Project description:After 5 days in culture with 2ng/ml of IL7, Pax5-/- transduced pro-B cells with PAX5A or PAX5B cDNA were sorted according to their EGFP positivity and total RNA was extracted.
Project description:VH-DJH recombination of the immunoglobulin heavy-chain (Igh) locus is temporally and spatially controlled during early B-cell development, and yet no regulatory elements other than the VH gene promoters have been identified throughout the entire 2.5-Mb VH gene cluster. Here we discovered novel regulatory sequences that are interspersed in the distal VH gene region. These conserved repeat elements were characterized by the presence of Pax5-dependent active chromatin, the binding of Pax5, E2A, CTCF and Rad21 as well as by Pax5-dependent antisense transcription in pro-B cells. The Pax5-activated intergenic repeat (PAIR) elements were no longer bound by Pax5 in pre-B and B cells consistent with the loss of antisense transcription, whereas E2A and CTCF interacted with PAIR elements throughout early B-cell development. The pro-B-cell-specific and Pax5-dependent activity of the PAIR elements suggests that they are involved in the regulation of distal VH-DJH recombination at the Igh locus. Analysis of chromatin and TF binding in rag2-/- and wt pro-B, DP T and Mature B cells. Chip-Seq of CTCF and Rad21. The provided data is in mm8 coordinates.
Project description:VH-DJH recombination of the immunoglobulin heavy-chain (Igh) locus is temporally and spatially controlled during early B-cell development, and yet no regulatory elements other than the VH gene promoters have been identified throughout the entire 2.5-Mb VH gene cluster. Here we discovered novel regulatory sequences that are interspersed in the distal VH gene region. These conserved repeat elements were characterized by the presence of Pax5-dependent active chromatin, the binding of Pax5, E2A, CTCF and Rad21 as well as by Pax5-dependent antisense transcription in pro-B cells. The Pax5-activated intergenic repeat (PAIR) elements were no longer bound by Pax5 in pre-B and B cells consistent with the loss of antisense transcription, whereas E2A and CTCF interacted with PAIR elements throughout early B-cell development. The pro-B-cell-specific and Pax5-dependent activity of the PAIR elements suggests that they are involved in the regulation of distal VH-DJH recombination at the Igh locus. Analysis of chromatin and TF binding in rag2-/- and pax5-/- rag2-/- pro-B cells. Chip-Chip with one experiment for each antibody, 12 samples.
Project description:Comparison of the RNA-expression pattern of Pax5-deficient progenitor-cell tumors with wild-type pro-B-cells Keywords: Genetic modification
Project description:The transcription factor Pax5 is essential for B cell commitment in the mouse, where it represses lineage-inappropriate gene expression, while simultaneously activating the B cell gene expression program. We have performed a global gene expression screen of wild type and Pax5-deficient pro-B cells in an attempt to identify the crucial Pax5 targets in early B-lymphopoiesis. We have also included Rag1-/- and wild type (+/+) proB cells starved of the cytokine IL-7 for 4 hours as controls. Rag1-/- proB cells are incapable of further differentiation due to an absence of immunoglobulin recombination and IL-7 is the major cytokine regulating proB cell growth. Keywords: comparison of genetically modified cell-lines
Project description:VH-DJH recombination of the immunoglobulin heavy-chain (Igh) locus is temporally and spatially controlled during early B-cell development, and yet no regulatory elements other than the VH gene promoters have been identified throughout the entire 2.5-Mb VH gene cluster. Here we discovered novel regulatory sequences that are interspersed in the distal VH gene region. These conserved repeat elements were characterized by the presence of Pax5-dependent active chromatin, the binding of Pax5, E2A, CTCF and Rad21 as well as by Pax5-dependent antisense transcription in pro-B cells. The Pax5-activated intergenic repeat (PAIR) elements were no longer bound by Pax5 in pre-B and B cells consistent with the loss of antisense transcription, whereas E2A and CTCF interacted with PAIR elements throughout early B-cell development. The pro-B-cell-specific and Pax5-dependent activity of the PAIR elements suggests that they are involved in the regulation of distal VH-DJH recombination at the Igh locus.
Project description:VH-DJH recombination of the immunoglobulin heavy-chain (Igh) locus is temporally and spatially controlled during early B-cell development, and yet no regulatory elements other than the VH gene promoters have been identified throughout the entire 2.5-Mb VH gene cluster. Here we discovered novel regulatory sequences that are interspersed in the distal VH gene region. These conserved repeat elements were characterized by the presence of Pax5-dependent active chromatin, the binding of Pax5, E2A, CTCF and Rad21 as well as by Pax5-dependent antisense transcription in pro-B cells. The Pax5-activated intergenic repeat (PAIR) elements were no longer bound by Pax5 in pre-B and B cells consistent with the loss of antisense transcription, whereas E2A and CTCF interacted with PAIR elements throughout early B-cell development. The pro-B-cell-specific and Pax5-dependent activity of the PAIR elements suggests that they are involved in the regulation of distal VH-DJH recombination at the Igh locus.
Project description:Gene expression profiling of pro-B cells in the bone marrow of WT and PAX5<Y351*/Y351*> mice and also of CD19+B220- cells found only in the BM of PAX5<Y351*/Y351*> mice. We used a modified single-cell protocol (4 wells of 50 cells each per sample) and then combined all data from the same sample for the analysis.