RESUMEN
Although only a fraction of CTCF motifs are bound in any cell type, and approximately half of the occupied sites overlap cohesin, the mechanisms underlying cell-type specific attachment and ability to function as a chromatin organizer remain unknown. To investigate the relationship between CTCF and chromatin we applied a combination of imaging, structural and molecular approaches, using a series of brain and cancer associated CTCF mutations that act as CTCF perturbations. We demonstrate that binding and the functional impact of WT and mutant CTCF depend not only on the unique properties of each protein, but also on the genomic context of bound sites. Our studies also highlight the reciprocal relationship between CTCF and chromatin, demonstrating that the unique binding properties of WT and mutant proteins have a distinct impact on accessibility, TF binding, cohesin overlap, chromatin interactivity and gene expression programs, providing insight into their cancer and brain related effects.
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PDZ domain mediated interactions with voltage-gated calcium (Ca V ) channel C-termini play important roles in localizing and compartmentalizing membrane Ca 2+ signaling. The first such interaction discovered was between the neuronal multi-domain protein Mint-1, and the presynaptc calcium channel Ca V 2.2 in mammals. Although the physiological significance of this interaction is unclear, its occurrence in vertebrates and bilaterian invertebrates suggests important and conserved functions. In this study, we explore the evolutionary origins of Mint and its interaction with Ca V 2 channels. Phylogenetic and structural in silico analyses revealed that Mint is an animal-specific gene, like Ca V 2 channels, which bears a highly divergent N-terminus but strongly conserved C-terminus comprised of a phosphotyrosine binding domain, two tandem PDZ domains (PDZ-1 and PDZ-2), and a C-terminal auto-inhibitory element that binds and inhibits PDZ-1. Also deeply conserved are other Mint interacting proteins, namely amyloid precursor and related proteins, presenilins, neurexin, as well as CASK and Veli which form a tripartite complex with Mint in bilaterians. Through yeast 2-hybrid and bacterial 2-hybrid experiments, we show that Mint and Ca V 2 channels from cnidarians and placozoans interact in vitro , and in situ hybridization revealed co-expression of corresponding transcripts in dissociated neurons from the cnidarian Nematostella vectensis . Unexpectedly, the Mint orthologue from the ctenophore Hormiphora californiensis was able to strongly bind the divergent C-terminal ligands of cnidarian and placozoan Ca V 2 channels, despite neither the ctenophore Mint, nor the placozoan and cnidarian orthologues, binding the ctenophore Ca V 2 channel C-terminus. Altogether, our analyses provide a model for the emergence of this interaction in early animals first via adoption of a PDZ ligand by Ca V 2 channels, followed by sequence changes in the ligand that caused a modality switch for binding to Mint.
RESUMEN
Although only a fraction of CTCF motifs are bound in any cell type, and approximately half of the occupied sites overlap cohesin, the mechanisms underlying cell-type specific attachment and ability to function as a chromatin organizer remain unknown. To investigate the relationship between CTCF and chromatin we applied a combination of imaging, structural and molecular approaches, using a series of brain and cancer associated CTCF mutations that act as CTCF perturbations. We demonstrate that binding and the functional impact of WT and mutant CTCF depend not only on the unique properties of each protein, but also on the genomic context of bound sites. Our studies also highlight the reciprocal relationship between CTCF and chromatin, demonstrating that the unique binding properties of WT and mutant proteins have a distinct impact on accessibility, TF binding, cohesin overlap, chromatin interactivity and gene expression programs, providing insight into their cancer and brain related effects.
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Whether synthetic genomes can power life has attracted broad interest in the synthetic biology field. Here, we report de novo synthesis of the largest eukaryotic chromosome thus far, synIV, a 1,454,621-bp yeast chromosome resulting from extensive genome streamlining and modification. We developed megachunk assembly combined with a hierarchical integration strategy, which significantly increased the accuracy and flexibility of synthetic chromosome construction. Besides the drastic sequence changes, we further manipulated the 3D structure of synIV to explore spatial gene regulation. Surprisingly, we found few gene expression changes, suggesting that positioning inside the yeast nucleoplasm plays a minor role in gene regulation. Lastly, we tethered synIV to the inner nuclear membrane via its hundreds of loxPsym sites and observed transcriptional repression of the entire chromosome, demonstrating chromosome-wide transcription manipulation without changing the DNA sequences. Our manipulation of the spatial structure of synIV sheds light on higher-order architectural design of the synthetic genomes.
