RESUMEN
Cancer is molecularly heterogeneous, with seemingly similar patients having different molecular landscapes and accordingly different clinical behaviors. In recent studies, gene expression networks have been shown as more effective/informative for cancer heterogeneity analysis than some simpler measures. Gene interconnections can be classified as "direct" and "indirect," where the latter can be caused by shared genomic regulators (such as transcription factors, microRNAs, and other regulatory molecules) and other mechanisms. It has been suggested that incorporating the regulators of gene expressions in network analysis and focusing on the direct interconnections can lead to a deeper understanding of the more essential gene interconnections. Such analysis can be seriously challenged by the large number of parameters (jointly caused by network analysis, incorporation of regulators, and heterogeneity) and often weak signals. To effectively tackle this problem, we propose incorporating prior information contained in the published literature. A key challenge is that such prior information can be partial or even wrong. We develop a two-step procedure that can flexibly accommodate different levels of prior information quality. Simulation demonstrates the effectiveness of the proposed approach and its superiority over relevant competitors. In the analysis of a breast cancer dataset, findings different from the alternatives are made, and the identified sample subgroups have important clinical differences.
RESUMEN
Recurrent upper tract urothelial carcinomas (UTUCs) arise in the context of nephropathy linked to exposure to the herbal carcinogen aristolochic acid (AA). Here we delineated the molecular programs underlying UTUC tumorigenesis in patients from endemic aristolochic acid nephropathy (AAN) regions in Southern Europe. We applied an integrative multiomics analysis of UTUCs, corresponding unaffected tissues and of patient urines. Quantitative microRNA (miRNA) and messenger ribonucleic acid (mRNA) expression profiling, immunohistochemical analysis by tissue microarrays and exome and transcriptome sequencing were performed in UTUC and nontumor tissues. Urinary miRNAs of cases undergoing surgery were profiled before and after tumor resection. Ribonucleic acid (RNA) and protein levels were analyzed using appropriate statistical tests and trend assessment. Dedicated bioinformatic tools were used for analysis of pathways, mutational signatures and result visualization. The results delineate UTUC-specific miRNA:mRNA networks comprising 89 miRNAs associated with 1,862 target mRNAs, involving deregulation of cell cycle, deoxyribonucleic acid (DNA) damage response, DNA repair, bladder cancer, oncogenes, tumor suppressors, chromatin structure regulators and developmental signaling pathways. Key UTUC-specific transcripts were confirmed at the protein level. Exome and transcriptome sequencing of UTUCs revealed AA-specific mutational signature SBS22, with 68% to 76% AA-specific, deleterious mutations propagated at the transcript level, a possible basis for neoantigen formation and immunotherapy targeting. We next identified a signature of UTUC-specific miRNAs consistently more abundant in the patients' urine prior to tumor resection, thereby defining biomarkers of tumor presence. The complex gene regulation programs of AAN-associated UTUC tumors involve regulatory miRNAs prospectively applicable to noninvasive urine-based screening of AAN patients for cancer presence and recurrence.
Asunto(s)
Ácidos Aristolóquicos/efectos adversos , Biomarcadores de Tumor/genética , Carcinoma de Células Transicionales/patología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , MicroARNs/orina , Mutación , Neoplasias de la Vejiga Urinaria/patología , Biomarcadores de Tumor/orina , Carcinoma de Células Transicionales/inducido químicamente , Carcinoma de Células Transicionales/genética , Carcinoma de Células Transicionales/orina , Exoma , Estudios de Seguimiento , Humanos , Pronóstico , Proteoma/análisis , Proteoma/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Neoplasias de la Vejiga Urinaria/inducido químicamente , Neoplasias de la Vejiga Urinaria/genética , Neoplasias de la Vejiga Urinaria/orinaRESUMEN
The exon junction complex controls the translation, degradation, and localization of spliced mRNAs, and three of its core subunits also play a role in splicing. Here, we show that a fourth subunit, Barentsz, has distinct functions within and separate from the exon junction complex in Drosophila neuromuscular development. The distribution of mitochondria in larval muscles requires Barentsz as well as other exon junction complex subunits and is not rescued by a Barentsz transgene in which residues required for binding to the core subunit eIF4AIII are mutated. In contrast, interactions with the exon junction complex are not required for Barentsz to promote the growth of neuromuscular synapses. We find that the Activin ligand Dawdle shows reduced expression in barentsz mutants and acts downstream of Barentsz to control synapse growth. Both barentsz and dawdle are required in motor neurons, muscles, and glia for normal synapse growth, and exogenous Dawdle can rescue synapse growth in the absence of barentsz. These results identify a biological function for Barentsz that is independent of the exon junction complex.
