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PubMed1999

Atm inactivation results in aberrant telomere clustering during meiotic prophase.

A-T (ataxia telangiectasia) individuals frequently display gonadal atrophy, and Atm-/- mice show spermatogenic failure due to arrest at prophase of meiosis I. Chromosomal movements take place during meiotic prophase, with telomeres congregating on the nuclear envelope to transiently form a cluster during the leptotene/zygotene transition (bouquet arrangement). Since the ATM protein has been implicated in telomere metabolism of somatic cells, we have set out to investigate the effects of Atm inactivation on meiotic telomere behavior. Fluorescent in situ hybridization and synaptonemal complex (SC) immunostaining of structurally preserved spermatocytes I revealed that telomere clustering occurs aberrantly in Atm-/- mice. Numerous spermatocytes of Atm-/- mice displayed locally accumulated telomeres with stretches of SC near the clustered chromosome ends. This contrasted with spermatogenesis of normal mice, where only a few leptotene/zygotene spermatocytes I with clustered telomeres were detected. Pachytene nuclei, which were much more abundant in normal mice, displayed telomeres scattered over the nuclear periphery. It appears that the timing and occurrence of chromosome polarization is altered in Atm-/- mice. When we examined telomere-nuclear matrix interactions in spermatocytes I, a significant difference was observed in the ratio of soluble versus matrix-associated telomeric DNA sequences between meiocytes of Atm-/- and control mice. We propose that the severe disruption of spermatogenesis during early prophase I in the absence of functional Atm may be partly due to altered interactions of telomeres with the nuclear matrix and distorted meiotic telomere clustering.

Animals↗
PubMed1984

The relationship of androgen receptor levels to androgen responsiveness in the Dunning R3327 rat prostate tumor sublines.

The objective of this study was to determine whether androgen receptor levels in a transplantable animal model of prostatic adenocarcinoma correlated with androgen responsiveness of the tumor. This is the first comparative study of androgen receptor levels in 3 subcellular compartments (cytosol, nuclear salt-extractable and nuclear salt-resistant fractions) of 4 Dunning R3327 rat prostatic adenocarcinoma sublines that vary in their response to androgen ablation. Tumors were harvested from intact adult male rats in order to best approximate the human clinical setting in which receptor levels are quantitated prior to androgen ablative therapy. Only the nuclear salt-resistant (nuclear matrix) and total nuclear androgen receptor contents were significantly different among all tumor sublines. The properties of the tumors studied and their nuclear salt-resistant androgen receptor levels were as follows: H tumor--well-differentiated, slow growing, androgen-dependent, 63 +/- 11 fmol./mg. DNA; HI tumor--well-differentiated, slow growing, androgen-insensitive, 19 +/- 8 fmol./mg. DNA; G tumor--poorly-differentiated, fast growing, androgen-sensitive, 195 +/- 42 fmol./mg. DNA; and AT-2 tumor--anaplastic, fast growing, androgen-insensitive, no detectable receptors. There was no apparent quantitative relationship between androgen receptor content and tumor growth rates, which varied considerably irrespective of the androgen responsiveness of the tumor. However, there was a qualitative relationship between nuclear salt-resistant or total nuclear receptor content and androgen responsiveness. Higher levels of receptor (H and G tumor sublines) were associated with responsiveness to androgen ablation (cessation or slowing of growth, respectively), whereas lower levels of receptor (HI and AT-2 sublines) were associated with androgen insensitivity. These observations, based on relatively homogeneous tumors, may have important implications for human prostatic cancers which appear to be composed of heterogeneous cell populations.

Adenocarcinoma↗
PubMed1989

Mapping replicational sites in the eucaryotic cell nucleus.

