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Identification of genes controlled by Ikaros and IL-7 in the mouse Ikaros-deficient pre-B cell line BH1

ABSTRACT: The aim of the experiment was to compare to single and combined effect of Ikaros activation and IL-7 withdrawal in the Ikaros-null pre-B cell line BH1

ORGANISM(S): Mus musculus

PROVIDER: GSE51350 | GEO | 2013/11/30

SECONDARY ACCESSION(S): PRJNA222807

REPOSITORIES: GEO

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Identification of genes controlled by Ikaros and IL-7 in the mouse Ikaros-deficient pre-B cell line BH1

Project description:The aim of the experiment was to compare to single and combined effect of Ikaros activation and IL-7 withdrawal in the Ikaros-null pre-B cell line BH1 The mouse BH1 pre-B cell line was transduced with retroviruses encoding Bcl2 and an Ikaros-estrogen receptor fusion protein. Doubly transduced cells were purified by FACS (BH1-Bcl2-Ik1ER). The transcriptome of these cells was analyzed at time 0, and after 6h and 24h of induction of Ikaros activity with 4-hydroxytamoxifen (4OHT) and/or withdrawal of IL-7. Samples from 2 independent experiments were analyzed (exp1 and exp2).

2013-11-30 | E-GEOD-51350 | biostudies-arrayexpress

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Ikaros-induced gene expression chages upon Ikaros re-expression in the ILC87 Ikaros-deficient tumor cell line

Project description:We used microarrays to analyze gene expression changes in the Ikaros null ILC87 T cell tumor line after re-expression of Ikaros. ILC87 cells were transduced to stably express the 4-hydroxytamoxifen-inducible (4OHT) Ikaros1-ER fusion protein. ILC87-Ik1-ER cells were treated with mock (EtOH) or 4OHT for 1 day and the global gene expression changes were assessed by microarray analysis.

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Gene expression in mouse pre-B cells transduced with Ikaros.

Project description:Ikaros family DNA binding proteins are critical regulators of B cell development. To identify Ikaros-regulated genes in pre-B cells we performed gene expression studies at enhanced temporal resolution. We used retroviral gene transfer to express Ikaros proteins in the pre-B cell line B3. Total RNA from 2 biological replicates of B3 cells transduced with wild type Ikaros (HA-Ikaros-IRES-GFP) and DNA binding-deficient Ikaros mutant 159A (HA-159A Ikaros-IRES-GFP) was isolated 48h after infection. To increase the temporal resolution of Ikaros-regulated gene expression we transduced B3 cells with inducible Ikaros (HA-Ikaros-ERt2-IRES-GFP) and isolated total RNA from 3 biological replicates after 2 h and 6 h of exposure to 4-hydroxytamoxifen. Vector transduced B3 cells (ERt2-IRES-GFP) treated with 4-hydroxytamoxifen were used as controls.

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Project description:Ikaros DNA binding proteins are important regulators of haematopoiesis and genetic deletion of Ikaros results in severe developmental disturbances, including delayed thymocyte differentiation and an early and complete block in B cell development. Although Ikaros ChIP-seq data are available for mouse thymocytes and human haematopoietic progenitors, it has not been achieved in B cell progenitors. The goal of this study was to identify Ikaros binding sites in pre-B cells to define Ikaros target genes which could explain the essential role of Ikaros proteins in B cell differentiation. We carried out chromatin immunoprecipitation and high throughput sequencing (ChIP-seq) with antibodies to the C-terminus of endogenous Ikaros in B3 cells and with anti-haemagglutinin (HA) in B3 cells transduced with epitope-tagged HA-Ikaros.

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Project description:We found that CD4+ T cells require Ikaros to promote IL-17 production when cultured in Th17 conditions. To understand why Ikaros null T cells do not produce IL-17, we analyzed the transcriptome of WT vs. Ikaros null naive CD4+ T cells both resting (day 0) or activated with anti-CD3 plus anti-CD28 antibodies, IL-6, TGFb1 and neutralizing anti-IFNg and anti-IL-4 antibodies (Th17 condition) for 1 or 2 days. Samples from 3 independent experiments were analyzed.

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