RESUMEN
The cardiac transcription factor (TF) gene NKX2-5 has been associated with electrocardiographic (EKG) traits through genome-wide association studies (GWASs), but the extent to which differential binding of NKX2-5 at common regulatory variants contributes to these traits has not yet been studied. We analyzed transcriptomic and epigenomic data from induced pluripotent stem cell-derived cardiomyocytes from seven related individuals, and identified ~2,000 single-nucleotide variants associated with allele-specific effects (ASE-SNVs) on NKX2-5 binding. NKX2-5 ASE-SNVs were enriched for altered TF motifs, for heart-specific expression quantitative trait loci and for EKG GWAS signals. Using fine-mapping combined with epigenomic data from induced pluripotent stem cell-derived cardiomyocytes, we prioritized candidate causal variants for EKG traits, many of which were NKX2-5 ASE-SNVs. Experimentally characterizing two NKX2-5 ASE-SNVs (rs3807989 and rs590041) showed that they modulate the expression of target genes via differential protein binding in cardiac cells, indicating that they are functional variants underlying EKG GWAS signals. Our results show that differential NKX2-5 binding at numerous regulatory variants across the genome contributes to EKG phenotypes.
Asunto(s)
Fibrilación Atrial/genética , Fibrilación Atrial/patología , Proteína Homeótica Nkx-2.5/genética , Proteína Homeótica Nkx-2.5/metabolismo , Polimorfismo de Nucleótido Simple , Sitios de Carácter Cuantitativo , Elementos Reguladores de la Transcripción , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Alelos , Niño , Electrocardiografía , Epigenómica , Femenino , Predisposición Genética a la Enfermedad , Genoma Humano , Estudio de Asociación del Genoma Completo , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/patología , Masculino , Persona de Mediana Edad , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , Fenotipo , Unión Proteica , Transcriptoma , Adulto JovenRESUMEN
Genetic variants affecting pancreatic islet enhancers are central to T2D risk, but the gene targets of islet enhancer activity are largely unknown. We generate a high-resolution map of islet chromatin loops using Hi-C assays in three islet samples and use loops to annotate target genes of islet enhancers defined using ATAC-seq and published ChIP-seq data. We identify candidate target genes for thousands of islet enhancers, and find that enhancer looping is correlated with islet-specific gene expression. We fine-map T2D risk variants affecting islet enhancers, and find that candidate target genes of these variants defined using chromatin looping and eQTL mapping are enriched in protein transport and secretion pathways. At IGF2BP2, a fine-mapped T2D variant reduces islet enhancer activity and IGF2BP2 expression, and conditional inactivation of IGF2BP2 in mouse islets impairs glucose-stimulated insulin secretion. Our findings provide a resource for studying islet enhancer function and identifying genes involved in T2D risk.
Asunto(s)
Cromatina/metabolismo , Diabetes Mellitus Tipo 2/genética , Redes Reguladoras de Genes/genética , Islotes Pancreáticos/metabolismo , Proteínas de Unión al ARN/genética , Adulto , Animales , Núcleo Celular/metabolismo , Ensamble y Desensamble de Cromatina/genética , Diabetes Mellitus Tipo 2/patología , Elementos de Facilitación Genéticos/genética , Femenino , Perfilación de la Expresión Génica , Predisposición Genética a la Enfermedad , Glucosa/metabolismo , Humanos , Insulina/metabolismo , Islotes Pancreáticos/citología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Persona de Mediana Edad , Conformación Molecular , Sitios de Carácter Cuantitativo/genética , Proteínas de Unión al ARN/metabolismoRESUMEN
Efforts to identify driver mutations in cancer have largely focused on genes, whereas non-coding sequences remain relatively unexplored. Here we develop a statistical method based on characteristics known to influence local mutation rate and a series of enrichment filters in order to identify distal regulatory elements harboring putative driver mutations in breast cancer. We identify ten DNase I hypersensitive sites that are significantly mutated in breast cancers and associated with the aberrant expression of neighboring genes. A pan-cancer analysis shows that three of these elements are significantly mutated across multiple cancer types and have mutation densities similar to protein-coding driver genes. Functional characterization of the most highly mutated DNase I hypersensitive sites in breast cancer (using in silico and experimental approaches) confirms that they are regulatory elements and affect the expression of cancer genes. Our study suggests that mutations of regulatory elements in tumors likely play an important role in cancer development.Cancer driver mutations can occur within noncoding genomic sequences. Here, the authors develop a statistical approach to identify candidate noncoding driver mutations in DNase I hypersensitive sites in breast cancer and experimentally demonstrate they are regulatory elements of known cancer genes.
