RESUMO
The conformational state of DNA fine-tunes the transcriptional rate and abundance of RNA. Here, we report that G-quadruplex DNA (G4-DNA) accumulates in neurons, in an experience-dependent manner, and that this is required for the transient silencing and activation of genes that are critically involved in learning and memory in male C57/BL6 mice. In addition, site-specific resolution of G4-DNA by dCas9-mediated deposition of the helicase DHX36 impairs fear extinction memory. Dynamic DNA structure states therefore represent a key molecular mechanism underlying memory consolidation.One-Sentence Summary: G4-DNA is a molecular switch that enables the temporal regulation of the gene expression underlying the formation of fear extinction memory.
Assuntos
Quadruplex G , Masculino , Animais , Camundongos , Extinção Psicológica , RNA Helicases DEAD-box/química , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Medo , DNA/metabolismoRESUMO
The RNA modification N6-methyladenosine (m6A) regulates the interaction between RNA and various RNA binding proteins within the nucleus and other subcellular compartments and has recently been shown to be involved in experience-dependent plasticity, learning, and memory. Using m6A RNA-sequencing, we have discovered a distinct population of learning-related m6A- modified RNAs at the synapse, which includes the long noncoding RNA metastasis-associated lung adenocarcinoma transcript 1 (Malat1). RNA immunoprecipitation and mass spectrometry revealed 12 new synapse-specific learning-induced m6A readers in the mPFC of male C57/BL6 mice, with m6A-modified Malat1 binding to a subset of these, including CYFIP2 and DPYSL2. In addition, a cell type- and synapse-specific, and state-dependent, reduction of m6A on Malat1 impairs fear-extinction memory; an effect that likely occurs through a disruption in the interaction between Malat1 and DPYSL2 and an associated decrease in dendritic spine formation. These findings highlight the critical role of m6A in regulating the functional state of RNA during the consolidation of fear-extinction memory, and expand the repertoire of experience-dependent m6A readers in the synaptic compartment.SIGNIFICANCE STATEMENT We have discovered that learning-induced m6A-modified RNA (including the long noncoding RNA, Malat1) accumulates in the synaptic compartment. We have identified several new m6A readers that are associated with fear extinction learning and demonstrate a causal relationship between m6A-modified Malat1 and the formation of fear-extinction memory. These findings highlight the role of m6A in regulating the functional state of an RNA during memory formation and expand the repertoire of experience-dependent m6A readers in the synaptic compartment.
Assuntos
Medo , RNA Longo não Codificante , Animais , Masculino , Camundongos , Extinção Psicológica , Medo/fisiologia , Aprendizagem/fisiologia , RNA Longo não Codificante/metabolismo , Sinapses/metabolismoRESUMO
We have identified a member of the growth arrest and DNA damage (Gadd45) protein family, Gadd45γ, which is known to be critically involved in DNA repair, as a key player in the regulation of immediate early gene (IEG) expression underlying the consolidation of associative fear memory in adult male C57BL/6 mice. Gadd45γ temporally influences learning-induced IEG expression in the prelimbic prefrontal cortex (PLPFC) through its interaction with DNA double-strand break (DSB)-mediated changes in DNA methylation. Our findings suggest a two-hit model of experience-dependent IEG activity and learning that comprises (1) a first wave of IEG expression governed by DSBs and followed by a rapid increase in DNA methylation, and (2) a second wave of IEG expression associated with the recruitment of Gadd45γ and active DNA demethylation at the same site, which is necessary for memory consolidation.SIGNIFICANCE STATEMENT How does the pattern of immediate early gene transcription in the brain relate to the storage and accession of information, and what controls these patterns? This paper explores how Gadd45γ, a gene that is known to be involved with DNA modification and repair, regulates the temporal coding of IEGs underlying associative learning and memory. We reveal that, during fear learning, Gadd45γ serves to act as a coordinator of IEG expression and subsequent memory consolidation by directing temporally specific changes in active DNA demethylation at the promoter of plasticity-related IEGs.
Assuntos
Reparo do DNA/genética , Medo/fisiologia , Genes Precoces/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Sistema Límbico/fisiologia , Consolidação da Memória/fisiologia , Memória/fisiologia , Córtex Pré-Frontal/fisiologia , Animais , Sinais (Psicologia) , Quebras de DNA de Cadeia Dupla , Metilação de DNA , Epigênese Genética , Masculino , Rememoração Mental/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Desempenho Psicomotor/fisiologiaRESUMO
The Piwi pathway is a conserved gene regulatory mechanism comprised of Piwi-like proteins and Piwi-interacting RNAs, which modulates gene expression via RNA interference and through interaction with epigenetic mechanisms. The mammalian Piwi pathway has been defined by its role in transposon control during spermatogenesis; however, despite an increasing number of studies demonstrating its expression in the nervous system, relatively little is known about its function in neurons or potential contribution to behavioural regulation. We have discovered that all three Piwi-like genes are expressed in the adult mouse brain, and that viral-mediated knockdown of the Piwi-like genes Piwil1 and Piwil2 in the dorsal hippocampus leads to enhanced contextual fear memory without affecting generalised anxiety. These results implicate the Piwi pathway in behavioural regulation in the adult mammalian brain, likely through modulation of plasticity-related gene expression.
