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
Human telencephalon is an evolutionarily advanced brain structure associated with many uniquely human behaviors and disorders. However, cell lineages and molecular pathways implicated in human telencephalic development remain largely unknown. We produce human telencephalic organoids from stem cell-derived single neural rosettes and investigate telencephalic development under normal and pathological conditions. We show that single neural rosette-derived organoids contain pallial and subpallial neural progenitors, excitatory and inhibitory neurons, as well as macroglial and periendothelial cells, and exhibit predictable organization and cytoarchitecture. We comprehensively characterize the properties of neurons in SNR-derived organoids and identify transcriptional programs associated with the specification of excitatory and inhibitory neural lineages from a common pool of NPs early in telencephalic development. We also demonstrate that neurons in organoids with a hemizygous deletion of an autism- and intellectual disability-associated gene SHANK3 exhibit intrinsic and excitatory synaptic deficits and impaired expression of several clustered protocadherins. Collectively, this study validates SNR-derived organoids as a reliable model for studying human telencephalic cortico-striatal development and identifies intrinsic, synaptic, and clustered protocadherin expression deficits in human telencephalic tissue with SHANK3 hemizygosity.
Assuntos
Transtorno Autístico , Transtorno Autístico/genética , Humanos , Proteínas do Tecido Nervoso/metabolismo , Organoides/metabolismo , Protocaderinas , TelencéfaloRESUMO
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
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 interplay of transcription factors and cis-regulatory elements (CREs) orchestrates the dynamic and diverse genetic programs that assemble the human central nervous system (CNS) during development and maintain its function throughout life. Genetic variation within CREs plays a central role in phenotypic variation in complex traits including the risk of developing disease. We took advantage of the retina, a well-characterized region of the CNS known to be affected by pathogenic variants in CREs, to establish a roadmap for characterizing regulatory variation in the human CNS. This comprehensive analysis of tissue-specific regulatory elements, transcription factor binding, and gene expression programs in three regions of the human visual system (retina, macula, and retinal pigment epithelium/choroid) reveals features of regulatory element evolution that shape tissue-specific gene expression programs and defines regulatory elements with the potential to contribute to Mendelian and complex disorders of human vision.
Assuntos
Evolução Molecular , Regulação da Expressão Gênica no Desenvolvimento , Sequências Reguladoras de Ácido Nucleico/genética , Retina/patologia , Doenças Retinianas/genética , Adulto , Animais , Análise Mutacional de DNA , Epigenômica , Feminino , Variação Genética , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Mutação , RNA-Seq , Retina/crescimento & desenvolvimento , Doenças Retinianas/patologia , Especificidade da EspécieRESUMO
Sensory experience influences the establishment of neural connectivity through molecular mechanisms that remain unclear. Here, we employ single-nucleus RNA sequencing to investigate the contribution of sensory-driven gene expression to synaptic refinement in the dorsal lateral geniculate nucleus of the thalamus, a region of the brain that processes visual information. We find that visual experience induces the expression of the cytokine receptor Fn14 in excitatory thalamocortical neurons. By combining electrophysiological and structural techniques, we show that Fn14 is dispensable for early phases of refinement mediated by spontaneous activity but that Fn14 is essential for refinement during a later, experience-dependent period of development. Refinement deficits in mice lacking Fn14 are associated with functionally weaker and structurally smaller retinogeniculate inputs, indicating that Fn14 mediates both functional and anatomical rearrangements in response to sensory experience. These findings identify Fn14 as a molecular link between sensory-driven gene expression and vision-sensitive refinement in the brain.
Assuntos
Corpos Geniculados/metabolismo , Células Ganglionares da Retina/metabolismo , Receptor de TWEAK/biossíntese , Percepção Visual/fisiologia , Animais , Feminino , Expressão Gênica , Corpos Geniculados/crescimento & desenvolvimento , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Trato Óptico/crescimento & desenvolvimento , Trato Óptico/metabolismo , Retina/metabolismo , Receptor de TWEAK/genéticaRESUMO
Enhancer elements are genomic regulatory sequences that direct the selective expression of genes so that genetically identical cells can differentiate and acquire the highly specialized forms and functions required to build a functioning animal. To differentiate, cells must select from among the â¼106 enhancers encoded in the genome the thousands of enhancers that drive the gene programs that impart their distinct features. We used a genetic approach to identify transcription factors (TFs) required for enhancer selection in fibroblasts. This revealed that the broadly expressed, growth-factor-inducible TFs FOS/JUN (AP-1) play a central role in enhancer selection. FOS/JUN selects enhancers together with cell-type-specific TFs by collaboratively binding to nucleosomal enhancers and recruiting the SWI/SNF (BAF) chromatin remodeling complex to establish accessible chromatin. These experiments demonstrate how environmental signals acting via FOS/JUN and BAF coordinate with cell-type-specific TFs to select enhancer repertoires that enable differentiation during development.