We have used fluorescent microscopy to map DNA replication sites in the interphase cell nucleus after incorporation of biotinylated dUTP into permeabilized PtK-1 kangaroo kidney or 3T3 mouse fibroblast cells. Discrete replication granules were found distributed throughout the nuclear interior and along the periphery. Three distinct patterns of replication sites in relationship to chromatin domains in the cell nucleus and the period of S phase were detected and termed type I (early to mid S), type II (mid to late S) and type III (late S). Similar patterns were seen with in vivo replicated DNA using antibodies to 5-bromodeoxyuridine. Extraction of the permeabilized cells with DNase I and 0.2 M ammonium sulfate revealed a striking maintenance of these replication granules and their distinct intranuclear arrangements with the remaining nuclear matrix structures despite the removal of greater than 90% of the total nuclear DNA. The in situ prepared nuclear matrix structures also incorporated biotinylated dUTP into replication granules that were indistinguishable from those detected within the intact nucleus.

Animals↗
PubMed1985

The anatomy of supercoiled loops in the Drosophila 7F locus.

The genome in eucaryotes is organized into a series of supercoiled loops, topologically anchored at their bases by components of the nuclear matrix. Previous studies have shown that active genes are associated with the nuclear matrix. We wished to know whether loops in general were solely organized by active genes. We therefore examined a locus of the Drosophila X-chromosome comprising 163,000 bp of continuous DNA sequences and devoid of known active genes. Of the 52 EcoRI restriction fragments comprising this region, we found 5 anchored fragments which non-randomly organized this region into 4 DNA loops. Each of the 5 anchored fragments contained a transcribed sequence. These results strongly suggest that supercoiled loops are organized in a specific fashion with respect to DNA sequence, with the anchorage points exclusively demarcated by transcriptionally active genes.

Animals↗
PubMed1998

Examination of determinants for intranuclear localization and transactivation within the RING finger of herpes simplex virus type 1 IE110k protein.

The herpesvirus regulatory protein IE110k possesses a cysteine-rich, RING finger motif required for its role in transactivation and virus replication. IE110k also localizes to subnuclear compartments termed PODs (PML oncogenic domains). Localization to PODs induces redistribution of the proteins associated with this nuclear compartment, including the cellular RING finger protein, PML. Here we construct a series of deletions, RING domain swaps and point mutations to analyse specific requirements within the IE110k RING finger for subnuclear localization, redistribution of PML and transactivation and we examine the relationship between these activities. We find that IE110k localizes to distinct nuclear subdomains that are more numerous than the cellular PODs and that mutation of two residues within a predicted loop of the RING finger, or replacing the IE110k RING finger with a RING finger from a cellular gene abrogates the ability of IE110k to localize to these extra compartments and traps IE110k in the original PODs. We further demonstrate that RING fingers from the cellular genes mdm-2 and Bmi I, when placed within IE110k, alter the nuclear distribution of IE110k, do not transactivate, and do not redistribute PML. We also demonstrate that the majority of wild-type IE110k, like PML, is associated with the nuclear matrix. Although substitutions and deletions within the RING finger abolish transactivation, these mutant proteins remain tightly associated with the matrix. These results further dissect the determinants involved in different aspects of nuclear compartmentalization of IE110k and are discussed in relation to PML, PODs and the IE110k RING finger.

Amino Acid Sequence↗
PubMed1984

Monoclonal antibodies binding to nuclear structures and axons in adult avian dorsal root ganglia.

Four monoclonal antibodies raised against embryonic chick dorsal root ganglia (5) recognize epitopes in neuronal and supporting cell nuclei, and in axons and contiguous cytoplasmic elements in some neurons of the adult chicken. Binding, analyzed at the ultrastructural level, is to reaction sites on the nuclear matrix, nucleolar complex, nuclear bodies, and filamentous elements in axons and some perikaryal regions.

Animals↗
PubMed1997

Direct interaction of the KRAB/Cys2-His2 zinc finger protein ZNF74 with a hyperphosphorylated form of the RNA polymerase II largest subunit.

