RESUMEN
RAF family kinases were among the first oncoproteins to be described more than 30 years ago. They primarily act as signalling relays downstream of RAS, and their close ties to cancer have fuelled a large number of studies. However, we still lack a systems-level understanding of their regulation and mode of action. The recent discovery that the catalytic activity of RAF depends on an allosteric mechanism driven by kinase domain dimerization is providing a vital new piece of information towards a comprehensive model of RAF function. The fact that current RAF inhibitors unexpectedly induce ERK signalling by stimulating RAF dimerization also calls for a deeper structural characterization of this family of kinases.
Asunto(s)
Sistema de Señalización de MAP Quinasas , Quinasas raf/metabolismo , Anomalías Múltiples/genética , Anomalías Múltiples/metabolismo , Animales , Humanos , Neoplasias/metabolismo , Quinasas raf/química , Quinasas raf/genéticaRESUMEN
Primary cilia are antenna-like structures protruding from the surface of various eukaryotic cells, and have distinct protein compositions in their membranes. This distinct protein composition is maintained by the presence of the transition zone (TZ) at the ciliary base, which acts as a diffusion barrier between the ciliary and plasma membranes. Defects in cilia and the TZ are known to cause a group of disorders collectively called the ciliopathies, which demonstrate a broad spectrum of clinical features, such as perinatally lethal Meckel syndrome (MKS), relatively mild Joubert syndrome (JBTS), and nonsyndromic nephronophthisis (NPHP). Proteins constituting the TZ can be grouped into the MKS and NPHP modules. The MKS module is composed of several transmembrane proteins and three soluble proteins. TMEM218 was recently reported to be mutated in individuals diagnosed as MKS and JBTS. However, little is known about how TMEM218 mutations found in MKS and JBTS affect the functions of cilia. In this study, we found that ciliary membrane proteins were not localized to cilia in TMEM218-knockout cells, indicating impaired barrier function of the TZ. Furthermore, the exogenous expression of JBTS-associated TMEM218 variants but not MKS-associated variants in TMEM218-knockout cells restored the localization of ciliary membrane proteins. In particular, when expressed in TMEM218-knockout cells, the TMEM218(R115H) variant found in JBTS was able to restore the barrier function of cells, whereas the MKS variant TMEM218(R115C) could not. Thus, the severity of symptoms of MKS and JBTS individuals appears to correlate with the degree of their ciliary defects at the cellular level.
Asunto(s)
Anomalías Múltiples , Cilios , Ciliopatías , Encefalocele , Anomalías del Ojo , Enfermedades Renales Quísticas , Proteínas de la Membrana , Mutación , Retina , Cilios/metabolismo , Cilios/genética , Cilios/patología , Humanos , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ciliopatías/genética , Ciliopatías/metabolismo , Ciliopatías/patología , Encefalocele/genética , Encefalocele/metabolismo , Encefalocele/patología , Enfermedades Renales Quísticas/genética , Enfermedades Renales Quísticas/metabolismo , Enfermedades Renales Quísticas/patología , Anomalías Múltiples/genética , Anomalías Múltiples/metabolismo , Anomalías Múltiples/patología , Anomalías del Ojo/genética , Anomalías del Ojo/patología , Anomalías del Ojo/metabolismo , Retina/metabolismo , Retina/anomalías , Retina/patología , Cerebelo/anomalías , Cerebelo/metabolismo , Cerebelo/patología , Enfermedades Cerebelosas/genética , Enfermedades Cerebelosas/metabolismo , Enfermedades Cerebelosas/patología , Animales , Membrana Celular/metabolismo , Ratones , Trastornos de la Motilidad Ciliar , Enfermedades Renales Poliquísticas , Retinitis PigmentosaRESUMEN
Centrosomal proteins play pivotal roles in orchestrating microtubule dynamics, and their dysregulation leads to disorders, including cancer and ciliopathies. Understanding the multifaceted roles of centrosomal proteins is vital to comprehend their involvement in disease development. Here, we report novel cellular functions of CEP41, a centrosomal and ciliary protein implicated in Joubert syndrome. We show that CEP41 is an essential microtubule-associated protein with microtubule-stabilizing activity. Purified CEP41 binds to preformed microtubules, promotes microtubule nucleation and suppresses microtubule disassembly. When overexpressed in cultured cells, CEP41 localizes to microtubules and promotes microtubule bundling. Conversely, shRNA-mediated knockdown of CEP41 disrupts the interphase microtubule network and delays microtubule reassembly, emphasizing its role in microtubule organization. Further, we demonstrate that the association of CEP41 with microtubules relies on its conserved rhodanese homology domain (RHOD) and the N-terminal region. Interestingly, a disease-causing mutation in the RHOD domain impairs CEP41-microtubule interaction. Moreover, depletion of CEP41 inhibits cell proliferation and disrupts cell cycle progression, suggesting its potential involvement in cell cycle regulation. These insights into the cellular functions of CEP41 hold promise for unraveling the impact of its mutations in ciliopathies.
