Showing papers on "Heterochromatin published in 1999"
TL;DR: It is shown that five unrelated ICF patients have mutations in both alleles of the gene that encodes DNA methyltransferase 3B (refs 5, 6), which is the only genetic disorder known to involve constitutive abnormalities of genomic methylation patterns.
Abstract: The recessive autosomal disorder known as ICF syndrome (for immunodeficiency, centromere instability and facial anomalies; Mendelian Inheritance in Man number 242860) is characterized by variable reductions in serum immunoglobulin levels which cause most ICF patients to succumb to infectious diseases before adulthood. Mild facial anomalies include hypertelorism, low-set ears, epicanthal folds and macroglossia. The cytogenetic abnormalities in lymphocytes are exuberant: juxtacentromeric heterochromatin is greatly elongated and thread-like in metaphase chromosomes, which is associated with the formation of complex multiradiate chromosomes. The same juxtacentromeric regions are subject to persistent interphase self-associations and are extruded into nuclear blebs or micronuclei. Abnormalities are largely confined to tracts of classical satellites 2 and 3 at juxtacentromeric regions of chromosomes 1, 9 and 16. Classical satellite DNA is normally heavily methylated at cytosine residues, but in ICF syndrome it is almost completely unmethylated in all tissues. ICF syndrome is the only genetic disorder known to involve constitutive abnormalities of genomic methylation patterns. Here we show that five unrelated ICF patients have mutations in both alleles of the gene that encodes DNA methyltransferase 3B (refs 5, 6). Cytosine methylation is essential for the organization and stabilization of a specific type of heterochromatin, and this methylation appears to be carried out by an enzyme specialized for the purpose.
1,144 citations
TL;DR: The release of yKu from telomeric chromatin may allow efficient scanning of the genome for DNA strand breaks and help identify hypersensitive strains of eukaryotic chromosomes that are hypersensitive to DNA-damaging agents.
487 citations
TL;DR: Immodetection of endogenous Suv39h1/SUV39H1 proteins in a variety of mammalian cell lines reveals enriched distribution at heterochromatic foci during interphase and centromere‐specific localization during metaphase, and indicates the existence of a mammalian SU(VAR) complex.
Abstract: The chromo and SET domains are conserved sequence motifs present in chromosomal proteins that function in epigenetic control of gene expression, presumably by modulating higher order chromatin. Based on sequence information from the SET domain, we have isolated human (SUV39H1) and mouse (Suv39h1) homologues of the dominant Drosophila modifier of position-effect-variegation (PEV) Su(var)3-9. Mammalian homologues contain, in addition to the SET domain, the characteristic chromo domain, a combination that is also preserved in the Schizosaccharyomyces pombe silencing factor clr4. Chromatin-dependent gene regulation is demonstrated by the potential of human SUV39H1 to increase repression of the pericentromeric white marker gene in transgenic flies. Immunodetection of endogenous Suv39h1/SUV39H1 proteins in a variety of mammalian cell lines reveals enriched distribution at heterochromatic foci during interphase and centromere-specific localization during metaphase. In addition, Suv39h1/SUV39H1 proteins associate with M31, currently the only other characterized mammalian SU(VAR) homologue. These data indicate the existence of a mammalian SU(VAR) complex and define Suv39h1/SUV39H1 as novel components of mammalian higher order chromatin.
459 citations
TL;DR: The results indicate that histones make Sir-independent contributions to telomeric silencing, and that the role of histones located elsewhere in chromosomes is gene specific rather than generally repressive.
Abstract: Eukaryotic genomes are packaged into nucleosomes, which are thought to repress gene expression generally1,2,3. Repression is particularly evident at yeast telomeres, where genes within the telomeric heterochromatin appear to be silenced by the histone-binding silent information regulator (SIR) complex (Sir2, Sir3, Sir4) and Rap1 (refs 4,5,6,7,8,9,10). Here, to investigate how nucleosomes and silencing factors influence global gene expression, we use high-density arrays to study the effects of depleting nucleosomal histones and silencing factors in yeast. Reducing nucleosome content by depleting histone H4 caused increased expression of 15% of genes and reduced expression of 10% of genes, but it had little effect on expression of the majority (75%) of yeast genes. Telomere-proximal genes were found to be de-repressed over regions extending 20 kilobases from the telomeres, well beyond the extent of Sir protein binding11,12 and the effects of loss of Sir function. These results indicate that histones make Sir-independent contributions to telomeric silencing, and that the role of histones located elsewhere in chromosomes is gene specific rather than generally repressive.
