RESUMO
Nuclear factor κB (NF-κB) plays roles in various diseases. Many inflammatory signals, such as circulating lipopolysaccharides (LPSs), activate NF-κB via specific receptors. Using whole-genome CRISPR-Cas9 screens of LPS-treated cells that express an NF-κB-driven suicide gene, we discovered that the LPS receptor Toll-like receptor 4 (TLR4) is specifically dependent on the oligosaccharyltransferase complex OST-A for N-glycosylation and cell-surface localization. The tool compound NGI-1 inhibits OST complexes in vivo, but the underlying molecular mechanism remained unknown. We did a CRISPR base-editor screen for NGI-1-resistant variants of STT3A, the catalytic subunit of OST-A. These variants, in conjunction with cryoelectron microscopy studies, revealed that NGI-1 binds the catalytic site of STT3A, where it traps a molecule of the donor substrate dolichyl-PP-GlcNAc2-Man9-Glc3, suggesting an uncompetitive inhibition mechanism. Our results provide a rationale for and an initial step toward the development of STT3A-specific inhibitors and illustrate the power of contemporaneous base-editor and structural studies to define drug mechanism of action.
Assuntos
Sistemas CRISPR-Cas , Hexosiltransferases , Lipopolissacarídeos , Proteínas de Membrana , NF-kappa B , Transdução de Sinais , Receptor 4 Toll-Like , Hexosiltransferases/metabolismo , Hexosiltransferases/genética , NF-kappa B/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Humanos , Receptor 4 Toll-Like/metabolismo , Animais , Sistemas CRISPR-Cas/genética , Lipopolissacarídeos/metabolismo , Lipopolissacarídeos/farmacologia , Camundongos , Células HEK293 , Inflamação/metabolismo , Inflamação/genética , Glicosilação , Microscopia Crioeletrônica , Domínio Catalítico , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genéticaRESUMO
Comparative studies of great apes provide a window into our evolutionary past, but the extent and identity of cellular differences that emerged during hominin evolution remain largely unexplored. We established a comparative loss-of-function approach to evaluate whether human cells exhibit distinct genetic dependencies. By performing genome-wide CRISPR interference screens in human and chimpanzee pluripotent stem cells, we identified 75 genes with species-specific effects on cellular proliferation. These genes comprised coherent processes, including cell-cycle progression and lysosomal signaling, which we determined to be human-derived by comparison with orangutan cells. Human-specific robustness to CDK2 and CCNE1 depletion persisted in neural progenitor cells and cerebral organoids, supporting the G1-phase length hypothesis as a potential evolutionary mechanism in human brain expansion. Our findings demonstrate that evolutionary changes in human cells reshaped the landscape of essential genes and establish a platform for systematically uncovering latent cellular and molecular differences between species.
Assuntos
Hominidae , Células-Tronco Neurais , Células-Tronco Pluripotentes , Células-Tronco , Animais , Humanos , Pan troglodytes/genéticaRESUMO
Schizophrenia (SCZ) is a highly heritable mental disorder with thousands of associated genetic variants located mostly in the noncoding space of the genome. Translating these associations into insights regarding the underlying pathomechanisms has been challenging because the causal variants, their mechanisms of action, and their target genes remain largely unknown. We implemented a massively parallel variant annotation pipeline (MVAP) to perform SCZ variant-to-function mapping at scale in disease-relevant neural cell types. This approach identified 620 functional variants (1.7%) that operate in a highly developmental context and neuronal-activity-dependent manner. Multimodal integration of epigenomic and CRISPRi screening data enabled us to link these functional variants to target genes, biological processes, and ultimately alterations of neuronal physiology. These results provide a multistage prioritization strategy to map functional single-nucleotide polymorphism (SNP)-to-gene-to-endophenotype relations and offer biological insights into the context-dependent molecular processes modulated by SCZ-associated genetic variation.
