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Identification of a cis-acting RNA synaptic localization element in Aplysia sensory neurons
Abstracts / Neuroscience Research 68S (2010) e55–e108
e59
O1-3-3-1 Identification of a cis-acting RNA synaptic localization element in Aplysia sensory neurons
O1-3-3-3 Epigenetic regulation of necdin gene expression during neural development
Dan Ohtan Wang 1 , Elliott Meer 2 , Sang Mok Kim 3 , Kelsey C. Martin 1,2,4
Tsuyoshi Ohkumo Kazuaki Yoshikawa
1 Department Psychiatry and Biobehavioral Sciences, University of California, Los Angeles 90095-1737 2 Department of Biological Chemistry, UCLA 3 Interdepartmental Program in Neurosciences, UCLA 4 Semel Institute for Neuroscience and Human Behavior, UCLA
RNA localization and regulated local translation allows gene expression to be spatially restricted to each of the thousands of synaptic compartments formed by a single neuron and has been implicated in learning and memory (Martin and Ephrussi, 2009). Hundreds of mRNAs have been identified in dendrites, although only a few have been confirmed to be bona fide synaptic mRNAs. One characterized synaptic mRNA in Aplysia encodes a sensory neuron-specific neuropeptide called sensorin. Sensorin mRNA is highly concentrated at sensory (SN)- motor (MN) neuron synapses, and thus offers an excellent molecular model for studying the localization mechanisms of synaptic mRNAs. In Aplysia neuronal cultures, we tested putative elements of sensorin mRNA for their sufficiency to localize reporter mRNA to synapses by expressing chimeric reporters. We found that the 3 UTR of sensorin is sufficient for localizing the reporter mRNA from the soma into distal processes while the 5 UTR is required for localizing the reporter mRNA to synapses. A 66 nt cis-element in the 5 UTR of sensorin was found to be required and sufficient for the concentration of the reporter mRNA at synapses. Deletion of a tandem 7mer-repeat within the 66 nt element from 5 UTR abolished synaptic localization of the reporter mRNA. Intriguingly, seven mutations induced in three out of four repeats within the tandem repeats did not disrupt synaptic localization of the reporter mRNA when compensatory mutations were simultaneously introduced to retain the secondary structure. This 66 nt element is, to our knowledge, the first identified mRNA synaptic localization element. Further experiments will be to identify the trans-acting factors such as binding proteins that recognize this element and mediate the mRNA localization.
, Koichi
Hasegawa, Kazushirou
Fujiwara,
Institute for Protein Research, Osaka University Necdin is expressed predominantly in postmitotic neurons and involved in neuronal differentiation. The necdin gene (Ndn) is located in human chromosome 15q11-q13, whose deletion causes Prader-Willi syndrome (PWS), a classic genomic imprinting-associated neurodevelopmental disorder. Genomic imprinting is an allele-specific gene silencing process through cytosine-specific methylation at CpG dinucleotides. Ndn is maternally imprinted and expressed only from the paternal allele. Although necdin is thought to play an important role in neuronal differentiation and maturation, the regulatory mechanism of Ndn gene expression is not well understood. We first examined Ndn expression in mouse neural stem cells (NSCs) and neurons differentiated from NSCs by real-time PCR. Ndn expression levels increased during neural differentiation, whereas Ndn was not expressed in paternal Ndn-deficient cells. We then examined whether the methylation status of the paternal Ndn affects its expression during neural differentiation. Ndn contains CpG-rich regions extending from the promoter region to the protein coding region. We prepared genomic DNA from undifferentiated and differentiated NSCs, and analyzed the methylation status of 23 CpG sites near the transcription start site in the paternal and maternal Ndn alleles by sodium bisulfite conversion analysis. The CpG sites in the paternal Ndn allele were more frequently methylated under undifferentiated conditions than under neurally differentiated conditions. Surprisingly, several CpG sites in the maternal Ndn allele were completely demethylated in NSCs and differentiated cells such as neurons and astrocytes. These results suggest that DNA methylation status of specific CpG sites of paternal Ndn regulates its expression levels by increasing the accessibility of transcription factors, and that maternal Ndn is silenced through DNA methylation-independent mechanisms such as allele-specific chromatin structure. doi:10.1016/j.neures.2010.07.029
