RESUMEN
Genome editing driven by zinc-finger nucleases (ZFNs) yields high gene-modification efficiencies (>10%) by introducing a recombinogenic double-strand break into the targeted gene. The cleavage event is induced using two custom-designed ZFNs that heterodimerize upon binding DNA to form a catalytically active nuclease complex. Using the current ZFN architecture, however, cleavage-competent homodimers may also form that can limit safety or efficacy via off-target cleavage. Here we develop an improved ZFN architecture that eliminates this problem. Using structure-based design, we engineer two variant ZFNs that efficiently cleave DNA only when paired as a heterodimer. These ZFNs modify a native endogenous locus as efficiently as the parental architecture, but with a >40-fold reduction in homodimer function and much lower levels of genome-wide cleavage. This architecture provides a general means for improving the specificity of ZFNs as gene modification reagents.
Asunto(s)
Biotecnología/métodos , Dedos de Zinc , Secuencia de Bases , Sitios de Unión , Catálisis , Desoxirribonucleasas de Localización Especificada Tipo II/química , Dimerización , Genoma , Proteínas Fluorescentes Verdes/química , Humanos , Células K562 , Modelos Biológicos , Conformación Molecular , Datos de Secuencia Molecular , Estructura Terciaria de ProteínaAsunto(s)
Biología Computacional/métodos , ADN/química , ADN/metabolismo , Endonucleasas/química , Endonucleasas/metabolismo , Modelos Químicos , Modelos Moleculares , Emparejamiento Base , Secuencia de Bases , Sitios de Unión , Simulación por Computador , Cristalografía por Rayos X , ADN/genética , Endonucleasas/genética , Intrones/genética , Método de Montecarlo , Conformación Proteica , Ingeniería de Proteínas/métodos , Especificidad por Sustrato , TermodinámicaRESUMEN
Designed zinc finger proteins (ZFPs) regulate expression of target genes when coupled to activator or repressor domains. Transfection of ZFPs into cell lines can create expression systems where the targeted endogenous gene is transcribed and the protein of interest can be investigated in its own cellular context. Here we describe the pharmacological investigation of an expression system generated using CCK2 receptor-selective ZFPs transfected into human embryonic kidney cells (HEKZFP system). The receptors expressed in this system, in response to ZFP expression, were functional in calcium mobilization studies and the potency of the agonists investigated was consistent with their action at CCK2 receptors (CCK-8S pA50 = 9.05+/-0.11, pentagastrin pA50 = 9.11+/-0.13). In addition, binding studies were conducted using [125I]-BH-CCK-8S as radioligand. The saturation binding analysis of this radioligand was consistent with a single population of high affinity CCK receptors (pK(D) = 10.24). Competition studies were also conducted using a number of previously well-characterized CCK-receptor selective ligands; JB93182, YF476, PD-134,308, SR27897, dexloxiglumide, L-365,260 and L-364,718. Overall, the estimated affinity values for these ligands were consistent with their interaction at CCK2 receptors. Therefore, CCK2 receptors up-regulated using zinc finger protein technology can provide an alternative to standard transfection techniques for the pharmacological analysis of compounds.
Asunto(s)
Riñón/metabolismo , Receptor de Colecistoquinina B/biosíntesis , Factores de Transcripción/metabolismo , Regulación hacia Arriba/efectos de los fármacos , Línea Celular , Evaluación Preclínica de Medicamentos/métodos , Humanos , Riñón/citología , Ligandos , Preparaciones Farmacéuticas/metabolismo , Receptor de Colecistoquinina B/antagonistas & inhibidores , Factores de Transcripción/genética , Transfección , Regulación hacia Arriba/genética , Dedos de Zinc/genética , Dedos de Zinc/fisiologíaRESUMEN
Drug discovery requires high-quality, high-throughput bioassays for lead identification and optimization. These assays are usually based on immortalized cell lines, which express the selected drug target either naturally or as a consequence of transfection with the cDNA encoding the target. Natural untransfected cell lines often fail to achieve the levels of expression required to provide assays of sufficient quality with a high enough signal-to-noise ratio. Unfortunately, the use of cDNA is increasingly restricted, as the sequences for more and more genes become subject to patent restrictions. To overcome these limitations, the authors demonstrate that engineered transcription factors with Cys2-His2 zinc finger DNA-binding domains can be used to effectively activate an endogenous gene of interest without the use of isolated cDNA of the target gene. Using this approach, the authors have generated a cell line that provides a high-quality and pharmacologically validated G-protein-coupled receptor bioassay. In principle, this technology is applicable to any gene of pharmaceutical importance in any cell type.
