeptide with mol. wt of 48,000 is a shuttling protein which may be involved in reorganization of nuclear matrix during neoplastic transformation.

Animals↗
PubMed1994

Radiation sensitivity correlates with changes in DNA supercoiling and nucleoid protein content in cells of three Chinese hamster cell lines.

We have investigated the composition of nuclear matrix proteins and DNA supercoiling characteristics of cell lines expressing altered radiation sensitivity. Chinese hamster ovary cell lines 4364 (wild-type), XR-1 (DSB repair-deficient, radiosensitive) and XR-122 (a radioresistant variant of XR-1 bearing human chromosome 5) were used as a model to study the relationship between intrinsic radiation sensitivity and the level of DNA supercoiling ability within chromatin loops and the composition of nuclear matrix proteins. Analysis of the ability of DNA loop domains to undergo changes in DNA supercoiling in the presence of DNA damage revealed that the degree of inhibition of loop rewinding was greater in the radiation-sensitive cells (XR-1) compared to the radiation-resistant cells (4364 and XR-122). Furthermore, the loop-rewinding characteristics correlated inversely with the clonogenic survival of these cells after exposure to ionizing radiation. Since DNA loops are anchored to the nuclear matrix by protein-DNA anchor points, a study of the nuclear matrix proteins by high-resolution 2D-PAGE was conducted for these cells to determine whether differential inhibition of loop rewinding could be due to differences in the DNA loop-protein anchor points in these cells. The XR-1 cells showed an overall absence of 13 proteins compared to the 4364 cells. Of these 13, 5 were restored in XR-122 cells. These results are consistent with the hypothesis that stability of the DNA loop domains in the presence of DNA damage contributes to the expression of potentially lethal damage by ionizing radiation.

Animals↗
PubMedDate not supplied

DNA organization in human spermatozoa.

Previous studies from this laboratory on hamster spermatozoa have demonstrated that rodent sperm DNA is packaged into the sperm nucleus in a specific manner by nuclear structures. The entire genome is organized into DNA loop domains attached at their bases to a sperm nuclear matrix, the skeletal structure of the nucleus. When nuclei are completely decondensed, the nuclear matrix dissipates, and the entire genome remains anchored to a single structure located at the base of the tail, termed the nuclear annulus. Here, we have extended these studies to human sperm nuclei, which were found to be similar to hamster. Human sperm DNA was found to be organized into loop domains attached at their bases to a nuclear matrix. The average size of the human sperm halo of DNA surrounding the extracted sperm nucleus (made up of DNA loop domains) was about 50% smaller than those that have been reported for somatic cells (this corresponds to an approximate loop domain size of 26.8 +/- 2.1 kb). Human sperm DNA also remained anchored to the base of the tail when completely decondensed, indicating the existence of a nuclear annulus-like structure in human spermatozoa; but, unlike the hamster nuclear annulus, the human annulus could not be isolated because of its structural instability when separated from the tail. Using human centromere repeats as a probe for in situ hybridization, we examined the packaging of individual DNA sequences within the sperm nucleus. These studies demonstrate that human sperm DNA is highly organized by nuclear structures.

Adult↗
PubMed2004

Large-scale identification and mapping of nuclear matrix-attachment regions in the distal imprinted domain of mouse chromosome 7.

Mammalian imprinted genes, which are expressed from only one of the parental alleles, have a tendency to form clusters and are regulated by long-range mechanisms. Nuclear matrix-attachment regions (MARs), the anchor points of loop domains, are involved in coordination of gene expression and could play a role in regulation of imprinted domains. We have identified and mapped a total of 52 MARs in a 1-Mb imprinted domain on mouse distal chromosome 7 using our cosmid contigs and an in vitro MAR assay. We find two MAR clusters (comprising 20 and 19 MARs), one of which is mapped in the Th-Ins2 intergenic region, coincident with the boundary between the two imprinted subdomains. However, the imprinted/non-imprinted boundaries are not associated with a MAR. Based on the sequence information, we find that many of the MARs are rich in long interspersed nuclear elements. In addition, comparisons of the results obtained with several MAR-prediction software programs reveal good performance of ChrClass in terms of both sensitivity and specificity. This study presents the first large-scale mapping of MARs in an imprinted domain and provides a platform for understanding the roles of MARs in imprinting.

Animals↗