Asunto(s)
Proteínas de Drosophila , Proteínas de Unión al ARN , Animales , Drosophila/metabolismo , Proteínas de Drosophila/metabolismo , Factor 4A Eucariótico de Iniciación/genética , Factor 4A Eucariótico de Iniciación/metabolismo , Exones/genética , Proteínas de Unión al ARN/metabolismo , Sinapsis/metabolismoRESUMEN
MOTIVATION: Retrotransposition is an important force in shaping the human genome and is involved in prenatal development, disease and aging. Current genome browsers are not optimized for visualizing the experimental evidence for retrotransposon insertions. RESULTS: We have developed a specialized browser to visualize the evidence for retrotransposon insertions for both targeted and whole-genome sequencing data. AVAILABILITY AND IMPLEMENTATION: TranspoScope's source code, as well as installation instructions, are available at https://github.com/FenyoLab/transposcope.
Asunto(s)
Retroelementos , Programas Informáticos , Genoma Humano , Humanos , Retroelementos/genética , Secuenciación Completa del GenomaRESUMEN
Long interspersed element-1 (LINE-1, L1) sequences, which comprise about 17% of human genome, are the product of one of the most active types of mobile DNAs in modern humans. LINE-1 insertion alleles can cause inherited and de novo genetic diseases, and LINE-1-encoded proteins are highly expressed in some cancers. Genome-wide LINE-1 mapping in single cells could be useful for defining somatic and germline retrotransposition rates, and for enabling studies to characterize tumour heterogeneity, relate insertions to transcriptional and epigenetic effects at the cellular level, or describe cellular phylogenies in development. Our laboratories have reported a genome-wide LINE-1 insertion site mapping method for bulk DNA, named transposon insertion profiling by sequencing (TIPseq). There have been significant barriers applying LINE-1 mapping to single cells, owing to the chimeric artefacts and features of repetitive sequences. Here, we optimize a modified TIPseq protocol and show its utility for LINE-1 mapping in single lymphoblastoid cells. Results from single-cell TIPseq experiments compare well to known LINE-1 insertions found by whole-genome sequencing and TIPseq on bulk DNA. Among the several approaches we tested, whole-genome amplification by multiple displacement amplification followed by restriction enzyme digestion, vectorette ligation and LINE-1-targeted PCR had the best assay performance. This article is part of a discussion meeting issue 'Crossroads between transposons and gene regulation'.
Asunto(s)
Elementos Transponibles de ADN/genética , Elementos de Nucleótido Esparcido Largo/genética , Mutagénesis Insercional , Línea Celular , Humanos , Análisis de Secuencia de ADN , Análisis de la Célula IndividualRESUMEN
Cross-species pathway transplantation enables insight into a biological process not possible through traditional approaches. We replaced the enzymes catalyzing the entire Saccharomyces cerevisiae adenine de novo biosynthesis pathway with the human pathway. While the 'humanized' yeast grew in the absence of adenine, it did so poorly. Dissection of the phenotype revealed that PPAT, the human ortholog of ADE4, showed only partial function whereas all other genes complemented fully. Suppressor analysis revealed other pathways that play a role in adenine de-novo pathway regulation. Phylogenetic analysis pointed to adaptations of enzyme regulation to endogenous metabolite level 'setpoints' in diverse organisms. Using DNA shuffling, we isolated specific amino acids combinations that stabilize the human protein in yeast. Thus, using adenine de novo biosynthesis as a proof of concept, we suggest that the engineering methods used in this study as well as the debugging strategies can be utilized to transplant metabolic pathway from any origin into yeast.
