RESUMO
To investigate the relationship between genome structure and function, we have developed a programmable CRISPR-Cas system for nuclear peripheral recruitment in yeast. We benchmarked this system at the HMR and GAL2 loci, both of which are well-characterized model systems for localization to the nuclear periphery. Using microscopy and gene silencing assays, we demonstrate that CRISPR-Cas-mediated tethering can recruit the HMR locus but does not detectably silence reporter gene expression. A previously reported Gal4-mediated tethering system does silence gene expression, and we demonstrate that the silencing effect has an unexpected dependence on the properties of the protein tether. The CRISPR-Cas system was unable to recruit GAL2 to the nuclear periphery. Our results reveal potential challenges for synthetic genome structure perturbations and suggest that distinct functional effects can arise from subtle structural differences in how genes are recruited to the periphery.
Assuntos
Sistemas CRISPR-Cas/genética , Núcleo Celular/genética , Expressão Gênica/genética , Inativação Gênica/fisiologia , Saccharomyces cerevisiae/genética , Proteínas de Ligação a DNA/genética , Genes Reporter/genética , Técnicas Genéticas , Genoma Bacteriano/genéticaRESUMO
To spatially control biochemical functions at specific sites within a genome, we have engineered a synthetic switch that activates when bound to its DNA target site. The system uses two CRISPR-Cas complexes to colocalize components of a de novo-designed protein switch (Co-LOCKR) to adjacent sites in the genome. Colocalization triggers a conformational change in the switch from an inactive closed state to an active open state with an exposed functional peptide. We prototype the system in yeast and demonstrate that DNA binding triggers activation of the switch, recruitment of a transcription factor, and expression of a downstream reporter gene. This DNA-triggered Co-LOCKR switch provides a platform to engineer sophisticated functions that should only be executed at a specific target site within the genome, with potential applications in a wide range of synthetic systems including epigenetic regulation, imaging, and genetic logic circuits.
Assuntos
Proteína 9 Associada à CRISPR/genética , DNA/metabolismo , Edição de Genes/métodos , DNA/química , Genes Reporter , RNA Guia de Cinetoplastídeos/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMO
In mammalian cells, programmed cell death (PCD) plays important roles in development, in the removal of damaged cells, and in fighting bacterial infections. Although widespread among multicellular organisms, there are relatively few documented instances of PCD in bacteria. Here we describe a potential PCD pathway in Pseudomonas aeruginosa that enhances the ability of the bacterium to cause disease in a lung infection model. Activation of the system can occur in a subset of cells in response to DNA damage through cleavage of an essential transcription regulator we call AlpR. Cleavage of AlpR triggers a cell lysis program through de-repression of the alpA gene, which encodes a positive regulator that activates expression of the alpBCDE lysis cassette. Although this is lethal to the individual cell in which it occurs, we find it benefits the population as a whole during infection of a mammalian host. Thus, host and pathogen each may use PCD as a survival-promoting strategy. We suggest that activation of the Alp cell lysis pathway is a disease-enhancing response to bacterial DNA damage inflicted by the host immune system.
Assuntos
Proteínas de Bactérias/genética , Bacteriólise/genética , Pseudomonas aeruginosa/genética , Transdução de Sinais/genética , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/metabolismo , Western Blotting , Regulação Bacteriana da Expressão Gênica , Masculino , Camundongos Endogâmicos C57BL , Viabilidade Microbiana/genética , Microscopia de Fluorescência , Dados de Sequência Molecular , Mutação , Óperon/genética , Infecções por Pseudomonas/genética , Infecções por Pseudomonas/metabolismo , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/metabolismo , Pseudomonas aeruginosa/patogenicidade , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de Aminoácidos , Imagem com Lapso de Tempo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Virulência/genéticaRESUMO
The perception and response to cellular death is an important aspect of multicellular eukaryotic life. For example, damage-associated molecular patterns activate an inflammatory cascade that leads to removal of cellular debris and promotion of healing. We demonstrate that lysis of Pseudomonas aeruginosa cells triggers a program in the remaining population that confers fitness in interspecies co-culture. We find that this program, termed P. aeruginosa response to antagonism (PARA), involves rapid deployment of antibacterial factors and is mediated by the Gac/Rsm global regulatory pathway. Type VI secretion, and, unexpectedly, conjugative type IV secretion within competing bacteria, induce P. aeruginosa lysis and activate PARA, thus providing a mechanism for the enhanced capacity of P. aeruginosa to target bacteria that elaborate these factors. Our finding that bacteria sense damaged kin and respond via a widely distributed pathway to mount a complex response raises the possibility that danger sensing is an evolutionarily conserved process.