407 citations
TL;DR: In this article, the identification and characterization of boundary elements that flank the transcriptionally repressed HMR locus in the yeast Saccharomyces cerevisiae was reported, and the boundary element contained a TY1 LTR, and a tRNA gene and mutational analysis has implicated the Smc proteins, which encode structural components of chromosomes, in boundary element function.
Abstract: The chromosomes of eukaryotes are organized into structurally and functionally discrete domains that provide a mechanism to compact the DNA as well as delineate independent units of gene activity. It is believed that insulator/boundary elements separate these domains. Here we report the identification and characterization of boundary elements that flank the transcriptionally repressed HMR locus in the yeast Saccharomyces cerevisiae. Deletion of these boundary elements led to the spread of silenced chromatin, whereas the ectopic insertion of these elements between a silencer and a promoter blocked the repressive effects of the silencer on that promoter at HMR and at telomeres. Sequence analysis indicated that the boundary element contained a TY1 LTR, and a tRNA gene and mutational analysis has implicated the Smc proteins, which encode structural components of chromosomes, in boundary element function.
388 citations
TL;DR: A mechanism for the recruitment of HP1-like gene products by the KRAB-ZFP–KAP-1 complex to specific loci within the genome through formation of heterochromatin-like complexes that silence gene activity is suggested.
Abstract: Kruppel-associated box (KRAB) domains are present in approximately one-third of all human zinc finger proteins (ZFPs) and are potent transcriptional repression modules. We have previously cloned a corepressor for the KRAB domain, KAP-1, which is required for KRAB-mediated repression in vivo. To characterize the repression mechanism utilized by KAP-1, we have analyzed the ability of KAP-1 to interact with murine (M31 and M32) and human (HP1α and HP1γ) homologues of the HP1 protein family, a class of nonhistone heterochromatin-associated proteins with a well-established epigenetic gene silencing function in Drosophila. In vitro studies confirmed that KAP-1 is capable of directly interacting with M31 and hHP1α, which are normally found in centromeric heterochromatin, as well as M32 and hHP1γ, both of which are found in euchromatin. Mapping of the region in KAP-1 required for HP1 interaction showed that amino acid substitutions which abolish HP1 binding in vitro reduce KAP-1 mediated repression in vivo. We observed colocalization of KAP-1 with M31 and M32 in interphase nuclei, lending support to the biochemical evidence that M31 and M32 directly interact with KAP-1. The colocalization of KAP-1 with M31 is sometimes found in subnuclear territories of potential pericentromeric heterochromatin, whereas colocalization of KAP-1 and M32 occurs in punctate euchromatic domains throughout the nucleus. This work suggests a mechanism for the recruitment of HP1-like gene products by the KRAB-ZFP–KAP-1 complex to specific loci within the genome through formation of heterochromatin-like complexes that silence gene activity. We speculate that gene-specific repression may be a consequence of the formation of such complexes, ultimately leading to silenced genes in newly formed heterochromatic chromosomal environments.
381 citations
TL;DR: An association of TIF1β with both heterochromatin and euchromatin in interphase nuclei is reported and it is shown that trichostatin A, an inhibitor of histone deacetylases, can interfere with both Tif1 and HP1 silencing.
Abstract: Mammalian TIF1alpha and TIF1beta (KAP-1/KRIP-1) are related transcriptional intermediary factors that possess intrinsic silencing activity. TIF1alpha is believed to be a euchromatic target for liganded nuclear receptors, while TIF1beta may serve as a co-repressor for the large family of KRAB domain-containing zinc finger proteins. Here, we report an association of TIF1beta with both heterochromatin and euchromatin in interphase nuclei. Co-immunoprecipitation of nuclear extracts shows that endogenous TIF1beta, but not TIF1alpha, is associated with members of the heterochromatin protein 1 (HP1) family. However, in vitro, both TIF1alpha and TIF1beta interact with and phosphorylate the HP1 proteins. This interaction involves a conserved amino acid motif, which is critical for the silencing activity of TIF1beta but not TIF1alpha. We further show that trichostatin A, an inhibitor of histone deacetylases, can interfere with both TIF1 and HP1 silencing. The silencing activity of TIF1alpha appears to result chiefly from histone deacetylation, whereas that of TIF1beta may be mediated via both HP1 binding and histone deacetylation.
360 citations
TL;DR: It is shown that Sir3p is released from telomeres in response to DNA double-strand breaks (DSBs), binds to DSBs, and mediates their repair, independent of cell mating type.
338 citations
01 Jan 1999
TL;DR: Biochemical analysis showed that HP1α and HP1γ are phosphorylated throughout the cell cycle, although more extensively in mitosis than in interphase, while HP1β apparently remains unphosphorylated.
