RESUMEN
Gene expression control occurs partially by modifications in chromatin structure, including the addition and removal of posttranslational modifications to histone tails. Histone post-translational modifications (HPTMs) can either facilitate gene expression or repression. For example, acetylation of histone tail lysine residues neutralizes the positive charge and reduces interactions between the tail and negatively charged DNA. The decrease in histone tail-DNA interactions results in increased accessibility of the underlying DNA, allowing for increased transcription factor access. The acetylation mark also serves as a recognition site for bromodomain-containing transcriptional activators, together resulting in enhanced gene expression. Histone marks can be dynamically regulated during cell differentiation and in response to different cellular environments and stimuli. While next-generation sequencing approaches have begun to characterize genomic locations for individual histone modifications, only one modification can be examined concurrently. Given that there are hundreds of different HPTMs, we have developed a high throughput, quantitative measure of global HPTMs that can be used to screen histone modifications prior to conducting more extensive genome sequencing approaches. This protocol describes a flow cytometry-based method to detect global HPTMs and can be conducted using cells in culture or isolated cells from in vivo tissues. We present example data from isolated mouse brain microglia to demonstrate the sensitivity of the assay to detect global shifts in HPTMs in response to a bacteria-derived immune stimulus (lipopolysaccharide). This protocol allows for the rapid and quantitative assessment of HPTMs and can be applied to any transcriptional or epigenetic regulator that can be detected by an antibody.
Asunto(s)
Encéfalo , Histonas , Microglía , Procesamiento Proteico-Postraduccional , Animales , Ratones , Acetilación , Encéfalo/metabolismo , ADN/genética , Citometría de Flujo , Histonas/genética , Histonas/metabolismo , Microglía/metabolismo , Procesamiento Proteico-Postraduccional/genética , Procesamiento Proteico-Postraduccional/fisiologíaRESUMEN
Down Syndrome (DS) is the most common genetic cause of intellectual disability, in which an extra copy of human chromosome 21 (HSA21) affects regional DNA methylation profiles across the genome. Although DNA methylation has been previously examined at select regulatory regions across the genome in a variety of DS tissues and cells, differentially methylated regions (DMRs) have yet to be examined in an unbiased sequencing-based approach. Here, we present the first analysis of DMRs from whole genome bisulfite sequencing (WGBS) data of human DS and matched control brain, specifically frontal cortex. While no global differences in DNA methylation were observed, we identified 3,152 DS-DMRs across the entire genome, the majority of which were hypermethylated in DS. DS-DMRs were significantly enriched at CpG islands and de-enriched at specific gene body and regulatory regions. Functionally, the hypermethylated DS-DMRs were enriched for one-carbon metabolism, membrane transport, and glutamatergic synaptic signalling, while the hypomethylated DMRs were enriched for proline isomerization, glial immune response, and apoptosis. Furthermore, in a cross-tissue comparison to previous studies of DNA methylation from diverse DS tissues and reference epigenomes, hypermethylated DS-DMRs showed a strong cross-tissue concordance, while a more tissue-specific pattern was observed for the hypomethylated DS-DMRs. Overall, this approach highlights that low-coverage WGBS of clinical samples can identify epigenetic alterations to known biological pathways, which are potentially relevant to therapeutic treatments and include metabolic pathways. These results also provide new insights into the genome-wide effects of genetic alterations on DNA methylation profiles indicative of altered neurodevelopment and brain function.
