RNA-expression profiling of mouse B cells coexpressing LMP1 with EBNA3A, Rel or Ebf1 in vitro
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE136072 | GEO | 2020/06/02
REPOSITORIES: GEO
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Project description:RNA-expression profiling of mouse B cells coexpressing LMP1 with EBNA3A, Rel or Ebf1 in vitro
| PRJNA561166 | ENA
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Project description:Early B cell factor 1 (EBF1) is one of the key transcription factors required for orchestrating B-cell lineage development. Although studies have shown that Ebf1 haploinsufficiency is involved in the development of leukemia, no study has been conducted that characterizes the global effect of Ebf1 heterozygosity on the proteome of pro-B lymphocytes. Here, we employ both DIA (Data Independent Acquisition) and shotgun DDA (Data Dependent Acquisition) workflows for profiling proteins that are differently expressed between Ebf1+/+ and Ebf1+/- cells. Both DDA and DIA were able to reveal the downregulation of the EBF1 transcription factor in Ebf1+/- pro-B lymphocytes. Further examination of differentially expressed proteins by DIA revealed that, similar to EBF1, the expression of other B-cell lineage regulators, such as TCF3 and Pax5, is also down-regulated in Ebf1 heterozygous cells. Functional DIA analysis of differentially expressed proteins showed that EBF1 heterozygosity resulted in the deregulation of at least 8 transcription factors involved in lymphopoiesis, and to the deregulation of key proteins playing crucial roles in survival, development and differentiation of pro-B lymphocytes.
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Project description:Foxo1 and Ebf1 deficiency leads to a similar disruption of normal B-cell development at the level of the common lymphoid progenitor (CLP). Both mouse strains display the existance of LY6D+ CLPs but a marked/complete lack of proB cells. To investigate similarities of the developmental defects observed we generated gene expression profiles from both genotypes (with corresponding WT controls). This illustrated a shared gene expression signature in Ebf1/Foxo1 deficient CLPs. Gene expression profiling was done using highly purified (FACS sorted) progenitor cells. Cells were purified from bone marrow of wild-type, Foxo1 deficient and Ebf1 deficient mice. Ebf1 deficient bone marrow was aquired by transplantation of Ebf1 deficient progenitors into irradiated hosts. Populations analysed were CLP LY6D-, CLP LY6D+ and proB cells.
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Project description:To study the function of EBF1-H240 residue, a gain-of function microarray analysis was performed by transducing Empty, Ebf1-wt and Ebf1-H240A expressing vectors into Ebf1-/- progenitor cells.
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Project description:Ebf1 is a key determinant of B-lymphocyte specification. In order to identify unknown transcriptional targets, endogenous Ebf1 was isolated by chromatin-IP from primary pro-B cells and the copurified DNA was hybridized to promoter tiling arrays. Ebf1 was ChIPed using a polyclonal antibody directed against an N-terminal peptide from primary pro-B cells grown in the presence of Il7. The resulting DNA was analyzed by tiling array hybridization.
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Project description:The Epstein Barr virus (EBV) encoded latent membrane protein-1 (LMP1) is a functional homologue of the tumor necrosis factor receptor family and contributes substantially to the oncogenic potential of EBV through activation of Nuclear Factor-kappaB (NF-kappaB). MicroRNAs (miRNAs) are a class of small RNA molecules that are involved in the regulation of cellular processes such as growth, development, and apoptosis, and have recently been linked to cancer phenotypes. Through miRNA microarray analysis, we demonstrate that LMP1 dysregulates the expression of several cellular miRNAs, including the most highly regulated of these, miR-146a. Quantitative RT-PCR analysis confirmed induced expression of miR-146a by LMP1. Analysis of miR-146a expression in EBV latency type III and type I cell lines revealed substantial expression of miR-146a in type III (which express LMP1) but not in type I cell lines. Reporter studies demonstrated that LMP1 induces miR-146a predominantly through two NF-kappaB binding sites in the miR-146a promoter and identified a role for an OCT-1 site in conferring basal and induced expression. Array analysis of cellular mRNAs expressed in Akata cells transduced with an miR-146a expressing retrovirus identified genes that are directly or indirectly regulated by miR-146a, including a group of interferon responsive genes that are inhibited by miR-146a. Since miR-146a is known to be induced by agents that activate the interferon response pathway (including LMP1), these results suggest that miR-146a functions in a negative feedback loop to modulate the intensity and/or duration of the interferon response. Keywords: microRNA expression modified by EBV encoded oncogene, LMP1 EBV negative Mutu E1dn Cl. 3 cells were transduced with either a control retrovirus (pEhyg) or an LMP1 containing retrovirus (pEhyg-FLAG-LMP1). Cells were selected with hygromycin for approximately 2 weeks afterwhich 5 pairs of RNA preps were carried out on 5 successive days. Arrays GSM255656 and GSM255659 are dye swaps in which LMP1 samples were labeled with Cy3 and control samples were labeled with Cy5.
2010-05-26 | E-GEOD-10105 | biostudies-arrayexpress
Project description:Gene expression profile of splenic B cells (CD19+) from transgenic mice expressing the Epstein-Barr virus (EBV) latent membrane proteins (LMP) 1 and/or LMP2A. Freshly harvested primary B cells were profiled. B lymphocytes from transgenic LMP1, LMP2A, LMP1/2A mice and negative littermates were profiled from 6 month old adult mice; lymphoma cells were passaged in SCID mice and profiled for three LMP1 positive lymphomas and one negative lymphoma. 12 total samples. 4 transgenic B lymphocyte samples pooled from multiple biological replicates were hybridized to duplicate microarrays: LMP1 (pooled from 2 replicates), LMP2A (pooled from 3 replicates); LMP1/2A (pooled from 5 replicates), negative littermates (pooled from 4 replicates). 3 biological replicates of LMP1 lymphomas expressing high, medium and low levels of LMP1 and; 1 negative lymphoma was hybridized to 1 microarray chip. The reference sample consisted of 4 biological replicates of splenic B cells (CD19+) pooled from 4-7 month old non-transgenic Balb/c mice. The same reference was used for all hybridizations.
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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.
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