ShRNA profiling of rat neurons to identify MEF2 Regulated Genes
ABSTRACT: Expression profiling in hippocampal neurons to identify activity-regulated genes controlled by MEF2; Experiments were conducted to identify activity-regulated MEF2 target genes. Experiment Overall Design: Neurons were depolarized with 55mM extracellular KCl for one or six hours in the presence of control or MEF2-specific shRNAs
Project description:To identify the activity-induced gene expression programs in inhibitory and excitatory neurons, we analyzed RNA extracted from cultured E14 mouse MGE- and CTX-derived neurons (DIV 10) after these cultures were membrane-depolarized for 0, 1 and 6 hrs with 55mM extracellular KCl. To identify the gene programs regulated in these cells by the activity-induced early-response transcription factor Npas4, we repeated the same experiment in the MGE- and CTX-cultures lacking Npas4 (Npas4-KO). Littermate mouse E14 MGE- or CTX-derived neurons (WT or KO for Npas4) were cultured for 9 days, quieted overnight with TTX and AP-5 and then membrane-depolarized for 0, 1 or 6 hours by raising the extracellular KCl-concentration to 55mM. RNA was then extracted and analyzed using Affymetrix GeneChip Mouse Expression Set 430 2.0 microarray platform.
Project description:Expression profiling in hippocampal neurons to identify genes upregulated in response to ectopic MEF2 activation by MEF2-VP16-ER; Experiments were conducted to identify activity-regulated MEF2 target genes. Experiment Overall Design: Neurons expressed either control MEF2deltaDBD-VP16-ER or MEF2-VP16-ER and expression profiling was conducted before and after 4-OH-Tamoxifen (4OHT) application.
Project description:To identify the activity-induced gene expression programs in inhibitory neurons, we analyzed RNA extracted from cultured E14 mouse MGE-derived neurons (DIV 10) after these cultures were membrane-depolarized for 0, 1 and 6 hrs with 55mM extracellular KCl. Mouse E14 MGE-derived neurons were cultured for 9 days, quieted overnight with TTX and AP-5 and then membrane-depolarized for 0, 1 or 6 hours by raising the extracellular KCl-concentration to 55mM. RNA was then extracted and WT RNA-Seq was performed on ABi SOLiD
Project description:Expression profiling in hippocampal neurons to identify activity-regulated genes controlled by MEF2 Experiments were conducted to identify activity-regulated MEF2 target genes. Keywords: Gene knockdown and depolarization time course Overall design: Neurons were depolarized with 55mM extracellular KCl for one or six hours in the presence of control or MEF2-specific shRNAs
Project description:Experience-dependent gene transcription is required for nervous system development and function. However, the DNA regulatory elements that control this program of gene expression are not well defined. Here we characterize the enhancers that function across the genome to mediate activity-dependent transcription in neurons. While ~12,000 putative activity-regulated enhancer sequences have previously been identified that are enriched for H3K4me1 and the histone acetyltransferase CBP, we find that this chromatin signature is not sufficient to distinguish which of these regulatory sequences are actively engaged in promoting activity-dependent transcription. We show here that a subset of H3K4me1/CBP positive enhancers that is enriched for H3K27 acetylation (H3K27ac) in vivo, and shows increased H3K27ac upon membrane depolarization of cortical neurons, function to regulate activity-dependent transcription. The function of many of these activity-regulated enhancers appears to be dependent on the binding of FOS, a protein that had previously been thought to interact primarily with the promoters of activity-regulated genes. Furthermore, many of these target genes in cortical neurons encode neuron specific proteins that regulate synaptic development and function. These findings suggest that FOS functions at enhancers to control activity-dependent gene programs that are critical for nervous system function, and provide a resource of activity-dependent enhancers that may give insight into genetic variation that contributes to brain development and disease. Genome-wide maps of H3K27ac and AP1 transcription factors (CFOS, FOSB, JUNB) before and after neuronal activity in mouse cortical neurons.
