RESUMO
3' untranslated region (3'UTR) variants are strongly associated with human traits and diseases, yet few have been causally identified. We developed the massively parallel reporter assay for 3'UTRs (MPRAu) to sensitively assay 12,173 3'UTR variants. We applied MPRAu to six human cell lines, focusing on genetic variants associated with genome-wide association studies (GWAS) and human evolutionary adaptation. MPRAu expands our understanding of 3'UTR function, suggesting that simple sequences predominately explain 3'UTR regulatory activity. We adapt MPRAu to uncover diverse molecular mechanisms at base pair resolution, including an adenylate-uridylate (AU)-rich element of LEPR linked to potential metabolic evolutionary adaptations in East Asians. We nominate hundreds of 3'UTR causal variants with genetically fine-mapped phenotype associations. Using endogenous allelic replacements, we characterize one variant that disrupts a miRNA site regulating the viral defense gene TRIM14 and one that alters PILRB abundance, nominating a causal variant underlying transcriptional changes in age-related macular degeneration.
Assuntos
Regiões 3' não Traduzidas/genética , Evolução Biológica , Doença/genética , Estudo de Associação Genômica Ampla , Algoritmos , Alelos , Regulação da Expressão Gênica , Genes Reporter , Variação Genética , Humanos , Fenótipo , Polimorfismo de Nucleotídeo Único/genética , Polirribossomos/metabolismo , Locos de Características Quantitativas/genética , RNA/genéticaRESUMO
We have produced gene expression profiles of all 302 neurons of the C. elegans nervous system that match the single-cell resolution of its anatomy and wiring diagram. Our results suggest that individual neuron classes can be solely identified by combinatorial expression of specific gene families. For example, each neuron class expresses distinct codes of â¼23 neuropeptide genes and â¼36 neuropeptide receptors, delineating a complex and expansive "wireless" signaling network. To demonstrate the utility of this comprehensive gene expression catalog, we used computational approaches to (1) identify cis-regulatory elements for neuron-specific gene expression and (2) reveal adhesion proteins with potential roles in process placement and synaptic specificity. Our expression data are available at https://cengen.org and can be interrogated at the web application CengenApp. We expect that this neuron-specific directory of gene expression will spur investigations of underlying mechanisms that define anatomy, connectivity, and function throughout the C. elegans nervous system.
Assuntos
Caenorhabditis elegans/metabolismo , Sistema Nervoso/metabolismo , Animais , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Corantes Fluorescentes/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Genes Reporter , Larva/metabolismo , Neurônios/metabolismo , Neuropeptídeos/genética , Neuropeptídeos/metabolismo , Motivos de Nucleotídeos/genética , RNA-Seq , Sequências Reguladoras de Ácido Nucleico/genética , Transdução de Sinais/genética , Fatores de Transcrição/metabolismo , Transcrição GênicaRESUMO
In vivo cell fate conversions have emerged as potential regeneration-based therapeutics for injury and disease. Recent studies reported that ectopic expression or knockdown of certain factors can convert resident astrocytes into functional neurons with high efficiency, region specificity, and precise connectivity. However, using stringent lineage tracing in the mouse brain, we show that the presumed astrocyte-converted neurons are actually endogenous neurons. AAV-mediated co-expression of NEUROD1 and a reporter specifically and efficiently induces reporter-labeled neurons. However, these neurons cannot be traced retrospectively to quiescent or reactive astrocytes using lineage-mapping strategies. Instead, through a retrograde labeling approach, our results reveal that endogenous neurons are the source for these viral-reporter-labeled neurons. Similarly, despite efficient knockdown of PTBP1 in vivo, genetically traced resident astrocytes were not converted into neurons. Together, our results highlight the requirement of lineage-tracing strategies, which should be broadly applied to studies of cell fate conversions in vivo.
