RESUMEN
During neural tube (NT) development, the notochord induces an organizer, the floorplate, which secretes Sonic Hedgehog (SHH) to pattern neural progenitors. Conversely, NT organoids (NTOs) from embryonic stem cells (ESCs) spontaneously form floorplates without the notochord, demonstrating that stem cells can self-organize without embryonic inducers. Here, we investigated floorplate self-organization in clonal mouse NTOs. Expression of the floorplate marker FOXA2 was initially spatially scattered before resolving into multiple clusters, which underwent competition and sorting, resulting in a stable "winning" floorplate. We identified that BMP signaling governed long-range cluster competition. FOXA2+ clusters expressed BMP4, suppressing FOXA2 in receiving cells while simultaneously expressing the BMP-inhibitor NOGGIN, promoting cluster persistence. Noggin mutation perturbed floorplate formation in NTOs and in the NT in vivo at mid/hindbrain regions, demonstrating how the floorplate can form autonomously without the notochord. Identifying the pathways governing organizer self-organization is critical for harnessing the developmental plasticity of stem cells in tissue engineering.
Asunto(s)
Proteína Morfogenética Ósea 4 , Tubo Neural , Notocorda , Organoides , Animales , Ratones , Organoides/metabolismo , Organoides/citología , Tubo Neural/metabolismo , Tubo Neural/citología , Notocorda/metabolismo , Notocorda/citología , Proteína Morfogenética Ósea 4/metabolismo , Transducción de Señal , Factor Nuclear 3-beta del Hepatocito/metabolismo , Factor Nuclear 3-beta del Hepatocito/genética , Proteínas Hedgehog/metabolismo , Proteínas Hedgehog/genética , Proteínas Portadoras/metabolismo , Proteínas Portadoras/genética , Regulación del Desarrollo de la Expresión Génica , Proteínas Morfogenéticas Óseas/metabolismoRESUMEN
Transcription factors (TFs) are important mediators of aberrant transcriptional programs in cancer cells. In this study, we focus on TF activity (TFa) as a biomarker for cell-line-selective anti-proliferative effects, in that high TFa predicts sensitivity to loss of function of a given gene (i.e., genetic dependencies [GDs]). Our linear-regression-based framework identifies 3,047 pan-cancer and 3,952 cancer-type-specific candidate TFa-GD associations from cell line data, which are then cross-examined for impact on survival in patient cohorts. One of the most prominent biomarkers is TEAD1 activity, whose associations with its predicted GDs are validated through experimental evidence as proof of concept. Overall, these TFa-GD associations represent an attractive resource for identifying innovative, biomarker-driven hypotheses for drug discovery programs in oncology.
Asunto(s)
Neoplasias , Factores de Transcripción , Humanos , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patología , Línea Celular Tumoral , Factores de Transcripción de Dominio TEA/metabolismo , Regulación Neoplásica de la Expresión Génica , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/genética , Biomarcadores de Tumor/metabolismo , Biomarcadores de Tumor/genética , Proliferación CelularRESUMEN
Loss-of-function genetic tools are widely applied for validating therapeutic targets, but their utility remains limited by incomplete on- and uncontrolled off-target effects. We describe artificial RNA interference (ARTi) based on synthetic, ultra-potent, off-target-free shRNAs that enable efficient and inducible suppression of any gene upon introduction of a synthetic target sequence into non-coding transcript regions. ARTi establishes a scalable loss-of-function tool with full control over on- and off-target effects.
