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1.
PLoS One ; 16(2): e0247258, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33592074

RESUMO

Health care workers (HCWs) are at higher risk for SARS-CoV-2 infection and may play a role in transmitting the infection to vulnerable patients and members of the community. This is particularly worrisome in the context of asymptomatic infection. We performed a cross-sectional study looking at asymptomatic SARS-CoV-2 infection in HCWs. We screened asymptomatic HCWs for SARS-CoV-2 via PCR. Complementary viral genome sequencing was performed on positive swab specimens. A seroprevalence analysis was also performed using multiple assays. Asymptomatic health care worker cohorts had a combined swab positivity rate of 29/5776 (0.50%, 95%CI 0.32-0.75) relative to a comparative cohort of symptomatic HCWs, where 54/1597 (3.4%) tested positive for SARS-CoV-2 (ratio of symptomatic to asymptomatic 6.8:1). SARS-CoV-2 seroprevalence among 996 asymptomatic HCWs with no prior known exposure to SARS-CoV-2 was 1.4-3.4%, depending on assay. A novel in-house Coronavirus protein microarray showed differing SARS-CoV-2 protein reactivities and helped define likely true positives vs. suspected false positives. Our study demonstrates the utility of routine screening of asymptomatic HCWs, which may help to identify a significant proportion of infections.


Assuntos
Infecções Assintomáticas/epidemiologia , /epidemiologia , Pessoal de Saúde/estatística & dados numéricos , /diagnóstico , Canadá , Humanos , Estudos Soroepidemiológicos , Centros de Atenção Terciária/estatística & dados numéricos
2.
Nat Commun ; 11(1): 3644, 2020 07 20.
Artigo em Inglês | MEDLINE | ID: mdl-32686686

RESUMO

Colorectal cancer (CRC) is a biologically heterogeneous disease. To characterize its mutational profile, we conduct targeted sequencing of 205 genes for 2,105 CRC cases with survival data. Our data shows several findings in addition to enhancing the existing knowledge of CRC. We identify PRKCI, SPZ1, MUTYH, MAP2K4, FETUB, and TGFBR2 as additional genes significantly mutated in CRC. We find that among hypermutated tumors, an increased mutation burden is associated with improved CRC-specific survival (HR = 0.42, 95% CI: 0.21-0.82). Mutations in TP53 are associated with poorer CRC-specific survival, which is most pronounced in cases carrying TP53 mutations with predicted 0% transcriptional activity (HR = 1.53, 95% CI: 1.21-1.94). Furthermore, we observe differences in mutational frequency of several genes and pathways by tumor location, stage, and sex. Overall, this large study provides deep insights into somatic mutations in CRC, and their potential relationships with survival and tumor features.


Assuntos
Neoplasias Colorretais/genética , Proteínas de Neoplasias/genética , Neoplasias do Colo/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Mutação INDEL , Mutação , Prognóstico , Proteína Supressora de Tumor p53/genética
3.
Nat Med ; 25(10): 1615-1626, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31591588

RESUMO

Oncogenesis is driven by germline, environmental and stochastic factors. It is unknown how these interact to produce the molecular phenotypes of tumors. We therefore quantified the influence of germline polymorphisms on the somatic epigenome of 589 localized prostate tumors. Predisposition risk loci influence a tumor's epigenome, uncovering a mechanism for cancer susceptibility. We identified and validated 1,178 loci associated with altered methylation in tumoral but not nonmalignant tissue. These tumor methylation quantitative trait loci influence chromatin structure, as well as RNA and protein abundance. One prominent tumor methylation quantitative trait locus is associated with AKT1 expression and is predictive of relapse after definitive local therapy in both discovery and validation cohorts. These data reveal intricate crosstalk between the germ line and the epigenome of primary tumors, which may help identify germline biomarkers of aggressive disease to aid patient triage and optimize the use of more invasive or expensive diagnostic assays.


