Studies on the function and regulation of BCL7A and BCL6 in germinal center B cells

Chromosome translocations are the genetic hallmark of lymphoid malignancies including B-cell non-Hodgkin's lymphoma (NHL) and have been shown to contribute to malignant transformation by deregulating the expression of target oncogenes. The human BCL7A gene was originally cloned from a Burkitt lymphoma cell carrying a chromosomal translocation that juxtaposed the immunoglobulin locus to the BCL7A coding region presumably leading to its deregulated expression. Furthermore, the BCL7A 5' regulatory region is mutated in 48% (15/31) of diffuse large B cell lymphoma cases, in 15% (3/19) of Burkitt lymphoma cases and in 25% (4/16) of follicular lymphoma cases by a lymphoma-associated mechanism named aberrant somatic hypermutation. In addition, BCL7A is expressed specifically in germinal center (GC) B cells, the largest structure from which B-cell NHL are derived. These observations suggest that BCL7A play a role in GC reaction and/or B-cell lymphomagenesis. Nevertheless, the function of BCL7A is unknown.; In order to study the normal function of BCL7A, we generated BCL7A deficient mice. Bcl7a-/- mice were born at the expected mendelian frequency and developed normally. In these mice, B and T cell populations in bone marrow, spleen, thymus and peritoneal cells are normal as shown by FACS analysis. Germinal center formation is comparable in wild type, Bcl7a+/- and Bcl7a-/- mice as shown by the analysis of aggregates of B cells expressing GC-specific markers (BCL6) in mice immunized with sheep red blood cells. Basal as well as antigen-specific Ig levels (after immunization with T cell-dependent antigen NP-KLH) were normal. The ability of B cells to undergo affinity maturation was also normal in Bcl7a-/- mice as evaluated by ELISA. In conclusion, Bcl7a-/- mice display normal lymphoid organ development and function including normal germinal center formation and T-cell dependent immune responses. Thus, the function of BCL7A in GC is either more subtle than detectable by standard assays or can be substituted by an alternative pathway in Bcl7a-/- mice.; Unlike BLC7A, much more is known about the function of BCL6 in GC biology. The proto-oncogene BCL6 was initially cloned from chromosome breakpoints observed in a subset of NHL. BCL6 is a transcription repressor specifically expressed in the GC within the B cell lineage. The role of BCL6 in GC has been recognized, as mice lacking BCL6 cannot form germinal centers and failed to mount T-cell dependent immune responses. The down-regulation of BCL6 is thought to be necessary for B cells to exit from GC and to differentiate into plasma and memory cells. It has been shown that CD40 signaling down-regulates BCL6 at the transcriptional level. However, the intermediate players in this signaling pathway are unknown.; Here, we demonstrate that CD40-induced BCL6 down-regulation is dependent on NF-kappaB activation. We identified IRF4, a member of the interferon-regulatory factor family of transcription factors, as a mediator of NF-kappaB activity on BCL6 expression. IRF4 was up-regulated by NF-kappaB complexes and then directly repressed BCL6 expression. The binding of NF-kappaB subunits to the IRF4 promoter and of IRF4 to the BCL6 5' regulatory region was demonstrated by chromatin immunoprecipitation assay. Using RNA interference to knockdown p50 and p65 abolished or severely reduced CD40-induced IRF4 up-regulation. The suppression of IRF4 expression by RNAi led to the derepression of BCL6. In addition, this CD40-induced and IRF4-mediated BCL6 down-regulation was blocked in lymphoma cell lines with chromosome translocations in the BCL6 5' regulatory region. These results revealed a signaling pathway important for the regulation of BCL6 in the GC function and in the B cell lymphomagenesis.