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1.
Cell ; 166(6): 1397-1410.e16, 2016 Sep 08.
Artigo em Inglês | MEDLINE | ID: mdl-27610566

RESUMO

Whereas domestication of livestock, pets, and crops is well documented, it is still unclear to what extent microbes associated with the production of food have also undergone human selection and where the plethora of industrial strains originates from. Here, we present the genomes and phenomes of 157 industrial Saccharomyces cerevisiae yeasts. Our analyses reveal that today's industrial yeasts can be divided into five sublineages that are genetically and phenotypically separated from wild strains and originate from only a few ancestors through complex patterns of domestication and local divergence. Large-scale phenotyping and genome analysis further show strong industry-specific selection for stress tolerance, sugar utilization, and flavor production, while the sexual cycle and other phenotypes related to survival in nature show decay, particularly in beer yeasts. Together, these results shed light on the origins, evolutionary history, and phenotypic diversity of industrial yeasts and provide a resource for further selection of superior strains. PAPERCLIP.


Assuntos
Cerveja/microbiologia , Microbiologia Industrial , Filogenia , Saccharomyces cerevisiae/classificação , Saccharomyces cerevisiae/fisiologia , Variações do Número de Cópias de DNA/genética , Genes Fúngicos/genética , Variação Genética , Genoma Fúngico/genética , Viabilidade Microbiana/genética , Fenótipo , Ploidias , Saccharomyces cerevisiae/genética , Seleção Genética
2.
Cell ; 157(2): 447-458, 2014 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-24725410

RESUMO

Maintenance of a constant cell volume in response to extracellular or intracellular osmotic changes is critical for cellular homeostasis. Activation of a ubiquitous volume-regulated anion channel (VRAC) plays a key role in this process; however, its molecular identity in vertebrates remains unknown. Here, we used a cell-based fluorescence assay and performed a genome-wide RNAi screen to find components of VRAC. We identified SWELL1 (LRRC8A), a member of a four-transmembrane protein family with unknown function, as essential for hypotonicity-induced iodide influx. SWELL1 is localized to the plasma membrane, and its knockdown dramatically reduces endogenous VRAC currents and regulatory cell volume decrease in various cell types. Furthermore, point mutations in SWELL1 cause a significant change in VRAC anion selectivity, demonstrating that SWELL1 is an essential VRAC component. These findings enable further molecular characterization of the VRAC channel complex and genetic studies for understanding the function of VRAC in normal physiology and disease.


Assuntos
Tamanho Celular , Proteínas de Membrana/metabolismo , Animais , Membrana Celular/química , Membrana Celular/metabolismo , Perfilação da Expressão Gênica , Técnicas de Silenciamento de Genes , Estudo de Associação Genômica Ampla , Células HEK293 , Células HeLa , Humanos , Iodetos/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/genética , Camundongos , Interferência de RNA
3.
Cell ; 139(1): 199-210, 2009 Oct 02.
Artigo em Inglês | MEDLINE | ID: mdl-19765810

RESUMO

Two decades of research identified more than a dozen clock genes and defined a biochemical feedback mechanism of circadian oscillator function. To identify additional clock genes and modifiers, we conducted a genome-wide small interfering RNA screen in a human cellular clock model. Knockdown of nearly 1000 genes reduced rhythm amplitude. Potent effects on period length or increased amplitude were less frequent; we found hundreds of these and confirmed them in secondary screens. Characterization of a subset of these genes demonstrated a dosage-dependent effect on oscillator function. Protein interaction network analysis showed that dozens of gene products directly or indirectly associate with known clock components. Pathway analysis revealed these genes are overrepresented for components of insulin and hedgehog signaling, the cell cycle, and the folate metabolism. Coupled with data showing many of these pathways are clock regulated, we conclude the clock is interconnected with many aspects of cellular function.


