RESUMO
During a large outbreak of Shiga toxin-producing Escherichia coli illness associated with an agricultural show in Australia, we used whole-genome sequencing to detect an IS1203v insertion in the Shiga toxin 2c subunit A gene of Shiga toxin-producing E. coli. Our study showed that clinical illness was mild, and hemolytic uremic syndrome was not detected.
Assuntos
Diarreia/epidemiologia , Surtos de Doenças , Infecções por Escherichia coli/epidemiologia , Escherichia coli O157/genética , Genoma Bacteriano , Toxina Shiga I/genética , Adolescente , Adulto , Idoso , Animais , Austrália/epidemiologia , Criança , Pré-Escolar , Busca de Comunicante , Diarreia/diagnóstico , Diarreia/microbiologia , Infecções por Escherichia coli/diagnóstico , Infecções por Escherichia coli/microbiologia , Escherichia coli O157/classificação , Escherichia coli O157/isolamento & purificação , Escherichia coli O157/patogenicidade , Fezes/microbiologia , Feminino , Cabras/microbiologia , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Sorotipagem , Índice de Gravidade de Doença , Ovinos/microbiologia , Toxina Shiga I/classificação , Toxina Shiga I/isolamento & purificação , Sequenciamento Completo do GenomaRESUMO
Aberrant DNA hypomethylation may play an important role in the growth rate of glioblastoma (GBM), but the functional impact on transcription remains poorly understood. We assayed the GBM methylome with MeDIP-seq and MRE-seq, adjusting for copy number differences, in a small set of non-glioma CpG island methylator phenotype (non-G-CIMP) primary tumors. Recurrent hypomethylated loci were enriched within a region of chromosome 5p15 that is specified as a cancer amplicon and also encompasses TERT, encoding telomerase reverse transcriptase, which plays a critical role in tumorigenesis. Overall, 76 gene body promoters were recurrently hypomethylated, including TERT and the oncogenes GLI3 and TP73. Recurring hypomethylation also affected previously unannotated alternative promoters, and luciferase reporter assays for three of four of these promoters confirmed strong promoter activity in GBM cells. Histone H3 lysine 4 trimethylation (H3K4me3) ChIP-seq on tissue from the GBMs uncovered peaks that coincide precisely with tumor-specific decrease of DNA methylation at 200 loci, 133 of which are in gene bodies. Detailed investigation of TP73 and TERT gene body hypomethylation demonstrated increased expression of corresponding alternate transcripts, which in TP73 encodes a truncated p73 protein with oncogenic function and in TERT encodes a putative reverse transcriptase-null protein. Our findings suggest that recurring gene body promoter hypomethylation events, along with histone H3K4 trimethylation, alter the transcriptional landscape of GBM through the activation of a limited number of normally silenced promoters within gene bodies, in at least one case leading to expression of an oncogenic protein.
Assuntos
Epigênese Genética , Regulação Neoplásica da Expressão Gênica , Glioblastoma/genética , Mutação , Regiões Promotoras Genéticas , Ilhas de CpG , Metilação de DNA , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Humanos , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Telomerase/genética , Telomerase/metabolismo , Ativação Transcricional , Proteína Tumoral p73 , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Proteína Gli3 com Dedos de ZincoRESUMO
In addition to their protein coding function, exons can also serve as transcriptional enhancers. Mutations in these exonic-enhancers (eExons) could alter both protein function and transcription. However, the functional consequence of eExon mutations is not well known. Here, using massively parallel reporter assays, we dissect the enhancer activity of three liver eExons (SORL1 exon 17, TRAF3IP2 exon 2, PPARG exon 6) at single nucleotide resolution in the mouse liver. We find that both synonymous and non-synonymous mutations have similar effects on enhancer activity and many of the deleterious mutation clusters overlap known liver-associated transcription factor binding sites. Carrying a similar massively parallel reporter assay in HeLa cells with these three eExons found differences in their mutation profiles compared to the liver, suggesting that enhancers could have distinct operating profiles in different tissues. Our results demonstrate that eExon mutations could lead to multiple phenotypes by disrupting both the protein sequence and enhancer activity and that enhancers can have distinct mutation profiles in different cell types.