Asunto(s)
Interferencia de ARN , ARN Interferente Pequeño/genéticaRESUMEN
Oncogenic alterations in human epidermal growth factor receptor 2 (HER2) occur in approximately 2% of patients with non-small cell lung cancer and predominantly affect the tyrosine kinase domain and cluster in exon 20 of the ERBB2 gene. Most clinical-grade tyrosine kinase inhibitors are limited by either insufficient selectivity against wild-type (WT) epidermal growth factor receptor (EGFR), which is a major cause of dose-limiting toxicity or by potency against HER2 exon 20 mutant variants. Here we report the discovery of covalent tyrosine kinase inhibitors that potently inhibit HER2 exon 20 mutants while sparing WT EGFR, which reduce tumor cell survival and proliferation in vitro and result in regressions in preclinical xenograft models of HER2 exon 20 mutant non-small cell lung cancer, concomitant with inhibition of downstream HER2 signaling. Our results suggest that HER2 exon 20 insertion-driven tumors can be effectively treated by a potent and highly selective HER2 inhibitor while sparing WT EGFR, paving the way for clinical translation.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Receptores ErbB/genética , Exones/genética , Genes erbB-2 , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacología , Receptor ErbB-2/genéticaRESUMEN
Genetic networks are characterized by extensive buffering. During tumor evolution, disruption of functional redundancies can create de novo vulnerabilities that are specific to cancer cells. Here, we systematically search for cancer-relevant paralog interactions using CRISPR screens and publicly available loss-of-function datasets. Our analysis reveals >2,000 candidate dependencies, several of which we validate experimentally, including CSTF2-CSTF2T, DNAJC15-DNAJC19, FAM50A-FAM50B, and RPP25-RPP25L. We provide evidence that RPP25L can physically and functionally compensate for the absence of RPP25 as a member of the RNase P/MRP complexes in tRNA processing. Our analysis also reveals unexpected redundancies between sex chromosome genes. We show that chrX- and chrY-encoded paralogs, such as ZFX-ZFY, DDX3X-DDX3Y, and EIF1AX-EIF1AY, are functionally linked. Tumor cell lines from male patients with loss of chromosome Y become dependent on the chrX-encoded gene. We propose targeting of chrX-encoded paralogs as a general therapeutic strategy for human tumors that have lost the Y chromosome.
Asunto(s)
Neoplasias , Oncogenes , ARN Helicasas DEAD-box/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Humanos , Masculino , Antígenos de Histocompatibilidad Menor/metabolismo , Neoplasias/genética , Proteínas de Unión al ARN/genética , Cromosomas Sexuales/metabolismo , Cromosoma X , Cromosoma YRESUMEN
SMARCA4/BRG1 and SMARCA2/BRM, the two mutually exclusive catalytic subunits of the BAF complex, display a well-established synthetic lethal relationship in SMARCA4-deficient cancers. Using CRISPR-Cas9 screening, we identify SMARCA4 as a novel dependency in SMARCA2-deficient esophageal squamous cell carcinoma (ESCC) models, reciprocal to the known synthetic lethal interaction. Restoration of SMARCA2 expression alleviates the dependency on SMARCA4, while engineered loss of SMARCA2 renders ESCC models vulnerable to concomitant depletion of SMARCA4. Dependency on SMARCA4 is linked to its ATPase activity, but not to bromodomain function. We highlight the relevance of SMARCA4 as a drug target in esophageal cancer using an engineered ESCC cell model harboring a SMARCA4 allele amenable to targeted proteolysis and identify SMARCA4-dependent cell models with low or absent SMARCA2 expression from additional tumor types. These findings expand the concept of SMARCA2/SMARCA4 paralog dependency and suggest that pharmacological inhibition of SMARCA4 represents a novel therapeutic opportunity for SMARCA2-deficient cancers.
