RESUMO
Neuronal activity is crucial for adaptive circuit remodelling but poses an inherent risk to the stability of the genome across the long lifespan of postmitotic neurons1-5. Whether neurons have acquired specialized genome protection mechanisms that enable them to withstand decades of potentially damaging stimuli during periods of heightened activity is unknown. Here we identify an activity-dependent DNA repair mechanism in which a new form of the NuA4-TIP60 chromatin modifier assembles in activated neurons around the inducible, neuronal-specific transcription factor NPAS4. We purify this complex from the brain and demonstrate its functions in eliciting activity-dependent changes to neuronal transcriptomes and circuitry. By characterizing the landscape of activity-induced DNA double-strand breaks in the brain, we show that NPAS4-NuA4 binds to recurrently damaged regulatory elements and recruits additional DNA repair machinery to stimulate their repair. Gene regulatory elements bound by NPAS4-NuA4 are partially protected against age-dependent accumulation of somatic mutations. Impaired NPAS4-NuA4 signalling leads to a cascade of cellular defects, including dysregulated activity-dependent transcriptional responses, loss of control over neuronal inhibition and genome instability, which all culminate to reduce organismal lifespan. In addition, mutations in several components of the NuA4 complex are reported to lead to neurodevelopmental and autism spectrum disorders. Together, these findings identify a neuronal-specific complex that couples neuronal activity directly to genome preservation, the disruption of which may contribute to developmental disorders, neurodegeneration and ageing.
Assuntos
Encéfalo , Reparo do DNA , Complexos Multiproteicos , Neurônios , Sinapses , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Encéfalo/metabolismo , Quebras de DNA de Cadeia Dupla , Regulação da Expressão Gênica , Lisina Acetiltransferase 5/metabolismo , Complexos Multiproteicos/metabolismo , Neurônios/metabolismo , Sinapses/metabolismo , Mutação , Longevidade/genética , Genoma , Envelhecimento/genética , Doenças NeurodegenerativasRESUMO
Mutations in the methyl-DNA-binding repressor protein MeCP2 cause the devastating neurodevelopmental disorder Rett syndrome. It has been challenging to understand how MeCP2 regulates transcription because MeCP2 binds broadly across the genome and MeCP2 mutations are associated with widespread small-magnitude changes in neuronal gene expression. We demonstrate here that MeCP2 represses nascent RNA transcription of highly methylated long genes in the brain through its interaction with the NCoR co-repressor complex. By measuring the rates of transcriptional initiation and elongation directly in the brain, we find that MeCP2 has no measurable effect on transcriptional elongation, but instead represses the rate at which Pol II initiates transcription of highly methylated long genes. These findings suggest a new model of MeCP2 function in which MeCP2 binds broadly across highly methylated regions of DNA, but acts at transcription start sites to attenuate transcriptional initiation.
Assuntos
Metilação de DNA/genética , Proteína 2 de Ligação a Metil-CpG/genética , Proteínas Repressoras/genética , Transcrição Gênica/genética , Animais , Encéfalo/fisiologia , DNA/genética , Masculino , Camundongos , Camundongos Knockout , Mutação/genética , Neurônios/fisiologia , RNA/genética , Síndrome de Rett/genéticaRESUMO
Behavioural experiences activate the FOS transcription factor in sparse populations of neurons that are critical for encoding and recalling specific events1-3. However, there is limited understanding of the mechanisms by which experience drives circuit reorganization to establish a network of Fos-activated cells. It is also not known whether FOS is required in this process beyond serving as a marker of recent neural activity and, if so, which of its many gene targets underlie circuit reorganization. Here we demonstrate that when mice engage in spatial exploration of novel environments, perisomatic inhibition of Fos-activated hippocampal CA1 pyramidal neurons by parvalbumin-expressing interneurons is enhanced, whereas perisomatic inhibition by cholecystokinin-expressing interneurons is weakened. This bidirectional modulation of inhibition is abolished when the function of the FOS transcription factor complex is disrupted. Single-cell RNA-sequencing, ribosome-associated mRNA profiling and chromatin analyses, combined with electrophysiology, reveal that FOS activates the transcription of Scg2, a gene that encodes multiple distinct neuropeptides, to coordinate these changes in inhibition. As parvalbumin- and cholecystokinin-expressing interneurons mediate distinct features of pyramidal cell activity4-6, the SCG2-dependent reorganization of inhibitory synaptic input might be predicted to affect network function in vivo. Consistent with this prediction, hippocampal gamma rhythms and pyramidal cell coupling to theta phase are significantly altered in the absence of Scg2. These findings reveal an instructive role for FOS and SCG2 in establishing a network of Fos-activated neurons via the rewiring of local inhibition to form a selectively modulated state. The opposing plasticity mechanisms acting on distinct inhibitory pathways may support the consolidation of memories over time.
