RESUMEN

The silencing of large chromosomal regions by epigenetic mechanisms has been reported to occur frequently in cancer. Epigenetic marks, such as histone methylation and acetylation, are altered at these loci. However, the mechanisms of formation of such aberrant gene clusters remain largely unknown. Here, we show that, in cancer cells, the epigenetic remodeling of chromatin into hypoacetylated domains covered with histone H3K27 trimethylation is paralleled by changes in higher-order chromatin structures. Using fluorescence in situ hybridization, we demonstrate that regional epigenetic silencing corresponds to the establishment of compact chromatin domains. We show that gene repression is tightly correlated to the state of chromatin compaction and not to the levels of H3K27me3-its removal through the knockdown of EZH2 does not induce significant gene expression nor chromatin decompaction. Moreover, transcription can occur with intact high-H3K27me3 levels; treatment with histone deacetylase inhibitors can relieve chromatin compaction and gene repression, without altering H3K27me3 levels. Our findings imply that compaction and subsequent repression of large chromatin domains are not direct consequences of PRC2 deregulation in cancer cells. By challenging the role of EZH2 in aberrant gene silencing in cancer, these findings have therapeutical implications, notably for the choice of epigenetic drugs for tumors with multiple regional epigenetic alterations.

Asunto(s)

Cromatina/genética , Metilación de ADN/genética , Complejo Represivo Polycomb 2/genética , Neoplasias de la Vejiga Urinaria/genética , Línea Celular Tumoral , Inmunoprecipitación de Cromatina , Proteína Potenciadora del Homólogo Zeste 2 , Epigénesis Genética/genética , Regulación Neoplásica de la Expresión Génica , Silenciador del Gen , N-Metiltransferasa de Histona-Lisina/biosíntesis , N-Metiltransferasa de Histona-Lisina/genética , Histonas/genética , Histonas/metabolismo , Humanos , Hibridación Fluorescente in Situ , Complejo Represivo Polycomb 2/antagonistas & inhibidores , Complejo Represivo Polycomb 2/biosíntesis , Neoplasias de la Vejiga Urinaria/patología

RESUMEN

Mechanisms controlling higher-order chromatin structure or chromatin compaction and linking this to gene regulation are poorly understood. Previously, we had shown that the PRC1 Polycomb repressive complex is required to maintain a compact chromatin state at Polycomb target loci in embryonic stem cells (ESCs) of the mouse and that this activity, together with the ability to repress target gene expression, is surprisingly independent of the histone ubiquitination activity of the Ring1B component of PRC1. Here we investigate and discuss the role of another histone modification--histone acetylation--in Polycomb function. We show that inhibition of histone deacetylases leads to some decompaction of Hox loci and suggest that histone deacetylation has a role in the pathway of PRC1-mediated chromatin compaction. We discuss whether PRC1 and histone hypoacetylation function together to establish a chromatin template at which stable nucleosomes act to antagonize transcriptional elongation.

Asunto(s)

Cromatina/metabolismo , Histonas/metabolismo , Proteínas Represoras/metabolismo , Acetilación/efectos de los fármacos , Animales , Inmunoprecipitación de Cromatina , Células Madre Embrionarias/metabolismo , Sitios Genéticos/genética , Proteínas de Homeodominio/genética , Ácidos Hidroxámicos/farmacología , Ratones , Mutación/genética , Complejo Represivo Polycomb 1 , Proteínas del Grupo Polycomb , Regiones Promotoras Genéticas/genética , Proteínas Represoras/deficiencia , Transcripción Genética/efectos de los fármacos , Ubiquitina-Proteína Ligasas , Ubiquitinación/efectos de los fármacos

Asunto(s)

Cromatina/química , Cromatina/genética , Genes Homeobox , Acetilación , Animales , Núcleo Celular/química , Núcleo Celular/genética , Regulación del Desarrollo de la Expresión Génica , Histonas/química , Histonas/genética , Técnicas In Vitro , Modelos Genéticos

