Methods: CaMKIIa-creERT2 (Erdmann et al., 2007) and Dicer1f/f (Harfe et al., 2005) were crossed to produce inducible forebrain-restricted Dicer1 knockout mice (Dicer-ifKO) mice. Hippocampal mRNA profiles of 3-month-old wild-type (WT) and (Dicer-ifKO) mice were generated by deep sequencing, in triplicate, using Illumina HiSeq 2500. Each sample included total RNA isolated from the hippocampus of 3 mice. In total, 12 mice per genotype were used. The sequence reads that passed quality filters were mapped to reference genome (GRCm38/mm10) using Bowtie 2 (2.0.5) and TopHat (2.0.6). SAM/BAM files were further processed with Samtools (0.1.18). Read count quantitations were obtained using Seqmonk (0.26.0). Normalization of read counts and differential expression analysis between genotypes was carried out using DESeq2 R package from Bioconductor (Release 2.13). qRT–PCR validation was performed using SYBR Green assays. Results: We mapped about 13-14 million sequence reads per sample to the mouse genome (build GRCm38/mm10) and quantified 76,938 annotated transcripts. DESeq2 R package was used to normalize the counts and perform the differential expression. Differential analysis output was filtered by FDR threshold (padj < 0.1). This approach led us to identify 641 gene isoforms, corresponding to 314 genes that were differentially regulated in the mouse hippocampus upon Dicer ablation. Conclusions: We extend here the characterization of inducible forebrain-restricted Dicer1 mutants confirming the initial memory improvement. Moreover, we describe several novel phenotypes associated with early Dicer loss in the mature brain including an exacerbated response to seizures, increased CA1 neuron excitability, a pronounced weight gain and enhanced induction of immediate early genes (IEGs) in relevant neuronal nuclei. To identify candidate genes that could explain these phenotypes, we conducted two complementary genomic screens for the miRNAs primarily affected and their targets. Overall, our results explain both the initial and late consequences of Dicer loss in excitatory neurons and indicate that Dicer and the miRNA system play a critical role regulating neuronal homeostasis and responsiveness. Overall design: Hippocampal mRNA profiles of 3-month-old wild-type (WT) and Dicer-ifKO (3 weeks upon tamoxifen administration) male mice were generated by deep sequencing, in triplicate, using Illumina HiSeq 2500. Each sample included total RNA isolated from the hippocampus of 3 mice. In total, 12 mice per genotype were used.
Blocking miRNA Biogenesis in Adult Forebrain Neurons Enhances Seizure Susceptibility, Fear Memory, and Food Intake by Increasing Neuronal Responsiveness.
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Xanthine oxidoreductase is a regulator of adipogenesis and PPARgamma activity.
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View Samples3T3-L1 fibroblasts are a commonly used in vitro model for adipogenesis. When induced with hormones, they differentiate into mature fat cells. Here, microarrays were used to study 3T3-L1 adipose differentiation through time.
Xanthine oxidoreductase is a regulator of adipogenesis and PPARgamma activity.
No sample metadata fields
View SamplesGene expression was studied from different mouse tissues
Xanthine oxidoreductase is a regulator of adipogenesis and PPARgamma activity.
No sample metadata fields
View SamplesWe sequenced mRNA from two preparations of isolated Notch-responsive ductal pancreas cells and compared transcript expression to all other non-Notch-responsive cells from each sample to charactarize zebrafish centroacinar cells. Overall design: Determination of gene expression levels in centroacinar cells and non-centroacinar cells from adult pancreas.
Centroacinar Cells Are Progenitors That Contribute to Endocrine Pancreas Regeneration.
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View SamplesTo comprehensively characterize microRNA (miRNA) expression in breast cancer, we performed the first extensive next-generation sequencing expression analysis of this disease. We sequenced small RNA from tumors with paired samples of normal and tumor-adjacent breast tissue. Our results indicate that tumor identity is achieved mainly by variation in the expression levels of a common set of miRNAs rather than by tissue-specific expression. We also report 361 new, well-supported miRNA precursors. Nearly two-thirds of these new genes were detected in other human tissues and 49% of the miRNAs were found associated with Ago2 in MCF7 cells. Ten percent of the new miRNAs are located in regions with high-level genomic amplifications in breast cancer. A new miRNA is encoded within the ERBB2/Her2 gene and amplification of this gene leads to overexpression of the new miRNA, indicating that this potent oncogene and important clinical marker may have two different biological functions. In summary, our work substantially expands the number of known miRNAs and highlights the complexity of small RNA expression in breast cancer. Overall design: Sequencing of approximately 18-35 nt small RNAs from paired samples of normal, tumor and tumor-adjacent tissue for five breast cancer patients
Identification of new microRNAs in paired normal and tumor breast tissue suggests a dual role for the ERBB2/Her2 gene.
Specimen part, Subject
View SamplesGene expression from MCF7 breast cancer cells at different times of TNFa incubation:pcs2 and 14-3-3 transduced cells
Inhibition of specific NF-κB activity contributes to the tumor suppressor function of 14-3-3σ in breast cancer.
Time
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Loss of neuronal 3D chromatin organization causes transcriptional and behavioural deficits related to serotonergic dysfunction.
Sex, Specimen part
View SamplesThe interior of the eukaryotic cell nucleus is a highly organized 3D structure. In mature hippocampal and cortical pyramidal neurons, transcriptionally silent DNA is typically compacted in a few clusters referred to as chromocenters that are strongly stained with DNA intercalating agents like DAPI and whose function is still uncertain. We found that this 3D structure was severely disrupted by the incorporation of the chimeric histone H2BGFP into neuronal chromatin. Experiments in inducible and forebrain restricted bitransgenic mice demonstrated that the expression of this histone alters the higher-order organization of neuronal heterochromatin and causes a complex behavioral phenotype that includes hyperactivity, and social interaction, prepulse inhibition and cognitive defects. This phenotype was associated with highly specific transcriptional deficits that affected several serotonin receptor genes located at the edge of gene desert regions. Pharmacological and electrophysiological experiments indicate that this epigenetically-induced hyposerotonergic state may underlie the behavioral defects. Our results suggest a new role for perinuclear heterochromatin and chromocenter organization in the epigenetic regulation of neuronal gene expression and mental illness.
Loss of neuronal 3D chromatin organization causes transcriptional and behavioural deficits related to serotonergic dysfunction.
Specimen part
View SamplesWe profiled total mRNA of pancreas and kidney tissues of 3 different strains (p53-null; In4a/Arf-null and WT) of reprogrammable mouse lines (they all express OCT4, SOX2, KLF4, C-MYC under the control of a tetracycline promoter, activated by doxycycline) Overall design: 5 mice of each genotype were treated with doxycycline to induce the expression of the reprogramming factors, they were sacrificed and total mRNA was extracted from pancreas and kidney tissues (we mapped >24M reads per sample)
Tissue damage and senescence provide critical signals for cellular reprogramming in vivo.
Specimen part, Cell line, Subject
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