Asunto(s)
Neoplasias de la Mama/genética , Desoxirribonucleasa I/metabolismo , Secuencias Reguladoras de Ácidos Nucleicos/genética , Cromatina/metabolismo , Ensamble y Desensamble de Cromatina , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Mutación/genética , Reproducibilidad de los Resultados , Eliminación de Secuencia , Telomerasa/metabolismoRESUMEN
During neuronal development, local mRNA translation is required for axon guidance and synaptogenesis, and dysregulation of this process contributes to multiple neurodevelopmental and cognitive disorders. However, regulation of local protein synthesis in developing axons remains poorly understood. Here, we uncover a novel role for the actin-regulatory protein Mena in the formation of a ribonucleoprotein complex that involves the RNA-binding proteins HnrnpK and PCBP1 and regulates local translation of specific mRNAs in developing axons. We find that translation of dyrk1a, a Down syndrome- and autism spectrum disorders-related gene, is dependent on Mena, both in steady-state conditions and upon BDNF stimulation. We identify hundreds of additional mRNAs that associate with the Mena complex, suggesting that it plays broader role(s) in post-transcriptional gene regulation. Our work establishes a dual role for Mena in neurons, providing a potential link between regulation of actin dynamics and local translation.
Asunto(s)
Actinas/metabolismo , Axones/fisiología , Neurogénesis/fisiología , Neuronas/metabolismo , ARN Mensajero/metabolismo , Animales , Movimiento Celular/fisiología , Proteínas del Citoesqueleto/metabolismo , Regulación de la Expresión Génica/fisiología , Biosíntesis de ProteínasRESUMEN
Large-scale collections of induced pluripotent stem cells (iPSCs) could serve as powerful model systems for examining how genetic variation affects biology and disease. Here we describe the iPSCORE resource: a collection of systematically derived and characterized iPSC lines from 222 ethnically diverse individuals that allows for both familial and association-based genetic studies. iPSCORE lines are pluripotent with high genomic integrity (no or low numbers of somatic copy-number variants) as determined using high-throughput RNA-sequencing and genotyping arrays, respectively. Using iPSCs from a family of individuals, we show that iPSC-derived cardiomyocytes demonstrate gene expression patterns that cluster by genetic background, and can be used to examine variants associated with physiological and disease phenotypes. The iPSCORE collection contains representative individuals for risk and non-risk alleles for 95% of SNPs associated with human phenotypes through genome-wide association studies. Our study demonstrates the utility of iPSCORE for examining how genetic variants influence molecular and physiological traits in iPSCs and derived cell lines.
