RESUMEN
Cornelia de Lange syndrome (CdLS) is a rare neurodevelopmental syndrome for which mutations in five causative genes that encode (SMC1A, SMC3, RAD21) or regulate (NIPBL, HDAC8) the cohesin complex, account for ~70% of cases. Herein we report on four female Subjects who were found to carry novel intragenic deletions in HDAC8. In one case, the deletion was found in mosaic state and it was determined to be present in ~38% of blood lymphocytes and in nearly all cells of a buccal sample. All deletions, for which parental blood samples were available, were shown to have arisen de novo. X-chromosome inactivation studies demonstrated marked skewing, suggesting strong selection against the mutated HDAC8 allele. Based on an investigation of the deletion breakpoints, we hypothesize that microhomology-mediated replicative mechanisms may be implicated in the formation of some of these rearrangements. This study broadens the mutational spectrum of HDAC8, provides the first description of a causative HDAC8 somatic mutation and increases the knowledge on possible mutational mechanisms underlying copy number variations in HDAC8. Moreover our findings highlight the clinical utility of considering copy number analysis in HDAC8 as well as the analysis on DNA from more than one tissue as an indispensable part of the routine molecular diagnosis of individuals with CdLS or CdLS-overlapping features.
Asunto(s)
Síndrome de Cornelia de Lange/diagnóstico , Síndrome de Cornelia de Lange/genética , Estudios de Asociación Genética , Histona Desacetilasas/genética , Fenotipo , Proteínas Represoras/genética , Eliminación de Secuencia , Secuencia de Bases , Niño , Preescolar , Puntos de Rotura del Cromosoma , Hibridación Genómica Comparativa , Variaciones en el Número de Copia de ADN , Exones , Facies , Femenino , Duplicación de Gen , Humanos , Análisis de Secuencia de ADN , Inactivación del Cromosoma XRESUMEN
Dietary restriction (DR) extends lifespan in a wide variety of species, yet the underlying mechanisms are not well understood. Here we show that the Caenorhabditis elegans HNF4α-related nuclear hormone receptor NHR-62 is required for metabolic and physiologic responses associated with DR-induced longevity. nhr-62 mediates the longevity of eat-2 mutants, a genetic mimetic of dietary restriction, and blunts the longevity response of DR induced by bacterial food dilution at low nutrient levels. Metabolic changes associated with DR, including decreased Oil Red O staining, decreased triglyceride levels, and increased autophagy are partly reversed by mutation of nhr-62. Additionally, the DR fatty acid profile is altered in nhr-62 mutants. Expression profiles reveal that several hundred genes induced by DR depend on the activity of NHR-62, including a putative lipase required for the DR response. This study provides critical evidence of nuclear hormone receptor regulation of the DR longevity response, suggesting hormonal and metabolic control of life span.
Asunto(s)
Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/genética , Restricción Calórica , Factor Nuclear 4 del Hepatocito/genética , Longevidad/genética , Receptores Citoplasmáticos y Nucleares/genética , Animales , Autofagia , Caenorhabditis elegans/fisiología , Proteínas de Caenorhabditis elegans/metabolismo , Ácidos Grasos/metabolismo , Factor Nuclear 4 del Hepatocito/metabolismo , Mutación , Transducción de SeñalRESUMEN
Endogenous small molecule metabolites that regulate animal longevity are emerging as a novel means to influence health and life span. In C. elegans, bile acid-like steroids called the dafachronic acids (DAs) regulate developmental timing and longevity through the conserved nuclear hormone receptor DAF-12, a homolog of mammalian sterol-regulated receptors LXR and FXR. Using metabolic genetics, mass spectrometry, and biochemical approaches, we identify new activities in DA biosynthesis and characterize an evolutionarily conserved short chain dehydrogenase, DHS-16, as a novel 3-hydroxysteroid dehydrogenase. Through regulation of DA production, DHS-16 controls DAF-12 activity governing longevity in response to signals from the gonad. Our elucidation of C. elegans bile acid biosynthetic pathways reveals the possibility of novel ligands as well as striking biochemical conservation to other animals, which could illuminate new targets for manipulating longevity in metazoans.