Assuntos
Relógios Biológicos , Ritmo Circadiano , Estudo de Associação Genômica Ampla , Linhagem Celular , Técnicas de Silenciamento de Genes , Humanos , Interferência de RNA , RNA Interferente Pequeno/metabolismo
4.
Proc Natl Acad Sci U S A ; 118(1)2021 01 05.
Artigo em Inglês | MEDLINE | ID: mdl-33443164

RESUMO

The regulatory mechanisms of circadian rhythms have been studied primarily at the level of the transcription-translation feedback loops of protein-coding genes. Regulatory modules involving noncoding RNAs are less thoroughly understood. In particular, emerging evidence has revealed the important role of microRNAs (miRNAs) in maintaining the robustness of the circadian system. To identify miRNAs that have the potential to modulate circadian rhythms, we conducted a genome-wide miRNA screen using U2OS luciferase reporter cells. Among 989 miRNAs in the library, 120 changed the period length in a dose-dependent manner. We further validated the circadian regulatory function of an miRNA cluster, miR-183/96/182, both in vitro and in vivo. We found that all three members of this miRNA cluster can modulate circadian rhythms. Particularly, miR-96 directly targeted a core circadian clock gene, PER2. The knockout of the miR-183/96/182 cluster in mice showed tissue-specific effects on circadian parameters and altered circadian rhythms at the behavioral level. This study identified a large number of miRNAs, including the miR-183/96/182 cluster, as circadian modulators. We provide a resource for further understanding the role of miRNAs in the circadian network and highlight the importance of miRNAs as a genome-wide layer of circadian clock regulation.


Assuntos
Ritmo Circadiano/genética , Regulação da Expressão Gênica/genética , MicroRNAs/metabolismo , Proteínas Circadianas Period/metabolismo , Animais , Linhagem Celular Tumoral , Ritmo Circadiano/efeitos da radiação , Regulação da Expressão Gênica/efeitos da radiação , Técnicas de Introdução de Genes , Técnicas de Inativação de Genes , Genômica , Humanos , Luciferases/genética , Luciferases/metabolismo , Pulmão/metabolismo , Pulmão/efeitos da radiação , Camundongos , MicroRNAs/genética , Família Multigênica , Especificidade de Órgãos , Proteínas Circadianas Period/genética , Retina/metabolismo , Retina/efeitos da radiação , Núcleo Supraquiasmático/metabolismo , Núcleo Supraquiasmático/efeitos da radiação , Fatores de Tempo
5.
Cell ; 135(1): 49-60, 2008 Oct 03.
Artigo em Inglês | MEDLINE | ID: mdl-18854154

RESUMO

Human Immunodeficiency Viruses (HIV-1 and HIV-2) rely upon host-encoded proteins to facilitate their replication. Here, we combined genome-wide siRNA analyses with interrogation of human interactome databases to assemble a host-pathogen biochemical network containing 213 confirmed host cellular factors and 11 HIV-1-encoded proteins. Protein complexes that regulate ubiquitin conjugation, proteolysis, DNA-damage response, and RNA splicing were identified as important modulators of early-stage HIV-1 infection. Additionally, over 40 new factors were shown to specifically influence the initiation and/or kinetics of HIV-1 DNA synthesis, including cytoskeletal regulatory proteins, modulators of posttranslational modification, and nucleic acid-binding proteins. Finally, 15 proteins with diverse functional roles, including nuclear transport, prostaglandin synthesis, ubiquitination, and transcription, were found to influence nuclear import or viral DNA integration. Taken together, the multiscale approach described here has uncovered multiprotein virus-host interactions that likely act in concert to facilitate the early steps of HIV-1 infection.


