RESUMEN
In somatic cells, recombination between the homologous chromosomes followed by equational segregation leads to loss of heterozygosity events (LOH), allowing the expression of recessive alleles and the production of novel allele combinations that are potentially beneficial upon Darwinian selection. However, inter-homolog recombination in somatic cells is rare, thus reducing potential genetic variation. Here, we explored the property of S. cerevisiae to enter the meiotic developmental program, induce meiotic Spo11-dependent double-strand breaks genome-wide and return to mitotic growth, a process known as Return To Growth (RTG). Whole genome sequencing of 36 RTG strains derived from the hybrid S288c/SK1 diploid strain demonstrates that the RTGs are bona fide diploids with mosaic recombined genome, derived from either parental origin. Individual RTG genome-wide genotypes are comprised of 5 to 87 homozygous regions due to the loss of heterozygous (LOH) events of various lengths, varying between a few nucleotides up to several hundred kilobases. Furthermore, we show that reiteration of the RTG process shows incremental increases of homozygosity. Phenotype/genotype analysis of the RTG strains for the auxotrophic and arsenate resistance traits validates the potential of this procedure of genome diversification to rapidly map complex traits loci (QTLs) in diploid strains without undergoing sexual reproduction.
Asunto(s)
Diploidia , Hibridación Genética , Meiosis/genética , Recombinación Genética , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/genética , Separación Celular , Mapeo Cromosómico , Intercambio Genético , Conversión Génica/genética , Variación Genética , Genoma Fúngico , Haplotipos/genética , Homocigoto , Fenotipo , Sitios de Carácter Cuantitativo/genéticaRESUMEN
The acquisition of mutations is relevant to every aspect of genetics, including cancer and evolution of species on Darwinian selection. Genome variations arise from rare stochastic imperfections of cellular metabolism and deficiencies in maintenance genes. Here, we established the genome-wide spectrum of mutations that accumulate in a WT and in nine Saccharomyces cerevisiae mutator strains deficient for distinct genome maintenance processes: pol32Δ and rad27Δ (replication), msh2Δ (mismatch repair), tsa1Δ (oxidative stress), mre11Δ (recombination), mec1Δ tel1Δ (DNA damage/S-phase checkpoints), pif1Δ (maintenance of mitochondrial genome and telomere length), cac1Δ cac3Δ (nucleosome deposition), and clb5Δ (cell cycle progression). This study reveals the diversity, complexity, and ultimate unique nature of each mutational spectrum, composed of punctual mutations, chromosomal structural variations, and/or aneuploidies. The mutations produced in clb5Δ/CCNB1, mec1Δ/ATR, tel1Δ/ATM, and rad27Δ/FEN1 strains extensively reshape the genome, following a trajectory dependent on previous events. It comprises the transmission of unstable genomes that lead to colony mosaicisms. This comprehensive analytical approach of mutator defects provides a model to understand how genome variations might accumulate during clonal evolution of somatic cell populations, including tumor cells.
Asunto(s)
Mutación/genética , Saccharomyces cerevisiae/genética , Aneuploidia , Cromosomas Fúngicos/genética , Reordenamiento Génico/genética , Genes Fúngicos/genética , Haploidia , Análisis de Secuencia de ADNRESUMEN
Genomes contain tandem repeats that are at risk of internal rearrangements and a threat to genome integrity. Here, we investigated the behavior of the human subtelomeric minisatellites HRAS1, CEB1, and CEB25 in Saccharomyces cerevisiae. In mitotically growing wild-type cells, these GC-rich tandem arrays stimulate the rate of gross chromosomal rearrangements (GCR) by 20, 1,620, and 276,000-fold, respectively. In the absence of the Pif1 helicase, known to inhibit GCR by telomere addition and to unwind G-quadruplexes, the GCR rate is further increased in the presence of CEB1, by 385-fold compared to the pif1Δ control strain. The behavior of CEB1 is strongly dependent on its capacity to form G-quadruplexes, since the treatment of WT cells with the Phen-DC(3) G-quadruplex ligand has a 52-fold stimulating effect while the mutation of the G-quadruplex-forming motif reduced the GCR rate 30-fold in WT and 100-fold in pif1Δ cells. The GCR events are telomere additions within CEB1. Differently, the extreme stimulation of CEB25 GCR depends on its affinity for Cdc13, which binds the TG-rich ssDNA telomere overhang. This property confers a biased orientation-dependent behavior to CEB25, while CEB1 and HRAS1 increase GCR similarly in either orientation. Furthermore, we analyzed the minisatellites' distribution in the human genome and discuss their potential role to trigger subtelomeric rearrangements.