Asunto(s)
ADN Helicasas/antagonistas & inhibidores , Neoplasias Esofágicas/tratamiento farmacológico , Carcinoma de Células Escamosas de Esófago/tratamiento farmacológico , Proteínas Nucleares/antagonistas & inhibidores , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/genética , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Sistemas CRISPR-Cas/genética , Línea Celular Tumoral , Supervivencia Celular/genética , ADN Helicasas/genética , Epigénesis Genética , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/patología , Carcinoma de Células Escamosas de Esófago/genética , Carcinoma de Células Escamosas de Esófago/patología , Edición Génica , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Técnicas de Inactivación de Genes , Humanos , Mutación con Pérdida de Función , Terapia Molecular Dirigida/métodos , Proteínas Nucleares/genética , ARN Guía de Kinetoplastida/genética , ARN Interferente Pequeño/metabolismo , Mutaciones Letales Sintéticas , Factores de Transcripción/deficienciaRESUMEN
Here, we report the fragment-based discovery of BI-9321, a potent, selective and cellular active antagonist of the NSD3-PWWP1 domain. The human NSD3 protein is encoded by the WHSC1L1 gene located in the 8p11-p12 amplicon, frequently amplified in breast and squamous lung cancer. Recently, it was demonstrated that the PWWP1 domain of NSD3 is required for the viability of acute myeloid leukemia cells. To further elucidate the relevance of NSD3 in cancer biology, we developed a chemical probe, BI-9321, targeting the methyl-lysine binding site of the PWWP1 domain with sub-micromolar in vitro activity and cellular target engagement at 1 µM. As a single agent, BI-9321 downregulates Myc messenger RNA expression and reduces proliferation in MOLM-13 cells. This first-in-class chemical probe BI-9321, together with the negative control BI-9466, will greatly facilitate the elucidation of the underexplored biological function of PWWP domains.
Asunto(s)
N-Metiltransferasa de Histona-Lisina/antagonistas & inhibidores , Proteínas Nucleares/antagonistas & inhibidores , Sistemas CRISPR-Cas , Línea Celular , Proliferación Celular/efectos de los fármacos , Supervivencia Celular , Regulación de la Expresión Génica/efectos de los fármacos , N-Metiltransferasa de Histona-Lisina/genética , N-Metiltransferasa de Histona-Lisina/metabolismo , Humanos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Dominios Proteicos , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismoRESUMEN
Targeted cancer therapy is based on exploiting selective dependencies of tumor cells. By leveraging recent functional screening data of cancer cell lines we identify Werner syndrome helicase (WRN) as a novel specific vulnerability of microsatellite instability-high (MSI-H) cancer cells. MSI, caused by defective mismatch repair (MMR), occurs frequently in colorectal, endometrial and gastric cancers. We demonstrate that WRN inactivation selectively impairs the viability of MSI-H but not microsatellite stable (MSS) colorectal and endometrial cancer cell lines. In MSI-H cells, WRN loss results in severe genome integrity defects. ATP-binding deficient variants of WRN fail to rescue the viability phenotype of WRN-depleted MSI-H cancer cells. Reconstitution and depletion studies indicate that WRN dependence is not attributable to acute loss of MMR gene function but might arise during sustained MMR-deficiency. Our study suggests that pharmacological inhibition of WRN helicase function represents an opportunity to develop a novel targeted therapy for MSI-H cancers.
Asunto(s)
Inestabilidad de Microsatélites , Neoplasias/terapia , Helicasa del Síndrome de Werner/antagonistas & inhibidores , Línea Celular Tumoral , Supervivencia Celular , Reparación de la Incompatibilidad de ADN , Humanos , Modelos Teóricos , Helicasa del Síndrome de Werner/genéticaRESUMEN
Genotype specific vulnerabilities of cancer cells constitute a promising strategy for the development of new therapeutics. Deletions of non-essential genes in tumors can generate unique vulnerabilities which could be exploited therapeutically. The MTAP gene is recurrently deleted in human cancers because of its chromosomal proximity to the tumor suppressor gene CDKN2A. Recent studies have uncovered an increased dependency of MTAP-deleted cancer cells on the function of a PRMT5 containing complex, including WDR77, PRMT5 and the kinase RIOK1. As RIOK1 kinase activity constitutes a potential therapeutic target, we wanted to test if MTAP deletion confers increased sensitivity to RIOK1 inhibition. Using CRISPR/Cas9-mediated genome engineering we generated analog sensitive alleles of RIOK1 in isogenic cell lines differing only by MTAP status. While we were able to independently confirm an increased dependency of MTAP-deleted cells on PRMT5, we did not detect a differential requirement for RIOK1 kinase activity between MTAP-proficient and deficient cells. Our results reveal that the kinase activity of RIOK1 is required for the survival of cancer cell lines irrespective of their MTAP status and cast doubt on the therapeutic exploitability of RIOK1 in the context of MTAP-deleted cancers.
