RESUMEN
Enhancers control the location and timing of gene expression and contain the majority of variants associated with disease1-3. The ZRS is arguably the most well-studied vertebrate enhancer and mediates the expression of Shh in the developing limb4. Thirty-one human single-nucleotide variants (SNVs) within the ZRS are associated with polydactyly4-6. However, how this enhancer encodes tissue-specific activity, and the mechanisms by which SNVs alter the number of digits, are poorly understood. Here we show that the ETS sites within the ZRS are low affinity, and identify a functional ETS site, ETS-A, with extremely low affinity. Two human SNVs and a synthetic variant optimize the binding affinity of ETS-A subtly from 15% to around 25% relative to the strongest ETS binding sequence, and cause polydactyly with the same penetrance and severity. A greater increase in affinity results in phenotypes that are more penetrant and more severe. Affinity-optimizing SNVs in other ETS sites in the ZRS, as well as in ETS, interferon regulatory factor (IRF), HOX and activator protein 1 (AP-1) sites within a wide variety of enhancers, cause gain-of-function gene expression. The prevalence of binding sites with suboptimal affinity in enhancers creates a vulnerability in genomes whereby SNVs that optimize affinity, even slightly, can be pathogenic. Searching for affinity-optimizing SNVs in genomes could provide a mechanistic approach to identify causal variants that underlie enhanceropathies.
Asunto(s)
Elementos de Facilitación Genéticos , Extremidades , Polidactilia , Proteínas Proto-Oncogénicas c-ets , Humanos , Elementos de Facilitación Genéticos/genética , Extremidades/embriología , Extremidades/patología , Mutación con Ganancia de Función , Proteínas de Homeodominio/metabolismo , Factores Reguladores del Interferón/metabolismo , Especificidad de Órganos/genética , Penetrancia , Fenotipo , Polidactilia/embriología , Polidactilia/genética , Polidactilia/patología , Polimorfismo de Nucleótido Simple , Unión Proteica , Proteínas Proto-Oncogénicas c-ets/metabolismo , Factor de Transcripción AP-1/metabolismoRESUMEN
Germ-line mutations in components of the Ras/MAPK pathway result in developmental disorders called RASopathies, affecting about 1/1,000 human births. Rapid advances in genome sequencing make it possible to identify multiple disease-related mutations, but there is currently no systematic framework for translating this information into patient-specific predictions of disease progression. As a first step toward addressing this issue, we developed a quantitative, inexpensive, and rapid framework that relies on the early zebrafish embryo to assess mutational effects on a common scale. Using this assay, we assessed 16 mutations reported in MEK1, a MAPK kinase, and provide a robust ranking of these mutations. We find that mutations found in cancer are more severe than those found in both RASopathies and cancer, which, in turn, are generally more severe than those found only in RASopathies. Moreover, this rank is conserved in other zebrafish embryonic assays and Drosophila-specific embryonic and adult assays, suggesting that our ranking reflects the intrinsic property of the mutant molecule. Furthermore, this rank is predictive of the drug dose needed to correct the defects. This assay can be readily used to test the strengths of existing and newly found mutations in MEK1 and other pathway components, providing the first step in the development of rational guidelines for patient-specific diagnostics and treatment of RASopathies.
Asunto(s)
Discapacidades del Desarrollo/genética , Proteínas ras/genética , Animales , Animales Modificados Genéticamente , Discapacidades del Desarrollo/tratamiento farmacológico , Discapacidades del Desarrollo/metabolismo , Relación Dosis-Respuesta a Droga , Proteínas de Drosophila/genética , Drosophila melanogaster/embriología , Drosophila melanogaster/genética , Humanos , MAP Quinasa Quinasa 1/antagonistas & inhibidores , MAP Quinasa Quinasa 1/genética , MAP Quinasa Quinasa 1/metabolismo , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/genética , Mutación , Fenotipo , Inhibidores de Proteínas Quinasas/administración & dosificación , Inhibidores de Proteínas Quinasas/farmacología , Pez Cebra/embriología , Pez Cebra/genética , Proteínas de Pez Cebra/antagonistas & inhibidores , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismoRESUMEN
The MEK1 kinase directly phosphorylates ERK2, after the activation loop of MEK1 is itself phosphorylated by Raf. Studies over the past decade have revealed a large number of disease-related mutations in the MEK1 gene that lead to tumorigenesis and abnormal development. Several of these mutations result in MEK1 constitutive activity, but how they affect MEK1 regulation and function remains largely unknown. Here, we address these questions focusing on two pathogenic variants of the Phe-53 residue, which maps to the well-characterized negative regulatory region of MEK1. We found that these variants are phosphorylated by Raf faster than the wild-type enzyme, and this phosphorylation further increases their enzymatic activity. However, the maximal activities of fully phosphorylated wild-type and mutant enzymes are indistinguishable. On the basis of available structural information, we propose that the activating substitutions destabilize the inactive conformation of MEK1, resulting in its constitutive activity and making it more prone to Raf-mediated phosphorylation. Experiments in zebrafish revealed that the effects of activating variants on embryonic development reflect the joint control of the negative regulatory region and activating phosphorylation. Our results underscore the complexity of the effects of activating mutations on signaling systems, even at the level of a single protein.