Assuntos
Infecções por HIV/virologia , HIV-1/fisiologia , Interações Hospedeiro-Patógeno , Proteínas/metabolismo , Replicação Viral , Linhagem Celular , Humanos , Interferência de RNA , Técnicas do Sistema de Duplo-Híbrido
6.
Nature ; 541(7638): 541-545, 2017 01 26.
Artigo em Inglês | MEDLINE | ID: mdl-28068668

RESUMO

Cell fate perturbations underlie many human diseases, including breast cancer. Unfortunately, the mechanisms by which breast cell fate are regulated are largely unknown. The mammary gland epithelium consists of differentiated luminal epithelial and basal myoepithelial cells, as well as undifferentiated stem cells and more restricted progenitors. Breast cancer originates from this epithelium, but the molecular mechanisms that underlie breast epithelial hierarchy remain ill-defined. Here, we use a high-content confocal image-based short hairpin RNA screen to identify tumour suppressors that regulate breast cell fate in primary human breast epithelial cells. We show that ablation of the large tumour suppressor kinases (LATS) 1 and 2 (refs 5, 6), which are part of the Hippo pathway, promotes the luminal phenotype and increases the number of bipotent and luminal progenitors, the proposed cells-of-origin of most human breast cancers. Mechanistically, we have identified a direct interaction between Hippo and oestrogen receptor-α (ERα) signalling. In the presence of LATS, ERα was targeted for ubiquitination and Ddb1-cullin4-associated-factor 1 (DCAF1)-dependent proteasomal degradation. Absence of LATS stabilized ERα and the Hippo effectors YAP and TAZ (hereafter YAP/TAZ), which together control breast cell fate through intrinsic and paracrine mechanisms. Our findings reveal a non-canonical (that is, YAP/TAZ-independent) effect of LATS in the regulation of human breast cell fate.


Assuntos
Mama/citologia , Mama/enzimologia , Diferenciação Celular , Linhagem da Célula , Receptor alfa de Estrogênio/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/agonistas , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Mama/patologia , Proteínas de Transporte/metabolismo , Células Cultivadas , Receptor alfa de Estrogênio/agonistas , Feminino , Genes Supressores de Tumor , Humanos , Fosfoproteínas/agonistas , Fosfoproteínas/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas Serina-Treonina Quinases/deficiência , Proteólise , Transdução de Sinais , Fatores de Transcrição , Proteínas Supressoras de Tumor/deficiência , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases , Proteínas de Sinalização YAP
7.
Nat Methods ; 15(11): 941-946, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30297964

RESUMO

CRISPR-Cas9 screening allows genome-wide interrogation of gene function. Currently, to achieve the high and uniform Cas9 expression desirable for screening, one needs to engineer stable and clonal Cas9-expressing cells-an approach that is not applicable in human primary cells. Guide Swap permits genome-scale pooled CRISPR-Cas9 screening in human primary cells by exploiting the unexpected finding that editing by lentivirally delivered, targeted guide RNAs (gRNAs) occurs efficiently when Cas9 is introduced in complex with nontargeting gRNA. We validated Guide Swap in depletion and enrichment screens in CD4+ T cells. Next, we implemented Guide Swap in a model of ex vivo hematopoiesis, and identified known and previously unknown regulators of CD34+ hematopoietic stem and progenitor cell (HSPC) expansion. We anticipate that this platform will be broadly applicable to other challenging cell types, and thus will enable discovery in previously inaccessible but biologically relevant human primary cell systems.


Assuntos
Linfócitos T CD8-Positivos/metabolismo , Proteína 9 Associada à CRISPR/metabolismo , Sistemas CRISPR-Cas , Edição de Genes , Genoma Humano , Células-Tronco Hematopoéticas/metabolismo , RNA Guia de Cinetoplastídeos/genética , Linfócitos T CD8-Positivos/citologia , Células Cultivadas , Células HEK293 , Células-Tronco Hematopoéticas/citologia , Humanos
8.
Biotechnol Bioeng ; 118(8): 3187-3199, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34050941