Assuntos
Rede Nervosa/citologia , Rede Nervosa/fisiologia , Inibição Neural , Plasticidade Neuronal/fisiologia , Proteínas Proto-Oncogênicas c-fos/metabolismo , Animais , Região CA1 Hipocampal/metabolismo , Colecistocinina/metabolismo , Comportamento Exploratório/fisiologia , Feminino , Ritmo Gama , Interneurônios/metabolismo , Masculino , Consolidação da Memória , Camundongos , Parvalbuminas/metabolismo , Células Piramidais/metabolismo , Secretogranina II/genética , Secretogranina II/metabolismo , Navegação Espacial/fisiologia , Ritmo TetaRESUMO
The advent of endothermy, which is achieved through the continuous homeostatic regulation of body temperature and metabolism1,2, is a defining feature of mammalian and avian evolution. However, when challenged by food deprivation or harsh environmental conditions, many mammalian species initiate adaptive energy-conserving survival strategies-including torpor and hibernation-during which their body temperature decreases far below its homeostatic set-point3-5. How homeothermic mammals initiate and regulate these hypothermic states remains largely unknown. Here we show that entry into mouse torpor, a fasting-induced state with a greatly decreased metabolic rate and a body temperature as low as 20 °C6, is regulated by neurons in the medial and lateral preoptic area of the hypothalamus. We show that restimulation of neurons that were activated during a previous bout of torpor is sufficient to initiate the key features of torpor, even in mice that are not calorically restricted. Among these neurons we identify a population of glutamatergic Adcyap1-positive cells, the activity of which accurately determines when mice naturally initiate and exit torpor, and the inhibition of which disrupts the natural process of torpor entry, maintenance and arousal. Taken together, our results reveal a specific neuronal population in the mouse hypothalamus that serves as a core regulator of torpor. This work forms a basis for the future exploration of mechanisms and circuitry that regulate extreme hypothermic and hypometabolic states, and enables genetic access to monitor, initiate, manipulate and study these ancient adaptations of homeotherm biology.
Assuntos
Metabolismo Energético/fisiologia , Hipotálamo/citologia , Vias Neurais/fisiologia , Neurônios/fisiologia , Torpor/fisiologia , Animais , Jejum , Feminino , Privação de Alimentos , Glutamina/metabolismo , Hipotálamo/fisiologia , Masculino , Camundongos , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/metabolismoRESUMO
Mutations in MECP2 give rise to Rett syndrome (RTT), an X-linked neurodevelopmental disorder that results in broad cognitive impairments in females. While the exact etiology of RTT symptoms remains unknown, one possible explanation for its clinical presentation is that loss of MECP2 causes miswiring of neural circuits due to defects in the brain's capacity to respond to changes in neuronal activity and sensory experience. Here, we show that MeCP2 is phosphorylated at four residues in the mouse brain (S86, S274, T308, and S421) in response to neuronal activity, and we generate a quadruple knock-in (QKI) mouse line in which all four activity-dependent sites are mutated to alanines to prevent phosphorylation. QKI mice do not display overt RTT phenotypes or detectable gene expression changes in two brain regions. However, electrophysiological recordings from the retinogeniculate synapse of QKI mice reveal that while synapse elimination is initially normal at P14, it is significantly compromised at P20. Notably, this phenotype is distinct from the synapse refinement defect previously reported for Mecp2 null mice, where synapses initially refine but then regress after the third postnatal week. We thus propose a model in which activity-induced phosphorylation of MeCP2 is critical for the proper timing of retinogeniculate synapse maturation specifically during the early postnatal period.