Asunto(s)
Encéfalo/metabolismo , Metilación de ADN/genética , Síndrome de Down/genética , Lóbulo Frontal/metabolismo , Encéfalo/patología , Islas de CpG/genética , Síndrome de Down/patología , Epigénesis Genética/genética , Lóbulo Frontal/patología , Genoma Humano/genética , Humanos , Masculino , Secuenciación Completa del GenomaRESUMEN
Mutations in the X-linked gene MECP2 cause the majority of Rett syndrome (RTT) cases. Two differentially spliced isoforms of exons 1 and 2 (MeCP2-e1 and MeCP2-e2) contribute to the diverse functions of MeCP2, but only mutations in exon 1, not exon 2, are observed in RTT. We previously described an isoform-specific MeCP2-e1-deficient male mouse model of a human RTT mutation that lacks MeCP2-e1 while preserving expression of MeCP2-e2. However, RTT patients are heterozygous females that exhibit delayed and progressive symptom onset beginning in late infancy, including neurologic as well as metabolic, immune, respiratory and gastrointestinal phenotypes. Consequently, we conducted a longitudinal assessment of symptom development in MeCP2-e1 mutant females and males. A delayed and progressive onset of motor impairments was observed in both female and male MeCP2-e1 mutant mice, including hind limb clasping and motor deficits in gait and balance. Because these motor impairments were significantly impacted by age-dependent increases in body weight, we also investigated metabolic phenotypes at an early stage of disease progression. Both male and female MeCP2-e1 mutants exhibited significantly increased body fat compared to sex-matched wild-type littermates prior to weight differences. Mecp2e1-/y males exhibited significant metabolic phenotypes of hypoactivity, decreased energy expenditure, increased respiratory exchange ratio, but decreased food intake compared to wild-type. Untargeted analysis of lipid metabolites demonstrated a distinguishable profile in MeCP2-e1 female mutant liver characterized by increased triglycerides. Together, these results demonstrate that MeCP2-e1 mutation in mice of both sexes recapitulates early and progressive metabolic and motor phenotypes of human RTT.
Asunto(s)
Proteína 2 de Unión a Metil-CpG/genética , Actividad Motora/genética , Síndrome de Rett/genética , Animales , Modelos Animales de Enfermedad , Exones/genética , Femenino , Regulación de la Expresión Génica/genética , Heterocigoto , Humanos , Masculino , Ratones , Actividad Motora/fisiología , Mutación , Fenotipo , Isoformas de Proteínas/genética , Síndrome de Rett/metabolismo , Síndrome de Rett/fisiopatologíaRESUMEN
Aging is accompanied by impairments in both circadian rhythmicity and long-term memory. Although it is clear that memory performance is affected by circadian cycling, it is unknown whether age-related disruption of the circadian clock causes impaired hippocampal memory. Here, we show that the repressive histone deacetylase HDAC3 restricts long-term memory, synaptic plasticity, and experience-induced expression of the circadian gene Per1 in the aging hippocampus without affecting rhythmic circadian activity patterns. We also demonstrate that hippocampal Per1 is critical for long-term memory formation. Together, our data challenge the traditional idea that alterations in the core circadian clock drive circadian-related changes in memory formation and instead argue for a more autonomous role for circadian clock gene function in hippocampal cells to gate the likelihood of long-term memory formation.
Asunto(s)
Envejecimiento/fisiología , Ritmo Circadiano/genética , Epigénesis Genética , Hipocampo/fisiología , Memoria/fisiología , Proteínas Circadianas Period/genética , Animales , Eliminación de Gen , Técnicas de Silenciamiento del Gen , Histona Desacetilasas/metabolismo , Potenciación a Largo Plazo , Trastornos de la Memoria/genética , Trastornos de la Memoria/fisiopatología , Ratones Endogámicos C57BL , Plasticidad Neuronal/genética , Proteínas Circadianas Period/metabolismoRESUMEN
Rhythmic oscillations of physiological processes depend on integrating the circadian clock and diurnal environment. DNA methylation is epigenetically responsive to daily rhythms, as a subset of CpG dinucleotides in brain exhibit diurnal rhythmic methylation. Here, we show a major genetic effect on rhythmic methylation in a mouse Snord116 deletion model of the imprinted disorder Prader-Willi syndrome (PWS). More than 23,000 diurnally rhythmic CpGs are identified in wild-type cortex, with nearly all lost or phase-shifted in PWS. Circadian dysregulation of a second imprinted Snord cluster at the Temple/Kagami-Ogata syndrome locus is observed at the level of methylation, transcription, and chromatin, providing mechanistic evidence of cross-talk. Genes identified by diurnal epigenetic changes in PWS mice overlapped rhythmic and PWS-specific genes in human brain and are enriched for PWS-relevant phenotypes and pathways. These results support the proposed evolutionary relationship between imprinting and sleep, and suggest possible chronotherapy in the treatment of PWS and related disorders.