RESUMO

Cancer is a disease of somatic mutations. These cellular mutations compete to dominate their microenvironment and dictate the disease outcome. While a therapeutic approach to target-specific oncogenic driver mutations helps to manage the disease, subsequent molecular evolution of tumor cells threatens to overtake therapeutic progress. There is a need for rapid, high-throughput, unbiased in vitro discovery screening platforms that capture the native complexities of the tumor and rapidly identify mutations that confer chemotherapeutic drug resistance. Taking the example of the CDK4/6 inhibitor (CDK4/6i) class of drugs, we show that the pooled in vitro CRISPR screening platform enables rapid discovery of drug resistance mutations in a three-dimensional (3D) setting. Gene-edited cancer cell clones assembled into an organotypic multicellular tumor spheroid (MCTS), exposed to CDK4/6i caused selection and enrichment of the most drug-resistant phenotypes, detectable by next-gen sequencing after a span of 28 days. The platform was sufficiently sensitive to enrich for even a single drug-resistant cell within a large, drug-responsive complex 3D tumor spheroid. The genome-wide 3D CRISPR-mediated knockout screen (>18,000 genes) identified several genes whose disruptions conferred resistance to CDK4/6i. Furthermore, multiple novel candidate genes were identified as top hits only in the microphysiological 3D enrichment assay platform and not the conventional 2D assays. Taken together, these findings suggest that including phenotypic 3D resistance profiling in decision trees could improve discovery and reconfirmation of drug resistance mechanisms and afford a platform for exploring noncell autonomous interactions, selection pressures, and clonal competition.


Assuntos
Antineoplásicos/farmacologia , Neoplasias da Mama , Sistemas CRISPR-Cas , Técnicas de Cultura de Células , Resistencia a Medicamentos Antineoplásicos , Esferoides Celulares/metabolismo , Microambiente Tumoral , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Humanos
9.
Mol Cell ; 50(3): 356-67, 2013 May 09.
Artigo em Inglês | MEDLINE | ID: mdl-23603119

RESUMO

A high-throughput RNA interference (RNAi) screen targeting 542 genes of the human kinome was used to discover regulators of RNAi. Here we report that the proto-oncogene Akt-3/PKBγ (Akt3) phosphorylates Argonaute 2 (Ago2) at S387, which downregulates cleavage and upregulates translational repression of endogenous microRNA (miRNA)-targeted messenger RNAs (mRNAs). We further demonstrate that Akt3 coimmunoprecipitates with Ago2 and phosphorylation of Ago2 at S387 facilitates its interaction with GW182 and localization to cytoplasmic processing bodies (P bodies), where miRNA-targeted mRNAs are thought to be stored and degraded. Therefore, Akt3-mediated phosphorylation of Ago2 is a molecular switch between target mRNA cleavage and translational repression activities of Ago2.


Assuntos
Proteínas Argonautas/genética , MicroRNAs/genética , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Argonautas/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Regulação para Baixo , Células HEK293 , Células HeLa , Humanos , Fosforilação , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas c-akt/metabolismo , Regulação para Cima
10.
Nature ; 463(7282): 813-7, 2010 Feb 11.
Artigo em Inglês | MEDLINE | ID: mdl-20027183

RESUMO

Influenza A virus is an RNA virus that encodes up to 11 proteins and this small coding capacity demands that the virus use the host cellular machinery for many aspects of its life cycle. Knowledge of these host cell requirements not only informs us of the molecular pathways exploited by the virus but also provides further targets that could be pursued for antiviral drug development. Here we use an integrative systems approach, based on genome-wide RNA interference screening, to identify 295 cellular cofactors required for early-stage influenza virus replication. Within this group, those involved in kinase-regulated signalling, ubiquitination and phosphatase activity are the most highly enriched, and 181 factors assemble into a highly significant host-pathogen interaction network. Moreover, 219 of the 295 factors were confirmed to be required for efficient wild-type influenza virus growth, and further analysis of a subset of genes showed 23 factors necessary for viral entry, including members of the vacuolar ATPase (vATPase) and COPI-protein families, fibroblast growth factor receptor (FGFR) proteins, and glycogen synthase kinase 3 (GSK3)-beta. Furthermore, 10 proteins were confirmed to be involved in post-entry steps of influenza virus replication. These include nuclear import components, proteases, and the calcium/calmodulin-dependent protein kinase (CaM kinase) IIbeta (CAMK2B). Notably, growth of swine-origin H1N1 influenza virus is also dependent on the identified host factors, and we show that small molecule inhibitors of several factors, including vATPase and CAMK2B, antagonize influenza virus replication.