Project description:To examine potential differences in activity-dependent gene expression, we analyzed mRNA expression in cultured neurons isolated from Wild-type vs MeCP2 S421A mice at 0 (unstimulated), 1 or 6 hours after membrane depolarization by exposure to high extracellular KCl (55mM) All mice were male littermates from one of three litters. We isolated RNA from dissociated cortical cultures (E16+7DIV) isolated from Wild-Type or MeCP2 S421A knock-in mice littermates. Cells were either left unstimulated or depolarized for 1 or 6 hours by addition of 55mM KCl to the media. mRNA expression was analyzed using the Affymetrix GeneChip Mouse Expression Set 430 2.0 microarray platform.
Project description:We analyzed the binding profiles of MEF2D, a member of MEF2 family transcription factors, in rat hippocampal neurons. Keywords: ChIP-chip We used custom-designed rat genome tiling array manufactured by Nimblegen Systems, Inc . This array contains probes that represent the following rat genomic regions: 182 genes identified by mRNA profiling experiments, 86 genes whose expression was decreased by both KCl-mediated depolarization and MEF2 RNAi, and 40 control genes whose expression was not altered by MEF2 RNAi or MEF2-VP16-ER. The array not only covers the entire gene regions but also contains probes that correspond to the 40 kb 5′and 3′ to each gene. Repeatmasking was conducted by Nimblegen to ensure that repetitive elements were not tiled on the microarray. Probe length and spacing between the probes were 50-75mer and 50 bp, respectively.
Project description:The transcription factor Mef2 regulates activity-dependent neuronal plasticity and morphology in mammals, and clock neurons are reported to experience activity-dependent circadian remodeling in Drosophila. We show here that Mef2 is required for this daily fasciculation-defasciculation cycle. Moreover, the master circadian transcription complex CLK/CYC directly regulates Mef2 transcription. ChIP-Chip analysis identified numerous Mef2 target genes implicated in neuronal plasticity, including the cell-adhesion gene Fas2. Genetic epistasis experiments support this transcriptional regulatory hierarchy, CLK/CYC->Mef2-> Fas2, indicate that it influences the circadian fasciculation cycle within pacemaker neurons and suggest that this cycle also contributes to circadian behavior. Mef2 therefore transmits clock information to machinery involved in neuronal remodeling, which contributes to locomotor activity rhythms. Mef2 ChIP-chip samples collected at 6 timepoints, input and IP samples
Project description:Transcription of immediate early genes (IEGs) in neurons is exquisitely sensitive to neuronal activity, but the mechanism underlying the earliest of these transcription events is largely unknown. Here we demonstrate that very fast IEGs (VF-IEGs) such as arc/arg3.1 are poised for rapid transcription by the stalling of RNA Polymerase II (Pol II) just downstream of the transcription start site. RNAi-depletion of two subunits of a mediator of Pol II stalling, Negative Elongation Factor, reduces Pol II occupancy of the arc promoter and compromises rapid induction of arc and other VF-IEGs. In contrast, reduction of Pol II stalling did not prevent expression of other fast IEGs (F-IEGs). These F-IEGs are expressed with comparatively slower kinetics and largely lack promoter proximal Pol II stalling. Taken together, our data strongly indicate that very fast kinetics of neuronal IEG expression require poised Pol II and suggest a role for this mechanism in transcription-dependent learning and memory. TTX withdrawal induced neuronal activity. To study activity-induced gene expression, neurons were treated with TTX for 48 hours and then TTX was washed out either for 15 minutes (W15) or for 45 minutes (W45). Gene expression was measured in these two groups in comparison to TTX treated neurons.
Project description:Microarray expression profiling of manually sorted m-citirin-labeled layer 4 visual cortex star pyramid neurons from deprived and non-deprived hemispheres. Monocular deprivation by TTX-injection (at P12-13 and again at P13-14), followed by manual sorting of m-Citrin-labeled Layer 4 Visual Cortex Star Pyramid neurons in deprived and non-deprived hemispheres. RNA was extracted using PicoPure RNA Isolation Kit, reverse transcribed, and amplified using a standard T7 IVT protocol (Affymetrix Small Sample Target Labeling Assay Version II).