Asunto(s)
Arritmias Cardíacas/genética , Bases de Datos Factuales , Estudios de Asociación Genética , Variación Genética , Células Madre Pluripotentes Inducidas/metabolismo , Miocitos Cardíacos/metabolismo , Arritmias Cardíacas/etnología , Arritmias Cardíacas/metabolismo , Arritmias Cardíacas/fisiopatología , Diferenciación Celular , Línea Celular , Reprogramación Celular/genética , Genotipo , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Células Madre Pluripotentes Inducidas/citología , Familia de Multigenes , Miocitos Cardíacos/citología , Análisis de Secuencia por Matrices de Oligonucleótidos , Fenotipo , Polimorfismo de Nucleótido Simple , Grupos RacialesRESUMEN
During a survey of clinical rectal prolapse (RP) cases in the mouse population at MIT animal research facilities, a high incidence of RP in the lamellipodin knock-out strain, C57BL/6-Raph1tm1Fbg (Lpd-/-) was documented. Upon further investigation, the Lpd-/- colony was found to be infected with multiple endemic enterohepatic Helicobacter species (EHS). Lpd-/- mice, a transgenic mouse strain produced at MIT, have not previously shown a distinct immune phenotype and are not highly susceptible to other opportunistic infections. Predominantly male Lpd-/- mice with RP exhibited lesions consistent with invasive rectal carcinoma concomitant to clinically evident RP. Multiple inflammatory cytokines, CD11b+Gr1+ myeloid-derived suppressor cell (MDSC) populations, and epithelial cells positive for a DNA damage biomarker, H2AX, were elevated in affected tissue, supporting their role in the neoplastic process. An evaluation of Lpd-/- mice with RP compared to EHS-infected, but clinically normal (CN) Lpd-/- animals indicated that all of these mice exhibit some degree of lower bowel inflammation; however, mice with prolapses had significantly higher degree of focal lesions at the colo-rectal junction. When Helicobacter spp. infections were eliminated in Lpd-/- mice by embryo transfer rederivation, the disease phenotype was abrogated, implicating EHS as a contributing factor in the development of rectal carcinoma. Here we describe lesions in Lpd-/- male mice consistent with a focal inflammation-induced neoplastic transformation and propose this strain as a mouse model of rectal carcinoma.
Asunto(s)
Proteínas Portadoras/genética , Modelos Animales de Enfermedad , Proteínas de la Membrana/genética , Neoplasias del Recto/genética , Animales , Daño del ADN , Humanos , Hibridación Fluorescente in Situ , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neoplasias del Recto/patologíaRESUMEN
The emergence of neurites from a symmetrical cell body is an essential feature of nervous system development. Neurites are the precursors of axons and dendrites and are tipped by growth cones, motile structures that guide elongating axons in the developing nervous system. Growth cones steer the axon along a defined path to its appropriate target in response to guidance cues. This navigation involves the dynamic extension and withdrawal of actin-filled finger-like protrusions called filopodia that continuously sample their environment. Ena/VASP proteins, a conserved family of actin-regulatory proteins, are crucial for filopodia formation and function downstream of several guidance cues. Here we review recent findings into Ena/VASP function in neurite initiation, axon outgrowth and guidance.
Asunto(s)
Diferenciación Celular/genética , Proteínas de Unión al ADN/metabolismo , Sistema Nervioso/embriología , Sistema Nervioso/metabolismo , Neuritas/metabolismo , Células Madre/metabolismo , Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/ultraestructura , Animales , Señales (Psicología) , Proteínas de Unión al ADN/genética , Conos de Crecimiento/metabolismo , Conos de Crecimiento/ultraestructura , Humanos , Sistema Nervioso/citología , Neuritas/ultraestructura , Seudópodos/metabolismo , Seudópodos/ultraestructura , Células Madre/citologíaRESUMEN
Cadherins are transmembrane adhesion molecules that mediate homotypic cell-cell contact. In adherens junctions, the cytoplasmic domain of cadherins is functionally linked to the actin cytoskeleton through a series of proteins known as catenins. E-cadherin binds to beta-catenin, which in turn binds to alpha-catenin to form a ternary complex. alpha-Catenin also binds to actin, and it was assumed previously that alpha-catenin links the cadherin-catenin complex to actin. However, biochemical, structural and live-cell imaging studies of the cadherin-catenin complex and its interaction with actin show that binding of beta-catenin to alpha-catenin prevents the latter from binding to actin. Biochemical and structural data indicate that alpha-catenin acts as an allosteric protein whose conformation and activity changes depending on whether or not it is bound to beta-catenin. Initial contacts between cells occur on dynamic lamellipodia formed by polymerization of branched actin networks, a process controlled by the Arp2/3 (actin-related protein 2/3) complex. alpha-Catenin can suppress the activity of Arp2/3 by competing for actin filaments. These findings lead to a model for adherens junction formation in which clustering of the cadherin-beta-catenin complex recruits high levels of alpha-catenin that can suppress the Arp2/3 complex, leading to cessation of lamellipodial movement and formation of a stable contact. Thus alpha-catenin appears to play a central role in cell-cell contact formation.