Asunto(s)
MAP Quinasa Quinasa 1/genética , MAP Quinasa Quinasa 1/metabolismo , Mutación Puntual , Animales , Cristalografía por Rayos X , Activación Enzimática , Humanos , MAP Quinasa Quinasa 1/química , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Modelos Moleculares , Neoplasias/genética , Neoplasias/metabolismo , Fosforilación , Conformación Proteica , Pez Cebra , Quinasas raf/metabolismoRESUMEN
Methods for cell-selective analysis of proteome dynamics will facilitate studies of biological processes in multicellular organisms. Here we describe a mutant murine methionyl-tRNA synthetase (designated L274GMmMetRS) that charges the noncanonical amino acid azidonorleucine (Anl) to elongator tRNA(Met) in hamster (CHO), monkey (COS7), and human (HeLa) cell lines. Proteins made in cells that express the synthetase can be labeled with Anl, tagged with dyes or affinity reagents, and enriched on affinity resin to facilitate identification by mass spectrometry. The method does not require expression of orthogonal tRNAs or depletion of canonical amino acids. Successful labeling of proteins with Anl in several mammalian cell lines demonstrates the utility of L274GMmMetRS as a tool for cell-selective analysis of mammalian protein synthesis.
Asunto(s)
Aminoacil-ARNt Sintetasas/metabolismo , Biosíntesis de Proteínas , Aminoácidos/análisis , Aminoácidos/química , Aminoacil-ARNt Sintetasas/genética , Animales , Cricetinae , Escherichia coli/metabolismo , Haplorrinos , Células HeLa , Humanos , Mamíferos , Metionina-ARNt Ligasa/metabolismo , Ratones , Proteoma/metabolismo , ARN de Transferencia de Metionina/metabolismoRESUMEN
Electroporation is a technique to introduce DNA constructs into cells using electric current. Here, we present a protocol to electroporate DNA plasmids into Ciona robusta embryos at the 1-cell stage. We describe steps for setting up and conducting electroporation. We then detail procedures for collecting, fixing, and mounting embryos and counting expression. This protocol can be used to study the expression of enhancers via reporter assays, manipulating cells using genes or modified genes such as dominant negatives, and genome editing. For complete details on the use and execution of this protocol, please refer to Song, et al.1.
RESUMEN
We describe a genetic AND gate for cell-targeted metabolic labeling and proteomic analysis in complex cellular systems. The centerpiece of the AND gate is a bisected methionyl-tRNA synthetase (MetRS) that charges the Met surrogate azidonorleucine (Anl) to tRNA(Met). Cellular protein labeling occurs only upon activation of two different promoters that drive expression of the N- and C-terminal fragments of the bisected MetRS. Anl-labeled proteins can be tagged with fluorescent dyes or affinity reagents via either copper-catalyzed or strain-promoted azide-alkyne cycloaddition. Protein labeling is apparent within 5 min after addition of Anl to bacterial cells in which the AND gate has been activated. This method allows spatial and temporal control of proteomic labeling and identification of proteins made in specific cellular subpopulations. The approach is demonstrated by selective labeling of proteins in bacterial cells immobilized in the center of a laminar-flow microfluidic channel, where they are exposed to overlapping, opposed gradients of inducers of the N- and C-terminal MetRS fragments. The observed labeling profile is predicted accurately from the strengths of the individual input signals.