Asunto(s)
Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Línea Celular , Cartilla de ADN , Humanos , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Ingeniería de Proteínas , Factores de Transcripción/química , Factores de Transcripción/genéticaRESUMEN
In recent years, two methods have been developed that may eventually allow the targeted regulation of a broad repertoire of genes. The engineered protein strategy involves selecting Cys(2)His(2) zinc finger proteins that will recognize specific sites in the major groove of DNA. The small molecule approach utilizes pairing rules for pyrrole-imidazole polyamides that target specific sites in the minor groove. To understand how these two methods might complement each other, we have begun exploring how polyamides and zinc fingers interact when they bind the same site on opposite grooves of DNA. Although structural comparisons show no obvious source of van der Waals collisions, we have found a significant "negative cooperativity" when the two classes of compounds are directed to the overlapping sites. Examining available crystal structures suggests that this may reflect differences in the precise DNA conformation, especially with regard to width and depth of the grooves, that is preferred for binding. These results may give new insights into the structural requirements for zinc finger and polyamide binding and may eventually lead to the development of even more powerful and flexible schemes for regulating gene expression.
Asunto(s)
Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/química , ADN/antagonistas & inhibidores , ADN/química , Conformación de Ácido Nucleico , Dedos de Zinc , Regulación Alostérica , Secuencia de Bases , Unión Competitiva , Simulación por Computador , Huella de ADN , Desoxirribonucleasa I/química , Proteína 1 de la Respuesta de Crecimiento Precoz , Ensayo de Cambio de Movilidad Electroforética , Proteínas Fúngicas/antagonistas & inhibidores , Proteínas Fúngicas/química , Humanos , Proteínas Inmediatas-Precoces/antagonistas & inhibidores , Proteínas Inmediatas-Precoces/química , Ligandos , Modelos Moleculares , Datos de Secuencia Molecular , Nylons/química , Unión Proteica , Proteína de Unión a TATA-Box/antagonistas & inhibidores , Proteína de Unión a TATA-Box/química , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/química , Proteína p53 Supresora de Tumor/antagonistas & inhibidores , Proteína p53 Supresora de Tumor/químicaRESUMEN
Angiogenic factors are necessary for tumor proliferation and thus are attractive therapeutic targets. In this study, we have used engineered zinc finger protein (ZFP) transcription factors (TFs) to repress expression of vascular endothelial growth factor (VEGF)-A in human cancer cell lines. We create potent transcriptional repressors by fusing a designed ZFP targeted to the VEGF-A promoter with either the ligand-binding domain of thyroid hormone receptor alpha or its viral relative, vErbA. Moreover, this ZFP-vErbA repressor binds its intended target site in vivo and mediates the specific deacetylation of histones H3 and H4 at the targeted promoter, a result that emulates the natural repression mechanism of these domains. The potential therapeutic relevance of ZFP-mediated VEGF-A repression was addressed using the highly tumorigenic glioblastoma cell line U87MG. Despite the aberrant overexpression of VEGF-A in this cell line, engineered ZFP TFs were able to repress the expression of VEGF-A by >20-fold. The VEGF-A levels observed after ZFP TF-mediated repression were comparable to those of a nonangiogenic cancer line (U251MG), suggesting that the degree of repression obtained with the ZFP TF would be sufficient to suppress tumor angiogenesis. Thus, engineered ZFP TFs are shown to be potent regulators of gene expression with therapeutic promise in the treatment of disease.
Asunto(s)
Glioblastoma/metabolismo , Glioblastoma/terapia , Factores de Transcripción/genética , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Dedos de Zinc/genética , Línea Celular Tumoral , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Regulación Neoplásica de la Expresión Génica/genética , Glioblastoma/irrigación sanguínea , Glioblastoma/genética , Humanos , Neovascularización Patológica/genética , Neovascularización Patológica/metabolismo , Proteínas Oncogénicas v-erbA/genética , Proteínas Oncogénicas v-erbA/metabolismo , Regiones Promotoras Genéticas , Factores de Transcripción/metabolismo , Transfección , Factor A de Crecimiento Endotelial Vascular/biosíntesis , Factor A de Crecimiento Endotelial Vascular/genéticaRESUMEN
Proteins that employ dimerization domains to bind cooperatively to DNA have a number of potential advantages over monomers with regards to gene regulation. Using a combination of structure-based design and phage display, a dimeric Cys(2)His(2) zinc finger protein has been created that binds cooperatively to DNA via an attached leucine zipper dimerization domain. This chimera, derived from components of Zif268 and GCN4, displayed excellent DNA-binding specificity, and we now report the 1.5 A resolution cocrystal structure of the Zif268-GCN4 homodimer bound to DNA. This structure shows how phage display has annealed the DNA binding and dimerization domains into a single functional unit. Moreover, this chimera provides a potential platform for the creation heterodimeric zinc finger proteins that can regulate a desired target gene through cooperative DNA recognition.
