RESUMEN
More than a decade of massive DNA sequencing efforts have generated a large body of genomic, transcriptomic and epigenomic information that has provided a more and more detailed view of the functional elements and transactions within the human genome. Considerable efforts have also focused on linking these elements with one another by mapping their interactions and by establishing 3-dimensional (3D) genomic landscapes in various cell and tissue types. In parallel, multiple studies have associated genomic deletions, duplications and other rearrangements with human pathologies. In this review, we explore recent progresses that have allowed connecting disease-causing alterations with perturbations of the 3D genome organization.
Asunto(s)
Predisposición Genética a la Enfermedad , Genoma Humano , Genómica , Salud , Cromosomas Humanos , Regulación de la Expresión Génica , Estudios de Asociación Genética/métodos , Genómica/métodos , Gónadas/embriología , Gónadas/metabolismo , Humanos , Desarrollo de Músculos/genéticaRESUMEN
Coating of the X chromosome by Xist RNA is an essential trigger for X inactivation. However, little is known about the early chromatin remodeling events that transform this signal into transcriptional silencing. Here we report that methylation of histone H3 lysine 9 on the inactive X chromosome occurs immediately after Xist RNA coating and before transcriptional inactivation of X-linked genes. X-chromosomal H3 Lys-9 methylation occurs during the same window of time as H3 Lys-9 hypoacetylation and H3 Lys-4 hypomethylation. Histone H3 modifications thus represent the earliest known chromatin changes during X inactivation. We also identify a unique "hotspot" of H3 Lys-9 methylation 5' to Xist, and we propose that this acts as a nucleation center for Xist RNA-dependent spread of inactivation along the X chromosome via H3 Lys-9 methylation.
Asunto(s)
Diferenciación Celular/fisiología , Cromatina/metabolismo , Proteínas Cromosómicas no Histona , Compensación de Dosificación (Genética) , Histonas/metabolismo , Proteínas Proto-Oncogénicas , Proteínas Represoras , Cromosoma X/metabolismo , Proteínas de Anclaje a la Quinasa A , Acetilación , Proteínas Adaptadoras Transductoras de Señales , Animales , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Células Cultivadas , Inhibidor p27 de las Quinasas Dependientes de la Ciclina , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Inhibidores Enzimáticos/metabolismo , Femenino , Fibroblastos/fisiología , Silenciador del Gen , Hibridación Fluorescente in Situ , Masculino , Proteína 2 de Unión a Metil-CpG , Metilación , Ratones , Antígenos de Histocompatibilidad Menor , Modelos Biológicos , Proteínas Oncogénicas/genética , Proteínas Oncogénicas/metabolismo , ARN/metabolismo , ARN Largo no Codificante , ARN no Traducido/genética , ARN no Traducido/metabolismo , Células Madre/fisiología , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismoRESUMEN
Image restoration approaches, such as digital deconvolution, are becoming widely used for improving the quality of microscopic images. However, no quantification of the gain in resolution of fluorescence images is available. We show that, after iterative constrained deconvolution, fluorescent cosmid signals appear to be 25% smaller, and 1.2-kb fragment signals on combed molecules faithfully display the expected length.
Asunto(s)
ADN de Hongos/metabolismo , Hibridación Fluorescente in Situ/métodos , Cromosomas Artificiales de Levadura/genética , Cromosomas Artificiales de Levadura/metabolismo , Cósmidos/genética , Sondas de ADN/genética , ADN de Hongos/genética , Fibroblastos , Fluorescencia , Humanos , Sensibilidad y Especificidad , Levaduras/citología , Levaduras/genéticaRESUMEN
The SAGA complex of Saccharomyces cerevisiae is required for the transcription of many RNA polymerase II-dependent genes. Previous studies have demonstrated that SAGA possesses histone acetyltransferase activity, catalyzed by the SAGA component Gcn5. However, the transcription of many genes, although SAGA dependent, is Gcn5 independent, suggesting the existence of distinct SAGA activities. We have studied the in vivo role of two other SAGA components, Spt3 and Spt20, at the well-characterized GAL1 promoter. Our results demonstrate that both Spt3 and Spt20 are required for the binding of TATA-binding protein but not of the activator Gal4 and that this role is Gcn5 independent. These results suggest a coactivator role for Spt3 and Spt20 in the recruitment of TBP.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/fisiología , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/fisiología , Galactoquinasa/genética , Regiones Promotoras Genéticas , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Factores de Transcripción/metabolismo , Factores de Transcripción/fisiología , Transcripción Genética , Acetilación , Proteínas de Unión al ADN/genética , Inducción Enzimática , Proteínas Fúngicas/genética , Galactoquinasa/biosíntesis , Eliminación de Gen , Regulación Fúngica de la Expresión Génica , Histona Acetiltransferasas , Sustancias Macromoleculares , Unión Proteica , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Proteína de Unión a TATA-Box , Factores de Transcripción/genéticaAsunto(s)
