Project description:The aim of experiment was to study on genome-wide level IRF4 target genes in chicken DT40 B cell line, by comparizon of gene expression profiles of IRF4-deficient DT40 cells with WT IRF4 DT40 cells .
Project description:Differentiation of B cells into antibody secreting cells (ASCs), plasmablasts and plasma cells, is regulated by a network of transcription factors. Within this network factors including PAX5 and BCL6 prevent ASC differentiation and maintain the B cell phenotype whereas BLIMP-1 and high IRF4 expression promote plasmacytic differentiation. BLIMP-1 is thought to induce immunoglobulin secretion. While IRF4 is needed for the survival of ASCs, its role in the regulation of antibody secretion has been controversial. BCL6-deficient DT40 B cell line has upregulated BLIMP-1 expression and secrete antibodies. In order to study the role of IRF4 in regulation of antibody secretion we have created a double knockout (DKO) DT40 B cell line deficient in both IRF4 and BCL6. This DKO cell line did not upregulate PRDM1 (the gene encoding for BLIMP-1) expression or secrete IgM. Even enforced BLIMP-1 expression in DKO cells or IRF4-deficient cells could not induce IgM secretion while it did in WT cells. However, enforced IRF4 expression in DKO cells induced strong IgM secretion. Our findings support a model where IRF4 expression in addition to BLIMP-1 expression is required to induce antibody secretion.
Project description:- transcription factor interferon regulatory factor 4 (IRF4) = crucial transcription factor for different immune cells, incl pro-inflammatory Th17 and anti-inflammatory Treg cells
- IRF4 is essential for the cell differentiation and fate determination
- however molecular mechanisms of IRF4-mediated gene expression in fully differentiated Th17/Treg cells are still not fully understood
- integration of data derived from affinity-purification and full mass spectrometry-based proteome analysis with chromatin immune precipitation sequencing (ChIP-Seq)
- characterization of proteins generally involved in the T cell development as well as subtype-specific differentiation and identification of novel, yet uncharted IRF4 interactors
Project description:The activation induced cytosine deaminase (AID) mediates diversification of B cell immunoglobulin genes by the three distinct yet related processes of somatic hypermutation (SHM), class switch recombination (CSR), and gene conversion (GCV). SHM occurs in germinal center B cells, and the transcription factor Bcl6 is a key regulator of the germinal center B cell gene expression program, including expression of AID. To test the hypothesis that Bcl6 function is important for the process of SHM, we compared WT chicken DT40 B cells, which constitutively perform SHM/GCV, to their Bcl6-deficient counterparts. We found that Bcl6-deficient DT40 cells were unable to perform SHM and GCV despite enforced high level expression of AID and substantial levels of AID in the nucleus of the cells. To gain mechanistic insight into the GCV/SHM dependency on Bcl6, transcriptional features of a highly expressed SHM target gene were analyzed in Bcl6-sufficient and -deficient DT40 cells. No defect was observed in the accumulation of single stranded DNA in the target gene as a result of Bcl6 deficiency. In contrast, association of Spt5, an RNA polymerase II (Pol II) and AID binding factor, was strongly reduced at the target gene body relative to the transcription start site in Bcl6-deficient cells as compared to WT cells. However, partial reconstitution of Bcl6 function substantially reconstituted Spt5 association with the target gene body but did not restore detectable SHM. Our observations suggest that in the absence of Bcl6, Spt5 fails to associate efficiently with Pol II at SHM targets, perhaps precluding robust AID action on the SHM target DNA. Our data also suggest, however, that Spt5 binding is not sufficient for SHM of a target gene even in DT40 cells with strong expression of AID. Sequencing of the IgL V region and mutationally active GFP transgene in WT, Bcl6-/- Pax5R, and Bcl6-/- Pax5R Bcl6R chicken DT40 cells for evidence of AID dependent mutations. ChIP-seq proiles of RNA Pol II, Spt5, and pSer5 Pol II at a GFP transgene in WT, Bcl6-/- Pax5R, and Bcl6-/- Pax5R Bcl6R chicken DT40 cells.