RESUMEN
Drosophila melanogaster neural stem cells (neuroblasts [NBs]) divide asymmetrically by differentially segregating protein determinants into their daughter cells. Although the machinery for asymmetric protein segregation is well understood, the events that reprogram one of the two daughter cells toward terminal differentiation are less clear. In this study, we use time-resolved transcriptional profiling to identify the earliest transcriptional differences between the daughter cells on their way toward distinct fates. By screening for coregulated protein complexes, we identify vacuolar-type H+-ATPase (v-ATPase) among the first and most significantly down-regulated complexes in differentiating daughter cells. We show that v-ATPase is essential for NB growth and persistent activity of the Notch signaling pathway. Our data suggest that v-ATPase and Notch form a regulatory loop that acts in multiple stem cell lineages both during nervous system development and in the adult gut. We provide a unique resource for investigating neural stem cell biology and demonstrate that cell fate changes can be induced by transcriptional regulation of basic, cell-essential pathways.
Asunto(s)
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/crecimiento & desarrollo , Células-Madre Neurales/metabolismo , Receptores Notch/metabolismo , Transcripción Genética/genética , ATPasas de Translocación de Protón Vacuolares/metabolismo , Animales , Células-Madre Neurales/citología , Neurogénesis/fisiología , Transducción de Señal , Transcriptoma/genéticaRESUMEN
Traditional loss-of-function studies in Drosophila suffer from a number of shortcomings, including off-target effects in the case of RNA interference (RNAi) or the stochastic nature of mosaic clonal analysis. Here, we describe minimal in vivo GFP interference (miGFPi) as a versatile strategy to characterize gene function and to conduct highly stringent, cell type-specific loss-of-function experiments in Drosophila miGFPi combines CRISPR/Cas9-mediated tagging of genes at their endogenous locus with an immunotag and an exogenous 21 nucleotide RNAi effector sequence with the use of a single reagent, highly validated RNAi line targeting this sequence. We demonstrate the utility and time effectiveness of this method by characterizing the function of the Polymerase I (Pol I)-associated transcription factor Tif-1a, and the previously uncharacterized gene MESR4, in the Drosophila female germline stem cell lineage. In addition, we show that miGFPi serves as a powerful technique to functionally characterize individual isoforms of a gene. We exemplify this aspect of miGFPi by studying isoform-specific loss-of-function phenotypes of the longitudinals lacking (lola) gene in neural stem cells. Altogether, the miGFPi strategy constitutes a generalized loss-of-function approach that is amenable to the study of the function of all genes in the genome in a stringent and highly time effective manner.
Asunto(s)
Sistemas CRISPR-Cas , Proteínas de Drosophila/genética , Drosophila/genética , Interferencia de ARN , Proteínas Represoras/genética , Factores de Transcripción/genética , Animales , Animales Modificados Genéticamente , Núcleo Celular/metabolismo , Proteínas de Drosophila/metabolismo , Femenino , Células Germinativas , Proteínas Fluorescentes Verdes/genética , Mutación , Isoformas de Proteínas/genética , ARN Guía de Kinetoplastida , Proteínas Represoras/metabolismo , Células MadreRESUMEN
The intestinal epithelium is the most rapidly self-renewing tissue in adult animals and maintained by intestinal stem cells (ISCs) in both Drosophila and mammals. To comprehensively identify genes and pathways that regulate ISC fates, we performed a genome-wide transgenic RNAi screen in adult Drosophila intestine and identified 405 genes that regulate ISC maintenance and lineage-specific differentiation. By integrating these genes into publicly available interaction databases, we further developed functional networks that regulate ISC self-renewal, ISC proliferation, ISC maintenance of diploid status, ISC survival, ISC-to-enterocyte (EC) lineage differentiation, and ISC-to-enteroendocrine (EE) lineage differentiation. By comparing regulators among ISCs, female germline stem cells, and neural stem cells, we found that factors related to basic stem cell cellular processes are commonly required in all stem cells, and stem-cell-specific, niche-related signals are required only in the unique stem cell type. Our findings provide valuable insights into stem cell maintenance and lineage-specific differentiation.