Abstract: Mammalian heterochromatin proteins 1 (HP1α, HP1β, and HP1γ) are nonhistone proteins that interact in vitro with a set of proteins that play a role in chromatin silencing, transcription, and chromatin re- modeling. Using antibodies specific for each HP1 iso- form, we showed that they segregate in distinct nuclear domains of human HeLa cells. By contrast, in mouse 3T3 interphase cells, HP1α and HP1β are strictly colo- calized. In mitotic HeLa cells, all of HP1 α and a fraction of HP1β and HP1γ remain associated with chromo- somes. Immunostaining of spread HeLa chromosomes showed that HP1α is mainly localized on centromeres as shown previously for HP1β, while HP1γ is distributed on discrete sites on the arms of chromosomes. Biochem- ical analysis showed that HP1α and HP1γ are phosphor- ylated throughout the cell cycle, although more exten- sively in mitosis than in interphase, while HP1 β appar- ently remains unphosphorylated. Therefore, despite their extensive sequence conservation, mammalian HP1 iso- forms differ widely in their nuclear localization, mitotic distribution and cell cycle-related phosphorylation. Thus, subtle differences in primary sequence and in posttranslational modifications may promote their tar- geting at different chromatin sites, generating pleiotropic effects.
306 citations
TL;DR: The unexpected association of a putative transcriptional regulator with highly repetitive DNA provides a potential explanation for the variability in phenotype of patients with identical mutations in the ATRX gene.
Abstract: ATRX is a member of the SNF2 family of helicase/ATPases that is thought to regulate gene expression via an effect on chromatin structure and/or function. Mutations in the hATRX gene cause severe syndromal mental retardation associated with α-thalassemia. Using indirect immunofluorescence and confocal microscopy we have shown that ATRX protein is associated with pericentromeric heterochromatin during interphase and mitosis. By coimmunofluorescence, ATRX localizes with a mouse homologue of the Drosophila heterochromatic protein HP1 in vivo, consistent with a previous two-hybrid screen identifying this interaction. From the analysis of a trap assay for nuclear proteins, we have shown that the localization of ATRX to heterochromatin is encoded by its N-terminal region, which contains a conserved plant homeodomain-like finger and a coiled-coil domain. In addition to its association with heterochromatin, at metaphase ATRX clearly binds to the short arms of human acrocentric chromosomes, where the arrays of ribosomal DNA are located. The unexpected association of a putative transcriptional regulator with highly repetitive DNA provides a potential explanation for the variability in phenotype of patients with identical mutations in the ATRX gene.
250 citations
TL;DR: It is proposed that perpetuation of heterochromatin involves self-maintenance factors, including local concentration of Hp1α and -β, and that a degree of plasticity is provided by the cycle of H4 acetylation/deacetylation assisted by CAF-1.
Abstract: To investigate the mechanisms that assure the maintenance of heterochromatin regions, we took advantage of the fact that clusters of heterochromatin DNA replicate late in S phase and are processed in discrete foci with a characteristic nuclear distribution. At the light microscopy level, within these entities, we followed DNA synthesis, histone H4 acetylation, heterochromatin protein 1 (Hp1α and -β), and chromatin assembly factor 1 (CAF-1). During replication, Hp1α and -β domains of concentration are stably maintained, whereas heterochromatin regions are enriched in both CAF-1 and replication-specific acetylated isoforms of histone H4 (H4Ac 5 and 12). We defined a time window of 20 min for the maintenance of this state. Furthermore, treatment with Trichostatin A (TSA), during and after replication, sustains the H4Ac 5 and 12 state in heterochromatin excluding H4Ac 8 and 16. In comparison, early replication foci, at the same level, did not display any specific enrichment in H4Ac 5 and 12. These data emphasize the specific importance for heterochromatin of the replication-associated H4 isoforms. We propose that perpetuation of heterochromatin involves self-maintenance factors, including local concentration of Hp1α and -β, and that a degree of plasticity is provided by the cycle of H4 acetylation/deacetylation assisted by CAF-1.
TL;DR: It is concluded that telomeric chromatin has a Sir3-dependent inhibitory effect on DNA replication.
Abstract: Different regions of the eukaryotic genome are known to replicate at distinct, reproducible times within the period of S phase. This is observed in plants, insects, and vertebrates, as well as in the yeast Saccharomyces cerevisiae (Hand 1978; Fangman and Brewer 1992). In species with large genomes, extensive regions of the chromosomes replicate early in S phase, whereas other domains do not initiate replication until after the early domains have completed synthesis. Frequently, late replication of DNA is associated with its assembly into heterochromatin, which is highly condensed chromatin that often contains repeated DNA sequences such as those found at centromeres and telomeres (John 1988). This condensed chromosome replicates much later in S phase than its transcriptionally active homolog.