Assuntos
Cromatina/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Elementos Facilitadores Genéticos , Proteínas Proto-Oncogênicas c-fos/fisiologia , Fatores de Transcrição/metabolismo , Fatores de Transcrição/fisiologia , Animais , Cromatina/genética , Proteínas Cromossômicas não Histona/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Nucleossomos , Regiões Promotoras Genéticas , Fatores de Transcrição/genéticaRESUMO
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
Bipolar disorder (BD) is a prevalent and severe mood disorder characterized by recurrent episodes of mania and depression. Both genetic and environmental factors have been implicated in BD etiology, but the biological underpinnings remain elusive. Recent genome-wide association studies (GWAS) for identifying genes conferring risk for schizophrenia, BD, and major depression, identified an association between single-nucleotide polymorphisms (SNPs) in the SYNE1 gene and increased risk of BD. SYNE1 has also been identified as a risk locus for multiple other neurological or neuromuscular genetic disorders. The BD associated SNPs map within the gene region homologous to part of rat Syne1 encompassing the brain specific transcripts encoding CPG2, a postsynaptic neuronal protein localized to excitatory synapses and an important regulator of glutamate receptor internalization. Here, we use RNA-seq, ChIP-seq and RACE to map the human SYNE1 transcriptome, focusing on the CPG2 locus. We validate several CPG2 transcripts, including ones not previously annotated in public databases, and identify and clone a full-length CPG2 cDNA expressed in human neocortex, hippocampus and striatum. Using lenti-viral gene knock down/replacement and surface receptor internalization assays, we demonstrate that human CPG2 protein localizes to dendritic spines in rat hippocampal neurons and is functionally equivalent to rat CPG2 in regulating glutamate receptor internalization. This study provides a valuable gene-mapping framework for relating multiple genetic disease loci in SYNE1 with their transcripts, and for evaluating the effects of missense SNPs identified by patient genome sequencing on neuronal function.
Assuntos
Genoma Humano , Proteínas do Tecido Nervoso/genética , Proteínas Nucleares/genética , Animais , Encéfalo/citologia , Encéfalo/metabolismo , Células Cultivadas , Mapeamento Cromossômico/métodos , Proteínas do Citoesqueleto , Espinhas Dendríticas/metabolismo , Endocitose , Células HEK293 , Humanos , Proteínas do Tecido Nervoso/metabolismo , Proteínas Nucleares/metabolismo , Polimorfismo de Nucleotídeo Único , Ratos , Receptores de Glutamato/metabolismo , Sinapses/metabolismo , TranscriptomaRESUMO
Disruption of the MECP2 gene leads to Rett syndrome (RTT), a severe neurological disorder with features of autism. MECP2 encodes a methyl-DNA-binding protein that has been proposed to function as a transcriptional repressor, but despite numerous mouse studies examining neuronal gene expression in Mecp2 mutants, no clear model has emerged for how MeCP2 protein regulates transcription. Here we identify a genome-wide length-dependent increase in gene expression in MeCP2 mutant mouse models and human RTT brains. We present evidence that MeCP2 represses gene expression by binding to methylated CA sites within long genes, and that in neurons lacking MeCP2, decreasing the expression of long genes attenuates RTT-associated cellular deficits. In addition, we find that long genes as a population are enriched for neuronal functions and selectively expressed in the brain. These findings suggest that mutations in MeCP2 may cause neurological dysfunction by specifically disrupting long gene expression in the brain.
