RESUMEN
In mammalian cells, chromosomal replication starts at thousands of origins at which replisomes are assembled. Replicative stress triggers additional initiation events from 'dormant' origins whose genomic distribution and regulation are not well understood. In this study, we have analyzed origin activity in mouse embryonic stem cells in the absence or presence of mild replicative stress induced by aphidicolin, a DNA polymerase inhibitor, or by deregulation of origin licensing factor CDC6. In both cases, we observe that the majority of stress-responsive origins are also active in a small fraction of the cell population in a normal S phase, and stress increases their frequency of activation. In a search for the molecular determinants of origin efficiency, we compared the genetic and epigenetic features of origins displaying different levels of activation, and integrated their genomic positions in three-dimensional chromatin interaction networks derived from high-depth Hi-C and promoter-capture Hi-C data. We report that origin efficiency is directly proportional to the proximity to transcriptional start sites and to the number of contacts established between origin-containing chromatin fragments, supporting the organization of origins in higher-level DNA replication factories.
Asunto(s)
Cromatina , Origen de Réplica , Animales , Ratones , Origen de Réplica/genética , Cromatina/genética , Células Madre Embrionarias de Ratones/metabolismo , Replicación del ADN/genética , Proteínas de Ciclo Celular/metabolismo , Mamíferos/genéticaRESUMEN
Cutaneous T-cell lymphoma (CTCL) is a heterogeneous group of primary cutaneous T-cell lymphoproliferative processes, mainly composed of mycosis fungoides and Sézary syndrome, the aggressive forms of which lack an effective treatment. The molecular pathogenesis of CTCL is largely unknown, although neoplastic cells show increased signaling from T-cell receptors (TCRs). DNAs from 11 patients with CTCL, both normal and tumoral, were target-enriched and sequenced by massive parallel sequencing for a selection of 524 TCR-signaling-related genes. Identified variants were validated by capillary sequencing. Multiple mutations were found that affected several signaling pathways, such as TCRs, nuclear factor κB, or Janus kinase/signal transducer and activator of transcription, but PLCG1 was found to be mutated in 3 samples, 2 of which featured a redundant mutation (c.1034T>C, S345F) in exon 11 that affects the PLCx protein catalytic domain. This mutation was further analyzed by quantitative polymerase chain reaction genotyping in a new cohort of 42 patients with CTCL, where it was found in 19% of samples. Immunohistochemical analysis for nuclear factor of activated T cells (NFAT) showed that PLCG1-mutated cases exhibited strong NFAT nuclear immunostaining. Functional studies demonstrated that PLCG1 mutants elicited increased downstream signaling toward NFAT activation, and inhibition of this pathway resulted in reduced CTCL cell proliferation and cell viability. Thus, increased proliferative and survival mechanisms in CTCL may partially depend on the acquisition of somatic mutations in PLCG1 and other genes that are essential for normal T-cell differentiation.
Asunto(s)
Linfoma de Células T/genética , Mutación , Fosfolipasa C gamma/genética , Neoplasias Cutáneas/genética , Animales , Línea Celular Tumoral , Supervivencia Celular/genética , Estudios de Cohortes , Análisis Mutacional de ADN , Femenino , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Linfoma de Células T/patología , Masculino , Ratones , Células 3T3 NIH , Neoplasias Cutáneas/patologíaRESUMEN
MOTIVATION: RUbioSeq has been developed to facilitate the primary and secondary analysis of re-sequencing projects by providing an integrated software suite of parallelized pipelines to detect exome variants (single-nucleotide variants and copy number variations) and to perform bisulfite-seq analyses automatically. RUbioSeq's variant analysis results have been already validated and published. AVAILABILITY: http://rubioseq.sourceforge.net/.
Asunto(s)
Exoma , Variación Genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Análisis de Secuencia de ADN/métodos , Programas Informáticos , Sulfitos , Variaciones en el Número de Copia de ADN , Metilación de ADN , Humanos , Polimorfismo de Nucleótido SimpleRESUMEN
BACKGROUND AND OBJECTIVE: To facilitate routine analysis and to improve the reproducibility of the results, next-generation sequencing (NGS) analysis requires intuitive, efficient and integrated data processing pipelines. METHODS: We have selected well-established software to construct a suite of automated and parallelized workflows to analyse NGS data for DNA-seq (single-nucleotide variants (SNVs) and indels), CNA-seq, bisulfite-seq and ChIP-seq experiments. RESULTS: Here, we present RUbioSeq+, an updated and extended version of RUbioSeq, a multiplatform application that incorporates a suite of automated and parallelized workflows to analyse NGS data. This new version includes: (i) an interactive graphical user interface (GUI) that facilitates its use by both biomedical researchers and bioinformaticians, (ii) a new pipeline for ChIP-seq experiments, (iii) pair-wise comparisons (case-control analyses) for DNA-seq experiments, (iv) and improvements in the parallelized and multithreaded execution options. Results generated by our software have been experimentally validated and accepted for publication. CONCLUSIONS: RUbioSeq+ is free and open to all users at http://rubioseq.bioinfo.cnio.es/.
