Project description:To investigate the molecular basis for the B cell developmental arrest in Pax5R31Q/– mice, we performed RNA-sequencing (RNA-seq) with ex vivo sorted Pax5+/+ and Pax5R31Q/– pro-B cells. We identified Pax5-activated and Pax5-repressed genes that were no longer properly regulated by the Pax5-R31Q protein in Pax5R31Q/– pro-B cells. Notably, these genes were only a subset of the activated and repressed genes identified by comparing Pax5+/+ and Pax5–/– pro-B cells, which raised the question whether binding of the Pax5-R31Q protein may be selectively lost at the subset of deregulated genes in Pax5R31Q/– pro-B cells. By using a Pax5 paired domain antibody for chromatin immunoprecipitation coupled with deep sequencing (ChIP-seq) of short-term cultured Pax5+/+ and Pax5R31Q/– pro-B cells, we identified all associated Pax5 binding sites. Common Pax5 peaks had a similar Pax5-binding density in contrast to the strong binding difference observed at the unique peaks present in Pax5+/+ pro-B cells. To investigate a correlation between the loss of Pax5 binding and gene expression, we focused our analysis on Pax5 peaks in the TSS region of activated genes. We systematically investigated the correlation between loss of Pax5 binding at the TSS and down-regulation of gene expression in Pax5R31Q/– pro-B cells. The ratio of Pax5 binding between Pax5R31Q/– and Pax5+/+ pro-B cells at the TSS of these activated genes was significantly reduced compared to that of expressed non-regulated genes. We next explored whether the Pax5-binding difference at the TSS also correlated with the magnitude of gene expression difference. The loss of Pax5 binding at the TSS also correlated with the degree of expression change in Pax5R31Q/– pro-B cells compared to Pax5+/+ pro-B cells. We conclude therefore that the selective DNA-binding of Pax5-R31Q is responsible for the observed gene expression differences in Pax5R31Q/– pro-B cells.

2022-07-06 | GSE182463 | GEO

The transcription factor Pax5 regulates its target genes by recruiting chromatin-modifying proteins in committed B cells

Project description:Pax5 is a critical regulator of B cell commitment. Here we identified direct Pax5 target genes by streptavidin-mediated ChIP-chip analysis of pro-B cells expressing in vivo biotinylated Pax5. By binding to promoters and enhancers, Pax5 directly regulates the expression of multiple transcription factor, cell surface receptor and signal transducer genes. One of the newly identified enhancers was shown by transgenic analysis to confer Pax5-dependent B-cell-specific activity to the Nedd9 gene controlling B cell trafficking. Profiling of histone modifications in Pax5-deficient and committed wild-type pro-B cells demonstrated that Pax5 induces active chromatin at activated target genes, while eliminating active chromatin at repressed genes in committed pro-B cells. Pax5 rapidly induces these chromatin and transcription changes by recruiting chromatin-remodeling, histone-modifying and basal transcription factor complexes to its target genes. These data provide novel insight into the regulatory network and epigenetic regulation, by which Pax5 controls B cell commitment. Analysis of chromatin and TF binding in rag2-/- and pax5-/- rag2-/- pro-B cells. Chip-Chip with 1-3 experiments for each antibody and celltype combination.

2012-03-29 | E-GEOD-28373 | biostudies-arrayexpress

Comparison of gene expression profiles of ex vivo sorted Common Lymphoid Progenitors (CLPs) and in vitro cultured cCLPs.

Project description:A common lymphoid progenitor cell culture system was established, maintaining a multipotent, proliferating state of CLPs. Cells can be cultured and expanded in vitro for several months, maintaining their in vitro and in vivo lymphoid differentiation potential. We were interested, whether cCLP are changing, with regards to their gene expression program, compared to in vivo CLPs, during their expansion in culture. Therefore we compared the gene expression profile of cultured cCLPs and their counterparts in vivo, by NextGenSeq. CLPs were sorted from bone marrow of 6-10 weeks old C57BL/6 mice (n=4). Cells were sorted for Lin-, IL7R+, Flk2+, CD27+, Ly6d- marker expressing CLPs by Flow Cytometry for gene expression analysis. In parallel the same cell population was sorted and cultured under cCLP culture conditions. After two months culture the cells sorted again for Lin-, CD27+, Ly6d- cells by Flow Cytometry for gene expression analysis (n=4). The analysis shows that cCLPs maintain a very similar transcription profile to ex vivo sorted common lymphoid progenitors.

2018-04-01 | GSE109805 | GEO

Effect of Lithum on adult white matter

Project description:Analysis of adult murine optic nerves cultured for 3 days in vitro (DIV) ex-vivo in organotypic media containing saline or Lithium Chloride. Results provide insight on the mechanism of action of Lithium on glial cells.

2019-09-18 | GSE132397 | GEO

The transcription factor Pax5 regulates its target genes by recruiting chromatin-modifying proteins in committed B cells

2012-03-30 | GSE28373 | GEO

Comparison of gene expression from thymocyte populations and equivalent OP9-DL1 cultured cells

Project description:Comparison between ex vivo immature, mature and stimulated T cells and in vitro generated counterparts. The T cells generated in vitro were cultured on OP9-DL1 stroma supplied with growth factors. Three in vivo (Thymus DPCD3+, Thymus SP8, PBMC CD8 POST) and three in vitro (CCd14 DPCD3+, CCd20 SP8, CCd27 Post) samples were collected. T-cells were sorted to high purity, RNA was extracted and hybridized on Affymetrix microarrays. No technical replicates were used. The expression profiles of in vivo samples were then compared to the ones of in vitro samples.

2014-07-19 | E-GEOD-49039 | biostudies-arrayexpress

E-MTAB-9540

Project description:RNA sequencing of native and ex vivo cultured murine tail tendon fascicles

2021-01-19 | E-MTAB-9540 | ExpressionAtlas

Transcription profiling of mouse B cells derived from bone marrow

Project description:Affymetrix GeneChip derived gene expression profiles of flow sorted murine B cell precursors, ex-vivo isolated, RNA-amplified

2007-12-13 | E-GEOD-13 | biostudies-arrayexpress