Assuntos
Fatores Biológicos/genética , Fatores Biológicos/fisiologia , Interações Hospedeiro-Patógeno/fisiologia , Vírus da Influenza A/crescimento & desenvolvimento , Influenza Humana/genética , Influenza Humana/virologia , Replicação Viral/fisiologia , Animais , Linhagem Celular , Chlorocebus aethiops , Biblioteca Gênica , Genoma Humano/genética , Interações Hospedeiro-Patógeno/genética , Humanos , Vírus da Influenza A Subtipo H1N1/classificação , Vírus da Influenza A Subtipo H1N1/crescimento & desenvolvimento , Vírus da Influenza A/classificação , Interferência de RNA , Células Vero , Internalização do Vírus
11.
Nat Genet ; 38(3): 312-9, 2006 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-16474406

RESUMO

Direct evidence for the requirement of transcriptional feedback repression in circadian clock function has been elusive. Here, we developed a molecular genetic screen in mammalian cells to identify mutants of the circadian transcriptional activators CLOCK and BMAL1, which were uncoupled from CRYPTOCHROME (CRY)-mediated transcriptional repression. Notably, mutations in the PER-ARNT-SIM domain of CLOCK and the C terminus of BMAL1 resulted in synergistic insensitivity through reduced physical interactions with CRY. Coexpression of these mutant proteins in cultured fibroblasts caused arrhythmic phenotypes in population and single-cell assays. These data demonstrate that CRY-mediated repression of the CLOCK/BMAL1 complex activity is required for maintenance of circadian rhythmicity and provide formal proof that transcriptional feedback is required for mammalian clock function.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Ritmo Circadiano/fisiologia , Regulação da Expressão Gênica , Transativadores/genética , Células 3T3 , Fatores de Transcrição ARNTL , Animais , Proteínas CLOCK , Linhagem Celular , Retroalimentação , Genes Reporter , Humanos , Luciferases/análise , Luciferases/genética , Luminescência , Camundongos , Plasmídeos , Tempo
12.
Proc Natl Acad Sci U S A ; 108(41): 17117-22, 2011 Oct 11.
Artigo em Inglês | MEDLINE | ID: mdl-21949408

RESUMO

Glucocorticoids can inhibit inflammation by abrogating the activity of NF-κB, a family of transcription factors that regulates the production of proinflammatory cytokines. To understand the molecular mechanism of repression of NF-κB activity by glucocorticoids, we performed a high-throughput siRNA oligo screen to identify novel genes involved in this process. Here, we report that loss of p53, a tumor suppressor protein, impaired repression of NF-κB target gene transcription by glucocorticoids. Additionally, loss of p53 also impaired transcription of glucocorticoid receptor (GR) target genes, whereas upstream NF-κB and glucocorticoid receptor signaling cascades remained intact. We further demonstrate that p53 loss severely impaired glucocorticoid rescue of death in a mouse model of LPS shock. Our findings unveil a new role for p53 in the repression of NF-κB by glucocorticoids and suggest important implications for treatment of the proinflammatory microenvironments found in tumors with aberrant p53 activity.