Asunto(s)
Proliferación Celular , Microtúbulos , Humanos , Microtúbulos/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas Asociadas a Microtúbulos/genética , Centrosoma/metabolismo , Retina/metabolismo , Retina/patología , Retina/anomalías , Ciliopatías/metabolismo , Ciliopatías/genética , Ciliopatías/patología , Cerebelo/metabolismo , Cerebelo/anomalías , Cerebelo/patología , Enfermedades Renales Quísticas/metabolismo , Enfermedades Renales Quísticas/genética , Enfermedades Renales Quísticas/patología , Cilios/metabolismo , Cilios/patología , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Animales , Anomalías Múltiples/metabolismo , Anomalías Múltiples/genética , Anomalías Múltiples/patología , Anomalías del Ojo/metabolismo , Anomalías del Ojo/genética , Anomalías del Ojo/patología , Unión Proteica , Ciclo Celular/genética , Células HEK293RESUMEN
PRC2 is a therapeutic target for several types of cancers currently undergoing clinical trials. Its activity is regulated by a positive feedback loop whereby its terminal enzymatic product, H3K27me3, is specifically recognized and bound by an aromatic cage present in its EED subunit. The ensuing allosteric activation of the complex stimulates H3K27me3 deposition on chromatin. Here we report a stepwise feedback mechanism entailing key residues within distinctive interfacing motifs of EZH2 or EED that are found to be mutated in cancers and/or Weaver syndrome. PRC2 harboring these EZH2 or EED mutants manifested little activity in vivo but, unexpectedly, exhibited similar chromatin association as wild-type PRC2, indicating an uncoupling of PRC2 activity and recruitment. With genetic and chemical tools, we demonstrated that targeting allosteric activation overrode the gain-of-function effect of EZH2Y646X oncogenic mutations. These results revealed critical implications for the regulation and biology of PRC2 and a vulnerability in tackling PRC2-addicted cancers.
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Regulación Alostérica/fisiología , Cromatina/metabolismo , Complejo Represivo Polycomb 2/metabolismo , Anomalías Múltiples/metabolismo , Línea Celular Tumoral , Hipotiroidismo Congénito/metabolismo , Anomalías Craneofaciales/metabolismo , Proteína Potenciadora del Homólogo Zeste 2/metabolismo , Deformidades Congénitas de la Mano/metabolismo , Histonas/metabolismo , Humanos , Neoplasias/metabolismoRESUMEN
The Joubert syndrome (JS), Meckel syndrome (MKS), and nephronophthisis (NPH) ciliopathy spectrum could be the poster child for advances and challenges in Mendelian human genetics over the past half century. Progress in understanding these conditions illustrates many core concepts of human genetics. The JS phenotype alone is caused by pathogenic variants in more than 40 genes; remarkably, all of the associated proteins function in and around the primary cilium. Primary cilia are near-ubiquitous, microtubule-based organelles that play crucial roles in development and homeostasis. Protruding from the cell, these cellular antennae sense diverse signals and mediate Hedgehog and other critical signaling pathways. Ciliary dysfunction causes many human conditions termed ciliopathies, which range from multiple congenital malformations to adult-onset single-organ failure. Research on the genetics of the JS-MKS-NPH spectrum has spurred extensive functional work exploring the broadly important role of primary cilia in health and disease. This functional work promises to illuminate the mechanisms underlying JS-MKS-NPH in humans, identify therapeutic targets across genetic causes, and generate future precision treatments.
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Anomalías Múltiples , Ciliopatías , Anomalías del Ojo , Enfermedades Renales Poliquísticas , Anomalías Múltiples/genética , Anomalías Múltiples/metabolismo , Anomalías Múltiples/patología , Cerebelo/anomalías , Cerebelo/metabolismo , Cerebelo/patología , Niño , Cilios/genética , Cilios/metabolismo , Cilios/patología , Trastornos de la Motilidad Ciliar , Ciliopatías/genética , Ciliopatías/metabolismo , Ciliopatías/patología , Encefalocele , Anomalías del Ojo/genética , Anomalías del Ojo/metabolismo , Anomalías del Ojo/patología , Proteínas Hedgehog/metabolismo , Humanos , Enfermedades Renales Quísticas , Enfermedades Renales Poliquísticas/genética , Enfermedades Renales Poliquísticas/metabolismo , Enfermedades Renales Poliquísticas/patología , Retina/anomalías , Retina/metabolismo , Retina/patología , Retinitis PigmentosaRESUMEN
Many recent advances have emerged in the telomere and telomerase fields. This Timeline article highlights the key advances that have expanded our views on the mechanistic underpinnings of telomeres and telomerase and their roles in ageing and disease. Three decades ago, the classic view was that telomeres protected the natural ends of linear chromosomes and that telomerase was a specific telomere-terminal transferase necessary for the replication of chromosome ends in single-celled organisms. While this concept is still correct, many diverse fields associated with telomeres and telomerase have substantially matured. These areas include the discovery of most of the key molecular components of telomerase, implications for limits to cellular replication, identification and characterization of human genetic disorders that result in premature telomere shortening, the concept that inhibiting telomerase might be a successful therapeutic strategy and roles for telomeres in regulating gene expression. We discuss progress in these areas and conclude with challenges and unanswered questions in the field.
