RESUMEN
Cellular heterogeneity confounds in situ assays of transcription factor (TF) binding. Single-cell RNA sequencing (scRNA-seq) deconvolves cell types from gene expression, but no technology links cell identity to TF binding sites (TFBS) in those cell types. We present self-reporting transposons (SRTs) and use them in single-cell calling cards (scCC), a novel assay for simultaneously measuring gene expression and mapping TFBS in single cells. The genomic locations of SRTs are recovered from mRNA, and SRTs deposited by exogenous, TF-transposase fusions can be used to map TFBS. We then present scCC, which map SRTs from scRNA-seq libraries, simultaneously identifying cell types and TFBS in those same cells. We benchmark multiple TFs with this technique. Next, we use scCC to discover BRD4-mediated cell-state transitions in K562 cells. Finally, we map BRD4 binding sites in the mouse cortex at single-cell resolution, establishing a new method for studying TF biology in situ.
Asunto(s)
Elementos Transponibles de ADN/genética , Análisis de la Célula Individual/métodos , Factores de Transcripción/metabolismo , Animales , Sitios de Unión , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Corteza Cerebral/metabolismo , Inmunoprecipitación de Cromatina , Expresión Génica , Factor Nuclear 3-beta del Hepatocito/genética , Factor Nuclear 3-beta del Hepatocito/metabolismo , Humanos , Ratones , Unión Proteica , Análisis de Secuencia de ARN , Factor de Transcripción Sp1/genética , Factor de Transcripción Sp1/metabolismo , Factores de Transcripción/genéticaRESUMEN
Nociceptin and its receptor are widely distributed throughout the brain in regions associated with reward behavior, yet how and when they act is unknown. Here, we dissected the role of a nociceptin peptide circuit in reward seeking. We generated a prepronociceptin (Pnoc)-Cre mouse line that revealed a unique subpopulation of paranigral ventral tegmental area (pnVTA) neurons enriched in prepronociceptin. Fiber photometry recordings during progressive ratio operant behavior revealed pnVTAPnoc neurons become most active when mice stop seeking natural rewards. Selective pnVTAPnoc neuron ablation, inhibition, and conditional VTA nociceptin receptor (NOPR) deletion increased operant responding, revealing that the pnVTAPnoc nucleus and VTA NOPR signaling are necessary for regulating reward motivation. Additionally, optogenetic and chemogenetic activation of this pnVTAPnoc nucleus caused avoidance and decreased motivation for rewards. These findings provide insight into neuromodulatory circuits that regulate motivated behaviors through identification of a previously unknown neuropeptide-containing pnVTA nucleus that limits motivation for rewards.
Asunto(s)
Motivación/efectos de los fármacos , Péptidos Opioides/farmacología , Recompensa , Área Tegmental Ventral/metabolismo , Potenciales de Acción , Animales , Conducta Animal/efectos de los fármacos , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Neuronas/fisiología , Técnicas de Placa-Clamp , Precursores de Proteínas/genética , Receptores Opioides/agonistas , Receptores Opioides/deficiencia , Receptores Opioides/genética , Receptor de Nociceptina , NociceptinaRESUMEN
Psychiatric genetic studies have drawn associations between human cognitive traits and noncoding genomic variants. However, the mechanistic effects of these variants are unclear. By weaving in strands of genomic data from developing human brains, de la Torre-Ubieta et al. tie disease-associated loci to functional enhancers, target genes, and putatively affected cell types.