Assuntos
Astrócitos/citologia , Diferenciação Celular , Linhagem da Célula , Neurônios/citologia , Animais , Astrócitos/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Encéfalo/patologia , Lesões Encefálicas/patologia , Linhagem Celular Tumoral , Reprogramação Celular , Dependovirus/metabolismo , Regulação para Baixo , Regulação da Expressão Gênica , Genes Reporter , Proteína Glial Fibrilar Ácida/genética , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Proteínas de Homeodomínio/metabolismo , Humanos , Integrases/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neurônios/metabolismo , Proteína de Ligação a Regiões Ricas em Polipirimidinas/metabolismo , Regiões Promotoras Genéticas/genética , Fatores de Transcrição/metabolismoRESUMO
Changes in appendage structure underlie key transitions in vertebrate evolution. Addition of skeletal elements along the proximal-distal axis facilitated critical transformations, including the fin-to-limb transition that permitted generation of diverse modes of locomotion. Here, we identify zebrafish mutants that form supernumerary long bones in their pectoral fins. These new bones integrate into musculature, form joints, and articulate with neighboring elements. This phenotype is caused by activating mutations in previously unrecognized regulators of appendage patterning, vav2 and waslb, that function in a common pathway. This pathway is required for appendage development across vertebrates, and loss of Wasl in mice causes defects similar to those seen in murine Hox mutants. Concordantly, formation of supernumerary bones requires Hox11 function, and mutations in the vav2/wasl pathway drive enhanced expression of hoxa11b, indicating developmental homology with the forearm. Our findings reveal a latent, limb-like pattern ability in fins that is activated by simple genetic perturbation.
Assuntos
Osso e Ossos/embriologia , Extremidades/embriologia , Peixe-Zebra/embriologia , Actinas/metabolismo , Nadadeiras de Animais/embriologia , Animais , Sequência de Bases , Padronização Corporal , Sistemas CRISPR-Cas/genética , Linhagem da Célula , Epistasia Genética , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Inativação de Genes , Genes Reporter , Células HeLa , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Camundongos , Mutação/genética , Fenótipo , Filogenia , Transdução de Sinais/genética , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
The binding affinity and kinetics of target engagement are fundamental to establishing structure-activity relationships (SARs) for prospective therapeutic agents. Enhancing these binding parameters for operative targets, while minimizing binding to off-target sites, can translate to improved drug efficacy and a widened therapeutic window. Compound activity is typically assessed through modulation of an observed phenotype in cultured cells. Quantifying the corresponding binding properties under common cellular conditions can provide more meaningful interpretation of the cellular SAR analysis. Consequently, methods for assessing drug binding in living cells have advanced and are now integral to medicinal chemistry workflows. In this review, we survey key technological advancements that support quantitative assessments of target occupancy in cultured cells, emphasizing generalizable methodologies able to deliver analytical precision that heretofore required reductionist biochemical approaches.
Assuntos
Química Farmacêutica/métodos , Corantes Fluorescentes/química , Ensaios de Triagem em Larga Escala , Técnicas de Sonda Molecular , Terapia de Alvo Molecular/métodos , Técnicas de Transferência de Energia por Ressonância de Bioluminescência , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Genes Reporter , Humanos , Cinética , Imagem Óptica/métodos , Bibliotecas de Moléculas Pequenas/síntese química , Bibliotecas de Moléculas Pequenas/farmacologia , Relação Estrutura-AtividadeRESUMO
Non-genetic factors can cause individual cells to fluctuate substantially in gene expression levels over time. It remains unclear whether these fluctuations can persist for much longer than the time of one cell division. Current methods for measuring gene expression in single cells mostly rely on single time point measurements, making the duration of gene expression fluctuations or cellular memory difficult to measure. Here, we combined Luria and Delbrück's fluctuation analysis with population-based RNA sequencing (MemorySeq) for identifying genes transcriptome-wide whose fluctuations persist for several divisions. MemorySeq revealed multiple gene modules that expressed together in rare cells within otherwise homogeneous clonal populations. These rare cell subpopulations were associated with biologically distinct behaviors like proliferation in the face of anti-cancer therapeutics. The identification of non-genetic, multigenerational fluctuations can reveal new forms of biological memory in single cells and suggests that non-genetic heritability of cellular state may be a quantitative property.