Telomeric chromatin in S. cerevisiae has several traits typical of heterochromatin (Grunstein 1998). In particular, telomeres confer epigenetic silencing of nearby genes (position effect variegation), and they replicate late in S phase (McCarroll and Fanagman 1988; Gottschling et al. 1990). The special chromatin found near telomeres is composed of hypoacetylated core histones as well as the SIR proteins, which are required for silencing telomeric genes (Grunstein 1998). Of the SIR proteins, Sir3p is probably the key component that defines a telomeric domain of transcriptional repression. It interacts with the tails of histones H3 and H4, spreading from the telomere inward along the chromosome, and its abundance in the cell determines how far a silent domain extends from the telomere (Renauld et al. 1993; Hecht et al. 1996).
In yeast, chromosome replication initiates at ARS (autonomous replicating sequences) elements (Fangman and Brewer 1991). ARS elements were originally identified as sequences that permit high-efficiency transformation of plasmids in yeast by serving as origins of DNA replication. However, in their normal chromosomal context, only a subset of the ARS elements initiate DNA replication within the ∼30 min duration of S phase (Fangman and Brewer 1992). Specific chromosomal origins of S. cerevisiae, like ARS1 on chromosome IV, initiate replication relatively early in S phase. Other ARS elements initiate later in S phase, such as ARS501 on chromosome V, which replicates ∼10 min after ARS1 (Ferguson et al. 1991).
It appears that telomeres can confer late replication on proximal origins. For instance, telomere-proximal middle repetitive sequences replicate relatively late in S phase (McCarroll and Fangman 1988; Louis 1995). In addition, whereas ARS elements on circular plasmids initiate replication early in S phase, they replicate late in S phase when the plasmid is linearized and telomeric sequences are added to its ends (Ferguson and Fangman 1992). In this study we investigated the role of silent chromatin in imposing late replication on origins near telomeres.
TL;DR: The findings are consistent with the hypothesis that one of the ways that genome-wide hypomethylation facilitates tumor development is that it often includes satellite hypometHylation which might predispose cells to structural and numerical chromosomal aberrations.
Abstract: Rearrangements in heterochromatin in the vicinity of the centromeres of chromosomes 1 and 16 are frequent in many types of cancer, including ovarian epithelial carcinomas. Satellite 2 DNA is the main sequence in the unusually long heterochromatin region adjacent to the centromere of each of these chromosomes. Rearrangements in these regions and hypomethylation of satellite 2 DNA are a characteristic feature of patients with a rare recessive genetic disease, ICF (immunodeficiency, centromeric region instability, and facial anomalies). In all normal tissues of postnatal somatic origin, satellite 2 DNA is highly methylated. We examined satellite 2 DNA methylation in ovarian tumors of different malignant potential, namely, ovarian cystadenomas, low malignant potential (LMP) tumors, and epithelial carcinomas. Most of the carcinomas and LMP tumors exhibited hypomethylation in satellite 2 DNA of both chromosomes 1 and 16. A comparison of methylation of these sequences in the three types of ovarian neoplasms demonstrated that there was a statistically significant correlation between the extent of this satellite DNA hypomethylation and the degree of malignancy (P<0.01). Also, there was a statistically significant association (P<0.005) between genome-wide hypomethylation and undermethylation of satellite 2 DNA among these 17 tumors. In addition, we found abnormal hypomethylation of satellite α DNA in the centromere of chromosome 1 in many of these tumors. Our findings are consistent with the hypothesis that one of the ways that genome-wide hypomethylation facilitates tumor development is that it often includes satellite hypomethylation which might predispose cells to structural and numerical chromosomal aberrations. Several of the proteins that bind to pericentromeric heterochromatin are known to be sensitive to the methylation status of their target sequences and so could be among the sensors for detecting abnormal demethylation and mediating effects on chromosome structure and stability.
TL;DR: A distinction between insulators and the boundary elements of the BX-C complex is implied by a recent analysis in which the Fab-7 boundary element was replaced by either the scs or gypsy insulator, implying a distinction between these types of boundaries.
TL;DR: A 149-bp segment consisting essentially of only three tandem Rap1-binding sites from the UASrpg of yeast TEF2 exhibits silencer-blocking activity and provides the first example of chromatin domain boundary or insulator elements in yeast.
