PU.1 and Spi-B oppose transformation of pre-B cells through activation of key genes involved in B cell receptor signaling
Ontology highlight
ABSTRACT: Deletion of genes encoding the E26 transformation-specific (ETS) transcription factors, PU.1 and Spi-B, in B cells (CD19-CreDPB mice) leads to acute lymphoblastic leukemia at 100% incidence and with a median survival of 21 weeks. To identify pathways of leukemic transformation, we compared gene expression in leukemia cells from CD19-CreDPB mice (CD19-CreDPB B220- B-ALL) with B cells from Spi-B knockout (Control DB) or B cells from CD19-CreDPB mice (CD19-CreDPB B220+ B cell)
Project description:Spi-B and PU.1 are highly related members of the E26-transformation-specific (ETS) family of transcription factors that have similar, but not identical, functions in B cell development. PU.1 and Spi-B are both expressed at high levels in lymphoma cell lines. We hypothesized that Spi-B and PU.1 occupy similar sites in the genome. To determine binding sites of Spi-B and PU.1, WEHI-279 mouse lymphoma cells were infected with retroviral vectors encoding 3XFLAG-tagged PU.1 or Spi-B. Anti-FLAG chromatin immunoprecipitation followed by next generation sequencing (ChIP-seq) was performed. Both transcription factors occupied approximately 2000 sites in the genome, and approximately half of these sites were bound by both factors while the other sites were unique to each factor.
Project description:Acute leukemia are characterized by deregulation of transcriptional networks that control the lineage specificity of gene expression. The aberrant overexpression of the Spi-1/PU.1 transcription factor leads to erythroleukemia. To determine how Spi-1 mechanistically influences the transcriptional program, we combined a ChIP-seq analysis with transcriptional profiling in cells from an erythroleukemic mouse model. We show that Spi-1 displays a selective DNA-binding that does not often cause transcriptional modulation. We report that Spi-1 controls transcriptional activation and repression through distinct Spi-1 recruitment to chromatin. We revealed several parameters impacting on Spi-1-mediated transcriptional activation. Gene activation is facilitated by Spi-1 occupancy close to transcriptional starting site of genes devoid of CGIs. Moreover, in those regions Spi-1 acts by binding to multiple motifs tightly clustered and with similar orientation. Finally, in contrast to the myeloid and lymphoid B cells in which Spi-1 exerts a physiological activity, in the erythroleukemic cells, lineage-specific cooperating factors do not play a prevalent role in Spi-1-mediated transcriptional activation. Thus, our work describes a new mechanism of gene activation through clustered site occupancy of Spi-1 particularly relevant in regard to the strong expression of Spi-1 in the erythroleukemic cells. Chromatin immunoprecipitations of Spi-1, H3K36me3, RNApolII,mouse IgG followed by sequencing were performed on spleen-derived erythroleukemic cells of spi-1 transgenic mice. In case of Spi-1, reads obtained from the two different mice represent biological replicates and were merged for bioinformatic analysis. Input DNA from each ChIP experiments have been pooled and sequenced as one control.
Project description:Acute leukemia are characterized by deregulation of transcriptional networks that control the lineage specificity of gene expression. The aberrant overexpression of the Spi-1/PU.1 transcription factor leads to erythroleukemia. To determine how Spi-1 mechanistically influences the transcriptional program, we combined a ChIP-seq analysis with transcriptional profiling in cells from an erythroleukemic mouse model. We show that Spi-1 displays a selective DNA-binding that does not often cause transcriptional modulation. We report that Spi-1 controls transcriptional activation and repression through distinct Spi-1 recruitment to chromatin. We revealed several parameters impacting on Spi-1-mediated transcriptional activation. Gene activation is facilitated by Spi-1 occupancy close to transcriptional starting site of genes devoid of CGIs. Moreover, in those regions Spi-1 acts by binding to multiple motifs tightly clustered and with similar orientation. Finally, in contrast to the myeloid and lymphoid B cells in which Spi-1 exerts a physiological activity, in the erythroleukemic cells, lineage-specific cooperating factors do not play a prevalent role in Spi-1-mediated transcriptional activation. Thus, our work describes a new mechanism of gene activation through clustered site occupancy of Spi-1 particularly relevant in regard to the strong expression of Spi-1 in the erythroleukemic cells.
Project description:Inflammatory (B220+ CD19+ CD44+ CD11c+ Tbet-AmCyanhi), GC (B220+ IgDlo CD95+ GL-7+), and naive follicular B cells (B220+ CD19+ IgDhi CD23+) were sorted from male Tbet-AmCyan reporter mice at day 12 post infection with LCMV-Armstrong. RNA-seq was employed to compare the transcriptomes of these three B cell populations.
Project description:We studied the role of the stable Igh mRNA in Igh chain check point. Here we generated RNA-seq data from sorted ProB (CD19+B220+c-Kit+CD25-) and PreB (CD19+B220+c-Kit-CD25+) populations from Bone marrow of mice.
Project description:We identified that IL-33 is expressed in pro-B (B220+ IgM- CD19+ CD43+) and large pre-B cells (B220+ IgM- CD19+ CD43- FSC-Ahi SSC-Ahi) in mice. We assessed for IL-33-dependent gene expression differences in these B cell stages by performing bulk RNA-sequencing of FACS-purified pro-B and large pre-B cells from WT and IL-33-deficient 8 week old adult, naive female mice.
Project description:To generate and compile data from ChIP-Seq libraries. Looking at genome-wide binding of transcription factors HilD, HilC, RtsA, RtsB, SprB, and InvF under SPI-1 inducing growth conditions in duplicate.
Project description:We determined the genome-wide binding of the ETS transcription factor Spi-C with target genes using chromatin immunoprecipitation and next generation sequencing (ChIP-seq)
Project description:To determine the functions of Spi-C in innate immune responses, we investigated the overall gene expression patterns in M-CSF-BMDMΦs prepared from Spicflox/flox and Lyz2-cre; Spicflox/flox mice. M-CSF-BMDMΦs were stimulated with or without LPS following heme treatment and used for RNA-seq analysis.