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Envejecimiento/genética , Genómica/historia , Neoplasias/genética , Telomerasa/genética , Telómero/química , Anomalías Múltiples/genética , Anomalías Múltiples/metabolismo , Anomalías Múltiples/patología , Envejecimiento/metabolismo , Animales , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , ADN/química , ADN/genética , ADN/metabolismo , Regulación de la Expresión Génica , Genómica/métodos , Historia del Siglo XX , Historia del Siglo XXI , Humanos , Chaperonas Moleculares , Neoplasias/metabolismo , Neoplasias/patología , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Progeria/genética , Progeria/metabolismo , Progeria/patología , Ribonucleoproteínas Nucleares Pequeñas/genética , Ribonucleoproteínas Nucleares Pequeñas/metabolismo , Ribonucleoproteínas Nucleolares Pequeñas/genética , Ribonucleoproteínas Nucleolares Pequeñas/metabolismo , Complejo Shelterina , Telomerasa/metabolismo , Telómero/metabolismo , Homeostasis del Telómero , Proteínas de Unión a Telómeros/genética , Proteínas de Unión a Telómeros/metabolismoRESUMEN
N-linked glycosylation is the most frequent modification of secreted and membrane-bound proteins in eukaryotic cells, disruption of which is the basis of the congenital disorders of glycosylation (CDGs). We describe a new type of CDG caused by mutations in the steroid 5alpha-reductase type 3 (SRD5A3) gene. Patients have mental retardation and ophthalmologic and cerebellar defects. We found that SRD5A3 is necessary for the reduction of the alpha-isoprene unit of polyprenols to form dolichols, required for synthesis of dolichol-linked monosaccharides, and the oligosaccharide precursor used for N-glycosylation. The presence of residual dolichol in cells depleted for this enzyme suggests the existence of an unexpected alternative pathway for dolichol de novo biosynthesis. Our results thus suggest that SRD5A3 is likely to be the long-sought polyprenol reductase and reveal the genetic basis of one of the earliest steps in protein N-linked glycosylation.
Asunto(s)
3-Oxo-5-alfa-Esteroide 4-Deshidrogenasa/metabolismo , Anomalías Múltiples/metabolismo , Dolicoles/metabolismo , Discapacidad Intelectual/metabolismo , Proteínas de la Membrana/metabolismo , Mutación , Proteínas de Saccharomyces cerevisiae/metabolismo , 3-Oxo-5-alfa-Esteroide 4-Deshidrogenasa/genética , Animales , Butadienos/metabolismo , Consanguinidad , Embrión de Mamíferos/metabolismo , Estudio de Asociación del Genoma Completo , Glicosilación , Hemiterpenos/metabolismo , Humanos , Proteínas de la Membrana/genética , Ratones , Pentanos/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Respuesta de Proteína DesplegadaRESUMEN
Genetic germline variants of PPP2R5D (encoding: phosphoprotein phosphatase 2 regulatory protein 5D) result in PPP2R5D-related disorder (Jordan's Syndrome), which is characterized by intellectual disability, hypotonia, seizures, macrocephaly, autism spectrum disorder, and delayed motor skill development. The disorder originates from de novo single nucleotide mutations, generating missense variants that act in a dominant manner. Pathogenic mutations altering 13 different amino acids have been identified, with the E198K variant accounting for â¼40% of reported cases. However, the generation of a heterozygous E198K variant cell line to study the molecular effects of the pathogenic mutation has been challenging. Here, we use CRISPR-PRIME genomic editing to introduce a transition (c.592G>A) in a single PPP2R5D allele in HEK293 cells, generating E198K-heterozygous lines to complement existing E420K variant lines. We generate global protein and phosphorylation profiles of WT, E198K, and E420K cell lines and find unique and shared changes between variants and WT cells in kinase- and phosphatase-controlled signaling cascades. We observed ribosomal protein S6 (RPS6) hyperphosphorylation as a shared signaling alteration, indicative of increased ribosomal protein S6-kinase activity. Treatment with rapamycin or an RPS6-kinase inhibitor (LY2584702) suppressed RPS6 phosphorylation in both, suggesting upstream activation of mTORC1/p70S6K. Intriguingly, our data suggests ERK-dependent activation of mTORC1 in both E198K and E420K variant cells, with additional AKT-mediated mTORC1 activation in the E420K variant. Thus, although upstream activation of mTORC1 differs between PPP2R5D-related disorder genotypes, inhibition of mTORC1 or RPS6 kinases warrants further investigation as potential therapeutic strategies for patients.