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Elementos Facilitadores Genéticos , Éxons/genética , Proteínas de Membrana Transportadoras/genética , PPAR gama/genética , Receptores de LDL/genética , Animais , Sítios de Ligação , Regulação da Expressão Gênica , Células HeLa , Humanos , Fígado/metabolismo , Camundongos , Mutação de Sentido Incorreto , Polimorfismo de Nucleotídeo Único , Splicing de RNA/genética , Fatores de Transcrição/biossínteseRESUMO
Studies of coat color mutants have greatly contributed to the discovery of genes that regulate melanocyte development and function. Here, we generated Yy1 conditional knockout mice in the melanocyte-lineage and observed profound melanocyte deficiency and premature gray hair, similar to the loss of melanocytes in human piebaldism and Waardenburg syndrome. Although YY1 is a ubiquitous transcription factor, YY1 interacts with M-MITF, the Waardenburg Syndrome IIA gene and a master transcriptional regulator of melanocytes. YY1 cooperates with M-MITF in regulating the expression of piebaldism gene KIT and multiple additional pigmentation genes. Moreover, ChIP-seq identified genome-wide YY1 targets in the melanocyte lineage. These studies mechanistically link genes implicated in human conditions of melanocyte deficiency and reveal how a ubiquitous factor (YY1) gains lineage-specific functions by co-regulating gene expression with a lineage-restricted factor (M-MITF)-a general mechanism which may confer tissue-specific gene expression in multiple lineages.
Assuntos
Cor de Cabelo , Melanócitos , Fator de Transcrição Associado à Microftalmia/metabolismo , Pigmentação , Síndrome de Waardenburg , Fator de Transcrição YY1/genética , Animais , Linhagem da Célula , Sobrevivência Celular , Modelos Animais de Doenças , Regulação da Expressão Gênica no Desenvolvimento , Cor de Cabelo/genética , Humanos , Melanócitos/citologia , Melanócitos/metabolismo , Camundongos , Camundongos Knockout , Fator de Transcrição Associado à Microftalmia/genética , Pigmentação/genética , Síndrome de Waardenburg/genética , Síndrome de Waardenburg/metabolismo , Fator de Transcrição YY1/metabolismoRESUMO
Accurate detection of epimutations in tumor cells is crucial for -understanding the molecular pathogenesis of cancer. Alterations in DNA methylation in cancer are functionally important and clinically relevant, but even this well-studied area is continually re-evaluated in light of unanticipated results, such as the strong association between aberrant DNA methylation in adult tumors and polycomb group profiles in embryonic stem cells, cancer-associated genetic mutations in epigenetic regulators such as DNMT3A and TET family genes, and the discovery of altered 5-hydroxymethylcytosine, a product of TET proteins acting on 5-methylcytosine, in human tumors with TET mutations. The abundance and distribution of covalent histone modifications in primary cancer tissues relative to normal cells is an important but largely uncharted area, although there is good evidence for a mechanistic role of cancer-specific alterations in histone modifications in tumor etiology, drug response, and tumor progression. Meanwhile, the discovery of new epigenetic marks continues, and there are many useful methods for epigenome analysis applicable to primary tumor samples, in addition to cancer cell lines. For DNA methylation and hydroxymethylation, next-generation sequencing allows increasingly inexpensive and quantitative whole-genome profiling. Similarly, the refinement and maturation of chromatin immunoprecipitation with next-generation sequencing (ChIP-seq) has made possible genome-wide mapping of histone modifications, open chromatin, and transcription factor binding sites. Computational tools have been developed apace with these epigenome methods to better enable accurate interpretation of the profiling data.
