RESUMEN
Acute myeloid leukemia (AML) is an aggressive disease with a poor prognosis (5-year survival rate of 30.5% in the United States). Designing cell therapies to target AML is challenging because no single tumor-associated antigen (TAA) is highly expressed on all cancer subpopulations. Furthermore, TAAs are also expressed on healthy cells, leading to toxicity risk. To address these targeting challenges, we engineer natural killer (NK) cells with a multi-input gene circuit consisting of chimeric antigen receptors (CARs) controlled by OR and NOT logic gates. The OR gate kills a range of AML cells from leukemic stem cells to blasts using a bivalent CAR targeting FLT3 and/or CD33. The NOT gate protects healthy hematopoietic stem cells (HSCs) using an inhibitory CAR targeting endomucin, a protective antigen unique to healthy HSCs. NK cells with the combined OR-NOT gene circuit kill multiple AML subtypes and protect primary HSCs, and the circuit also works in vivo.
Asunto(s)
Células Asesinas Naturales , Leucemia Mieloide Aguda , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/metabolismo , Humanos , Leucemia Mieloide Aguda/terapia , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/inmunología , Animales , Ratones , Receptores Quiméricos de Antígenos/metabolismo , Receptores Quiméricos de Antígenos/inmunología , Redes Reguladoras de Genes , Células Madre Hematopoyéticas/metabolismo , Línea Celular Tumoral , Medicina de Precisión/métodos , Tratamiento Basado en Trasplante de Células y Tejidos/métodosRESUMEN
We have developed a platform for quantitative genetic interaction mapping using viral infectivity as a functional readout and constructed a viral host-dependency epistasis map (vE-MAP) of 356 human genes linked to HIV function, comprising >63,000 pairwise genetic perturbations. The vE-MAP provides an expansive view of the genetic dependencies underlying HIV infection and can be used to identify drug targets and study viral mutations. We found that the RNA deadenylase complex, CNOT, is a central player in the vE-MAP and show that knockout of CNOT1, 10, and 11 suppressed HIV infection in primary T cells by upregulating innate immunity pathways. This phenotype was rescued by deletion of IRF7, a transcription factor regulating interferon-stimulated genes, revealing a previously unrecognized host signaling pathway involved in HIV infection. The vE-MAP represents a generic platform that can be used to study the global effects of how different pathogens hijack and rewire the host during infection.
Asunto(s)
Epistasis Genética , Infecciones por VIH/genética , Factor 7 Regulador del Interferón/genética , Factores de Transcripción/genética , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/patología , Infecciones por VIH/inmunología , Infecciones por VIH/patología , Infecciones por VIH/virología , VIH-1/genética , VIH-1/patogenicidad , Interacciones Huésped-Patógeno/genética , Interacciones Huésped-Patógeno/inmunología , Humanos , Inmunidad Innata/genética , Interferones/genética , Mutación , Transducción de Señal/genéticaRESUMEN
Epigenetic modifications can maintain or alter the inherent symmetry of the nucleosome. However, the mechanisms that deposit and/or propagate symmetry or asymmetry are not understood. Here we report that yeast Set1C/COMPASS (complex of proteins associated with Set1) is dimeric and, consequently, symmetrically trimethylates histone 3 Lys4 (H3K4me3) on promoter nucleosomes. Mutation of the dimer interface to make Set1C monomeric abolished H3K4me3 on most promoters. The most active promoters, particularly those involved in the oxidative phase of the yeast metabolic cycle, displayed H3K4me2, which is normally excluded from active promoters, and a subset of these also displayed H3K4me3. In wild-type yeast, deletion of the sole H3K4 demethylase, Jhd2, has no effect. However, in monomeric Set1C yeast, Jhd2 deletion increased H3K4me3 levels on the H3K4me2 promoters. Notably, the association of Set1C with the elongating polymerase was not perturbed by monomerization. These results imply that symmetrical H3K4 methylation is an embedded consequence of Set1C dimerism and that Jhd2 demethylates asymmetric H3K4me3. Consequently, rather than methylation and demethylation acting in opposition as logic would suggest, a dimeric methyltransferase and monomeric demethylase cooperate to eliminate asymmetry and focus symmetrical H3K4me3 onto selected nucleosomes. This presents a new paradigm for the establishment of epigenetic detail.