Assuntos
Proteína 2 de Ligação a Metil-CpG , Síndrome de Rett , Feminino , Camundongos , Animais , Fosforilação , Proteína 2 de Ligação a Metil-CpG/genética , Proteína 2 de Ligação a Metil-CpG/metabolismo , Síndrome de Rett/genética , Síndrome de Rett/metabolismo , Encéfalo/metabolismo , Sinapses/metabolismo , Neurônios/metabolismo , Camundongos Knockout , Modelos Animais de DoençasRESUMO
Angelman Syndrome is a debilitating neurological disorder caused by mutation of the E3 ubiquitin ligase Ube3A, a gene whose mutation has also recently been associated with autism spectrum disorders (ASDs). The function of Ube3A during nervous system development and how Ube3A mutations give rise to cognitive impairment in individuals with Angleman Syndrome and ASDs are not clear. We report here that experience-driven neuronal activity induces Ube3A transcription and that Ube3A then regulates excitatory synapse development by controlling the degradation of Arc, a synaptic protein that promotes the internalization of the AMPA subtype of glutamate receptors. We find that disruption of Ube3A function in neurons leads to an increase in Arc expression and a concomitant decrease in the number of AMPA receptors at excitatory synapses. We propose that this deregulation of AMPA receptor expression at synapses may contribute to the cognitive dysfunction that occurs in Angelman Syndrome and possibly other ASDs.
Assuntos
Síndrome de Angelman/fisiopatologia , Proteínas do Citoesqueleto/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Células Cultivadas , Cognição , Humanos , Camundongos , Camundongos Knockout , Receptores de AMPA/metabolismo , Sinapses/metabolismo , UbiquitinaçãoRESUMO
Sensory stimuli drive the maturation and function of the mammalian nervous system in part through the activation of gene expression networks that regulate synapse development and plasticity. These networks have primarily been studied in mice, and it is not known whether there are species- or clade-specific activity-regulated genes that control features of brain development and function. Here we use transcriptional profiling of human fetal brain cultures to identify an activity-dependent secreted factor, Osteocrin (OSTN), that is induced by membrane depolarization of human but not mouse neurons. We find that OSTN has been repurposed in primates through the evolutionary acquisition of DNA regulatory elements that bind the activity-regulated transcription factor MEF2. In addition, we demonstrate that OSTN is expressed in primate neocortex and restricts activity-dependent dendritic growth in human neurons. These findings suggest that, in response to sensory input, OSTN regulates features of neuronal structure and function that are unique to primates.
Assuntos
Evolução Molecular , Proteínas Musculares/metabolismo , Neocórtex/metabolismo , Neurônios/metabolismo , Fatores de Transcrição/metabolismo , Transcriptoma , Animais , Sequência de Bases , Osso e Ossos/metabolismo , Dendritos/metabolismo , Elementos Facilitadores Genéticos/genética , Feminino , Humanos , Fatores de Transcrição MEF2/metabolismo , Macaca mulatta , Masculino , Camundongos , Dados de Sequência Molecular , Proteínas Musculares/genética , Músculos/metabolismo , Neocórtex/citologia , Neurônios/citologia , Especificidade de Órgãos , Especificidade da Espécie , Fatores de Transcrição/genéticaRESUMO
DNA methylation at CpG dinucleotides is an important epigenetic regulator common to virtually all mammalian cell types, but recent evidence indicates that during early postnatal development neuronal genomes also accumulate uniquely high levels of two alternative forms of methylation, non-CpG methylation and hydroxymethylation. Here we discuss the distinct landscape of DNA methylation in neurons, how it is established, and how it might affect the binding and function of protein readers of DNA methylation. We review studies of one critical reader of DNA methylation in the brain, the Rett syndrome protein methyl CpG-binding protein 2 (MeCP2), and discuss how differential binding affinity of MeCP2 for non-CpG and hydroxymethylation may affect the function of this methyl-binding protein in the nervous system.