RESUMEN

The Nuclear Protein Database (NPD) is a curated database that contains information on more than 1300 vertebrate proteins that are thought, or are known, to localise to the cell nucleus. Each entry is annotated with information on predicted protein size and isoelectric point, as well as any repeats, motifs or domains within the protein sequence. In addition, information on the sub-nuclear localisation of each protein is provided and the biological and molecular functions are described using Gene Ontology (GO) terms. The database is searchable by keyword, protein name, sub-nuclear compartment and protein domain/motif. Links to other databases are provided (e.g. Entrez, SWISS-PROT, OMIM, PubMed, PubMed Central). Thus, NPD provides a gateway through which the nuclear proteome may be explored. The database can be accessed at http://npd.hgu.mrc.ac.uk and is updated monthly.

Asunto(s)

Núcleo Celular/química , Bases de Datos de Proteínas , Proteínas Nucleares , Secuencia de Aminoácidos , Animales , Almacenamiento y Recuperación de la Información , Proteínas Nucleares/análisis , Proteínas Nucleares/química , Proteínas Nucleares/genética , Proteínas Nucleares/fisiología , Conformación Proteica , Proteoma/fisiología , Vertebrados/genética

Asunto(s)

Prejuicio , Investigación/educación , Investigación/tendencias , Mujeres , Femenino , Humanos , Masculino , Publicaciones , Factores Sexuales

RESUMEN

Chromatin structure is important for regulating gene expression and for the proper condensation and segregation of chromosomes during cell division. Several human genetic diseases have been found to be due to mutations in genes producing proteins known or suspected to be involved in maintaining or modifying chromatin structure. Here we describe these 'chromatin diseases' and review what is known about the associated chromatin proteins in light of recent advances in the understanding of chromatin components, modification and function.

Asunto(s)

Cromatina/genética , Enfermedades Genéticas Congénitas/genética , ADN/química , ADN/genética , ADN/metabolismo , Regulación de la Expresión Génica , Silenciador del Gen , Histonas/genética , Humanos , Conformación Molecular , Transcripción Genética

RESUMEN

Combining fluorescence in situ hybridization (FISH) and indirect immunofluorescence staining of protein markers provides a highly specific method for identifying chromosomes in phenotypically defined cells and tissues. We developed a technique enabling dual chromosome painting and immunofluorescence staining of archival formalin-fixed, paraffin-embedded material, and used this to phenotype chimeric cells in female-to-male human liver transplants.

Asunto(s)

Trasplante de Hígado , Hígado/ultraestructura , Cromosoma Y , Pintura Cromosómica , Femenino , Técnica del Anticuerpo Fluorescente Indirecta , Humanos , Masculino , Fenotipo

RESUMEN

Many nuclear components participating in related pathways appear concentrated in specific areas of the mammalian nucleus. The importance of this organization is attested to by the dysfunction that correlates with mis-localization of nuclear proteins in human disease and cancer. Determining the sub-nuclear localization of proteins is therefore important for understanding genome regulation and function, and it also provides clues to function for novel proteins. However, the complexity of proteins in the mammalian nucleus is too large to tackle this on a protein by protein basis. Large-scale approaches to determining protein function and sub-cellular localization are required. We have used a visual gene trap screen to identify more than 100 proteins, many of which are normal, located within compartments of the mouse nucleus. The most common discrete localizations detected are at the nucleolus and the splicing speckles and on chromosomes. Proteins at the nuclear periphery, or in other nuclear foci, have also been identified. Several of the proteins have been implicated in human disease or cancer, e.g. ATRX, HMGI-C, NBS1 and EWS, and the gene-trapped proteins provide a route into further understanding their function. We find that sequence motifs are often shared amongst proteins co-localized within the same sub-nuclear compartment. Conversely, some generally abundant motifs are lacking from the proteins concentrated in specific areas of the nucleus. This suggests that we may be able to predict sub-nuclear localization for proteins in databases based on their sequence.