Asunto(s)
Adenina/biosíntesis , Vías Biosintéticas/genética , Carboxiliasas/genética , Cromosomas Artificiales Humanos/química , Péptido Sintasas/genética , Saccharomyces cerevisiae/genética , Secuencia de Aminoácidos , Sistemas CRISPR-Cas , Carboxiliasas/metabolismo , Cromosomas Artificiales Humanos/metabolismo , Prueba de Complementación Genética , Ingeniería Genética/métodos , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Péptido Sintasas/metabolismo , Filogenia , Plásmidos/química , Plásmidos/metabolismo , Saccharomyces cerevisiae/clasificación , Saccharomyces cerevisiae/metabolismo , Alineación de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
BACKGROUND: Transposable elements make up a significant portion of the human genome. Accurately locating these mobile DNAs is vital to understand their role as a source of structural variation and somatic mutation. To this end, laboratories have developed strategies to selectively amplify or otherwise enrich transposable element insertion sites in genomic DNA. RESULTS: Here we describe a technique, Transposon Insertion Profiling by sequencing (TIPseq), to map Long INterspersed Element 1 (LINE-1, L1) retrotransposon insertions in the human genome. This method uses vectorette PCR to amplify species-specific L1 (L1PA1) insertion sites followed by paired-end Illumina sequencing. In addition to providing a step-by-step molecular biology protocol, we offer users a guide to our pipeline for data analysis, TIPseqHunter. Our recent studies in pancreatic and ovarian cancer demonstrate the ability of TIPseq to identify invariant (fixed), polymorphic (inherited variants), as well as somatically-acquired L1 insertions that distinguish cancer genomes from a patient's constitutional make-up. CONCLUSIONS: TIPseq provides an approach for amplifying evolutionarily young, active transposable element insertion sites from genomic DNA. Our rationale and variations on this protocol may be useful to those mapping L1 and other mobile elements in complex genomes.
RESUMEN
Transposable elements (TEs) represent a substantial fraction of many eukaryotic genomes, and transcriptional regulation of these factors is important to determine TE activities in human cells. However, due to the repetitive nature of TEs, identifying transcription factor (TF)-binding sites from ChIP-sequencing (ChIP-seq) datasets is challenging. Current algorithms are focused on subtle differences between TE copies and thus bias the analysis to relatively old and inactive TEs. Here we describe an approach termed "MapRRCon" (mapping repeat reads to a consensus) which allows us to identify proteins binding to TE DNA sequences by mapping ChIP-seq reads to the TE consensus sequence after whole-genome alignment. Although this method does not assign binding sites to individual insertions in the genome, it provides a landscape of interacting TFs by capturing factors that bind to TEs under various conditions. We applied this method to screen TFs' interaction with L1 in human cells/tissues using ENCODE ChIP-seq datasets and identified 178 of the 512 TFs tested as bound to L1 in at least one biological condition with most of them (138) localized to the promoter. Among these L1-binding factors, we focused on Myc and CTCF, as they play important roles in cancer progression and 3D chromatin structure formation. Furthermore, we explored the transcriptomes of The Cancer Genome Atlas breast and ovarian tumor samples in which a consistent anti-/correlation between L1 and Myc/CTCF expression was observed, suggesting that these two factors may play roles in regulating L1 transcription during the development of such tumors.