Asunto(s)
Cromatina , Estudio de Asociación del Genoma Completo , Cognición , Humanos , Neurogénesis , Fenotipo , Polimorfismo de Nucleótido SimpleRESUMEN
Astrotactin 2 (ASTN2) is a transmembrane neuronal protein highly expressed in the cerebellum that functions in receptor trafficking and modulates cerebellar Purkinje cell (PC) synaptic activity. Individuals with ASTN2 mutations exhibit neurodevelopmental disorders, including autism spectrum disorder (ASD), attention-deficit/hyperactivity disorder (ADHD), learning difficulties, and language delay. To provide a genetic model for the role of the cerebellum in ASD-related behaviors and study the role of ASTN2 in cerebellar circuit function, we generated global and PC-specific conditional Astn2 knockout (KO and cKO, respectively) mouse lines. Astn2 KO mice exhibit strong ASD-related behavioral phenotypes, including a marked decrease in separation-induced pup ultrasonic vocalization calls, hyperactivity, repetitive behaviors, altered behavior in the three-chamber test, and impaired cerebellar-dependent eyeblink conditioning. Hyperactivity and repetitive behaviors are also prominent in Astn2 cKO animals, but they do not show altered behavior in the three-chamber test. By Golgi staining, Astn2 KO PCs have region-specific changes in dendritic spine density and filopodia numbers. Proteomic analysis of Astn2 KO cerebellum reveals a marked upregulation of ASTN2 family member, ASTN1, a neuron-glial adhesion protein. Immunohistochemistry and electron microscopy demonstrate a significant increase in Bergmann glia volume in the molecular layer of Astn2 KO animals. Electrophysiological experiments indicate a reduced frequency of spontaneous excitatory postsynaptic currents (EPSCs), as well as increased amplitudes of both spontaneous EPSCs and inhibitory postsynaptic currents in the Astn2 KO animals, suggesting that pre- and postsynaptic components of synaptic transmission are altered. Thus, ASTN2 regulates ASD-like behaviors and cerebellar circuit properties.
Asunto(s)
Trastorno del Espectro Autista , Cerebelo , Ratones Noqueados , Células de Purkinje , Animales , Ratones , Trastorno del Espectro Autista/metabolismo , Trastorno del Espectro Autista/genética , Trastorno del Espectro Autista/fisiopatología , Células de Purkinje/metabolismo , Cerebelo/metabolismo , Conducta Animal/fisiología , Proteínas del Tejido Nervioso/metabolismo , Proteínas del Tejido Nervioso/genética , Modelos Animales de Enfermedad , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genética , MasculinoRESUMEN
In vitro studies indicate the neurodevelopmental disorder gene myelin transcription factor 1-like (MYT1L) suppresses non-neuronal lineage genes during fibroblast-to-neuron direct differentiation. However, MYT1L's molecular and cellular functions in the adult mammalian brain have not been fully characterized. Here, we found that MYT1L loss leads to up-regulated deep layer (DL) gene expression, corresponding to an increased ratio of DL/UL neurons in the adult mouse cortex. To define potential mechanisms, we conducted Cleavage Under Targets & Release Using Nuclease (CUT&RUN) to map MYT1L binding targets and epigenetic changes following MYT1L loss in mouse developing cortex and adult prefrontal cortex (PFC). We found MYT1L mainly binds to open chromatin, but with different transcription factor co-occupancies between promoters and enhancers. Likewise, multiomic data set integration revealed that, at promoters, MYT1L loss does not change chromatin accessibility but increases H3K4me3 and H3K27ac, activating both a subset of earlier neuronal development genes as well as Bcl11b, a key regulator for DL neuron development. Meanwhile, we discovered that MYT1L normally represses the activity of neurogenic enhancers associated with neuronal migration and neuronal projection development by closing chromatin structures and promoting removal of active histone marks. Further, we showed that MYT1L interacts with HDAC2 and transcriptional repressor SIN3B in vivo, providing potential mechanisms underlying repressive effects on histone acetylation and gene expression. Overall, our findings provide a comprehensive map of MYT1L binding in vivo and mechanistic insights into how MYT1L loss leads to aberrant activation of earlier neuronal development programs in the adult mouse brain.
Asunto(s)
Cromatina , Factores de Transcripción , Animales , Ratones , Encéfalo/metabolismo , Cromatina/genética , Cromatina/metabolismo , Mamíferos/genética , Neuronas/metabolismo , Regiones Promotoras Genéticas , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteínas Supresoras de Tumor/metabolismoRESUMEN
A classic example of experience-dependent plasticity is ocular dominance (OD) shift, in which the responsiveness of neurons in the visual cortex is profoundly altered following monocular deprivation (MD). It has been postulated that OD shifts also modify global neural networks, but such effects have never been demonstrated. Here, we use wide-field fluorescence optical imaging (WFOI) to characterize calcium-based resting-state functional connectivity during acute (3â d) MD in female and male mice with genetically encoded calcium indicators (Thy1-GCaMP6f). We first establish the fundamental performance of WFOI by computing signal to noise properties throughout our data processing pipeline. Following MD, we found that Δ band (0.4-4â Hz) GCaMP6 activity in the deprived visual cortex decreased, suggesting that excitatory activity in this region was reduced by MD. In addition, interhemispheric visual homotopic functional connectivity decreased following MD, which was accompanied by a reduction in parietal and motor homotopic connectivity. Finally, we observed enhanced internetwork connectivity between the visual and parietal cortex that peaked 2â d after MD. Together, these findings support the hypothesis that early MD induces dynamic reorganization of disparate functional networks including the association cortices.