Assuntos
Esquizofrenia , Humanos , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Neurônios/metabolismo , Polimorfismo de Nucleotídeo Único/genética , Esquizofrenia/genética , Animais , Camundongos , Sequenciamento de Nucleotídeos em Larga EscalaRESUMO
Despite remarkable clinical efficacy of immune checkpoint blockade (ICB) in cancer treatment, ICB benefits for triple-negative breast cancer (TNBC) remain limited. Through pooled in vivo CRISPR knockout (KO) screens in syngeneic TNBC mouse models, we found that deletion of the E3 ubiquitin ligase Cop1 in cancer cells decreases secretion of macrophage-associated chemokines, reduces tumor macrophage infiltration, enhances anti-tumor immunity, and strengthens ICB response. Transcriptomics, epigenomics, and proteomics analyses revealed that Cop1 functions through proteasomal degradation of the C/ebpδ protein. The Cop1 substrate Trib2 functions as a scaffold linking Cop1 and C/ebpδ, which leads to polyubiquitination of C/ebpδ. In addition, deletion of the E3 ubiquitin ligase Cop1 in cancer cells stabilizes C/ebpδ to suppress expression of macrophage chemoattractant genes. Our integrated approach implicates Cop1 as a target for improving cancer immunotherapy efficacy in TNBC by regulating chemokine secretion and macrophage infiltration in the tumor microenvironment.
Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Imunoterapia , Macrófagos/enzimologia , Neoplasias/imunologia , Neoplasias/terapia , Proteínas Nucleares/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Proteína delta de Ligação ao Facilitador CCAAT/metabolismo , Proteína 9 Associada à CRISPR/metabolismo , Linhagem Celular Tumoral , Quimiocinas/metabolismo , Quimiotaxia , Modelos Animais de Doenças , Biblioteca Gênica , Humanos , Evasão da Resposta Imune , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Proteólise , Especificidade por Substrato , Neoplasias de Mama Triplo Negativas/imunologia , Neoplasias de Mama Triplo Negativas/terapiaRESUMO
How early events in effector T cell (TEFF) subsets tune memory T cell (TMEM) responses remains incompletely understood. Here, we systematically investigated metabolic factors in fate determination of TEFF and TMEM cells using in vivo pooled CRISPR screening, focusing on negative regulators of TMEM responses. We found that amino acid transporters Slc7a1 and Slc38a2 dampened the magnitude of TMEM differentiation, in part through modulating mTORC1 signaling. By integrating genetic and systems approaches, we identified cellular and metabolic heterogeneity among TEFF cells, with terminal effector differentiation associated with establishment of metabolic quiescence and exit from the cell cycle. Importantly, Pofut1 (protein-O-fucosyltransferase-1) linked GDP-fucose availability to downstream Notch-Rbpj signaling, and perturbation of this nutrient signaling axis blocked terminal effector differentiation but drove context-dependent TEFF proliferation and TMEM development. Our study establishes that nutrient uptake and signaling are key determinants of T cell fate and shape the quantity and quality of TMEM responses.
Assuntos
Aminoácidos/metabolismo , Linfócitos T CD8-Positivos/citologia , Memória Imunológica , Transdução de Sinais , Sistemas de Transporte de Aminoácidos/metabolismo , Animais , Linfócitos T CD8-Positivos/imunologia , Sistemas CRISPR-Cas , Ciclo Celular , Diferenciação Celular , Modelos Animais de Doenças , Feminino , Técnicas de Introdução de Genes , Coriomeningite Linfocítica/imunologia , Masculino , Camundongos , Camundongos Transgênicos , Células Precursoras de Linfócitos T/citologiaRESUMO
Mitochondrial dysfunction is associated with a spectrum of human conditions, ranging from rare, inborn errors of metabolism to the aging process. To identify pathways that modify mitochondrial dysfunction, we performed genome-wide CRISPR screens in the presence of small-molecule mitochondrial inhibitors. We report a compendium of chemical-genetic interactions involving 191 distinct genetic modifiers, including 38 that are synthetic sick/lethal and 63 that are suppressors. Genes involved in glycolysis (PFKP), pentose phosphate pathway (G6PD), and defense against lipid peroxidation (GPX4) scored high as synthetic sick/lethal. A surprisingly large fraction of suppressors are pathway intrinsic and encode mitochondrial proteins. A striking example of such "intra-organelle" buffering is the alleviation of a chemical defect in complex V by simultaneous inhibition of complex I, which benefits cells by rebalancing redox cofactors, increasing reductive carboxylation, and promoting glycolysis. Perhaps paradoxically, certain forms of mitochondrial dysfunction may best be buffered with "second site" inhibitors to the organelle.