Asunto(s)
Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Programas Informáticos , Inmunoprecipitación de Cromatina , Variaciones en el Número de Copia de ADN , Polimorfismo de Nucleótido Simple , Interfaz Usuario-ComputadorRESUMEN
Purpose: We aimed to maximize the performance of detecting genetic alterations in lung cancer using high-throughput sequencing for patient-derived xenografts (PDXs).Experimental Design: We undertook an integrated RNA and whole-exome sequencing of 14 PDXs. We focused on the genetic and functional analysis of ß2-microglobulin (B2M), a component of the HLA class-I complex.Results: We identified alterations in genes involved in various functions, such as B2M involved in immunosurveillance. We extended the mutational analysis of B2M to about 230 lung cancers. Five percent of the lung cancers carried somatic mutations, most of which impaired the correct formation of the HLA-I complex. We also report that genes such as CALR, PDIA3, and TAP1, which are involved in the maturation of the HLA-I complex, are altered in lung cancer. By gene expression microarrays, we observed that restitution of B2M in lung cancer cells upregulated targets of IFNα/IFNγ. Furthermore, one third of the lung cancers lacked the HLA-I complex, which was associated with lower cytotoxic CD8+ lymphocyte infiltration. The levels of B2M and HLA-I proteins correlated with those of PD-L1. Finally, a deficiency in HLA-I complex and CD8+ infiltration tended to correlate with reduced survival of patients with lung cancer treated with anti-PD-1/anti-PD-L1.Conclusions: Here, we report recurrent inactivation of B2M in lung cancer. These observations, coupled with the mutations found at CALR, PDIA3, and TAP1, and the downregulation of the HLA-I complex, indicate that an abnormal immunosurveillance axis contributes to lung cancer development. Finally, our observations suggest that an impaired HLA-I complex affects the response to anti-PD-1/anti-PD-L1 therapies. Clin Cancer Res; 23(12); 3203-13. ©2016 AACR.
Asunto(s)
Genómica , Antígenos de Histocompatibilidad Clase I/genética , Neoplasias Pulmonares/genética , Microglobulina beta-2/genética , Animales , Antígeno B7-H1/antagonistas & inhibidores , Antígeno B7-H1/inmunología , Linfocitos T CD8-positivos/inmunología , Regulación Neoplásica de la Expresión Génica , Antígenos de Histocompatibilidad Clase I/inmunología , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/inmunología , Neoplasias Pulmonares/patología , Ratones , Mutación , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Receptor de Muerte Celular Programada 1/inmunología , Secuenciación del Exoma , Ensayos Antitumor por Modelo de Xenoinjerto , Microglobulina beta-2/antagonistas & inhibidores , Microglobulina beta-2/inmunologíaRESUMEN
We sought to identify genetic variants associated with disease relapse and failure to hormonal treatment in hormone-receptor positive breast cancer (HRPBC). We analyzed a series of HRPBC with distant relapse, by sequencing pairs (n = 11) of tumors (primary and metastases) at >800X. Comparative genomic hybridization was performed as well. Top hits, based on the frequency of alteration and severity of the changes, were tested in the TCGA series. Genes determining the most parsimonious prognostic signature were studied for their functional role in vitro, by performing cell growth assays in hormonal-deprivation conditions, a setting that mimics treatment with aromatase inhibitors. Severe alterations were recurrently found in 18 genes in the pairs. However, only MYC, DNAH5, CSFR1, EPHA7, ARID1B, and KMT2C preserved an independent prognosis impact and/or showed a significantly different incidence of alterations between relapsed and non-relapsed cases in the TCGA series. The signature composed of MYC, KMT2C, and EPHA7 best discriminated the clinical course, (overall survival 90,7 vs. 144,5 months; p = 0.0001). Having an alteration in any of the genes of the signature implied a hazard ratio of death of 3.25 (p<0.0001), and early relapse during the adjuvant hormonal treatment. The presence of the D348N mutation in KMT2C and/or the T666I mutation in the kinase domain of EPHA7 conferred hormonal resistance in vitro. Novel inactivating mutations in KMT2C and EPHA7, which confer hormonal resistance, are linked to adverse clinical course in HRPBC.
Asunto(s)
Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Resistencia a Antineoplásicos/genética , Hormonas/metabolismo , Antineoplásicos Hormonales/uso terapéutico , Inhibidores de la Aromatasa/farmacología , Mama/patología , Hibridación Genómica Comparativa , Femenino , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Variación Genética , Humanos , Mutación , Recurrencia Local de Neoplasia/genética , Recurrencia Local de Neoplasia/patología , Análisis de Secuencia por Matrices de Oligonucleótidos , Pronóstico , Receptores de Estrógenos/metabolismo , Recurrencia , Estudios Retrospectivos , Riesgo , Análisis de Secuencia de ADN , Tamoxifeno/farmacología , Resultado del TratamientoRESUMEN
BACKGROUND: Genomic studies of endangered species provide insights into their evolution and demographic history, reveal patterns of genomic erosion that might limit their viability, and offer tools for their effective conservation. The Iberian lynx (Lynx pardinus) is the most endangered felid and a unique example of a species on the brink of extinction. RESULTS: We generate the first annotated draft of the Iberian lynx genome and carry out genome-based analyses of lynx demography, evolution, and population genetics. We identify a series of severe population bottlenecks in the history of the Iberian lynx that predate its known demographic decline during the 20th century and have greatly impacted its genome evolution. We observe drastically reduced rates of weak-to-strong substitutions associated with GC-biased gene conversion and increased rates of fixation of transposable elements. We also find multiple signatures of genetic erosion in the two remnant Iberian lynx populations, including a high frequency of potentially deleterious variants and substitutions, as well as the lowest genome-wide genetic diversity reported so far in any species. CONCLUSIONS: The genomic features observed in the Iberian lynx genome may hamper short- and long-term viability through reduced fitness and adaptive potential. The knowledge and resources developed in this study will boost the research on felid evolution and conservation genomics and will benefit the ongoing conservation and management of this emblematic species.