Asunto(s)
Epigénesis Genética/genética , Histona Demetilasas con Dominio de Jumonji/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Desmetilación , Dimerización , Eliminación de Gen , Histonas/metabolismo , Metilación , Mutagénesis , Nucleosomas/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Transcripción Genética/genéticaRESUMEN
This protocol describes an optimized high-throughput procedure for generating double deletion mutants in Schizosaccharomyces pombe using the colony replicating robot ROTOR HDA and the PEM (pombe epistasis mapper) system. The method is based on generating high-density colony arrays (1536 colonies per agar plate) and passaging them through a series of antidiploid and mating-type selection (ADS-MTS) and double-mutant selection (DMS) steps. Detailed program parameters for each individual replication step are provided. Using this procedure, batches of 25 or more screens can be routinely performed.
Asunto(s)
Mapeo Cromosómico/instrumentación , Mapeo Cromosómico/métodos , Epistasis Genética , Genes Fúngicos , Genética Microbiana/instrumentación , Genética Microbiana/métodos , Schizosaccharomyces/genética , Eliminación de Gen , Ensayos Analíticos de Alto Rendimiento/instrumentación , Ensayos Analíticos de Alto Rendimiento/métodos , Robótica/instrumentación , Robótica/métodos , Schizosaccharomyces/crecimiento & desarrollo , Selección GenéticaRESUMEN
This protocol describes chemical transformation of Schizosaccharomyces pombe with linear DNA in a 96-well format. This procedure has been successfully used for large-scale strain construction in fission yeast.
Asunto(s)
Genética Microbiana/métodos , Biología Molecular/métodos , Schizosaccharomyces/genética , Transformación Genética , Ensayos Analíticos de Alto Rendimiento/métodosRESUMEN
Epistasis mapping, in which the phenotype that emerges from combining pairs of mutations is measured quantitatively, is a powerful tool for unbiased study of gene function. When performed at a large scale, this approach has been used to assign function to previously uncharacterized genes, define functional modules and pathways, and study their cross talk. These experiments rely heavily on methods for rapid sampling of binary combinations of mutant alleles by systematic generation of a series of double mutants. Epistasis mapping technologies now exist in various model systems. Here we provide an overview of different epistasis mapping technologies, including the pombe epistasis mapper (PEM) system designed for the collection of quantitative genetic interaction data in fission yeast Schizosaccharomyces pombe Comprising a series of high-throughput selection steps for generation and characterization of double mutants, the PEM system has provided insight into a wide range of biological processes as well as facilitated evolutionary analysis of genetic interactomes across different species.
Asunto(s)
Mapeo Cromosómico/métodos , Epistasis Genética , Genes Fúngicos , Genética Microbiana/métodos , Schizosaccharomyces/genética , Mutación , Selección GenéticaRESUMEN
This protocol describes computational analysis of genetic interaction screens, ranging from data capture (plate imaging) to downstream analyses. Plate imaging approaches using both digital camera and office flatbed scanners are included, along with a protocol for the extraction of colony size measurements from the resulting images. A commonly used genetic interaction scoring method, calculation of the S-score, is discussed. These methods require minimal computer skills, but some familiarity with MATLAB and Linux/Unix is a plus. Finally, an outline for using clustering and visualization software for analysis of resulting data sets is provided.
Asunto(s)
Bioestadística/métodos , Mapeo Cromosómico/métodos , Epistasis Genética , Genética Microbiana/métodos , Procesamiento de Imagen Asistido por Computador/métodos , Imagen Óptica/métodos , Schizosaccharomyces/genética , Análisis por Conglomerados , Genes Fúngicos , Mutación , Schizosaccharomyces/crecimiento & desarrollo , Selección GenéticaRESUMEN
This protocol includes two methods for genomic DNA preparation from Schizosaccharomyces pombe The first is a quick method for preparation of DNA suitable for polymerase chain reaction (PCR) genotyping. The second, longer method yields high-quality DNA that can be used for amplification of targeting cassettes.