Asunto(s)
Núcleo Celular , Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Cromosomas/genética , Genoma Fúngico , Biología Sintética/métodosRESUMEN
DNA targeting Class 2 CRISPR-Cas effector nucleases, including the well-studied Cas9 proteins, evolved protospacer-adjacent motif (PAM) and guide RNA interactions that sequentially license their binding and cleavage activities at protospacer target sites. Both interactions are nucleic acid sequence specific but function constitutively; thus, they provide intrinsic spatial control over DNA targeting activities but naturally lack temporal control. Here we show that engineered Cas9 fusion proteins which bind to nascent RNAs near a protospacer can facilitate spatiotemporal coupling between transcription and DNA targeting at that protospacer: Transcription-associated Cas9 Targeting (TraCT). Engineered TraCT is enabled when suboptimal PAM interactions limit basal activity in vivo and when one or more nascent RNA substrates are still tethered to the actively transcribing target DNA in cis. We further show that this phenomenon can be exploited for selective editing at one of two identical targets in distinct gene loci, or, in diploid allelic loci that are differentially transcribed. Our work demonstrates that temporal control over Cas9's targeting activity at specific DNA sites may be engineered without modifying Cas9's core domains and guide RNA components or their expression levels. More broadly, it establishes RNA binding in cis as a mechanism that can conditionally stimulate CRISPR-Cas DNA targeting in eukaryotes.
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Cys2His2 zinc finger (ZF) domains engineered to bind specific target sequences in the genome provide an effective strategy for programmable regulation of gene expression, with many potential therapeutic applications. However, the structurally intricate engagement of ZF domains with DNA has made their design challenging. Here we describe the screening of 49 billion protein-DNA interactions and the development of a deep-learning model, ZFDesign, that solves ZF design for any genomic target. ZFDesign is a modern machine learning method that models global and target-specific differences induced by a range of library environments and specifically takes into account compatibility of neighboring fingers using a novel hierarchical transformer architecture. We demonstrate the versatility of designed ZFs as nucleases as well as activators and repressors by seamless reprogramming of human transcription factors. These factors could be used to upregulate an allele of haploinsufficiency, downregulate a gain-of-function mutation or test the consequence of regulation of a single gene as opposed to the many genes that a transcription factor would normally influence.
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Aprendizaje Profundo , Factores de Transcripción , Humanos , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Dedos de Zinc/genética , Regulación de la Expresión Génica , ADN/genéticaRESUMEN
The discovery of regulatory domains has been limited to the investigation of transcription factors and homologous protein sequences. In this issue of Cell Genomics, motivated by an oncogenic protein fusion, Tak et al.1 direct the regulatory potential of a nontraditional effector domain to novel genomic loci with fusions to programmable DNA-binding domains.
RESUMEN
The widely used Streptococcus pyogenes Cas9 (SpCas9) nuclease derives its DNA targeting specificity from protein-DNA contacts with protospacer adjacent motif (PAM) sequences, in addition to base-pairing interactions between its guide RNA and target DNA. Previous reports have established that the PAM specificity of SpCas9 can be altered via positive selection procedures for directed evolution or other protein engineering strategies. Here we exploit in vivo directed evolution systems that incorporate simultaneous positive and negative selection to evolve SpCas9 variants with commensurate or improved activity on NAG PAMs relative to wild type and reduced activity on NGG PAMs, particularly YGG PAMs. We also show that the PAM preferences of available evolutionary intermediates effectively determine whether similar counterselection PAMs elicit different selection stringencies, and demonstrate that negative selection can be specifically increased in a yeast selection system through the fusion of compensatory zinc fingers to SpCas9.