We previously identified ZNF74 as a developmentally expressed gene commonly deleted in DiGeorge syndrome. ZNF74 encodes an RNA-binding protein tightly associated with the nuclear matrix and belongs to a large subfamily of Cys2-His2 zinc finger proteins containing a KRAB (Kruppel-associated box) repressor motif. We now report on the multifunctionality of the zinc finger domain of ZNF74. This nucleic acid binding domain is shown here to function as a nuclear matrix targeting sequence and to be involved in protein-protein interaction. By far-Western analysis and coimmunoprecipitation studies, we demonstrate that ZNF74 interacts, via its zinc finger domain, with the hyperphosphorylated largest subunit of RNA polymerase II (pol IIo) but not with the hypophosphorylated form. The importance of the phosphorylation in this interaction is supported by the observation that phosphatase treatment inhibits ZNF74 binding. Double immunofluorescence experiments indicate that ZNF74 colocalizes with the pol IIo and the SC35 splicing factor in irregularly shaped subnuclear domains. Thus, ZNF74 sublocalization in nuclear domains enriched in pre-mRNA maturating factors, its RNA binding activity, and its direct phosphodependent interaction with the pol IIo, a form of the RNA polymerase functionally associated with pre- mRNA processing, suggest a role for this member of the KRAB multifinger protein family in RNA processing.

Animals↗
PubMed2001

Breakpoints of t(4;11) translocations in the human MLL and AF4 genes in ALL patients are preferentially clustered outside of high-affinity matrix attachment regions.

Chromosomal translocations t(4;11) are based on illegitimate recombinations between the human MLL and AF4 genes, and are associated with high-risk acute leukemias of infants and young children. Here, the question was asked, whether a correlation exists between the location of translocation breakpoints within both genes and the location of S/MARs. In "halo mapping experiments" (to define SARs), about 20 kb of MLL DNA was found to be attached to the nuclear matrix. Similar experiments performed for the translocation partner gene AF4 revealed that SARs are spanning nearly the complete breakpoint cluster region of the AF4 gene. By using short DNA fragments in "scaffold reassociation experiments" (to define MARs), similar results were obtained for both genes. However, Distamycin A competition experiments in combination with "scaffold reassociation experiments" revealed specific differences in the affinity of each tested DNA fragment to bind the isolated nuclear matrix proteins. When the latter data were compared with the known location of chromosomal breakpoints for both genes, an unexpected correlation was observed. DNA areas with strong MAR affinity contained fewer translocation breakpoints, while areas with weak or absent MAR affinity showed a higher density of chromosomal breakpoints.

Chromosome Breakage↗
PubMed1996

Dissection of the ability of the chicken lysozyme gene 5' matrix attachment region to stimulate transgene expression and to dampen position effects.

The chicken lysozyme gene domain is flanked by nuclear matrix attachment regions (MARs) on each side. We have previously shown that bilaterally flanking 5'MARs in stably transfected artificial genetic units enhance expression of a reporter transgene and dampen position effects of the chromatin structure at the site of integration. The 5' MAR was now dissected into smaller fragments that were monitored for effects on transgene expression in mouse 3T3 cells by a similar assay. Fragments, which contain 1.32 and 1.45 kb and represent the upstream and the downstream half, respectively, of the 5' MAR, retained the ability to stimulate transgene expression as well as the ability to reduce the variation in the level of expression. However, a 452 bp subfragment (H1-HaeII), which still exhibits specific binding to nuclear matrices and contains two high-affinity binding sites for the abundant nuclear matrix protein ARBP, lost both of those abilities. A dimerized 177 bp sequence from fragment H1-HaeII, which also binds selectively to nuclear matrices and includes a duplicated ARBP binding site, was also unable to stimulate reporter gene expression. Furthermore, a 0.65 kb subfragment containing an intrinsically bent sequence did not affect an elevated reporter gene expression and its dampening. Our results show that the ability of MAR fragments to bind to nuclear matrices is not sufficient to enhance and insulate transgene expression in stably transfected cells.

3T3 Cells↗
PubMed2004

Expression and functional significance of mouse paraspeckle protein 1 on spermatogenesis.