Assuntos
Metilação de DNA/genética , Epigenoma/genética , Mutação em Linhagem Germinativa/genética , Neoplasias da Próstata/genética , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Predisposição Genética para Doença , Genoma Humano/genética , Humanos , Masculino , Recidiva Local de Neoplasia/genética , Recidiva Local de Neoplasia/patologia , Neoplasias da Próstata/patologia , Proteínas Proto-Oncogênicas c-akt/genética , Locos de Características Quantitativas/genética
4.
Genetics ; 212(3): 711-728, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31092540

RESUMO

The Polymerase Associated Factor 1 complex (Paf1C) is a multifunctional regulator of eukaryotic gene expression important for the coordination of transcription with chromatin modification and post-transcriptional processes. In this study, we investigated the extent to which the functions of Paf1C combine to regulate the Saccharomyces cerevisiae transcriptome. While previous studies focused on the roles of Paf1C in controlling mRNA levels, here, we took advantage of a genetic background that enriches for unstable transcripts, and demonstrate that deletion of PAF1 affects all classes of Pol II transcripts including multiple classes of noncoding RNAs (ncRNAs). By conducting a de novo differential expression analysis independent of gene annotations, we found that Paf1 positively and negatively regulates antisense transcription at multiple loci. Comparisons with nascent transcript data revealed that many, but not all, changes in RNA levels detected by our analysis are due to changes in transcription instead of post-transcriptional events. To investigate the mechanisms by which Paf1 regulates protein-coding genes, we focused on genes involved in iron and phosphate homeostasis, which were differentially affected by PAF1 deletion. Our results indicate that Paf1 stimulates phosphate gene expression through a mechanism that is independent of any individual Paf1C-dependent histone modification. In contrast, the inhibition of iron gene expression by Paf1 correlates with a defect in H3 K36 trimethylation. Finally, we showed that one iron regulon gene, FET4, is coordinately controlled by Paf1 and transcription of upstream noncoding DNA. Together, these data identify roles for Paf1C in controlling both coding and noncoding regions of the yeast genome.


Assuntos
Regulação Fúngica da Expressão Gênica , Proteínas Nucleares/genética , Proteínas de Saccharomyces cerevisiae/genética , Transcriptoma , Cromatina/metabolismo , Proteínas de Transporte de Cobre/genética , Proteínas de Transporte de Cobre/metabolismo , Histonas/metabolismo , Proteínas de Ligação ao Ferro/genética , Proteínas de Ligação ao Ferro/metabolismo , Proteínas Nucleares/metabolismo , Processamento de Proteína Pós-Traducional , RNA não Traduzido/genética , RNA não Traduzido/metabolismo , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/metabolismo
5.
Cancer Cell ; 35(3): 414-427.e6, 2019 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-30889379

RESUMO

DNA sequencing has identified recurrent mutations that drive the aggressiveness of prostate cancers. Surprisingly, the influence of genomic, epigenomic, and transcriptomic dysregulation on the tumor proteome remains poorly understood. We profiled the genomes, epigenomes, transcriptomes, and proteomes of 76 localized, intermediate-risk prostate cancers. We discovered that the genomic subtypes of prostate cancer converge on five proteomic subtypes, with distinct clinical trajectories. ETS fusions, the most common alteration in prostate tumors, affect different genes and pathways in the proteome and transcriptome. Globally, mRNA abundance changes explain only ∼10% of protein abundance variability. As a result, prognostic biomarkers combining genomic or epigenomic features with proteomic ones significantly outperform biomarkers comprised of a single data type.


Assuntos
Neoplasias da Próstata/patologia , Proteogenômica/métodos , Proteínas Proto-Oncogênicas c-ets/genética , Proteínas Proto-Oncogênicas c-ets/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Linhagem Celular Tumoral , Epigenômica , Perfilação da Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Prognóstico , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Translocação Genética , Sequenciamento Completo do Genoma
6.
Cell ; 176(4): 831-843.e22, 2019 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-30735634

RESUMO

The cancer transcriptome is remarkably complex, including low-abundance transcripts, many not polyadenylated. To fully characterize the transcriptome of localized prostate cancer, we performed ultra-deep total RNA-seq on 144 tumors with rich clinical annotation. This revealed a linear transcriptomic subtype associated with the aggressive intraductal carcinoma sub-histology and a fusion profile that differentiates localized from metastatic disease. Analysis of back-splicing events showed widespread RNA circularization, with the average tumor expressing 7,232 circular RNAs (circRNAs). The degree of circRNA production was correlated to disease progression in multiple patient cohorts. Loss-of-function screening identified 11.3% of highly abundant circRNAs as essential for cell proliferation; for ∼90% of these, their parental linear transcripts were not essential. Individual circRNAs can have distinct functions, with circCSNK1G3 promoting cell growth by interacting with miR-181. These data advocate for adoption of ultra-deep RNA-seq without poly-A selection to interrogate both linear and circular transcriptomes.