Asunto(s)
ADN de Hongos/aislamiento & purificación , Genética Microbiana/métodos , Biología Molecular/métodos , Schizosaccharomyces/genética , Técnicas de Genotipaje , Técnicas de Tipificación Micológica , Reacción en Cadena de la PolimerasaRESUMEN
A single gene can partake in several biological processes, and therefore gene deletions can lead to different-sometimes unexpected-phenotypes. However, it is not always clear whether such pleiotropy reflects the loss of a unique molecular activity involved in different processes or the loss of a multifunctional protein. Here, using Saccharomyces cerevisiae metabolism as a model, we systematically test the null hypothesis that enzyme phenotypes depend on a single annotated molecular function, namely their catalysis. We screened a set of carefully selected genes by quantifying the contribution of catalysis to gene deletion phenotypes under different environmental conditions. While most phenotypes were explained by loss of catalysis, slow growth was readily rescued by a catalytically inactive protein in about one-third of the enzymes tested. Such noncatalytic phenotypes were frequent in the Alt1 and Bat2 transaminases and in the isoleucine/valine biosynthetic enzymes Ilv1 and Ilv2, suggesting novel "moonlighting" activities in these proteins. Furthermore, differential genetic interaction profiles of gene deletion and catalytic mutants indicated that ILV1 is functionally associated with regulatory processes, specifically to chromatin modification. Our systematic study shows that gene loss phenotypes and their genetic interactions are frequently not driven by the loss of an annotated catalytic function, underscoring the moonlighting nature of cellular metabolism.
Asunto(s)
Fenotipo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Catálisis , Biología Computacional/métodos , Epistasis Genética , Perfilación de la Expresión Génica , Regulación Fúngica de la Expresión Génica , Ontología de Genes , Estudios de Asociación Genética , Genoma Fúngico , Genómica/métodos , Sistemas de Lectura Abierta , Saccharomyces cerevisiae/enzimología , Selección Genética , Eliminación de SecuenciaRESUMEN
We describe a combinatorial CRISPR interference (CRISPRi) screening platform for mapping genetic interactions in mammalian cells. We targeted 107 chromatin-regulation factors in human cells with pools of either single or double single guide RNAs (sgRNAs) to downregulate individual genes or gene pairs, respectively. Relative enrichment analysis of individual sgRNAs or sgRNA pairs allowed for quantitative characterization of genetic interactions, and comparison with protein-protein-interaction data revealed a functional map of chromatin regulation.
Asunto(s)
Mapeo Cromosómico/métodos , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Epistasis Genética/genética , Mapeo de Interacción de Proteínas/métodos , Animales , Secuenciación de Nucleótidos de Alto Rendimiento , RatonesRESUMEN
We developed a systematic approach to map human genetic networks by combinatorial CRISPR-Cas9 perturbations coupled to robust analysis of growth kinetics. We targeted all pairs of 73 cancer genes with dual guide RNAs in three cell lines, comprising 141,912 tests of interaction. Numerous therapeutically relevant interactions were identified, and these patterns replicated with combinatorial drugs at 75% precision. From these results, we anticipate that cellular context will be critical to synthetic-lethal therapies.