Assuntos
Análise de Célula Única/métodos , Transcriptoma , Divisão Celular , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/genética , Genes Reporter , Humanos , Hibridização in Situ Fluorescente , Microscopia de Fluorescência , Análise de Sequência de RNA , Imagem com Lapso de TempoRESUMO
In order to analyze how a signal transduction network converts cellular inputs into cellular outputs, ideally one would measure the dynamics of many signals within the network simultaneously. We found that, by fusing a fluorescent reporter to a pair of self-assembling peptides, it could be stably clustered within cells at random points, distant enough to be resolved by a microscope but close enough to spatially sample the relevant biology. Because such clusters, which we call signaling reporter islands (SiRIs), can be modularly designed, they permit a set of fluorescent reporters to be efficiently adapted for simultaneous measurement of multiple nodes of a signal transduction network within single cells. We created SiRIs for indicators of second messengers and kinases and used them, in hippocampal neurons in culture and intact brain slices, to discover relationships between the speed of calcium signaling, and the amplitude of PKA signaling, upon receiving a cAMP-driving stimulus.
Assuntos
Corantes Fluorescentes/metabolismo , Genes Reporter , Imagem Óptica , Transdução de Sinais , Animais , Cálcio/metabolismo , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Feminino , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Hipocampo/metabolismo , Humanos , Camundongos , Neurônios/metabolismo , Peptídeos/metabolismo , Proteínas/metabolismo , Células Piramidais/metabolismoRESUMO
Thousands of proteins localize to the nucleus; however, it remains unclear which contain transcriptional effectors. Here, we develop HT-recruit, a pooled assay where protein libraries are recruited to a reporter, and their transcriptional effects are measured by sequencing. Using this approach, we measure gene silencing and activation for thousands of domains. We find a relationship between repressor function and evolutionary age for the KRAB domains, discover that Homeodomain repressor strength is collinear with Hox genetic organization, and identify activities for several domains of unknown function. Deep mutational scanning of the CRISPRi KRAB maps the co-repressor binding surface and identifies substitutions that improve stability/silencing. By tiling 238 proteins, we find repressors as short as ten amino acids. Finally, we report new activator domains, including a divergent KRAB. These results provide a resource of 600 human proteins containing effectors and demonstrate a scalable strategy for assigning functions to protein domains.
Assuntos
Ensaios de Triagem em Larga Escala , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Sistemas CRISPR-Cas/genética , Feminino , Inativação Gênica , Genes Reporter , Células HEK293 , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Células K562 , Lentivirus/fisiologia , Anotação de Sequência Molecular , Mutação/genética , Proteínas Nucleares/metabolismo , Regiões Promotoras Genéticas/genética , Domínios Proteicos , Proteínas Repressoras/química , Proteínas Repressoras/metabolismo , Reprodutibilidade dos Testes , Transcrição Gênica , Dedos de ZincoRESUMO
Transforming the vast knowledge from genetics, biochemistry, and structural biology into detailed molecular descriptions of biological processes inside cells remains a major challenge-one in sore need of better imaging technologies. For example, transcription involves the complex interplay between RNA polymerase II (Pol II), regulatory factors (RFs), and chromatin, but visualizing these dynamic molecular transactions in their native intracellular milieu remains elusive. Here, we zoom into single tagged genes using nanoscopy techniques, including an active target-locking, ultra-sensitive system that enables single-molecule detection in addressable sub-diffraction volumes, within crowded intracellular environments. We image, track, and quantify Pol II with single-molecule resolution, unveiling its dynamics during the transcription cycle. Further probing multiple functionally linked events-RF-chromatin interactions, Pol II dynamics, and nascent transcription kinetics-reveals detailed operational parameters of gene-regulatory mechanisms hitherto-unseen in vivo. Our approach sets the stage for single-molecule studies of complex molecular processes in live cells.