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Proteína 9 Asociada a CRISPR/metabolismo , Sistemas CRISPR-Cas , ADN/metabolismo , Edición Génica/métodos , ARN Guía de Kinetoplastida/metabolismo , Streptococcus pyogenes/metabolismo , Secuencia de Aminoácidos , Proteína 9 Asociada a CRISPR/genética , Línea Celular Tumoral , ADN/química , ADN/genética , Evolución Molecular Dirigida/métodos , Humanos , Mutación , Conformación de Ácido Nucleico , Motivos de Nucleótidos/genética , Ingeniería de Proteínas/métodos , ARN Guía de Kinetoplastida/genética , Streptococcus pyogenes/genética , Especificidad por SustratoRESUMEN
The Cys2His2 zinc finger is the most common DNA-binding domain expanding in metazoans since the fungi human split. A proposed catalyst for this expansion is an arms race to silence transposable elements yet it remains poorly understood how this domain is able to evolve the required specificities. Likewise, models of its DNA binding specificity remain error prone due to a lack of understanding of how adjacent fingers influence each other's binding specificity. Here, we use a synthetic approach to exhaustively investigate binding geometry, one of the dominant influences on adjacent finger function. By screening over 28 billion protein-DNA interactions in various geometric contexts we find the plasticity of the most common natural geometry enables more functional amino acid combinations across all targets. Further, residues that define this geometry are enriched in genomes where zinc fingers are prevalent and specificity transitions would be limited in alternative geometries. Finally, these results demonstrate an exhaustive synthetic screen can produce an accurate model of domain function while providing mechanistic insight that may have assisted in the domains expansion.
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Modelos Moleculares , Dominios Proteicos/fisiología , Dedos de Zinc/fisiología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , ADN/síntesis química , ADN/genética , ADN/metabolismo , Aprendizaje Profundo , Humanos , Enlace de Hidrógeno , Dominios Proteicos/genética , Reproducibilidad de los Resultados , Especificidad por Sustrato/genética , Factores de Transcripción/química , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Dedos de Zinc/genéticaRESUMEN
Optogenetic tools for controlling gene expression are ideal for tuning synthetic biological networks due to the exquisite spatiotemporal control available with light. Here we develop an optogenetic system for gene expression control integrated with an existing yeast toolkit allowing for rapid, modular assembly of light-controlled circuits in the important chassis organism Saccharomyces cerevisiae. We reconstitute activity of a split synthetic zinc-finger transcription factor (TF) using light-induced dimerization mediated by the proteins CRY2 and CIB1. We optimize function of this split TF and demonstrate the utility of the toolkit workflow by assembling cassettes expressing the TF activation domain and DNA-binding domain at different levels. Utilizing this TF and a synthetic promoter we demonstrate that light intensity and duty cycle can be used to modulate gene expression over the range currently available from natural yeast promoters. This study allows for rapid generation and prototyping of optogenetic circuits to control gene expression in S. cerevisiae.
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Regulación Fúngica de la Expresión Génica , Optogenética/métodos , Regiones Promotoras Genéticas , Saccharomyces cerevisiae/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Clonación Molecular , Criptocromos/genética , Criptocromos/metabolismo , Regulación Fúngica de la Expresión Génica/genética , Regulación Fúngica de la Expresión Génica/efectos de la radiación , Regiones Promotoras Genéticas/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Dedos de Zinc/genéticaRESUMEN
The accurate determination of protein-protein interactions has been an important focus of molecular biology toward which much progress has been made due to the continuous development of existing and new technologies. However, current methods can have limitations, including scale and restriction to high affinity interactions, limiting our understanding of a large subset of these interactions. Here, we describe a modified bacterial-hybrid assay that employs combined selectable and scalable reporters that enable the sensitive screening of large peptide libraries followed by the sorting of positive interactions by the level of reporter output. We have applied this tool to characterize a set of human and E. coli PDZ domains. Our results are consistent with prior characterization of these proteins, and the improved sensitivity increases our ability to predict known and novel in vivo binding partners. This approach allows for the recovery of a wide range of affinities with a high throughput method that does not sacrifice the scale of the screen.