Assuntos
Encéfalo/metabolismo , Metilação de DNA/fisiologia , Regulação da Expressão Gênica/fisiologia , Proteína 2 de Ligação a Metil-CpG/metabolismo , Modelos Biológicos , Neurônios/metabolismo , Animais , Citosina/química , Humanos , Estrutura Molecular , Ligação ProteicaRESUMO
Torpor and hibernation are extreme physiological adaptations of homeotherms associated with pro-longevity effects. Yet the underlying mechanisms of how torpor affects aging, and whether hypothermic and hypometabolic states can be induced to slow aging and increase health span, remain unknown. We demonstrate that the activity of a spatially defined neuronal population in the avMLPA, which has previously been identified as a torpor-regulating brain region, is sufficient to induce a torpor like state (TLS) in mice. Prolonged induction of TLS slows epigenetic aging across multiple tissues and improves health span. We isolate the effects of decreased metabolic rate, long-term caloric restriction, and decreased core body temperature (Tb) on blood epigenetic aging and find that the pro-longevity effect of torpor-like states is mediated by decreased Tb. Taken together, our findings provide novel mechanistic insight into the pro-longevity effects of torpor and hibernation and support the growing body of evidence that Tb is an important mediator of aging processes.
RESUMO
Cells must adapt to environmental changes to maintain homeostasis. One of the most striking environmental adaptations is entry into hibernation during which core body temperature can decrease from 37°C to as low at 4°C. How mammalian cells, which evolved to optimally function within a narrow range of temperatures, adapt to this profound decrease in temperature remains poorly understood. In this study, we conducted the first genome-scale CRISPR-Cas9 screen in cells derived from Syrian hamster, a facultative hibernator, as well as human cells to investigate the genetic basis of cold tolerance in a hibernator and a non-hibernator in an unbiased manner. Both screens independently revealed glutathione peroxidase 4 (GPX4), a selenium-containing enzyme, and associated proteins as critical for cold tolerance. We utilized genetic and pharmacological approaches to demonstrate that GPX4 is active in the cold and its catalytic activity is required for cold tolerance. Furthermore, we show that the role of GPX4 as a suppressor of cold-induced cell death extends across hibernating species, including 13-lined ground squirrels and greater horseshoe bats, highlighting the evolutionary conservation of this mechanism of cold tolerance. This study identifies GPX4 as a central modulator of mammalian cold tolerance and advances our understanding of the evolved mechanisms by which cells mitigate cold-associated damage-one of the most common challenges faced by cells and organisms in nature.
RESUMO
Mutations in MECP2 give rise to Rett syndrome (RTT), an X-linked neurodevelopmental disorder that results in broad cognitive impairments in females. While the exact etiology of RTT symptoms remains unknown, one possible explanation for its clinical presentation is that loss of MeCP2 causes miswiring of neural circuits due to defects in the brain's capacity to respond to changes in neuronal activity and sensory experience. Here we show that MeCP2 is phosphorylated at four residues in the brain (S86, S274, T308, and S421) in response to neuronal activity, and we generate a quadruple knock-in (QKI) mouse line in which all four activity-dependent sites are mutated to alanines to prevent phosphorylation. QKI mice do not display overt RTT phenotypes or detectable gene expression changes in two brain regions. However, electrophysiological recordings from the retinogeniculate synapse of QKI mice reveal that while synapse elimination is initially normal at P14, it is significantly compromised at P20. Notably, this phenotype is distinct from that previously reported for Mecp2 null mice, where synapses initially refine but then regress after the third postnatal week. We thus propose a model in which activity-induced phosphorylation of MeCP2 is critical for the proper timing of retinogeniculate synapse maturation specifically during the early postnatal period. SIGNIFICANCE STATEMENT: Rett syndrome (RTT) is an X-linked neurodevelopmental disorder that predominantly affects girls. RTT is caused by loss of function mutations in a single gene MeCP2. Girls with RTT develop normally during their first year of life, but then experience neurological abnormalities including breathing and movement difficulties, loss of speech, and seizures. This study investigates the function of the MeCP2 protein in the brain, and how MeCP2 activity is modulated by sensory experience in early life. Evidence is presented that sensory experience affects MeCP2 function, and that this is required for synaptic pruning in the brain. These findings provide insight into MeCP2 function, and clues as to what goes awry in the brain when the function of MeCP2 is disrupted.
