The role of AID in class switch recombination and somatic hypermutation of immunoglobulin genes

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B-lymphocytes modify their immunoglobulin genes upon antigen stimulation in helper T-lymphocyte dependent manner. The modifications include somatic hypermutation (SHM) and class switch recombination (CSR), which are molecular basis's of affinity maturation and isotype switch of immunoglobulin, respectively. Recently, knockout studies have provided evidence for indispensable role of AID (activation induced cytidine deaminase) in these reactions, but the mechanism how AID induces SHM and CSR is still unclear.To map the portion for regulating the subcellular localization of AID, and to examine the correlation between AID function and its subcellular localization, many mutant AID proteins fused with GFP were generated and examined. We found that AID has weak nuclear localization signal and nuclear export signal at N- and C- terminus, respectively. The region containing the nuclear export signal match the consensus for ORM1 binding site. Consistently, CRM1 inhibitor leptomycin B causes the nuclear accumulation of AID, suggesting direct binding of CRM1 at the C-terminus.The C-terminus of AID has been shown to be essential for CSR but not for SHM. Complementarily, we found that the N-terminus region of AID is essential for SHM but not for CSR by mutagenesis study. These results suggest that CSR and SHM require different co-factors, which bind C- and N- termini, respectively.To determine if de novo protein synthesis after AID induction is required for CSR and SHM, we have established an inducible AID system in which AID is fused with estrogen receptor hormone binding domain thereby hormone dependent AID regulation is achieved. When protein synthesis is blocked, AID dependent DNA strand breaks are significantly reduced in both CSR and SHM, suggesting the involvement of newly synthesized protein after AID expression in these reactions. The result is consistent with the notion that the RNA editing by AID generates proteins essential for CSR and SHM.