Asunto(s)
Cadherinas/fisiología , Comunicación Celular/fisiología , alfa Catenina/química , alfa Catenina/fisiología , Complejo 2-3 Proteico Relacionado con la Actina/química , Complejo 2-3 Proteico Relacionado con la Actina/fisiología , Actinas/química , Actinas/fisiología , Animales , Cadherinas/química , Citoesqueleto/química , Citoesqueleto/fisiología , Modelos Biológicos , Unión Proteica , Conformación Proteica , Estructura Terciaria de Proteína , beta Catenina/química , beta Catenina/fisiologíaRESUMEN
Spatial and functional organization of cells in tissues is determined by cell-cell adhesion, thought to be initiated through trans-interactions between extracellular domains of the cadherin family of adhesion proteins, and strengthened by linkage to the actin cytoskeleton. Prevailing dogma is that cadherins are linked to the actin cytoskeleton through beta-catenin and alpha-catenin, although the quaternary complex has never been demonstrated. We test this hypothesis and find that alpha-catenin does not interact with actin filaments and the E-cadherin-beta-catenin complex simultaneously, even in the presence of the actin binding proteins vinculin and alpha-actinin, either in solution or on isolated cadherin-containing membranes. Direct analysis in polarized cells shows that mobilities of E-cadherin, beta-catenin, and alpha-catenin are similar, regardless of the dynamic state of actin assembly, whereas actin and several actin binding proteins have higher mobilities. These results suggest that the linkage between the cadherin-catenin complex and actin filaments is more dynamic than previously appreciated.
Asunto(s)
Actinas/metabolismo , Cadherinas/metabolismo , Adhesión Celular/fisiología , alfa Catenina/metabolismo , beta Catenina/metabolismo , Actinas/genética , Animales , Antineoplásicos/metabolismo , Cadherinas/genética , Línea Celular , Membrana Celular/metabolismo , Citocalasina D/metabolismo , Citoesqueleto/metabolismo , Depsipéptidos/metabolismo , Colorantes Fluorescentes/metabolismo , Ratones , Proteínas de Microfilamentos/metabolismo , Complejos Multiproteicos , Inhibidores de la Síntesis del Ácido Nucleico/metabolismo , Unión Proteica , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Vinculina/metabolismo , alfa Catenina/genética , beta Catenina/genéticaRESUMEN
Epithelial cell-cell junctions, organized by adhesion proteins and the underlying actin cytoskeleton, are considered to be stable structures maintaining the structural integrity of tissues. Contrary to the idea that alpha-catenin links the adhesion protein E-cadherin through beta-catenin to the actin cytoskeleton, in the accompanying paper we report that alpha-catenin does not bind simultaneously to both E-cadherin-beta-catenin and actin filaments. Here we demonstrate that alpha-catenin exists as a monomer or a homodimer with different binding properties. Monomeric alpha-catenin binds more strongly to E-cadherin-beta-catenin, whereas the dimer preferentially binds actin filaments. Different molecular conformations are associated with these different binding states, indicating that alpha-catenin is an allosteric protein. Significantly, alpha-catenin directly regulates actin-filament organization by suppressing Arp2/3-mediated actin polymerization, likely by competing with the Arp2/3 complex for binding to actin filaments. These results indicate a new role for alpha-catenin in local regulation of actin assembly and organization at sites of cadherin-mediated cell-cell adhesion.