Impresión Genómica , Ligasas/genética , ARN sin Sentido/genética , ARN Mensajero/genética , Síndrome de Angelman/genética , Secuencia de Bases , Cromosomas Humanos Par 15 , Femenino , Humanos , Reacción en Cadena de la Polimerasa , Síndrome de Prader-Willi/genética , Ubiquitina-Proteína LigasasRESUMEN
The clinical features of Angelman syndrome (AS) include microcephaly, severe mental retardation, "puppet-like" ataxic gait with jerky arm movements, hyperactivity, bouts of inappropriate laughter, EEG abnormalities, and seizures. The frequency of occurrence of AS is in the range of 1/10,000 to 1/20,000 births. The AS locus maps to the imprinted chromosome 15q11-q13 region and the disease is caused by the absence of a normal maternal contribution to this region. The genetic complexity of AS is revealed by the existence of at least four molecular classes. A candidate AS gene, ubiquitin protein ligase 3A (UBE3A/E6-AP), has been identified, and mutations of this gene have been detected in several cases of AS. Moreover, UBE3A is expressed predominantly from the maternal allele in brain, strongly supporting its causative role in AS. However, there is evidence to suggest that, in addition to UBE3A, another gene(s) may be involved either directly in AS and/or indirectly by regulating UBE3A expression.
Asunto(s)
Síndrome de Angelman/enzimología , Síndrome de Angelman/genética , Cromosomas Humanos Par 15/genética , Silenciador del Gen/fisiología , Impresión Genómica/genética , Ligasas/genética , Alelos , Empalme Alternativo , Síndrome de Angelman/etiología , Animales , Humanos , Isoenzimas/biosíntesis , Isoenzimas/genética , Ligasas/biosíntesis , Ratones , Mutación , Especificidad de Órganos , ARN Mensajero/biosíntesis , Ubiquitina-Proteína LigasasRESUMEN
Prader-Willi syndrome (PWS) is a neurogenetic disorder that results from the absence of a normal paternal contribution to the 15q11-13 region. The clinical manifestations of PWS are a transient severe hypotonia in the newborn period, with mental retardation, hypogonadism and obesity observed later in development. Five transcripts with exclusive expression from the paternal allele have been isolated, but none of these has been shown to be involved in PWS. In this study, we report the isolation and characterization of NDN, a new human imprinted gene. NDN is exclusively expressed from the paternal allele in the tissues analysed and is located in the PWS region. It encodes a putative protein homologous to the mouse brain-specific NECDIN protein, NDN; as in mouse, expression in brain is restricted to post-mitotic neurons. NDN displays several characteristics of an imprinted locus, including allelic DNA methylation and asynchronous DNA replication. A complete lack of NDN expression in PWS brain and fibroblasts indicates that the gene is expressed exclusively from the paternal allele in these tissues and suggests a possible role of this new gene in PWS.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Impresión Genómica , Proteínas del Tejido Nervioso/genética , Proteínas Nucleares/genética , Síndrome de Prader-Willi/genética , Síndrome de Angelman/genética , Animales , Northern Blotting , Mapeo Cromosómico , Cromosomas Humanos Par 15 , Metilación de ADN , Desoxirribonucleasas de Localización Especificada Tipo II/genética , Femenino , Humanos , Hibridación in Situ/métodos , Hibridación Fluorescente in Situ , Masculino , Ratones , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/metabolismo , Fenómenos Fisiológicos del Sistema Nervioso , Proteínas Nucleares/metabolismo , Distribución TisularRESUMEN
The X inactivation center candidate region (Xic/XIC in mouse and human) is poorly characterized for the presence of transcription units. Only two conserved genes have been isolated to date, Xist/XIST and Cdx4/CDX4. The other known gene lying within this region, Tsx, has been identified so far only in rodents by analyzing the complete genomic sequence of a 94-kb region distal to Xist. Here, we report the characterization of an additional gene lying within this 94-kb sequenced region. Brx, for Brain X-linked gene, is a rare transcript preferentially expressed in the brain. It is normally X-inactivated in the mouse. Localisation of BRX, its human homolog has shown the gene to be located within the orthologous but inverted human CDX4-XIST segment. These results suggest that the gene order of the region encompassing the Cdx4-Xist interval in the mouse is similar in human. Comparison of the Xist-Brx and Brx-Cdx4 regions in mouse and human indicates that these intervals are three times longer in human than in mouse. BRX is a new potential candidate for one of the X-linked mental retardation syndromes mapped within the pericentromeric region of the human X Chromosome (Chr).