Assuntos
Genes Modificadores , Mitocôndrias/genética , Mitocôndrias/patologia , Autoantígenos/metabolismo , Morte Celular/efeitos dos fármacos , Citosol/efeitos dos fármacos , Citosol/metabolismo , Complexo I de Transporte de Elétrons/metabolismo , Epistasia Genética/efeitos dos fármacos , Ferroptose/efeitos dos fármacos , Ferroptose/genética , Genoma , Glutationa Peroxidase/metabolismo , Glicólise/efeitos dos fármacos , Glicólise/genética , Humanos , Células K562 , Mitocôndrias/efeitos dos fármacos , Oligomicinas/toxicidade , Oxirredução , Fosforilação Oxidativa/efeitos dos fármacos , Via de Pentose Fosfato/efeitos dos fármacos , Via de Pentose Fosfato/genética , Espécies Reativas de Oxigênio/metabolismo , Ribonucleoproteínas/metabolismo , Antígeno SS-BRESUMO
A primary cause of disease progression in type 2 diabetes (T2D) is ß cell dysfunction due to inflammatory stress and insulin resistance. However, preventing ß cell exhaustion under diabetic conditions is a major therapeutic challenge. Here, we identify the vitamin D receptor (VDR) as a key modulator of inflammation and ß cell survival. Alternative recognition of an acetylated lysine in VDR by bromodomain proteins BRD7 and BRD9 directs association to PBAF and BAF chromatin remodeling complexes, respectively. Mechanistically, ligand promotes VDR association with PBAF to effect genome-wide changes in chromatin accessibility and enhancer landscape, resulting in an anti-inflammatory response. Importantly, pharmacological inhibition of BRD9 promotes PBAF-VDR association to restore ß cell function and ameliorate hyperglycemia in murine T2D models. These studies reveal an unrecognized VDR-dependent transcriptional program underpinning ß cell survival and identifies the VDR:PBAF/BAF association as a potential therapeutic target for T2D.
Assuntos
Proteínas Cromossômicas não Histona/metabolismo , Células Secretoras de Insulina/efeitos dos fármacos , Receptores de Calcitriol/metabolismo , Fatores de Transcrição/metabolismo , Vitamina D/farmacologia , Animais , Calcitriol/análogos & derivados , Calcitriol/farmacologia , Montagem e Desmontagem da Cromatina , Diabetes Mellitus Experimental/induzido quimicamente , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patologia , Humanos , Insulina/sangue , Insulina/metabolismo , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Obesos , Mutagênese Sítio-Dirigida , Fosforilação Oxidativa/efeitos dos fármacos , Ligação Proteica , Interferência de RNA , RNA Guia de Cinetoplastídeos/genética , RNA Interferente Pequeno/metabolismo , Receptores de Calcitriol/antagonistas & inibidores , Receptores de Calcitriol/genética , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/genética , Transcrição Gênica/efeitos dos fármacosRESUMO
Cancer cells can evade natural killer (NK) cell activity, thereby limiting anti-tumor immunity. To reveal genetic determinants of susceptibility to NK cell activity, we examined interacting NK cells and blood cancer cells using single-cell and genome-scale functional genomics screens. Interaction of NK and cancer cells induced distinct activation and type I interferon (IFN) states in both cell types depending on the cancer cell lineage and molecular phenotype, ranging from more sensitive myeloid to less sensitive B-lymphoid cancers. CRISPR screens in cancer cells uncovered genes regulating sensitivity and resistance to NK cell-mediated killing, including adhesion-related glycoproteins, protein fucosylation genes, and transcriptional regulators, in addition to confirming the importance of antigen presentation and death receptor signaling pathways. CRISPR screens with a single-cell transcriptomic readout provided insight into underlying mechanisms, including regulation of IFN-γ signaling in cancer cells and NK cell activation states. Our findings highlight the diversity of mechanisms influencing NK cell susceptibility across different cancers and provide a resource for NK cell-based therapies.