Paraspeckle protein 1 (PSP1) in humans is a recently identified component protein of a novel nuclear body, paraspeckle. The protein has a DBHS (Drosophila behavior, human splicing) motif that is found in PSF and p54(nrb)/NonO proteins. These DBHS-containing proteins have been reported to be involved in various nuclear events such as DNA replication, transcription, and mRNA processing. Here we show that mouse paraspeckle protein 1 (mPSP1; encoded by the Pspc1 gene) has two isoforms with different C-termini lengths. Abundant expression of the longer isoform (mPSP1-alpha) and the shorter one (mPSP1-beta) were observed in testis and kidney, respectively. Transiently expressed mPSP1-alpha was localized in nuclei, but mPSP1-beta was localized in both nuclei and cytoplasm. These observations suggest that alternative splicing regulates tissue distribution and subcellular localization. Like other DBHS-containing proteins, mPSP1 has RNA-binding activity. In mouse testis, mPSP1-alpha was found in the nuclear matrix fraction. Furthermore, by coimmunoprecipitation, we confirmed that mPSP1 interacts with other DBHS-containing proteins, PSF and p54(nrb)/NonO. Therefore, we conclude that mPSP1 may regulate multiple phases of important nuclear events during spermatogenesis.

Animals↗
PubMed2002

DNase I sensitive site in the core region of the human beta-globin origin of replication.

HeLa cells were synchronized at late G1, early S, and late S phase of the cell cycle by nocodazole treatment. The cells were permeabilized with Triton X-100, digested with DNAse I, and extracted with 0.2 M ammonium sulfate to remove the digested chromatin. DNA was isolated from the residual chromatin attached to the nuclear matrix, digested with Hind III, and subjected to hybridization with [(32)P] labeled probe located upstream of the core region of the human beta-globin replication origin. The hybridization pattern revealed the existence of a DNase I sensitive site in the core region of the beta-globin replicator. The results suggest that association with the nuclear matrix induce alteration in the chromatin structure of the origin of replication that represents a more open chromatin configuration.

Binding Sites↗
PubMed2006

Functional characterization of human mesenchymal stem cells that maintain osteochondral fates.

Adult stem cells are essential for tissue renewal, regeneration and repair, and their expansion in defined culture medium is on focus for regenerative medicine and genetic pathologies. The bone marrow has been shown to be very rich is pluripotent mesenchymal stem cells (MSCs) capable of forming bone, cartilage and also may give rise, to neurons and astrocytes in vivo and in vitro. MSCs can be isolated and expanded in culture, but human cells cannot be verified for a cartilage or a bone fate by transfer experiments. Accordingly, here we used different approaches to characterize hMSCs osteoblastic differentiation in vitro. hMSCs grown in culture in the presence of fetal bovine serum (FBS) expressed the bone-specific transcription factor Runx2/AML3. When cells were incubated in osteoblastic differentiation medium, cells expressed transcripts belonging to the signaling of Indian HH-PTHrP axis, GLI transcription factors, and bone target genes including osteopontin. The HH pathway proved to be functional since it induced cells to grow. Cells growing or differentiating to osteoblasts presented the Runx2/AML3 transcription factor, its partner CBFB, and Smad2/3 at the nuclei associated with the nuclear matrix. Furthermore, Runx2/AML3 was observed to co-localize with SC35 to the nuclear intermediary filaments. These data support the notion that hMSCs isolated from human bone are or become bone progenitor cells upon culture. In the absence of FBS and in the presence of insulin or prolactin, cells show cytoskeletal organization and an AP-1 transcription site activity resembling proliferative osteochondrocytes while cells in the presence of dexamethasone and added prolactin or TGF-beta resembled differentiated osteoblasts. These specific cellular conditions match those observed during endochondral bone formation.

Cell Differentiation↗
PubMed2001

The human brm protein is cleaved during apoptosis: the role of cathepsin G.