Asunto(s)
Mapeo Cromosómico/métodos , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Técnicas Químicas Combinatorias , Epistasis Genética/genética , Proteínas de Neoplasias/genética , Células A549 , Línea Celular Tumoral , Células HeLa , Secuenciación de Nucleótidos de Alto Rendimiento , HumanosRESUMEN
Although numerous regulatory connections between pre-mRNA splicing and chromatin have been demonstrated, the precise mechanisms by which chromatin factors influence spliceosome assembly and/or catalysis remain unclear. To probe the genetic network of pre-mRNA splicing in the fission yeast Schizosaccharomyces pombe, we constructed an epistatic mini-array profile (E-MAP) and discovered many new connections between chromatin and splicing. Notably, the nucleosome remodeler SWI/SNF had strong genetic interactions with components of the U2 snRNP SF3 complex. Overexpression of SF3 components in ΔSWI/SNF cells led to inefficient splicing of many fission yeast introns, predominantly those with non-consensus splice sites. Deletion of SWI/SNF decreased recruitment of the splicing ATPase Prp2, suggesting that SWI/SNF promotes co-transcriptional spliceosome assembly prior to first step catalysis. Importantly, defects in SWI/SNF as well as SF3 overexpression each altered nucleosome occupancy along intron-containing genes, illustrating that the chromatin landscape both affects--and is affected by--co-transcriptional splicing.
Asunto(s)
Proteínas Cromosómicas no Histona/genética , Redes Reguladoras de Genes , Nucleosomas/genética , Empalme del ARN/genética , Ribonucleoproteína Nuclear Pequeña U2/genética , Empalmosomas/genética , Factores de Transcripción/genética , Adenosina Trifosfatasas/genética , Cromatina/genética , Regulación Fúngica de la Expresión Génica , Intrones/genética , Nucleosomas/metabolismo , Regiones Promotoras Genéticas , Schizosaccharomyces/genética , Empalmosomas/metabolismo , Transcripción GenéticaRESUMEN
Brc1, which was first identified as a high-copy, allele-specific suppressor of a mutation impairing the Smc5-Smc6 holocomplex in Schizosaccharomyces pombe, protects genome integrity during normal DNA replication and when cells are exposed to toxic compounds that stall or collapse replication forks. The C-terminal tandem BRCT (BRCA1 C-terminus) domain of fission yeast Brc1 docks with phosphorylated histone H2A (γH2A)-marked chromatin formed by ATR/Rad3 checkpoint kinase at arrested and damaged replication forks; however, how Brc1 functions in relation to other genome protection modules remains unclear. Here, an epistatic mini-array profile reveals critical requirements for Brc1 in mutants that are defective in multiple DNA damage response pathways, including checkpoint signaling by Rad3-Rad26/ATR-ATRIP kinase, DNA repair by Smc5-Smc6 holocomplex, replication fork stabilization by Mrc1/claspin and Swi1-Swi3/Timeless-Tipin, and control of ubiquitin-regulated proteolysis by the COP9 signalosome (CSN). Exogenous genotoxins enhance these negative genetic interactions. Rad52 and RPA foci are increased in CSN-defective cells, and loss of γH2A increases genotoxin sensitivity, indicating a critical role for the γH2A-Brc1 module in stabilizing replication forks in CSN-defective cells. A negative genetic interaction with the Nse6 subunit of Smc5-Smc6 holocomplex indicates that the DNA repair functions of Brc1 and Smc5-Smc6 holocomplex are at least partially independent. Rtt107, the Brc1 homolog in Saccharomyces cerevisiae, has a very different pattern of genetic interactions, indicating evolutionary divergence of functions and DNA damage responses.
Asunto(s)
Daño del ADN , Epistasis Genética , Mutación , Proteínas de Schizosaccharomyces pombe/genética , Schizosaccharomyces/genética , Epistasis Genética/efectos de los fármacos , Expresión Génica , Técnicas de Inactivación de Genes , Ontología de Genes , Histonas/metabolismo , Mutágenos/farmacología , Proteína Recombinante y Reparadora de ADN Rad52/genética , Proteína Recombinante y Reparadora de ADN Rad52/metabolismo , Proteína de Replicación A/genética , Proteína de Replicación A/metabolismo , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismoRESUMEN
We combine a genome-scale RNAi screen in mouse epiblast stem cells (EpiSCs) with genetic interaction, protein localization, and "protein-level dependency" studies-a systematic technique that uncovers post-transcriptional regulation-to delineate the network of factors that control the expression of Oct4, a key regulator of pluripotency. Our data signify that there are similarities, but also fundamental differences in Oct4 regulation in EpiSCs versus embryonic stem cells (ESCs). Through multiparametric data analyses, we predict that Tox4 is associating with the Paf1C complex, which maintains cell identity in both cell types, and validate that this protein-protein interaction exists in ESCs and EpiSCs. We also identify numerous knockdowns that increase Oct4 expression in EpiSCs, indicating that, in stark contrast to ESCs, Oct4 is under active repressive control in EpiSCs. These studies provide a framework for better understanding pluripotency and for dissecting the molecular events that govern the transition from the pre-implantation to the post-implantation state.