Asunto(s)
Ratones Endogámicos C57BL , Red Nerviosa , Privación Sensorial , Corteza Visual , Animales , Ratones , Masculino , Femenino , Privación Sensorial/fisiología , Corteza Visual/fisiología , Red Nerviosa/fisiología , Plasticidad Neuronal/fisiología , Predominio Ocular/fisiología , Período Crítico Psicológico , Vías Visuales/fisiologíaRESUMEN
MOTIVATION: Unraveling the transcriptional programs that control how cells divide, differentiate, and respond to their environments requires a precise understanding of transcription factors' (TFs) DNA-binding activities. Calling cards (CC) technology uses transposons to capture transient TF binding events at one instant in time and then read them out at a later time. This methodology can also be used to simultaneously measure TF binding and mRNA expression from single-cell CC and to record and integrate TF binding events across time in any cell type of interest without the need for purification. Despite these advantages, there has been a lack of dedicated bioinformatics tools for the detailed analysis of CC data. RESULTS: We introduce Pycallingcards, a comprehensive Python module specifically designed for the analysis of single-cell and bulk CC data across multiple species. Pycallingcards introduces two innovative peak callers, CCcaller and MACCs, enhancing the accuracy and speed of pinpointing TF binding sites from CC data. Pycallingcards offers a fully integrated environment for data visualization, motif finding, and comparative analysis with RNA-seq and ChIP-seq datasets. To illustrate its practical application, we have reanalyzed previously published mouse cortex and glioblastoma datasets. This analysis revealed novel cell-type-specific binding sites and potential sex-linked TF regulators, furthering our understanding of TF binding and gene expression relationships. Thus, Pycallingcards, with its user-friendly design and seamless interface with the Python data science ecosystem, stands as a critical tool for advancing the analysis of TF functions via CC data. AVAILABILITY AND IMPLEMENTATION: Pycallingcards can be accessed on the GitHub repository: https://github.com/The-Mitra-Lab/pycallingcards.
Asunto(s)
Ecosistema , Factores de Transcripción , Animales , Ratones , Inmunoprecipitación de Cromatina , Factores de Transcripción/metabolismo , Sitios de Unión , Unión Proteica , Análisis de Secuencia de ADNRESUMEN
As a regressive neurodevelopmental disorder with a well-established genetic cause, Rett syndrome and its Mecp2 loss-of-function mouse model provide an excellent opportunity to define potentially translatable functional signatures of disease progression, as well as offer insight into the role of Mecp2 in functional circuit development. Thus, we applied widefield optical fluorescence imaging to assess mesoscale calcium functional connectivity (FC) in the Mecp2 cortex both at postnatal day (P)35 in development and during the disease-related decline. We found that FC between numerous cortical regions was disrupted in Mecp2 mutant males both in juvenile development and early adulthood. Female Mecp2 mice displayed an increase in homotopic contralateral FC in the motor cortex at P35 but not in adulthood, where instead more posterior parietal regions were implicated. An increase in the amplitude of connection strength, both with more positive correlations and more negative anticorrelations, was observed across the male cortex in numerous functional regions. Widespread rescue of MeCP2 protein in GABAergic neurons rescued none of these functional deficits, nor, surprisingly, the expected male lifespan. Altogether, the female results identify early signs of disease progression, while the results in males indicate MeCP2 protein is required for typical FC in the brain.
