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

B淋巴细胞在抗原刺激下以辅助性T淋巴细胞依赖的方式修饰其免疫球蛋白基因。这些修饰包括体细胞超突变(SHM)和类开关重组(CSR)，它们分别是免疫球蛋白亲和成熟和同型切换的分子基础。最近的基因敲除研究证实了AID(激活诱导胞苷脱氨酶)在这些反应中起着不可或缺的作用，但AID诱导SHM和CSR的机制尚不清楚。为了定位AID的亚细胞定位调控部分，并研究AID功能与其亚细胞定位的相关性，我们构建并检测了许多与GFP融合的突变AID蛋白。我们发现AID在N-端和C-端分别有微弱的核定位信号和核输出信号。含有核输出信号的区域与ORM1结合位点的共识一致。一直以来，CRM1抑制剂Leptomycin B导致AID的核积聚，提示C端与CRM1直接结合。AID的C端被证明是CSR所必需的，但对SHM不是必需的。另外，我们通过突变研究发现，AID的N-末端区域对SHM是必需的，而对CSR不是必需的。这些结果表明，CSR和SHM需要不同的辅助因子，分别结合C-端和N-端。为了确定CSR和SHM是否需要AID诱导后从头合成蛋白质，我们建立了一个诱导型AID系统，其中AID与雌激素受体激素结合域融合，从而实现激素依赖的AID调节。当蛋白质合成受阻时，依赖AID的DNA链断裂在CSR和SHM中都显著减少，这表明AID表达后新合成的蛋白质参与了这些反应。这一结果与以下观点一致，即AID编辑的RNA产生了CSR和SHM所必需的蛋白质。

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.