Asunto(s)
3-Hidroxiesteroide Deshidrogenasas/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/enzimología , Caenorhabditis elegans/crecimiento & desarrollo , Longevidad , 3-Hidroxiesteroide Deshidrogenasas/genética , Animales , Ácidos y Sales Biliares/metabolismo , Ácidos y Sales Biliares/fisiología , Vías Biosintéticas , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Colestenos/metabolismo , Colesterol/metabolismo , Colesterol/fisiología , Sistema Enzimático del Citocromo P-450/genética , Sistema Enzimático del Citocromo P-450/metabolismo , Epistasis Genética , Retroalimentación Fisiológica , Perfilación de la Expresión Génica , Homeostasis , Insulina/fisiología , Factor I del Crecimiento Similar a la Insulina/fisiología , Cetosteroides/metabolismo , Especificidad de Órganos , Fenotipo , Receptores Citoplasmáticos y Nucleares/metabolismo , Reproducción , Transducción de SeñalRESUMEN
Pathways of mutagenesis are induced in microbes under adverse conditions controlled by stress responses. Control of mutagenesis by stress responses may accelerate evolution specifically when cells are maladapted to their environments, i.e. are stressed. Stress-induced mutagenesis in the Escherichia coli Lac assay occurs either by 'point' mutation or gene amplification. Point mutagenesis is associated with DNA double-strand-break (DSB) repair and requires DinB error-prone DNA polymerase and the SOS DNA-damage- and RpoS general-stress responses. We report that the RpoE envelope-protein-stress response is also required. In a screen for mutagenesis-defective mutants, we isolated a transposon insertion in the rpoE P2 promoter. The insertion prevents rpoE induction during stress, but leaves constitutive expression intact, and allows cell viability. rpoE insertion and suppressed null mutants display reduced point mutagenesis and maintenance of amplified DNA. Furthermore, sigma(E) acts independently of stress responses previously implicated: SOS/DinB and RpoS, and of sigma(32), which was postulated to affect mutagenesis. I-SceI-induced DSBs alleviated much of the rpoE phenotype, implying that sigma(E) promoted DSB formation. Thus, a third stress response and stress input regulate DSB-repair-associated stress-induced mutagenesis. This provides the first report of mutagenesis promoted by sigma(E), and implies that extracytoplasmic stressors may affect genome integrity and, potentially, the ability to evolve.
Asunto(s)
Proteínas de Escherichia coli/metabolismo , Escherichia coli/genética , Respuesta SOS en Genética , Factor sigma/metabolismo , Roturas del ADN de Doble Cadena , Reparación del ADN , Elementos Transponibles de ADN , ADN Bacteriano/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Mutagénesis Insercional , Mutación Puntual , Regiones Promotoras Genéticas , Factor sigma/genética , Estrés FisiológicoRESUMEN
Mutations in the dystrophin gene (DMD) cause Duchenne and Becker muscular dystrophies and the majority of cases are due to DMD gene rearrangements. Despite the high incidence of these aberrations, little is known about their causative molecular mechanism(s). We examined 792 DMD/BMD clinical samples by oligonucleotide array-CGH and report on the junction sequence analysis of 15 unique deletion cases and three complex intragenic rearrangements to elucidate potential underlying mechanism(s). Furthermore, we present three cases with intergenic rearrangements involving DMD and neighboring loci. The cases with intragenic rearrangements include an inversion with flanking deleted sequences; a duplicated segment inserted in direct orientation into a deleted region; and a splicing mutation adjacent to a deletion. Bioinformatic analysis demonstrated that 7 of 12 breakpoints combined among 3 complex cases aligned with repetitive sequences, as compared to 4 of 30 breakpoints for the 15 deletion cases. Moreover, the inversion/deletion case may involve a stem-loop structure that has contributed to the initiation of this rearrangement. For the duplication/deletion and splicing mutation/deletion cases, the presence of the first mutation, either a duplication or point mutation, may have elicited the deletion events in an attempt to correct preexisting mutations. While NHEJ is one potential mechanism for these complex rearrangements, the highly complex junction sequence of the inversion/deletion case suggests the involvement of a replication-based mechanism. Our results support the notion that regional genomic instability, aided by the presence of repetitive elements, a stem-loop structure, and possibly preexisting mutations, may elicit complex rearrangements of the DMD gene.
Asunto(s)
Distrofina/genética , Reordenamiento Génico , Mutación , Empalme Alternativo , Hibridación Genómica Comparativa , Biología Computacional/métodos , Femenino , Eliminación de Gen , Humanos , Recién Nacido , Masculino , Hibridación de Ácido Nucleico , Análisis de Secuencia por Matrices de Oligonucleótidos , Embarazo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Nuclear receptors are a class of hormone-gated transcription factors found in metazoans that regulate global changes in gene expression when bound to their cognate ligands. Despite species diversification, nuclear receptors function similarly across taxa, having fundamental roles in detecting intrinsic and environmental signals, and subsequently in coordinating transcriptional cascades that direct reproduction, development, metabolism and homeostasis. These endocrine receptors function in vivo in part as molecular switches and timers that regulate transcriptional cascades. Several Caenorhabditis elegans nuclear receptors integrate intrinsic and extrinsic signals to regulate the dauer diapause and longevity, molting, and heterochronic circuits of development, and are comparable to similar in vivo endocrine regulated processes in other animals.