Asunto(s)
Aberraciones Cromosómicas , G-Cuádruplex , Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Unión a Telómeros/genética , Telómero/genética , Composición de Base , ADN Helicasas/genética , Replicación del ADN , Humanos , Repeticiones de Minisatélite/genética , Mutación , Proteínas Proto-Oncogénicas p21(ras)/genética , Saccharomyces cerevisiae/genética , Secuencias Repetidas en Tándem/genéticaRESUMEN
Acute leukemias are characterized by deregulation of transcriptional networks that control the lineage specificity of gene expression. The aberrant overexpression of the Spi-1/PU.1 transcription factor leads to erythroleukemia. To determine how Spi-1 mechanistically influences the transcriptional program, we combined a ChIP-seq analysis with transcriptional profiling in cells from an erythroleukemic mouse model. We show that Spi-1 displays a selective DNA-binding that does not often cause transcriptional modulation. We report that Spi-1 controls transcriptional activation and repression partially through distinct Spi-1 recruitment to chromatin. We revealed several parameters impacting on Spi-1-mediated transcriptional activation. Gene activation is facilitated by Spi-1 occupancy close to transcriptional starting site of genes devoid of CGIs. Moreover, in those regions Spi-1 acts by binding to multiple motifs tightly clustered and with similar orientation. Finally, in contrast to the myeloid and lymphoid B cells in which Spi-1 exerts a physiological activity, in the erythroleukemic cells, lineage-specific cooperating factors do not play a prevalent role in Spi-1-mediated transcriptional activation. Thus, our work describes a new mechanism of gene activation through clustered site occupancy of Spi-1 particularly relevant in regard to the strong expression of Spi-1 in the erythroleukemic cells.
Asunto(s)
Leucemia Eritroblástica Aguda/genética , Proteínas Proto-Oncogénicas/metabolismo , Elementos Reguladores de la Transcripción , Transactivadores/metabolismo , Activación Transcripcional , Animales , Sitios de Unión , Línea Celular Tumoral , Inmunoprecipitación de Cromatina , Islas de CpG , ADN/química , ADN/metabolismo , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Genoma , Leucemia Eritroblástica Aguda/metabolismo , Ratones , Ratones Transgénicos , Motivos de Nucleótidos , Análisis de Secuencia de ADN , Sitio de Iniciación de la TranscripciónRESUMEN
SUMMARY: We present SVDetect, a program designed to identify genomic structural variations from paired-end and mate-pair next-generation sequencing data produced by the Illumina GA and ABI SOLiD platforms. Applying both sliding-window and clustering strategies, we use anomalously mapped read pairs provided by current short read aligners to localize genomic rearrangements and classify them according to their type, e.g. large insertions-deletions, inversions, duplications and balanced or unbalanced inter-chromosomal translocations. SVDetect outputs predicted structural variants in various file formats for appropriate graphical visualization. AVAILABILITY: Source code and sample data are available at http://svdetect.sourceforge.net/
Asunto(s)
Genoma/genética , Variación Estructural del Genoma , Genómica/métodos , Programas InformáticosRESUMEN
BAP1 whose protein interacts with BRCA1 was analysed in a series of 47 French familial breast cancer cases negatively tested for BRCA1/2 mutations. The lack of detection of deleterious mutations suggests that BAP1 is not a high risk breast cancer predisposing gene. However, a common identified variant, rs123602, may be tested in sporadic cases as candidate for moderate risk.
Asunto(s)
Neoplasias de la Mama/genética , Predisposición Genética a la Enfermedad , Proteínas Supresoras de Tumor/genética , Ubiquitina Tiolesterasa/genética , Adulto , Análisis Mutacional de ADN , Femenino , Humanos , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa , Polimorfismo de Nucleótido Simple , Factores de RiesgoRESUMEN
The majority of patients with neuroblastoma have tumors that initially respond to chemotherapy, but a large proportion will experience therapy-resistant relapses. The molecular basis of this aggressive phenotype is unknown. Whole-genome sequencing of 23 paired diagnostic and relapse neuroblastomas showed clonal evolution from the diagnostic tumor, with a median of 29 somatic mutations unique to the relapse sample. Eighteen of the 23 relapse tumors (78%) showed mutations predicted to activate the RAS-MAPK pathway. Seven of these events were detected only in the relapse tumor, whereas the others showed clonal enrichment. In neuroblastoma cell lines, we also detected a high frequency of activating mutations in the RAS-MAPK pathway (11/18; 61%), and these lesions predicted sensitivity to MEK inhibition in vitro and in vivo. Our findings provide a rationale for genetic characterization of relapse neuroblastomas and show that RAS-MAPK pathway mutations may function as a biomarker for new therapeutic approaches to refractory disease.