Asunto(s)
Encéfalo/fisiología , Corteza Cerebral/metabolismo , Ritmo Circadiano , Síndrome de Prader-Willi/genética , ARN Nucleolar Pequeño/genética , ARN Nucleolar Pequeño/metabolismo , Animales , Cromatina/genética , Cromatina/metabolismo , Metilación de ADN , Femenino , Eliminación de Gen , Humanos , Masculino , Ratones , Síndrome de Prader-Willi/metabolismoRESUMEN
Augmented maternal care during the first postnatal week promotes life-long stress resilience and improved memory compared with the outcome of routine rearing conditions. Recent evidence suggests that this programming commences with altered synaptic connectivity of stress sensitive hypothalamic neurons. However, the epigenomic basis of the long-lived consequences is not well understood. Here, we employed whole-genome bisulfite sequencing (WGBS), RNA-sequencing (RNA-seq), and a multiplex microRNA (miRNA) assay to examine the effects of augmented maternal care on DNA cytosine methylation, gene expression, and miRNA expression. A total of 9,439 differentially methylated regions (DMRs) associated with augmented maternal care were identified in male offspring hypothalamus, as well as a modest but significant decrease in global DNA methylation. Differentially methylated and expressed genes were enriched for functions in neurotransmission, neurodevelopment, protein synthesis, and oxidative phosphorylation, as well as known stress response genes. Twenty prioritized genes were identified as highly relevant to the stress resiliency phenotype. This combined unbiased approach enabled the discovery of novel genes and gene pathways that advance our understanding of the epigenomic mechanisms underlying the effects of maternal care on the developing brain.
Asunto(s)
Metilación de ADN/genética , Desarrollo Embrionario/genética , Epigenómica , Hipotálamo/crecimiento & desarrollo , Animales , Islas de CpG/genética , Femenino , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica/genética , Humanos , Hipotálamo/metabolismo , Masculino , MicroARNs/genética , Relaciones Madre-Hijo , Plasticidad Neuronal/genética , Ratas , Análisis de Secuencia de ADN , Análisis de Secuencia de ARN , Estrés Psicológico/genética , Secuenciación Completa del GenomaRESUMEN
Dysregulation in immune responses during pregnancy increases the risk of a having a child with an autism spectrum disorder (ASD). Asthma is one of the most common chronic diseases among pregnant women, and symptoms often worsen during pregnancy. We recently developed a mouse model of maternal allergic asthma (MAA) that induces changes in sociability, repetitive, and perseverative behaviors in the offspring. Since epigenetic changes help a static genome adapt to the maternal environment, activation of the immune system may epigenetically alter fetal microglia, the brain's resident immune cells. We therefore tested the hypothesis that epigenomic alterations to microglia may be involved in behavioral abnormalities observed in MAA offspring. We used the genome-wide approaches of whole genome bisulfite sequencing to examine DNA methylation and RNA sequencing to examine gene expression in microglia from juvenile MAA offspring. Differentially methylated regions were enriched for immune signaling pathways and important microglial developmental transcription factor binding motifs. Differential expression analysis identified genes involved in controlling microglial sensitivity to the environment and shaping neuronal connections in the developing brain. Differentially expressed genes significantly overlapped genes with altered expression in human ASD cortex, supporting a role for microglia in the pathogenesis of ASD.
Asunto(s)
Asma/metabolismo , Trastorno Autístico/metabolismo , Epigénesis Genética , Hipersensibilidad/metabolismo , Microglía/metabolismo , Efectos Tardíos de la Exposición Prenatal , Animales , Trastorno Autístico/genética , Metilación de ADN , Modelos Animales de Enfermedad , Femenino , Expresión Génica/fisiología , Ratones Endogámicos C57BL , Embarazo , Distribución Aleatoria , Análisis de Secuencia de ARN , Transcriptoma/fisiología , Secuenciación Completa del GenomaRESUMEN
The dysregulation of genes in neurodevelopmental disorders that lead to social and cognitive phenotypes is a complex, multilayered process involving both genetics and epigenetics. Parent-of-origin effects of deletion and duplication of the 15q11-q13 locus leading to Angelman, Prader-Willi, and Dup15q syndromes are due to imprinted genes, including UBE3A, which is maternally expressed exclusively in neurons. UBE3A encodes a ubiquitin E3 ligase protein with multiple downstream targets, including RING1B, which in turn monoubiquitinates histone variant H2A.Z. To understand the impact of neuronal UBE3A levels on epigenome-wide marks of DNA methylation, histone variant H2A.Z positioning, active H3K4me3 promoter marks, and gene expression, we took a multi-layered genomics approach. We performed an siRNA knockdown of UBE3A in two human neuroblastoma cell lines, including parental SH-SY5Y and the SH(15M) model of Dup15q. Genes differentially methylated across cells with differing UBE3A levels were enriched for functions in gene regulation, DNA binding, and brain morphology. Importantly, we found that altering UBE3A levels had a profound epigenetic effect on the methylation levels of up to half of known imprinted genes. Genes with differential H2A.Z peaks in SH(15M) compared to SH-SY5Y were enriched for ubiquitin and protease functions and associated with autism, hypoactivity, and energy expenditure. Together, these results support a genome-wide epigenetic consequence of altered UBE3A levels in neurons and suggest that UBE3A regulates an imprinted gene network involving DNA methylation patterning and H2A.Z deposition.