Asunto(s)
Anomalías Múltiples , Humanos , Trastorno del Espectro Autista , Células HEK293 , Diana Mecanicista del Complejo 1 de la Rapamicina/genética , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Fosforilación , Proteína Fosfatasa 2/genética , Proteína Fosfatasa 2/metabolismo , Proteómica , Proteína S6 Ribosómica/genética , Proteína S6 Ribosómica/metabolismo , Anomalías Múltiples/metabolismo , Anomalías Múltiples/patologíaRESUMEN
Mutations in genes that lead to dysfunctional cilia can cause a broad spectrum of human disease phenotypes referred to as ciliopathies. Many ciliopathy-associated proteins are localized to the evolutionary conserved ciliary transition zone (TZ) subdomain. We identified biallelic missense and nonsense mutations in the gene encoding the transmembrane protein TMEM218 in unrelated patients with features related to Bardet-Biedl, Joubert and Meckel-Gruber syndrome (MKS) and characterized TMEM218 as a major component of the ciliary TZ module. Co-immunoprecipitation assays resulted in the physical interaction of TMEM218 with the MKS module member TMEM67/Meckelin that was significantly reduced by the TMEM218 missense change harboured by one of our patients. We could further validate its pathogenicity by functional in vivo analysis in zebrafish (Danio rerio) as a well-established vertebrate model for ciliopathies. Notably, ciliopathy-related phenotypes were most prominent by genetic interactions with the NPHP module component Nphp4. Conclusively, we describe TMEM218 as a new disease gene for patients with a wide spectrum of syndromic ciliopathy phenotypes and provide evidence for a synergistic interaction of TMEM218 and the NPHP module crucial for proper ciliary function.
Asunto(s)
Anomalías Múltiples , Ciliopatías , Enfermedades Renales Poliquísticas , Anomalías Múltiples/genética , Anomalías Múltiples/metabolismo , Animales , Caenorhabditis elegans/genética , Cilios/genética , Cilios/metabolismo , Trastornos de la Motilidad Ciliar , Ciliopatías/genética , Ciliopatías/metabolismo , Encefalocele , Humanos , Mutación , Enfermedades Renales Poliquísticas/genética , Retinitis Pigmentosa , Pez Cebra/genéticaRESUMEN
Glypicans are a family of cell surface heparan sulfate proteoglycans that play critical roles in multiple cell signaling pathways. Glypicans consist of a globular core, an unstructured stalk modified with sulfated glycosaminoglycan chains, and a glycosylphosphatidylinositol anchor. Though these structural features are conserved, their individual contribution to glypican function remains obscure. Here, we investigate how glypican 3 (GPC3), which is mutated in Simpson-Golabi-Behmel tissue overgrowth syndrome, regulates Hedgehog signaling. We find that GPC3 is necessary for the Hedgehog response, surprisingly controlling a downstream signal transduction step. Purified GPC3 ectodomain rescues signaling when artificially recruited to the surface of GPC3-deficient cells but has dominant-negative activity when unattached. Strikingly, the purified stalk, modified with heparan sulfate but not chondroitin sulfate, is necessary and sufficient for activity. Our results demonstrate a novel function for GPC3-associated heparan sulfate and provide a framework for the functional dissection of glycosaminoglycans by in vivo biochemical complementation. This article has an associated First Person interview with the first author of the paper.
Asunto(s)
Anomalías Múltiples , Glipicanos , Proteínas Hedgehog , Heparitina Sulfato , Anomalías Múltiples/genética , Anomalías Múltiples/metabolismo , Arritmias Cardíacas , Enfermedades Genéticas Ligadas al Cromosoma X , Gigantismo , Glipicanos/genética , Glipicanos/metabolismo , Cardiopatías Congénitas , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Proteoglicanos de Heparán Sulfato , Humanos , Discapacidad Intelectual/genética , Discapacidad Intelectual/metabolismo , Transducción de SeñalRESUMEN
Cilia are organelles extend from cells to sense external signals for tuning intracellular signaling for optimal cellular functioning. They have evolved sensory and motor roles in various cells for tissue organization and homeostasis in development and post-development. More than a thousand genes are required for cilia function. Mutations in them cause multisystem disorders termed ciliopathies. The null mutations in CC2D2A result in Meckel syndrome (MKS), which is embryonic lethal, whereas patients who have missense mutations in the C2 domain of CC2D2A display Joubert syndrome (JBTS). They survive with blindness and mental retardation. How C2 domain defects cause disease conditions is not understood. To answer this question, C2 domain of Cc2d2a (mice gene) was knocked down (KD) in IMCD-3 cells by shRNA. This resulted in defective cilia morphology observed by immunofluorescence analysis. To further probe the cellular signaling alteration in affected cells, gene expression profiling was done by RNAseq and compared with the controls. Bioinformatics analysis revealed that the differentially expressed genes (DEGs) have functions in cilia. Among the 61 cilia DEGs identified, 50 genes were downregulated and 11 genes were upregulated. These cilia genes are involved in cilium assembly, protein trafficking to the cilium, intraflagellar transport (IFT), cellular signaling like polarity patterning, and Hedgehog signaling pathway. This suggests that the C2 domain of CC2D2A plays a critical role in cilia assembly and molecular signaling hosted in cilia for cellular homeostasis. Taken together, the missense mutations in the C2 domain of CC2D2A seen in JBTS might have affected cilia-mediated signaling in neurons of the retina and brain.