Asunto(s)
Ingeniería Genética , Proteínas/metabolismo , Catálisis , Ciclización , Metionina-ARNt Ligasa/metabolismo , Proteínas/genéticaRESUMEN
The notochord is a defining feature of all chordates. The transcription factors Zic and ETS regulate enhancer activity within the notochord. We conduct high-throughput screens of genomic elements within developing Ciona embryos to understand how Zic and ETS sites encode notochord activity. Our screen discovers an enhancer located near Lama, a gene critical for notochord development. Reversing the orientation of an ETS site within this enhancer abolishes expression, indicating that enhancer grammar is critical for notochord activity. Similarly organized clusters of Zic and ETS sites occur within mouse and human Lama1 introns. Within a Brachyury (Bra) enhancer, FoxA and Bra, in combination with Zic and ETS binding sites, are necessary and sufficient for notochord expression. This binding site logic also occurs within other Ciona and vertebrate Bra enhancers. Collectively, this study uncovers the importance of grammar within notochord enhancers and discovers signatures of enhancer logic and grammar conserved across chordates.
Asunto(s)
Ciona intestinalis , Notocorda , Animales , Humanos , Ratones , Ciona intestinalis/genética , Secuencias Reguladoras de Ácidos Nucleicos , Proteínas de Dominio T Box/metabolismo , Factores de Transcripción/metabolismo , Regulación del Desarrollo de la Expresión Génica , Elementos de Facilitación Genéticos/genéticaRESUMEN
Transcriptional enhancers direct precise gene expression patterns during development and harbor the majority of variants associated with phenotypic diversity, evolutionary adaptations, and disease. Pinpointing which enhancer variants contribute to changes in gene expression and phenotypes is a major challenge. Here, we find that suboptimal or low-affinity binding sites are necessary for precise gene expression during heart development. Single-nucleotide variants (SNVs) can optimize the affinity of ETS binding sites, causing gain-of-function (GOF) gene expression, cell migration defects, and phenotypes as severe as extra beating hearts in the marine chordate Ciona robusta. In human induced pluripotent stem cell (iPSC)-derived cardiomyocytes, a SNV within a human GATA4 enhancer increases ETS binding affinity and causes GOF enhancer activity. The prevalence of suboptimal-affinity sites within enhancers creates a vulnerability whereby affinity-optimizing SNVs can lead to GOF gene expression, changes in cellular identity, and organismal-level phenotypes that could contribute to the evolution of novel traits or diseases.
Asunto(s)
Elementos de Facilitación Genéticos , Células Madre Pluripotentes Inducidas , Humanos , Elementos de Facilitación Genéticos/genética , Miocitos Cardíacos/metabolismo , Sitios de Unión , NucleótidosRESUMEN
Each language has standard books describing that language's grammatical rules. Biologists have searched for similar, albeit more complex, principles relating enhancer sequence to gene expression. Here, we review the literature on enhancer grammar. We introduce dependency grammar, a model where enhancers encode information based on dependencies between enhancer features shaped by mechanistic, evolutionary, and biological constraints. Classifying enhancers based on the types of dependencies may identify unifying principles relating enhancer sequence to gene expression. Such rules would allow us to read the instructions for development within genomes and pinpoint causal enhancer variants underlying disease and evolutionary changes.
Asunto(s)
Enfermedad/genética , Elementos de Facilitación Genéticos , Evolución Molecular , Regulación del Desarrollo de la Expresión Génica , HumanosRESUMEN
Terminal regions of Drosophila embryos are patterned by signaling through ERK, which is genetically deregulated in multiple human diseases. Quantitative studies of terminal patterning have been recently used to investigate gain-of-function variants of human MEK1, encoding the MEK kinase that directly activates ERK by dual phosphorylation. Unexpectedly, several mutations reduced ERK activation by extracellular signals, possibly through a negative feedback triggered by signal-independent activity of the mutant variants. Here we present experimental evidence supporting this model. Using a MEK variant that combines a mutation within the negative regulatory region with alanine substitutions in the activation loop, we prove that pathogenic variants indeed acquire signal-independent kinase activity. We also demonstrate that signal-dependent activation of these variants is independent of kinase suppressor of Ras, a conserved adaptor that is indispensable for activation of normal MEK. Finally, we show that attenuation of ERK activation by extracellular signals stems from transcriptional induction of Mkp3, a dual specificity phosphatase that deactivates ERK by dephosphorylation. These findings in the Drosophila embryo highlight its power for investigating diverse effects of human disease mutations.