Asunto(s)
Proteína 2 de Unión a Metil-CpG , Síndrome de Rett , Masculino , Femenino , Ratones , Animales , Proteína 2 de Unión a Metil-CpG/genética , Síndrome de Rett/genética , Síndrome de Rett/metabolismo , Encéfalo , Neuronas GABAérgicas/fisiología , Modelos Animales de Enfermedad , Ratones Endogámicos C57BLRESUMEN
The first evidence of local translation in the CNS appeared nearly 40 years ago, when electron microscopic studies showed polyribosomes localized to the base of dendritic spines. Since then, local translation has been established as an important regulatory mechanism for gene expression in polarized or functionally compartmentalized cells. While much attention has been placed on characterizing the local transcriptome and regulatory "grammar" directing mRNA localization in neurons and glia, less is understood about how these cells subsequently de-repress mRNA translation in their peripheral processes to produce a rapid translational response to stimuli. MicroRNA-mediated translation regulation offers a possible solution to this question. Not only do miRNAs provide the specificity needed for targeted gene regulation, but association and dynamic interactions between Argonaute (AGO) with sequence-specific RNA-binding proteins may provide a molecular switch to allow for de-repression of target mRNAs. Here, we review the expression and activity of different AGO proteins in miRNA-induced silencing complexes in neurons and glia and discuss known pathways of miRNA-mediated regulation, including activity-dependent pre-miRNA maturation in dendrites. We further detail work on AGO and RNA-binding protein interactions that allow for the reversal of miRNA-mediated translational silencing, and we propose a model for how intercellular communication may play a role in the regulation of local translation.
Asunto(s)
Proteínas Argonautas , MicroARNs , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , MicroARNs/genética , MicroARNs/metabolismo , Neuroglía/metabolismo , Neuronas/metabolismo , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismoRESUMEN
The overwhelming success of exome- and genome-wide association studies in discovering thousands of disease-associated genes necessitates developing novel high-throughput functional genomics approaches to elucidate the molecular mechanisms of these genes. Here, we have coupled multiplexed repression of neurodevelopmental disease-associated genes to single-cell transcriptional profiling in differentiating human neurons to rapidly assay the functions of multiple genes in a disease-relevant context, assess potentially convergent mechanisms, and prioritize genes for specific functional assays. For a set of 13 autism spectrum disorder (ASD)-associated genes, we show that this approach generated important mechanistic insights, revealing two functionally convergent modules of ASD genes: one that delays neuron differentiation and one that accelerates it. Five genes that delay neuron differentiation (ADNP, ARID1B, ASH1L, CHD2, and DYRK1A) mechanistically converge, as they all dysregulate genes involved in cell-cycle control and progenitor cell proliferation. Live-cell imaging after individual ASD-gene repression validated this functional module, confirming that these genes reduce neural progenitor cell proliferation and neurite growth. Finally, these functionally convergent ASD gene modules predicted shared clinical phenotypes among individuals with mutations in these genes. Altogether, these results show the utility of a novel and simple approach for the rapid functional elucidation of neurodevelopmental disease-associated genes.
Asunto(s)
Trastorno del Espectro Autista/genética , Neurogénesis/genética , Neuronas/metabolismo , Análisis de la Célula Individual/métodos , Sistemas CRISPR-Cas , Línea Celular , Proliferación Celular , Regulación del Desarrollo de la Expresión Génica , Técnicas de Silenciamiento del Gen/métodos , Células HEK293 , Humanos , Procesamiento de Imagen Asistido por Computador , Modelos Genéticos , Neurogénesis/fisiología , Proyección Neuronal/genética , Fenotipo , RNA-Seq , TranscriptomaRESUMEN
Alzheimer's disease is initiated by the toxic aggregation of amyloid-ß. Immunotherapeutics aimed at reducing amyloid beta are in clinical trials but with very limited success to date. Identification of orthogonal approaches for clearing amyloid beta may complement these approaches for treating Alzheimer's disease. In the brain, the astrocytic water channel Aquaporin 4 is involved in clearance of amyloid beta, and the fraction of Aquaporin 4 found perivascularly is decreased in Alzheimer's disease. Further, an unusual stop codon readthrough event generates a conserved C-terminally elongated variant of Aquaporin 4 (AQP4X), which is exclusively perivascular. However, it is unclear whether the AQP4X variant specifically mediates amyloid beta clearance. Here, using Aquaporin 4 readthrough-specific knockout mice that still express normal Aquaporin 4, we determine that this isoform indeed mediates amyloid beta clearance. Further, with high-throughput screening and counterscreening, we identify small molecule compounds that enhance readthrough of the Aquaporin 4 sequence and validate a subset on endogenous astrocyte Aquaporin 4. Finally, we demonstrate these compounds enhance brain amyloid-ß clearance in vivo, which depends on AQP4X. This suggests derivatives of these compounds may provide a viable pharmaceutical approach to enhance clearance of amyloid beta and potentially other aggregating proteins in neurodegenerative disease.