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Anomalías Múltiples , Cerebelo , Anomalías del Ojo , Enfermedades Renales Quísticas , Enfermedades Renales Poliquísticas , Retina , Animales , Ratones , Anomalías Múltiples/genética , Anomalías Múltiples/metabolismo , Dominios C2 , Cerebelo/metabolismo , Cerebelo/anomalías , Cilios/genética , Cilios/metabolismo , Proteínas del Citoesqueleto/genética , Anomalías del Ojo/genética , Anomalías del Ojo/metabolismo , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Mutación/genética , Enfermedades Renales Poliquísticas/genética , Enfermedades Renales Poliquísticas/metabolismo , Retina/anomalíasRESUMEN
The inability to maintain a strictly regulated endo(lyso)somal acidic pH through the proton-pumping action of the vacuolar-ATPases (v-ATPases) has been associated with various human diseases including heritable connective tissue disorders. Autosomal recessive (AR) cutis laxa (CL) type 2C syndrome is associated with genetic defects in the ATP6V1E1 gene and is characterized by skin wrinkles or loose redundant skin folds with pleiotropic systemic manifestations. The underlying pathological mechanisms leading to the clinical presentations remain largely unknown. Here, we show that loss of atp6v1e1b in zebrafish leads to early mortality, associated with craniofacial dysmorphisms, vascular anomalies, cardiac dysfunction, N-glycosylation defects, hypotonia, and epidermal structural defects. These features are reminiscent of the phenotypic manifestations in ARCL type 2C patients. Our data demonstrates that loss of atp6v1e1b alters endo(lyso)somal protein levels, and interferes with non-canonical v-ATPase pathways in vivo. In order to gain further insights into the processes affected by loss of atp6v1e1b, we performed an untargeted analysis of the transcriptome, metabolome, and lipidome in early atp6v1e1b-deficient larvae. We report multiple affected pathways including but not limited to oxidative phosphorylation, sphingolipid, fatty acid, and energy metabolism together with profound defects on mitochondrial respiration. Taken together, our results identify complex pathobiological effects due to loss of atp6v1e1b in vivo.
Asunto(s)
Anomalías Múltiples/genética , Cutis Laxo/genética , Células Epiteliales/metabolismo , Piel/metabolismo , ATPasas de Translocación de Protón Vacuolares/genética , Proteínas de Pez Cebra/genética , Anomalías Múltiples/metabolismo , Anomalías Múltiples/patología , Animales , Cutis Laxo/metabolismo , Cutis Laxo/patología , Modelos Animales de Enfermedad , Endosomas/metabolismo , Endosomas/patología , Células Epiteliales/patología , Regulación de la Expresión Génica , Humanos , Larva/genética , Larva/crecimiento & desarrollo , Larva/metabolismo , Lipidómica , Longevidad/genética , Lisosomas/metabolismo , Lisosomas/patología , Metaboloma/genética , Mitocondrias/metabolismo , Mitocondrias/patología , Fosforilación Oxidativa , Isoformas de Proteínas/deficiencia , Isoformas de Proteínas/genética , Piel/patología , Síndrome , Transcriptoma , ATPasas de Translocación de Protón Vacuolares/deficiencia , Pez Cebra/embriología , Pez Cebra/genética , Pez Cebra/metabolismo , Proteínas de Pez Cebra/deficienciaRESUMEN
Loss-of-function mutations in chromatin remodeler gene ARID1A are a cause of Coffin-Siris syndrome, a developmental disorder characterized by dysgenesis of corpus callosum. Here, we characterize Arid1a function during cortical development and find unexpectedly selective roles for Arid1a in subplate neurons (SPNs). SPNs, strategically positioned at the interface of cortical gray and white matter, orchestrate multiple developmental processes indispensable for neural circuit wiring. We find that pancortical deletion of Arid1a leads to extensive mistargeting of intracortical axons and agenesis of corpus callosum. Sparse Arid1a deletion, however, does not autonomously misroute callosal axons, implicating noncell-autonomous Arid1a functions in axon guidance. Supporting this possibility, the ascending axons of thalamocortical neurons, which are not autonomously affected by cortical Arid1a deletion, are also disrupted in their pathfinding into cortex and innervation of whisker barrels. Coincident with these miswiring phenotypes, which are reminiscent of subplate ablation, we unbiasedly find a selective loss of SPN gene expression following Arid1a deletion. In addition, multiple characteristics of SPNs crucial to their wiring functions, including subplate organization, subplate axon-thalamocortical axon cofasciculation ("handshake"), and extracellular matrix, are severely disrupted. To empirically test Arid1a sufficiency in subplate, we generate a cortical plate deletion of Arid1a that spares SPNs. In this model, subplate Arid1a expression is sufficient for subplate organization, subplate axon-thalamocortical axon cofasciculation, and subplate extracellular matrix. Consistent with these wiring functions, subplate Arid1a sufficiently enables normal callosum formation, thalamocortical axon targeting, and whisker barrel development. Thus, Arid1a is a multifunctional regulator of subplate-dependent guidance mechanisms essential to cortical circuit wiring.