RESUMO
The precise regulation of gene expression is fundamental to neurodevelopment, plasticity and cognitive function. Although several studies have profiled transcription in the developing human brain, there is a gap in understanding of accompanying translational regulation. In this study, we performed ribosome profiling on 73 human prenatal and adult cortex samples. We characterized the translational regulation of annotated open reading frames (ORFs) and identified thousands of previously unknown translation events, including small ORFs that give rise to human-specific and/or brain-specific microproteins, many of which we independently verified using proteomics. Ribosome profiling in stem-cell-derived human neuronal cultures corroborated these findings and revealed that several neuronal activity-induced non-coding RNAs encode previously undescribed microproteins. Physicochemical analysis of brain microproteins identified a class of proteins that contain arginine-glycine-glycine (RGG) repeats and, thus, may be regulators of RNA metabolism. This resource expands the known translational landscape of the human brain and illuminates previously unknown brain-specific protein products.
Assuntos
Regulação da Expressão Gênica , Biossíntese de Proteínas , Adulto , Arginina/genética , Arginina/metabolismo , Encéfalo/metabolismo , Glicina , Humanos , RNA Mensageiro/metabolismoRESUMO
We report the results of a genetic screen to identify molecules important for synapse formation and/or maintenance. siRNAs were used to decrease the expression of candidate genes in neurons, and synapse development was assessed. We surveyed 22 cadherin family members and demonstrated distinct roles for cadherin-11 and cadherin-13 in synapse development. Our screen also revealed roles for the class 4 Semaphorins Sema4B and Sema4D in the development of glutamatergic and/or GABAergic synapses. We found that Sema4D affects the formation of GABAergic, but not glutamatergic, synapses. Our screen also identified the activity-regulated small GTPase Rem2 as a regulator of synapse development. A known calcium channel modulator, Rem2 may function as part of a homeostatic mechanism that controls synapse number. These experiments establish the feasibility of RNAi screens to characterize the mechanisms that control mammalian neuronal development and to identify components of the genetic program that regulate synapse formation and/or maintenance.
Assuntos
Ácido Glutâmico/metabolismo , Proteínas do Tecido Nervoso/fisiologia , Interferência de RNA , Sinapses/fisiologia , Ácido gama-Aminobutírico/metabolismo , Animais , Caderinas/fisiologia , Estudos de Viabilidade , Humanos , Biologia Molecular , Proteínas Monoméricas de Ligação ao GTP/fisiologia , RNA Interferente Pequeno , Semaforinas/classificação , Semaforinas/fisiologiaRESUMO
Neuronal activity-dependent gene expression is essential for brain development. Although transcriptional and epigenetic effects of neuronal activity have been explored in mice, such an investigation is lacking in humans. Because alterations in GABAergic neuronal circuits are implicated in neurological disorders, we conducted a comprehensive activity-dependent transcriptional and epigenetic profiling of human induced pluripotent stem cell-derived GABAergic neurons similar to those of the early developing striatum. We identified genes whose expression is inducible after membrane depolarization, some of which have specifically evolved in primates and/or are associated with neurological diseases, including schizophrenia and autism spectrum disorder (ASD). We define the genome-wide profile of human neuronal activity-dependent enhancers, promoters and the transcription factors CREB and CRTC1. We found significant heritability enrichment for ASD in the inducible promoters. Our results suggest that sequence variation within activity-inducible promoters of developing human forebrain GABAergic neurons contributes to ASD risk.