Assuntos
Biomarcadores Tumorais/genética , Epigênese Genética/genética , Epigenômica/métodos , Regulação Neoplásica da Expressão Gênica , Neoplasias/genética , Animais , HumanosRESUMO
The majority of common variants associated with common diseases, as well as an unknown proportion of causal mutations for rare diseases, fall in noncoding regions of the genome. Although catalogs of noncoding regulatory elements are steadily improving, we have a limited understanding of the functional effects of mutations within them. Here, we perform saturation mutagenesis in conjunction with massively parallel reporter assays on 20 disease-associated gene promoters and enhancers, generating functional measurements for over 30,000 single nucleotide substitutions and deletions. We find that the density of putative transcription factor binding sites varies widely between regulatory elements, as does the extent to which evolutionary conservation or integrative scores predict functional effects. These data provide a powerful resource for interpreting the pathogenicity of clinically observed mutations in these disease-associated regulatory elements, and comprise a rich dataset for the further development of algorithms that aim to predict the regulatory effects of noncoding mutations.
Assuntos
Biologia Computacional/métodos , Doença/genética , Mutagênese , Elementos Reguladores de Transcrição/genética , Linhagem Celular , Clonagem Molecular , Genoma Humano/genética , Biblioteca Genômica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Polimorfismo de Nucleotídeo ÚnicoRESUMO
TERT promoter mutations reactivate telomerase, allowing for indefinite telomere maintenance and enabling cellular immortalization. These mutations specifically recruit the multimeric ETS factor GABP, which can form two functionally independent transcription factor species: a dimer or a tetramer. We show that genetic disruption of GABPß1L (ß1L), a tetramer-forming isoform of GABP that is dispensable for normal development, results in TERT silencing in a TERT promoter mutation-dependent manner. Reducing TERT expression by disrupting ß1L culminates in telomere loss and cell death exclusively in TERT promoter mutant cells. Orthotopic xenografting of ß1L-reduced, TERT promoter mutant glioblastoma cells rendered lower tumor burden and longer overall survival in mice. These results highlight the critical role of GABPß1L in enabling immortality in TERT promoter mutant glioblastoma.
Assuntos
Neoplasias Encefálicas/genética , Fator de Transcrição de Proteínas de Ligação GA/metabolismo , Glioblastoma/patologia , Regiões Promotoras Genéticas/genética , Telomerase/genética , Animais , Neoplasias Encefálicas/mortalidade , Neoplasias Encefálicas/patologia , Feminino , Fator de Transcrição de Proteínas de Ligação GA/genética , Técnicas de Silenciamento de Genes , Glioblastoma/genética , Glioblastoma/mortalidade , Humanos , Masculino , Camundongos , Camundongos Nus , Mutação , Cultura Primária de Células , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Multimerização Proteica/genética , RNA Interferente Pequeno/metabolismo , Análise de Sobrevida , Telomerase/metabolismo , Telômero/metabolismo , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Telomerase (TERT) activation is a fundamental step in tumorigenesis. By maintaining telomere length, telomerase relieves a main barrier on cellular lifespan, enabling limitless proliferation driven by oncogenes. The recently discovered, highly recurrent mutations in the promoter of TERT are found in over 50 cancer types, and are the most common mutation in many cancers. Transcriptional activation of TERT, via promoter mutation or other mechanisms, is the rate-limiting step in production of active telomerase. Although TERT is expressed in stem cells, it is naturally silenced upon differentiation. Thus, the presence of TERT promoter mutations may shed light on whether a particular tumor arose from a stem cell or more differentiated cell type. It is becoming clear that TERT mutations occur early during cellular transformation, and activate the TERT promoter by recruiting transcription factors that do not normally regulate TERT gene expression. This review highlights the fundamental and widespread role of TERT promoter mutations in tumorigenesis, including recent progress on their mechanism of transcriptional activation. These somatic promoter mutations, along with germline variation in the TERT locus also appear to have significant value as biomarkers of patient outcome. Understanding the precise molecular mechanism of TERT activation by promoter mutation and germline variation may inspire novel cancer cell-specific targeted therapies for a large number of cancer patients.