Asunto(s)
Corteza Cerebral/metabolismo , Cromatina/química , Cuerpo Calloso/metabolismo , Proteínas de Unión al ADN/genética , Mutación con Pérdida de Función , Tálamo/metabolismo , Factores de Transcripción/genética , Anomalías Múltiples/genética , Anomalías Múltiples/metabolismo , Anomalías Múltiples/patología , Animales , Corteza Cerebral/patología , Cromatina/metabolismo , Conectoma , Cuerpo Calloso/patología , Proteínas de Unión al ADN/deficiencia , Cara/anomalías , Cara/patología , Eliminación de Gen , Regulación de la Expresión Génica , Sustancia Gris/metabolismo , Sustancia Gris/patología , Deformidades Congénitas de la Mano/genética , Deformidades Congénitas de la Mano/metabolismo , Deformidades Congénitas de la Mano/patología , Humanos , Discapacidad Intelectual/genética , Discapacidad Intelectual/metabolismo , Discapacidad Intelectual/patología , Ratones , Ratones Transgénicos , Micrognatismo/genética , Micrognatismo/metabolismo , Micrognatismo/patología , Cuello/anomalías , Cuello/patología , Vías Nerviosas/metabolismo , Vías Nerviosas/patología , Neuronas/metabolismo , Neuronas/patología , Tálamo/patología , Factores de Transcripción/deficiencia , Vibrisas/metabolismo , Vibrisas/patología , Sustancia Blanca/metabolismo , Sustancia Blanca/patologíaRESUMEN
Heterogeneous nuclear ribonucleoprotein K (hnRNPK) is an RNA-binding protein that regulates multiple biological processes, including paraspeckles formation and cellular signal transduction. Recently, hnRNPK has been shown to interact with SINE-derived nuclear RNA localization (SIRLOIN)-containing RNAs, and orchestrate nuclear enrichment and cellular functions of long noncoding RNAs (lncRNAs). hnRNPK-lncRNAs interaction is potentially implicated in various pathogenic disorders including tumorigenesis, and Kabuki-like, Au-Kline, and Okamoto syndromes.
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Fisura del Paladar/metabolismo , Ribonucleoproteína Heterogénea-Nuclear Grupo K/metabolismo , Hidronefrosis/metabolismo , Discapacidad Intelectual/metabolismo , Hipotonía Muscular/metabolismo , Síndromes Paraneoplásicos/metabolismo , ARN Largo no Codificante/metabolismo , Anomalías Múltiples/metabolismo , Cara/anomalías , Facies , Enfermedades Hematológicas/metabolismo , Humanos , Enfermedades Vestibulares/metabolismoRESUMEN
BACKGROUND: The pathogenic missense variant p.G125R in TBX5 (T-box transcription factor 5) causes Holt-Oram syndrome (also known as hand-heart syndrome) and early onset of atrial fibrillation. Revealing how an altered key developmental transcription factor modulates cardiac physiology in vivo will provide unique insights into the mechanisms underlying atrial fibrillation in these patients. METHODS: We analyzed ECGs of an extended family pedigree of Holt-Oram syndrome patients. Next, we introduced the TBX5-p.G125R variant in the mouse genome (Tbx5G125R) and performed electrophysiologic analyses (ECG, optical mapping, patch clamp, intracellular calcium measurements), transcriptomics (single-nuclei and tissue RNA sequencing), and epigenetic profiling (assay for transposase-accessible chromatin using sequencing, H3K27ac [histone H3 lysine 27 acetylation] CUT&RUN [cleavage under targets and release under nuclease sequencing]). RESULTS: We discovered high incidence of atrial extra systoles and atrioventricular conduction disturbances in Holt-Oram syndrome patients. Tbx5G125R/+ mice were morphologically unaffected and displayed variable RR intervals, atrial extra systoles, and susceptibility to atrial fibrillation, reminiscent of TBX5-p.G125R patients. Atrial conduction velocity was not affected but systolic and diastolic intracellular calcium concentrations were decreased and action potentials were prolonged in isolated cardiomyocytes of Tbx5G125R/+ mice compared with controls. Transcriptional profiling of atria revealed the most profound transcriptional changes in cardiomyocytes versus other cell types, and identified over a thousand coding and noncoding transcripts that were differentially expressed. Epigenetic profiling uncovered thousands of TBX5-p.G125R-sensitive, putative regulatory elements (including enhancers) that gained accessibility in atrial cardiomyocytes. The majority of sites with increased accessibility were occupied by Tbx5. The small group of sites with reduced accessibility was enriched for DNA-binding motifs of members of the SP (specificity protein) and KLF (Krüppel-like factor) families of transcription factors. These data show that Tbx5-p.G125R induces changes in regulatory element activity, alters transcriptional regulation, and changes cardiomyocyte behavior, possibly caused by altered DNA binding and cooperativity properties. CONCLUSIONS: Our data reveal that a disease-causing missense variant in TBX5 induces profound changes in the atrial transcriptional regulatory network and epigenetic state in vivo, leading to arrhythmia reminiscent of those seen in human TBX5-p.G125R variant carriers.