Asunto(s)
Impresión Genómica , Neuronas/metabolismo , Ubiquitina-Proteína Ligasas/genética , Línea Celular Tumoral , Metilación de ADN , Código de Histonas , Humanos , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Recent human exome-sequencing studies have implicated polymorphic Brg1-associated factor (BAF) complexes (mammalian SWI/SNF chromatin remodeling complexes) in several intellectual disabilities and cognitive disorders, including autism. However, it remains unclear how mutations in BAF complexes result in impaired cognitive function. Post-mitotic neurons express a neuron-specific assembly, nBAF, characterized by the neuron-specific subunit BAF53b. Subdomain 2 of BAF53b is essential for the differentiation of neuronal precursor cells into neurons. We generated transgenic mice lacking subdomain 2 of Baf53b (BAF53bΔSB2). Long-term synaptic potentiation (LTP) and long-term memory, both of which are associated with phosphorylation of the actin severing protein cofilin, were assessed in these animals. A phosphorylation mimic of cofilin was stereotaxically delivered into the hippocampus of BAF53bΔSB2 mice in an effort to rescue LTP and memory. BAF53bΔSB2 mutant mice show impairments in phosphorylation of synaptic cofilin, LTP, and memory. Both the synaptic plasticity and memory deficits are rescued by overexpression of a phosphorylation mimetic of cofilin. Baseline physiology and behavior were not affected by the mutation or the experimental treatment. This study suggests a potential link between nBAF function, actin cytoskeletal remodeling at the dendritic spine, and memory formation. This work shows that a targeted manipulation of synaptic function can rescue adult plasticity and memory deficits caused by manipulations of nBAF, and thereby provides potential novel avenues for therapeutic development for multiple intellectual disability disorders.
Asunto(s)
Ensamble y Desensamble de Cromatina/genética , Proteínas Cromosómicas no Histona/metabolismo , Memoria/fisiología , Mutación/genética , Plasticidad Neuronal/genética , Fosfopiruvato Hidratasa/metabolismo , Factores Despolimerizantes de la Actina/genética , Factores Despolimerizantes de la Actina/metabolismo , Animales , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/genética , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Nucléolo Celular/metabolismo , Proteínas Cromosómicas no Histona/genética , Hipocampo/citología , Hipocampo/metabolismo , Técnicas In Vitro , Potenciación a Largo Plazo/genética , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Red Nerviosa/fisiología , Neuronas/ultraestructura , Fosfopiruvato Hidratasa/genética , Fosforilación/genética , Eliminación de Secuencia/genética , Transducción GenéticaRESUMEN
Rett syndrome (RTT) is an X-linked neurodevelopmental disorder caused by mutations in the gene encoding methyl CpG binding protein 2 (MeCP2) that occur sporadically in 1:10,000 female births. RTT is characterized by a period of largely normal development followed by regression in language and motor skills at 6-18 months of age. Mecp2 mutant mice recapitulate many of the clinical features of RTT, but the majority of behavioral assessments have been conducted in male Mecp2 hemizygous null mice as offspring of heterozygous dams. Given that RTT patients are predominantly female, we conducted a systematic analysis of developmental milestones, sensory abilities, and motor deficits, following the longitudinal decline of function from early postnatal to adult ages in female Mecp2 heterozygotes of the conventional Bird line (Mecp2tm1.1bird-/+), as compared to their female wildtype littermate controls. Further, we assessed the impact of postnatal maternal environment on developmental milestones and behavioral phenotypes. Cross-fostering to CD1 dams accelerated several developmental milestones independent of genotype, and induced earlier onset of weight gain in adult female Mecp2tm1.1bird-/+ mice. Cross-fostering improved the sensitivity of a number of motor behaviors that resulted in observable deficits in Mecp2tm1.1bird-/+ mice at much earlier (6-7 weeks) ages than were previously reported (6-9 months). Our findings indicate that female Mecp2tm1.1bird-/+ mice recapitulate many of the motor aspects of RTT syndrome earlier than previously appreciated. In addition, rearing conditions may impact the phenotypic severity and improve the ability to detect genotype differences in female Mecp2 mutant mice.