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Escherichia coli/metabolismo , Ensayos Analíticos de Alto Rendimiento/métodos , Péptidos/metabolismo , Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Secuencia de Aminoácidos , Genes Reporteros , Humanos , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/metabolismo , Dominios PDZ , Biblioteca de Péptidos , Péptidos/química , Unión ProteicaRESUMEN
The sensory nervous system of C. elegans comprises cells with varied molecular and functional characteristics, and is, therefore, a powerful model for understanding mechanisms that generate neuronal diversity. We report here that VAB-3, a C. elegans homolog of the homeodomain-containing protein Pax6, has opposing functions in regulating expression of a specific chemosensory fate. A homeodomain-only short isoform of VAB-3 is expressed in BAG chemosensory neurons, where it promotes gene expression and cell function. In other cells, a long isoform of VAB-3, comprising a Paired homology domain and a homeodomain, represses expression of ETS-5, a transcription factor required for expression of BAG fate. Repression of ets-5 requires the Eyes Absent homolog EYA-1 and the Six-class homeodomain protein CEH-32. We determined sequences that mediate high-affinity binding of ETS-5, VAB-3 and CEH-32. The ets-5 locus is enriched for ETS-5-binding sites but lacks sequences that bind VAB-3 and CEH-32, suggesting that these factors do not directly repress ets-5 expression. We propose that a promoter-selection system together with lineage-specific expression of accessory factors allows VAB-3/Pax6 to either promote or repress expression of specific cell fates in a context-dependent manner. This article has an associated 'The people behind the papers' interview.
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Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Proteínas de Homeodominio/metabolismo , Factores de Transcripción/metabolismo , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/metabolismo , Bleomicina/metabolismo , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Clorambucilo/metabolismo , Cisplatino/metabolismo , Ciclofosfamida/metabolismo , Dactinomicina/metabolismo , Regulación del Desarrollo de la Expresión Génica/genética , Proteínas de Homeodominio/genética , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Neuronas/citología , Neuronas/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Regiones Promotoras Genéticas/genética , Proteínas Tirosina Fosfatasas/genética , Proteínas Tirosina Fosfatasas/metabolismo , Proteínas Proto-Oncogénicas c-ets/genética , Proteínas Proto-Oncogénicas c-ets/metabolismo , Factores de Transcripción/genética , Vinblastina/metabolismoRESUMEN
The developmental accumulation of proliferative germ cells in the C. elegans hermaphrodite is sensitive to the organismal environment. Previously, we found that the TGFß signaling pathway links the environment and proliferative germ cell accumulation. Neuronal DAF-7/TGFß causes a DAF-1/TGFßR signaling cascade in the gonadal distal tip cell (DTC), the germline stem cell niche, where it negatively regulates a DAF-3 SMAD and DAF-5 Sno-Ski. LAG-2, a founding DSL ligand family member, is produced in the DTC and activates the GLP-1/Notch receptor on adjacent germ cells to maintain germline stem cell fate. Here, we show that DAF-7/TGFß signaling promotes expression of lag-2 in the DTC in a daf-3-dependent manner. Using ChIP and one-hybrid assays, we find evidence for direct interaction between DAF-3 and the lag-2 promoter. We further identify a 25â bp DAF-3 binding element required for the DTC lag-2 reporter response to the environment and to DAF-7/TGFß signaling. Our results implicate DAF-3 repressor complex activity as a key molecular mechanism whereby the environment influences DSL ligand expression in the niche to modulate developmental expansion of the germline stem cell pool.
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Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Nicho de Células Madre/fisiología , Factor de Crecimiento Transformador beta/metabolismo , Animales , Caenorhabditis elegans/citología , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Inmunoprecipitación de Cromatina , Hibridación in Situ , Transducción de Señal/genética , Transducción de Señal/fisiología , Nicho de Células Madre/genética , Factor de Crecimiento Transformador beta/genéticaRESUMEN
Cell Systems invited 16 experts to share their views on the field of systems genetics. In questions repeated in the headings, we asked them to define systems genetics, highlight its relevance to researchers outside the field, discuss what makes a strong systems genetics paper, and paint a picture of where the field is heading in the coming years. Their responses, ordered by the journal but otherwise unedited, make it clear that deciphering genotype to phenotype relationships is a central challenge of systems genetics and will require understanding how networks and higher-order properties of biological systems underlie complex traits. In addition, our experts illuminate the applications and relevance of systems genetics to human disease, the gut microbiome, development of tools that connect the global research community, sustainability, drug discovery, patient-specific disease and network models, and personalized treatments. Finally, a table of suggested reading provides a sample of influential work in the field.