Asunto(s)
Drosophila/metabolismo , Genoma , Intestinos/citología , Interferencia de ARN , Células Madre/citología , Animales , Animales Modificados Genéticamente/genética , Animales Modificados Genéticamente/metabolismo , Diferenciación Celular , Linaje de la Célula , Proliferación Celular , Supervivencia Celular , Bases de Datos Factuales , Drosophila/genética , Proteínas de Drosophila/antagonistas & inhibidores , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Femenino , Redes Reguladoras de Genes , Células Germinativas/citología , Células Germinativas/metabolismo , Células-Madre Neurales/citología , Células-Madre Neurales/metabolismo , Fenotipo , ARN Bicatenario/metabolismo , Receptores de Interleucina/antagonistas & inhibidores , Receptores de Interleucina/genética , Receptores de Interleucina/metabolismo , Factores de Transcripción STAT/antagonistas & inhibidores , Factores de Transcripción STAT/genética , Factores de Transcripción STAT/metabolismo , Células Madre/metabolismoRESUMEN
Gene silencing through sequence-specific targeting of mRNAs by RNAi has enabled genome-wide functional screens in cultured cells and in vivo in model organisms. These screens have resulted in the identification of new cellular pathways and potential drug targets. Considerable progress has been made to improve the quality of RNAi screen data through the development of new experimental and bioinformatics approaches. The recent availability of genome-editing strategies, such as the CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 system, when combined with RNAi, could lead to further improvements in screen data quality and follow-up experiments, thus promoting our understanding of gene function and gene regulatory networks.
Asunto(s)
Redes Reguladoras de Genes/fisiología , Pruebas Genéticas/métodos , Secuencias Invertidas Repetidas/fisiología , Interferencia de ARN , ARN Interferente Pequeño/genética , Animales , Pruebas Genéticas/tendencias , HumanosRESUMEN
Stem cells possess the capacity to generate two cells of distinct fate upon division: one cell retaining stem cell identity and the other cell destined to differentiate. These cell fates are established by cell-type-specific genetic networks. To comprehensively identify components of these networks, we performed a large-scale RNAi screen in Drosophila female germline stem cells (GSCs) covering â¼25% of the genome. The screen identified 366 genes that affect GSC maintenance, differentiation, or other processes involved in oogenesis. Comparison of GSC regulators with neural stem cell self-renewal factors identifies common and cell-type-specific self-renewal genes. Importantly, we identify the histone methyltransferase Set1 as a GSC-specific self-renewal factor. Loss of Set1 in neural stem cells does not affect cell fate decisions, suggesting a differential requirement of H3K4me3 in different stem cell lineages. Altogether, our study provides a resource that will help to further dissect the networks underlying stem cell self-renewal.
Asunto(s)
Diferenciación Celular , División Celular/fisiología , Linaje de la Célula/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Células Germinativas/citología , Células Madre/citología , Animales , Diferenciación Celular/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/citología , Femenino , Células Germinativas/metabolismo , Ovario/citología , Ovario/metabolismo , Interferencia de ARN/fisiología , Transducción de Señal/fisiología , Células Madre/metabolismoRESUMEN
Here we report the development of an in vivo system to study the interaction of stem cells with drugs using a tumor model in the adult Drosophila intestine. Strikingly, we find that some Food and Drug Administration-approved chemotherapeutics that can inhibit the growth of Drosophila tumor stem cells can paradoxically promote the hyperproliferation of their wild-type counterparts. These results reveal an unanticipated side effect on stem cells that may contribute to tumor recurrence. We propose that the same side effect may occur in humans based on our finding that it is driven in Drosophila by the evolutionarily conserved Janus kinase-signal transducers and activators of transcription (JAK-STAT) pathway. An immediate implication of our findings is that supplementing traditional chemotherapeutics with anti-inflammatories may reduce tumor recurrence.