Asunto(s)

Núcleo Celular/metabolismo , Proteínas Nucleares/metabolismo , Animales , Proteínas Reguladoras de la Apoptosis , Transporte Biológico , Ciclo Celular/fisiología , Diferenciación Celular/fisiología , Línea Celular , Nucléolo Celular/metabolismo , Bases de Datos de Ácidos Nucleicos , Embrión de Mamíferos/citología , Embrión de Mamíferos/metabolismo , Embrión no Mamífero , Regulación de la Expresión Génica , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Transcripción Genética , Células Tumorales Cultivadas , beta-Galactosidasa/genética , beta-Galactosidasa/metabolismo

RESUMEN

Human ribosomal gene repeats are distributed among five nucleolar organizer regions (NORs) on the p arms of acrocentric chromosomes. On exit from mitosis, nucleoli form around individual active NORs. As cells progress through the cycle, these mini-nucleoli fuse to form large nucleoli incorporating multiple NORs. It is generally assumed that nucleolar incorporation of individual NORs is dependent on ribosomal gene transcription. To test this assumption, we determined the nuclear location of individual human acrocentric chromosomes, and their associated NORs, in mouse> human cell hybrids. Human ribosomal genes are transcriptionally silent in this context. Combined immunofluorescence and in situ hybridization (immuno-FISH) on three-dimensional preserved nuclei showed that human acrocentric chromosomes associate with hybrid cell nucleoli. Analysis of purified nucleoli demonstrated that human and mouse NORs are equally likely to be within a hybrid cell nucleolus. This is supported further by the observation that murine upstream binding factor can associate with human NORs. Incorporation of silent NORs into mature nucleoli raises interesting issues concerning the maintenance of the activity status of individual NORs.

Asunto(s)

Cromosomas Humanos/genética , ADN Ribosómico/genética , Silenciador del Gen , Región Organizadora del Nucléolo/genética , ARN Ribosómico 28S/genética , Transcripción Genética , Animales , Línea Celular , Núcleo Celular/genética , Núcleo Celular/ultraestructura , Cromosomas Humanos/ultraestructura , Técnica del Anticuerpo Fluorescente , Células HeLa , Humanos , Células Híbridas , Hibridación Fluorescente in Situ , Metafase , Ratones , Región Organizadora del Nucléolo/ultraestructura , Reacción en Cadena de la Polimerasa

RESUMEN

To fully understand genome function, the linear genome map must be integrated with a spatial map of chromosomes in the nucleus. Distinct nuclear addresses for a few human chromosomes have been described. Previously we have demonstrated that the gene-rich human chromosome 19 is located in a more central position in the nucleus than the similarly sized, but gene-poor, chromosome 18. To determine whether these two chromosomes are a paradigm for the organization of chromatin in the human nucleus, we have now analysed the nuclear organization of every human chromosome in diploid lymphoblasts and primary fibroblasts. We find that the most gene-rich chromosomes concentrate at the centre of the nucleus, whereas the more gene-poor chromosomes are located towards the nuclear periphery. In contrast, we find no significant relationship between chromosome size and position within the nucleus. Proteins of the nuclear membrane or lamina are candidates for molecules that might anchor regions of the genome at the nuclear periphery and it has been suggested that disruption of this organization may play a role in some disease pathologies. We show that the intranuclear organization of chromosomes is not altered in cells that lack the integral nuclear membrane protein emerin, from an individual with X-linked Emery--Dreifuss muscular dystrophy. This suggests that emerin is not necessary for localizing chromosomes at the nuclear periphery and that the muscular dystrophy phenotype in such individuals is not due to grossly altered nuclear organization of chromatin.