Assuntos
Proteínas Argonautas/metabolismo , Comportamento Animal/fisiologia , Medo/fisiologia , Hipocampo/metabolismo , Memória/fisiologia , RNA Interferente Pequeno/metabolismo , Animais , Ansiedade/genética , Proteínas Argonautas/genética , Técnicas de Cultura de Células , Condicionamento Operante/fisiologia , Epigênese Genética/fisiologia , Expressão Gênica/fisiologia , Masculino , Aprendizagem em Labirinto/fisiologia , Redes e Vias Metabólicas , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Agitação Psicomotora/genéticaRESUMO
Closed-loop paradigms provide an effective approach for studying visual choice behaviour and attention in small animals. Different flying and walking paradigms have been developed to investigate behavioural and neuronal responses to competing stimuli in insects such as bees and flies. However, the variety of stimulus choices that can be presented over one experiment is often limited. Current choice paradigms are mostly constrained as single binary choice scenarios that are influenced by the linear structure of classical conditioning paradigms. Here, we present a novel behavioural choice paradigm that allows animals to explore a closed geometry of interconnected binary choices by repeatedly selecting among competing objects, thereby revealing stimulus preferences in an historical context. We used our novel paradigm to investigate visual flicker preferences in honeybees (Apis mellifera) and found significant preferences for 20-25â Hz flicker and avoidance of higher (50-100â Hz) and lower (2-4â Hz) flicker frequencies. Similar results were found when bees were presented with three simultaneous choices instead of two, and when they were given the chance to select previously rejected choices. Our results show that honeybees can discriminate among different flicker frequencies and that their visual preferences are persistent even under different experimental conditions. Interestingly, avoided stimuli were more attractive if they were novel, suggesting that novelty salience can override innate preferences. Our recursive virtual reality environment provides a new approach to studying visual discrimination and choice behaviour in animals.
Assuntos
Abelhas/fisiologia , Animais , Comportamento Animal , Comportamento de Escolha , Simulação por Computador , Discriminação Psicológica , Movimento , Percepção VisualRESUMO
Long noncoding RNAs (lncRNAs) represent a multidimensional class of regulatory molecules that are involved in many aspects of brain function. Emerging evidence indicates that lncRNAs are localized to the synapse; however, a direct role for their activity in this subcellular compartment in memory formation has yet to be demonstrated. Using lncRNA capture-seq, we identified a specific set of lncRNAs that accumulate in the synaptic compartment within the infralimbic prefrontal cortex of adult male C57/Bl6 mice. Among these was a splice variant related to the stress-associated lncRNA, Gas5. RNA immunoprecipitation followed by mass spectrometry and single-molecule imaging revealed that this Gas5 isoform, in association with the RNA binding proteins G3BP2 and CAPRIN1, regulates the activity-dependent trafficking and clustering of RNA granules. In addition, we found that cell-type-specific, activity-dependent, and synapse-specific knockdown of the Gas5 variant led to impaired fear extinction memory. These findings identify a new mechanism of fear extinction that involves the dynamic interaction between local lncRNA activity and RNA condensates in the synaptic compartment.
Assuntos
Medo , RNA Longo não Codificante , Camundongos , Masculino , Animais , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Extinção Psicológica , Córtex Pré-Frontal/metabolismo , Sinapses/metabolismoRESUMO
Here, we used RNA capture-seq to identify a large population of lncRNAs that are expressed in the infralimbic prefrontal cortex of adult male mice in response to fear-related learning. Combining these data with cell-type-specific ATAC-seq on neurons that had been selectively activated by fear extinction learning, we find inducible 434 lncRNAs that are derived from enhancer regions in the vicinity of protein-coding genes. In particular, we discover an experience-induced lncRNA we call ADRAM (activity-dependent lncRNA associated with memory) that acts as both a scaffold and a combinatorial guide to recruit the brain-enriched chaperone protein 14-3-3 to the promoter of the memory-associated immediate-early gene Nr4a2 and is required fear extinction memory. This study expands the lexicon of experience-dependent lncRNA activity in the brain and highlights enhancer-derived RNAs (eRNAs) as key players in the epigenomic regulation of gene expression associated with the formation of fear extinction memory.
Assuntos
Medo , RNA Longo não Codificante , Proteínas 14-3-3/genética , Proteínas 14-3-3/metabolismo , Animais , Extinção Psicológica/fisiologia , Medo/fisiologia , Masculino , Camundongos , Córtex Pré-Frontal/metabolismo , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismoRESUMO
Higher-order organisms possess information processing capabilities that are only made possible by their biological complexity. Emerging evidence indicates a critical role for regulatory RNAs in coordinating many aspects of cellular function that are directly involved in experience-dependent neural plasticity. Here, we focus on a structurally distinct class of RNAs known as circular RNAs. These closed loop, single-stranded RNA molecules are highly stable, enriched in the brain, and functionally active in both healthy and disease conditions. Current evidence implicating this ancient class of RNA as a contributor toward higher-order functions such as cognition and memory is discussed.