Assuntos
Metilação de DNA/genética , Proteína 2 de Ligação a Metil-CpG/genética , Proteína 2 de Ligação a Metil-CpG/metabolismo , Mutação/genética , Síndrome de Rett/genética , Animais , Sequência de Bases , Encéfalo/metabolismo , DNA (Citosina-5-)-Metiltransferases/metabolismo , DNA Metiltransferase 3A , Modelos Animais de Doenças , Feminino , Regulação da Expressão Gênica , Humanos , Masculino , Proteína 2 de Ligação a Metil-CpG/deficiência , Camundongos , Dados de Sequência Molecular , Neurônios/metabolismoRESUMO
Organismal development requires the precise coordination of genetic programs to regulate cell fate and function. MEF2 transcription factors (TFs) play essential roles in this process but how these broadly expressed factors contribute to the generation of specific cell types during development is poorly understood. Here we show that despite being expressed in virtually all mammalian tissues, in the retina MEF2D binds to retina-specific enhancers and controls photoreceptor cell development. MEF2D achieves specificity by cooperating with a retina-specific factor CRX, which recruits MEF2D away from canonical MEF2 binding sites and redirects it to retina-specific enhancers that lack the consensus MEF2-binding sequence. Once bound to retina-specific enhancers, MEF2D and CRX co-activate the expression of photoreceptor-specific genes that are critical for retinal function. These findings demonstrate that broadly expressed TFs acquire specific functions through competitive recruitment to enhancers by tissue-specific TFs and through selective activation of these enhancers to regulate tissue-specific genes.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento/genética , Proteínas de Homeodomínio/metabolismo , Células Fotorreceptoras/fisiologia , Retina/citologia , Transativadores/metabolismo , Adaptação Ocular/genética , Fatores Etários , Animais , Animais Recém-Nascidos , Imunoprecipitação da Cromatina , Eletrorretinografia , Embrião de Mamíferos , Proteínas do Olho/metabolismo , Genoma , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Fatores de Transcrição MEF2/genética , Fatores de Transcrição MEF2/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutação/genética , Retina/crescimento & desenvolvimentoRESUMO
Experience-dependent gene transcription is required for nervous system development and function. However, the DNA regulatory elements that control this program of gene expression are not well defined. Here we characterize the enhancers that function across the genome to mediate activity-dependent transcription in mouse cortical neurons. We find that the subset of enhancers enriched for monomethylation of histone H3 Lys4 (H3K4me1) and binding of the transcriptional coactivator CREBBP (also called CBP) that shows increased acetylation of histone H3 Lys27 (H3K27ac) after membrane depolarization of cortical neurons functions to regulate activity-dependent transcription. A subset of these enhancers appears to require binding of FOS, which was previously thought to bind primarily to promoters. These findings suggest that FOS functions at enhancers to control activity-dependent gene programs that are critical for nervous system function and provide a resource of functional cis-regulatory elements that may give insight into the genetic variants that contribute to brain development and disease.
Assuntos
Regulação da Expressão Gênica/genética , Neurônios/fisiologia , 2-Amino-5-fosfonovalerato/farmacologia , Animais , Proteína de Ligação a CREB/metabolismo , Embrião de Mamíferos , Antagonistas de Aminoácidos Excitatórios/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Estudo de Associação Genômica Ampla , Humanos , Histona Desmetilases com o Domínio Jumonji/metabolismo , Fatores de Transcrição MEF2/genética , Fatores de Transcrição MEF2/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Mutação/genética , Neurônios/efeitos dos fármacos , Proteínas Oncogênicas v-fos/metabolismo , Cloreto de Potássio/farmacologia , Bloqueadores dos Canais de Sódio/farmacologia , Tetrodotoxina/farmacologia , Fatores de Tempo , Córtex Visual/citologiaRESUMO