Asunto(s)

Núcleo Celular/genética , Cromosomas Humanos/genética , Proteínas de la Membrana/genética , Timopoyetinas/genética , Mapeo Cromosómico , Pintura Cromosómica , Femenino , Fibroblastos/citología , Fibroblastos/metabolismo , Genes/genética , Ligamiento Genético , Genoma Humano , Humanos , Hibridación Fluorescente in Situ , Linfocitos/citología , Linfocitos/metabolismo , Masculino , Distrofia Muscular de Emery-Dreifuss/genética , Distrofia Muscular de Emery-Dreifuss/patología , Mutación , Proteínas Nucleares , Cromosoma X/genética

Asunto(s)

Impresión Genómica/fisiología , Fase S/fisiología , Animales , Mamíferos , Schizosaccharomyces

RESUMEN

Amplification of sequences within mammalian chromosomes is often accompanied by the formation of homogeneously staining regions (HSRs). The arrangement of DNA sequences within such amplicons has been investigated, but little is known about the chromosome structure or behaviour of these unusual regions. We have analysed the metaphase chromosome structure of the dihydrofolate reductase (DHFR) amplicon of CHOC400 cells. The chromatin in this region contains hyperacetylated nucleosomes yet, at the same time, appears to be densely packed like heterochromatin. The region does not bind heterochromatin proteins. We show that the dense packing of the region is restricted to DNA located close to the chromosome core/scaffold. In contrast, levels of the chromosome scaffold protein topoisomerase II at HSRs are the same as those found at other euchromatic locations. Metaphase chromosome condensation of the HSR is shown to be sensitive to topoisomerase II inhibitors, and sister chromatids often appear to remain attached within the HSRs at metaphase. We suggest that these features underlie anaphase bridging and the aberrant interphase structure of the HSR. The DHFR amplicon is widely used as a model system to study mammalian DNA replication. We conclude that the higher-order chromosome structure of this amplicon is unusual and suggest that caution needs to be exercised in extrapolating data from HSRs to normal chromosomal loci.

Asunto(s)

Cromosomas/genética , ADN-Topoisomerasas de Tipo II , ADN/análisis , Secuencias Repetitivas de Ácidos Nucleicos/genética , Tetrahidrofolato Deshidrogenasa/genética , Anafase/genética , Animales , Antígenos de Neoplasias , Células CHO , Segregación Cromosómica , Cricetinae , ADN/aislamiento & purificación , Replicación del ADN , ADN-Topoisomerasas de Tipo II/metabolismo , Proteínas de Unión al ADN , Técnica del Anticuerpo Fluorescente , Hibridación Fluorescente in Situ , Isoenzimas/metabolismo , Cariotipificación , Metafase/genética , Replicón , Intercambio de Cromátides Hermanas

RESUMEN

Chromatin remodelling complexes containing the nucleosome-dependent ATPase ISWI were first isolated from Drosophila embryos (NURF, CHRAC and ACF). ISWI was the only common component reported in these complexes. Our purification of human CHRAC (HuCHRAC) shows that ISWI chromatin remodelling complexes can have a conserved subunit composition in completely different cell types, suggesting a conserved function of ISWI. We show that the human homologues of two novel putative histone-fold proteins in Drosophila CHRAC are present in HuCHRAC. The two human histone-fold proteins form a stable complex that binds naked DNA but not nucleosomes. HuCHRAC also contains human ACF1 (hACF1), the homologue of Acf1, a subunit of Drosophila ACF. The N-terminus of mouse ACF1 was reported as a heterochromatin-targeting domain. hACF1 is a member of a family of proteins with a related domain structure that all may target heterochromatin. We discuss a possible function for HuCHRAC in heterochromatin dynamics. HuCHRAC does not contain topoisomerase II, which was reported earlier as a subunit of Drosophila CHRAC.

