Cell Cycle Regulates Nuclear Stability of AID and Determines the Cellular Response to AID

AID (Activation Induced Deaminase) deaminates cytosines in DNA to initiate immunoglobulin gene diversification and to reprogram CpG methylation in early development. AID is potentially highly mutagenic, and it causes genomic instability evident as translocations in B cell malignancies. Here we show that AID is cell cycle regulated. By high content screening microscopy, we demonstrate that AID undergoes nuclear degradation more slowly in G1 phase than in S or G2-M phase, and that mutations that affect regulatory phosphorylation or catalytic activity can alter AID stability and abundance. We directly test the role of cell cycle regulation by fusing AID to tags that destabilize nuclear protein outside of G1 or S-G2/M phases. We show that enforced nuclear localization of AID in G1 phase accelerates somatic hypermutation and class switch recombination, and is well-tolerated; while nuclear AID compromises viability in S-G2/M phase cells. We identify AID derivatives that accelerate somatic hypermutation with minimal impact on viability, which will be useful tools for engineering genes and proteins by iterative mutagenesis and selection. Our results further suggest that use of cell cycle tags to regulate nuclear stability may be generally applicable to studying DNA repair and to engineering the genome.

活化诱导胞嘧啶脱氨酶（Activation Induced Deaminase，AID）可对DNA中的胞嘧啶进行脱氨基修饰，以启动免疫球蛋白基因多样化进程，并在早期发育阶段重编程CpG甲基化模式。AID具有潜在的高度致突变性，可引发基因组不稳定，其表现为B细胞恶性肿瘤中的染色体易位。本研究发现AID的表达受细胞周期调控。通过高内涵筛选显微术，我们证实AID在G1期的核内降解速率慢于S期或G2-M期；且影响调控性磷酸化或催化活性的突变可改变AID的稳定性与表达丰度。我们通过将AID与特定标签融合（该标签可导致G1期或S-G2/M期之外的核蛋白降解不稳定），直接验证了细胞周期调控的功能。研究表明，在G1期强制使AID定位于细胞核，可加速体细胞超突变与类别转换重组，且细胞对此具有良好耐受性；而在S-G2/M期细胞中，核定位的AID会降低细胞活力。我们筛选得到了可加速体细胞超突变且对细胞活力影响极小的AID突变体，这类突变体将成为通过迭代诱变与筛选工程化改造基因与蛋白质的实用工具。本研究结果进一步表明，利用细胞周期标签调控核蛋白稳定性的策略，或可广泛应用于DNA修复研究与基因组工程改造领域。