Asunto(s)
Enfermedad de Alzheimer , Acuaporina 4/metabolismo , Enfermedades Neurodegenerativas , Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Animales , Acuaporina 4/genética , Encéfalo/metabolismo , Codón de Terminación , Ratones , Enfermedades Neurodegenerativas/metabolismoRESUMEN
Comparative analysis can provide important insights into complex biological systems. As demonstrated in the accompanying paper, translating ribosome affinity purification (TRAP) permits comprehensive studies of translated mRNAs in genetically defined cell populations after physiological perturbations. To establish the generality of this approach, we present translational profiles for 24 CNS cell populations and identify known cell-specific and enriched transcripts for each population. We report thousands of cell-specific mRNAs that were not detected in whole-tissue microarray studies and provide examples that demonstrate the benefits deriving from comparative analysis. To provide a foundation for further biological and in silico studies, we provide a resource of 16 transgenic mouse lines, their corresponding anatomic characterization, and translational profiles for cell types from a variety of central nervous system structures. This resource will enable a wide spectrum of molecular and mechanistic studies of both well-known and previously uncharacterized neural cell populations.
Asunto(s)
Encéfalo/metabolismo , Técnicas Genéticas , Biosíntesis de Proteínas , Animales , Sistema Nervioso Central/metabolismo , Cromosomas Artificiales Bacterianos/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Inmunohistoquímica/métodos , Ratones , Ratones Transgénicos , Modelos Biológicos , Neuronas/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Ribosomas/metabolismoRESUMEN
Cross-sectional studies have established a variety of structural, synaptic, and cell physiological changes corresponding to critical periods in cortical development. However, the emergence of functional connectivity (FC) in development has not been fully characterized, and hemodynamic-based measures are vulnerable to any neurovascular coupling changes occurring in parallel. We therefore used optical fluorescence imaging to trace longitudinal calcium FC in the awake, resting-state mouse cortex at 5 developmental timepoints beginning at postnatal day 15 (P15) and ending in early adulthood at P60. Calcium FC displayed coherent functional maps as early as P15, and FC significantly varied in connections between many regions across development, with the developmental trajectory's shape specific to the functional region. Evaluating 325 seed-seed connections, we found that there was a significant increase in FC between P15 and P22 over the majority of the cortex as well as bilateral connectivity and node degree differences in frontal, motor, and retrosplenial cortices after P22. A rebalancing of inter- and intrahemispheric FC and local-distal FC dominance was also observed during development. This longitudinal developmental calcium FC study therefore provides a resource dataset to the field and identifies periods of dynamic change which cross-sectional studies may target for examination of disease states.
Asunto(s)
Calcio , Acoplamiento Neurovascular , Animales , Corteza Cerebral/diagnóstico por imagen , Estudios Transversales , Giro del Cíngulo , Imagen por Resonancia Magnética , Ratones , Acoplamiento Neurovascular/fisiologíaRESUMEN
Transcription factors (TFs) enact precise regulation of gene expression through site-specific, genome-wide binding. Common methods for TF-occupancy profiling, such as chromatin immunoprecipitation, are limited by requirement of TF-specific antibodies and provide only end-point snapshots of TF binding. Alternatively, TF-tagging techniques, in which a TF is fused to a DNA-modifying enzyme that marks TF-binding events across the genome as they occur, do not require TF-specific antibodies and offer the potential for unique applications, such as recording of TF occupancy over time and cell type specificity through conditional expression of the TF-enzyme fusion. Here, we create a viral toolkit for one such method, calling cards, and demonstrate that these reagents can be delivered to the live mouse brain and used to report TF occupancy. Further, we establish a Cre-dependent calling cards system and, in proof-of-principle experiments, show utility in defining cell type-specific TF profiles and recording and integrating TF-binding events across time. This versatile approach will enable unique studies of TF-mediated gene regulation in live animal models.
Asunto(s)
Cromatina/genética , Elementos Transponibles de ADN/genética , Proteínas de Unión al ADN/genética , Epigenómica/métodos , Factores de Transcripción/genética , Algoritmos , Animales , Anticuerpos/genética , Sitios de Unión/genética , Cromatina/virología , Dependovirus/genética , Regulación de la Expresión Génica/genética , Genoma/genética , Humanos , Integrasas/genética , Ratones , Distribución Tisular/genéticaRESUMEN
Mutations in the transcription factor Forkhead box p1 (FOXP1) are causative for neurodevelopmental disorders such as autism. However, the function of FOXP1 within the brain remains largely uncharacterized. Here, we identify the gene expression program regulated by FoxP1 in both human neural cells and patient-relevant heterozygous Foxp1 mouse brains. We demonstrate a role for FoxP1 in the transcriptional regulation of autism-related pathways as well as genes involved in neuronal activity. We show that Foxp1 regulates the excitability of striatal medium spiny neurons and that reduction of Foxp1 correlates with defects in ultrasonic vocalizations. Finally, we demonstrate that FoxP1 has an evolutionarily conserved role in regulating pathways involved in striatal neuron identity through gene expression studies in human neural progenitors with altered FOXP1 levels. These data support an integral role for FoxP1 in regulating signaling pathways vulnerable in autism and the specific regulation of striatal pathways important for vocal communication.