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Anomalías Múltiples , Regulación de la Expresión Génica , Cardiopatías Congénitas , Defectos del Tabique Interatrial , Heterocigoto , Deformidades Congénitas de las Extremidades Inferiores , Mutación Missense , Linaje , Proteínas de Dominio T Box , Deformidades Congénitas de las Extremidades Superiores , Anomalías Múltiples/genética , Anomalías Múltiples/metabolismo , Sustitución de Aminoácidos , Animales , Fibrilación Atrial/genética , Fibrilación Atrial/metabolismo , Femenino , Atrios Cardíacos/metabolismo , Cardiopatías Congénitas/genética , Cardiopatías Congénitas/metabolismo , Defectos del Tabique Interatrial/genética , Defectos del Tabique Interatrial/metabolismo , Humanos , Deformidades Congénitas de las Extremidades Inferiores/genética , Deformidades Congénitas de las Extremidades Inferiores/metabolismo , Masculino , Ratones , Ratones Mutantes , Proteínas de Dominio T Box/genética , Proteínas de Dominio T Box/metabolismo , Deformidades Congénitas de las Extremidades Superiores/genética , Deformidades Congénitas de las Extremidades Superiores/metabolismoRESUMEN
Temple and Kagami-Ogata syndromes are genomic imprinting diseases caused by maternal and paternal duplication of human chromosome 14, respectively. They exhibit different postnatal muscle-related symptoms as well as prenatal placental problems. Using the mouse models for these syndromes, it has been demonstrated that retrotransposon gag like 1 [Rtl1, also known as paternally expressed 11 (Peg11)] located in the mouse orthologous imprinted region is responsible for the prenatal placental problems because it is an essential placental gene for maintenance of fetal capillary network during gestation. However, the causative imprinted gene for the postnatal muscle-related symptoms remains unknown. Here, we demonstrate that Rtl1 also plays an important role in fetal/neonatal skeletal muscle development: its deletion and overproduction in mice lead to neonatal lethality associated with severe but distinct skeletal muscle defects, similar to those of Temple and Kagami-Ogata syndromes, respectively. Thus, it is strongly suggested that RTL1 is the major gene responsible for the muscle defects in addition to the placental defects in these two genomic imprinting diseases. This is the first example of an LTR retrotransposon-derived gene specific to eutherians contributing to eutherian skeletal muscle development.
Asunto(s)
Anomalías Múltiples/metabolismo , Anomalías Múltiples/patología , Músculos/anomalías , Proteínas Gestacionales/deficiencia , Animales , Animales Recién Nacidos , Diferenciación Celular , Proliferación Celular , Desmina/metabolismo , Femenino , Feto/metabolismo , Regulación del Desarrollo de la Expresión Génica , Humanos , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Modelos Genéticos , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/patología , Músculos/embriología , Músculos/patología , Mutación/genética , Proteínas Gestacionales/genética , Proteínas Gestacionales/metabolismo , Células Satélite del Músculo Esquelético/metabolismo , Síndrome , Factores de TiempoRESUMEN
Joubert syndrome (JBTS) is a Mendelian disorder of the primary cilium defined by the clinical triad of hypotonia, developmental delay, and a distinct cerebellar malformation called the molar tooth sign. JBTS is inherited in an autosomal recessive, autosomal dominant, or X-linked recessive manner. Though over 40 genes have been identified as causal for JBTS, molecular diagnosis is not made in 30%-40% of individuals who meet clinical criteria. TOPORS encodes topoisomerase I-binding arginine/serine-rich protein, and homozygosity for a TOPORS missense variant (c.29C > A; p.(Pro10Gln)) was identified in individuals with the ciliopathy oral-facial-digital syndrome in two families of Dominican descent. Here, we report an additional proband of Dominican ancestry with JBTS found by exome sequencing to be homozygous for the identical p.(Pro10Gln) TOPORS missense variant. Query of the Mount Sinai BioMe biobank, which includes 1880 individuals of Dominican ancestry, supports a high carrier frequency of the TOPORS p.(Pro10Gln) variant in individuals of Dominican descent. Our data nominates TOPORS as a novel causal gene for JBTS and suggests that TOPORS variants should be considered in the differential of ciliopathy-spectrum disease in individuals of Dominican ancestry.
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Anomalías Múltiples , Ciliopatías , Anomalías del Ojo , Enfermedades Renales Quísticas , Malformaciones del Sistema Nervioso , Humanos , Cerebelo/anomalías , Anomalías Múltiples/diagnóstico , Anomalías Múltiples/genética , Anomalías Múltiples/metabolismo , Anomalías del Ojo/diagnóstico , Anomalías del Ojo/genética , Retina/anomalías , Enfermedades Renales Quísticas/diagnóstico , Enfermedades Renales Quísticas/genética , Mutación , Ciliopatías/genéticaRESUMEN
Kabuki syndrome is an autosomal dominant developmental disorder with high similarities to CHARGE syndrome. It is characterized by a typical facial gestalt in combination with short stature, intellectual disability, skeletal findings and additional features like cardiac and urogenital malformations, cleft palate, hearing loss and ophthalmological anomalies. The major cause of Kabuki syndrome are mutations in KMT2D, a gene encoding a histone H3 lysine 4 (H3K4) methyltransferase belonging to the group of chromatin modifiers. Here we provide evidence that Kabuki syndrome is a neurocrestopathy, by showing that Kmt2d loss-of-function inhibits specific steps of neural crest (NC) development. Using the Xenopus model system, we find that Kmt2d loss-of-function recapitulates major features of Kabuki syndrome including severe craniofacial malformations. A detailed marker analysis revealed defects in NC formation as well as migration. Transplantation experiments confirm that Kmt2d function is required in NC cells. Furthermore, analyzing in vivo and in vitro NC migration behavior demonstrates that Kmt2d is necessary for cell dispersion but not protrusion formation of migrating NC cells. Importantly, Kmt2d knockdown correlates with a decrease in H3K4 monomethylation and H3K27 acetylation supporting a role of Kmt2d in the transcriptional activation of target genes. Consistently, using a candidate approach, we find that Kmt2d loss-of-function inhibits Xenopus Sema3F expression, and overexpression of Sema3F can partially rescue Kmt2d loss-of-function defects. Taken together, our data reveal novel functions of Kmt2d in multiple steps of NC development and support the hypothesis that major features of Kabuki syndrome are caused by defects in NC development.
