RESUMO
Long noncoding RNAs (lncRNAs) are transcribed elements increasingly recognized for their roles in regulating gene expression. Thus far, however, we have little understanding of how lncRNAs contribute to evolution and adaptation. Here, we show that a conserved lncRNA, ivory, is an important color patterning gene in the buckeye butterfly Junonia coenia. ivory overlaps with cortex, a locus linked to multiple cases of crypsis and mimicry in Lepidoptera. Along with a companion paper by Livraghi et al., we argue that ivory, not cortex, is the color pattern gene of interest at this locus. In J. coenia, a cluster of cis-regulatory elements (CREs) in the first intron of ivory are genetically associated with natural variation in seasonal color pattern plasticity, and targeted deletions of these CREs phenocopy seasonal phenotypes. Deletions of different ivory CREs produce other distinct phenotypes as well, including loss of melanic eyespot rings, and positive and negative changes in overall wing pigmentation. We show that the color pattern transcription factors Spineless, Bric-a-brac, and Ftz-f1 bind to the ivory promoter during wing pattern development, suggesting that they directly regulate ivory. This case study demonstrates how cis-regulation of a single noncoding RNA can exert diverse and nuanced effects on the evolution and development of color patterns, including modulating seasonally plastic color patterns.
Assuntos
Borboletas , Pigmentação , RNA Longo não Codificante , Estações do Ano , Animais , Borboletas/genética , Borboletas/fisiologia , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Pigmentação/genética , Asas de Animais , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Fenótipo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
There is evidence that transcription factor (TF) encoding genes, which temporally control development in multiple cell types, can have tens of enhancers that regulate their expression. The NR2F1 TF developmentally promotes caudal and ventral cortical regional fates. Here, we epigenomically compared the activity of Nr2f1's enhancers during mouse cortical development with their activity in a transgenic assay. We identified at least six that are likely to be important in prenatal cortical development, with three harboring de novo mutants identified in ASD individuals. We chose to study the function of two of the most robust enhancers by deleting them singly or together. We found that they have distinct and overlapping functions in driving Nr2f1's regional and laminar expression in the developing cortex. Thus, these two enhancers, probably in combination with the others that we defined epigenetically, precisely tune Nr2f1's regional, cell type, and temporal expression during corticogenesis.
Assuntos
Fator I de Transcrição COUP , Córtex Cerebral , Elementos Facilitadores Genéticos , Regulação da Expressão Gênica no Desenvolvimento , Animais , Fator I de Transcrição COUP/metabolismo , Fator I de Transcrição COUP/genética , Camundongos , Córtex Cerebral/metabolismo , Córtex Cerebral/embriologia , Camundongos Transgênicos , Humanos , FemininoRESUMO
Using machine learning (ML), we interrogated the function of all human-chimpanzee variants in 2,645 human accelerated regions (HARs), finding 43% of HARs have variants with large opposing effects on chromatin state and 14% on neurodevelopmental enhancer activity. This pattern, consistent with compensatory evolution, was confirmed using massively parallel reporter assays in chimpanzee and human neural progenitor cells. The species-specific enhancer activity of HARs was accurately predicted from the presence and absence of transcription factor footprints in each species. Despite these striking cis effects, activity of a given HAR sequence was nearly identical in human and chimpanzee cells. This suggests that HARs did not evolve to compensate for changes in the trans environment but instead altered their ability to bind factors present in both species. Thus, ML prioritized variants with functional effects on human neurodevelopment and revealed an unexpected reason why HARs may have evolved so rapidly.
Assuntos
Encéfalo , Elementos Facilitadores Genéticos , Pan troglodytes , Animais , Humanos , Cromatina , Aprendizado de Máquina , Pan troglodytes/metabolismo , Fatores de Transcrição/genética , Encéfalo/crescimento & desenvolvimentoRESUMO
Deleterious genetic variants in POGZ, which encodes the chromatin regulator Pogo Transposable Element with ZNF Domain protein, are strongly associated with autism spectrum disorder (ASD). Although it is a high-confidence ASD risk gene, the neurodevelopmental functions of POGZ remain unclear. Here we reveal the genomic binding of POGZ in the developing forebrain at euchromatic loci and gene regulatory elements (REs). We profile chromatin accessibility and gene expression in Pogz-/- mice and show that POGZ promotes the active chromatin state and transcription of clustered synaptic genes. We further demonstrate that POGZ forms a nuclear complex and co-occupies loci with ADNP, another high-confidence ASD risk gene, and provide evidence that POGZ regulates other neurodevelopmental disorder risk genes as well. Our results reveal a neurodevelopmental function of an ASD risk gene and identify molecular targets that may elucidate its function in ASD.