Assuntos
Neoplasias da Próstata/genética , RNA/genética , RNA/metabolismo , Perfilação da Expressão Gênica/métodos , Perfil Genético , Células HEK293 , Humanos , Masculino , MicroRNAs/metabolismo , Próstata/metabolismo , Processamento de RNA/genética , RNA Circular , RNA não Traduzido/genética , Análise de Sequência de RNA/métodos , Transcriptoma
7.
Nat Genet ; 51(2): 308-318, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30643250

RESUMO

Many primary-tumor subregions have low levels of molecular oxygen, termed hypoxia. Hypoxic tumors are at elevated risk for local failure and distant metastasis, but the molecular hallmarks of tumor hypoxia remain poorly defined. To fill this gap, we quantified hypoxia in 8,006 tumors across 19 tumor types. In ten tumor types, hypoxia was associated with elevated genomic instability. In all 19 tumor types, hypoxic tumors exhibited characteristic driver-mutation signatures. We observed widespread hypoxia-associated dysregulation of microRNAs (miRNAs) across cancers and functionally validated miR-133a-3p as a hypoxia-modulated miRNA. In localized prostate cancer, hypoxia was associated with elevated rates of chromothripsis, allelic loss of PTEN and shorter telomeres. These associations are particularly enriched in polyclonal tumors, representing a constellation of features resembling tumor nimbosus, an aggressive cellular phenotype. Overall, this work establishes that tumor hypoxia may drive aggressive molecular features across cancers and shape the clinical trajectory of individual tumors.


Assuntos
Hipóxia/genética , Neoplasias da Próstata/genética , Hipóxia Tumoral/genética , Alelos , Linhagem Celular Tumoral , Cromotripsia , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica/genética , Instabilidade Genômica/genética , Humanos , Masculino , MicroRNAs/genética , Células PC-3 , PTEN Fosfo-Hidrolase/genética , Telômero/genética
8.
Cell ; 173(4): 1003-1013.e15, 2018 05 03.
Artigo em Inglês | MEDLINE | ID: mdl-29681457

RESUMO

The majority of newly diagnosed prostate cancers are slow growing, with a long natural life history. Yet a subset can metastasize with lethal consequences. We reconstructed the phylogenies of 293 localized prostate tumors linked to clinical outcome data. Multiple subclones were detected in 59% of patients, and specific subclonal architectures associate with adverse clinicopathological features. Early tumor development is characterized by point mutations and deletions followed by later subclonal amplifications and changes in trinucleotide mutational signatures. Specific genes are selectively mutated prior to or following subclonal diversification, including MTOR, NKX3-1, and RB1. Patients with low-risk monoclonal tumors rarely relapse after primary therapy (7%), while those with high-risk polyclonal tumors frequently do (61%). The presence of multiple subclones in an index biopsy may be necessary, but not sufficient, for relapse of localized prostate cancer, suggesting that evolution-aware biomarkers should be studied in prospective studies of low-risk tumors suitable for active surveillance.


Assuntos
Neoplasias da Próstata/patologia , Biomarcadores Tumorais/sangue , Sequenciamento de Nucleotídeos em Larga Escala , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Masculino , Gradação de Tumores , Recidiva Local de Neoplasia , Polimorfismo de Nucleotídeo Único , Modelos de Riscos Proporcionais , Estudos Prospectivos , Neoplasias da Próstata/classificação , Neoplasias da Próstata/genética , Proteínas de Ligação a Retinoblastoma/genética , Proteínas de Ligação a Retinoblastoma/metabolismo , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo
9.
Nat Commun ; 8(1): 656, 2017 09 22.
Artigo em Inglês | MEDLINE | ID: mdl-28939825