Asunto(s)
Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , ADN/metabolismo , Dedos de Zinc , Secuencia de Aminoácidos , Secuencia de Bases , Cristalografía por Rayos X , ADN/química , Proteínas de Unión al ADN/genética , Dimerización , Leucina Zippers , Modelos Moleculares , Datos de Secuencia Molecular , Biblioteca de Péptidos , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
Stem cells are functionally defined as progenitor cells that can self-renew and differentiate. Critical transitions in these cells are controlled via signaling pathways and subsequent transcriptional regulation. Technologies capable of modulating the levels of gene expression, especially those of transcription factors, represent powerful tools for research and could potentially be used in therapeutic applications. In this study, we evaluated the ability of synthetic zinc finger protein transcription factors (ZFP-TFs) to cause the differentiation of embryonic stem (ES) cells. We constructed ZFP-TFs that target the mouse Oct-4 gene (which is a major regulator of ES cell pluripotency and self-renewal). These designed transcription factors were able to regulate the transcription of Oct-4, affecting the expression of downstream genes and thus regulating ES cell differentiation.
Asunto(s)
Diferenciación Celular , Proteínas de Unión al ADN/genética , Embrión de Mamíferos/citología , Marcación de Gen , Ingeniería de Proteínas/métodos , Células Madre/fisiología , Factores de Transcripción/genética , Dedos de Zinc/genética , Animales , Técnicas de Cultivo de Célula , Regulación del Desarrollo de la Expresión Génica , Técnicas de Transferencia de Gen , Ratones , Factor 3 de Transcripción de Unión a Octámeros , Células Madre/metabolismo , Factores de Transcripción/metabolismoRESUMEN
Engineered Cys2His2 zinc finger proteins (ZFPs) can mediate regulation of endogenous gene expression in mammalian cells. Ideally, all zinc fingers in an engineered multifinger protein should be optimized concurrently because cooperative and context-dependent contacts can affect DNA recognition. However, the simultaneous selection of key contacts in even three fingers from fully randomized libraries would require the consideration of >10(24) possible combinations. To address this challenge, we have developed a novel strategy that utilizes directed domain shuffling and rapid cell-based selections. Unlike previously described methods, our strategy is amenable to scale-up and does not sacrifice combinatorial diversity. Using this approach, we have successfully isolated multifinger proteins with improved in vitro and in vivo function. Our results demonstrate that both DNA binding affinity and specificity are important for cellular function and also provide a general approach for optimizing multidomain proteins.
Asunto(s)
Dedos de Zinc/genética , Secuencia de Aminoácidos , Secuencia de Bases , Línea Celular , ADN Recombinante/genética , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Proteínas de Fusión bcr-abl/química , Proteínas de Fusión bcr-abl/genética , Proteínas de Fusión bcr-abl/metabolismo , VIH/genética , Humanos , Técnicas In Vitro , Datos de Secuencia Molecular , Biblioteca de Péptidos , Ingeniería de Proteínas , Estructura Terciaria de Proteína , Receptor ErbB-2/química , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Selección Genética , Técnicas del Sistema de Dos HíbridosRESUMEN
Zinc-finger protein transcription factors (ZFP TFs) can be designed to control the expression of any desired target gene, and thus provide potential therapeutic tools for the study and treatment of disease. Here we report that a ZFP TF can repress target gene expression with single-gene specificity within the human genome. A ZFP TF repressor that binds an 18-bp recognition sequence within the promoter of the endogenous CHK2 gene gives a >10-fold reduction in CHK2 mRNA and protein. This level of repression was sufficient to generate a functional phenotype, as demonstrated by the loss of DNA damage-induced CHK2-dependent p53 phosphorylation. We determined the specificity of repression by using DNA microarrays and found that the ZFP TF repressed a single gene (CHK2) within the monitored genome in two different cell types. These data demonstrate the utility of ZFP TFs as precise tools for target validation, and highlight their potential as clinical therapeutics.