Assuntos
Transtorno Autístico/enzimologia , Encéfalo/enzimologia , Proteínas de Ciclo Celular/fisiologia , Montagem e Desmontagem da Cromatina , Proteínas de Ligação a DNA/fisiologia , Eucromatina/metabolismo , Sinapses/enzimologia , Transposases/metabolismo , Animais , Transtorno Autístico/genética , Transtorno Autístico/fisiopatologia , Sítios de Ligação , Encéfalo/crescimento & desenvolvimento , Proteínas de Ciclo Celular/genética , Elementos de DNA Transponíveis , Proteínas de Ligação a DNA/genética , Elementos Facilitadores Genéticos , Eucromatina/genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Predisposição Genética para Doença , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Neurogênese , Regiões Promotoras Genéticas , Sinapses/genética , Transposases/genéticaRESUMO
Many genes have been linked to autism. However, it remains unclear what long-term changes in neural circuitry result from disruptions in these genes, and how these circuit changes might contribute to abnormal behaviors. To address these questions, we studied behavior and physiology in mice heterozygous for Pogz, a high confidence autism gene. Pogz+/- mice exhibit reduced anxiety-related avoidance in the elevated plus maze (EPM). Theta-frequency communication between the ventral hippocampus (vHPC) and medial prefrontal cortex (mPFC) is known to be necessary for normal avoidance in the EPM. We found deficient theta-frequency synchronization between the vHPC and mPFC in vivo. When we examined vHPC-mPFC communication at higher resolution, vHPC input onto prefrontal GABAergic interneurons was specifically disrupted, whereas input onto pyramidal neurons remained intact. These findings illustrate how the loss of a high confidence autism gene can impair long-range communication by causing inhibitory circuit dysfunction within pathways important for specific behaviors.
Assuntos
Ansiedade/genética , Transtorno Autístico/genética , Transposases/genética , Animais , Ansiedade/fisiopatologia , Transtorno Autístico/fisiopatologia , Aprendizagem da Esquiva , Comunicação , Feminino , Heterozigoto , Hipocampo/fisiopatologia , Interneurônios , Masculino , Camundongos , Neurociências , Córtex Pré-Frontal/fisiopatologia , Células Piramidais , Ritmo Teta , Transposases/metabolismoRESUMO
To discover regulatory elements driving the specificity of gene expression in different cell types and regions of the developing human brain, we generated an atlas of open chromatin from nine dissected regions of the mid-gestation human telencephalon, as well as microdissected upper and deep layers of the prefrontal cortex. We identified a subset of open chromatin regions (OCRs), termed predicted regulatory elements (pREs), that are likely to function as developmental brain enhancers. pREs showed temporal, regional, and laminar differences in chromatin accessibility and were correlated with gene expression differences across regions and gestational ages. We identified two functional de novo variants in a pRE for autism risk gene SLC6A1, and using CRISPRa, demonstrated that this pRE regulates SCL6A1. Additionally, mouse transgenic experiments validated enhancer activity for pREs proximal to FEZF2 and BCL11A. Thus, this atlas serves as a resource for decoding neurodevelopmental gene regulation in health and disease.
Assuntos
Cromatina/genética , Cromatina/metabolismo , Elementos Facilitadores Genéticos , Regulação da Expressão Gênica no Desenvolvimento/genética , Córtex Pré-Frontal/embriologia , Telencéfalo/embriologia , Animais , Transtorno Autístico/genética , Linhagem Celular , Sequenciamento de Cromatina por Imunoprecipitação , Eucromatina/genética , Proteínas da Membrana Plasmática de Transporte de GABA/genética , Ontologia Genética , Predisposição Genética para Doença , Idade Gestacional , Humanos , Camundongos , Camundongos Transgênicos , Motivos de Nucleotídeos , Mutação Puntual , Córtex Pré-Frontal/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Análise Espaço-Temporal , Telencéfalo/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Whole-genome sequencing (WGS) has facilitated the first genome-wide evaluations of the contribution of de novo noncoding mutations to complex disorders. Using WGS, we identified 255,106 de novo mutations among sample genomes from members of 1902 quartet families in which one child, but not a sibling or their parents, was affected by autism spectrum disorder (ASD). In contrast to coding mutations, no noncoding functional annotation category, analyzed in isolation, was significantly associated with ASD. Casting noncoding variation in the context of a de novo risk score across multiple annotation categories, however, did demonstrate association with mutations localized to promoter regions. We found that the strongest driver of this promoter signal emanates from evolutionarily conserved transcription factor binding sites distal to the transcription start site. These data suggest that de novo mutations in promoter regions, characterized by evolutionary and functional signatures, contribute to ASD.