Asunto(s)
Química Encefálica/genética , Expresión Génica/genética , Cromosoma X/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Mapeo Cromosómico , Cromosomas Artificiales de Levadura , Secuencia Conservada , ADN/aislamiento & purificación , ADN Complementario , Ligamiento Genético , Humanos , Ratones , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , ARN/aislamiento & purificación , Alineación de Secuencia , Análisis de Secuencia de ADNAsunto(s)
Síndrome de Angelman/genética , Encéfalo/enzimología , Ligasas/genética , Adulto , Síndrome de Angelman/enzimología , Preescolar , Inversión Cromosómica , Cromosomas Humanos Par 15 , Elementos Transponibles de ADN , Femenino , Mutación del Sistema de Lectura , Impresión Genómica , Humanos , Ligasas/biosíntesis , Linfocitos/enzimología , Especificidad de Órganos , Mutación Puntual , Valores de Referencia , Eliminación de Secuencia , Transcripción Genética , Ubiquitina-Proteína LigasasAsunto(s)
Mapeo Cromosómico , Seudogenes , ARN no Traducido , Factores de Transcripción/genética , Animales , Secuencia de Bases , Secuencia de Consenso , Humanos , Ratones , Datos de Secuencia Molecular , ARN Largo no Codificante , Ratas , Mapeo Restrictivo , Homología de Secuencia de Ácido Nucleico , Cromosoma XRESUMEN
The initiation of X-chromosome inactivation in female mammals is controlled by a key locus, the X-inactivation centre (Xic). The Xist gene, which maps to the candidate region for Xic and is expressed exclusively from the inactive X chromosome, is thought to be an essential component of the Xic. To test whether sequences spanning several hundred kilobases and including Xist from the Xic region are capable of initiating inactivation, we have created a series of transgenic mice using a 460 kb yeast artificial chromosome (YAC). Analysis in these mice of the expression of Xist, of a LacZ reporter gene and of two genes in the region that are normally silent on the inactive X chromosome, suggests that essential sequences for Xist expression and X-inactivation may be absent in these transgenic animals.
Asunto(s)
Cromosomas Artificiales de Levadura , Compensación de Dosificación (Genética) , ARN no Traducido , Factores de Transcripción/genética , Animales , Secuencia de Bases , Cartilla de ADN , Femenino , Dosificación de Gen , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/genética , Humanos , Operón Lac , Masculino , Ratones , Ratones Endogámicos C3H , Ratones Transgénicos , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/genética , ARN Largo no Codificante , TransgenesRESUMEN
The X inactivation centre (Xic) is a cis-acting locus thought to play a key role in the initiation of X-inactivation. We have cloned and characterized a new gene, Bpx, lying distal to the murine Xist. Bpx, which is specifically expressed in the brain, shows strong homology to genes encoding nucleosome assembly proteins and is normally X-inactivated in mice. Isolation and localization of BPX, its human homologue, has shown the gene to be located centromeric to XIST in man. The Xq13 region, whose orientation is apparently globally conserved between man and mouse, must therefore contain an inversion of at least 600 kb spanning the XIST sequence and including the CDX4 and BPX genes.
Asunto(s)
Química Encefálica , Compensación de Dosificación (Genética) , Genes/genética , Proteínas del Tejido Nervioso/genética , ARN no Traducido , Homología de Secuencia de Aminoácido , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Mapeo Cromosómico , Clonación Molecular , Femenino , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/genética , Humanos , Masculino , Ratones , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/química , Especificidad de Órganos , ARN Largo no Codificante , ARN Mensajero/análisis , ARN Mensajero/genética , Análisis de Secuencia de ADN , Homología de Secuencia de Ácido Nucleico , Factores de Transcripción/genética , Cromosoma X/genéticaRESUMEN
The Xist sequence has several characteristics that make it a potential candidate for the X-inactivation center. To investigate the role of Xist and adjacent sequences lying within the X-inactivation center candidate region, a 460-kb region surrounding the murine Xist sequence has been arrayed in lambda contigs with a combination of IRS-PCR-based hybridization and YAC fragmentation. The orientation of the Xist sequence in relation to the telomere and centromere of the X Chromosome (Chr) has been established with this contig and shown to be inverted compared to that in human.