Asunto(s)
Antineoplásicos/uso terapéutico , Neoplasias Experimentales/tratamiento farmacológico , Células Madre Neoplásicas/efectos de los fármacos , Animales , Antineoplásicos/farmacología , Proliferación Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Drosophila , Ensayos de Selección de Medicamentos Antitumorales , Neoplasias Experimentales/patología , Microambiente TumoralRESUMEN
A major objective of systems biology is to organize molecular interactions as networks and to characterize information flow within networks. We describe a computational framework to integrate protein-protein interaction (PPI) networks and genetic screens to predict the 'signs' of interactions (i.e., activation-inhibition relationships). We constructed a Drosophila melanogaster signed PPI network consisting of 6,125 signed PPIs connecting 3,352 proteins that can be used to identify positive and negative regulators of signaling pathways and protein complexes. We identified an unexpected role for the metabolic enzymes enolase and aldo-keto reductase as positive and negative regulators of proteolysis, respectively. Characterization of the activation-inhibition relationships between physically interacting proteins within signaling pathways will affect our understanding of many biological functions, including signal transduction and mechanisms of disease.
Asunto(s)
Drosophila melanogaster/metabolismo , Mapeo de Interacción de Proteínas , Oxidorreductasas de Alcohol/metabolismo , Aldehído Reductasa , Aldo-Ceto Reductasas , Animales , Biología Computacional/métodos , Drosophila melanogaster/genética , Regulación de la Expresión Génica , Fenotipo , Complejo de la Endopetidasa Proteasomal/química , Mapas de Interacción de Proteínas , Proteínas/metabolismo , Interferencia de ARN , ARN Bicatenario/metabolismo , Transducción de Señal , Biología de Sistemas/métodosRESUMEN
Regulation of cell growth is a fundamental process in development and disease that integrates a vast array of extra- and intracellular information. A central player in this process is RNA polymerase I (Pol I), which transcribes ribosomal RNA (rRNA) genes in the nucleolus. Rapidly growing cancer cells are characterized by increased Pol I-mediated transcription and, consequently, nucleolar hypertrophy. To map the genetic network underlying the regulation of nucleolar size and of Pol I-mediated transcription, we performed comparative, genome-wide loss-of-function analyses of nucleolar size in Saccharomyces cerevisiae and Drosophila melanogaster coupled with mass spectrometry-based analyses of the ribosomal DNA (rDNA) promoter. With this approach, we identified a set of conserved and nonconserved molecular complexes that control nucleolar size. Furthermore, we characterized a direct role of the histone information regulator (HIR) complex in repressing rRNA transcription in yeast. Our study provides a full-genome, cross-species analysis of a nuclear subcompartment and shows that this approach can identify conserved molecular modules.
Asunto(s)
Nucléolo Celular/metabolismo , ARN Polimerasa I/metabolismo , ARN de Hongos/biosíntesis , ARN Ribosómico/biosíntesis , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiología , Transcripción Genética/fisiología , Nucléolo Celular/genética , ADN de Hongos/genética , ADN de Hongos/metabolismo , ADN Ribosómico/genética , ADN Ribosómico/metabolismo , Genes Fúngicos/fisiología , Genes de ARNr/fisiología , Histonas/genética , Histonas/metabolismo , ARN Polimerasa I/genética , ARN de Hongos/genética , ARN Ribosómico/genética , Saccharomyces cerevisiae/citología , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
In Drosophila collections of green fluorescent protein (GFP) trap lines have been used to probe the endogenous expression patterns of trapped genes or the subcellular localization of their protein products. Here, we describe a method, based on nonoverlapping, highly specific, shRNA transgenes directed against GFP, that extends the utility of these collections to loss-of-function studies. Furthermore, we used a MiMIC transposon to generate GFP traps in Drosophila cell lines with distinct subcellular localization patterns, which will permit high-throughput screens using fluorescently tagged proteins. Finally, we show that fluorescent traps, paired with recombinant nanobodies and mass spectrometry, allow the study of endogenous protein complexes in Drosophila.