Asunto(s)
Regulación de la Expresión Génica/genética , Elementos Reguladores de la Transcripción/genética , Retroelementos/genética , Factores de Transcripción/genética , Algoritmos , Neoplasias de la Mama/genética , Cromatina/genética , Femenino , Genoma/genética , Humanos , Elementos de Nucleótido Esparcido Largo/genética , Neoplasias Ováricas/genética , Regiones Promotoras Genéticas/genética , Unión Proteica/genética , Transcriptoma/genéticaRESUMEN
Male mammals must simultaneously produce prodigious numbers of sperm and maintain an adequate reserve of stem cells to ensure continuous production of gametes throughout life. Failures in the mechanisms responsible for balancing germ cell differentiation and spermatogonial stem cell (SSC) self-renewal can result in infertility. We discovered a novel requirement for Ubiquitous Expressed Transcript (UXT) in spermatogenesis by developing the first knockout mouse model for this gene. Constitutive deletion of Uxt is embryonic lethal, while conditional knockout in the male germline results in a Sertoli cell-only phenotype during the first wave of spermatogenesis that does not recover in the adult. This phenotype begins to manifest between 6 and 7 days post-partum, just before meiotic entry. Gene expression analysis revealed that Uxt deletion downregulates the transcription of genes governing SSC self-renewal, differentiation, and meiosis, consistent with its previously defined role as a transcriptional co-factor. Our study has revealed the first in vivo function for UXT in the mammalian germline as a regulator of distinct transcriptional programs in SSCs and differentiating spermatogonia.
Asunto(s)
Chaperonas Moleculares/genética , Espermatogénesis/genética , Animales , Caspasa 3/metabolismo , Ciclo Celular , Proteínas de Ciclo Celular , Diferenciación Celular/genética , Eliminación de Gen , Genes Letales , Inmunohistoquímica , Aprendizaje Automático , Masculino , Meiosis/genética , Ratones , Ratones Noqueados , Chaperonas Moleculares/metabolismo , Fenotipo , Espermatogonias/metabolismo , Testículo/metabolismo , Testículo/patologíaRESUMEN
We describe design, rapid assembly, and characterization of synthetic yeast Sc2.0 chromosome VI (synVI). A mitochondrial defect in the synVI strain mapped to synonymous coding changes within PRE4 (YFR050C), encoding an essential proteasome subunit; Sc2.0 coding changes reduced Pre4 protein accumulation by half. Completing Sc2.0 specifies consolidation of 16 synthetic chromosomes into a single strain. We investigated phenotypic, transcriptional, and proteomewide consequences of Sc2.0 chromosome consolidation in poly-synthetic strains. Another "bug" was discovered through proteomic analysis, associated with alteration of the HIS2 transcription start due to transfer RNA deletion and loxPsym site insertion. Despite extensive genetic alterations across 6% of the genome, no major global changes were detected in the poly-synthetic strain "omics" analyses. This work sets the stage for completion of a designer, synthetic eukaryotic genome.
Asunto(s)
Cromosomas Artificiales de Levadura/química , Cromosomas Artificiales de Levadura/genética , Saccharomyces cerevisiae/genética , Biología Sintética/métodos , Células Artificiales/metabolismo , Mapeo Físico de Cromosoma , Complejo de la Endopetidasa Proteasomal/genética , Proteómica , Saccharomyces cerevisiae/crecimiento & desarrollo , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
As the use of synthetic biology both in industry and in academia grows, there is an increasing need to ensure biocontainment. There is growing interest in engineering bacterial- and yeast-based safeguard (SG) strains. First-generation SGs were based on metabolic auxotrophy; however, the risk of cross-feeding and the cost of growth-controlling nutrients led researchers to look for other avenues. Recent strategies include bacteria engineered to be dependent on nonnatural amino acids and yeast SG strains that have both transcriptional- and recombinational-based biocontainment. We describe improving yeast Saccharomyces cerevisiae-based transcriptional SG strains, which have near-WT fitness, the lowest possible escape rate, and nanomolar ligands controlling growth. We screened a library of essential genes, as well as the best-performing promoter and terminators, yielding the best SG strains in yeast. The best constructs were fine-tuned, resulting in two tightly controlled inducible systems. In addition, for potential use in the prevention of industrial espionage, we screened an array of possible "decoy molecules" that can be used to mask any proprietary supplement to the SG strain, with minimal effect on strain fitness.