Assuntos
Transtorno do Espectro Autista/genética , Mutação , Regiões Promotoras Genéticas/genética , Sítios de Ligação/genética , Sequência Conservada , Análise Mutacional de DNA , Loci Gênicos , Variação Genética , Humanos , Linhagem , Risco , Fatores de Transcrição/metabolismoRESUMO
Genomic association studies of common or rare protein-coding variation have established robust statistical approaches to account for multiple testing. Here we present a comparable framework to evaluate rare and de novo noncoding single-nucleotide variants, insertion/deletions, and all classes of structural variation from whole-genome sequencing (WGS). Integrating genomic annotations at the level of nucleotides, genes, and regulatory regions, we define 51,801 annotation categories. Analyses of 519 autism spectrum disorder families did not identify association with any categories after correction for 4,123 effective tests. Without appropriate correction, biologically plausible associations are observed in both cases and controls. Despite excluding previously identified gene-disrupting mutations, coding regions still exhibited the strongest associations. Thus, in autism, the contribution of de novo noncoding variation is probably modest in comparison to that of de novo coding variants. Robust results from future WGS studies will require large cohorts and comprehensive analytical strategies that consider the substantial multiple-testing burden.
Assuntos
Transtorno do Espectro Autista/genética , Predisposição Genética para Doença/genética , Mutação INDEL/genética , Polimorfismo de Nucleotídeo Único/genética , Isoformas de Proteínas/genética , Feminino , Genoma/genética , Estudo de Associação Genômica Ampla/métodos , Humanos , MasculinoRESUMO
The realization that nuclear distribution of DNA, RNA, and proteins differs between cell types and developmental stages suggests that nuclear organization serves regulatory functions. Understanding the logic of nuclear architecture and how it contributes to differentiation and cell fate commitment remains challenging. Here, we use soft X-ray tomography (SXT) to image chromatin organization, distribution, and biophysical properties during neurogenesis in vivo. Our analyses reveal that chromatin with similar biophysical properties forms an elaborate connected network throughout the entire nucleus. Although this interconnectivity is present in every developmental stage, differentiation proceeds with concomitant increase in chromatin compaction and re-distribution of condensed chromatin toward the nuclear core. HP1ß, but not nucleosome spacing or phasing, regulates chromatin rearrangements because it governs both the compaction of chromatin and its interactions with the nuclear envelope. Our experiments introduce SXT as a powerful imaging technology for nuclear architecture.
Assuntos
Cromatina/metabolismo , Neurogênese , Tomografia por Raios X , Animais , Diferenciação Celular , Nucléolo Celular/metabolismo , Homólogo 5 da Proteína Cromobox , Proteínas Cromossômicas não Histona/metabolismo , Células Epiteliais/metabolismo , Heterocromatina/metabolismo , Imageamento Tridimensional , Camundongos Knockout , Neurônios/citologia , Neurônios/metabolismo , Bulbo Olfatório/citologiaRESUMO
Sprinkled throughout the genome are a million regulatory sequences called transcriptional enhancers that activate gene promoters in the right cells, at the right time. Enhancers endow the brain with its incredible diversity of cell types and also translate neural activity into gene induction. Thanks to rapid advances in genomic technologies, it is now possible to identify thousands of enhancers rapidly, test their transcriptional function en masse, and address their neurobiological functions via genome editing. Enhancers also promise to be a great technological opportunity for neuroscience, offering the potential for cell-type-specific genetic labeling and manipulation without the need for transgenesis. The objective of this review and the accompanying 2015 SfN mini-symposium is to highlight the use of new and emerging genomic technologies to probe enhancer function in the nervous system. SIGNIFICANCE STATEMENT: Transcriptional enhancers turn on genes in the right cells, at the right time. Enhancers are also the genomic sequences that encode the incredible diversity of cell types in the brain and enable the brain to turn genes on in response to new experiences. New technology enables enhancers to be found and manipulated. The study of enhancers promises to inform our understanding of brain development and function. The application of enhancer technology holds promise in accelerating basic neuroscience research and enabling gene therapies to be targeted to specific cell types in the brain.
Assuntos
Sistema Nervoso Central/fisiologia , Elementos Facilitadores Genéticos/genética , Genômica , Regiões Promotoras Genéticas/genética , Fatores de Transcrição/genética , Animais , Sistema Nervoso Central/citologia , Expressão Gênica/genética , Humanos , Modelos BiológicosRESUMO
The transcriptional activation of one out of ?2800 olfactory receptor (OR) alleles is a poorly understood process. Here, we identify a plethora of putative OR enhancers and study their in vivo activity in olfactory neurons. Distinguished by an unusual epigenetic signature, candidate OR enhancers are characterized by extensive interchromosomal interactions associated with OR transcription and share a similar pattern of transcription factor footprints. In particular, we establish the role of the transcription factor Bptf as a facilitator of both enhancer interactions and OR transcription. Our observations agree with the model whereby OR transcription occurs in the context of multiple interacting enhancers. Disruption of these interchromosomal interactions results in weak and multigenic OR expression, suggesting that the rare coincidence of numerous enhancers over a stochastically chosen OR may account for the singularity and robustness in OR transcription.