Asunto(s)
Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila/genética , Drosophila/metabolismo , Colorantes Fluorescentes , Proteínas Fluorescentes Verdes , Mapeo de Interacción de Proteínas/métodos , Animales , Línea Celular , Supervivencia Celular/genética , Embrión no Mamífero/metabolismo , Femenino , Orden Génico , Silenciador del Gen , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Complejos Multiproteicos/aislamiento & purificación , Complejos Multiproteicos/metabolismo , Factores de Elongación de Péptidos/genética , Factores de Elongación de Péptidos/metabolismo , Unión Proteica/fisiología , ARN Interferente Pequeño/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Células Madre/metabolismoRESUMEN
The balance between stem cell self-renewal and differentiation is precisely controlled to ensure tissue homeostasis and prevent tumorigenesis. Here we use genome-wide transgenic RNAi to identify 620 genes potentially involved in controlling this balance in Drosophila neuroblasts. We quantify all phenotypes and derive measurements for proliferation, lineage, cell size, and cell shape. We identify a set of transcriptional regulators essential for self-renewal and use hierarchical clustering and integration with interaction data to create functional networks for the control of neuroblast self-renewal and differentiation. Our data identify key roles for the chromatin remodeling Brm complex, the spliceosome, and the TRiC/CCT-complex and show that the alternatively spliced transcription factor Lola and the transcriptional elongation factors Ssrp and Barc control self-renewal in neuroblast lineages. As our data are strongly enriched for genes highly expressed in murine neural stem cells, they are likely to provide valuable insights into mammalian stem cell biology as well.
Asunto(s)
Chaperonina con TCP-1/metabolismo , Drosophila/genética , Células-Madre Neurales/metabolismo , ARN Mensajero/análisis , Factores de Transcripción/metabolismo , Empalme Alternativo/genética , Animales , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Diferenciación Celular/genética , Supervivencia Celular/genética , Células Cultivadas , Chaperonina con TCP-1/genética , Ensamble y Desensamble de Cromatina/genética , Biología Computacional , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Estudio de Asociación del Genoma Completo , Proteínas del Grupo de Alta Movilidad/genética , Proteínas del Grupo de Alta Movilidad/metabolismo , Larva/genética , Familia de Multigenes/genética , Células-Madre Neurales/citología , Empalmosomas/genética , Transactivadores/genética , Transactivadores/metabolismo , Factores de Transcripción/genética , Factores de Elongación Transcripcional/genética , Factores de Elongación Transcripcional/metabolismoRESUMEN
Systems biology aims to describe the complex interplays between cellular building blocks which, in their concurrence, give rise to the emergent properties observed in cellular behaviors and responses. This approach tries to determine the molecular players and the architectural principles of their interactions within the genetic networks that control certain biological processes. Large-scale loss-of-function screens, applicable in various different model systems, have begun to systematically interrogate entire genomes to identify the genes that contribute to a certain cellular response. In particular, RNA interference (RNAi)-based high-throughput screens have been instrumental in determining the composition of regulatory systems and paired with integrative data analyses have begun to delineate the genetic networks that control cell biological and developmental processes. Through the creation of tools for both, in vitro and in vivo genome-wide RNAi screens, Drosophila melanogaster has emerged as one of the key model organisms in systems biology research and over the last years has massively contributed to and hence shaped this discipline. WIREs Syst Biol Med 2011 3 471-478 DOI: 10.1002/wsbm.127