Assuntos
NF-kappa B/metabolismo , Receptores de Glucocorticoides/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Animais , Dexametasona/farmacologia , Ensaios de Triagem em Larga Escala , Lipopolissacarídeos/toxicidade , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mapas de Interação de Proteínas , Interferência de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , Choque Séptico/induzido quimicamente , Choque Séptico/genética , Choque Séptico/metabolismo , Transdução de Sinais , Proteína Supressora de Tumor p53/deficiência , Proteína Supressora de Tumor p53/genética
13.
Proc Natl Acad Sci U S A ; 107(34): 15087-92, 2010 Aug 24.
Artigo em Inglês | MEDLINE | ID: mdl-20696907

RESUMO

DNA methylation is an important epigenetic modification involved in transcriptional regulation, nuclear organization, development, aging, and disease. Although DNA methyltransferases have been characterized, the mechanisms for DNA demethylation remain poorly understood. Using a cell-based reporter assay, we performed a functional genomics screen to identify genes involved in DNA demethylation. Here we show that RNF4 (RING finger protein 4), a SUMO-dependent ubiquitin E3-ligase previously implicated in maintaining genome stability, plays a key role in active DNA demethylation. RNF4 reactivates methylation-silenced reporters and promotes global DNA demethylation. Rnf4 deficiency is embryonic lethal with higher levels of methylation in genomic DNA. Mechanistic studies show that RNF4 interacts with and requires the base excision repair enzymes TDG and APE1 for active demethylation. This activity appears to occur by enhancing the enzymatic activities that repair DNA G:T mismatches generated from methylcytosine deamination. Collectively, our study reveals a unique function for RNF4, which may serve as a direct link between epigenetic DNA demethylation and DNA repair in mammalian cells.


Assuntos
Metilação de DNA , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Animais , Sequência de Bases , Linhagem Celular , DNA/química , DNA/genética , DNA/metabolismo , Metilação de DNA/genética , Metilação de DNA/fisiologia , Reparo de Erro de Pareamento de DNA/genética , Reparo de Erro de Pareamento de DNA/fisiologia , Feminino , Genes Letais , Genes Reporter , Genes p16 , Genômica , Humanos , Camundongos , Camundongos Knockout , Proteínas Nucleares/deficiência , Proteínas Nucleares/genética , Gravidez , Regiões Promotoras Genéticas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Fatores de Transcrição/deficiência , Fatores de Transcrição/genética , Ubiquitina-Proteína Ligases
14.
Proc Natl Acad Sci U S A ; 106(40): 17025-30, 2009 Oct 06.
Artigo em Inglês | MEDLINE | ID: mdl-19805117

RESUMO

Malignant melanoma is the most aggressive form of cutaneous carcinoma, accounting for 75% of all deaths caused by skin cancers. Microphthalmia-associated transcription factor (MITF) is a master gene regulating melanocyte development and functions as a "lineage addiction" oncogene in malignant melanoma. We have identified the receptor protein tyrosine kinase TYRO3 as an upstream regulator of MITF expression by a genome-wide gain-of-function cDNA screen and show that TYRO3 induces MITF-M expression in a SOX10-dependent manner in melanoma cells. Expression of TYRO3 is significantly elevated in human primary melanoma tissue samples and melanoma cell lines and correlates with MITF-M mRNA levels. TYRO3 overexpression bypasses BRAF(V600E)-induced senescence in primary melanocytes, inducing transformation of non-tumorigenic cell lines. Furthermore, TYRO3 knockdown represses cellular proliferation and colony formation in melanoma cells, and sensitizes them to chemotherapeutic agent-induced apoptosis; TYRO3 knockdown in melanoma cells also inhibits tumorigenesis in vivo. Taken together, these data indicate that TYRO3 may serve as a target for the development of therapeutic agents for melanoma.