Asunto(s)
Síndrome de Rett/diagnóstico , Animales , Conducta Animal , Modelos Animales de Enfermedad , Ambiente , Femenino , Estudios de Asociación Genética , Genotipo , Heterocigoto , Masculino , Proteína 2 de Unión a Metil-CpG/metabolismo , Ratones , Ratones Noqueados , Destreza Motora/fisiología , Fenotipo , Síndrome de Rett/genética , Síndrome de Rett/veterinariaRESUMEN
Rare variants enriched for functions in chromatin regulation and neuronal synapses have been linked to autism. How chromatin and DNA methylation interact with environmental exposures at synaptic genes in autism etiologies is currently unclear. Using whole-genome bisulfite sequencing in brain tissue and a neuronal cell culture model carrying a 15q11.2-q13.3 maternal duplication, we find that significant global DNA hypomethylation is enriched over autism candidate genes and affects gene expression. The cumulative effect of multiple chromosomal duplications and exposure to the pervasive persistent organic pollutant PCB 95 altered methylation of more than 1,000 genes. Hypomethylated genes were enriched for H2A.Z, increased maternal UBE3A in Dup15q corresponded to reduced levels of RING1B, and bivalently modified H2A.Z was altered by PCB 95 and duplication. These results demonstrate the compounding effects of genetic and environmental insults on the neuronal methylome that converge upon dysregulation of chromatin and synaptic genes.
Asunto(s)
Trastorno Autístico/genética , Duplicación Cromosómica/genética , Metilación de ADN/efectos de los fármacos , Epigénesis Genética , Trastorno Autístico/patología , Secuencia de Bases/efectos de los fármacos , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Encéfalo/patología , Cromatina/efectos de los fármacos , Metilación de ADN/genética , Regulación de la Expresión Génica/efectos de los fármacos , Interacción Gen-Ambiente , Estudios de Asociación Genética , Genoma Humano , Impresión Genómica/genética , Humanos , Bifenilos Policlorados/toxicidad , Complejo Represivo Polycomb 1/genética , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
Increasing evidence points to a complex interplay between genes and the environment in autism spectrum disorder (ASD), including rare de novo mutations in chromatin genes such as methyl-CpG binding protein 2 (MECP2) in Rett syndrome. Epigenetic mechanisms such as DNA methylation act at this interface, reflecting the plasticity in metabolic and neurodevelopmentally regulated gene pathways. Genome-wide studies of gene sequences, gene pathways and DNA methylation are providing valuable mechanistic insights into ASD. The dynamic developmental landscape of DNA methylation is vulnerable to numerous genetic and environmental insults: therefore, understanding pathways that are central to this 'perfect storm' will be crucial to improving the diagnosis and treatment of ASD.