RESUMEN
BACKGROUND: The evolutionarily conserved DNA mismatch repair (MMR) system corrects base-substitution and insertion-deletion mutations generated during erroneous replication. The mutation or inactivation of many MMR factors strongly predisposes to cancer, where the resulting tumors often display resistance to standard chemotherapeutics. A new direction to develop targeted therapies is the harnessing of synthetic genetic interactions, where the simultaneous loss of two otherwise non-essential factors leads to reduced cell fitness or death. High-throughput screening in human cells to directly identify such interactors for disease-relevant genes is now widespread, but often requires extensive case-by-case optimization. Here we asked if conserved genetic interactors (CGIs) with MMR genes from two evolutionary distant yeast species (Saccharomyces cerevisiae and Schizosaccharomyzes pombe) can predict orthologous genetic relationships in higher eukaryotes. METHODS: High-throughput screening was used to identify genetic interaction profiles for the MutSα and MutSß heterodimer subunits (msh2Δ, msh3Δ, msh6Δ) of fission yeast. Selected negative interactors with MutSß (msh2Δ/msh3Δ) were directly analyzed in budding yeast, and the CGI with SUMO-protease Ulp2 further examined after RNA interference/drug treatment in MSH2-deficient and -proficient human cells. RESULTS: This study identified distinct genetic profiles for MutSα and MutSß, and supports a role for the latter in recombinatorial DNA repair. Approximately 28% of orthologous genetic interactions with msh2Δ/msh3Δ are conserved in both yeasts, a degree consistent with global trends across these species. Further, the CGI between budding/fission yeast msh2 and SUMO-protease Ulp2 is maintained in human cells (MSH2/SENP6), and enhanced by Olaparib, a PARP inhibitor that induces the accumulation of single-strand DNA breaks. This identifies SENP6 as a promising new target for the treatment of MMR-deficient cancers. CONCLUSION: Our findings demonstrate the utility of employing evolutionary distance in tractable lower eukaryotes to predict orthologous genetic relationships in higher eukaryotes. Moreover, we provide novel insights into the genome maintenance functions of a critical DNA repair complex and propose a promising targeted treatment for MMR deficient tumors.
RESUMEN
Chromatin regulatory proteins affect diverse developmental and environmental response pathways via their influence on nuclear processes such as the regulation of gene expression. Through a genome-wide genetic screen, we implicate a novel protein called X-chromosome-associated protein 5 (Xap5) in chromatin regulation. We show that Xap5 is a chromatin-associated protein acting in a similar manner as the histone variant H2A.Z to suppress expression of antisense and repeat element transcripts throughout the fission yeast genome. Xap5 is highly conserved across eukaryotes, and a plant homolog rescues xap5 mutant yeast. We propose that Xap5 likely functions as a chromatin regulator in diverse organisms.