Asunto(s)
MAP Quinasa Quinasa 1/genética , Animales , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Fosfatasas de Especificidad Dual , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Humanos , MAP Quinasa Quinasa 1/metabolismo , Sistema de Señalización de MAP Quinasas/genética , Sistema de Señalización de MAP Quinasas/fisiología , Mutación , Fosforilación/efectos de los fármacos , Transducción de SeñalRESUMEN
Vertebrate embryonic patterning depends on signaling from Nodal, a TGFß superfamily member. There are three Nodal orthologs in zebrafish; southpaw directs left-right asymmetries, while squint and cyclops function earlier to pattern mesendoderm. TGFß member Vg1 is implicated in mesoderm formation but the role of the zebrafish ortholog, Growth differentiation factor 3 (Gdf3), has not been fully explored. We show that zygotic expression of gdf3 is dispensable for embryonic development, while maternally deposited gdf3 is required for mesendoderm formation and dorsal-ventral patterning. We further show that Gdf3 can affect left-right patterning at multiple stages, including proper development of regional cell morphology in Kupffer's vesicle and the establishment of southpaw expression in the lateral plate mesoderm. Collectively, our data indicate that gdf3 is critical for robust Nodal signaling at multiple stages in zebrafish embryonic development.
Asunto(s)
Tipificación del Cuerpo , Estratos Germinativos/embriología , Proteína Nodal/metabolismo , Transducción de Señal , Factor de Crecimiento Transformador beta/metabolismo , Proteínas de Pez Cebra/metabolismo , Pez Cebra/embriología , AnimalesRESUMEN
Germline mutations in Ras pathway components are associated with a large class of human developmental abnormalities, known as RASopathies, that are characterized by a range of structural and functional phenotypes, including cardiac defects and neurocognitive delays. Although it is generally believed that RASopathies are caused by altered levels of pathway activation, the signaling changes in developing tissues remain largely unknown. We used assays with spatiotemporal resolution in Drosophila melanogaster (fruit fly) and Danio rerio (zebrafish) to quantify signaling changes caused by mutations in MAP2K1 (encoding MEK), a core component of the Ras pathway that is mutated in both RASopathies and cancers in humans. Surprisingly, we discovered that intrinsically active MEK variants can both increase and reduce the levels of pathway activation in vivo. The sign of the effect depends on cellular context, implying that some of the emerging phenotypes in RASopathies may be caused by increased, as well as attenuated, levels of Ras signaling.
Asunto(s)
Mutación de Línea Germinal/genética , Proteínas Quinasas Activadas por Mitógenos/genética , Transducción de Señal/genética , Proteínas ras/genética , Animales , Drosophila melanogaster/genética , Cardiopatías/genética , Humanos , Neoplasias/genética , Trastornos Neurocognitivos/genética , Fenotipo , Pez Cebra/genéticaRESUMEN
RASopathies are developmental disorders caused by germline mutations in the Ras-MAPK pathway, and are characterized by a broad spectrum of functional and morphological abnormalities. The high incidence of these disorders (â¼1/1000 births) motivates the development of systematic approaches for their efficient diagnosis and potential treatment. Recent advances in genome sequencing have greatly facilitated the genotyping and discovery of mutations in affected individuals, but establishing the causal relationships between molecules and disease phenotypes is non-trivial and presents both technical and conceptual challenges. Here, we discuss how these challenges could be addressed using genetically modified model organisms that have been instrumental in delineating the Ras-MAPK pathway and its roles during development. Focusing on studies in mice, zebrafish and Drosophila, we provide an up-to-date review of animal models of RASopathies at the molecular and functional level. We also discuss how increasingly sophisticated techniques of genetic engineering can be used to rigorously connect changes in specific components of the Ras-MAPK pathway with observed functional and morphological phenotypes. Establishing these connections is essential for advancing our understanding of RASopathies and for devising rational strategies for their management and treatment.