Assuntos
Carcinogênese/genética , Mutação , Regiões Promotoras Genéticas , Telomerase/genética , Fatores de Transcrição/metabolismo , Biomarcadores Tumorais/genética , Carcinogênese/metabolismo , Ativação Enzimática , Regulação Neoplásica da Expressão Gênica , Mutação em Linhagem Germinativa , Humanos , Telomerase/metabolismo , Homeostase do Telômero , Transcrição GênicaRESUMO
Mutations in the isocitrate dehydrogenase gene IDH1 are common in low-grade glioma, where they result in the production of 2-hydroxyglutarate (2HG), disrupted patterns of histone methylation, and gliomagenesis. IDH1 mutations also cosegregate with mutations in the ATRX gene and the TERT promoter, suggesting that IDH mutation may drive the creation or selection of telomere-stabilizing events as part of immortalization/transformation process. To determine whether and how this may occur, we investigated the phenotype of pRb-/p53-deficient human astrocytes engineered with IDH1 wild-type (WT) or R132H-mutant (IDH1mut) genes as they progressed through their lifespan. IDH1mut expression promoted 2HG production and altered histone methylation within 20 population doublings (PD) but had no effect on telomerase expression or telomere length. Accordingly, cells expressing either IDH1WT or IDH1mut entered a telomere-induced crisis at PD 70. In contrast, only IDH1mut cells emerged from crisis, grew indefinitely in culture, and formed colonies in soft agar and tumors in vivo Clonal populations of postcrisis IDH1mut cells displayed shared genetic alterations, but no mutations in ATRX or the TERT promoter were detected. Instead, these cells reactivated telomerase and stabilized their telomeres in association with increased histone lysine methylation (H3K4me3) and c-Myc/Max binding at the TERT promoter. Overall, these results show that although IDH1mut does not create or select for ATRX or TERT promoter mutations, it can indirectly reactivate TERT, and in doing so contribute to astrocytic immortalization and transformation. Cancer Res; 76(22); 6680-9. ©2016 AACR.
Assuntos
Isocitrato Desidrogenase/metabolismo , Telomerase/metabolismo , Animais , Metilação de DNA , Humanos , Camundongos , TransfecçãoRESUMO
UNLABELLED: Regulation of mRNA splicing, a critical and tightly regulated cellular function, underlies the majority of proteomic diversity and is frequently disrupted in disease. Using an integrative genomics approach, we combined both genomic data and exon-level transcriptome data in two somatic tissues (cerebella and peripheral ganglia) from a transgenic mouse model of neuroblastoma, a tumor that arises from the peripheral neural crest. Here, we describe splicing quantitative trait loci associated with differential splicing across the genome that we use to identify genes with previously unknown functions within the splicing pathway and to define de novo intronic splicing motifs that influence splicing from hundreds of bases away. Our results show that these splicing motifs represent sites for functional recurrent mutations and highlight novel candidate genes in human cancers, including childhood neuroblastoma. SIGNIFICANCE: Somatic mutations with predictable downstream effects are largely relegated to coding regions, which comprise less than 2% of the human genome. Using an unbiased in vivo analysis of a mouse model of neuroblastoma, we have identified intronic splicing motifs that translate into sites for recurrent somatic mutations in human cancers.
Assuntos
Regulação Neoplásica da Expressão Gênica , Neuroblastoma/genética , Splicing de RNA , Processamento Alternativo , Animais , Cerebelo/metabolismo , Modelos Animais de Doenças , Epistasia Genética , Éxons , Redes Reguladoras de Genes , Estudo de Associação Genômica Ampla , Genômica , Íntrons , Camundongos , Mutação , Neuroblastoma/metabolismo , Motivos de Nucleotídeos , Locos de Características Quantitativas , Isoformas de RNA , Especificidade da Espécie , Gânglio Cervical Superior/metabolismoRESUMO
Reactivation of telomerase reverse transcriptase (TERT) expression enables cells to overcome replicative senescence and escape apoptosis, which are fundamental steps in the initiation of human cancer. Multiple cancer types, including up to 83% of glioblastomas (GBMs), harbor highly recurrent TERT promoter mutations of unknown function but specific to two nucleotide positions. We identified the functional consequence of these mutations in GBMs to be recruitment of the multimeric GA-binding protein (GABP) transcription factor specifically to the mutant promoter. Allelic recruitment of GABP is consistently observed across four cancer types, highlighting a shared mechanism underlying TERT reactivation. Tandem flanking native E26 transformation-specific motifs critically cooperate with these mutations to activate TERT, probably by facilitating GABP heterotetramer binding. GABP thus directly links TERT promoter mutations to aberrant expression in multiple cancers.