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Genética/tendencias , Biología de Sistemas/tendencias , Animales , Descubrimiento de Drogas , Genómica , Genotipo , Humanos , Microbiota/genética , Herencia Multifactorial , Fenotipo , Biología de Sistemas/métodosRESUMEN
Engineered nucleases have transformed biological research and offer great therapeutic potential by enabling the straightforward modification of desired genomic sequences. While many nuclease platforms have proven functional, all can produce unanticipated off-target lesions and have difficulty discriminating between homologous sequences, limiting their therapeutic application. Here we describe a multi-reporter selection system that allows the screening of large protein libraries to uncover variants able to discriminate between sequences with substantial homology. We have used this system to identify zinc-finger nucleases that exhibit high cleavage activity (up to 60% indels) at their targets within the CCR5 and HBB genes and strong discrimination against homologous sequences within CCR2 and HBD. An unbiased screen for off-target lesions using a novel set of CCR5-targeting nucleases confirms negligible CCR2 activity and demonstrates minimal off-target activity genome wide. This system offers a straightforward approach to generate nucleases that discriminate between similar targets and provide exceptional genome-wide specificity.
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Desoxirribonucleasas/metabolismo , Regulación Enzimológica de la Expresión Génica/fisiología , Receptores CCR5/metabolismo , Dedos de Zinc , Animales , Proteínas de Unión al ADN/genética , Desoxirribonucleasas/genética , Genes Reporteros , Genoma , Humanos , Biblioteca de Péptidos , Receptores CCR2/metabolismoRESUMEN
Cys2His2 zinc fingers (C2H2-ZFs) comprise the largest class of metazoan DNA-binding domains. Despite this domain's well-defined DNA-recognition interface, and its successful use in the design of chimeric proteins capable of targeting genomic regions of interest, much remains unknown about its DNA-binding landscape. To help bridge this gap in fundamental knowledge and to provide a resource for design-oriented applications, we screened large synthetic protein libraries to select binding C2H2-ZF domains for each possible three base pair target. The resulting data consist of >160 000 unique domain-DNA interactions and comprise the most comprehensive investigation of C2H2-ZF DNA-binding interactions to date. An integrated analysis of these independent screens yielded DNA-binding profiles for tens of thousands of domains and led to the successful design and prediction of C2H2-ZF DNA-binding specificities. Computational analyses uncovered important aspects of C2H2-ZF domain-DNA interactions, including the roles of within-finger context and domain position on base recognition. We observed the existence of numerous distinct binding strategies for each possible three base pair target and an apparent balance between affinity and specificity of binding. In sum, our comprehensive data help elucidate the complex binding landscape of C2H2-ZF domains and provide a foundation for efforts to determine, predict and engineer their DNA-binding specificities.
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Cisteína/química , ADN/metabolismo , Histidina/química , Dedos de Zinc , Sitios de Unión , ADN/química , Recolección de DatosRESUMEN
Understanding how sequence-specific protein-DNA interactions direct cellular function is of great interest to the research community. High-throughput methods have been developed to determine DNA-binding specificities; one such technique, the bacterial one-hybrid (B1H) system, confers advantages including ease of use, sensitivity and throughput. In this review, we describe the evolution of the B1H system as a tool capable of screening large DNA libraries to investigate protein-DNA interactions of interest. We discuss how DNA-binding specificities produced by the B1H system have been used to predict regulatory targets. Additionally, we examine how this approach has been applied to characterize two common DNA-binding domain families-homeodomains and Cys2His2 zinc fingers-both in organism-wide studies and with synthetic approaches. In the case of the former, the B1H system has produced large catalogs of protein specificity and nuanced information about previously recovered DNA targets, thereby improving our understanding of these proteins' functions in vivo and increasing our capacity to predict similar interactions in other species. In the latter, synthetic screens of the same DNA-binding domains have further refined our models of specificity, through analyzing comprehensive libraries to uncover all proteins able to bind a complete set of targets, and, for instance, exploring how context-in the form of domain position within the parent protein-may affect specificity. Finally, we recognize the limitations of the B1H system and discuss its potential for use in the production of designer proteins and in studies of protein-protein interactions.