Assuntos
Estudo de Associação Genômica Ampla/métodos , Melanoma/patologia , Fator de Transcrição Associado à Microftalmia/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Animais , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Western Blotting , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Análise por Conglomerados , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Melanoma/genética , Melanoma/metabolismo , Melanoma Experimental/genética , Melanoma Experimental/metabolismo , Melanoma Experimental/patologia , Camundongos , Camundongos Nus , Fator de Transcrição Associado à Microftalmia/genética , Análise de Sequência com Séries de Oligonucleotídeos , Interferência de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores Proteína Tirosina Quinases/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa
15.
Commun Biol ; 4(1): 1085, 2021 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-34526658

RESUMO

We present two high-throughput compatible methods to detect the interaction of ectopically expressed (RT-Bind) or endogenously tagged (EndoBind) proteins of interest. Both approaches provide temporal evaluation of dimer formation over an extended duration. Using examples of the Nrf2-KEAP1 and the CRAF-KRAS-G12V interaction, we demonstrate that our method allows for the detection of signal for more than 2 days after substrate addition, allowing for continuous monitoring of endogenous protein-protein interactions in real time.


Assuntos
Ensaios de Triagem em Larga Escala/métodos , Proteína 1 Associada a ECH Semelhante a Kelch/química , Fator 2 Relacionado a NF-E2/química , Proteínas Proto-Oncogênicas p21(ras)/química , Células HEK293 , Humanos , Ligação Proteica
16.
Proc Natl Acad Sci U S A ; 104(51): 20314-9, 2007 Dec 18.
Artigo em Inglês | MEDLINE | ID: mdl-18077367

RESUMO

Signal transduction pathways often use a transcriptional component to mediate adaptive cellular responses. Coactivator proteins function prominently in these pathways as the conduit to the basic transcriptional machinery. Here we present a high-throughput cell-based screening strategy, termed the "coactivator trap," to study the functional interactions of coactivators with transcription factors. We applied this strategy to the cAMP signaling pathway, which utilizes two families of coactivators, the cAMP response element binding protein (CREB) binding protein (CBP)/p300 family and the recently identified transducers of regulated CREB activity family (TORCs1-3). In addition to identifying numerous known interactions of these coactivators, this analysis identified NONO (p54(nrb)) as a TORC-interacting protein. RNA interference experiments demonstrate that NONO is necessary for cAMP-dependent activation of CREB target genes in vivo. Furthermore, TORC2 and NONO complex on cAMP-responsive promoters, and NONO acts as a bridge between the CREB/TORC complex and RNA polymerase II. These data demonstrate the utility of the coactivator trap by identification of a component of cAMP-mediated transcription.


Assuntos
AMP Cíclico/metabolismo , Proteínas Associadas à Matriz Nuclear/metabolismo , Fatores de Transcrição de Octâmero/metabolismo , Mapeamento de Interação de Proteínas/métodos , Proteínas de Ligação a RNA/metabolismo , Linhagem Celular , Proteínas de Ligação a DNA , Humanos , Proteínas Associadas à Matriz Nuclear/antagonistas & inibidores , Proteínas Associadas à Matriz Nuclear/genética , Fatores de Transcrição de Octâmero/antagonistas & inibidores , Fatores de Transcrição de Octâmero/genética , Interferência de RNA , Proteínas de Ligação a RNA/antagonistas & inibidores , Proteínas de Ligação a RNA/genética , Transdução de Sinais , Fatores de Transcrição/metabolismo , Transcrição Gênica
17.
Neuron ; 43(4): 527-37, 2004 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-15312651

RESUMO

The mammalian circadian clock plays an integral role in timing rhythmic physiology and behavior, such as locomotor activity, with anticipated daily environmental changes. The master oscillator resides within the suprachiasmatic nucleus (SCN), which can maintain circadian rhythms in the absence of synchronizing light input. Here, we describe a genomics-based approach to identify circadian activators of Bmal1, itself a key transcriptional activator that is necessary for core oscillator function. Using cell-based functional assays, as well as behavioral and molecular analyses, we identified Rora as an activator of Bmal1 transcription within the SCN. Rora is required for normal Bmal1 expression and consolidation of daily locomotor activity and is regulated by the core clock in the SCN. These results suggest that opposing activities of the orphan nuclear receptors Rora and Rev-erb alpha, which represses Bmal1 expression, are important in the maintenance of circadian clock function.


