Gene regulation by Early B-cell Factor (EBF)

Sammanfattning: In this study we aimed at defining genes that are controlled by Early B-cell Factor (EBF), that could explain the severe block in B-cell development observed in EBF-/- mice. The fact that EBF appears to be important for the transcriptional control of genes encoding proteins in the pre-B and B-cell receptor complex implied to us that EBF could be a common regulator of other components in the pre-B and B-cell receptor. In addition to the immunoglobulin (Ig) heavy chain, the surrogate light chains and the Ig associated alfa protein the pre-B cell receptor also consists of the second Ig associated beta protein as well as B-cell specific kinase (Blk). The hypothesis that EBF is involved in regulating other components in the B-cell receptor was further strengthened by information from the EBF-/- mouse indicating that its pro-B cells completely lacked B29 transcripts. This was an intriguing finding, since B29 is expressed in normal pro-B cells as well as in pro-B cells from E2A-/- mice displaying a similar B-cell differentiation block as the EBF-/- mouse. We examined the promoter DNA sequence of murine B29 (Ig-beta) and Blk and found potential EBF DNA binding sites. In electrophoretic mobility shift assay (EMSA) EBF interacted with three independent sites in the B29 promoter and with a single site in the Blk promoter. EBF could also activate both the B29 and the Blk promoters in transfected epitheloid HeLa cells, indicating that EBF functionally interacted with these promoters. The importance of the EBF binding sites for the function of the Blk and B29 promoters appeared to be stage dependent, since mutation of the EBF sites had an impact on promoter function in pre-B cell lines, but not in a B-cell line. However, B29 and Blk as well as EBF are expressed in mature B-cells and B29 even in plasma cells. Thus, there seems to be a shift in transcription factor usage, from EBF to other factors, at the transition from pre-B to B-cells. We also show data suggesting that EBF may also have additional roles as a negative regulator of the Ig gene expression in late B cell development. This may signify that transcription factors considered to be of importance mainly in the early differentiation stages can have important, unrevealed roles during late development.