Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Quantitative Analysis of Wild Type and Dicer1-ifKO Hippocampal Transcriptomes (mRNA and small RNA) Through Next Generation Sequencing (smallRNA-Seq).

ABSTRACT: 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 small RNA 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. After trimming of the adapter sequences and masking for low-complexity or low-quality sequence, the inserts were sorted in separated files according to their lengths. The reads in which no match of the adapter sequences is found were discarded. Inserts of length 18-50 were pooled together and mapped to reference genome (GRCm38/mm10) using Burrows-Wheeler Alignment Tool (BWA) v.0.5.9. We used BWA to map the inserts with a maximum of 2 mismatches. Inserts mapping to several positions on the reference sequences with the same mapping quality were attributed at random to one of the position with a mapping quality of 0. Files were further processed using Samtools v.0.1.18 to get binary files (BAM). Read count quantitations were obtained using Seqmonk (0.26.0). We counted reads that mapped to annotated features (miRNAs, 2035; miscRNA, 491; mRNA 76938; rRNA, 341; snoRNA, 1602; snRNA, 1431; tRNAs, 26248; tRNA, 22). The DESeq2 R package from Bioconductor was used to normalize the counts and perform the differential expression analysis. Results: We mapped about 17-22 million sequence reads per sample to the mouse genome (build GRCm38/mm10) and quantified 109,108 annotated features. 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 119 annotated features (including 96 miRNAs) 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. Hippocampal small RNA 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.

ORGANISM(S): Mus musculus

SUBMITTER: jose lopez

PROVIDER: E-GEOD-60262 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Blocking miRNA Biogenesis in Adult Forebrain Neurons Enhances Seizure Susceptibility, Fear Memory, and Food Intake by Increasing Neuronal Responsiveness.

Fiorenza Anna A Lopez-Atalaya Jose P JP Rovira Victor V Scandaglia Marilyn M Geijo-Barrientos Emilio E Barco Angel A

Cerebral cortex (New York, N.Y. : 1991) 20150116 4

The RNase Dicer is essential for the maturation of most microRNAs, a molecular system that plays an essential role in fine-tuning gene expression. To gain molecular insight into the role of Dicer and the microRNA system in brain function, we conducted 2 complementary RNA-seq screens in the hippocampus of inducible forebrain-restricted Dicer1 mutants aimed at identifying the microRNAs primarily affected by Dicer loss and their targets, respectively. Functional genomics analyses predicted the main ...[more]

PMID: 25595182

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Project description: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. 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.

Project description: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 small RNA 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. After trimming of the adapter sequences and masking for low-complexity or low-quality sequence, the inserts were sorted in separated files according to their lengths. The reads in which no match of the adapter sequences is found were discarded. Inserts of length 18-50 were pooled together and mapped to reference genome (GRCm38/mm10) using Burrows-Wheeler Alignment Tool (BWA) v.0.5.9. We used BWA to map the inserts with a maximum of 2 mismatches. Inserts mapping to several positions on the reference sequences with the same mapping quality were attributed at random to one of the position with a mapping quality of 0. Files were further processed using Samtools v.0.1.18 to get binary files (BAM). Read count quantitations were obtained using Seqmonk (0.26.0). We counted reads that mapped to annotated features (miRNAs, 2035; miscRNA, 491; mRNA 76938; rRNA, 341; snoRNA, 1602; snRNA, 1431; tRNAs, 26248; tRNA, 22). The DESeq2 R package from Bioconductor was used to normalize the counts and perform the differential expression analysis. Results: We mapped about 17-22 million sequence reads per sample to the mouse genome (build GRCm38/mm10) and quantified 109,108 annotated features. 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 119 annotated features (including 96 miRNAs) 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.

Project description:MiRNAs have the potential to regulate cellular differentiation programs. However, miRNA-deficiency in primary hematopoietic stem cells (HSCs) results in HSC depletion in mice, leaving the question of whether miRNAs play a role in early-lineage decisions unanswered. To address this issue, we deleted Dicer1, which encodes an essential RNaseIII enzyme for miRNA biogenesis, in murine CCAAT/enhancer-binding protein alpha (C/EBPA)-positive myeloid-committed progenitors in vivo. In contrast to the results in HSCs, we found that miRNA depletion affected neither the number of myeloid progenitors nor the percentage of C/EBPA-positive progenitor cells. Analysis of gene-expression profiles from wild type and Dicer1-deficient granulocyte-macrophage progenitors (GMPs) revealed that 20 miRNA families were active in GMPs. Of the derepressed miRNA targets in Dicer1-null GMPs, 27% are normally exclusively expressed in HSCs or are specific for multi-potent progenitors and erythropoiesis, indicating an altered gene-expression landscape. Dicer1-deficient GMPs were defective in myeloid development in vitro and exhibited an increased replating capacity, indicating a regained self-renewal potential of these cells. In mice, Dicer1 deletion blocked monocytic differentiation, depleted macrophages and caused myeloid dysplasia with morphological features of Pelger-Huët anomaly. These results provide evidence for a miRNA-controlled switch for a cellular program of self-renewal and expansion towards myeloid differentiation in GMPs. To generate Cebpa-Cre;R26-LSL-Eyfp;Dicer1wt/fl/Dicer1fl/fl mice, we crossed mice that contain floxed Dicer1 alleles (Dicer1fl) with Cebpa-Cre;R26-LSL-Eyfp reporter mice 2. Fetal livers were obtained on embryonic day (E) 13.5. Routine genotyping of Dicer1; Cebpa-Cre;R26-LSL-Eyfp mice was performed by PCR assays of DNA from tail or toe biopsies. For transplantation, 6 to 8-week-old recipient mice (C57Bl/6, Jackson Laboratories) were irradiated (8.5 Gy) and tail-vein injected with fetal liver single-cell suspensions. Typically, cells from each fetal liver were transplanted into two recipient mice. Hematopoietic tissues were analyzed 6-10 weeks post transplantation. EYFP positive GMPs from the bone marrow of Dicer wt control (n=3), Dicer -/wt (n=3 and Dicer fl/fl (n=3) were sorted and analyzed for gene expression profiles.

Dataset Information

Quantitative Analysis of Wild Type and Dicer1-ifKO Hippocampal Transcriptomes (mRNA and small RNA) Through Next Generation Sequencing (smallRNA-Seq).

Publications

Blocking miRNA Biogenesis in Adult Forebrain Neurons Enhances Seizure Susceptibility, Fear Memory, and Food Intake by Increasing Neuronal Responsiveness.

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