The nervous system adapts to experience by inducing a transcriptional program that controls important aspects of synaptic plasticity. Although the molecular mechanisms of experience-dependent plasticity are well characterized in excitatory neurons, the mechanisms that regulate this process in inhibitory neurons are only poorly understood. Here, we describe a transcriptional program that is induced by neuronal activity in inhibitory neurons. We find that, while neuronal activity induces expression of early-response transcription factors such as Npas4 in both excitatory and inhibitory neurons, Npas4 activates distinct programs of late-response genes in inhibitory and excitatory neurons. These late-response genes differentially regulate synaptic input to these two types of neurons, promoting inhibition onto excitatory neurons while inducing excitation onto inhibitory neurons. These findings suggest that the functional outcomes of activity-induced transcriptional responses are adapted in a cell-type-specific manner to achieve a circuit-wide homeostatic response.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Regulação da Expressão Gênica , Neurônios/metabolismo , Transcrição Gênica , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Técnicas de Cultura de Células , Embrião de Mamíferos/citologia , Camundongos , Camundongos Knockout , Sinapses/metabolismoRESUMO
mRNA synthesis, processing, and destruction involve a complex series of molecular steps that are incompletely understood. Because the RNA intermediates in each of these steps have finite lifetimes, extensive mechanistic and dynamical information is encoded in total cellular RNA. Here we report the development of SnapShot-Seq, a set of computational methods that allow the determination of in vivo rates of pre-mRNA synthesis, splicing, intron degradation, and mRNA decay from a single RNA-Seq snapshot of total cellular RNA. SnapShot-Seq can detect in vivo changes in the rates of specific steps of splicing, and it provides genome-wide estimates of pre-mRNA synthesis rates comparable to those obtained via labeling of newly synthesized RNA. We used SnapShot-Seq to investigate the origins of the intrinsic bimodality of metazoan gene expression levels, and our results suggest that this bimodality is partly due to spillover of transcriptional activation from highly expressed genes to their poorly expressed neighbors. SnapShot-Seq dramatically expands the information obtainable from a standard RNA-Seq experiment.
Assuntos
RNA Mensageiro/metabolismo , Processamento Alternativo , Biflavonoides/farmacologia , Células HeLa/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Íntrons , Modelos Teóricos , Método de Monte Carlo , RNA/genética , Precursores de RNA , Splicing de RNA , Estabilidade de RNA , RNA Mensageiro/genética , Análise de Sequência de RNA/métodos , Transcrição GênicaRESUMO
Despite significant heritability of autism spectrum disorders (ASDs), their extreme genetic heterogeneity has proven challenging for gene discovery. Studies of primarily simplex families have implicated de novo copy number changes and point mutations, but are not optimally designed to identify inherited risk alleles. We apply whole-exome sequencing (WES) to ASD families enriched for inherited causes due to consanguinity and find familial ASD associated with biallelic mutations in disease genes (AMT, PEX7, SYNE1, VPS13B, PAH, and POMGNT1). At least some of these genes show biallelic mutations in nonconsanguineous families as well. These mutations are often only partially disabling or present atypically, with patients lacking diagnostic features of the Mendelian disorders with which these genes are classically associated. Our study shows the utility of WES for identifying specific genetic conditions not clinically suspected and the importance of partial loss of gene function in ASDs.
Assuntos
Transtorno Autístico/diagnóstico , Transtorno Autístico/genética , Exoma/genética , Estudo de Associação Genômica Ampla/métodos , Adolescente , Animais , Células Cultivadas , Criança , Pré-Escolar , Estudos de Coortes , Feminino , Humanos , Masculino , Linhagem , Ratos , Análise de Sequência de DNA/métodos , Adulto JovemRESUMO
Autism spectrum disorders such as Rett syndrome (RTT) have been hypothesized to arise from defects in experience-dependent synapse maturation. RTT is caused by mutations in MECP2, a nuclear protein that becomes phosphorylated at S421 in response to neuronal activation. We show here that disruption of MeCP2 S421 phosphorylation in vivo results in defects in synapse development and behavior, implicating activity-dependent regulation of MeCP2 in brain development and RTT. We investigated the mechanism by which S421 phosphorylation regulates MeCP2 function and show by chromatin immunoprecipitation-sequencing that this modification occurs on MeCP2 bound across the genome. The phosphorylation of MeCP2 S421 appears not to regulate the expression of specific genes; rather, MeCP2 functions as a histone-like factor whose phosphorylation may facilitate a genome-wide response of chromatin to neuronal activity during nervous system development. We propose that RTT results in part from a loss of this experience-dependent chromatin remodeling.