Assuntos
Fator de Transcrição de Proteínas de Ligação GA/metabolismo , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Glioblastoma/genética , Telomerase/genética , Alelos , Linhagem Celular Tumoral , Humanos , Regiões Promotoras Genéticas , Ligação Proteica , Multimerização ProteicaRESUMO
The role of intermediate methylation states in DNA is unclear. Here, to comprehensively identify regions of intermediate methylation and their quantitative relationship with gene activity, we apply integrative and comparative epigenomics to 25 human primary cell and tissue samples. We report 18,452 intermediate methylation regions located near 36% of genes and enriched at enhancers, exons and DNase I hypersensitivity sites. Intermediate methylation regions average 57% methylation, are predominantly allele-independent and are conserved across individuals and between mouse and human, suggesting a conserved function. These regions have an intermediate level of active chromatin marks and their associated genes have intermediate transcriptional activity. Exonic intermediate methylation correlates with exon inclusion at a level between that of fully methylated and unmethylated exons, highlighting gene context-dependent functions. We conclude that intermediate DNA methylation is a conserved signature of gene regulation and exon usage.
Assuntos
Metilação de DNA , Regulação da Expressão Gênica , Genoma Humano , Sintenia , Animais , Epigenômica , Evolução Molecular , Código das Histonas , Humanos , CamundongosRESUMO
The evolutionary history of tumor cell populations can be reconstructed from patterns of genetic alterations. In contrast to stable genetic events, epigenetic states are reversible and sensitive to the microenvironment, prompting the question whether epigenetic information can similarly be used to discover tumor phylogeny. We examined the spatial and temporal dynamics of DNA methylation in a cohort of low-grade gliomas and their patient-matched recurrences. Genes transcriptionally upregulated through promoter hypomethylation during malignant progression to high-grade glioblastoma were enriched in cell cycle function, evolving in parallel with genetic alterations that deregulate the G1/S cell cycle checkpoint. Moreover, phyloepigenetic relationships robustly recapitulated phylogenetic patterns inferred from somatic mutations. These findings highlight widespread co-dependency of genetic and epigenetic events throughout brain tumor evolution.
Assuntos
Neoplasias Encefálicas/genética , Metilação de DNA/genética , Pontos de Checagem da Fase G1 do Ciclo Celular/genética , Mutação/genética , Epigênese Genética/genética , Regulação Neoplásica da Expressão Gênica/genética , Glioblastoma/genética , Glioma/genética , Humanos , Filogenia , Regiões Promotoras Genéticas/genética , Transcrição Gênica/genética , Regulação para Cima/genéticaRESUMO
Psoriasis is an immune-mediated skin disorder that is inherited as a complex genetic trait. Although genome-wide association scans (GWAS) have identified 36 disease susceptibility regions, more than 50% of the genetic variance can be attributed to a single Major Histocompatibility Complex (MHC) locus, known as PSORS1. Genetic studies indicate that HLA-C is the strongest PSORS1 candidate gene, since markers tagging HLA-Cw*0602 consistently generate the most significant association signals in GWAS. However, it is unclear whether HLA-Cw*0602 is itself the causal PSORS1 allele, especially as the role of SNPs that may affect its expression has not been investigated. Here, we have undertaken an in-depth molecular characterization of the PSORS1 interval, with a view to identifying regulatory variants that may contribute to disease susceptibility. By analysing high-density SNP data, we refined PSORS1 to a 179 kb region encompassing HLA-C and the neighbouring HCG27 pseudogene. We compared multiple MHC sequences spanning this refined locus and identified 144 candidate susceptibility variants, which are unique to chromosomes bearing HLA-Cw*0602. In parallel, we investigated the epigenetic profile of the critical PSORS1 interval and uncovered three enhancer elements likely to be active in T lymphocytes. Finally we showed that nine candidate susceptibility SNPs map within a HLA-C enhancer and that three of these variants co-localise with binding sites for immune-related transcription factors. These data indicate that SNPs affecting HLA-Cw*0602 expression are likely to contribute to psoriasis susceptibility and highlight the importance of integrating multiple experimental approaches in the investigation of complex genomic regions such as the MHC.