Assuntos
RNA Circular , RNA , Encéfalo , HumanosRESUMO
DNA forms conformational states beyond the right-handed double helix; however, the functional relevance of these noncanonical structures in the brain remains unknown. Here we show that, in the prefrontal cortex of mice, the formation of one such structure, Z-DNA, is involved in the regulation of extinction memory. Z-DNA is formed during fear learning and reduced during extinction learning, which is mediated, in part, by a direct interaction between Z-DNA and the RNA-editing enzyme Adar1. Adar1 binds to Z-DNA during fear extinction learning, which leads to a reduction in Z-DNA at sites where Adar1 is recruited. Knockdown of Adar1 leads to an inability to modify a previously acquired fear memory and blocks activity-dependent changes in DNA structure and RNA state-effects that are fully rescued by the introduction of full-length Adar1. These findings suggest a new mechanism of learning-induced gene regulation that is dependent on proteins that recognize alternate DNA structure states, which are required for memory flexibility.
Assuntos
Adenosina Desaminase/metabolismo , Adenosina Desaminase/fisiologia , DNA Forma Z/fisiologia , Extinção Psicológica/fisiologia , Edição de RNA/fisiologia , Animais , DNA Forma Z/metabolismo , Medo , Aprendizagem/fisiologia , Camundongos , Córtex Pré-Frontal/metabolismo , RNA Interferente Pequeno/farmacologiaRESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMO
DNA modification is known to regulate experience-dependent gene expression. However, beyond cytosine methylation and its oxidated derivatives, very little is known about the functional importance of chemical modifications on other nucleobases in the brain. Here we report that in adult mice trained in fear extinction, the DNA modification N6-methyl-2'-deoxyadenosine (m6dA) accumulates along promoters and coding sequences in activated prefrontal cortical neurons. The deposition of m6dA is associated with increased genome-wide occupancy of the mammalian m6dA methyltransferase, N6amt1, and this correlates with extinction-induced gene expression. The accumulation of m6dA is associated with transcriptional activation at the brain-derived neurotrophic factor (Bdnf) P4 promoter, which is required for Bdnf exon IV messenger RNA expression and for the extinction of conditioned fear. These results expand the scope of DNA modifications in the adult brain and highlight changes in m6dA as an epigenetic mechanism associated with activity-induced gene expression and the formation of fear extinction memory.
Assuntos
Metilação de DNA , Desoxiadenosinas/metabolismo , Extinção Psicológica/fisiologia , Medo , Regulação da Expressão Gênica , Neurônios/metabolismo , Córtex Pré-Frontal/metabolismo , Animais , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Epigênese Genética , Masculino , Camundongos Endogâmicos C57BL , RNA Mensageiro/metabolismoRESUMO
Epigenetic regulation of activity-induced gene expression involves multiple levels of molecular interaction, including histone and DNA modifications, as well as mechanisms of DNA repair. Here we demonstrate that the genome-wide deposition of inhibitor of growth family member 1 (ING1), which is a central epigenetic regulatory protein, is dynamically regulated in response to activity in primary cortical neurons. ING1 knockdown leads to decreased expression of genes related to synaptic plasticity, including the regulatory subunit of calcineurin, Ppp3r1. In addition, ING1 binding at a site upstream of the transcription start site (TSS) of Ppp3r1 depends on yet another group of neuroepigenetic regulatory proteins, the Piwi-like family, which are also involved in DNA repair. These findings provide new insight into a novel mode of activity-induced gene expression, which involves the interaction between different epigenetic regulatory mechanisms traditionally associated with gene repression and DNA repair.
Assuntos
Córtex Cerebral/metabolismo , Epigênese Genética , Proteína 1 Inibidora do Crescimento/metabolismo , Neurônios/metabolismo , Animais , Proteínas Argonautas/metabolismo , Proteínas de Ligação ao Cálcio , Células Cultivadas , Fibroblastos/metabolismo , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Proteína 1 Inibidora do Crescimento/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Camundongos Endogâmicos C57BL , Proteínas Musculares/metabolismo , RNA Mensageiro/metabolismoRESUMO
Transcriptome-wide expression profiling of neurons has provided important insights into the underlying molecular mechanisms and gene expression patterns that transpire during learning and memory formation. However, there is a paucity of tools for profiling stimulus-induced RNA within specific neuronal cell populations. A bioorthogonal method to chemically label nascent (i.e., newly transcribed) RNA in a cell-type-specific and temporally controlled manner, which is also amenable to bioconjugation via click chemistry, was recently developed and optimized within conventional immortalized cell lines. However, its value within a more fragile and complicated cellular system such as neurons, as well as for transcriptome-wide expression profiling, has yet to be demonstrated. Here, we report the visualization and sequencing of activity-dependent nascent RNA derived from neurons using this labeling method. This work has important implications for improving transcriptome-wide expression profiling and visualization of nascent RNA in neurons, which has the potential to provide valuable insights into the mechanisms underlying neural plasticity, learning, and memory.