doi:10.1016/j.neures.2010.07.027
O1-3-3-2 Epigenetic control of the critical period in sensory development Judy CG Sng 1 , Patrick Lee 1 , Siti Norfiza Bte Rahmat 1 , Vania Lim 1 , Tendy Ching 1 , Marco Bezzi 2 , Ernesto Guccione 2
O1-3-3-4 The spatiotemporal regulation of activitydependent genes in post-mitotic neurons Takumi Takizawa , Misato Takagi, Hirotoshi Sasaoka, Kenji Itoh, Kinichi Nakashima Mol Neurosci, Grad Sch of Bio Sci, NAIST, Nara
1
Growth, Development and Metabolism, Singapore Institute for Clinical Sciences, A*Star 2 Institute of Molecular Biology, A*Star, 60 Biopolis Way, Singapore The capacity for plasticity in the adult is limited by the anatomical traces laid down during sensitive periods in early postnatal life. These sensitive periods are called critical periods. In the mouse visual cortex, the critical period have a clear onset and closure in dictating plasticity. We found that epigenetics play a role in critical period window formations and perturbing the epigenome enable reactivation of adult visual cortical plasticity (Sng et al, in submission). To better understand how epigenetics interact with genes in critical period, our lab focuses on deciphering the histone code hypothesis for critical period window formation. Here we describe the role the role of Arginine Methyltransferases (PRMTs), a family of transcriptional co-activators/repressors that target histones, in shaping of epigenetic landscapes and synaptic plasticity. Real-time quantitative PCR was used to show that gene expression profiles differ at various postnatal ages and brain regions. One such enzyme is PRMT8 and is specifically expressed in the brain. PRMT8 expression is concomitant with the onset of critical period and is specific in the visual cortex but lower in other parts of the brain such as the hippocampus or the cerebral cortex, which exhibit lifelong plasticity. Immunohistochemistry results show that PRMT8 exhibits nuclear subcellular localization and is predominantly localized in the neurons. To investigate the binding partners of PRMT8, we are using chromatin immunoprecipitation (ChIP) assays to first validate various synaptic plasticity genes such as CBP, GluR1 and 2, and NR2A and B known to be responsible for CP onset and using ChIP-seq as a tool for genome-wide analysis. These findings suggest that the importance of the protein arginine methylation in the onset of the critical period in the visual cortex and our studies are now targeting at this modification in reactivating adult plasticity. doi:10.1016/j.neures.2010.07.028
An increasing body of evidence shows that sub-nuclear spatial gene positioning is of great relevance in a wide range of cellular functions such as differentiation and gene expression. For instance, the nuclear periphery has been shown to constitute a repressive environment for gene transcription in mammalian cells. Neurons express a number of specific genes upon depolarization in response to external stimuli. Although signaling pathways and transcription factors involved in activity-dependent gene expression in neurons have been intensively studied, spatial positioning and chromatin regulation of activity-dependent neuronal genes remain elusive. We have mapped the sub-nuclear spatial positioning and chromatin states of activity-dependent genes in mouse hippocampal neurons and delineated their spatio-temporal regulation. Using microarray analysis we have identified sets of genes which are upregulated within 30 minutes (designated as the early genes) while another set of genes is upregulated around180 minutes (the late genes) after depolarization. DNA fluorescence in situ hybridization revealed that the late genes are preferentially located at the nuclear periphery while the early genes are not. Surprisingly the late genes were transcribed at the periphery after depolarization, indicating the nuclear periphery in neurons is a transcriptionally permissive environment. The late genes are enriched in a repressive histone marker, di-methyl lysine 9 of histone H3, and get phosphorylated on serine 10 of H3 upon depolarization. The early genes are enriched in promoter-initiated RNA polymerase II (RNAP II) phosphorylated on serine 5 of its C-terminal domain. Negative elongation factor (NELF) also occupies the proximal region of the early genes and plays a critical role in RNAP II stalling. These results demonstrate that the temporal regulation of activity-dependent genes in post-mitotic neurons correlates with the sub-nuclear spatial positioning and chromatin states. doi:10.1016/j.neures.2010.07.030
