Bach2 regulates AID-mediated immunoglobulin gene conversion and somatic hypermutation in DT40 B cells.

The transcription factor Bach2 is required for germinal center formation, somatic hypermutation (SHM), and class-switch recombination (CSR) of immunoglobulins. SHM and CSR are initiated by activation-induced cytidine deaminase (AID) which has potential to induce human B cell lymphoma. To understand the role of Bach2 in AID-mediated immunoglobulin gene diversification processes, we established a BACH2-deficient DT40 B cell line. We show that in addition to allowing SHM, Bach2 drives immunoglobulin gene conversion (GCV), another AID-dependent antibody gene diversification process. We demonstrate that Bach2 promotes GCV by increasing the expression of AID. Importantly, we found that the regulation of AID is independent of Blimp-1 and that BACH2-deficient cells have altered expression of several genes regulating AID expression, stability and function. Furthermore, re-expression of BACH2 or AID in Bach2KO cells restored the SHM and GCV defects. These results demonstrate that Bach2 has a previously unappreciated role in the production of high-affinity antibodies.

Alternate pathways for Bcl6-mediated regulation of B cell to plasma cell differentiation.

The transcription factor Bcl6 regulates germinal center formation and differentiation of B cells into high-affinity antibody-producing plasma cells. The direct double-negative regulatory circuit between Bcl6 and Blimp-1 is well established. We now reveal alternative mechanisms for Bcl6-mediated regulation of B-cell differentiation to plasma cells and show with DT40 cells that Bcl6 directly promotes the expression of Bach2, a known suppressor of Blimp-1. Moreover, Bcl6 suppresses Blimp-1 expression through direct binding to the IRF4 gene, as well as by promoting the expression of MITF, a known suppressor of IRF4. We also provide evidence that Bcl6 is needed for the expression of AID and UNG, the indispensable proteins for somatic hypermutation and class-switch recombination, and UNG appears to be a direct Bcl6 target. Our findings reveal a complex regulatory network in which Bcl6 acts as a key element dictating the transition of DT40 B cells to plasma cells.

Concerted action of Helios and Ikaros controls the expression of the inositol 5-phosphatase SHIP.

Ikaros family transcription factors have a key role in lymphoid development, and their aberrant function contributes to a multitude of lymphoid malignancies. Ikaros and Helios bind to similar DNA sequences, and Helios associates with Ikaros-containing chromatin remodeling complexes. Previously, we have shown that loss of Ikaros leads to diminished BCR-signaling strength. In this study, we describe a Helios-deficient chicken DT40 B-cell line with a BCR signaling phenotype that is the opposite to that of Ikaros-deficient cells. In contrast to Ikaros-deficient cells, Helios(-/-) B cells exhibit increased calcium release to the cytoplasm after BCR crosslinking, but diminished BCR-induced phosphorylation of signaling molecules. The inositol 5-phosphatase SHIP, an important regulator in several signaling pathways, is differentially expressed in Ikaros- and Helios-deficient cells. In the absence of Ikaros, SHIP is upregulated, whereas Helios deficiency leads to the downregulation of SHIP expression. We also show with ChIP that Ikaros binds to the promoter of the INPP5D gene-encoding SHIP. Considering the critical role of SHIP in the BCR signaling pathway, our findings provide insight into the mechanism of how both Helios and Ikaros are involved in the regulation of BCR signaling.

DT40 mutants: a model to study transcriptional regulation of B cell development and function.

A key issue in understanding the hematopoietic system and B cell biology is to define the function of transcription factors. B lymphocyte development and function is controlled by a hierarchy of transcription factors including PU.1, Ikaros, E2A, EBF, Pax5 and Aiolos. Mouse knockout models provide information about the developmental and physiological importance of the disrupted gene. However, an early block in the development or a lethal phenotype prevents the studies of the functional importance of the gene at the later developing system such as the immune system. The chicken B cell line DT40 is used to circumvent these problems. Studies with DT40 have revealed a role for Ikaros transcription factor in B cell receptor signaling and Aiolos has been shown to regulate immunoglobulin gene conversion and cell survival. On the other hand, findings with Pax5 deficient mutants support DT40 targeting system as a valid model for the plasma cell differentiation and demonstrate the genetic plasticity of the cell line. This system is an excellent model to study transcription factors in B cell specific functions, antibody production and B cell differentiation.

Loss of Pax5 promotes plasma cell differentiation.

Pax5 is indispensable for the commitment of early lymphoid progenitors to the B cell lineage as well as for the development of B cells. To better understand the functional importance of Pax5 at the later stages of B cell differentiation, we established a Pax5-deficient DT40 B cell line. The Pax5(-/-) cells exhibited slower growth, decreased surface IgM expression, and total loss of B cell receptor signaling. Moreover, the expression of the plasma cell-characteristic transcription factors Blimp-1 and XBP-1 were significantly upregulated and the expression of Bcl-6 diminished in the Pax5(-/-) cells, and this alteration was normalized by restored Pax5 expression. The Pax5-deficient cells further manifested substantially elevated secretion of IgM into the supernatant, another characteristic of plasma cells. These results indicate that downregulation of Pax5 function promotes the plasma cell differentiation of B cells.

Ikaros has a crucial role in regulation of B cell receptor signaling.

The transcription factor Ikaros, a key regulator of hematopoiesis, has an essential role in lymphocyte development. In mice, fetal lymphoid differentiation is blocked in the absence of Ikaros, and whereas T cells develop postnatally, B cells are totally absent. The significance of Ikaros in the B cell development is evident, but how Ikaros regulates B cell function has neither been established nor previously been studied with B cells that lack Ikaros expression. Here we show that disruption of Ikaros in the chicken B cell line DT40 induces a B cell receptor (BCR) signaling defect with reduced phospholipase Cgamma2 phosphorylation and impaired intracellular calcium mobilization, which is restored by Ikaros reintroduction. Furthermore, we show that lack of Ikaros induces hyperphosphorylation of Casitas B lymphoma protein subsequent to BCR activation. These results indicate that the absolute need of Ikaros for development, cell fate decisions and maintenance of B cells is due to the enhancement of BCR signaling.