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Anomalías Múltiples/enzimología , Cara/anomalías , Enfermedades Hematológicas/enzimología , N-Metiltransferasa de Histona-Lisina/genética , N-Metiltransferasa de Histona-Lisina/metabolismo , Cresta Neural/metabolismo , Enfermedades Vestibulares/enzimología , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo , Anomalías Múltiples/genética , Anomalías Múltiples/metabolismo , Anomalías Múltiples/patología , Acetilación , Animales , Movimiento Celular/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Cara/patología , Enfermedades Hematológicas/genética , Enfermedades Hematológicas/metabolismo , Enfermedades Hematológicas/patología , Histonas/metabolismo , Mutación con Pérdida de Función , Metilación , Mutación , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Cresta Neural/enzimología , Cresta Neural/patología , Placa Neural/crecimiento & desarrollo , Placa Neural/metabolismo , Placa Neural/patología , Semaforinas/genética , Semaforinas/metabolismo , Enfermedades Vestibulares/genética , Enfermedades Vestibulares/metabolismo , Enfermedades Vestibulares/patología , Xenopus/embriología , Xenopus/genética , Xenopus/metabolismo , Proteínas de Xenopus/fisiologíaRESUMEN
RTL1 (also termed paternal expressed 11 (PEG11)) is considered the major imprinted gene responsible for the placental and fetal/neonatal muscle defects that occur in the Kagami-Ogata and Temple syndromes (KOS14 and TS14, respectively). However, it remains elusive whether RTL1 is also involved in their neurological symptoms, such as behavioral and developmental delay/intellectual disability, feeding difficulties, motor delay, and delayed speech. Here, we demonstrate that the mouse RTL1 protein is widely expressed in the central nervous system (CNS), including the limbic system. Importantly, two disease model mice with over- and under-expression of Rtl1 exhibited reduced locomotor activity, increased anxiety, and impaired amygdala-dependent cued fear, demonstrating that Rtl1 also plays an important role in the CNS. These results indicate that the KOS14 and TS14 are neuromuscular as well as neuropsychiatric diseases caused by irregular CNS RTL1 expression, presumably leading to impaired innervation of motor neurons to skeletal muscles as well as malfunction of the hippocampus-amygdala complex. It is of considerable interest that eutherian-specific RTL1 is expressed in mammalian- and eutherian-specific brain structures, that is, the corticospinal tract and corpus callosum, respectively, suggesting that RTL1 might have contributed to the acquisition of both these structures themselves and fine motor skill in eutherian brain evolution.
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Anomalías Múltiples/metabolismo , Euterios/metabolismo , Sistema Nervioso/metabolismo , Proteínas Gestacionales/metabolismo , Animales , Animales Recién Nacidos , Ansiedad/metabolismo , Conducta Animal , Encéfalo/metabolismo , Condicionamiento Clásico , Miedo , Femenino , Regulación del Desarrollo de la Expresión Génica , Humanos , Masculino , Ratones Endogámicos C57BL , Actividad Motora , Proteínas Gestacionales/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Especificidad de la Especie , SíndromeRESUMEN
Kabuki syndrome (KS) is a rare genetic disorder caused primarily by mutations in the histone modifier genes KMT2D and KDM6A. The genes have broad temporal and spatial expression in many organs, resulting in complex phenotypes observed in KS patients. Hypotonia is one of the clinical presentations associated with KS, yet detailed examination of skeletal muscle samples from KS patients has not been reported. We studied the consequences of loss of KMT2D function in both mouse and human muscles. In mice, heterozygous loss of Kmt2d resulted in reduced neuromuscular junction (NMJ) perimeter, decreased muscle cell differentiation in vitro and impaired myofiber regeneration in vivo. Muscle samples from KS patients of different ages showed presence of increased fibrotic tissue interspersed between myofiber fascicles, which was not seen in mouse muscles. Importantly, when Kmt2d-deficient muscle stem cells were transplanted in vivo in a physiologic non-Kabuki environment, their differentiation potential is restored to levels undistinguishable from control cells. Thus, the epigenetic changes due to loss of function of KMT2D appear reversible through a change in milieu, opening a potential therapeutic avenue.