Asunto(s)
Genoma/genética , Saccharomyces cerevisiae/genética , Genes Esenciales/genética , Ingeniería Metabólica/métodos , Regiones Promotoras Genéticas/genética , Biología Sintética/métodos , Regiones Terminadoras Genéticas/genética , Transcripción Genética/genéticaRESUMEN
Mammalian genomes are replete with interspersed repeats reflecting the activity of transposable elements. These mobile DNAs are self-propagating, and their continued transposition is a source of both heritable structural variation as well as somatic mutation in human genomes. Tailored approaches to map these sequences are useful to identify insertion alleles. Here, we describe in detail a strategy to amplify and sequence long interspersed element-1 (LINE-1, L1) retrotransposon insertions selectively in the human genome, transposon insertion profiling by next-generation sequencing (TIPseq). We also report the development of a machine-learning-based computational pipeline, TIPseqHunter, to identify insertion sites with high precision and reliability. We demonstrate the utility of this approach to detect somatic retrotransposition events in high-grade ovarian serous carcinoma.
Asunto(s)
Elementos de Nucleótido Esparcido Largo/genética , Neoplasias Ováricas/genética , Algoritmos , Femenino , Genoma Humano , Humanos , Aprendizaje Automático , Reacción en Cadena de la Polimerasa/métodosRESUMEN
BACKGROUND: Gliomas are the most common primary brain tumors in adults. We sought to understand the roles of endogenous transposable elements in these malignancies by identifying evidence of somatic retrotransposition in glioblastomas (GBM). We performed transposon insertion profiling of the active subfamily of Long INterspersed Element-1 (LINE-1) elements by deep sequencing (TIPseq) on genomic DNA of low passage oncosphere cell lines derived from 7 primary GBM biopsies, 3 secondary GBM tissue samples, and matched normal intravenous blood samples from the same individuals. RESULTS: We found and PCR validated one somatically acquired tumor-specific insertion in a case of secondary GBM. No LINE-1 insertions present in primary GBM oncosphere cultures were missing from corresponding blood samples. However, several copies of the element (11) were found in genomic DNA from blood and not in the oncosphere cultures. SNP 6.0 microarray analysis revealed deletions or loss of heterozygosity in the tumor genomes over the intervals corresponding to these LINE-1 insertions. CONCLUSIONS: These findings indicate that LINE-1 retrotransposon can act as an infrequent insertional mutagen in secondary GBM, but that retrotransposition is uncommon in these central nervous system tumors as compared to other neoplasias.
RESUMEN
Improvements in mass spectrometry (MS)-based peptide sequencing provide a new opportunity to determine whether polymorphisms, mutations, and splice variants identified in cancer cells are translated. Herein, we apply a proteogenomic data integration tool (QUILTS) to illustrate protein variant discovery using whole genome, whole transcriptome, and global proteome datasets generated from a pair of luminal and basal-like breast-cancer-patient-derived xenografts (PDX). The sensitivity of proteogenomic analysis for singe nucleotide variant (SNV) expression and novel splice junction (NSJ) detection was probed using multiple MS/MS sample process replicates defined here as an independent tandem MS experiment using identical sample material. Despite analysis of over 30 sample process replicates, only about 10% of SNVs (somatic and germline) detected by both DNA and RNA sequencing were observed as peptides. An even smaller proportion of peptides corresponding to NSJ observed by RNA sequencing were detected (<0.1%). Peptides mapping to DNA-detected SNVs without a detectable mRNA transcript were also observed, suggesting that transcriptome coverage was incomplete (â¼80%). In contrast to germline variants, somatic variants were less likely to be detected at the peptide level in the basal-like tumor than in the luminal tumor, raising the possibility of differential translation or protein degradation effects. In conclusion, this large-scale proteogenomic integration allowed us to determine the degree to which mutations are translated and identify gaps in sequence coverage, thereby benchmarking current technology and progress toward whole cancer proteome and transcriptome analysis.