Abstract: The HM loci in Saccharomyces cerevisiae constitute region-specific but gene-nonspecific repression domains, as a number of heterologous genes transcribed by RNA polymerase II or III are silenced when placed at these loci. The promoters of the Ashbya gossypii TEF gene and the S. cerevisiae TEF1 and TEF2 genes, however, are resistant to transcriptional silencing by the HM silencers in yeast. Moreover, when interposed between the HML α genes and the E silencer, certain segments of these promoters block the repression effect of the silencer on the α genes. All of these fragments contain UASrpg (upstream activation sequence of ribosome protein genes) composed of multiple binding sites for Rap1. In fact, a 149-bp segment consisting essentially of only three tandem Rap1-binding sites from the UASrpg of yeast TEF2 exhibits silencer-blocking activity. This element also exhibits insulating activity and orientation dependence characteristic of known chromatin boundary elements. Finally, the element blocks the physical spread of heterochromatin initiated at a silencer. This segment provides the first example of chromatin domain boundary or insulator elements in yeast.
TL;DR: The data indicate that both nuclear organization and local chromatin structure play a role in this telomeric PEV, where the variegating phenotype of the white transgene is suppressed, sensitivity to a mutation in HP1 is retained.
Abstract: Transgenes inserted into the telomeric regions of Drosophila melanogaster chromosomes exhibit position effect variegation (PEV), a mosaic silencing characteristic of euchromatic genes brought into juxtaposition with heterochromatin. Telomeric transgenes on the second and third chromosomes are flanked by telomeric associated sequences (TAS), while fourth chromosome telomeric transgenes are most often associated with repetitious transposable elements. Telomeric PEV on the second and third chromosomes is suppressed by mutations in Su(z)2, but not by mutations in Su(var)2-5 (encoding HP1), while the converse is true for telomeric PEV on the fourth chromosome. This genetic distinction allowed for a spatial and molecular analysis of telomeric PEV. Reciprocal translocations between the fourth chromosome telomeric region containing a transgene and a second chromosome telomeric region result in a change in nuclear location of the transgene. While the variegating phenotype of the white transgene is suppressed, sensitivity to a mutation in HP1 is retained. Corresponding changes in the chromatin structure and inducible activity of an associated hsp26 transgene are observed. The data indicate that both nuclear organization and local chromatin structure play a role in this telomeric PEV.
TL;DR: The bulk of the eukaryotic genome is composed of families of repetitive sequences that are genetically silent and exhibit various types of instability, and transposable elements are particularly common in heterochromatic regions of the genome.
TL;DR: It is shown that PEV can also occur when the transgene is non-centromeric and that the extent of variegation can be developmentally regulated and that components or modifiers of heterochromatin may have a chromosomal-context-dependent role in gene silencing and activation decisions in mammals.
Abstract: Locus control regions (LCRs) are gene regulatory elements in mammals that can overcome the highly repressive effects normally associated with heterochromatic transgene locations (for example the centromere) in mice1,2,3. Deletion of essential LCR sequences renders the cognate gene susceptible to this form of repression, so a proportion of the cells from transgenic mice that would normally express the transgene are silenced—a phenomenon known as position effect variegation4,5,6 (PEV). We show here that PEV can also occur when the transgene is non-centromeric and that the extent of variegation can be developmentally regulated. Furthermore, by overexpressing a mammalian homologue (M31) of Drosophila melanogaster heterochromatin protein 1 (HP1; refs 7,8) in transgenic mouse lines that exhibit PEV, it is possible to modify the proportion of cells that silence the transgene in a dose-dependent manner. Thus, we show M31 overexpression to have two contrasting effects which are dependent on chromosomal context: (i) it enhanced PEV in those lines with centromeric or pericentromeric transgene locations; and (ii) it suppressed PEV when the transgene was non-centromeric. Our results indicate that components or modifiers of heterochromatin may have a chromosomal-context-dependent role in gene silencing and activation decisions in mammals.
TL;DR: It is determined that normal human telomeres replicate throughout S phase rather than being very late replicating, and there is no significant delay between synthesis and the processing events that contribute to the establishment of asymmetric overhangs.
TL;DR: To investigate the origins of trans-silencing ability and susceptibility, the structures, flanking DNA sequences and chromosomal locations of a nopaline synthase promoter silencing locus, H2, and a sensitive target locu, K81, are analyzed.