Assuntos
Nanotecnologia , RNA Polimerase II/metabolismo , Imagem Individual de Molécula/métodos , Transcrição Gênica , Linhagem Celular Tumoral , Cromatina/metabolismo , Genes Reporter , Vetores Genéticos/genética , Vetores Genéticos/metabolismo , Humanos , Cinética , Mutagênese , RNA Polimerase II/genética , Anticorpos de Domínio Único/química , Anticorpos de Domínio Único/imunologia , Imagem com Lapso de Tempo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
mRNA translation is a key step in decoding genetic information. Genetic decoding is surprisingly heterogeneous because multiple distinct polypeptides can be synthesized from a single mRNA sequence. To study translational heterogeneity, we developed the MoonTag, a fluorescence labeling system to visualize translation of single mRNAs. When combined with the orthogonal SunTag system, the MoonTag enables dual readouts of translation, greatly expanding the possibilities to interrogate complex translational heterogeneity. By placing MoonTag and SunTag sequences in different translation reading frames, each driven by distinct translation start sites, start site selection of individual ribosomes can be visualized in real time. We find that start site selection is largely stochastic but that the probability of using a particular start site differs among mRNA molecules and can be dynamically regulated over time. This study provides key insights into translation start site selection heterogeneity and provides a powerful toolbox to visualize complex translation dynamics.
Assuntos
Corantes Fluorescentes/química , RNA Mensageiro/metabolismo , Imagem Individual de Molécula/métodos , Regiões 3' não Traduzidas , Regiões 5' não Traduzidas , Linhagem Celular Tumoral , Genes Reporter , Células HEK293 , Humanos , Iniciação Traducional da Cadeia Peptídica , RNA Mensageiro/química , Ribossomos/metabolismo , Anticorpos de Domínio Único/química , Anticorpos de Domínio Único/imunologiaRESUMO
How signaling dynamics encode information is a central question in biology. During vertebrate development, dynamic Notch signaling oscillations control segmentation of the presomitic mesoderm (PSM). In mouse embryos, this molecular clock comprises signaling oscillations of several pathways, i.e., Notch, Wnt, and FGF signaling. Here, we directly address the role of the relative timing between Wnt and Notch signaling oscillations during PSM patterning. To this end, we developed a new experimental strategy using microfluidics-based entrainment that enables specific control of the rhythm of segmentation clock oscillations. Using this approach, we find that Wnt and Notch signaling are coupled at the level of their oscillation dynamics. Furthermore, we provide functional evidence that the oscillation phase shift between Wnt and Notch signaling is critical for PSM segmentation. Our work hence reveals that dynamic signaling, i.e., the relative timing between oscillatory signals, encodes essential information during multicellular development.
Assuntos
Padronização Corporal , Mesoderma/embriologia , Receptores Notch/metabolismo , Transdução de Sinais , Proteínas Wnt/metabolismo , Animais , Genes Reporter , Mesoderma/metabolismo , Camundongos , Microfluídica , Somitos/embriologia , Somitos/metabolismoRESUMO
Modern genetic approaches are powerful in providing access to diverse cell types in the brain and facilitating the study of their function. Here, we report a large set of driver and reporter transgenic mouse lines, including 23 new driver lines targeting a variety of cortical and subcortical cell populations and 26 new reporter lines expressing an array of molecular tools. In particular, we describe the TIGRE2.0 transgenic platform and introduce Cre-dependent reporter lines that enable optical physiology, optogenetics, and sparse labeling of genetically defined cell populations. TIGRE2.0 reporters broke the barrier in transgene expression level of single-copy targeted-insertion transgenesis in a wide range of neuronal types, along with additional advantage of a simplified breeding strategy compared to our first-generation TIGRE lines. These novel transgenic lines greatly expand the repertoire of high-precision genetic tools available to effectively identify, monitor, and manipulate distinct cell types in the mouse brain.