RESUMO

Nuclear mutations are well known to drive tumor incidence, aggression and response to therapy. By contrast, the frequency and roles of mutations in the maternally inherited mitochondrial genome are poorly understood. Here we sequence the mitochondrial genomes of 384 localized prostate cancer patients, and identify a median of one mitochondrial single-nucleotide variant (mtSNV) per patient. Some of these mtSNVs occur in recurrent mutational hotspots and associate with aggressive disease. Younger patients have fewer mtSNVs than those who diagnosed at an older age. We demonstrate strong links between mitochondrial and nuclear mutational profiles, with co-occurrence between specific mutations. For example, certain control region mtSNVs co-occur with gain of the MYC oncogene, and these mutations are jointly associated with patient survival. These data demonstrate frequent mitochondrial mutation in prostate cancer, and suggest interplay between nuclear and mitochondrial mutational profiles in prostate cancer.In prostate cancer, the role of mutations in the maternally-inherited mitochondrial genome are not well known. Here, the authors demonstrate frequent, age-dependent mitochondrial mutation in prostate cancer. Strong links between mitochondrial and nuclear mutational profiles are associated with clinical aggressivity.


Assuntos
Adenocarcinoma/genética , DNA Mitocondrial/genética , Mutação Puntual , Neoplasias da Próstata/genética , Adenocarcinoma/patologia , Adulto , Fatores Etários , Idoso , Idoso de 80 Anos ou mais , Genes myc , Estudos de Associação Genética , Genoma Mitocondrial , Humanos , Masculino , Pessoa de Meia-Idade , Invasividade Neoplásica/genética , Neoplasias da Próstata/patologia , Análise de Sobrevida
10.
Nat Commun ; 8: 13671, 2017 01 09.
Artigo em Inglês | MEDLINE | ID: mdl-28067867

RESUMO

Germline mutations in the BRCA2 tumour suppressor are associated with both an increased lifetime risk of developing prostate cancer (PCa) and increased risk of aggressive disease. To understand this aggression, here we profile the genomes and methylomes of localized PCa from 14 carriers of deleterious germline BRCA2 mutations (BRCA2-mutant PCa). We show that BRCA2-mutant PCa harbour increased genomic instability and a mutational profile that more closely resembles metastastic than localized disease. BRCA2-mutant PCa shows genomic and epigenomic dysregulation of the MED12L/MED12 axis, which is frequently dysregulated in metastatic castration-resistant prostate cancer (mCRPC). This dysregulation is enriched in BRCA2-mutant PCa harbouring intraductal carcinoma (IDC). Microdissection and sequencing of IDC and juxtaposed adjacent non-IDC invasive carcinoma in 10 patients demonstrates a common ancestor to both histopathologies. Overall we show that localized castration-sensitive BRCA2-mutant tumours are uniquely aggressive, due to de novo aberration in genes usually associated with metastatic disease, justifying aggressive initial treatment.


Assuntos
Proteína BRCA2/genética , Carcinoma Ductal/genética , Regulação Neoplásica da Expressão Gênica , Mutação em Linhagem Germinativa , Complexo Mediador/genética , Neoplasias da Próstata/genética , Idoso , Proteína BRCA2/deficiência , Carcinoma Ductal/metabolismo , Carcinoma Ductal/patologia , Carcinoma Ductal/cirurgia , Análise Mutacional de DNA , Epigênese Genética , Evolução Molecular , Perfilação da Expressão Gênica , Predisposição Genética para Doença , Instabilidade Genômica , Heterozigoto , Humanos , Masculino , Complexo Mediador/metabolismo , Pessoa de Meia-Idade , Invasividade Neoplásica , Próstata/metabolismo , Próstata/patologia , Próstata/cirurgia , Prostatectomia , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Neoplasias da Próstata/cirurgia , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estudos Retrospectivos , Sequenciamento Completo do Genoma
11.
Nature ; 541(7637): 359-364, 2017 01 19.
Artigo em Inglês | MEDLINE | ID: mdl-28068672

RESUMO

Prostate tumours are highly variable in their response to therapies, but clinically available prognostic factors can explain only a fraction of this heterogeneity. Here we analysed 200 whole-genome sequences and 277 additional whole-exome sequences from localized, non-indolent prostate tumours with similar clinical risk profiles, and carried out RNA and methylation analyses in a subset. These tumours had a paucity of clinically actionable single nucleotide variants, unlike those seen in metastatic disease. Rather, a significant proportion of tumours harboured recurrent non-coding aberrations, large-scale genomic rearrangements, and alterations in which an inversion repressed transcription within its boundaries. Local hypermutation events were frequent, and correlated with specific genomic profiles. Numerous molecular aberrations were prognostic for disease recurrence, including several DNA methylation events, and a signature comprised of these aberrations outperformed well-described prognostic biomarkers. We suggest that intensified treatment of genomically aggressive localized prostate cancer may improve cure rates.