Assuntos
Elementos Facilitadores Genéticos/genética , Predisposição Genética para Doença/genética , Antígenos HLA-C/genética , Psoríase/genética , Alelos , Mapeamento Cromossômico/métodos , Cromossomos Humanos Par 6/genética , Epigênese Genética , Loci Gênicos/genética , Estudo de Associação Genômica Ampla , Haplótipos , Humanos , Desequilíbrio de Ligação , Dados de Sequência Molecular , Polimorfismo de Nucleotídeo Único , Proteínas/genética , RNA Longo não Codificante , Análise de Sequência de DNA , Linfócitos T/metabolismoRESUMO
Technical advances in lipidomic analysis have generated tremendous amounts of quantitative lipid molecular species data, whose value has not been fully explored. We describe a novel computational method to infer mechanisms of de novo lipid synthesis and remodeling from lipidomic data. We focus on the mitochondrial-specific lipid cardiolipin (CL), a polyglycerol phospholipid with four acyl chains. The lengths and degree of unsaturation of these acyl chains vary across CL molecules, and regulation of these differences is important for mitochondrial energy metabolism. We developed a novel mathematical approach to determine mechanisms controlling the steady-state distribution of acyl chain combinations in CL . We analyzed mitochondrial lipids from 18 types of steady-state samples, each with at least 3 replicates, from mouse brain, heart, lung, liver, tumor cells, and tumors grown in vitro. Using a mathematical model for the CL remodeling mechanisms and a maximum likelihood approach to infer parameters, we found that for most samples the four chain positions have an independent and identical distribution, indicating they are remodeled by the same processes. Furthermore, for most brain samples and liver, the distribution of acyl chains is well-fit by a simple linear combination of the pools of acyl chains in phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylglycerol (PG). This suggests that headgroup chemistry is the key determinant of acyl donation into CL, with chain length/saturation less important. This canonical remodeling behavior appears damaged in some tumor samples, which display a consistent excess of CL molecules having particular masses. For heart and lung, the "proportional incorporation" assumption is not adequate to explain the CL distribution, suggesting additional acyl CoA-dependent remodeling that is chain-type specific. Our findings indicate that CL remodeling processes can be described by a small set of quantitative relationships, and that bioinformatic approaches can help determine these processes from high-throughput lipidomic data.
Assuntos
Cardiolipinas/metabolismo , Biologia Computacional , Modelos Biológicos , Animais , Cardiolipinas/química , Ácidos Graxos/química , Funções Verossimilhança , Masculino , Espectrometria de Massas , Camundongos , Reprodutibilidade dos TestesRESUMO
This paper describes outbreaks of Salmonella Typhimurium phage type 197 (STm197) linked to eggs from the farm of a single egg producer. Epidemiological and microbiological investigations (genotyping by multiple locus variable number tandem repeats analysis [MLVA]) identified outbreaks of STm197 with the same or closely-related MLVA profiles in a series of restaurants across Brisbane over 2 months. Environmental health investigations revealed that these restaurants were supplied with eggs from the same egg producer and that cross-contamination may have contributed to the outbreak. Environmental swabs taken from restaurant kitchens and the farm of the egg producer identified a number of salmonellas including STm197, many with MLVA profiles matching or closely related to the human strains from outbreak cases. A case-to-case comparison study showed a significant association between illness with 1 MLVA type and attending a restaurant during the 5 days before onset of illness (odds ratio [OR] 8.1, 95% confidence interval [CI] 1.8, 35.4). MLVA has become a valuable tool for S. Typhimurium surveillance and outbreak investigation. This outbreak further justifies the Commonwealth Government's decision to develop a draft national primary production and processing standard for eggs and egg products to address food safety risks posed by cracked and dirty eggs.