Assuntos
Encéfalo/metabolismo , Epigênese Genética , Neurônios GABAérgicos/metabolismo , Regulação da Expressão Gênica , Estudo de Associação Genômica Ampla , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Regiões Promotoras GenéticasRESUMO
Maternal infection and inflammation during pregnancy are associated with neurodevelopmental disorders in offspring, but little is understood about the molecular mechanisms underlying this epidemiologic phenomenon. Here, we leveraged single-cell RNA sequencing to profile transcriptional changes in the mouse fetal brain in response to maternal immune activation (MIA) and identified perturbations in cellular pathways associated with mRNA translation, ribosome biogenesis and stress signaling. We found that MIA activates the integrated stress response (ISR) in male, but not female, MIA offspring in an interleukin-17a-dependent manner, which reduced global mRNA translation and altered nascent proteome synthesis. Moreover, blockade of ISR activation prevented the behavioral abnormalities as well as increased cortical neural activity in MIA male offspring. Our data suggest that sex-specific activation of the ISR leads to maternal inflammation-associated neurodevelopmental disorders.
Assuntos
Encéfalo/imunologia , Feto/imunologia , Imunidade Inata/genética , Proteostase/genética , Animais , Comportamento Animal , Deficiências do Desenvolvimento/genética , Feminino , Perfilação da Expressão Gênica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Gravidez , Biossíntese de Proteínas/genética , Proteoma/biossíntese , RNA/biossíntese , RNA/genética , RNA Interferente Pequeno , Caracteres Sexuais , Transdução de Sinais , Estresse Psicológico/genética , Estresse Psicológico/psicologiaRESUMO
Mutations or duplications in MECP2 cause Rett and Rett-like syndromes, neurodevelopmental disorders characterized by mental retardation, motor dysfunction, and autistic behaviors. MeCP2 is expressed in many mammalian tissues and functions as a global repressor of transcription; however, the molecular mechanisms by which MeCP2 dysfunction leads to the neural-specific phenotypes of RTT remain poorly understood. Here, we show that neuronal activity and subsequent calcium influx trigger the de novo phosphorylation of MeCP2 at serine 421 (S421) by a CaMKII-dependent mechanism. MeCP2 S421 phosphorylation is induced selectively in the brain in response to physiological stimuli. Significantly, we find that S421 phosphorylation controls the ability of MeCP2 to regulate dendritic patterning, spine morphogenesis, and the activity-dependent induction of Bdnf transcription. These findings suggest that, by triggering MeCP2 phosphorylation, neuronal activity regulates a program of gene expression that mediates nervous system maturation and that disruption of this process in individuals with mutations in MeCP2 may underlie the neural-specific pathology of RTT.
Assuntos
Fator Neurotrófico Derivado do Encéfalo/biossíntese , Encéfalo/crescimento & desenvolvimento , Encéfalo/metabolismo , Diferenciação Celular/fisiologia , Espinhas Dendríticas/metabolismo , Proteína 2 de Ligação a Metil-CpG/metabolismo , Animais , Encéfalo/citologia , Fator Neurotrófico Derivado do Encéfalo/genética , Sinalização do Cálcio/fisiologia , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Espinhas Dendríticas/ultraestrutura , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Proteína 2 de Ligação a Metil-CpG/genética , Vias Neurais/citologia , Vias Neurais/crescimento & desenvolvimento , Vias Neurais/metabolismo , Plasticidade Neuronal/fisiologia , Técnicas de Cultura de Órgãos , Especificidade de Órgãos/fisiologia , Fosforilação , Ratos , Síndrome de Rett/genética , Síndrome de Rett/metabolismo , Síndrome de Rett/fisiopatologia , Serina/metabolismo , Transmissão Sináptica/fisiologiaRESUMO
Ephrin signaling through Eph receptor tyrosine kinases can promote attraction or repulsion of axonal growth cones during development. However, the mechanisms that determine whether Eph signaling promotes attraction or repulsion are not known. We show here that the Rho family GEF Vav2 plays a key role in this process. We find that, during axon guidance, ephrin binding to Ephs triggers Vav-dependent endocytosis of the ligand-receptor complex, thus converting an initially adhesive interaction into a repulsive event. In the absence of Vav proteins, ephrin-Eph endocytosis is blocked, leading to defects in growth cone collapse in vitro and significant defects in the ipsilateral retinogeniculate projections in vivo. These findings suggest an important role for Vav family GEFs as regulators of ligand-receptor endocytosis and determinants of repulsive signaling during axon guidance.