Abstract: One type of homology-dependent gene silencing in transgenic plants involves a silencing locus that is able to transcriptionally inactivate and methylate an unlinked target locus with which it shares sequence identity in promoter regions. In a manner resembling paramutation of endogenous genes, the target locus reactivates and loses methylation progressively over several generations after segregating away from the silencing locus, which autonomously acquires stable methylation. To investigate the origins of trans-silencing ability and susceptibility, we have analyzed the structures, flanking DNA sequences and chromosomal locations of a nopaline synthase promoter silencing locus, H2, and a sensitive target locus, K81. A partially resistant target locus, K alpha has been characterized molecularly. The complex and scrambled H2 locus comprises six copies of the nopaline synthase promoter, two of which are collinear with prokaryotic non-T-DNA sequences, and is integrated close to a region of intercalary heterochromatin. These features probably contribute collectively to the silencing ability because H2 subclones reintroduced into random locations in the K81 genome did not frequently induce silencing. Both the K81 and K alpha loci have simple structures, although the former contains non-T-DNA prokaryotic sequences that are also present at H2, and they are flanked by low copy plant DNA. H2 and K81 might interact effectively because they are present on morphologically similar chromosomes from the T subgenome of allotetraploid tobacco.
TL;DR: The mating-type locus in the yeast Saccharomyces cerevisiae provides information about whether cells are of the a or α mating type, and genes at this locus encode transcriptional regulators that determine the phenotypes associated with the different cell types.
Abstract: The mating-type locus (MAT) in the yeast Saccharomyces cerevisiae provides information about whether cells are of the a or α mating type, and genes at this locus encode transcriptional regulators that determine the phenotypes associated with the different cell types1. In a/α diploid cells, the a1/α2 repressor is formed, which inhibits haploid-specific gene expression and indirectly promotes meiosis. Mutations in SIR (silent information regulator) genes cause a loss of both heterochromatin and transcriptional silencing, resulting in the expression of cryptic a and α genes resident at the HML and HMR loci. As a result, sir mutant strains have the properties of a/α diploids.
TL;DR: The amino acid sequence of MENT reveals a close structural relationship with serpins, a large family of proteins known for their ability to undergo dramatic conformational transitions, and Conservation of the “hinge region” consensus in MENT indicates that this ability is retained by the protein.
TL;DR: Subnuclear distribution of the human herpesvirus-8 (HHV-8)- encoded nuclear protein LNA-1 was analysed at high resolution in body cavity (BC) lymphoma-derived cell lines, in cell hybrids between BC cells and various human and mouse cells and in freshly infected K562 and ECV cell lines to show that LNAs associates preferentially with the border of heterochromatin in the interphase nuclei.
Abstract: Subnuclear distribution of the human herpesvirus-8 (HHV-8)- encoded nuclear protein LNA-1 was analysed at high resolution in body cavity (BC) lymphoma-derived cell lines, in cell hybrids between BC cells and various human and mouse cells and in freshly infected K562 and ECV cell lines. Three-dimensional reconstruction of nuclei from optical sections and quantitative analysis of the distribution of LNA-1 fluorescence in relation to chromatin showed that LNA-1 associates preferentially with the border of heterochromatin in the interphase nuclei. This was further confirmed in the following systems: in endo- and exonuclease-digested nuclei, in human–mouse (BC-1–Sp2- 0) hybrids and on chromatin spreads. LNA-1 was found to bind to mitotic chromosomes at random. Epstein–Barr virus (EBV), but not HHV-8, was rapidly lost from mouse–human hybrid cells in parallel with the loss of human chromosomes. HHV-8 could persist on the residual mouse background for more than 8 months. In early human–mouse hybrids that contain a single fused nucleus, LNA-1 preferentially associates with human chromatin. After the gradual loss of the human chromosomes, LNA-1 becomes associated with the murine pericentromeric heterochromatin. In human–human hybrids derived from the fusion of the HHV-8-carrying BCBL-1 cells and the EBV-immortalized lymphoblastoid cell line IB4, LNA-1 did not co-localize with EBNA-1, EBNA-2, EBNA-5 or EBNA-6. LNA-1 was not associated with PML containing ND10 bodies either. DNase but not RNase or detergent treatment of isolated nuclei destroys LNA-1 bodies. In advanced apoptotic cells LNA- 1 bodies remain intact but are not included in the apoptotic bodies themselves.
TL;DR: It is demonstrated that TIF1alpha is a non-histone chromosomal protein tightly associated with highly accessible euchromatic regions of the genome and could belong to a novel class of chromatin-associated TIFs that facilitate the access of transregulators (e.g. liganded nuclear receptors) to their cognate sites in target genes, thereby participitating in the epigenetic control of transcription during embryonic development and cell differentiation.