e59
O1-3-3-1 Identification of a cis-acting RNA synaptic localization element in Aplysia sensory neurons
O1-3-3-3 Epigenetic regulation of necdin gene expression during neural development
Dan Ohtan Wang 1 , Elliott Meer 2 , Sang Mok Kim 3 , Kelsey C. Martin 1,2,4
Tsuyoshi Ohkumo Kazuaki Yoshikawa
1 Department Psychiatry and Biobehavioral Sciences, University of California, Los Angeles 90095-1737 2 Department of Biological Chemistry, UCLA 3 Interdepartmental Program in Neurosciences, UCLA 4 Semel Institute for Neuroscience and Human Behavior, UCLA
RNA localization and regulated local translation allows gene expression to be spatially restricted to each of the thousands of synaptic compartments formed by a single neuron and has been implicated in learning and memory (Martin and Ephrussi, 2009). Hundreds of mRNAs have been identified in dendrites, although only a few have been confirmed to be bona fide synaptic mRNAs. One characterized synaptic mRNA in Aplysia encodes a sensory neuron-specific neuropeptide called sensorin. Sensorin mRNA is highly concentrated at sensory (SN)- motor (MN) neuron synapses, and thus offers an excellent molecular model for studying the localization mechanisms of synaptic mRNAs. In Aplysia neuronal cultures, we tested putative elements of sensorin mRNA for their sufficiency to localize reporter mRNA to synapses by expressing chimeric reporters. We found that the 3 UTR of sensorin is sufficient for localizing the reporter mRNA from the soma into distal processes while the 5 UTR is required for localizing the reporter mRNA to synapses. A 66 nt cis-element in the 5 UTR of sensorin was found to be required and sufficient for the concentration of the reporter mRNA at synapses. Deletion of a tandem 7mer-repeat within the 66 nt element from 5 UTR abolished synaptic localization of the reporter mRNA. Intriguingly, seven mutations induced in three out of four repeats within the tandem repeats did not disrupt synaptic localization of the reporter mRNA when compensatory mutations were simultaneously introduced to retain the secondary structure. This 66 nt element is, to our knowledge, the first identified mRNA synaptic localization element. Further experiments will be to identify the trans-acting factors such as binding proteins that recognize this element and mediate the mRNA localization.
, Koichi
Hasegawa, Kazushirou
Fujiwara,
Institute for Protein Research, Osaka University Necdin is expressed predominantly in postmitotic neurons and involved in neuronal differentiation. The necdin gene (Ndn) is located in human chromosome 15q11-q13, whose deletion causes Prader-Willi syndrome (PWS), a classic genomic imprinting-associated neurodevelopmental disorder. Genomic imprinting is an allele-specific gene silencing process through cytosine-specific methylation at CpG dinucleotides. Ndn is maternally imprinted and expressed only from the paternal allele. Although necdin is thought to play an important role in neuronal differentiation and maturation, the regulatory mechanism of Ndn gene expression is not well understood. We first examined Ndn expression in mouse neural stem cells (NSCs) and neurons differentiated from NSCs by real-time PCR. Ndn expression levels increased during neural differentiation, whereas Ndn was not expressed in paternal Ndn-deficient cells. We then examined whether the methylation status of the paternal Ndn affects its expression during neural differentiation. Ndn contains CpG-rich regions extending from the promoter region to the protein coding region. We prepared genomic DNA from undifferentiated and differentiated NSCs, and analyzed the methylation status of 23 CpG sites near the transcription start site in the paternal and maternal Ndn alleles by sodium bisulfite conversion analysis. The CpG sites in the paternal Ndn allele were more frequently methylated under undifferentiated conditions than under neurally differentiated conditions. Surprisingly, several CpG sites in the maternal Ndn allele were completely demethylated in NSCs and differentiated cells such as neurons and astrocytes. These results suggest that DNA methylation status of specific CpG sites of paternal Ndn regulates its expression levels by increasing the accessibility of transcription factors, and that maternal Ndn is silenced through DNA methylation-independent mechanisms such as allele-specific chromatin structure. doi:10.1016/j.neures.2010.07.029
doi:10.1016/j.neures.2010.07.027