Assuntos
Encéfalo/metabolismo , Técnicas de Inativação de Genes/métodos , Genes Reporter , Animais , Encéfalo/citologia , Cálcio/metabolismo , Linhagem Celular , Hibridização in Situ Fluorescente , Luz , Camundongos , Camundongos Transgênicos , Microscopia de Fluorescência , Neurônios/metabolismo , Optogenética , RNA não Traduzido/genética , Transgenes/genéticaRESUMO
Genetic changes causing brain size expansion in human evolution have remained elusive. Notch signaling is essential for radial glia stem cell proliferation and is a determinant of neuronal number in the mammalian cortex. We find that three paralogs of human-specific NOTCH2NL are highly expressed in radial glia. Functional analysis reveals that different alleles of NOTCH2NL have varying potencies to enhance Notch signaling by interacting directly with NOTCH receptors. Consistent with a role in Notch signaling, NOTCH2NL ectopic expression delays differentiation of neuronal progenitors, while deletion accelerates differentiation into cortical neurons. Furthermore, NOTCH2NL genes provide the breakpoints in 1q21.1 distal deletion/duplication syndrome, where duplications are associated with macrocephaly and autism and deletions with microcephaly and schizophrenia. Thus, the emergence of human-specific NOTCH2NL genes may have contributed to the rapid evolution of the larger human neocortex, accompanied by loss of genomic stability at the 1q21.1 locus and resulting recurrent neurodevelopmental disorders.
Assuntos
Encéfalo/embriologia , Córtex Cerebral/fisiologia , Neurogênese/fisiologia , Receptor Notch2/metabolismo , Transdução de Sinais , Animais , Diferenciação Celular , Células-Tronco Embrionárias/metabolismo , Feminino , Deleção de Genes , Genes Reporter , Gorilla gorilla , Células HEK293 , Humanos , Neocórtex/citologia , Células-Tronco Neurais/metabolismo , Neuroglia/metabolismo , Neurônios/metabolismo , Pan troglodytes , Receptor Notch2/genética , Análise de Sequência de RNARESUMO
The brain's synaptic networks endow an animal with powerfully adaptive biological behavior. Maps of such synaptic circuits densely reconstructed in those model brains that can be examined and manipulated by genetic means offer the best prospect for understanding the underlying biological bases of behavior. That prospect is now technologically feasible and a scientifically enabling possibility in neurobiology, much as genomics has been in molecular biology and genetics. In Drosophila, two major advances are in electron microscopic technology, using focused ion beam-scanning electron microscopy (FIB-SEM) milling to capture and align digital images, and in computer-aided reconstruction of neuron morphologies. The last decade has witnessed enormous progress in detailed knowledge of the actual synaptic circuits formed by real neurons. Advances in various brain regions that heralded identification of the motion-sensing circuits in the optic lobe are now extending to other brain regions, with the prospect of encompassing the fly's entire nervous system, both brain and ventral nerve cord.
Assuntos
Drosophila/fisiologia , Neurônios/citologia , Animais , Comportamento Animal/fisiologia , Encéfalo/citologia , Encéfalo/fisiologia , Biologia Computacional , Drosophila/citologia , Drosophila/genética , Expressão Gênica , Genes Reporter , Microscopia Eletrônica de Varredura/métodos , Microscopia de Fluorescência , Neuroanatomia , Neurônios/metabolismo , Neurônios/ultraestrutura , Sinapses/fisiologia , Sinapses/ultraestruturaRESUMO
Microglia and central nervous system (CNS)-associated macrophages (CAMs), such as perivascular and meningeal macrophages, are implicated in virtually all diseases of the CNS. However, little is known about their cell-type-specific roles in the absence of suitable tools that would allow for functional discrimination between the ontogenetically closely related microglia and CAMs. To develop a new microglia gene targeting model, we first applied massively parallel single-cell analyses to compare microglia and CAM signatures during homeostasis and disease and identified hexosaminidase subunit beta (Hexb) as a stably expressed microglia core gene, whereas other microglia core genes were substantially downregulated during pathologies. Next, we generated HexbtdTomato mice to stably monitor microglia behavior in vivo. Finally, the Hexb locus was employed for tamoxifen-inducible Cre-mediated gene manipulation in microglia and for fate mapping of microglia but not CAMs. In sum, we provide valuable new genetic tools to specifically study microglia functions in the CNS.