Abstract: Ligand-dependent transcriptional regulation by nuclear receptors is believed to be mediated by intermediary factors (TIFs) acting on remodelling of the chromatin structure and/or the activity of the transcriptional machinery. The putative transcriptional mediator TIF1alpha is a nuclear protein kinase that has been identified via its interaction with liganded nuclear receptors, including retinoic acid (RAR), retinoid X (RXR) and estrogen (ER) receptors. Here, we demonstrate that TIF1alpha is a non-histone chromosomal protein tightly associated with highly accessible euchromatic regions of the genome. Immunofluorescence confocal microscopy reveals that TIF1alpha exhibits a finely granular distribution in euchromatin of interphase nuclei, while it is mostly excluded from condensed chromatin and metaphase chromosomes. Immunoelectron microscopy shows that, in contrast to the heterochromatin protein HP1alpha, most of TIF1alpha is associated with euchromatin, where it is preferentially localised on regions known to be sites for RNA polymerase II (perichromatin fibrils and borders between euchromatin and heterochromatin). Early mouse embryos as well as embryonal carcinoma (EC) and embryonic stem (ES) cells express high levels of TIF1alpha. These levels dramatically decrease during organogenesis and upon differentiation of P19 EC cells, indicating that TIF1alpha is preferentially expressed in undifferentiated pluripotent cells in the course of development. Therefore, TIF1alpha could belong to a novel class of chromatin-associated TIFs that facilitate the access of transregulators (e.g. liganded nuclear receptors) to their cognate sites in target genes, thereby participitating in the epigenetic control of transcription during embryonic development and cell differentiation.
TL;DR: A striking karyotypic similarity, shown by both fluorochrome banding and FISH patterns, implies close genome relationships among oak species no matter their geographic origin (European or American) or their ecophysiology (deciduous or evergreens).
Abstract: Genomes of 11 Quercus species were characterized using cytogenetic (Giemsa C-banding, fluorochrome banding), molecular-cytogenetic (fluorescence in situ hybridization, FISH, to ribosomal genes) and molecular (dot-blot for ribosomal gene-copy number assessment) techniques. Ribosomal genes are the first DNA sequences to be physically mapped in oaks, and the copy number of the 18S-5.8S-26 S rRNA genes is estimated for the first time. Oak karyotypes were analysed on the basis of DAPI banding and FISH patterns; five marker chromosomes were found. In addition, chromosomal organization of ribosomal genes with respect to AT- and GC-differentiated heterochromatin was studied. Fluorochrome staining produced very similar CMA/DAPI banding patterns, and the position and number of ribosomal loci were identical for all the species studied. The 18S-5.8S-26 S rRNA genes in oak complements were represented by a major locus at the subterminal secondary constriction (SC) of the only subtelocentric chromosome pair and a minor locus at paracentromeric SC of one metacentric pair. The only 5 S rDNA locus was revealed at the paracentromeric region of the second largest metacentric pair. A striking karyotypic similarity, shown by both fluorochrome banding and FISH patterns, implies close genome relationships among oak species no matter their geographic origin (European or American) or their ecophysiology (deciduous or evergreens). Dot-blot analysis gave preliminary evidence for different copy numbers of 18S-5.8S-26 S rRNA genes in diploid genomes of Q. cerris, Q. ilex, Q. petraea, Q. pubescens and Q. robur (2700, 1300, 2200, 4000 and 2200 copies, respectively) that was correlated with the size polymorphism of the major locus.
TL;DR: It is reported here that HP1 is phosphorylated by casein kinase II in vivo at three serine residues located at the N and C termini of the protein.
TL;DR: Five additional genes encoding putative DNA methyltransferases are identified, very similar to METI throughout the coding region; these genes probably arose by a series of gene duplication events, the most recent giving rise to METIIa and METIIb.
Abstract: Methylation of plant DNA occurs at cytosines in any sequence context, and as the Arabidopsis methyltransferase, METI, preferentially methylates cytosines in CG dinucleotides, it is likely that Arabidopsis has other methyltransferases with different target specificities. We have identified five additional genes encoding putative DNA methyltransferases. Three of these genes are very similar to METI throughout the coding region; these genes probably arose by a series of gene duplication events, the most recent giving rise to METIIa and METIIb. METIIa and b are expressed at low levels in vegetative and floral organs and the level of transcripts is not affected by the introduction of a METI antisense transgene, nor do the METII enzymes substitute for the reduced activity of METI in methylating CG dinucleotides. METIII is not essential as it encodes a truncated protein. Two other genes encode a second class of DNA methyltransferase with the conserved motifs characteristic of cytosine methyltransferases, but with little homology to the METI-like methyltransferases through the remainder of the protein. These two methyltransferases are characterized by the presence of a chromodomain inserted within the methyltransferase domain, suggesting that they may be associated with heterochromatin. Both these genes are transcribed at low levels in vegetative and reproductive tissues.
