Figure 5.

DEF6 regulates the expression of AID in antigen-engaged B cells by controlling IRF4 function. (A) Purified B cells from indicated 8–10-wk-old female mice were stimulated with single-round pulse αIgM (pIgM) followed by stimulation with an αCD40 antibody in the presence or absence of 50 ng/ml IL-21. 48 h after stimulation, Aicda mRNA expression was measured by real-time RT-PCR. The data are representative of three independent experiments. (B) Purified B cells from female WT and Def6^tr/tr mice were stimulated as in A. 48 h later, extracts were obtained and analyzed by Western blot using an anti-AID antibody (top). The blot was reprobed with an antibody against β-actin as a loading control (bottom). The data are representative of three independent experiments. (C) Purified B cells from female WT mice were stimulated as in A. Nuclear extracts were then prepared and immunoprecipitated with anti-IRF4 (αIRF4 Ab IP) or with a control antibody (Contrl IP). The immunoprecipitates were then analyzed by immunoblotting using an antiserum against DEF6 (top). The blot was later stripped and reprobed with an IRF4 antibody (bottom). The data are representative of four independent experiments. (D) ChIP assays were performed on purified B cells from female WT and Def6^tr/tr mice stimulated as in A with either an anti-IRF4 antibody or a control antibody, and real-time PCR for the Aicda promoter was performed as indicated. The data are representative of two independent experiments. (E) Purified B cells from female WT and Def6^tr/tr mice were stimulated as in A. 48 h later, extracts were obtained and analyzed by Western blot using an antibody recognizing phospho-STAT3 (Y705; top). The blot was reprobed with an antibody recognizing total STAT3 (bottom). The data are representative of two independent experiments. (F) ChIP assays were performed on purified B cells from female WT and Def6^tr/tr mice stimulated as in A with either an anti-STAT3 antibody or a control antibody, and real-time PCR for the Aicda promoter was performed as indicated. The data are representative of two independent experiments. (G) Purified B cells from 8–10-wk-old WT, Def6^tr/tr, Irf4^−/−, and Def6^tr/trIrf4^−/− female mice were stimulated as described in A. 48 h after stimulation, Aicda mRNA expression was measured by real-time PCR. The data are representative of three independent experiments. (H) Purified B cells from female WT and Def6^tr/tr mice were retrovirally transduced with a control vector or with vectors expressing either a full-length form of DEF6 (DEF6 FL) or DEF6 (1–385) in presence of stimulation with pIgM+αCD40. 5 d after stimulation, cells were harvested and sorted for YFP⁺ cells. Sorted YFP⁺ cells were then subjected to real-time RT-PCR for Aicda mRNA expression. The data are representative of two independent experiments. Throughout the figure white bars represent unstimulated cells, gray bars represent cells stimulated with pIgM+αCD40 and black bars represent cells stimulated with pIgM+αCD40+IL-21. *, P < 0.05; **, P < 0.01; ***, P < 0.005; ns, not statistically significant.