Asunto(s)
Regulación de la Expresión Génica , Proteínas Quinasas/genética , Proteínas Serina-Treonina Quinasas , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Dedos de Zinc/genética , Secuencia de Bases , Sitios de Unión/genética , Línea Celular , Quinasa de Punto de Control 2 , ADN/genética , ADN/metabolismo , Daño del ADN , Regulación Enzimológica de la Expresión Génica , Genoma Humano , Humanos , Regiones Promotoras Genéticas , Ingeniería de Proteínas , Proteínas Quinasas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/metabolismoRESUMEN
Zinc-finger proteins offer a versatile and effective framework for the recognition of DNA binding sites. By connecting multiple fingers together with canonical TGEKP linkers, a protein may be designed to recognize almost any desired target DNA sequence. However, proteins containing more than three zinc-fingers do not bind as tightly as one might predict, and it appears that some type of strain is introduced when a six-finger protein is constructed with canonical linkers. In an attempt to understand the sources of this strain, we have solved the 2.2A resolution X-ray crystallographic structure of a complex that has two copies of the three-finger Zif268 protein bound to adjacent sites on one duplex DNA. Conceptually, this is equivalent to a six-finger protein in which the central linker has been removed and the complex has been allowed to "relax" to its most stable conformation. As in other Zif268-DNA complexes, the DNA is approximately linear and is slightly underwound. Surprisingly, the structure of the complex is similar (within 0.5A) to an arrangement that would allow a canonical linker at the center of the complex, and it seems possible that entropic effects (involving the librational degrees of freedom in the complex) could be important in determining optimal linker length.
Asunto(s)
Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , ADN/metabolismo , Factores de Transcripción/química , Factores de Transcripción/metabolismo , Dedos de Zinc , Secuencia de Aminoácidos , Sitios de Unión , Cristalografía por Rayos X , ADN/química , Proteínas de Unión al ADN/genética , Sustancias Macromoleculares , Modelos Moleculares , Datos de Secuencia Molecular , Ácidos Nucleicos Heterodúplex/química , Ácidos Nucleicos Heterodúplex/metabolismo , Conformación Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Factores de Transcripción/genéticaRESUMEN
Zinc-finger proteins (ZFPs) that recognize novel DNA sequences are the basis of a powerful technology platform with many uses in drug discovery and therapeutics. These proteins have been used as the DNA-binding domains of novel transcription factors (ZFP TFs), which are useful for validating genes as drug targets and for engineering cell lines for small-molecule screening and protein production. Recently, they have also been used as a basis for novel human therapeutics. Most of our advances in the design and application of these ZFP TFs rely on our ability to engineer ZFPs that bind short stretches of DNA (typically 9-18 base pairs) located within the promoters of target genes. Here, we summarize the methods used to design these DNA-binding domains, explain how they are incorporated into novel transcription factors (and other useful molecules) and describe some key applications in drug discovery.
Asunto(s)
Diseño de Fármacos , Ingeniería de Proteínas , Factores de Transcripción/metabolismo , Dedos de Zinc , Secuencia de Bases , Sitios de Unión , ADN/química , ADN/metabolismo , Regulación de la Expresión Génica , HumanosRESUMEN
Covalent modifications of chromatin have emerged as key determinants of the genome's transcriptional competence. Histone H3 lysine 9 (H3K9) methylation is an epigenetic signal that is recognized by HP1 and correlates with gene silencing in a variety of organisms. Discovery of the enzymes that catalyze H3K9 methylation has identified a second gene-specific function for this modification in transcriptional repression. Whether H3K9 methylation is causative in the initiation and establishment of gene repression or is a byproduct of the process leading to the repressed state remains unknown. To investigate the role of HMTs and specifically H3K9 methylation in gene repression, we have employed engineered zinc-finger transcription factors (ZFPs) to target HMT activity to a specific endogenous gene. By utilizing ZFPs that recognize the promoter of the endogenous VEGF-A gene, and thus employing this chromosomal locus as an in vivo reporter, we show that ZFPs linked to a minimal catalytic HMT domain affect local methylation of histone H3K9 and the consequent repression of target gene expression. Furthermore, amino acid substitutions within the HMT that ablate its catalytic activity effectively eliminate the ability of the ZFP fusions to repress transcription. Thus, H3K9 methylation is a primary signal that is sufficient for initiating a gene repression pathway in vivo.