The human brm (hbrm) protein (homologue of the Drosophila melanogaster brahma and Saccharomyces cervisiae SNF-2 proteins) is part of a polypeptide complex believed to regulate chromatin conformation. We have shown that the hbrm protein is cleaved in NB4 leukemic cells after induction of apoptosis by UV-irradiation, DNA damaging agents, or staurosporine. Because hbrm is found only in the nucleus, we have investigated the nature of the proteases that may regulate the degradation of this protein during apoptosis. In an in vitro assay, the hbrm protein could not be cleaved by caspase-3, -7, or -6, the "effector" caspases generally believed to carry out the cleavage of nuclear protein substrates. In contrast, we find that cathepsin G, a granule enzyme found in NB4 cells, cleaves hbrm in a pattern similar to that observed in vivo during apoptosis. In addition, a peptide inhibitor of cathepsin G blocks hbrm cleavage during apoptosis but does not block activation of caspases or cleavage of the nuclear protein polyADP ribose polymerase (PARP). Although localized in granules and in the Golgi complex in untreated cells, cathepsin G becomes diffusely distributed during apoptosis. Cleavage by cathepsin G removes a 20-kDa fragment containing a bromodomain from the carboxyl terminus of hbrm. This cleavage disrupts the association between hbrm and the nuclear matrix; the 160-kDa hbrm cleavage fragment is less tightly associated with the nuclear matrix than full-length hbrm.

Animals↗
PubMed1989

Effects of hyperthermia on chromatin condensation and nucleoli disintegration as visualized by induction of premature chromosome condensation in interphase mammalian cells.

The effects of hyperthermia on chromatin condensation and nucleoli disintegration, as visualized by induction of premature chromosome condensation in interphase mammalian cells, was studied in exponentially growing and plateau phase Chinese hamster ovary cells. Exposure to heat reduced the ability of interphase chromatin to condense and the ability of the nucleolar organizing region to disintegrate under the influence of factors provided by mitotic cells when fused to interphase cells. Based on these effects treated cells were classified in three categories. Category 1 contained cells able to condense their chromatin and disintegrate the nucleolar organizing region. Category 2 contained cells able to only partly condense their chromatin and unable to disintegrate the nucleolar organizing region. Category 3 contained cells unable to condense their chromatin and unable to disintegrate the nucleolar organizing region. The fraction of cells with nondisintegrated nucleoli increased with increasing exposure time at 45.5 degrees C and reached a plateau at almost 100% after about 20 min. Exponentially growing and plateau phase cells showed similar response. Recovery from the effects of heat on chromatin condensation and disintegration of the nucleolar organizing region depended upon the duration of the heat treatment. For exposures up to 15 min at 45.5 degrees C, a gradual reduction in the fraction of cells with nondisintegrated nucleoli was observed when cells were allowed for repair at 37 degrees C. However, only a very limited amount of repair was observed after a 30-min exposure to 45.5 degrees C. The repair times observed at the chromosome level were similar to those reported for the removal of excess protein accumulating in chromatin or the nuclear matrix, suggesting a causal relationship between the two phenomena. It is proposed that nuclear protein accumulation on chromatin or in the nuclear matrix reduces the accessibility of chromatin to enzymes responsible for the phosphorylation reactions necessary for chromatin condensation and disintegration of the nucleolus.

Animals↗
PubMed1996

Two splice variants of a tyrosine phosphatase differ in substrate specificity, DNA binding, and subcellular location.

Four different forms of a non-receptor type protein-tyrosine phosphatase are generated by alternative splicing; two of these forms (PTP-S2 and PTP-S4) are major forms, which are expressed in rat as well as human cells. Here we report that PTP-S2 binds to nonspecific DNA in vitro and localizes in the nucleus upon transfection in HeLa cells. PTP-S4 does not bind to nonspecific DNA and shows perinuclear and cytoplasmic localization. Removal of the C-terminal 34 amino acids of PTP-S4 gives rise to a truncated protein, which binds to nonspecific DNA and localizes to the nucleus. PTP-S4, but not PTP-S2, interacts strongly with the isolated nuclear matrix. The two forms of this tyrosine phosphatase show different substrate specificity in vitro, a feature novel to splice variants of tyrosine phosphatases. Mitogenic stimulation induces mRNAs for PTP-S2 as well as for PTP-S4 in the G1 phase during liver regeneration. These results suggest that alternative splicing gives rise to two protein-tyrosine phosphatases with distinct substrate specificities and subcellular locations. The 34 amino acids at the C terminus of PTP-S4 play a critical role in determining substrate specificity, subcellular location, and interaction with nuclear matrix and DNA.

