B-cell receptor activation inhibits AID expression through calmodulin inhibition of E-proteins

Abstract

Upon encountering antigens, B-lymphocytes can adapt to produce a highly specific and potent antibody response. Somatic hypermutation, which introduces point mutations in the variable regions of antibody genes, can increase the affinity for antigen, and antibody effector functions can be altered by class switch recombination (CSR), which changes the expressed constant region exons. Activation-induced cytidine deaminase (AID) is the mutagenic antibody diversification enzyme that is essential for both somatic hypermutation and CSR. The mutagenic AID enzyme has to be tightly controlled. Here, we show that engagement of the membranebound antibodies of the B-cell receptor (BCR), which signals that good antibody affinity has been reached, inhibits AID gene expression and that calcium (Ca 2؉ ) signaling is essential for this inhibition. Moreover, we show that overexpression of the Ca 2؉ sensor protein calmodulin inhibits AID gene expression, and that the transcription factor E2A is required for regulation of the AID gene by the BCR. E2A mutated in the binding site for calmodulin, and thus showing calmodulin-resistant DNA binding, makes AID expression resistant to the inhibition through BCR activation. Thus, BCR activation inhibits AID gene expression through Ca 2؉ /calmodulin inhibition of E2A.activation-induced cytidine deaminase ͉ BCR ͉ calcium ͉ E2A P rocesses that diversify antibodies play a major role in protecting higher organisms from pathogens. Upon encountering antigens, antibody-expressing B-lineage lymphocytes adapt to produce a highly specific and potent antibody response. Somatic hypermutation, which introduces point mutations in the variable regions of antibody genes, and gene conversion can increase the affinity for antigen, and antibody effector functions can be altered by class switch recombination (CSR), which changes the expressed constant region exons (1, 2). Activationinduced cytidine deaminase (AID) is the antibody diversification enzyme that is essential for somatic hypermutation, gene conversion, and CSR (3-5). The mutagenic AID enzyme has to be tightly controlled, and both aberrant AID activity and chromosomal translocations involving switch regions of antibody genes are hallmark features of B-cell malignancies (2, 6-9).AID is specifically expressed in a subset of germinal center B-lymphocytes (10, 11). Specific stimulatory signals activate their proliferation and the diversification of the variable regions of their Ig genes, followed by expression of mutated surface Ig and selection of the mutated B-cell receptors (BCRs) for reactivity with antigen (12-14). E-protein, NF-B, Pax5, STAT6, and IRF-8 transcription factors participate in expression of the AID gene (15-18). The gene is induced by IL-4 and ligation of CD40, and, in mice, other inducers have also been identified, including LPS (11,17,19). Only ligation of CD45 has been reported to lead to signaling that inhibits AID gene expression, and this was shown to involve inhibition of signaling to STAT6 (19).In the present study, we show that a...

The MAPK/ERK and PI(3)K Pathways Additively Coordinate the Transcription of Recombination-Activating Genes in B Lineage Cells

“…Moreover, we show that the MEK/ERK and PI(3)K pathways coordinate in regulating Rag, and an additive effect in the induction or suppression of Rag is obtained when both pathways are specifically inhibited. There are many examples in which expression of genes is regulated through multiple pathways, including NF-kB (55), BCL-2 family proteins (56), and activationinduced cytidine deaminase (57), which in many cases function for tuning expression levels and/or balancing functional properties of the target genes. Thus, the BCR activates two independent pathways that coordinate for induction of Rag and for tuning its expression level in B lineage cells.…”

High-Affinity B Cell Receptor Ligation by Cognate Antigen Induces Cytokine-Independent Isotype Switching

“…In similar findings, Jabara et al (44) demonstrated that inclusion of an anti-IgM Ab in B cell cultures stimulated with LPS and IL-4 inhibits IgG1 expression and secretion and that this inhibition is not due to either suppressed or delayed proliferation or increased apoptosis. In another example of this phenomenon, Hauser et al (45) found that addition of an anti-IgM Ab decreased AID protein levels by 73% 1 h after addition to a culture of B cells that had been previously stimulated for 48 h with LPS and IL-4. This reduction in protein was due to inhibition of AID mRNA transcription rather than degradation of existing AID protein.…”

Negative Feedback Regulation of Antigen Receptors through Calmodulin Inhibition of E2A

“…The MAPK inhibitor PD98059, but not the protein kinase C inhibitor bisindolylmaleimide, blocked the reduction in CD19 mRNA (Supplemental Fig. 1C Previous studies showed that the main Ca 2+ -sensor protein CaM, when Ca 2+ loaded, can bind to and inhibit transcriptional activities of E2A transcription factors (5,7,9,14,16,17). The E2A proteins have important roles in the transcriptional regulatory network that promotes commitment to and differentiation of B cells (18,19).…”

“…3C) for the indicated times. The MSCV-IRES-GFP-based retroviruses encoding wild-type and CaM-resistant m8N47 mutant of the E12 splice form of mouse E2A were described previously (5). Retrovirus concentrated by centrifugation was added with 5 mg/ml polybrene to 0.5 3 10 6 purified B cells after activation with CD40L plus IL-4 for 14 h (Figs.…”

“…Primary B lymphocytes were purified from mouse spleens, maintained as previously described (5), and stimulated with 5 ng/ml IL-4 (PeproTech) and either 200 ng/ml CD40L (R&D Systems) or 10 mg/ml LPS (Calbiochem) for 48 h. The BCR of stimulated cells at 10 6 cells/ml, where indicated, was activated by incubation with goat F(ab9) 2 anti-mouse IgM at 2.5 mg/ml (5 mg/ ml for Fig. 3A and 3B, and various concentrations, as indicated, for Fig.…”