Assuntos
Encéfalo/patologia , Encefalomielite Autoimune Experimental/patologia , Traumatismos do Nervo Facial/patologia , Microglia/metabolismo , Cadeia beta da beta-Hexosaminidase/metabolismo , Animais , Encéfalo/citologia , Encéfalo/imunologia , Sistemas CRISPR-Cas/genética , Encefalomielite Autoimune Experimental/imunologia , Traumatismos do Nervo Facial/imunologia , Técnicas de Introdução de Genes , Genes Reporter/genética , Loci Gênicos/genética , Humanos , Microscopia Intravital , Substâncias Luminescentes/química , Proteínas Luminescentes/química , Proteínas Luminescentes/genética , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Microglia/imunologia , Células NIH 3T3 , RNA-Seq , Análise de Célula Única , Transfecção , Cadeia beta da beta-Hexosaminidase/genética , Proteína Vermelha FluorescenteRESUMO
Multiple genome-wide studies have identified associations between outcome of human immunodeficiency virus (HIV) infection and polymorphisms in and around the gene encoding the HIV co-receptor CCR5, but the functional basis for the strongest of these associations, rs1015164A/G, is unknown. We found that rs1015164 marks variation in an activating transcription factor 1 binding site that controls expression of the antisense long noncoding RNA (lncRNA) CCR5AS. Knockdown or enhancement of CCR5AS expression resulted in a corresponding change in CCR5 expression on CD4+ T cells. CCR5AS interfered with interactions between the RNA-binding protein Raly and the CCR5 3' untranslated region, protecting CCR5 messenger RNA from Raly-mediated degradation. Reduction in CCR5 expression through inhibition of CCR5AS diminished infection of CD4+ T cells with CCR5-tropic HIV in vitro. These data represent a rare determination of the functional importance of a genome-wide disease association where expression of a lncRNA affects HIV infection and disease progression.
Assuntos
Regulação da Expressão Gênica , Variação Genética , Infecções por HIV/genética , Infecções por HIV/virologia , HIV-1 , RNA Antissenso/genética , RNA Longo não Codificante/genética , Receptores CCR5/genética , Regiões 3' não Traduzidas , Alelos , Biomarcadores , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD4-Positivos/virologia , Membrana Celular/metabolismo , Genes Reporter , Genótipo , Infecções por HIV/metabolismo , Humanos , Desequilíbrio de Ligação , Polimorfismo de Nucleotídeo Único , Grupos Populacionais/genética , Prognóstico , Estabilidade de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores CCR5/metabolismo , Carga ViralRESUMO
Genome-wide association studies (GWAS) have successfully identified thousands of associations between common genetic variants and human disease phenotypes, but the majority of these variants are non-coding, often requiring genetic fine-mapping, epigenomic profiling, and individual reporter assays to delineate potential causal variants. We employ a massively parallel reporter assay (MPRA) to simultaneously screen 2,756 variants in strong linkage disequilibrium with 75 sentinel variants associated with red blood cell traits. We show that this assay identifies elements with endogenous erythroid regulatory activity. Across 23 sentinel variants, we conservatively identified 32 MPRA functional variants (MFVs). We used targeted genome editing to demonstrate endogenous enhancer activity across 3 MFVs that predominantly affect the transcription of SMIM1, RBM38, and CD164. Functional follow-up of RBM38 delineates a key role for this gene in the alternative splicing program occurring during terminal erythropoiesis. Finally, we provide evidence for how common GWAS-nominated variants can disrupt cell-type-specific transcriptional regulatory pathways.