Asunto(s)

Adenosina Trifosfatasas/metabolismo , ADN Polimerasa III , Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila , Histonas/metabolismo , Nucleoproteínas/metabolismo , Factores de Transcripción/metabolismo , Adenosina Trifosfatasas/química , Secuencia de Aminoácidos , Animales , Proteínas Cromosómicas no Histona , Proteínas de Unión al ADN/química , Drosophila , Células HeLa , Humanos , Ratones , Datos de Secuencia Molecular , Nucleoproteínas/química , Nucleosomas/metabolismo , Unión Proteica , Pliegue de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Factores de Transcripción/química

RESUMEN

CpG islands are found at the 5' end of approximately 60% of human genes and so are important genomic landmarks. They are concentrated in early-replicating, highly acetylated gene-rich regions. With respect to CpG island content, human Chrs 18 and 22 are very different from each other: Chr 18 appears to be CpG island poor, whereas Chr 22 appears to be CpG island rich. We have constructed and validated CpG island libraries from flow-sorted Chrs 18 and 22 and used these to estimate the difference in number of CpG islands found on these two chromosomes. These libraries contain normalized collections of sequences from the 5' end of genes. Clones from the libraries were sequenced and compared with the sequence databases; one third matched ESTs, thus anchoring these ESTs at the 5' end of their gene. However, it was striking that many clones either had no match or matched only existing CpG island clones. This suggests that a significant proportion of 5' gene sequences are absent from databases, presumably either because they are difficult to clone or the gene is poorly expressed and/or has a restricted expression pattern. This point should be taken into consideration if the currently available libraries are those used for the elucidation of complete, as opposed to partial, gene sequences. The Chr 18 and 22 CpG island libraries are a sequence resource for the isolation of such 5' gene sequences from specific human chromosomes.

Asunto(s)

Cromosomas Humanos Par 18 , Cromosomas Humanos Par 22 , Islas de CpG/genética , Secuencia de Bases , Mapeo Cromosómico , Clonación Molecular , Cartilla de ADN , Humanos , Hibridación de Ácido Nucleico , Reacción en Cadena de la Polimerasa

RESUMEN

Spatial organisation of the genome within the nucleus can play a role in maintaining the expressed or silent state of some genes [1]. There are distinct addresses for specific chromosomes, which have different functional characteristics, within the nuclei of dividing populations of human cells [2]. Here, we demonstrate that this level of nuclear architecture is altered in cells that have become either quiescent or senescent. Upon cell cycle exit, a gene-poor human chromosome moves from a location at the nuclear periphery to a more internal site in the nucleus, and changes its associations with nuclear substructures. The chromosome moves back toward the edge of the nucleus at a distinctive time after re-entry into the cell cycle. There is a 2-4 hour period at the beginning of G1 when the spatial organisation of these human chromosomes is established. Lastly, these experiments provide evidence that temporal control of DNA replication can be independent of spatial chromosome organisation. We conclude that the sub-nuclear organisation of chromosomes in quiescent or senescent mammalian somatic cells is fundamentally different from that in proliferating cells and that the spatial organisation of the genome is plastic.

Asunto(s)

División Celular/fisiología , Núcleo Celular/genética , Núcleo Celular/ultraestructura , Senescencia Celular/fisiología , Núcleo Celular/química , Células Cultivadas , Cromosomas Humanos Par 18/genética , Cromosomas Humanos Par 18/fisiología , Cromosomas Humanos Par 19/genética , Cromosomas Humanos Par 19/fisiología , Replicación del ADN , Fibroblastos , Humanos , Microscopía Confocal

RESUMEN

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 alpha-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.