Asunto(s)
Sistema de Señalización de MAP Quinasas/genética , Proteínas Quinasas Activadas por Mitógenos/genética , Mutación , Recurrencia Local de Neoplasia/genética , Neuroblastoma/genética , Proteínas ras/genética , Quinasa de Linfoma Anaplásico , Animales , Bencimidazoles/farmacología , Western Blotting , Línea Celular Tumoral , Niño , Preescolar , Aberraciones Cromosómicas , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Femenino , Regulación Neoplásica de la Expresión Génica , Células HEK293 , Humanos , Lactante , Masculino , Ratones SCID , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Neuroblastoma/tratamiento farmacológico , Neuroblastoma/patología , Fosforilación/efectos de los fármacos , Proteínas Tirosina Quinasas Receptoras/genética , Proteínas Tirosina Quinasas Receptoras/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Ensayos Antitumor por Modelo de Xenoinjerto , Proteínas ras/metabolismoRESUMEN
To meet challenges in terms of throughput and turnaround time, many diagnostic laboratories are shifting from Sanger sequencing to higher throughput next-generation sequencing (NGS) platforms. Bearing in mind that the performance and quality criteria expected from NGS in diagnostic or research settings are strikingly different, we have developed an Ion Torrent's PGM-based routine diagnostic procedure for BRCA1/2 sequencing. The procedure was first tested on a training set of 62 control samples, and then blindly validated on 77 samples in parallel with our routine technique. The training set was composed of difficult cases, for example, insertions and/or deletions of various sizes, large-scale rearrangements and, obviously, mutations occurring in homopolymer regions. We also compared two bioinformatic solutions in this diagnostic context, an in-house academic pipeline and the commercially available NextGene software (Softgenetics). NextGene analysis provided higher sensitivity, as four previously undetected single-nucleotide variations were found. Regarding specificity, an average of 1.5 confirmatory Sanger sequencings per patient was needed for complete BRCA1/2 screening. Large-scale rearrangements were identified by two distinct analyses, that is, bioinformatics and fragment analysis with electrophoresis profile comparison. Turnaround time was enhanced, as a series of 30 patients were sequenced by one technician, making the results available for the clinician in 10 working days following blood sampling. BRCA1/2 genes are a good model, representative of the difficulties commonly encountered in diagnostic settings, which is why we believe our findings are of interest for the whole community, and the pipeline described can be adapted by any user of PGM for diagnostic purposes.
Asunto(s)
Proteína BRCA1/genética , Proteína BRCA2/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Neoplasias de la Mama/diagnóstico , Neoplasias de la Mama/genética , Biología Computacional , Femenino , Reordenamiento Génico , Pruebas Genéticas , Humanos , Neoplasias Ováricas/diagnóstico , Neoplasias Ováricas/genética , Sensibilidad y Especificidad , Análisis de Secuencia de ADN , Eliminación de Secuencia , Programas InformáticosRESUMEN
Epithelial-to-mesenchymal transition-like (EMT-like) is a critical process allowing initiation of metastases during tumour progression. Here, to investigate its role in intestinal cancer, we combine computational network-based and experimental approaches to create a mouse model with high metastatic potential. Construction and analysis of this network map depicting molecular mechanisms of EMT regulation based on the literature suggests that Notch activation and p53 deletion have a synergistic effect in activating EMT-like processes. To confirm this prediction, we generate transgenic mice by conditionally activating the Notch1 receptor and deleting p53 in the digestive epithelium (NICD/p53(-/-)). These mice develop metastatic tumours with high penetrance. Using GFP lineage tracing, we identify single malignant cells with mesenchymal features in primary and metastatic tumours in vivo. The development of such a model that recapitulates the cellular features observed in invasive human colorectal tumours is appealing for innovative drug discovery.