Alternative Splicing↗
PubMed2000

An upstream Oct-1- and Oct-2-binding silencer governs B29 (Ig beta) gene expression.

The B cell-specific B29 (Igbeta) gene is activated in the earliest B cell precursors and is expressed throughout B cell development. Tissue-specific expression of the murine B29 gene is controlled by a B cell-specific promoter whose activity is governed by a cassette of upstream transcriptional silencers. This study describes a potent new silencer that is located 5' of the previously identified B29 silencer elements, FROG and TOAD. Like these known elements, the new B29 silencer is not restricted to the B29 promoter. Nuclear proteins from all cell lines tested interacted with this A+T-rich sequence, which closely resembled a noncanonical octamer binding motif and also conformed to the consensus sequence for nuclear matrix attachment regions. Interaction of Oct-1 and Oct-2 with the B29 A+T-rich sequence was confirmed using octamer-specific Abs. Oct-1/Oct-2 binding was required for the inhibitory activity of this sequence because mutations that blocked Oct-1/Oct-2 binding also eliminated inhibition of the B29 promoter. This B29 A+T-rich sequence specifically interacted with isolated nuclear matrix proteins in vitro, suggesting that it may also function as a matrix attachment region element. Maintenance of the level of B29 gene expression through the interaction of the minimal promoter and the upstream silencer elements FROG, TOAD, and the A+T-rich Oct-1/Oct-2 binding motif may be essential for normal B cell development and/or function.

5' Untranslated Regions↗
PubMed1999

Molecular shape and ATP binding activity of rat p50, a putative mammalian homologue of RuvB DNA helicase.

Based on partial amino acid sequences of p50 purified from a high-salt buffer extract of a rat liver nuclear matrix fraction, p50 cDNA was cloned and sequenced, and its amino acid sequence was predicted. The sequence contained helicase motifs, and showed homology with RuvB DNA helicase of Thermus thermophilus and an open reading frame for an unknown 50.5 k protein of Saccharomyces cerevisiae. p50 was expressed as a GST-fusion protein and antiserum against the protein was generated. p50 was localized to the nuclear matrix by cell fractionation and immunoblotting. p50 bound to ATP-Sepharose beads. Ultracentrifugation and gel filtration analyses showed that p50 in rat liver and Xenopus egg mitotic extracts exists as large complexes corresponding to 697 k and 447 k, respectively. A 50 k protein reactive with p50 antibodies was detected not only in rat liver nuclei, but also in a Xenopus egg cytoplasm fraction and a S. cerevisiae extract. This suggests that this putative DNA helicase is present in a wide variety of species ranging from yeast to mammals.

ATPases Associated with Diverse Cellular Activitie↗
PubMed1984

Ca2+-calmodulin-dependent protein kinase in neuronal nuclei.

Ca2+-calmodulin-dependent protein kinase represents a major enzyme activity in neuronal nuclei. Most of the protein kinase activity is associated with the nuclear matrix but it can phosphorylate a broad range of substrates which derive from different nuclear compartments. Notable among these substrates is a Mr = 50,000 component of the nuclear matrix and a high mobility group chromatin protein which behaves like HMG17. The high level of activity, age- and tissue-specificity, and sensitivity to low concentrations of calmodulin suggest a role for this Ca2+-calmodulin-dependent protein kinase in regulating neuronal function at the level of the nucleus.

Age Factors↗