IgH transcription and AID-ChIP in 3′RR-deficient and WT mice. (A and B) Analysis by real-time PCR of IgH (A) and κ (B) primary transcripts in B220⁺PNA^highFas⁺ cells from Peyer’s patches of immunized 3′RR-deficient and WT mice. Transcript levels were normalized to GAPDH transcripts. Results are the mean ± SEM of six WT and six 3′RR-deficient mice. (C) Accessibility of IgH locus downstream of J_H4 to AID. ChIP assays were performed with splenic B cells isolated from immunized AID-deficient, 3′RR-deficient, and WT mice. For each sample, AID-Chip values were normalized to the input control, and AID-Chip signal in WT B cells was assigned an arbitrary value of 1. Data presented are from one primer pair and error bars corresponding to technical replicates. One representative experiment out of two is shown. (D and E) Analysis by real-time PCR of IgH (D) and κ (E) primary transcripts in B220⁺PNA^high splenic B cells of immunized 3′RR-deficient and WT mice. Results are the mean ± SEM of four WT and five 3′RR-deficient mice. (F) Accessibility of IgH locus to RNA polymerase II (Pol II). ChIP assays were performed with 3-d LPS-stimulated splenic B cells isolated from 3′RR-deficient and WT mice. The relative enrichments (percent input) were analyzed by real-time PCR (same primers as for analysis of IgH primary transcripts) and compared with those of negative controls obtained without antibody (mock). Results are the mean ± SEM of three 3′RR-deficient and three WT mice. ns, not significant. (G) ChIP analysis for AID occupancy at the S_μ region in B splenocytes of immunized WT, 3′RR-deficient, and AID-deficient mice. For each sample, AID-Chip values (mean ± SD) were normalized to the input control, and AID-Chip signal in WT B cells was assigned an arbitrary value of 1. Data represent results obtained using two different primer pairs (error bars are indicative of the variation between the two PCRs). One representative experiment out of two is shown. *, P < 0.05; Mann–Whitney U test.