Asunto(s)
Trastorno del Espectro Autista/genética , Trastorno Autístico/genética , Metilación de ADN/genética , Epigénesis Genética/genética , Síndrome de Rett/genética , Animales , Cromatina/metabolismo , HumanosRESUMEN
Designer receptors exclusively activated by designer drug (DREADDs) are a novel tool with the potential to bidirectionally drive cellular, circuit, and ultimately, behavioral changes. We used DREADDs to evaluate memory formation in a hippocampus-dependent task in mice and effects on synaptic physiology in the dorsal hippocampus. We expressed neuron-specific (hSyn promoter) DREADDs that were either excitatory (HM3D) or inhibitory (HM4D) in the dorsal hippocampus. As predicted, hSyn-HM3D was able to transform a subthreshold learning event into long-term memory (LTM), and hSyn-HM4D completely impaired LTM formation. Surprisingly, the opposite was observed during experiments examining the effects on hippocampal long-term potentiation (LTP). hSyn-HM3D impaired LTP and hSyn-HM4D facilitated LTP. Follow-up experiments indicated that the hSyn-HM3D-mediated depression of fEPSP appears to be driven by presynaptic activation of inhibitory currents, whereas the hSyn-HM4D-mediated increase of fEPSP is induced by a reduction in GABAA receptor function. To determine whether these observations were promoter specific, we next examined the effects of using the CaMKIIα promoter that limits expression to forebrain excitatory neurons. CaMKIIα-HM3D in the dorsal hippocampus led to the transformation of a subthreshold learning event into LTM, whereas CaMKIIα-HM4D blocked LTM formation. Consistent with these findings, baseline synaptic transmission and LTP was increased in CaMKIIα-HM3D hippocampal slices, whereas slices from CaMKIIα-HM4D mice produced expected decreases in baseline synaptic transmission and LTP. Together, these experiments further demonstrate DREADDs as being a robust and reliable means of modulating neuronal function to manipulate long-term changes in behavior, while providing evidence for specific dissociations between LTM and LTP. SIGNIFICANCE STATEMENT: This study evaluates the efficacy of designer receptors exclusively activated by designer drug (DREADDs) as a means of bidirectionally modulating the hippocampus in not only a hippocampus-dependent task but also in hippocampal synaptic plasticity. This is the first study to evaluate the effects of DREADD-mediated inhibition and excitation in hippocampal long-term potentiation. More specifically, this study evaluates the effect of promoter-specific expression of DREADD viruses in a heterogenic cell population, which revealed surprising effects of different promoters. With chemogenetics becoming a more ubiquitous tool throughout studies investigating circuit-specific function, these data are of broad interest to the neuroscientific community because we have shown that promoter-specific effects can drastically alter synaptic function within a specific region, without parallel changes at the level of behavior.
Asunto(s)
Drogas de Diseño/administración & dosificación , Hipocampo/fisiología , Potenciación a Largo Plazo/fisiología , Memoria/fisiología , Proteínas del Tejido Nervioso/genética , Plasticidad Neuronal/fisiología , Animales , Hipocampo/efectos de los fármacos , Potenciación a Largo Plazo/efectos de los fármacos , Masculino , Memoria/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Proteínas del Tejido Nervioso/metabolismo , Plasticidad Neuronal/efectos de los fármacos , Regiones Promotoras Genéticas/efectos de los fármacos , Regiones Promotoras Genéticas/genética , Hipofraccionamiento de la Dosis de Radiación , Transmisión Sináptica/efectos de los fármacos , Transmisión Sináptica/fisiologíaRESUMEN
Three-dimensional chromosomal conformations regulate transcription by moving enhancers and regulatory elements into spatial proximity with target genes. Here we describe activity-regulated long-range loopings bypassing up to 0.5 Mb of linear genome to modulate NMDA glutamate receptor GRIN2B expression in human and mouse prefrontal cortex. Distal intronic and 3' intergenic loop formations competed with repressor elements to access promoter-proximal sequences, and facilitated expression via a "cargo" of AP-1 and NRF-1 transcription factors and TALE-based transcriptional activators. Neuronal deletion or overexpression of Kmt2a/Mll1 H3K4- and Kmt1e/Setdb1 H3K9-methyltransferase was associated with higher-order chromatin changes at distal regulatory Grin2b sequences and impairments in working memory. Genetic polymorphisms and isogenic deletions of loop-bound sequences conferred liability for cognitive performance and decreased GRIN2B expression. Dynamic regulation of chromosomal conformations emerges as a novel layer for transcriptional mechanisms impacting neuronal signaling and cognition.
Asunto(s)
Cromatina/metabolismo , Cognición/fisiología , Regulación de la Expresión Génica/fisiología , Receptores de N-Metil-D-Aspartato/metabolismo , Anciano , Anciano de 80 o más Años , Animales , Animales Recién Nacidos , Antipsicóticos/farmacología , Antipsicóticos/uso terapéutico , Células Cultivadas , Corteza Cerebral/citología , Corteza Cerebral/ultraestructura , Cromatina/efectos de los fármacos , Cognición/efectos de los fármacos , Femenino , Células HEK293 , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Persona de Mediana Edad , Neuronas/metabolismo , Neuronas/ultraestructura , Polimorfismo de Nucleótido Simple/genética , Receptores de N-Metil-D-Aspartato/química , Receptores de N-Metil-D-Aspartato/genética , Esquizofrenia/tratamiento farmacológico , Esquizofrenia/genética , Esquizofrenia/patología , Transducción de Señal/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
This unit is designed to provide sufficient instruction for the setup and execution of tests for object location and object recognition in adult mice. This task is ideally suited for the study of a variety of mouse models that examine disease mechanisms and novel therapeutic targets. By altering several key parameters, the experimenter can investigate short-term or long-term memory and look for either memory impairments or enhancements. Object location and object recognition memory tasks rely on a rodent's innate preference for novelty, and can be conducted sequentially in the same cohort of animals. These two tasks avoid the inherent stress induced with other common measures of rodent memory such as fear conditioning and the Morris water maze. This protocol covers detailed instructions on conducting both tasks, as well as key points concerning data collection, analysis, and interpretation.