Assuntos
Genoma Humano/genética , Genômica , Mutação , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Cromotripsia , Variações do Número de Cópias de DNA , Metilação de DNA , Exoma/genética , Humanos , Masculino , Metástase Neoplásica/genética , Prognóstico , Neoplasias de Próstata Resistentes à Castração/genética , Neoplasias de Próstata Resistentes à Castração/patologia , Recidiva
12.
Nat Commun ; 6: 10001, 2015 Dec 09.
Artigo em Inglês | MEDLINE | ID: mdl-26647970

RESUMO

As whole-genome sequencing for cancer genome analysis becomes a clinical tool, a full understanding of the variables affecting sequencing analysis output is required. Here using tumour-normal sample pairs from two different types of cancer, chronic lymphocytic leukaemia and medulloblastoma, we conduct a benchmarking exercise within the context of the International Cancer Genome Consortium. We compare sequencing methods, analysis pipelines and validation methods. We show that using PCR-free methods and increasing sequencing depth to ∼ 100 × shows benefits, as long as the tumour:control coverage ratio remains balanced. We observe widely varying mutation call rates and low concordance among analysis pipelines, reflecting the artefact-prone nature of the raw data and lack of standards for dealing with the artefacts. However, we show that, using the benchmark mutation set we have created, many issues are in fact easy to remedy and have an immediate positive impact on mutation detection accuracy.


Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/métodos , Leucemia Linfoide/genética , Meduloblastoma/genética , Mutação , Genoma Humano , Humanos
13.
Genome Med ; 6(4): 32, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24944581

RESUMO

Target identification is a critical step in the lengthy and expensive process of drug development. Here, we describe a genome-wide screening platform that uses systematic overexpression of pooled human ORFs to understand drug mode-of-action and resistance mechanisms. We first calibrated our screen with the well-characterized drug methotrexate. We then identified new genes involved in the bioactivity of diverse drugs including antineoplastic agents and biologically active molecules. Finally, we focused on the transcription factor RHOXF2 whose overexpression conferred resistance to DNA damaging agents. This approach represents an orthogonal method for functional screening and, to our knowledge, has never been reported before.

14.
Science ; 344(6180): 208-11, 2014 Apr 11.
Artigo em Inglês | MEDLINE | ID: mdl-24723613

RESUMO

Genome-wide characterization of the in vivo cellular response to perturbation is fundamental to understanding how cells survive stress. Identifying the proteins and pathways perturbed by small molecules affects biology and medicine by revealing the mechanisms of drug action. We used a yeast chemogenomics platform that quantifies the requirement for each gene for resistance to a compound in vivo to profile 3250 small molecules in a systematic and unbiased manner. We identified 317 compounds that specifically perturb the function of 121 genes and characterized the mechanism of specific compounds. Global analysis revealed that the cellular response to small molecules is limited and described by a network of 45 major chemogenomic signatures. Our results provide a resource for the discovery of functional interactions among genes, chemicals, and biological processes.


Assuntos
Células/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos/métodos , Resistência a Medicamentos/genética , Redes Reguladoras de Genes , Estudo de Associação Genômica Ampla/métodos , Bibliotecas de Moléculas Pequenas/farmacologia , Linhagem Celular Tumoral , Haploinsuficiência , Humanos , Farmacogenética , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética
15.
Biochem Biophys Res Commun ; 445(4): 746-56, 2014 Mar 21.
Artigo em Inglês | MEDLINE | ID: mdl-24561123