Assuntos
Encéfalo/crescimento & desenvolvimento , Comportamento Exploratório/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Genoma/fisiologia , Proteína 2 de Ligação a Metil-CpG/genética , Proteína 2 de Ligação a Metil-CpG/fisiologia , Animais , Encéfalo/metabolismo , Encéfalo/fisiologia , Cromatina/metabolismo , Imunoprecipitação da Cromatina/métodos , Dendritos/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/genética , Técnicas de Introdução de Genes/métodos , Proteína 2 de Ligação a Metil-CpG/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/citologia , Neurônios/metabolismo , Neurônios/fisiologia , FosforilaçãoRESUMO
We used genome-wide sequencing methods to study stimulus-dependent enhancer function in mouse cortical neurons. We identified approximately 12,000 neuronal activity-regulated enhancers that are bound by the general transcriptional co-activator CBP in an activity-dependent manner. A function of CBP at enhancers may be to recruit RNA polymerase II (RNAPII), as we also observed activity-regulated RNAPII binding to thousands of enhancers. Notably, RNAPII at enhancers transcribes bi-directionally a novel class of enhancer RNAs (eRNAs) within enhancer domains defined by the presence of histone H3 monomethylated at lysine 4. The level of eRNA expression at neuronal enhancers positively correlates with the level of messenger RNA synthesis at nearby genes, suggesting that eRNA synthesis occurs specifically at enhancers that are actively engaged in promoting mRNA synthesis. These findings reveal that a widespread mechanism of enhancer activation involves RNAPII binding and eRNA synthesis.
Assuntos
Elementos Facilitadores Genéticos/genética , Regulação da Expressão Gênica/genética , Neurônios/metabolismo , Transcrição Gênica/genética , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Proteína de Ligação a CREB/metabolismo , Sequência Consenso/genética , Proteínas do Citoesqueleto/genética , Genes Reporter , Genes fos/genética , Histonas/metabolismo , Metilação , Camundongos , Camundongos Endogâmicos C57BL , Proteínas do Tecido Nervoso/genética , RNA Polimerase II/metabolismo , RNA não Traduzido/biossíntese , RNA não Traduzido/genéticaRESUMO
Although many transcription factors are known to control important aspects of neural development, the genome-wide programs that are directly regulated by these factors are not known. We have characterized the genetic program that is activated by MEF2, a key regulator of activity-dependent synapse development. These MEF2 target genes have diverse functions at synapses, revealing a broad role for MEF2 in synapse development. Several of the MEF2 targets are mutated in human neurological disorders including epilepsy and autism spectrum disorders, suggesting that these disorders may be caused by disruption of an activity-dependent gene program that controls synapse development. Our analyses also reveal that neuronal activity promotes alternative polyadenylation site usage at many of the MEF2 target genes, leading to the production of truncated mRNAs that may have different functions than their full-length counterparts. Taken together, these analyses suggest that the ubiquitously expressed transcription factor MEF2 regulates an intricate transcriptional program in neurons that controls synapse development.
Assuntos
Genômica , Neurônios/fisiologia , Poliadenilação/genética , Sinapses/genética , Transcrição Gênica/fisiologia , Análise de Variância , Animais , Mapeamento Encefálico , Núcleo Celular/genética , Células Cultivadas , Imunoprecipitação da Cromatina , Biologia Computacional , RNA Polimerases Dirigidas por DNA/metabolismo , Embrião de Mamíferos , Comportamento Exploratório , Hipocampo/citologia , Humanos , Fatores de Transcrição MEF2 , Masculino , Fatores de Regulação Miogênica/metabolismo , Doenças do Sistema Nervoso/genética , Neurônios/citologia , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Estimulação Luminosa/métodos , Ratos , Ratos Long-Evans , Córtex Visual/fisiologiaRESUMO
UNLABELLED: The serial analysis of chromatin occupancy technique (SACO) promises to become a widely used method for the unbiased genome-wide experimental identification of loci bound by a transcription factor of interest. We describe the first web-based automatic tool, termed sequence tag analysis and reporting tool (START), for processing SACO data generated by experiments performed for the yeast, fruit fly, mouse, rat or human genomes. The program uses as input sequences of inserts from a SACO library from which it extracts all SACO tags, maps them to genomic locations and annotates them. START returns detailed information about these tags including the genes, the genomic elements and the miRNA precursors found in their vicinity, and makes use of the MAPPER database to identify putative transcription factor binding sites located close to the tags. AVAILABILITY: The program is available at http://bio.chip.org/start/. SUPPLEMENTARY INFORMATION: SUPPLEMENTARY INFORMATION is available at http://bio.chip.org/doc/start/START-supplementary.pdf