DNA damage checkpoints in immunoglobulin diversification and the germinal center reaction

检查点信号通路是维持我们身体所有细胞基因组完整性的工具。对于脊椎动物适应性免疫系统中发生的靶向基因组变化，这些途径需要以一种特殊的方式进行调节，以允许基因修饰，同时防止恶性转化。本研究评估了检查点信号传导对艾滋病诱导的免疫球蛋白多样化事件（体细胞超突变、类开关重组和免疫球蛋白基因转换）和生发中心反应的影响。在第一个资助期，我们可以证明在生发中心B细胞的继发性免疫球蛋白多样化过程中，Chk1和Chk2的检查点信号在调节体细胞超突变和其他继发性免疫球蛋白多样化途径中发挥作用。一方面，它们通过同源重组调节无错误DNA修复，从而调节体细胞杂交易出错的修复途径。另一方面，常见的Chk1/2靶蛋白p53以一种方式调节免疫球蛋白诱变，这意味着p53在生发中心的两个区域具有不同的功能：发生体细胞超突变的暗区和发生类开关重组的亮区。在第二个资助期，我们希望评估影响的上游事件，以及通过检查点信号调节免疫球蛋白多样化的下游目标。首先，我们将通过将AID的活性限制在细胞周期的单个阶段来研究继发性Ig多样化的单个途径如何与细胞周期控制相关联。其次，我们将评估它们在不同细胞周期阶段的活性是如何通过生发中心的Chk1/2的差异调节来调节的。第三，我们将探讨复制分叉处同源重组的限制如何影响体细胞超突变。这项研究将对免疫球蛋白多样化的机制和进化，以及其失调导致人类B细胞淋巴瘤发病的途径提供重要的见解。

Checkpoint signalling pathways are instrumental for maintenance of genome integrity in all cells of our body. For targeted genome changes occuring in the adaptive immune system of vertebrates, these pathways need to be modulated in a peculiar manner to allow genetic modification but prevent malignant transformation at the same time. The present proposal assesses the influence of checkpoint signalling on AID-induced immunoglobulin diversification events - somatic hypermutation, class switch recombination and immunoglobulin gene conversion - and the germinal center reaction. In the first funding period, we could show that during secondary immunoglobulin diversification in germinal center B cells, checkpoint signalling by Chk1 and Chk2 plays a role in the regulation of somatic hypermutation and other pathways of secondary immunoglobulin diversification. On the one hand, this occurs via their regulation of error-free DNA repair by homologous recombination, which modulates the error-prone repair pathways of somatic hyypermutation. On the other hand, the common Chk1/2 target protein p53 regulates immunoglobulin mutagenesis in a manner implying differential p53 functionality in two areas of the germinal center: the dark zone where somatic hypermutation occurs, and the light zone where class switch recombination takes place. In the second funding period, we wish to assess the upstream events that affect, as well as the downstream targets that mediate the regulation of immunoglobulin diversification by checkpoint signalling. First, we will investigate how the individual pathways of secondary Ig diversification are linked to cell cycle control, by restricting the activity of AID to individual phases of the cell cycle. Second, we will assess how their activity in different cell cycle phases is modulated by differential regulation of Chk1/2 in the germinal center. Third, we will ask how restriction of homologous recombination at replication forks affects somatic hypermutation. This study will allow important insights into the mechanism and evolution of immunoglobulin diversification, as well as into pathways of its deregulation that lead to the pathogenesis of human B cell lymphoma.