Asunto(s)
Caenorhabditis elegans/fisiología , Receptores Citoplasmáticos y Nucleares/fisiología , AnimalesRESUMEN
Gene amplification is a collection of processes whereby a DNA segment is reiterated to multiple copies per genome. It is important in carcinogenesis and resistance to chemotherapeutic agents, and can underlie adaptive evolution via increased expression of an amplified gene, evolution of new gene functions, and genome evolution. Though first described in the model organism Escherichia coli in the early 1960s, only scant information on the mechanism(s) of amplification in this system has been obtained, and many models for mechanism(s) were possible. More recently, some gene amplifications in E. coli were shown to be stress-inducible and to confer a selective advantage to cells under stress (adaptive amplifications), potentially accelerating evolution specifically when cells are poorly adapted to their environment. We focus on stress-induced amplification in E. coli and report several findings that indicate a novel molecular mechanism, and we suggest that most amplifications might be stress-induced, not spontaneous. First, as often hypothesized, but not shown previously, certain proteins used for DNA double-strand-break repair and homologous recombination are required for amplification. Second, in contrast with previous models in which homologous recombination between repeated sequences caused duplications that lead to amplification, the amplified DNAs are present in situ as tandem, direct repeats of 7-32 kilobases bordered by only 4 to 15 base pairs of G-rich homology, indicating an initial non-homologous recombination event. Sequences at the rearrangement junctions suggest nonhomologous recombination mechanisms that occur via template switching during DNA replication, but unlike previously described template switching events, these must occur over long distances. Third, we provide evidence that 3'-single-strand DNA ends are intermediates in the process, supporting a template-switching mechanism. Fourth, we provide evidence that lagging-strand templates are involved. Finally, we propose a novel, long-distance template-switching model for the mechanism of adaptive amplification that suggests how stress induces the amplifications. We outline its possible applicability to amplification in humans and other organisms and circumstances.
Asunto(s)
Escherichia coli/genética , Secuencia de Bases , ADN/química , Daño del ADN , Reparación del ADN , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Amplificación de Genes , Genes Bacterianos , Genómica , Datos de Secuencia Molecular , Mutación , Recombinación Genética , Homología de Secuencia de Ácido NucleicoRESUMEN
Hormone-gated nuclear receptors (NRs) are conserved transcriptional regulators of metabolism, reproduction, and homeostasis. Here we show that C. elegans NHR-8 NR, a homolog of vertebrate liver X and vitamin D receptors, regulates nematode cholesterol balance, fatty acid desaturation, apolipoprotein production, and bile acid metabolism. Loss of nhr-8 results in a deficiency in bile acid-like steroids, called the dafachronic acids, which regulate the related DAF-12/NR, thus controlling entry into the long-lived dauer stage through cholesterol availability. Cholesterol supplementation rescues various nhr-8 phenotypes, including developmental arrest, unsaturated fatty acid deficiency, reduced fertility, and shortened life span. Notably, nhr-8 also interacts with daf-16/FOXO to regulate steady-state cholesterol levels and is synthetically lethal in combination with insulin signaling mutants that promote unregulated growth. Our studies provide important insights into nuclear receptor control of cholesterol balance and metabolism and their impact on development, reproduction, and aging in the context of larger endocrine networks.
Asunto(s)
Ácidos y Sales Biliares/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Colesterol/metabolismo , Metabolismo de los Lípidos/genética , Receptores Citoplasmáticos y Nucleares/metabolismo , Secuencia de Aminoácidos , Animales , Apolipoproteínas/biosíntesis , Transporte Biológico , Caenorhabditis elegans/genética , Colestenos/metabolismo , Ácidos Grasos/metabolismo , Fertilidad/genética , Factores de Transcripción Forkhead , Regulación de la Expresión Génica , Homeostasis , Longevidad/genética , Datos de Secuencia Molecular , Oxigenasas/metabolismo , Alineación de Secuencia , Transducción de Señal/genética , Factores de Transcripción/metabolismoRESUMEN
Bile acids are cholesterol-derived signaling molecules that regulate mammalian metabolism through sterol-sensing nuclear receptor transcription factors. In C. elegans, bile acid-like steroids called dafachronic acids (DAs) control developmental timing and longevity by activating the nuclear receptor DAF-12. However, little is known about the biosynthesis of these molecules. Here, we show that the DAF-36/Rieske oxygenase works at the first committed step, converting cholesterol to 7-dehydrocholesterol. Its elucidation as a cholesterol 7-desaturase provides crucial biochemical evidence that such oxygenases are key steroidogenic enzymes. By controlling DA production, DAF-36 regulates DAF-12 activities for reproductive development and longevity and may illuminate related pathways in metazoans.
