Unknown,Transcriptomics,Genomics,Proteomics

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MicroRNA expression profile of activated versus non activated primary B cells

ABSTRACT: microRNA expression profile in 5 indepent sets of paired samples of LPS+IL-4 activated versus non activated primary mouse spleen B cells Keywords: cell type comparison Activated B cells for miRNA array analysis were obtained after CFSE labelling (Molecular Probes), LPS+IL4 stimulation for 3 days and sorting of cells that had undergone at least 1 cell division. Sortings were performed with a FACSAria cell sorter (BD Biosciences).

ORGANISM(S): Mus musculus

SUBMITTER: Almudena Ramiro

PROVIDER: E-GEOD-12176 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

miR-181b negatively regulates activation-induced cytidine deaminase in B cells.

de Yébenes Virginia G VG Belver Laura L Pisano David G DG González Susana S Villasante Aranzazu A Croce Carlo C He Lin L Ramiro Almudena R AR

The Journal of experimental medicine 20080901 10

Activated B cells reshape their primary antibody repertoire after antigen encounter by two molecular mechanisms: somatic hypermutation (SHM) and class switch recombination (CSR). SHM and CSR are initiated by activation-induced cytidine deaminase (AID) through the deamination of cytosine residues on the immunoglobulin loci, which leads to the generation of DNA mutations or double-strand break intermediates. As a bystander effect, endogenous AID levels can also promote the generation of chromosome ...[more]

PMID: 18762567

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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.

Dataset Information

MicroRNA expression profile of activated versus non activated primary B cells

Publications

miR-181b negatively regulates activation-induced cytidine deaminase in B cells.

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