Assuntos
Neoplasias Hematológicas , Neoplasias , Humanos , Células Matadoras Naturais , Neoplasias/genética , Apresentação de Antígeno , Genômica , Citotoxicidade Imunológica/genética , Linhagem Celular TumoralRESUMO
Mitochondria are essential regulators of innate immunity. They generate long mitochondrial double-stranded RNAs (mt-dsRNAs) and release them into the cytosol to trigger an immune response under pathological stress conditions. Yet the regulation of these self-immunogenic RNAs remains largely unknown. Here, we employ CRISPR screening on mitochondrial RNA (mtRNA)-binding proteins and identify NOP2/Sun RNA methyltransferase 4 (NSUN4) as a key regulator of mt-dsRNA expression in human cells. We find that NSUN4 induces 5-methylcytosine (m5C) modification on mtRNAs, especially on the termini of light-strand long noncoding RNAs. These m5C-modified RNAs are recognized by complement C1q-binding protein (C1QBP), which recruits polyribonucleotide nucleotidyltransferase to facilitate RNA turnover. Suppression of NSUN4 or C1QBP results in increased mt-dsRNA expression, while C1QBP deficiency also leads to increased cytosolic mt-dsRNAs and subsequent immune activation. Collectively, our study unveils the mechanism underlying the selective degradation of light-strand mtRNAs and establishes a molecular mark for mtRNA decay and cytosolic release.
Assuntos
5-Metilcitosina , Citosol , Mitocôndrias , Estabilidade de RNA , RNA de Cadeia Dupla , RNA Mitocondrial , Humanos , Citosol/metabolismo , 5-Metilcitosina/metabolismo , Mitocôndrias/metabolismo , Mitocôndrias/genética , RNA de Cadeia Dupla/metabolismo , RNA de Cadeia Dupla/genética , RNA Mitocondrial/genética , RNA Mitocondrial/metabolismo , Células HEK293 , Células HeLa , Metiltransferases/metabolismo , Metiltransferases/genética , Imunidade Inata , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Animais , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/genética , Sistemas CRISPR-CasRESUMO
Highly coordinated changes in gene expression underlie T cell activation and exhaustion. However, the mechanisms by which such programs are regulated and how these may be targeted for therapeutic benefit remain poorly understood. Here, we comprehensively profile the genomic occupancy of mSWI/SNF chromatin remodeling complexes throughout acute and chronic T cell stimulation, finding that stepwise changes in localization over transcription factor binding sites direct site-specific chromatin accessibility and gene activation leading to distinct phenotypes. Notably, perturbation of mSWI/SNF complexes using genetic and clinically relevant chemical strategies enhances the persistence of T cells with attenuated exhaustion hallmarks and increased memory features in vitro and in vivo. Finally, pharmacologic mSWI/SNF inhibition improves CAR-T expansion and results in improved anti-tumor control in vivo. These findings reveal the central role of mSWI/SNF complexes in the coordination of T cell activation and exhaustion and nominate small-molecule-based strategies for the improvement of current immunotherapy protocols.
Assuntos
Montagem e Desmontagem da Cromatina , Proteínas Cromossômicas não Histona , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Fatores de Transcrição/metabolismo , Cromatina/genética , Ativação TranscricionalRESUMO
RNA-binding proteins (RBPs) are critical regulators of post-transcriptional gene expression, and aberrant RBP-RNA interactions can promote cancer progression. Here, we interrogate the function of RBPs in cancer using pooled CRISPR-Cas9 screening and identify 57 RBP candidates with distinct roles in supporting MYC-driven oncogenic pathways. We find that disrupting YTHDF2-dependent mRNA degradation triggers apoptosis in triple-negative breast cancer (TNBC) cells and tumors. eCLIP and m6A sequencing reveal that YTHDF2 interacts with mRNAs encoding proteins in the MAPK pathway that, when stabilized, induce epithelial-to-mesenchymal transition and increase global translation rates. scRibo-STAMP profiling of translating mRNAs reveals unique alterations in the translatome of single cells within YTHDF2-depleted solid tumors, which selectively contribute to endoplasmic reticulum stress-induced apoptosis in TNBC cells. Thus, our work highlights the therapeutic potential of RBPs by uncovering a critical role for YTHDF2 in counteracting the global increase of mRNA synthesis in MYC-driven breast cancers.
Assuntos
Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Proteínas de Ligação a RNA/genética , Adenosina/análogos & derivados , Adenosina/metabolismo , Animais , Morte Celular/genética , Transição Epitelial-Mesenquimal/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Genes myc , Humanos , Camundongos Nus , Camundongos Transgênicos , Biossíntese de Proteínas , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/patologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Cancer-associated mutations that stabilize NRF2, an oxidant defense transcription factor, are predicted to promote tumor development. Here, utilizing 3D cancer spheroid models coupled with CRISPR-Cas9 screens, we investigate the molecular pathogenesis mediated by NRF2 hyperactivation. NRF2 hyperactivation was necessary for proliferation and survival in lung tumor spheroids. Antioxidant treatment rescued survival but not proliferation, suggesting the presence of distinct mechanisms. CRISPR screens revealed that spheroids are differentially dependent on the mammalian target of rapamycin (mTOR) for proliferation and the lipid peroxidase GPX4 for protection from ferroptosis of inner, matrix-deprived cells. Ferroptosis inhibitors blocked death from NRF2 downregulation, demonstrating a critical role of NRF2 in protecting matrix-deprived cells from ferroptosis. Interestingly, proteomics analyses show global enrichment of selenoproteins, including GPX4, by NRF2 downregulation, and targeting NRF2 and GPX4 killed spheroids overall. These results illustrate the value of spheroid culture in revealing environmental or spatial differential dependencies on NRF2 and reveal exploitable vulnerabilities of NRF2-hyperactivated tumors.
Assuntos
Sistemas CRISPR-Cas , Técnicas de Cultura de Células , Proliferação de Células , Ferroptose , Fator 2 Relacionado a NF-E2/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , Esferoides Celulares/metabolismo , Células A549 , Humanos , Fator 2 Relacionado a NF-E2/genética , Proteínas de Neoplasias/genética , Neoplasias/genética , Neoplasias/patologia , Fosfolipídeo Hidroperóxido Glutationa Peroxidase/genética , Fosfolipídeo Hidroperóxido Glutationa Peroxidase/metabolismo , Esferoides Celulares/patologia , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismoRESUMO
Single-cell clustered regularly interspaced short palindromic repeats-sequencing (scCRISPR-seq) is an emerging high-throughput CRISPR screening technology where the true cellular response to perturbation is coupled with infected proportion bias of guide RNAs (gRNAs) across different cell clusters. The mixing of these effects introduces noise into scCRISPR-seq data analysis and thus obstacles to relevant studies. We developed scDecouple to decouple true cellular response of perturbation from the influence of infected proportion bias. scDecouple first models the distribution of gene expression profiles in perturbed cells and then iteratively finds the maximum likelihood of cell cluster proportions as well as the cellular response for each gRNA. We demonstrated its performance in a series of simulation experiments. By applying scDecouple to real scCRISPR-seq data, we found that scDecouple enhances the identification of biologically perturbation-related genes. scDecouple can benefit scCRISPR-seq data analysis, especially in the case of heterogeneous samples or complex gRNA libraries.
Assuntos
Ensaios de Triagem em Larga Escala , RNA Guia de Sistemas CRISPR-CasRESUMO
The metabolic pathways fueling tumor growth have been well characterized, but the specific impact of transforming events on network topology and enzyme essentiality remains poorly understood. To this end, we performed combinatorial CRISPR-Cas9 screens on a set of 51 carbohydrate metabolism genes that represent glycolysis and the pentose phosphate pathway (PPP). This high-throughput methodology enabled systems-level interrogation of metabolic gene dispensability, interactions, and compensation across multiple cell types. The metabolic impact of specific combinatorial knockouts was validated using 13C and 2H isotope tracing, and these assays together revealed key nodes controlling redox homeostasis along the KEAP-NRF2 signaling axis. Specifically, targeting KEAP1 in combination with oxidative PPP genes mitigated the deleterious effects of these knockouts on growth rates. These results demonstrate how our integrated framework, combining genetic, transcriptomic, and flux measurements, can improve elucidation of metabolic network alterations and guide precision targeting of metabolic vulnerabilities based on tumor genetics.
Assuntos
Sistemas CRISPR-Cas , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Redes e Vias Metabólicas , Fator 2 Relacionado a NF-E2/metabolismo , Transcriptoma , Glicólise , Células HeLa , Homeostase , Humanos , Proteína 1 Associada a ECH Semelhante a Kelch/antagonistas & inibidores , Proteína 1 Associada a ECH Semelhante a Kelch/genética , Fator 2 Relacionado a NF-E2/antagonistas & inibidores , Fator 2 Relacionado a NF-E2/genética , Oxirredução , Via de Pentose Fosfato , Transdução de SinaisRESUMO
The placenta serves as the interface between the mother and fetus, facilitating the exchange of gases and nutrients between their separate blood circulation systems. Trophoblasts in the placenta play a central role in this process. Our current understanding of mammalian trophoblast development relies largely on mouse models. However, given the diversification of mammalian placentas, findings from the mouse placenta cannot be readily extrapolated to other mammalian species, including humans. To fill this knowledge gap, we performed CRISPR knockout screening in human trophoblast stem cells (hTSCs). We targeted genes essential for mouse placental development and identified more than 100 genes as critical regulators in both human hTSCs and mouse placentas. Among them, we further characterized in detail two transcription factors, DLX3 and GCM1, and revealed their essential roles in hTSC differentiation. Moreover, a gene function-based comparison between human and mouse trophoblast subtypes suggests that their relationship may differ significantly from previous assumptions based on tissue localization or cellular function. Notably, our data reveal that hTSCs may not be analogous to mouse TSCs or the extraembryonic ectoderm (ExE) in which in vivo TSCs reside. Instead, hTSCs may be analogous to progenitor cells in the mouse ectoplacental cone and chorion. This finding is consistent with the absence of ExE-like structures during human placental development. Our data not only deepen our understanding of human trophoblast development but also facilitate cross-species comparison of mammalian placentas.
Assuntos
Placenta , Placentação , Humanos , Gravidez , Camundongos , Feminino , Animais , Placentação/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Trofoblastos , Diferenciação Celular , Células-Tronco , MamíferosRESUMO
The infection of mammalian cells by viruses and innate immune responses to infection are spatiotemporally organized processes. Cytosolic RNA sensors trigger nuclear translocation of the transcription factor interferon regulatory factor 3 (IRF3) and consequent induction of host immune responses to RNA viruses. Previous genetic screens for factors involved in viral sensing did not resolve changes in the subcellular localization of host or viral proteins. Here, we increased the throughput of our optical pooled screening technology by over fourfold. This allowed us to carry out a genome-wide CRISPR knockout screen using high-resolution multiparameter imaging of cellular responses to Sendai virus infection coupled with in situ cDNA sequencing by synthesis (SBS) to identify 80,408 single guide RNAs (sgRNAs) in 10,366,390 cells-over an order of magnitude more genomic perturbations than demonstrated previously using an in situ SBS readout. By ranking perturbations using human-designed and deep learning image feature scores, we identified regulators of IRF3 translocation, Sendai virus localization, and peroxisomal biogenesis. Among the hits, we found that ATP13A1, an ER-localized P5A-type ATPase, is essential for viral sensing and is required for targeting of mitochondrial antiviral signaling protein (MAVS) to mitochondrial membranes where MAVS must be localized for effective signaling through retinoic acid-inducible gene I (RIG-I). The ability to carry out genome-wide pooled screens with complex high-resolution image-based phenotyping dramatically expands the scope of functional genomics approaches.
Assuntos
Vírus de RNA , Transdução de Sinais , Animais , Humanos , RNA , Imunidade Inata/genética , Vírus de RNA/genética , Antivirais , Fator Regulador 3 de Interferon/metabolismo , Mamíferos/genéticaRESUMO
Porcine deltacoronavirus (PDCoV) is an emerging enteropathogenic coronavirus. It causes mortality in neonatal piglets and is of growing concern because of its broad host range, including humans. To date, the mechanism of PDCoV infection remains poorly understood. Here, based on a genome-wide CRISPR screen of PDCoV-infected cells, we found that HSP90AB1 (heat shock protein 90 alpha family class B1) promotes PDCoV infection. Knockdown or KO of HSP90AB1 in LLC-PK cells resulted in a significantly suppressed PDCoV infection. Infected cells treated with HSP90 inhibitors 17-AAG and VER-82576 also showed a significantly suppressed PDCoV infection, although KW-2478, which does not affect the ATPase activity of HSP90AB1, had no effect on PDCoV infection. We found that HSP90AB1 interacts with the N, NS7, and NSP10 proteins of PDCoV. We further evaluated the interaction between N and HSP90AB1 and found that the C-tail domain of the N protein is the HSP90AB1-interacting domain. Further studies showed that HSP90AB1 protects N protein from degradation via the proteasome pathway. In summary, our results reveal a key role for HSP90AB1 in the mechanism of PDCoV infection and contribute to provide new host targets for PDCoV antiviral research.
Assuntos
Proteínas de Choque Térmico HSP90 , Replicação Viral , Animais , Humanos , Deltacoronavirus , Especificidade de Hospedeiro , Proteínas de Choque Térmico HSP90/genética , Proteínas de Choque Térmico HSP90/metabolismo , Suínos , Células HEK293RESUMO
Tumor suppressor p53 (TP53) is frequently mutated in cancer, often resulting not only in loss of its tumor-suppressive function but also acquisition of dominant-negative and even oncogenic gain-of-function traits. While wild-type p53 levels are tightly regulated, mutants are typically stabilized in tumors, which is crucial for their oncogenic properties. Here, we systematically profiled the factors that regulate protein stability of wild-type and mutant p53 using marker-based genome-wide CRISPR screens. Most regulators of wild-type p53 also regulate p53 mutants, except for p53 R337H regulators, which are largely private to this mutant. Mechanistically, FBXO42 emerged as a positive regulator for a subset of p53 mutants, working with CCDC6 to control USP28-mediated mutant p53 stabilization. Additionally, C16orf72/HAPSTR1 negatively regulates both wild-type p53 and all tested mutants. C16orf72/HAPSTR1 is commonly amplified in breast cancer, and its overexpression reduces p53 levels in mouse mammary epithelium leading to accelerated breast cancer. This study offers a network perspective on p53 stability regulation, potentially guiding strategies to reinforce wild-type p53 or target mutant p53 in cancer.
Assuntos
Mutação , Estabilidade Proteica , Proteína Supressora de Tumor p53 , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Animais , Humanos , Camundongos , Feminino , Sistemas CRISPR-Cas , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Ubiquitina Tiolesterase/genética , Ubiquitina Tiolesterase/metabolismo , Regulação Neoplásica da Expressão Gênica , Repetições Palindrômicas Curtas Agrupadas e Regularmente EspaçadasRESUMO
Chemical libraries paired with phenotypic screens can now readily identify compounds with therapeutic potential. A central limitation to exploiting these compounds, however, has been in identifying their relevant cellular targets. Here, we present a two-tiered CRISPR-mediated chemical-genetic strategy for target identification: combined genome-wide knockdown and overexpression screening as well as focused, comparative chemical-genetic profiling. Application of these strategies to rigosertib, a drug in phase 3 clinical trials for high-risk myelodysplastic syndrome whose molecular target had remained controversial, pointed singularly to microtubules as rigosertib's target. We showed that rigosertib indeed directly binds to and destabilizes microtubules using cell biological, in vitro, and structural approaches. Finally, expression of tubulin with a structure-guided mutation in the rigosertib-binding pocket conferred resistance to rigosertib, establishing that rigosertib kills cancer cells by destabilizing microtubules. These results demonstrate the power of our chemical-genetic screening strategies for pinpointing the physiologically relevant targets of chemical agents.
Assuntos
Antineoplásicos/farmacologia , Regulação Neoplásica da Expressão Gênica , Testes Genéticos/métodos , Glicina/análogos & derivados , Microtúbulos/efeitos dos fármacos , Sulfonas/farmacologia , Moduladores de Tubulina/farmacologia , Tubulina (Proteína)/genética , Antineoplásicos/química , Sistemas CRISPR-Cas , Colchicina/farmacologia , Resistencia a Medicamentos Antineoplásicos , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Glicina/química , Glicina/farmacologia , Células HeLa , Humanos , Células K562 , Cinesinas/genética , Cinesinas/metabolismo , Lentivirus/genética , Lentivirus/metabolismo , Microtúbulos/metabolismo , Microtúbulos/ultraestrutura , Mutação , Síndromes Mielodisplásicas/genética , Síndromes Mielodisplásicas/metabolismo , Síndromes Mielodisplásicas/patologia , RNA Guia de Cinetoplastídeos/genética , RNA Guia de Cinetoplastídeos/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Sulfonas/química , Tubulina (Proteína)/química , Tubulina (Proteína)/metabolismo , Moduladores de Tubulina/química , Vimblastina/farmacologiaRESUMO
The genome-wide perturbation of transcriptional networks with CRISPR-Cas technology has primarily involved systematic and targeted gene modulation. Here, we developed PRISM (Perturbing Regulatory Interactions by Synthetic Modulators), a screening platform that uses randomized CRISPR-Cas transcription factors (crisprTFs) to globally perturb transcriptional networks. By applying PRISM to a yeast model of Parkinson's disease (PD), we identified guide RNAs (gRNAs) that modulate transcriptional networks and protect cells from alpha-synuclein (αSyn) toxicity. One gRNA identified in this screen outperformed the most protective suppressors of αSyn toxicity reported previously, highlighting PRISM's ability to identify modulators of important phenotypes. Gene expression profiling revealed genes differentially modulated by this strong protective gRNA that rescued yeast from αSyn toxicity when overexpressed. Human homologs of top-ranked hits protected against αSyn-induced cell death in a human neuronal PD model. Thus, high-throughput and unbiased perturbation of transcriptional networks via randomized crisprTFs can reveal complex biological phenotypes and effective disease modulators.