Asunto(s)
Aprendizaje por Laberinto/fisiología , Memoria/fisiología , Reconocimiento en Psicología/fisiología , Animales , Ratones , Ratones Endogámicos C57BLRESUMEN
Long-term memory formation requires the coordinated regulation of gene expression. Until recently nucleosome remodeling, one of the major epigenetic mechanisms for controlling gene expression, had been largely unexplored in the field of neuroscience. Nucleosome remodeling is carried out by chromatin remodeling complexes (CRCs) that interact with DNA and histones to physically alter chromatin structure and ultimately regulate gene expression. Human exome sequencing and gene wide association studies have linked mutations in CRC subunits to intellectual disability disorders, autism spectrum disorder and schizophrenia. However, how mutations in CRC subunits were related to human cognitive disorders was unknown. There appears to be both developmental and adult specific roles for the neuron specific CRC nBAF (neuronal Brg1/hBrm Associated Factor). nBAF regulates gene expression required for dendritic arborization during development, and in the adult, contributes to long-term potentiation, a form of synaptic plasticity, and long-term memory. We propose that the nBAF complex is a novel epigenetic mechanism for regulating transcription required for long-lasting forms of synaptic plasticity and memory processes and that impaired nBAF function may result in human cognitive disorders.
Asunto(s)
Ensamble y Desensamble de Cromatina , Epigénesis Genética , Discapacidad Intelectual/etiología , Memoria , Modelos Biológicos , Plasticidad Neuronal , Neuronas/metabolismo , Actinas/genética , Actinas/metabolismo , Animales , Trastorno Autístico/genética , Trastorno Autístico/metabolismo , Trastorno Autístico/fisiopatología , Encéfalo/enzimología , Encéfalo/metabolismo , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , Trastornos del Conocimiento/genética , Trastornos del Conocimiento/metabolismo , Trastornos del Conocimiento/fisiopatología , ADN Helicasas/genética , ADN Helicasas/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica , Humanos , Trastornos de la Memoria/genética , Trastornos de la Memoria/metabolismo , Trastornos de la Memoria/fisiopatología , Mutación , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Neuronas/enzimología , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Nucleosomas/enzimología , Nucleosomas/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Transcriptional dysregulation is an early feature of Huntington disease (HD). We observed gene-specific changes in histone H3 lysine 4 trimethylation (H3K4me3) at transcriptionally repressed promoters in R6/2 mouse and human HD brain. Genome-wide analysis showed a chromatin signature for this mark. Reducing the levels of the H3K4 demethylase SMCX/Jarid1c in primary neurons reversed down-regulation of key neuronal genes caused by mutant Huntingtin expression. Finally, reduction of SMCX/Jarid1c in primary neurons from BACHD mice or the single Jarid1 in a Drosophila HD model was protective. Therefore, targeting this epigenetic signature may be an effective strategy to ameliorate the consequences of HD.
Asunto(s)
Encéfalo/metabolismo , Histonas/metabolismo , Enfermedad de Huntington/metabolismo , Lisina/metabolismo , Animales , Animales Modificados Genéticamente , Western Blotting , Encéfalo/patología , Factor Neurotrófico Derivado del Encéfalo/genética , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Células Cultivadas , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Femenino , Perfilación de la Expresión Génica , Histona Demetilasas , Humanos , Proteína Huntingtina , Enfermedad de Huntington/genética , Masculino , Metilación , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Neuronas/citología , Neuronas/metabolismo , Oxidorreductasas N-Desmetilantes/genética , Oxidorreductasas N-Desmetilantes/metabolismo , Regiones Promotoras Genéticas/genética , Interferencia de ARN , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Recent exome sequencing studies have implicated polymorphic Brg1-associated factor (BAF) complexes (mammalian SWI/SNF chromatin remodeling complexes) in several human intellectual disabilities and cognitive disorders. However, it is currently unknown how mutations in BAF complexes result in impaired cognitive function. Postmitotic neurons express a neuron-specific assembly, nBAF, characterized by the neuron-specific subunit BAF53b. Mice harboring selective genetic manipulations of BAF53b have severe defects in long-term memory and long-lasting forms of hippocampal synaptic plasticity. We rescued memory impairments in BAF53b mutant mice by reintroducing BAF53b in the adult hippocampus, which suggests a role for BAF53b beyond neuronal development. The defects in BAF53b mutant mice appeared to derive from alterations in gene expression that produce abnormal postsynaptic components, such as spine structure and function, and ultimately lead to deficits in synaptic plasticity. Our results provide new insight into the role of dominant mutations in subunits of BAF complexes in human intellectual and cognitive disorders.