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Bacterias/metabolismo , ADN/metabolismo , Técnicas del Sistema de Dos Híbridos , Unión Proteica , Estructura Terciaria de Proteína , Factores de Transcripción/química , Factores de Transcripción/metabolismoRESUMEN
The Cys2His2 zinc finger (ZF) is the most frequently found sequence-specific DNA-binding domain in eukaryotic proteins. The ZF's modular protein-DNA interface has also served as a platform for genome engineering applications. Despite decades of intense study, a predictive understanding of the DNA-binding specificities of either natural or engineered ZF domains remains elusive. To help fill this gap, we developed an integrated experimental-computational approach to enrich and recover distinct groups of ZFs that bind common targets. To showcase the power of our approach, we built several large ZF libraries and demonstrated their excellent diversity. As proof of principle, we used one of these ZF libraries to select and recover thousands of ZFs that bind several 3-nt targets of interest. We were then able to computationally cluster these recovered ZFs to reveal several distinct classes of proteins, all recovered from a single selection, to bind the same target. Finally, for each target studied, we confirmed that one or more representative ZFs yield the desired specificity. In sum, the described approach enables comprehensive large-scale selection and characterization of ZF specificities and should be a great aid in furthering our understanding of the ZF domain.
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Proteínas de Unión al ADN/química , Factores de Transcripción/química , Dedos de Zinc , Sitios de Unión , Biología Computacional/métodos , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Biblioteca de Genes , Secuenciación de Nucleótidos de Alto Rendimiento , Mutagénesis , Reacción en Cadena de la Polimerasa , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Synthetic biology aims to rationally design and build synthetic circuits with desired quantitative properties, as well as provide tools to interrogate the structure of native control circuits. In both cases, the ability to program gene expression in a rapid and tunable fashion, with no off-target effects, can be useful. We have constructed yeast strains containing the ACT1 promoter upstream of a URA3 cassette followed by the ligand-binding domain of the human estrogen receptor and VP16. By transforming this strain with a linear PCR product containing a DNA binding domain and selecting against the presence of URA3, a constitutively expressed artificial transcription factor (ATF) can be generated by homologous recombination. ATFs engineered in this fashion can activate a unique target gene in the presence of inducer, thereby eliminating both the off-target activation and nonphysiological growth conditions found with commonly used conditional gene expression systems. A simple method for the rapid construction of GFP reporter plasmids that respond specifically to a native or artificial transcription factor of interest is also provided.
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Proteínas Fluorescentes Verdes/genética , Ingeniería de Proteínas/métodos , Biología Sintética/métodos , Factores de Transcripción/genética , Secuencia de Bases , Conexina 43/genética , Citometría de Flujo/métodos , Proteínas Fluorescentes Verdes/biosíntesis , Proteínas Fluorescentes Verdes/química , Humanos , Datos de Secuencia Molecular , Fragmentos de Péptidos/genética , Plásmidos/genética , Factores de Transcripción/biosíntesis , Factores de Transcripción/química , Levaduras/genética , Levaduras/metabolismoRESUMEN
A general method for the dynamic control of single gene expression in eukaryotes, with no off-target effects, is a long-sought tool for molecular and systems biologists. We engineered two artificial transcription factors (ATFs) that contain Cys(2)His(2) zinc-finger DNA-binding domains of either the mouse transcription factor Zif268 (9 bp of specificity) or a rationally designed array of four zinc fingers (12 bp of specificity). These domains were expressed as fusions to the human estrogen receptor and VP16 activation domain. The ATFs can rapidly induce a single gene driven by a synthetic promoter in response to introduction of an otherwise inert hormone with no detectable off-target effects. In the absence of inducer, the synthetic promoter is inactive and the regulated gene product is not detected. Following addition of inducer, transcripts are induced >50-fold within 15 min. We present a quantitative characterization of these ATFs and provide constructs for making their implementation straightforward. These new tools allow for the elucidation of regulatory network elements dynamically, which we demonstrate with a major metabolic regulator, Gcn4p.