Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Investigating GPR34 expression regulation using whole transcriptome sequencing of spleens and dendritic cells from wildtype and GPR34 knockout mice

ABSTRACT: Naive spleens as well as naive and LPS-treated dendritic cells from wildtype and GPR34-/- mice were sequenced to integrate expression profiles with protein interaction networks and find functional modules that are affected by GPR34 Expression profiles of dendritic cells and whole spleens were generated using Illumina HiSeq 2500/ Illumina HiScan

ORGANISM(S): Mus musculus

SUBMITTER: Torsten Schoneberg

PROVIDER: E-GEOD-68155 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

ACCESS DATA

Publications

Dendritic Cells Regulate GPR34 through Mitogenic Signals and Undergo Apoptosis in Its Absence.

Jäger Elisabeth E Schulz Angela A Lede Vera V Lin Chen-Ching CC Schöneberg Torsten T Le Duc Diana D

Journal of immunology (Baltimore, Md. : 1950) 20160205 6

Dendritic cells (DCs) are specifically equipped with the G protein-coupled receptor 34 (GPR34). Tight regulation of GPR34 gene expression seems highly important for proper immunological functions, because the absence of this receptor leads to an alteration of the immune response, whereas overexpression was reported to be involved in neuroinflammation. However, the regulatory mechanism of GPR34 expression has not yet been investigated. Whole-transcriptome RNA sequencing analysis from spleens and ...[more]

PMID: 26851221

Similar Datasets

Project description:Purpose: the goal of this study is to investigate the consequences of USP3 deletion on gene expression in mouse LSK hematopoietic progenitors and in splenic B cells Methods: mRNA profiles of 8 weeks-old wild-type (WT) and ubiquitin specific protease 3 knockout (Usp3−/−) mice were generated by deep sequencing, in duplicate, using Illumina Hiseq2000. The sequence reads that passed quality filters were mapped with TopHat and the gene expressions were calculated using HTSeq-count. qRT–PCR validation was performed using SYBR Green assays Results: We assigned about 8-16 million reads per sample uniquely to a gene of the mouse reference genome (mm9). We identified 23,429 genes in the LSKs, naive B cells and activate B cells of WT and USP3−/− mice using TopHat in combination with HTSeq-count. Comparison of the RNAseq data from LSK with naive or activated B cells show that both the wt and the Usp3-/- LSKs largely exibited a gene expression profile that is specific for wt LSK and distinct from B cells (as supported by statistical significant difference between the transciptional profile of LSK versus naive or activated B cells, p value<0.0001 by Student t test). Comparison of normalized gene expression data for Wt LSKs versus naive B cells of one representative experiment shows Pearson coefficient of r=0.874, and R2=0.763. Distinct LSK-specific expressed genes (such as the MlI receptor and the Kit receptor) and B cells specific genes (such as the MS4A1/CD20 and Spi-B transcription factors) are identified. Expression of a set of 19 genes was assessed by RT-qPCR in three independent LSK mRNA per each genotype. qRT-PCR and the RNA-seq normalized expression data for these genes had a good linear relationship, validating the RNAseq analysis. Comparison of normalized gene expression data for Usp3-/- versus Wt LSK show Pearson coefficient r=0.986; R2=0.9738), naive B cells (Pearson coefficient r=0.987, R2=0.974) and LPS activated B cells (Pearson coefficient r=0.991, R2=0.983). RT-qPCR of a subset of hematopoietic stem cell genes, including Mlp2, ENg, Tek and Fdzl3, show no significant difference beteewn wt and Usp3-/- LSK cells. Less than 100 genes showed differential expression (up or down regulated) between the Wt and Usp3-/- LSK, with a fold change ≥1.5 and p value <0.05. Conclusions: Our results represent the first detailed analyis of the consequences of USP3 deletion on gene expression in hematopoietic populations such as LSKs progenitors and B cells by genome wide expression profiling in wt and Usp3-/- mice. RNAseq of two freshly isolated biological replicas of sorted LSKs from 8 weeks old Usp3-/- animals showed a very limited number of genes either slighly up or down regulated (<100 out of about 25.000) in Usp3-/- LSKs, none of which are reported to be directly involved in hematopoietic stem cell maintenance or to be linked to premature differentiation. We confirmed that Usp3-/- and wt LSKs express hematopoietic stem cell-specific genes to a similar extent. We conclude that young adult hematopoietic stem and progenitor cells (LSKs) perpetuated a stable gene expression program regardless of the homozygous deletion of USP3. mRNA profiles of 8 weeks-old wild type (WT) and Usp3-/- mice were generated by deep sequencing, in duplicate, using Illumina Hiseq2000. For each experiment wt n=4, Usp3-/- n=4 mice were analized. FACS sorted cells from from individual animals were pooled and subjected to deep sequencing. Cells were: LSK (Lin- Sca1+ cKit+) from bone marrow, sorted naive B cells from spleens (CD19+) and activated B cells harvested and FACS sorted after 4 days stimulation with lipopolysaccharide (LPS) in culture.

Project description:Dendritic cells (DCs) are a special class of leukocytes able to activate both innate and adaptive immune responses. They interact with microbes through germline-encoded pattern-recognition receptors (PRRs), which recognize molecular patterns expressed by various microorganisms. Upon antigen binding, PRRs instruct DCs for the appropriate priming of natural killer cells, followed by specific T-cell responses. Once completed the effector phase, DCs reach the terminal differentiation stage and eventually die by apoptosis. We have observed that following lipopolysaccharide (LPS)-stimulation the initiation of the apoptotic pathway in DCs is due the activation of NFAT proteins. Indeed, LPS induces Src-family kinase and phospholipase C (PLC)Î³2 activation, influx of extracellular Ca2+ and calcineurin-dependent nuclear NFAT translocation. The initiation of this pathway is independent of TLR4 engagement, and dependent exclusively on CD14. According with this observation CD14-deficient DCs do not die following LPS stimulation. Nevertheless, CD14-deficient DC death following LPS activation can be restored by co-stimulating DCs with LPS and thapsigargin. Thapsigargin empties the intracellular calcium stores by blocking calcium pumping into the sarcoplasmic and endoplasmic reticulum and thereby activates plasma membrane calcium channels. This, in turn, allows an influx of calcium into the cytosol and NFAT activation. To identify the NFAT controlled apoptosis genes in LPS activated DCs we have performed a kinetic microarray analysis (0, 48 and 60 h) in conditions allowing or inhibiting NFAT activation. Four genes have been selected: Nur77, Gadd45g, Ddit3/Gadd153/Chop-10 and Tia1. To identify apoptosis genes selectively modulated by NFAT, a comparative kinetic (time points 0, 48 and 60 h) microarray analysis was performed in the following conditions: 1) wild type bone marrow derived DCs (wtBMDCs) stimulated with LPS; 2) CD14-deficient BMDCs stimulated with LPS; 3) wtBMDCs stimulated with LPS in presence of thapsigargin; 4) CD14-deficient BMDCs stimulated with LPS in presence of thapsigargin.

Dataset Information

Investigating GPR34 expression regulation using whole transcriptome sequencing of spleens and dendritic cells from wildtype and GPR34 knockout mice

Publications

Dendritic Cells Regulate GPR34 through Mitogenic Signals and Undergo Apoptosis in Its Absence.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure