Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Gene expression analysis after reconstitution of Ebf1-deficient pre-pro-B cells with Ebf1

ABSTRACT: 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.

ORGANISM(S): Mus musculus

SUBMITTER: Elizabeth Mandel

PROVIDER: E-GEOD-21455 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Early B cell factor 1 regulates B cell gene networks by activation, repression, and transcription- independent poising of chromatin.

Treiber Thomas T Mandel Elizabeth M EM Pott Sebastian S Györy Ildiko I Firner Sonja S Liu Edison T ET Grosschedl Rudolf R

Immunity 20100506 5

The transcription factor early B cell factor-1 (Ebf1) is a key determinant of B lineage specification and differentiation. To gain insight into the molecular basis of Ebf1 function in early-stage B cells, we combined a genome-wide ChIP sequencing analysis with gain- and loss-of-function transcriptome analyses. Among 565 genes that are occupied and transcriptionally regulated by Ebf1, we identified large sets involved in (pre)-B cell receptor and Akt signaling, cell adhesion, and migration. Inter ...[more]

PMID: 20451411

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Project description:Ebf1 is a transcription factor with documented, and dose dependent, functions in both normal and malignant B-lymphocyte development. In order to understand more about the role of Ebf1 in malignant transformation, we have investigated the impact of reduced functional Ebf1 dose on early B-cell progenitors. Gene expression analysis in loss and gain of function analysis suggested that Ebf1 was involved in the regulation of genes of importance for DNA repair as well as cell survival. Investigation of the level of DNA damage in steady state as well as after induction of DNA damage by UV light supported that pro-B cells lacking one functional allele of Ebf1 display a reduced ability to repair DNA damage. This was correlated to a reduction in expression of Rad51 and combined analysis of published 4C and chromatin Immuno precipitation data suggested that this gene is a direct target for Ebf1. Even though the lack of one allele of Ebf1 did not result in any dramatic increase of tumor formation, we noted a dramatic increase in the formation of pro-B cell leukemia in mice carrying a combined heterozygote mutation in the Ebf1 and Pax5 genes. Even though the tumors were phenotypically similar and stable, we noted a large degree of molecular heterogeneity well in line with a mechanism involving impaired DNA repair. Our data support the idea that Ebf1 controls homologous DNA repair in a dose dependent manner and that this may explain the frequent involvement of Ebf1 in human leukemia 230238: Briefly, 230238 preB-cell line was infected with pMIG-Ebf1-engrailed or pMIG-control (empty vector). Cells were GFP sorted and 500.000 cells were resuspended in RLT for subsequent RNA-extraction. Analysis were perfomred in triplicate. FL Ebf1-KO: E14.5 fetal liver (FL) Ebf1-/- were isolated and expanded expanded in vitro on OP9 stroma cells on conditions permissive for B-cell development. Expanded cells were infected with pMIG-Ebf1-ER and sorted on GFP. Cells were reseeded on OP9 stroma cells and tamoxifen was added to a final concentration of 0.1 uM. 500.000 cells were harvested and RNA extracted 0, 6, 12 and 24h. Gene expression levels were compared to 0h and analysis were performed in duplicates. Tumor LNs: Enlarged lymphnodes from approximately 30 weeks old C57BL/6 Ebf1+/-Pax5+/- were dissected, homogenized and live frozen. After thawing, 1 million lymphnodes cells were live sorted and resuspended in RLT prior to RNA extraction. 8 samples were analyzed in duplicates. 1 million proB cells from wt C57BL/6 were sorted and used as controls.

Project description:In order to investigate how transcription factor dose impacts B-lymphocyte development, we generated mice carrying transheterozygous mutations in the Pax5 and Ebf1 genes. While combined reduction of Pax5 and Ebf1 dose had minimal impact on the development of the earliest CD19+ progenitors, these cells displayed an increased T-cell potential in vivo and in vitro. Alteration in lineage fate depended on a Notch1 mediated conversion process while no signs of de-differentiation could be detected. The differences in functional response to Notch signaling in Wt and Pax5+/-Ebf1+/- pro-B cells was reflected in the transcriptional response because even though cells of both genotypes responded by the generation of intracellular Notch1 and activation of a set of target genes, only the Pax5+/-Ebf1+/- pro-B cells down-regulated genes central for the preservation of stable B-cell identity. This report stresses the importance of transcription factor dose in lymphocyte development and suggests that Pax5 and Ebf1 collaborate to modulate the transcriptional response to Notch signaling after the generation of activated intracellular Notch1. This provides an insight to how transcription factors like Ebf1 and Pax5 preserve cellular identity during differentiation. EBF-1 ChIP-seq: Cultivated CD43+IgM- cells (ProB) cells from Wt, EBF-1 +/-, PAX-5 +/- and EBF-1 +/- PAX-5 +/- (TH) were assessed for EBF-1 binding by ChIP-seq. Replicate Ebf1 ChIP-seq runs on each genotype (Wt, TH, Ebf1+/- and Pax5+/-) and corresponding inputs were pooled into one dataset and analyzed as one combined sample per genotype. RNA-seq no treatment: Briefly, ProB-cells from C57BL/6J Ebf1+/-Pax5+/- (n=4), WT (n=4), Ebf1+/- (n=2) and Pax5+/- (n=2) were sorted and RNA extracted with Qiagen RNeasy Micro Kit. RNA was sent to UCLA Microarray Core for library preparation and were subsequently for 50 cycles of HiSeq 2000 SBS sequencing generating 20-30 million reads/sample. Data analysis was performed with Arraystar® (DNASTAR)). RNA-seq 1 day on OP9DL1 and OP9: In short, in vitro expanded ProB-cells from C57BL/6J Ebf1+/-Pax5+/- (n=4) and WT (n=4) were exposed either on OP9 or OP9-DL1 stromal cells for 24 hours and RNA extracted with Qiagen RNeasy Micro Kit. Due to low reads, two Wt and Ebf1+/-Pax5+/- were sequenced twice. Libraries were constructed using Nugen's Ovation Ultralow Library systems and were subsequently for 50 cycles of NextSeq500 sequencing generating 20-30 million reads/sample. Data analysis was performed with Arraystar® (DNASTAR)).

Dataset Information

Gene expression analysis after reconstitution of Ebf1-deficient pre-pro-B cells with Ebf1

Publications

Early B cell factor 1 regulates B cell gene networks by activation, repression, and transcription- independent poising of chromatin.

Similar Datasets

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