RESUMO

G-protein coupled receptors (GPCRs) are involved in a variety of disease processes and comprise major drug targets. However, the complexity of integral membrane proteins such as GPCRs makes the identification of their interacting partners and subsequent drug development challenging. A comprehensive understanding of GPCR protein interaction networks is needed to design effective therapeutic strategies to inhibit these drug targets. Here, we developed a novel split-ubiquitin membrane yeast two-hybrid (MYTH) technology called CHIP-MYTH, which allows the unbiased characterization of interaction partners of full-length GPCRs in a drug-dependent manner. This was achieved by coupling DNA microarray technology to the MYTH approach, which allows a quantitative evaluation of interacting partners of a given integral membrane protein in the presence or absence of drug. As a proof of principle, we applied the CHIP-MYTH approach to the human ß2-adrenergic receptor (ß2AR), a target of interest in the treatment of asthma, chronic obstructive pulmonary disease (COPD), neurological disease, cardiovascular disease, and obesity. A CHIP-MYTH screen was performed in the presence or absence of salmeterol, a long-acting ß2AR-agonist. Our results suggest that ß2AR activation with salmeterol can induce the dissociation of heterotrimeric G-proteins, Gαßγ, into Gα and Gßγ subunits, which in turn activates downstream signaling cascades. Using CHIP-MYTH, we confirmed previously known and identified novel ß2AR interactors involved in GPCR-mediated signaling cascades. Several of these interactions were confirmed in mammalian cells using LUminescence-based Mammalian IntERactome (LUMIER) and co-immunoprecipitation assays. In summary, the CHIP-MYTH approach is ideal for conducting comprehensive protein-protein interactions (PPI) screenings of full-length GPCRs in the presence or absence of drugs, thus providing a valuable tool to further our understanding of GPCR-mediated signaling.


Assuntos
Agonistas de Receptores Adrenérgicos beta 2/farmacologia , Albuterol/análogos & derivados , Mapeamento de Interação de Proteínas/métodos , Mapas de Interação de Proteínas/efeitos dos fármacos , Proteômica/métodos , Receptores Adrenérgicos beta 2/metabolismo , Albuterol/farmacologia , Animais , Células HEK293 , Humanos , Modelos Moleculares , Receptores Acoplados a Proteínas-G/metabolismo , Xinafoato de Salmeterol , Transdução de Sinais/efeitos dos fármacos , Técnicas do Sistema de Duplo-Híbrido , Ubiquitina/metabolismo
16.
G3 (Bethesda) ; 3(8): 1375-87, 2013 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-23797109

RESUMO

The application of new proteomics and genomics technologies support a view in which few drugs act solely by inhibiting a single cellular target. Indeed, drug activity is modulated by complex, often incompletely understood cellular mechanisms. Therefore, efforts to decipher mode of action through genetic perturbation such as RNAi typically yields "hits" that fall into several categories. Of particular interest to the present study, we aimed to characterize secondary activities of drugs on cells. Inhibiting a known target can result in clinically relevant synthetic phenotypes. In one scenario, drug perturbation could, for example, improperly activate a protein that normally inhibits a particular kinase. In other cases, additional, lower affinity targets can be inhibited as in the example of inhibition of c-Kit observed in Bcr-Abl-positive cells treated with Gleevec. Drug transport and metabolism also play an important role in the way any chemicals act within the cells. Finally, RNAi per se can also affect cell fitness by more general off-target effects, e.g., via the modulation of apoptosis or DNA damage repair. Regardless of the root cause of these unwanted effects, understanding the scope of a drug's activity and polypharmacology is essential for better understanding its mechanism(s) of action, and such information can guide development of improved therapies. We describe a rapid, cost-effective approach to characterize primary and secondary effects of small-molecules by using small-scale libraries of virally integrated short hairpin RNAs. We demonstrate this principle using a "minipool" composed of shRNAs that target the genes encoding the reported protein targets of approved drugs. Among the 28 known reported drug-target pairs, we successfully identify 40% of the targets described in the literature and uncover several unanticipated drug-target interactions based on drug-induced synthetic lethality. We provide a detailed protocol for performing such screens and for analyzing the data. This cost-effective approach to mammalian knockdown screens, combined with the increasing maturation of RNAi technology will expand the accessibility of similar approaches in academic settings.


