Project description:Ebf1 deficient pre-pro B-cells (Fraction A) can be cultured in the presence of stromal feeders and cytokines. The retroviral transduction of these cells with Ebf1 was used as gain-of-function experiment for the analysis of direct and functional target genes of Ebf1. Ebf1 deficient pre-pro B-cells were retrovirally transduced with an Ebf1-expressing or control retrovirus. 24h after transduction the infected cells were isolated and their gene expression profile was compared.
Project description:An assessment of a role of Ebf1 in committed B lineage cells. In this study, we adopted the strategy of deleting Ebf1 after reconstitution of Rag2-/-Il2rg-/- mice and found that committed pre-B cells could be converted into T cells and ILCs upon deletion of Ebf1. We used µ-arrays to gain insight into changes in gene expression of CD19+ Ebf1-deficient cells. The dataset was compared with microarray data available for different hematopoietic developmental stages (GSE15907). The complete dataset is linked below as a supplementary file.
Project description:Ebf1 deficient pre-pro B-cells (Fraction A) can be cultured in the presence of stromal feeders and cytokines. The retroviral transduction of these cells with Ebf1 was used as gain-of-function experiment for the analysis of direct and functional target genes of Ebf1.
Project description:Loss of one allele of Ebf1 impairs pre-B cell (B220+CD19+CD43low/negIgM-) expansion. In order to better understand the underlying cause of the reduced pre-B cell compartment in Ebf1+/- mice, we sorted pro-B (B220+CD19+CD43highIgM- ) as well as pre-B cells from Wt and Ebf1 heterozygote mutant mice and performed Affymetrix based microarray gene expression analysis. While the overall gene expression patterns as well as Pax5 expression in Wt and Ebf1 pro-B cells were similar, gene set enrichment (GSE) analysis of the microarray data suggested a reduced expression of cell division (p<0.001) and mitosis (p<0.001) genes in the Ebf1+/- pre-B cells as compared to their Wt counterparts. This in combination with rather normal expression of B-lineage genes in Ebf1+/- pre-B cells opens for the possibility that the phenotypic loss of pre-B cells in Ebf1+/- mice could be a result of reduced expansion of progenitors rather than by a differentiation block.
Project description:Loss of one allele of Ebf1 impairs pre-B cell (B220+CD19+CD43low/negIgM-) expansion. In order to better understand the underlying cause of the reduced pre-B cell compartment in Ebf1+/- mice, we sorted pro-B (B220+CD19+CD43highIgM- ) as well as pre-B cells from Wt and Ebf1 heterozygote mutant mice and performed Affymetrix based microarray gene expression analysis. While the overall gene expression patterns as well as Pax5 expression in Wt and Ebf1 pro-B cells were similar, gene set enrichment (GSE) analysis of the microarray data suggested a reduced expression of cell division (p<0.001) and mitosis (p<0.001) genes in the Ebf1+/- pre-B cells as compared to their Wt counterparts. This in combination with rather normal expression of B-lineage genes in Ebf1+/- pre-B cells opens for the possibility that the phenotypic loss of pre-B cells in Ebf1+/- mice could be a result of reduced expansion of progenitors rather than by a differentiation block. RNA was extracted from 20,000 or 25000 purified adult bone marrow cells using the RNAeasy microkit. RNA was labeled and amplified by dual amplification and hybridized to Affymetrix microarray MOE430_2, according to AffymetrixTM GeneChip Expression Analysis Technical Manual. Probe level expression values were calculated using the RMA algorithm.
Project description:An assessment of a role of Ebf1 in committed B lineage cells. In this study, we adopted the strategy of deleting Ebf1 after reconstitution of Rag2-/-Il2rg-/- mice and found that committed pre-B cells could be converted into T cells and ILCs upon deletion of Ebf1.
Project description:Transcription factor Ebf1 is an important determinant of early B lymphopoiesis. To gain insight into differentiation stage-specific functions of Ebf1, we conditionally inactivated Ebf1. We found that Ebf1 is required for proliferation, survival and signaling of pro-B cells and peripheral B cell subsets. The proliferation defect of Ebf1-deficient pro-B cells, including the impaired expression of IL-7Ra and several cell cycle regulators, is overcome by transformation with v-Abl. The survival defect of transformed Ebf1fl/fl pro-B cells can be rescued by the forced expression of the Ebf1 targets c-Myb or Bcl-xL. In mature B cells, Ebf1 deficiency interferes with the BAFF-R and BCR-dependent Akt signaling pathways, as well as with germinal center formation and class switch recombination. Genome-wide analyses of Ebf1 binding and Ebf1-mediated gene expression in mature B cells and comparison with reported data sets in pro-B cells provide insight into the basis for lineage- and stage-specific functions of Ebf1. EBF1 binding in splenic B cells in mice
Project description:Transcription factor Ebf1 is an important determinant of early B lymphopoiesis. To gain insight into differentiation stage-specific functions of Ebf1, we conditionally inactivated Ebf1. We found that Ebf1 is required for proliferation, survival and signaling of pro-B cells and peripheral B cell subsets. The proliferation defect of Ebf1-deficient pro-B cells, including the impaired expression of IL-7Ra and several cell cycle regulators, is overcome by transformation with v-Abl. The survival defect of transformed Ebf1fl/fl pro-B cells can be rescued by the forced expression of the Ebf1 targets c-Myb or Bcl-xL. In mature B cells, Ebf1 deficiency interferes with the BAFF-R and BCR-dependent Akt signaling pathways, as well as with germinal center formation and class switch recombination. Genome-wide analyses of Ebf1 binding and Ebf1-mediated gene expression in mature B cells and comparison with reported data sets in pro-B cells provide insight into the basis for lineage- and stage-specific functions of Ebf1. Localistaion of histone modification (H3K4me2 and H3K4me3) in splenic B cells in mice by ChIP-seq
Project description:The transcription factor EBF1 is essential for lineage specification in early B cell development. Here we demonstrate by conditional mutagenesis that EBF1 was required for B cell commitment, pro-B cell development and subsequent transition to pre-B cells. Later in B cell development, EBF1 was essential for the generation and maintenance of distinct mature B cell types. Marginal zone and B-1 B cells were lost, whereas follicular and germinal center B cells were reduced in the absence of EBF1. Activation of the B cell receptor resulted in impaired intracellular signaling, proliferation and survival of EBF1-deficient follicular B cells. Immune responses were severely reduced upon Ebf1 inactivation, as germinal centers were formed but not maintained. ChIP- and RNA-sequencing of follicular B cells identified EBF1-activated genes that code for receptors, signal transducers and transcriptional regulators implicated in B cell signaling. Notably, ectopic expression of EBF1 efficiently induced the development of B-1 cells at the expense of conventional B cells. These gain- and loss-of-function analyses uncovered novel important functions of EBF1 in controlling B cell immunity. 29 samples (4 ChIP-seq,16 input, 9 RNA-seq), All ChIP-seq in 2 biological replicates, but EBF1 ChIP-seq Pro-B in 2 technical replicates; All RNA-seq in 2 biological replicates but RNA-seq mature B_FO B_Cd23-Cre Ebf1(fl/–). WT and experimental samples are provided.