Asunto(s)
Actinas/metabolismo , Cadherinas/metabolismo , Adhesión Celular/fisiología , Conformación Proteica , alfa Catenina/química , alfa Catenina/metabolismo , beta Catenina/metabolismo , Complejo 2-3 Proteico Relacionado con la Actina/metabolismo , Actinas/química , Actinas/genética , Animales , Cadherinas/genética , Membrana Celular/metabolismo , Citoesqueleto/metabolismo , Dimerización , Ratones , Modelos Moleculares , Complejos Multiproteicos , Unión Proteica , Subunidades de Proteína/química , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Vinculina/química , Vinculina/genética , Vinculina/metabolismo , alfa Catenina/genética , beta Catenina/genéticaRESUMEN
Cadherins regulate cell-cell adhesion throughout embryonic development and in the adult organism, and defects in cadherin expression and function are characteristic of many disease states including cancer. Although extracellular binding between cadherins specifies adhesion between cells, the strength of the interaction is thought to be regulated by cadherin clustering through reorganization of the actin cytoskeleton. Protein-protein interactions have been described that could link cadherins either directly or indirectly to the actin cytoskeleton. Here, we describe these protein interactions, and examine critically the evidence that they link cadherins to the actin cytoskeleton.
Asunto(s)
Actinas/metabolismo , Cadherinas/metabolismo , Citoesqueleto/metabolismo , Animales , Cateninas/metabolismo , Adhesión Celular , Proteínas del Citoesqueleto/metabolismo , Citoesqueleto/química , Unión ProteicaRESUMEN
Cell adhesion between cells and with the extracellular matrix (ECM) results in dramatic changes in cell organization and, in particular, the cytoskeleton and plasma membrane domains involved in adhesion. However, current methods to analyze these changes are limited because of the small areas of membrane involved in adhesion, compared to the areas of membrane not adhering (a signal to noise problem), and the difficulty in accessing native protein complexes directly for imaging or reconstitution with purified proteins. The methods described here overcome these problems. Using a mammalian expression system, a chimeric protein comprising the extracellular domain of E-cadherin fused at its C-terminus to the Fc domain of human IgG1 (E-cadherin:Fc) is expressed and purified. A chemical bridge of biotin-NeutrAvidin-biotinylated Protein G bound to a silanized glass cover slip is fabricated to which the E-cadherin:Fc chimera binds in the correct orientation for adhesion by cells. After cell attachment, the basal membrane (a contact formed between cellular E-cadherin and the E-cadherin:Fc substratum) is isolated by sonication; a similar method is described to isolate basal membranes of cells attached to ECM. These membrane patches provide direct access to protein complexes formed on the membrane following cell-cell or cell-ECM adhesion.
Asunto(s)
Cadherinas/fisiología , Adhesión Celular/fisiología , Membrana Celular/fisiología , Movimiento Celular/fisiología , Animales , Línea Celular , Membrana Celular/ultraestructura , Polaridad Celular/fisiología , Perros , Matriz Extracelular/fisiología , Humanos , Fragmentos Fc de Inmunoglobulinas , Riñón , RatasRESUMEN
alpha-Catenin is an integral component of adherens junctions, where it links cadherins to the actin cytoskeleton. alpha-Catenin is also required for the colocalization of the nectin/afadin/ponsin adhesion system to adherens junctions, and it specifically associates with the nectin-binding protein afadin. A proteolytic fragment of alpha-catenin, residues 385-651, contains the afadin-binding site. The three-dimensional structure of this fragment comprises two side-by-side four-helix bundles, both of which are required for afadin binding. The alpha-catenin fragment 385-651 binds afadin more strongly than the full-length protein, suggesting that the full-length protein harbors a cryptic binding site for afadin. Comparison of the alpha-catenin 385-651 structure with the recently solved structure of the alpha-catenin M-fragment (Yang, J., Dokurno, P., Tonks, N. K., and Barford, D. (2001) EMBO J. 20, 3645-3656) reveals a surprising flexibility in the orientation of the two four-helix bundles. alpha-Catenin and the actin-binding protein vinculin share sequence and most likely structural similarity within their actin-binding domains. Despite this homology, actin binding requires additional sequences adjacent to this region.