Asunto(s)
Trastorno del Espectro Autista/fisiopatología , Cuerpo Estriado/fisiopatología , Factores de Transcripción Forkhead/metabolismo , Proteínas Represoras/metabolismo , Transducción de Señal/genética , Animales , Trastorno del Espectro Autista/genética , Células Cultivadas , Modelos Animales de Enfermedad , Factores de Transcripción Forkhead/genética , Regulación de la Expresión Génica/genética , Haploinsuficiencia , Hipocampo/fisiopatología , Humanos , Ratones , Ratones Endogámicos C57BL , Mutación , Neuronas/patología , Proteínas Represoras/genética , Conducta Verbal/fisiologíaRESUMEN
Gtf2ird1 and Gtf2i are two transcription factors (TFs) among the 28 genes deleted in Williams syndrome, and prior mouse models of each TF show behavioral phenotypes. Here we identify their genomic binding sites in the developing brain and test for additive effects of their mutation on transcription and behavior. GTF2IRD1 binding targets were enriched for transcriptional and chromatin regulators and mediators of ubiquitination. GTF2I targets were enriched for signal transduction proteins, including regulators of phosphorylation and WNT. Both TFs are highly enriched at promoters, strongly overlap CTCF binding and topological associating domain boundaries and moderately overlap each other, suggesting epistatic effects. Shared TF targets are enriched for reactive oxygen species-responsive genes, synaptic proteins and transcription regulators such as chromatin modifiers, including a significant number of highly constrained genes and known ASD genes. We next used single and double mutants to test whether mutating both TFs will modify transcriptional and behavioral phenotypes of single Gtf2ird1 mutants, though with the caveat that our Gtf2ird1 mutants, like others previously reported, do produce low levels of a truncated protein product. Despite little difference in DNA binding and transcriptome-wide expression, homozygous Gtf2ird1 mutation caused balance, marble burying and conditioned fear phenotypes. However, mutating Gtf2i in addition to Gtf2ird1 did not further modify transcriptomic or most behavioral phenotypes, suggesting Gtf2ird1 mutation alone was sufficient for the observed phenotypes.
Asunto(s)
Factor de Unión a CCCTC/genética , Proteínas Musculares/genética , Transactivadores/genética , Factores de Transcripción TFII/genética , Síndrome de Williams/genética , Animales , Encéfalo/metabolismo , Encéfalo/patología , Sistemas CRISPR-Cas/genética , Proteínas de Unión al ADN/genética , Modelos Animales de Enfermedad , Desarrollo Embrionario/genética , Edición Génica , Regulación del Desarrollo de la Expresión Génica/genética , Humanos , Ratones , Transcripción Genética/genética , Síndrome de Williams/patologíaRESUMEN
SUMMARY: Whole genome sequencing of patient populations is identifying thousands of new variants in untranslated regions (UTRs). While the consequences of UTR mutations are not as easily predicted from primary sequence as coding mutations are, there are some known features of UTRs that modulate their function. utr.annotation is an R package that can be used to annotate potential deleterious variants in the UTR regions for both human and mouse species. Given a CSV or VCF format variant file, utr.annotation provides information of each variant on whether and how it alters known translational regulators including upstream open reading frames, upstream Kozak sequences, polyA signals, Kozak sequences at the annotated translation start site, start codons and stop codons, conservation scores in the variant position, and whether and how it changes ribosome loading based on a model derived from empirical data. AVAILABILITY AND IMPLEMENTATION: utr.annotation is freely available on Bitbucket (https://bitbucket.org/jdlabteam/utr.annotation/src/master/) and CRAN (https://cran.r-project.org/web/packages/utr.annotation/index.html). SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
Asunto(s)
Regulación de la Expresión Génica , Genómica , Ratones , Humanos , Animales , Regiones no TraducidasRESUMEN