Assuntos
Elementos Facilitadores Genéticos , Receptores Odorantes/genética , Ativação Transcricional , Animais , Animais Geneticamente Modificados , Antígenos Nucleares/metabolismo , Camundongos , Proteínas do Tecido Nervoso/metabolismo , Nucleoproteínas/metabolismo , Neurônios Receptores Olfatórios/metabolismo , Fatores de Transcrição/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/metabolismoRESUMO
During differentiation, neurons exhibit a reorganization of DNA modification patterns across their genomes. The de novo DNA methyltransferase Dnmt3a is implicated in this process, but the effects of its absence have not been fully characterized in a purified neuronal population. To better understand how DNA modifications contribute to neuronal function, we performed a comprehensive analysis of the epigenetic and transcriptional landscapes of Dnmt3a-deficient mature olfactory sensory neurons (mOSNs), the primary sensory neurons of the olfactory epithelium. Dnmt3a is required for both 5-methylcytosine and 5-hydroxymethylcytosine patterning within accessible genomic regions, including hundreds of neurodevelopmental genes and neural enhancers. Loss of Dnmt3a results in the global disruption of gene expression via activation of silent genes and reduction of mOSN-expressed transcripts. Importantly, the DNA modification state and inducibility of odorant-activated genes are markedly impaired in Dnmt3a knockouts, suggesting a crucial role for this enzyme in establishing an epigenetic landscape compatible with neuronal plasticity.
Assuntos
DNA (Citosina-5-)-Metiltransferases/metabolismo , Regulação da Expressão Gênica , Percepção Olfatória/genética , Neurônios Receptores Olfatórios/metabolismo , Olfato/genética , Animais , Células Cultivadas , Metilação de DNA/genética , DNA Metiltransferase 3A , Epigenômica , Camundongos , Plasticidade Neuronal/genética , Neurônios Receptores Olfatórios/crescimento & desenvolvimentoRESUMO
Constitutive heterochromatin is traditionally viewed as the static form of heterochromatin that silences pericentromeric and telomeric repeats in a cell cycle- and differentiation-independent manner. Here, we show that, in the mouse olfactory epithelium, olfactory receptor (OR) genes are marked in a highly dynamic fashion with the molecular hallmarks of constitutive heterochromatin, H3K9me3 and H4K20me3. The cell type and developmentally dependent deposition of these marks along the OR clusters are, most likely, reversed during the process of OR choice to allow for monogenic and monoallelic OR expression. In contrast to the current view of OR choice, our data suggest that OR silencing takes place before OR expression, indicating that it is not the product of an OR-elicited feedback signal. Our findings suggest that chromatin-mediated silencing lays a molecular foundation upon which singular and stochastic selection for gene expression can be applied.
Assuntos
Montagem e Desmontagem da Cromatina , Inativação Gênica , Mucosa Olfatória/metabolismo , Receptores Odorantes/genética , Animais , Imunoprecipitação da Cromatina , Expressão Gênica , Heterocromatina , Código das Histonas , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Análise de Sequência com Séries de OligonucleotídeosRESUMO
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
The b1012 operon of Escherichia coli K-12, which is composed of seven unidentified ORFs, is one of the most highly expressed operons under control of nitrogen regulatory protein C. Examination of strains with lesions in this operon on Biolog Phenotype MicroArray (PM3) plates and subsequent growth tests indicated that they failed to use uridine or uracil as the sole nitrogen source and that the parental strain could use them at room temperature but not at 37 degrees C. A strain carrying an ntrB(Con) mutation, which elevates transcription of genes under nitrogen regulatory protein C control, could also grow on thymidine as the sole nitrogen source, whereas strains with lesions in the b1012 operon could not. Growth-yield experiments indicated that both nitrogens of uridine and thymidine were available. Studies with [(14)C]uridine indicated that a three-carbon waste product from the pyrimidine ring was excreted. After trimethylsilylation and gas chromatography, the waste product was identified by mass spectrometry as 3-hydroxypropionic acid. In agreement with this finding, 2-methyl-3-hydroxypropionic acid was released from thymidine. Both the number of available nitrogens and the waste products distinguished the pathway encoded by the b1012 operon from pyrimidine catabolic pathways described previously. We propose that the genes of this operon be named rutA-G for pyrimidine utilization. The product of the divergently transcribed gene, b1013, is a tetracycline repressor family regulator that controls transcription of the b1012 operon negatively.