Asunto(s)
Empalme Alternativo , Neoplasias Mamarias Experimentales/genética , Mutación , Proteómica/métodos , Análisis de Secuencia de ADN/métodos , Análisis de Secuencia de ARN/métodos , Animales , Biología Computacional/métodos , Bases de Datos Genéticas , Femenino , Genoma , Humanos , Neoplasias Mamarias Experimentales/metabolismo , Ratones , Polimorfismo de Nucleótido Simple , Espectrometría de Masas en Tándem , TranscriptomaRESUMEN
Cleft palate is a common birth defect in humans. Therefore, understanding the molecular genetics of palate development is important from both scientific and medical perspectives. Lhx6 and Lhx8 encode LIM homeodomain transcription factors, and inactivation of both genes in mice resulted in profound craniofacial defects including cleft secondary palate. The initial outgrowth of the palate was severely impaired in the mutant embryos, due to decreased cell proliferation. Through genome-wide transcriptional profiling, we discovered that p57(Kip2) (Cdkn1c), encoding a cell cycle inhibitor, was up-regulated in the prospective palate of Lhx6(-/-);Lhx8(-/-) mutants. p57(Kip2) has been linked to Beckwith-Wiedemann syndrome and IMAGe syndrome in humans, which are developmental disorders with increased incidents of palate defects among the patients. To determine the molecular mechanism underlying the regulation of p57(Kip2) by the Lhx genes, we combined chromatin immunoprecipitation, in silico search for transcription factor-binding motifs, and in vitro reporter assays with putative cis-regulatory elements. The results of these experiments indicated that LHX6 and LHX8 regulated p57(Kip2) via both direct and indirect mechanisms, with the latter mediated by Forkhead box (FOX) family transcription factors. Together, our findings uncovered a novel connection between the initiation of palate development and a cell cycle inhibitor via LHX. We propose a model in which Lhx6 and Lhx8 negatively regulate p57(Kip2) expression in the prospective palate area to allow adequate levels of cell proliferation and thereby promote normal palate development. This is the first report elucidating a molecular genetic pathway downstream of Lhx in palate development.
Asunto(s)
Inhibidor p57 de las Quinasas Dependientes de la Ciclina/genética , Regulación del Desarrollo de la Expresión Génica , Proteínas con Homeodominio LIM/genética , Proteínas del Tejido Nervioso/genética , Hueso Paladar/embriología , Hueso Paladar/metabolismo , Factores de Transcripción/genética , Animales , Proliferación Celular , Inhibidor p57 de las Quinasas Dependientes de la Ciclina/metabolismo , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismo , Expresión Génica , Perfilación de la Expresión Génica , Estudio de Asociación del Genoma Completo , Genotipo , Humanos , Proteínas con Homeodominio LIM/metabolismo , Maxilar/embriología , Maxilar/metabolismo , Ratones , Mutación , Proteínas del Tejido Nervioso/metabolismo , Organogénesis/genética , Hueso Paladar/patología , Unión Proteica , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Factores de Transcripción/metabolismo , Activación TranscripcionalRESUMEN
Development of the mammalian face requires a large number of genes that are expressed with spatio-temporal specificity, and transcriptional regulation mediated by enhancers plays a key role in the precise control of gene expression. Using chromatin immunoprecipitation for a histone marker of active enhancers, we generated a genome-wide map of candidate enhancers from the maxillary arch (primordium for the upper jaw) of mouse embryos. Furthermore, we confirmed multiple novel craniofacial enhancers near the genes implicated in human palate defects through functional assays. We characterized in detail one of the enhancers (Lhx8_enh1) located upstream of Lhx8, a key regulatory gene for craniofacial development. Lhx8_enh1 contained an evolutionarily conserved binding site for lymphoid enhancer factor/T-cell factor family proteins, which mediate the transcriptional regulation by the WNT/ß-catenin signaling pathway. We demonstrated in vitro that WNT/ß-catenin signaling was indeed essential for the expression of Lhx8 in the maxillary arch cells and that Lhx8_enh1 was a direct target of the WNT/ß-catenin pathway. Together, we uncovered a molecular mechanism for the regulation of Lhx8, and we provided valuable resources for further investigation into the gene regulatory network of craniofacial development.