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DNA damage checkpoints in immunoglobulin diversification and the germinal center reaction

Subject Area General Genetics and Functional Genome Biology
Immunology
Term from 2012 to 2022
Project identifier Deutsche Forschungsgemeinschaft (DFG) - Project number 227483551
 
Final Report Year 2023

Final Report Abstract

Somatic hypermutation and class switch recombination of immunoglobulin genes in germinal center B cells are essential for an efficient humoral immune response. However, both genetic processes may also lead to genetic aberrations and lymphomagenesis and must thus be tightly regulated. The focus of the research project this final report summarizes is the regulation of immunoglobulin diversification by checkpoint signalling in vitro and in vivo, by the cell cycle and by homologous recombination activity. In the first funding period, we have finished the analysis of Chk1 and Chk2 effects on immunoglobulin diversification in vitro, the preliminary data of which formed the basis of the first proposal. We could show that a decrease in Chk1 activity led to increased somatic hypermutation in cell lines, apparently due to decreases in homologous recombination activity, while Chk2 inactivation led to decreased somatic hypermutation by increased Chk1 function. Moreover, we have analysed the impact of inactivation of p53, a common downstream target of both Chk1 and Chk2, as well as of Chk1 in vivo in adequate mouse models. Here, we found that p53 inactivation in germinal center B cells did not affect somatic hypermutation of the variable region of the Ig genes, but did affect mutagenesis in the switch region. This indicated differential activity of p53 during hypermutation and class switching. Analysing Chk1 function in the germinal center in vivo, we found that decreasing its activity in the germinal center lead to decreased somatic hypermutation activity, implying that a complex regulation of somatic hypermutation downstream of Chk1 function occurs. We have also analysed the role of the cell cycle in regulating immunoglobulin diversification. To this end, we have generated fusions of AID, the initiating enzyme of both somatic hypermutation and class switch recombination, with tags that allowed its degradation specifically in the G1 or S/G2 phase of the cell cycle. We found that G:C mutagenesis during hypermutation, as well as immunoglobulin gene conversion, could proceed with AID generated lesions both in G1 as well as S/G2, while A:T mutagenesis during hypermutation as well as class switch recombination required AID-induced lesions only in G1. Finally, we have analyzed the role of homologous recombination in regulating immunoglobulin diversification and the germinal center reaction. We could show that inactivation of homologous recombination led to a proliferation and survival defect of activated B cells and thus decreased class switch recombination. B cells deficient in homologous recombination could form germinal centers, though, and undergo affinity maturation. Somatic hypermutation was significantly decreased in these mice, mostly due to a depletion in highly mutated sequences with higher A:T mutagenesis. Subsequently, we have analysed mice with a defect in the homologous recombination inhibitor PARI. Here, we found increased hypermutation activity mostly due to an increase in highly mutated sequences with more A:T mutagenesis. Also, we found increased hypermutation activity at non-immunoglobulin genes in these mice.

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