Asunto(s)
Caenorhabditis elegans/enzimología , Deshidrocolesteroles/metabolismo , Longevidad , Oxigenasas/metabolismo , Animales , Caenorhabditis elegans/citología , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Línea Celular , Colesterol/metabolismo , Cromatografía Liquida , Cromatografía de Gases y Espectrometría de Masas , Regulación del Desarrollo de la Expresión Génica , Insectos/citología , Microsomas/metabolismo , Células Neuroendocrinas/citología , Células Neuroendocrinas/metabolismo , Oxigenasas/genética , Fenotipo , Receptores Citoplasmáticos y Nucleares/genética , Receptores Citoplasmáticos y Nucleares/metabolismo , Transducción de SeñalRESUMEN
Maintenance of genomic stability is critical for all cells. Homologous recombination (HR) pathways promote genome stability using evolutionarily conserved proteins such as RecA, SSB, and RecQ, the Escherichia coli homologue of five human proteins at least three of which suppress genome instability and cancer. A previous report indicated that RecQ promotes the net accumulation in cells of intermolecular HR intermediates (IRIs), a net effect opposite that of the yeast and two human RecQ homologues. Here we extend those conclusions. We demonstrate that cells that lack both UvrD, an inhibitor of RecA-mediated strand exchange, and RecG, a DNA helicase implicated in IRI resolution, are inviable. We show that the uvrD recG cells die a "death-by-recombination" in which IRIs accumulate blocking chromosome segregation. First, their death requires RecA HR protein. Second, the death is accompanied by cytogenetically visible failure to segregate chromosomes. Third, FISH analyses show that the unsegregated chromosomes have completed replication, supporting the hypothesis that unresolved IRIs prevented the segregation. Fourth, we show that RecQ and induction of the SOS response are required for the accumulation of replicated, unsegregated chromosomes and death, as are RecF, RecO, and RecJ. ExoI exonuclease and MutL mismatch-repair protein are partially required. This set of genes is similar but not identical to those that promote death-by-recombination of DeltauvrD Deltaruv cells. The data support models in which RecQ promotes the net accumulation in cells of IRIs and RecG promotes resolution of IRIs that form via pathways not wholly identical to those that produce the IRIs resolved by RuvABC. This implies that RecG resolves intermediates other than or in addition to standard Holliday junctions resolved by RuvABC. The role of RecQ in net accumulation of IRIs may be shared by one or more of its human homologues.
Asunto(s)
ADN Helicasas/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , RecQ Helicasas/metabolismo , Recombinación Genética , ADN Helicasas/genética , Proteínas de Escherichia coli/genética , MutaciónRESUMEN
The RecQ-helicase family is widespread, is highly conserved, and includes human orthologs that suppress genomic instability and cancer. In vivo, some RecQ homologs promote reduction of steady-state levels of bimolecular recombination intermediates (BRIs), which block chromosome segregation if not resolved. We find that, in vivo, E. coli RecQ can promote the opposite: the net accumulation of BRIs. We report that cells lacking Ruv and UvrD BRI-resolution and -prevention proteins die and display failed chromosome segregation attributable to accumulation of BRIs. Death and segregation failure require RecA and RecF strand exchange proteins. FISH data show that replication is completed during chromosome-segregation failure/death of ruv uvrD recA(Ts) cells. Surprisingly, RecQ (and RecJ) promotes this death. The data imply that RecQ promotes the net accumulation of BRIs in vivo, indicating a second paradigm for the in vivo effect of RecQ-like proteins. The E. coli RecQ paradigm may provide a useful model for some human RecQ homologs.
Asunto(s)
RecQ Helicasas/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , ADN Helicasas/genética , ADN Helicasas/metabolismo , Reparación del ADN , Momento de Replicación del ADN , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Endodesoxirribonucleasas/genética , Endodesoxirribonucleasas/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Exodesoxirribonucleasas/genética , Exodesoxirribonucleasas/metabolismo , Hibridación Fluorescente in Situ , Modelos Biológicos , Mutación , Rec A Recombinasas/genética , Rec A Recombinasas/metabolismo , Recombinación Genética , TemperaturaRESUMEN
Apparently conflicting data regarding the role of SOS-inducible, error-prone DNA polymerase IV (DinB) in spontaneous mutation are resolved by the finding that mutation is reduced by a polar allele with which dinB and neighboring yafN are deleted but not by two nonpolar dinB alleles. We demonstrate the existence of a dinB operon that contains four genes, dinB-yafN-yafO-yafP. The results imply a role for yafN, yafO, and/or yafP in spontaneous mutation.