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Proteínas con Homeodominio LIM/genética , Factores de Transcripción/genética , Vía de Señalización Wnt , Animales , Secuencia de Bases , Sitios de Unión , Región Branquial/embriología , Región Branquial/metabolismo , Línea Celular , Mapeo Cromosómico , Secuencia de Consenso , Redes Reguladoras de Genes , Histonas/metabolismo , Humanos , Proteínas con Homeodominio LIM/metabolismo , Maxilar/embriología , Maxilar/metabolismo , Mesodermo/embriología , Mesodermo/metabolismo , Ratones Endogámicos C3H , Ratones Transgénicos , Datos de Secuencia Molecular , Hueso Paladar/anomalías , Cultivo Primario de Células , Factores de Transcripción/metabolismoRESUMEN
Promoters and enhancers establish precise gene transcription patterns. The development of functional approaches for their identification in mammalian cells has been complicated by the size of these genomes. Here we report a high-throughput functional assay for directly identifying active promoter and enhancer elements called FIREWACh (Functional Identification of Regulatory Elements Within Accessible Chromatin), which we used to simultaneously assess over 80,000 DNA fragments derived from nucleosome-free regions within the chromatin of embryonic stem cells (ESCs) and identify 6,364 active regulatory elements. Many of these represent newly discovered ESC-specific enhancers, showing enriched binding-site motifs for ESC-specific transcription factors including SOX2, POU5F1 (OCT4) and KLF4. The application of FIREWACh to additional cultured cell types will facilitate functional annotation of the genome and expand our view of transcriptional network dynamics.
Asunto(s)
Elementos de Facilitación Genéticos , Regulación de la Expresión Génica , Secuenciación de Nucleótidos de Alto Rendimiento , Regiones Promotoras Genéticas , Animales , Sitios de Unión , Cromatina/química , Biología Computacional , Desoxirribonucleasa I/metabolismo , Células Madre Embrionarias/citología , Citometría de Flujo , Biblioteca de Genes , Genes Reporteros , Técnicas Genéticas , Genoma , Proteínas Fluorescentes Verdes/metabolismo , Factor 4 Similar a Kruppel , Lentivirus/genética , Lentivirus/metabolismo , Luciferasas/metabolismo , Ratones , Plásmidos/metabolismo , Transcripción Genética , TransgenesRESUMEN
Relapsed childhood acute lymphoblastic leukemia (ALL) carries a poor prognosis, despite intensive retreatment, owing to intrinsic drug resistance. The biological pathways that mediate resistance are unknown. Here, we report the transcriptome profiles of matched diagnosis and relapse bone marrow specimens from ten individuals with pediatric B-lymphoblastic leukemia using RNA sequencing. Transcriptome sequencing identified 20 newly acquired, novel nonsynonymous mutations not present at initial diagnosis, with 2 individuals harboring relapse-specific mutations in the same gene, NT5C2, encoding a 5'-nucleotidase. Full-exon sequencing of NT5C2 was completed in 61 further relapse specimens, identifying additional mutations in 5 cases. Enzymatic analysis of mutant proteins showed that base substitutions conferred increased enzymatic activity and resistance to treatment with nucleoside analog therapies. Clinically, all individuals who harbored NT5C2 mutations relapsed early, within 36 months of initial diagnosis (P = 0.03). These results suggest that mutations in NT5C2 are associated with the outgrowth of drug-resistant clones in ALL.