Assuntos
Antineoplásicos/farmacologia , Benzamidas/farmacologia , Proliferação de Células/efeitos dos fármacos , Piperazinas/farmacologia , Pirimidinas/farmacologia , RNA Interferente Pequeno/genética , Linhagem Celular Tumoral , Proteínas de Fusão bcr-abl/genética , Proteínas de Fusão bcr-abl/metabolismo , Vetores Genéticos/genética , Vetores Genéticos/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Mesilato de Imatinib , Lentivirus/genética , Miniaturização , Proteínas/antagonistas & inibidores , Proteínas/genética , Proteínas/metabolismo , Proteínas Proto-Oncogênicas c-kit/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-kit/genética , Proteínas Proto-Oncogênicas c-kit/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo
17.
Mol Cell Biol ; 33(1): 170-82, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23109428

RESUMO

Across diverse eukaryotes, the Paf1 complex (Paf1C) plays critical roles in RNA polymerase II transcription elongation and regulation of histone modifications. Beyond these roles, the human and Saccharomyces cerevisiae Paf1 complexes also interact with RNA 3'-end processing components to affect transcript 3'-end formation. Specifically, the Saccharomyces cerevisiae Paf1C functions with the RNA binding proteins Nrd1 and Nab3 to regulate the termination of at least two small nucleolar RNAs (snoRNAs). To determine how Paf1C-dependent functions regulate snoRNA formation, we used high-density tiling arrays to analyze transcripts in paf1Δ cells and uncover new snoRNA targets of Paf1. Detailed examination of Paf1-regulated snoRNA genes revealed locus-specific requirements for Paf1-dependent posttranslational histone modifications. We also discovered roles for the transcriptional regulators Bur1-Bur2, Rad6, and Set2 in snoRNA 3'-end formation. Surprisingly, at some snoRNAs, this function of Rad6 appears to be primarily independent of its role in histone H2B monoubiquitylation. Cumulatively, our work reveals a broad requirement for the Paf1C in snoRNA 3'-end formation in S. cerevisiae, implicates the participation of transcriptional proteins and histone modifications in this process, and suggests that the Paf1C contributes to the fine tuning of nuanced levels of regulation that exist at individual loci.


Assuntos
Regulação Fúngica da Expressão Gênica , Histonas/metabolismo , Proteínas Nucleares/metabolismo , RNA Nucleolar Pequeno/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Quinases Ciclina-Dependentes/genética , Quinases Ciclina-Dependentes/metabolismo , Ciclinas/genética , Ciclinas/metabolismo , Histonas/genética , Metiltransferases/genética , Metiltransferases/metabolismo , Mutação , Proteínas Nucleares/genética , Processamento de Proteína Pós-Traducional , RNA Nucleolar Pequeno/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteína de Ligação a TATA-Box/genética , Proteína de Ligação a TATA-Box/metabolismo , Fatores de Elongação da Transcrição/genética , Fatores de Elongação da Transcrição/metabolismo , Enzimas de Conjugação de Ubiquitina/genética , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitinação
18.
G3 (Bethesda) ; 2(10): 1279-89, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23050238

RESUMO

Systematic analysis of gene overexpression phenotypes provides an insight into gene function, enzyme targets, and biological pathways. Here, we describe a novel functional genomics platform that enables a highly parallel and systematic assessment of overexpression phenotypes in pooled cultures. First, we constructed a genome-level collection of ~5100 yeast barcoder strains, each of which carries a unique barcode, enabling pooled fitness assays with a barcode microarray or sequencing readout. Second, we constructed a yeast open reading frame (ORF) galactose-induced overexpression array by generating a genome-wide set of yeast transformants, each of which carries an individual plasmid-born and sequence-verified ORF derived from the Saccharomyces cerevisiae full-length EXpression-ready (FLEX) collection. We combined these collections genetically using synthetic genetic array methodology, generating ~5100 strains, each of which is barcoded and overexpresses a specific ORF, a set we termed "barFLEX." Additional synthetic genetic array allows the barFLEX collection to be moved into different genetic backgrounds. As a proof-of-principle, we describe the properties of the barFLEX overexpression collection and its application in synthetic dosage lethality studies under different environmental conditions.