Assuntos
Relógios Biológicos/genética , Ritmo Circadiano/genética , Genômica/métodos , Receptores do Ácido Retinoico/genética , Fatores de Transcrição ARNTL , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Células HeLa , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes Neurológicos , Membro 1 do Grupo F da Subfamília 1 de Receptores Nucleares , Regiões Promotoras Genéticas , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Receptores Citoplasmáticos e Nucleares , Receptores do Ácido Retinoico/biossíntese , Elementos de Resposta/genética , Transativadores , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
18.
Methods Mol Biol ; 1755: 89-105, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29671265

RESUMO

Functional genomics is the study of the function of genes on a genome-wide level. Reporter gene assays can be utilized in this context to dissect signaling cascades, find new drug targets, or decipher the function of gene expression. The genome-wide scale of these experiments necessitates a different approach toward science than traditional single hypothesis driven research. High-throughput experimentation requires large project teams, automation, and discrete validation of each step in the automation and assay process. The purpose of this chapter is to provide a general outline of a standard functional genomics project with a reporter gene assay as readout, give an overview of the methodologies employed and familiarize the reader with the subsequent data analysis. The advantages of such high throughput experimentation are speed, quantitative results, and insights into biology on a genome-wide scale all of which enable a more rapid progress of science.


Assuntos
Automação Laboratorial/métodos , Genes Reporter/genética , Genômica/métodos , Ensaios de Triagem em Larga Escala/métodos , RNA Interferente Pequeno/metabolismo , Animais , Automação Laboratorial/instrumentação , Bioensaio/instrumentação , Bioensaio/métodos , Linhagem Celular , Análise de Dados , Biblioteca Gênica , Genômica/instrumentação , Ensaios de Triagem em Larga Escala/instrumentação , Humanos , Camundongos , Interferência de RNA , RNA Interferente Pequeno/genética , Fatores de Tempo
19.
Methods Mol Biol ; 1755: 107-120, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29671266

RESUMO

Transfectable functional genomics libraries are traditionally the workhorses of functional genomics screening using reporter gene assays. These libraries offer insight into fundamental cellular processes governing health and disease and can be utilized in an arrayed fashion which makes them uniquely suited to deconvolute complicated disease phenotypes and dissect biological networks that would otherwise be inaccessible. Here we give an overview of the principles for the generation, screening and data analysis of such arrayed libraries. Specifically we cover the differences between the various transfectable reagents, library selection and handling, and data analysis to offer a comprehensive understanding of these important technologies and how to apply them.


Assuntos
Genes Reporter/genética , Biblioteca Genômica , Genômica/métodos , DNA Complementar/agonistas , DNA Complementar/genética , DNA Complementar/metabolismo , Análise de Dados , Genômica/instrumentação , Células HEK293 , Humanos , MicroRNAs/antagonistas & inibidores , MicroRNAs/genética , MicroRNAs/metabolismo , Interferência de RNA , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transfecção/instrumentação , Transfecção/métodos
20.
Methods Mol Biol ; 1755: 121-133, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29671267

RESUMO

While transfectable libraries are the workhorse for many screening cores, there is one obvious area where these reagents are not useful-hard to transfect cell lines and primary cells. One solution to this problem is the use of virus to introduce genomic reagents. This strategy is more commonplace now than ever before with libraries covering cDNAs, shDNAs, miRNAs, and guide RNAs readily available. Maintenance and use of these libraries are more challenging than the transient transfection approach due to the viral production step, and the infrastructure necessary to generate them. The following pages will delve into the details for working with arrayed well formats for both lentiviral and retroviral libraries.


Assuntos
DNA Viral/genética , Biblioteca Gênica , Genes Reporter/genética , Vetores Genéticos/genética , Genômica/métodos , Lentivirus/genética , Bioensaio , Linhagem Celular , DNA Complementar/genética , Genômica/instrumentação , Células HEK293 , Humanos , Transdução Genética/instrumentação , Transdução Genética/métodos , Transfecção/instrumentação , Transfecção/métodos
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