Asunto(s)
5'-Nucleotidasa/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , 5'-Nucleotidasa/metabolismo , Secuencia de Bases , Niño , Resistencia a Antineoplásicos , Exones , Humanos , Datos de Secuencia Molecular , Mutación , Conformación Proteica , RecurrenciaRESUMEN
BACKGROUND: Oncogenic mechanisms in small-cell lung cancer remain poorly understood leaving this tumor with the worst prognosis among all lung cancers. Unlike other cancer types, sequencing genomic approaches have been of limited success in small-cell lung cancer, i.e., no mutated oncogenes with potential driver characteristics have emerged, as it is the case for activating mutations of epidermal growth factor receptor in non-small-cell lung cancer. Differential gene expression analysis has also produced SCLC signatures with limited application, since they are generally not robust across datasets. Nonetheless, additional genomic approaches are warranted, due to the increasing availability of suitable small-cell lung cancer datasets. Gene co-expression network approaches are a recent and promising avenue, since they have been successful in identifying gene modules that drive phenotypic traits in several biological systems, including other cancer types. RESULTS: We derived an SCLC-specific classifier from weighted gene co-expression network analysis (WGCNA) of a lung cancer dataset. The classifier, termed SCLC-specific hub network (SSHN), robustly separates SCLC from other lung cancer types across multiple datasets and multiple platforms, including RNA-seq and shotgun proteomics. The classifier was also conserved in SCLC cell lines. SSHN is enriched for co-expressed signaling network hubs strongly associated with the SCLC phenotype. Twenty of these hubs are actionable kinases with oncogenic potential, among which spleen tyrosine kinase (SYK) exhibits one of the highest overall statistical associations to SCLC. In patient tissue microarrays and cell lines, SCLC can be separated into SYK-positive and -negative. SYK siRNA decreases proliferation rate and increases cell death of SYK-positive SCLC cell lines, suggesting a role for SYK as an oncogenic driver in a subset of SCLC. CONCLUSIONS: SCLC treatment has thus far been limited to chemotherapy and radiation. Our WGCNA analysis identifies SYK both as a candidate biomarker to stratify SCLC patients and as a potential therapeutic target. In summary, WGCNA represents an alternative strategy to large scale sequencing for the identification of potential oncogenic drivers, based on a systems view of signaling networks. This strategy is especially useful in cancer types where no actionable mutations have emerged.
Asunto(s)
Perfilación de la Expresión Génica , Redes Reguladoras de Genes , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Proteínas Oncogénicas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Carcinoma Pulmonar de Células Pequeñas/metabolismo , Carcinoma Pulmonar de Células Pequeñas/patología , Línea Celular Tumoral , Proliferación Celular , Supervivencia Celular , Técnicas de Silenciamiento del Gen , Humanos , Péptidos y Proteínas de Señalización Intracelular/deficiencia , Péptidos y Proteínas de Señalización Intracelular/genética , Terapia Molecular Dirigida , Proteínas Oncogénicas/deficiencia , Proteínas Oncogénicas/genética , Proteínas Tirosina Quinasas/deficiencia , Proteínas Tirosina Quinasas/genética , Proteómica , Quinasa SykRESUMEN
Defining the architecture of a specific cancer genome, including its structural variants, is essential for understanding tumor biology, mechanisms of oncogenesis, and for designing effective personalized therapies. Short read paired-end sequencing is currently the most sensitive method for detecting somatic mutations that arise during tumor development. However, mapping structural variants using this method leads to a large number of false positive calls, mostly due to the repetitive nature of the genome and the difficulty of assigning correct mapping positions to short reads. This study describes a method to efficiently identify large tumor-specific deletions, inversions, duplications and translocations from low coverage data using SVDetect or BreakDancer software and a set of novel filtering procedures designed to reduce false positive calls. Applying our method to a spontaneous T cell lymphoma arising in a core RAG2/p53-deficient mouse, we identified 40 validated tumor-specific structural rearrangements supported by as few as 2 independent read pairs.