Assuntos
Endocitose/fisiologia , Cones de Crescimento/metabolismo , Receptores da Família Eph/metabolismo , Transdução de Sinais/fisiologia , Proteínas rho de Ligação ao GTP/metabolismo , Animais , Efrinas/metabolismo , Camundongos , Camundongos Knockout , Microscopia Confocal , Técnicas do Sistema de Duplo-HíbridoRESUMO
Enhancers are the primary DNA regulatory elements that confer cell type specificity of gene expression. Recent studies characterizing individual enhancers have revealed their potential to direct heterologous gene expression in a highly cell-type-specific manner. However, it has not yet been possible to systematically identify and test the function of enhancers for each of the many cell types in an organism. We have developed PESCA, a scalable and generalizable method that leverages ATAC- and single-cell RNA-sequencing protocols, to characterize cell-type-specific enhancers that should enable genetic access and perturbation of gene function across mammalian cell types. Focusing on the highly heterogeneous mammalian cerebral cortex, we apply PESCA to find enhancers and generate viral reagents capable of accessing and manipulating a subset of somatostatin-expressing cortical interneurons with high specificity. This study demonstrates the utility of this platform for developing new cell-type-specific viral reagents, with significant implications for both basic and translational research.
Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , Biologia Molecular/métodos , Neurônios/efeitos dos fármacos , Neurofisiologia/métodos , Proteínas Recombinantes/biossíntese , Somatostatina/metabolismo , Vírus/genética , Animais , Animais Geneticamente Modificados , Córtex Cerebral/fisiologia , Genes Reguladores , Vetores Genéticos , Interneurônios/fisiologia , Camundongos , Proteínas Recombinantes/genéticaRESUMO
The transcription factor CREB mediates diverse responses in the nervous system. It is not known how CREB induces specific patterns of gene expression in response to different extracellular stimuli. We find that Ca(2+) influx into neurons induces CREB phosphorylation at Ser133 and two additional sites, Ser142 and Ser143. While CREB Ser133 phosphorylation is induced by many stimuli, phosphorylation at Ser142 and Ser143 is selectively activated by Ca(2+) influx. The triple phosphorylation of CREB is required for effective Ca(2+) stimulation of CREB-dependent transcription, but the phosphorylation of Ser142 and Ser143, in addition to Ser133, disrupts the interaction of CREB with its cofactor CBP. These results suggest that Ca(2+) influx triggers a specific program of gene expression in neurons by selectively regulating CREB phosphorylation.
Assuntos
Cálcio/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Neurônios/metabolismo , Sequência de Aminoácidos/genética , Proteína de Ligação a CREB , Células Cultivadas , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Eletrofisiologia , Imuno-Histoquímica , Neurônios/fisiologia , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Nucleares/fisiologia , Fosforilação , Transativadores/genética , Transativadores/metabolismo , Transativadores/fisiologia , Transcrição Gênica/fisiologiaRESUMO
In females with X-linked genetic disorders, wild-type and mutant cells coexist within brain tissue because of X-chromosome inactivation, posing challenges for interpreting the effects of X-linked mutant alleles on gene expression. We present a single-nucleus RNA sequencing approach that resolves mosaicism by using single-nucleotide polymorphisms in genes expressed in cis with the X-linked mutation to determine which nuclei express the mutant allele even when the mutant gene is not detected. This approach enables gene expression comparisons between mutant and wild-type cells within the same individual, eliminating variability introduced by comparisons to controls with different genetic backgrounds. We apply this approach to mosaic female mouse models and humans with Rett syndrome, an X-linked neurodevelopmental disorder caused by mutations in the gene encoding the methyl-DNA-binding protein MECP2, and observe that cell-type-specific DNA methylation predicts the degree of gene upregulation in MECP2-mutant neurons. This approach can be broadly applied to study gene expression in mosaic X-linked disorders.