For CSR assays, LPS (30 g/mL, no. the locus in an activation-induced cytidine deaminase (AID) and H2AX-dependent fashion. Finally, we demonstrate that depletion of another BRCT-domain protein, MDC1, in BRIT1-deleted B cells increases the severity of CSR defect over what is observed upon loss of either protein alone. Our results identify BRIT1 as a factor in CSR and demonstrate that multiple BRCT-domain proteins contribute to optimal resolution of AID-induced DSBs. Mature B cells responding to antigenic challenge undergo Ig heavy chain (breaks. Results BRIT1 Promotes CSR in CH12 Cells. shRNAs against mRNAs encoding 12 known BRCT domain-containing proteins (Table S1) with no reported activity in CSR were individually transduced into CH12 cells, a murine B-lymphoma line that undergoes CSR from IgM to IgA in response to a combination of anti-CD40, interleukin-4 (IL-4), and transforming growth factor (TGF-). CSR to IgA was assessed relative to cells transduced with a scrambled shRNA by quantification of cells expressing surface IgA using flow cytometry (Fig. 1and and Fig. S1). DBF4 participates in initiation of DNA replication (33), and the CSR defect in shDBF4 cells likely reflects impaired proliferation. Because loss of BRIT1 leads to defects in DNA repair during meiotic recombination and increased genomic instability (27), we decided to investigate its role in CSR. Open in a separate window Fig. 1. BRIT1 promotes CSR in CH12 cells. (= 2). Exp, experiment; Scr, scrambled shRNA control. (= 8; ** 0.005). (= 6). Table S1. List of qPCR primers and shRNA clones for target genes 0.0001; Fig. 1and and and = 6) mice. Frequency represents live singlets in the lymphocyte gate. (= 6). (= 6). (= 6) mice. (= 6). (= 6). (and and = 7; ** 0.005). ( 0.005. (= 6). Max, maximum. (variable domain (5 labeled for green signal) and sequences immediately downstream of the constant region exons (3 0.005. Error bars represent means SD. (locus (break ((36). Although nearly all metaphases from AID KO B cells showed colocalization of the two probes, split signals (separated red and green signals) were significantly higher in BRIT1 KO B cells compared with control B cells (8% versus 2.4%, = 4; 0.001; Fig. 2DSBs was left unrepaired in the absence of BRIT1. Thus, BRIT1 appears Oridonin (Isodonol) to influence CSR by promoting the resolution of AID-induced DSBs. BRCT Domains of BRIT1 Are Required for Efficient CSR. The influence of BRIT1 on the DNA repair phase of CSR suggested that the BRCT domains might play a crucial role in this process. The correlation between BRCT domains, per se, and molecular functions in CSR is not straightforward. For example, the N terminus Eltd1 BRCT domain of PTIP is required for CSR, whereas the C terminus BRCT repeats of 53BP1 are dispensable (37, 38). BRIT1 contains an N-terminal BRCT domain and two C-terminal BRCT domains (Fig. 3and = 3). (= 3; ** 0.005, * 0.05). (= 4; ** 0.005, * 0.05). (locus. Antibodies against histone H3 and nonspecific IgG were used as positive and negative controls, respectively. ChIP-quantitative PCR (qPCR) analysis demonstrated that BRIT1 was significantly enriched at S compared with the IgG control and not detected in BRIT1 KO B cells (Fig. 4and = 3). (= 3). (= 3). (= 3). (= 4), Oridonin (Isodonol) BRIT1 KO (= 4), and AID KO (= 4) mice following NP-CGG immunization on day 28 by ELISA. Conc., concentration. The DDR protein ATM is a major effector of CSR, in part, through its ability to phosphorylate H2AX. To explore the ATMC-H2AXCBRIT1 axis in CSR further, we treated splenic B cells with the small-molecule ATM inhibitor Oridonin (Isodonol) ATMi (39). In control cells, ATMi addition led to an 50% reduction in CSR to IgG1 over what was observed upon vehicle treatment without altering levels of BRIT1 or AID (Fig. 5 and = 3; ** 0.005, * 0.05). (= 3; ** 0.005, * 0.05). Besides interacting with BRIT1, -H2AX also interacts with the BRCT-domain protein MDC1 to facilitate its recruitment to DSBs (22). In fact, the BRCT domains of both BRIT1 and MDC1 interact with -H2AX peptide with similar.