Williams syndrome (WS) is a neurodevelopmental disorder caused by a 1.5-1.8 Mbp deletion on chromosome 7q11.23, affecting the copy number of 26-28 genes. Phenotypes of WS include cardiovascular problems, craniofacial dysmorphology, deficits in visual-spatial cognition and a characteristic hypersocial personality. There are still no genes in the region that have been consistently linked to the cognitive and behavioral phenotypes, although human studies and mouse models have led to the current hypothesis that the general transcription factor 2 I family of genes, GTF2I and GTF2IRD1, are responsible. Here we test the hypothesis that these two transcription factors are sufficient to reproduce the phenotypes that are caused by deletion of the WS critical region (WSCR). We compare a new mouse model with loss of function mutations in both Gtf2i and Gtf2ird1 to an established mouse model lacking the complete WSCR. We show that the complete deletion (CD) model has deficits across several behavioral domains including social communication, motor functioning and conditioned fear that are not explained by loss of function mutations in Gtf2i and Gtf2ird1. Furthermore, transcriptome profiling of the hippocampus shows changes in synaptic genes in the CD model that are not seen in the double mutants. Thus, we have thoroughly defined a set of molecular and behavioral consequences of complete WSCR deletion and shown that genes or combinations of genes beyond Gtf2i and Gtf2ird1 are necessary to produce these phenotypic effects.
Asunto(s)
Proteínas Musculares/genética , Mutación/genética , Transactivadores/genética , Factores de Transcripción TFII/genética , Síndrome de Williams/genética , Síndrome de Williams/patología , Animales , Femenino , Hipocampo/metabolismo , Masculino , Ratones , Fenotipo , Vocalización Animal/fisiologíaRESUMEN
Genetic disruptions of the forkhead box transcription factor FOXP2 in humans cause an autosomal-dominant speech and language disorder. While FOXP2 expression pattern are highly conserved, its role in specific brain areas for mammalian social behaviors remains largely unknown. Here we studied mice carrying a homozygous cortical Foxp2 deletion. The postnatal development and gross morphological architecture of mutant mice was indistinguishable from wildtype (WT) littermates. Unbiased behavioral profiling of adult mice revealed abnormalities in approach behavior towards conspecifics as well as in the reciprocal responses of WT interaction partners. Furthermore mutant mice showed alterations in acoustical parameters of ultrasonic vocalizations, which also differed in function of the social context. Cell type-specific gene expression profiling of cortical pyramidal neurons revealed aberrant regulation of genes involved in social behavior. In particular Foxp2 mutants showed the downregulation of Mint2 (Apba2), a gene involved in approach behavior in mice and autism spectrum disorder in humans. Taken together these data demonstrate that cortical Foxp2 is required for normal social behaviors in mice.
Asunto(s)
Conducta Animal , Corteza Cerebral/metabolismo , Corteza Cerebral/fisiopatología , Factores de Transcripción Forkhead/deficiencia , Eliminación de Gen , Proteínas Represoras/deficiencia , Conducta Social , Animales , Corteza Cerebral/diagnóstico por imagen , Corteza Cerebral/patología , Homocigoto , Ratones , Ratones Noqueados , Neuronas/metabolismoRESUMEN
The nervous system of the bowel regulates the inflammatory phenotype of tissue resident muscularis macrophages (MM), and in adult mice, enteric neurons are the main local source of colony stimulating factor 1 (CSF1), a protein required for MM survival. Surprisingly, we find that during development MM colonize the bowel before enteric neurons. This calls into question the requirement for neuron-derived CSF1 for MM colonization of the bowel. To determine if intestinal innervation is required for MM development, we analyzed MM of neonatal Ret-/- (Ret KO) mice that have no enteric nervous system in small bowel or colon. We found normal numbers of well-patterned MM in Ret KO bowel. Similarly, the abundance and distribution of MM in aganglionic human colon obtained from Hirschsprung disease patients was normal. We also identify endothelial cells and interstitial cells of Cajal as the main sources of CSF1 in the developing bowel. Additionally, MM from neonatal Ret KOs do not differ from controls in baseline activation status or cytokine-production in response to lipopolysaccharide. Unexpectedly, these data demonstrate that the enteric nervous system is dispensable for MM colonization and patterning in the bowel, and suggest that modulatory interactions between MM and the bowel nervous system are established postnatally.