O1-3-3-2 Epigenetic control of the critical period in sensory development Judy CG Sng 1 , Patrick Lee 1 , Siti Norfiza Bte Rahmat 1 , Vania Lim 1 , Tendy Ching 1 , Marco Bezzi 2 , Ernesto Guccione 2
O1-3-3-4 The spatiotemporal regulation of activitydependent genes in post-mitotic neurons Takumi Takizawa , Misato Takagi, Hirotoshi Sasaoka, Kenji Itoh, Kinichi Nakashima Mol Neurosci, Grad Sch of Bio Sci, NAIST, Nara
1
Growth, Development and Metabolism, Singapore Institute for Clinical Sciences, A*Star 2 Institute of Molecular Biology, A*Star, 60 Biopolis Way, Singapore The capacity for plasticity in the adult is limited by the anatomical traces laid down during sensitive periods in early postnatal life. These sensitive periods are called critical periods. In the mouse visual cortex, the critical period have a clear onset and closure in dictating plasticity. We found that epigenetics play a role in critical period window formations and perturbing the epigenome enable reactivation of adult visual cortical plasticity (Sng et al, in submission). To better understand how epigenetics interact with genes in critical period, our lab focuses on deciphering the histone code hypothesis for critical period window formation. Here we describe the role the role of Arginine Methyltransferases (PRMTs), a family of transcriptional co-activators/repressors that target histones, in shaping of epigenetic landscapes and synaptic plasticity. Real-time quantitative PCR was used to show that gene expression profiles differ at various postnatal ages and brain regions. One such enzyme is PRMT8 and is specifically expressed in the brain. PRMT8 expression is concomitant with the onset of critical period and is specific in the visual cortex but lower in other parts of the brain such as the hippocampus or the cerebral cortex, which exhibit lifelong plasticity. Immunohistochemistry results show that PRMT8 exhibits nuclear subcellular localization and is predominantly localized in the neurons. To investigate the binding partners of PRMT8, we are using chromatin immunoprecipitation (ChIP) assays to first validate various synaptic plasticity genes such as CBP, GluR1 and 2, and NR2A and B known to be responsible for CP onset and using ChIP-seq as a tool for genome-wide analysis. These findings suggest that the importance of the protein arginine methylation in the onset of the critical period in the visual cortex and our studies are now targeting at this modification in reactivating adult plasticity. doi:10.1016/j.neures.2010.07.028
An increasing body of evidence shows that sub-nuclear spatial gene positioning is of great relevance in a wide range of cellular functions such as differentiation and gene expression. For instance, the nuclear periphery has been shown to constitute a repressive environment for gene transcription in mammalian cells. Neurons express a number of specific genes upon depolarization in response to external stimuli. Although signaling pathways and transcription factors involved in activity-dependent gene expression in neurons have been intensively studied, spatial positioning and chromatin regulation of activity-dependent neuronal genes remain elusive. We have mapped the sub-nuclear spatial positioning and chromatin states of activity-dependent genes in mouse hippocampal neurons and delineated their spatio-temporal regulation. Using microarray analysis we have identified sets of genes which are upregulated within 30 minutes (designated as the early genes) while another set of genes is upregulated around180 minutes (the late genes) after depolarization. DNA fluorescence in situ hybridization revealed that the late genes are preferentially located at the nuclear periphery while the early genes are not. Surprisingly the late genes were transcribed at the periphery after depolarization, indicating the nuclear periphery in neurons is a transcriptionally permissive environment. The late genes are enriched in a repressive histone marker, di-methyl lysine 9 of histone H3, and get phosphorylated on serine 10 of H3 upon depolarization. The early genes are enriched in promoter-initiated RNA polymerase II (RNAP II) phosphorylated on serine 5 of its C-terminal domain. Negative elongation factor (NELF) also occupies the proximal region of the early genes and plays a critical role in RNAP II stalling. These results demonstrate that the temporal regulation of activity-dependent genes in post-mitotic neurons correlates with the sub-nuclear spatial positioning and chromatin states. doi:10.1016/j.neures.2010.07.030