Asunto(s)
Proteínas de Unión al ADN/fisiología , Histonas/fisiología , Proteínas de Schizosaccharomyces pombe/fisiología , Schizosaccharomyces/genética , Elementos sin Sentido (Genética) , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Genes Fúngicos , Prueba de Complementación Genética , Unión Proteica , ARN de Hongos/genética , ARN de Hongos/metabolismo , ARN Mensajero/metabolismo , Secuencias Repetitivas de Ácidos Nucleicos , Schizosaccharomyces/metabolismo , Transcripción Genética , Regulación hacia ArribaRESUMEN
Cells are regularly exposed to stress conditions that may lead to protein misfolding. To cope with this challenge, molecular chaperones selectively target structurally perturbed proteins for degradation via the ubiquitin-proteasome pathway. In mammals the co-chaperone BAG-1 plays an important role in this system. BAG-1 has two orthologues, Bag101 and Bag102, in the fission yeast Schizosaccharomyces pombe. We show that both Bag101 and Bag102 interact with 26S proteasomes and Hsp70. By epistasis mapping we identify a mutant in the conserved kinetochore component Spc7 (Spc105/Blinkin) as a target for a quality control system that also involves, Hsp70, Bag102, the 26S proteasome, Ubc4 and the ubiquitin-ligases Ubr11 and San1. Accordingly, chromosome missegregation of spc7 mutant strains is alleviated by mutation of components in this pathway. In addition, we isolated a dominant negative version of the deubiquitylating enzyme, Ubp3, as a suppressor of the spc7-23 phenotype, suggesting that the proteasome-associated Ubp3 is required for this degradation system. Finally, our data suggest that the identified pathway is also involved in quality control of other kinetochore components and therefore likely to be a common degradation mechanism to ensure nuclear protein homeostasis and genome integrity.
Asunto(s)
Inestabilidad Genómica , Cinetocoros , Chaperonas Moleculares/genética , Proteolisis , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Proteínas HSP70 de Choque Térmico/genética , Proteínas HSP70 de Choque Térmico/metabolismo , Chaperonas Moleculares/metabolismo , Complejo de la Endopetidasa Proteasomal/genética , Schizosaccharomyces , Proteínas de Schizosaccharomyces pombe/genética , Transducción de Señal , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Ubiquitina/genética , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
Condensin is a central regulator of mitotic genome structure with mutants showing poorly condensed chromosomes and profound segregation defects. Here, we identify NCT, a complex comprising the Nrc1 BET-family tandem bromodomain protein (SPAC631.02), casein kinase II (CKII), and several TAFs, as a regulator of condensin function. We show that NCT and condensin bind similar genomic regions but only briefly colocalize during the periods of chromosome condensation and decondensation. This pattern of NCT binding at the core centromere, the region of maximal condensin enrichment, tracks the abundance of acetylated histone H4, as regulated by the Hat1-Mis16 acetyltransferase complex and recognized by the first Nrc1 bromodomain. Strikingly, mutants in NCT or Hat1-Mis16 restore the formation of segregation-competent chromosomes in cells containing defective condensin. These results are consistent with a model where NCT targets CKII to chromatin in a cell-cycle-directed manner in order to modulate the activity of condensin during chromosome condensation and decondensation.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Quinasa de la Caseína II/metabolismo , Cromatina/metabolismo , Proteínas de Unión al ADN/metabolismo , Complejos Multiproteicos/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Unión al ARN/metabolismo , Acetilación , Centrómero/metabolismo , Histona Acetiltransferasas/metabolismo , Histonas/metabolismo , Mitosis/fisiología , Levaduras/metabolismoRESUMEN
Stable compartments of the plasma membrane promote a wide range of cellular functions. In yeast cells, cytosolic structures called eisosomes generate prominent cortical invaginations of unknown function. Through a series of genetic screens in fission yeast, we found that the eisosome proteins Pil1 and Sle1 function with the synaptojanin-like lipid phosphatase Syj1 and its ligand Tax4. This genetic pathway connects eisosome function with the hydrolysis of phosphatidylinositol (4,5)-bisphosphate [PI(4,5)P2] in cells. Defects in PI(4,5)P2 regulation led to eisosome defects, and we found that the core eisosome protein Pil1 can bind to and tubulate liposomes containing PI(4,5)P2. Mutations in components of the Pil1-Sle1-Syj1-Tax4 pathway suppress the growth and morphology defects of TORC2 mutants, indicating that eisosome-dependent regulation of PI(4,5)P2 feeds into signal transduction pathways. We propose that the geometry of membrane invaginations generates spatial and temporal signals for lipid-mediated signaling events in cells.