Asunto(s)

Centrómero/química , ADN Helicasas , Proteínas de Unión al ADN/análisis , Heterocromatina/química , Proteínas Nucleares/análisis , Factores de Transcripción/análisis , Animales , Anticuerpos Monoclonales/inmunología , Células COS , Fraccionamiento Celular , Línea Celular Transformada , Homólogo de la Proteína Chromobox 5 , Proteínas Cromosómicas no Histona/metabolismo , Cromosomas/química , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/inmunología , Células HeLa , Humanos , Ratones , Ratones Endogámicos BALB C , Proteínas Nucleares/genética , Proteínas Nucleares/inmunología , Ovinos , Factores de Transcripción/genética , Factores de Transcripción/inmunología , Proteína Nuclear Ligada al Cromosoma X

Asunto(s)

Cromosomas/genética , Genoma , Animales , Ciclo Celular , Núcleo Celular/genética , Humanos , Plantas , Recombinación Genética , Escocia , Levaduras

RESUMEN

Using fluorescence in situ hybridization we show striking differences in nuclear position, chromosome morphology, and interactions with nuclear substructure for human chromosomes 18 and 19. Human chromosome 19 is shown to adopt a more internal position in the nucleus than chromosome 18 and to be more extensively associated with the nuclear matrix. The more peripheral localization of chromosome 18 is established early in the cell cycle and is maintained thereafter. We show that the preferential localization of chromosomes 18 and 19 in the nucleus is reflected in the orientation of translocation chromosomes in the nucleus. Lastly, we show that the inhibition of transcription can have gross, but reversible, effects on chromosome architecture. Our data demonstrate that the distribution of genomic sequences between chromosomes has implications for nuclear structure and we discuss our findings in relation to a model of the human nucleus that is functionally compartmentalized.

Asunto(s)

Núcleo Celular/genética , Cromosomas Humanos Par 18/ultraestructura , Cromosomas Humanos Par 19/ultraestructura , Ciclo Celular/efectos de los fármacos , Línea Celular , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Células Cultivadas , Centrómero/metabolismo , Centrómero/ultraestructura , Cromosomas Humanos Par 18/química , Cromosomas Humanos Par 18/genética , Cromosomas Humanos Par 18/metabolismo , Cromosomas Humanos Par 19/química , Cromosomas Humanos Par 19/genética , Cromosomas Humanos Par 19/metabolismo , ADN/metabolismo , Dactinomicina/farmacología , Diclororribofuranosil Benzoimidazol/farmacología , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Inhibidores de Histona Desacetilasas , Histona Desacetilasas/metabolismo , Humanos , Ácidos Hidroxámicos/farmacología , Hibridación Fluorescente in Situ , Linfocitos/citología , Linfocitos/efectos de los fármacos , Linfocitos/metabolismo , Matriz Nuclear/efectos de los fármacos , Matriz Nuclear/genética , Matriz Nuclear/metabolismo , ARN Polimerasa II/antagonistas & inhibidores , ARN Polimerasa II/metabolismo , Telómero/metabolismo , Telómero/ultraestructura , Transcripción Genética/efectos de los fármacos , Translocación Genética

Asunto(s)

Cromatina/genética , Cromatina/ultraestructura , Cromosomas/genética , Cromosomas/ultraestructura , Hibridación Fluorescente in Situ/métodos , Animales , Fraccionamiento Celular/métodos , Cromosomas Humanos/genética , Cromosomas Humanos/ultraestructura , ADN/análisis , ADN/genética , Humanos , Indicadores y Reactivos , Linfocitos/citología , Metafase , Ratones , Mitosis , Reacción en Cadena de la Polimerasa/métodos

RESUMEN

The first complete genomic sequence of a eukaryote (Saccharomyces cerevisiae) has already been accomplished. It is estimated that the sequence of the human genome will be known early in the next millennium. Yet it is already apparent that, despite their immense length, these linear primary sequence maps will be inadequate descriptions of the eukaryotic genome, be it of a budding yeast or a human. To reflect our growing awareness of the importance of spatial context in chromosome function and in gene expression we argue that a more complete map of the genome should seek to embody the richness of information that we expect of the maps we use to navigate our way around the outside world.

Asunto(s)

Mapeo Cromosómico , Animales , Secuencia de Bases , ADN , Genoma Humano , Humanos , Mitosis , Datos de Secuencia Molecular , Saccharomyces cerevisiae/genética