Assuntos
Eritrócitos , Técnicas Genéticas , Variação Genética , Processamento Alternativo , Linhagem Celular , Linhagem da Célula/genética , Eritropoese/genética , Biblioteca Gênica , Genes Reporter , Humanos , Sequências Reguladoras de Ácido Nucleico , Transcrição GênicaRESUMO
Although studies have identified hundreds of loci associated with human traits and diseases, pinpointing causal alleles remains difficult, particularly for non-coding variants. To address this challenge, we adapted the massively parallel reporter assay (MPRA) to identify variants that directly modulate gene expression. We applied it to 32,373 variants from 3,642 cis-expression quantitative trait loci and control regions. Detection by MPRA was strongly correlated with measures of regulatory function. We demonstrate MPRA's capabilities for pinpointing causal alleles, using it to identify 842 variants showing differential expression between alleles, including 53 well-annotated variants associated with diseases and traits. We investigated one in detail, a risk allele for ankylosing spondylitis, and provide direct evidence of a non-coding variant that alters expression of the prostaglandin EP4 receptor. These results create a resource of concrete leads and illustrate the promise of this approach for comprehensively interrogating how non-coding polymorphism shapes human biology.
Assuntos
Regulação da Expressão Gênica , Genes Reporter , Doenças Genéticas Inatas/genética , Técnicas Genéticas , Variação Genética , Alelos , Biblioteca Gênica , Células Hep G2 , Humanos , Locos de Características Quantitativas , Sensibilidade e Especificidade , Espondilite Anquilosante/genéticaRESUMO
Transcription is episodic, consisting of a series of discontinuous bursts. Using live-imaging methods and quantitative analysis, we examine transcriptional bursting in living Drosophila embryos. Different developmental enhancers positioned downstream of synthetic reporter genes produce transcriptional bursts with similar amplitudes and duration but generate very different bursting frequencies, with strong enhancers producing more bursts than weak enhancers. Insertion of an insulator reduces the number of bursts and the corresponding level of gene expression, suggesting that enhancer regulation of bursting frequency is a key parameter of gene control in development. We also show that linked reporter genes exhibit coordinated bursting profiles when regulated by a shared enhancer, challenging conventional models of enhancer-promoter looping.
Assuntos
Cromossomos/metabolismo , Drosophila melanogaster/metabolismo , Elementos Facilitadores Genéticos , Transcrição Gênica , Ativação Transcricional , Animais , Drosophila melanogaster/genética , Embrião não Mamífero/metabolismo , Feminino , Regulação da Expressão Gênica , Genes Reporter , Proteínas de Fluorescência Verde/análise , Proteínas de Fluorescência Verde/genética , Elementos Isolantes , Masculino , Regiões Promotoras GenéticasRESUMO
Regulation of mRNA translation, the process by which ribosomes decode mRNAs into polypeptides, is used to tune cellular protein levels. Currently, methods for observing the complete process of translation from single mRNAs in vivo are unavailable. Here, we report the long-term (>1 hr) imaging of single mRNAs undergoing hundreds of rounds of translation in live cells, enabling quantitative measurements of ribosome initiation, elongation, and stalling. This approach reveals a surprising heterogeneity in the translation of individual mRNAs within the same cell, including rapid and reversible transitions between a translating and non-translating state. Applying this method to the cell-cycle gene Emi1, we find strong overall repression of translation initiation by specific 5' UTR sequences, but individual mRNA molecules in the same cell can exhibit dramatically different translational efficiencies. The ability to observe translation of single mRNA molecules in live cells provides a powerful tool to study translation regulation.