TL;DR: An integrated cytogenetic-molecular map of chromosome 6 is presented that provides a framework for physical mapping and suggests that Mi-1 and Aps-1 are at least 40 Mb apart, a base pair-to-centiMorgan relationship that is more than 50-fold higher than the average value of 750 kb/cM of the tomato genome.
Abstract: The root-knot nematode resistance gene Mi-1 in tomato has long been thought to be located in the pericentromeric heterochromatin region of the long arm of chromosome 6 because of its very tight genetic linkage (approx. 1 cM) to the markers Aps-1 (Acid phosphatase 1) and yv (yellow virescent). Using Mi-BAC clones and an Aps-1 YAC clone in fluorescence in situ hybridisation (FISH) to pachytene chromosomes we now provide direct physical evidence showing that Mi-1 is located at the border of the euchromatin and heterochromatin regions in the short arm (6S) and Aps-1 in the pericentromeric heterochromatin of the long arm (6L) close to the euchromatin. Taking into account both the estimated DNA content of hetero- and euchromatin regions and the compactness of the tomato chromosomes at pachytene (2 Mb/μm), our data suggest that Mi-1 and Aps-1 are at least 40 Mb apart, a base pair-to-centiMorgan relationship that is more than 50-fold higher than the average value of 750 kb/cM of the tomato genome. An integrated cytogenetic-molecular map of chromosome 6 is presented that provides a framework for physical mapping.
TL;DR: The data suggest that the two haploid sets are imprinted by parent-of-origin-specific DNA methylation with no correlation with the known gene-silencing properties of this base modification.
Abstract: The phenomenon of chromosome, or genomic, imprinting indicates the relevance of parental origin in determining functional differences between alleles, homologous chromosomes, or haploid sets. In mealybug males (Homoptera, Coccoidea), the haploid set of paternal origin undergoes heterochromatization at midcleavage and remains so in most of the tissues. This different behavior of the two haploid sets, which depends on their parental origin, represents one of the most striking examples of chromosome imprinting. In mammals, DNA methylation has been postulated as a possible molecular mechanism to differentially imprint DNA sequences during spermatogenesis or oogenesis. In the present article we addressed the role of DNA methylation in the imprinting of whole haploid sets as it occurs in Coccids. We investigated the DNA methylation patterns at both the molecular and chromosomal level in the mealybug Planococcus citri. We found that in both males and females the paternally derived haploid set is hypomethylated with respect to the maternally derived one. Therefore, in males, it is the paternally derived hypomethylated haploid set that is heterochromatized. Our data suggest that the two haploid sets are imprinted by parent-of-origin-specific DNA methylation with no correlation with the known gene-silencing properties of this base modification.
01 Jan 1999
TL;DR: Differences in the distribution of chromosome polymorphisms imply that intervarietal changes to these highly repetitive DNA families occurred independently, despite their juxtaposition or even overlapping locations in subtelomeric heterochromatic regions.
Abstract: In a study of polymorphism and stability in rye chromosomes, three rye varieties and the sets of wheat-rye addition and substitution lines were compared using two non-homologous highly repetitive DNA families, pSc200 and pSc250. The rye varieties, Petkus, Imperial and Onohoiskaya, showed polymorphism for the presence and the size of the pSc200 in-situ hybridization signals on chromosome pairs, 2R, 4R and 7R, and the pSc250 signals on chromosomes, 5R, 6R and 7R. Chromosome 1R was heteromorphic within the Onohoiskaya variety. Differences in the distribution of chromosome polymorphisms imply that intervarietal changes to these highly repetitive DNA families occurred independently, despite their juxtaposition or even overlapping locations in subtelomeric heterochromatic regions. In the set of Saratovskaya 29 wheat/Onohoiskaya substitution lines, only chromosome 2R was altered relative to its counterpart in the parental rye variety due to amplification of the pSc250 signal on the long arm, although this did not exceed intervarietal polymorphism. In the set of Chinese Spring wheat/Imperial addition lines, only two Imperial chromosomes, 4R and 6R, were unchanged. We detected the loss of one or both rye homologous chromosomes, the loss of one arm, and the deletion of subtelomeric heterochromatin accompanied by the loss of the pSc200 signal. The results show that Saratovskaya 29/Onohoiskaya chromosome substitution lines possess increased chromosome stability compared with Chinese Spring/Imperial addition lines.