Asunto(s)
Actinas/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica/genética , Plasticidad Neuronal/fisiología , Neuronas/citología , Neuronas/metabolismo , Reconocimiento en Psicología/fisiología , Factores Despolimerizantes de la Actina/metabolismo , Actinas/genética , Animales , Proteínas Cromosómicas no Histona/genética , Condicionamiento Psicológico/fisiología , Proteínas de Unión al ADN/genética , Espinas Dendríticas/fisiología , Espinas Dendríticas/ultraestructura , Dependovirus/genética , Homólogo 4 de la Proteína Discs Large , Potenciales Postsinápticos Excitadores/genética , Miedo/fisiología , Guanilato-Quinasas/metabolismo , Hipocampo/citología , Técnicas In Vitro , Aprendizaje por Laberinto/fisiología , Proteínas de la Membrana/metabolismo , Trastornos de la Memoria/genética , Trastornos de la Memoria/fisiopatología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Mutación/genética , Plasticidad Neuronal/genética , Factores de Tiempo , TranscriptomaRESUMEN
Nr4a1 and Nr4a2 are transcription factors and immediate early genes belonging to the nuclear receptor Nr4a family. In this study, we examine their role in long-term memory formation for object location and object recognition. Using siRNA to block expression of either Nr4a1 or Nr4a2, we found that Nr4a2 is necessary for both long-term memory for object location and object recognition. In contrast, Nr4a1 appears to be necessary only for object location. Indeed, their roles in these different types of long-term memory may be dependent on their expression in the brain, as NR4A2 was found to be expressed in hippocampal neurons (associated with object location memory) as well as in the insular and perirhinal cortex (associated with object recognition memory), whereas NR4A1 showed minimal neuronal expression in these cortical areas. These results begin to elucidate how NR4A1 and NR4A2 differentially contribute to object location versus object recognition memory.
Asunto(s)
Memoria a Largo Plazo/efectos de la radiación , Miembro 1 del Grupo A de la Subfamilia 4 de Receptores Nucleares/metabolismo , Miembro 2 del Grupo A de la Subfamilia 4 de Receptores Nucleares/metabolismo , Reconocimiento en Psicología/fisiología , Animales , Corteza Cerebral/efectos de los fármacos , Corteza Cerebral/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/fisiología , Hipocampo/citología , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Aprendizaje , Masculino , Memoria a Largo Plazo/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Miembro 1 del Grupo A de la Subfamilia 4 de Receptores Nucleares/genética , Miembro 2 del Grupo A de la Subfamilia 4 de Receptores Nucleares/genética , Fosfopiruvato Hidratasa/metabolismo , ARN Mensajero/metabolismo , ARN Interferente Pequeño/farmacología , Reconocimiento en Psicología/efectos de los fármacos , Factores de TiempoRESUMEN
Under basal conditions histone deacetylases(HDACs) and their associated co-repressor complexes serve as molecular 'brake pads' to prevent the gene expression required for long-term memory formation. Following a learning event, HDACs and their co-repressor complexes are removed from a subset of specific gene promoters, allowing the histone acetylation and active gene expression required for long-term memory formation.Inhibition of HDACs increases histone acetylation,extends gene expression profiles, and allows for the formation of persistent long-term memories for training events that are otherwise forgotten. We propose that emotionally salient experiences have utilized this system to form strong and persistent memories for behaviorally significant events. Consequently, the presence or absence of HDACs at a selection of specific gene promoters could serve as a critical barrier for permitting the formation of long-term memories.