Assuntos
Código de Barras de DNA Taxonômico , Proteínas Fúngicas/genética , Expressão Gênica , Genômica/métodos , Saccharomyces cerevisiae/genética , Biologia Computacional/métodos , Proteínas Fúngicas/metabolismo , Perfilação da Expressão Gênica , Genoma Fúngico , Saccharomyces cerevisiae/metabolismo
19.
ACS Chem Biol ; 7(11): 1892-901, 2012 Nov 16.
Artigo em Inglês | MEDLINE | ID: mdl-22928710

RESUMO

Platinum-based drugs have been used to successfully treat diverse cancers for several decades. Cisplatin, the original compound of this class, cross-links DNA, resulting in cell cycle arrest and cell death via apoptosis. Cisplatin is effective against several tumor types, yet it exhibits toxic side effects and tumors often develop resistance. To mitigate these liabilities while maintaining potency, we generated a library of non-classical platinum-acridine hybrid agents and assessed their mechanisms of action using a validated genome-wide screening approach in Saccharomyces cerevisiae and in the distantly related yeast Schizosaccharomyces pombe. Chemogenomic profiles from both S. cerevisiae and S. pombe demonstrate that several of the platinum-acridines damage DNA differently than cisplatin based on their requirement for distinct modules of DNA repair.


Assuntos
Acridinas/química , Acridinas/farmacologia , Antineoplásicos/química , Antineoplásicos/farmacologia , Dano ao DNA/efeitos dos fármacos , Compostos Organoplatínicos/química , Compostos Organoplatínicos/farmacologia , Cisplatino/farmacologia , DNA Fúngico/genética , Genômica , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Schizosaccharomyces/efeitos dos fármacos , Schizosaccharomyces/genética
20.
BMC Genomics ; 13: 267, 2012 Jun 22.
Artigo em Inglês | MEDLINE | ID: mdl-22727066

RESUMO

BACKGROUND: Chitosan oligosaccharide (COS), a deacetylated derivative of chitin, is an abundant, and renewable natural polymer. COS has higher antimicrobial properties than chitosan and is presumed to act by disrupting/permeabilizing the cell membranes of bacteria, yeast and fungi. COS is relatively non-toxic to mammals. By identifying the molecular and genetic targets of COS, we hope to gain a better understanding of the antifungal mode of action of COS. RESULTS: Three different chemogenomic fitness assays, haploinsufficiency (HIP), homozygous deletion (HOP), and multicopy suppression (MSP) profiling were combined with a transcriptomic analysis to gain insight in to the mode of action and mechanisms of resistance to chitosan oligosaccharides. The fitness assays identified 39 yeast deletion strains sensitive to COS and 21 suppressors of COS sensitivity. The genes identified are involved in processes such as RNA biology (transcription, translation and regulatory mechanisms), membrane functions (e.g. signalling, transport and targeting), membrane structural components, cell division, and proteasome processes. The transcriptomes of control wild type and 5 suppressor strains overexpressing ARL1, BCK2, ERG24, MSG5, or RBA50, were analyzed in the presence and absence of COS. Some of the up-regulated transcripts in the suppressor overexpressing strains exposed to COS included genes involved in transcription, cell cycle, stress response and the Ras signal transduction pathway. Down-regulated transcripts included those encoding protein folding components and respiratory chain proteins. The COS-induced transcriptional response is distinct from previously described environmental stress responses (i.e. thermal, salt, osmotic and oxidative stress) and pre-treatment with these well characterized environmental stressors provided little or any resistance to COS. CONCLUSIONS: Overexpression of the ARL1 gene, a member of the Ras superfamily that regulates membrane trafficking, provides protection against COS-induced cell membrane permeability and damage. We found that the ARL1 COS-resistant over-expression strain was as sensitive to Amphotericin B, Fluconazole and Terbinafine as the wild type cells and that when COS and Fluconazole are used in combination they act in a synergistic fashion. The gene targets of COS identified in this study indicate that COS's mechanism of action is different from other commonly studied fungicides that target membranes, suggesting that COS may be an effective fungicide for drug-resistant fungal pathogens.


Assuntos
Quitosana/farmacologia , Saccharomyces cerevisiae/efeitos dos fármacos , Anfotericina B/farmacologia , Antifúngicos/farmacologia , Permeabilidade da Membrana Celular/efeitos dos fármacos , Farmacorresistência Fúngica/efeitos dos fármacos , Fluconazol/farmacologia , Perfilação da Expressão Gênica , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Haploinsuficiência/efeitos dos fármacos , Proteínas Monoméricas de Ligação ao GTP/genética , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Naftalenos/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Terbinafina , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo
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