Fig 2: Aff3 deficiency increases the sensitivity of mice to P. yoelii infection.(A) Wild-type (dark blue), Aff3+/- (light blue), Aff3-/- (red), and Aicda-/- (brown) mice were infected with P. yoelii, and the level of parasitemia was monitored every 4 days (n = 4 to 5 in each group). Aicda-/- mice died after (B) anti–P. yoelii antibodies in serum were measured by ELISA at 50 and 14 days after inoculation (n = 4 to 5 in each group, except n = 2 in naïve group of day 14). In (A and B), the data are shown as means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.005, and ****P < 0.001. The P values were calculated using multiple Welch’s t test with Holm-Sidak correction (A), one-way analysis of variance (ANOVA) with Tukey’s multiple comparisons test [(B), day 50] or unpaired t tests with Welch’s correction [(B), day 14]. The asterisks indicating significant differences compared with naïve mice are not shown in (B).

Fig 3: AFF3 protein directly binds to the switch region.(A) CD43- B cells were stained with CFSE and cultured. The cells were analyzed by flow cytometry. (B) Transcriptome analysis of Aff3-deficient B cells. CD43- B cells were cultured with LPS and IL-4. After 3 days of culture, total RNA was prepared and analyzed on a cDNA microarray. (C) Quantitative reverse transcription polymerase chain reaction (qRT-PCR) and Western blotting analyses of Aicda (n = 3 in each group for RT-PCR). CD43- B cells were cultured with LPS (10 µg/ml) and IL-4 (10 ng/ml). Western blotting was performed with anti-AID antibody, and the membrane was reprobed with anti–extracellular signal–regulated kinase 1/2 (ERK1/2) antibody to assess the loading amounts. nt, nucleotide. (D) AFF3 binds to switch regions. CD43- B cells were cultured with LPS (10 µg/ml) and IL-4 (10 ng/ml) for 3 days. Empty (purple) or 3xFLAG-tagged cDNA of the C-terminal half of Aff3 (red) was transduced. The efficiency of transduction was estimated on the basis of the expression of enhanced GFP (EGFP), which was coexpressed with AFF3 by IRES (internal ribosomal entry site) insertion. Then, the cells were analyzed by ChIP with an anti-FLAG antibody. The amount of DNA precipitated was measured by real-time PCR (n = 3 in each group), with primers specific to the switch regions or the Acta (encoding a-actin) gene serving as a negative control. Sµ-SR indicates the switch region of IgM (table S4) (70). S?1-c and S?1-d indicate the switch region of IgG1 (fig. S7 and table S4). In (C and D), the data are shown as means ± SEM. ****P < 0.001. The P values were calculated using unpaired t tests with Welch’s correction. The data shown in this figure are representative of at least two experiments except for those in (B). The primers used in the experiments are listed in table S4.