Asunto(s)
ARN no Traducido , Factores de Transcripción/genética , Cromosoma X , Animales , Bacteriófago lambda/genética , Secuencia de Bases , Cromosomas Artificiales de Levadura , Clonación Molecular , Compensación de Dosificación (Genética) , Femenino , Masculino , Ratones , Ratones Endogámicos C3H , Ratones Transgénicos , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos , Reacción en Cadena de la Polimerasa , ARN Largo no CodificanteRESUMEN
Cytochrome P-450 (Cyp) 51 or lanosterol-C14-demethylase is the main target for antifungal compounds of the triazole family like ketoconazole (Kz). Disruption of the associated NADPH-P-450 reductase-encoding gene (YRED) is not lethal, but decreases by about 20-fold the Kz resistance (KzR) of wild-type (wt) Saccharomyces cerevisiae. Transformation of a YRED-disrupted strain by a yeast genomic library based on a multicopy vector allowed us to identify a suppressor of Kz hypersensitivity. Deletion analysis of the 5-kb cloned fragment indicated that yeast cytochrome b5-encoding gene (CYB5), which encodes a 120-amino-acid (aa) protein, is required and sufficient for the suppressor effect. The encoded polypeptide shares about 30% aa identity with mammalian cytochromes b5 (Cyb5). CYB5 disruption and tetrad analysis demonstrate that yeast Cyb5 is not required for growth in a Yred+ strain. Determination of the microsomal content of b-type cytochromes by differential spectra indicated the presence of a strongly decreased or null Cyb5 level in the disrupted strain. This confirms that we have cloned the gene encoding the major microsomal form of Cyb5 which appears not to be essential. Minor Cyb5 isoforms could also be present in yeast or other redox proteins could substitute for the pleiotropic roles of Cyb5 in the sterol and lipid biosynthesis pathways.
Asunto(s)
Citocromos b5/genética , Cetoconazol/farmacología , NADPH-Ferrihemoproteína Reductasa/metabolismo , Saccharomyces cerevisiae/genética , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , ADN de Hongos , Farmacorresistencia Microbiana/genética , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Fenotipo , Mapeo Restrictivo , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/enzimología , Homología de Secuencia de Aminoácido , Transformación GenéticaRESUMEN
The X Chromosome (Chr) controlling element locus (Xce) in the mouse has been shown to influence the X inactivation process. Xce maps to the central region of the X Chr, which also contains the Xist sequence, itself possibly implicated in the X inactivation process. Three microsatellite markers spanning the Xist locus have been isolated from an Xist containing YAC. All three microsatellite markers showed complete linkage with Xce in recombinants for the central span of the mouse X Chr between Ta and Moblo and strong linkage disequilibrium with Xce in all but one of the inbred mouse strains tested. In the standard Xceb typing strain JU/Ct, the two microsatellites most closely flanking Xist fail to carry the allelic forms expected if Xist and Xce are synonymous. Alternative explanations for this finding are presented in the context of our search for understanding the relation between Xist and Xce.
Asunto(s)
Mapeo Cromosómico , Secuencias Repetitivas de Ácidos Nucleicos , Cromosoma X , Animales , Secuencia de Bases , Cromosomas Artificiales de Levadura , ADN Satélite , Compensación de Dosificación (Genética) , Marcadores Genéticos , Ratones , Ratones Endogámicos , Datos de Secuencia Molecular , MuridaeRESUMEN
The Xist sequence has been proposed as a potential candidate for the X-inactivation center based both on its localization within the candidate region for the X-inactivation center in man and mouse and on its unique pattern of expression from the inactive X chromosome. We have cloned 550 kb of DNA surrounding the mouse Xist sequence in contiguously overlapping YAC clones and have developed a long-range restriction map that spans almost 1 Mb of this region and includes this YAC contig. The detailed restriction map we have established provides a framework for the identification of expressed sequences other than Xist that may equally exhibit unusual expression characteristics associated with X inactivation. The presence of possible structural or methylation differences within this region between the active and inactive X chromosomes has been investigated through comparative analysis of male and female genomic DNA, and we report here the identification of certain CpG-containing restriction sites around Xist that have an interesting differential methylation status on the inactive and active X chromosomes.
Asunto(s)
Compensación de Dosificación (Genética) , Cromosoma X , Animales , Cromosomas Fúngicos , ADN , Fosfatos de Dinucleósidos/metabolismo , Femenino , Eliminación de Gen , Reordenamiento Génico , Biblioteca Genómica , Humanos , Células Híbridas , Masculino , Metilación , Ratones , Mapeo RestrictivoRESUMEN
We have developed a novel strategy, based on the random insertion by homologous recombination of artificial I-Sce I sites within mammalian repetitive DNA sequences, which should greatly facilitate the high resolution physical mapping of large DNA fragments cloned in YAC. A set of transgenic yeast strains containing appropriately spaced I-Sce I sites within the YAC insert defines a series of nested physical intervals against which new genes, clones or DNA fragments can be mapped by simple hybridisation. Sequential hybridisation using such a series of nested YAC fragments as probes can also allow the rapid sorting of phage or cosmid libraries into contigs. This approach, which has been applied to a YAC containing a 460 kb insert from the mouse X chromosome, may also have applications for the restriction mapping of large genomic segments, mapping of exons and the search for homologous genes.