AID from bony fish catalyzes class switch recombination

Abstract

Class switch recombination was the last of the lymphocyte-specific DNA modification reactions to appear in the evolution of the adaptive immune system. It is absent in cartilaginous and bony fish, and it is common to all tetrapods. Class switching is initiated by activation-induced cytidine deaminase (AID), an enzyme expressed in cartilaginous and bony fish that is also required for somatic hypermutation. Fish AID differs from orthologs found in tetrapods in several respects, including its catalytic domain and carboxy-terminal region, both of which are essential for the switching reaction. To determine whether evolution of class switch recombination required alterations in AID, we assayed AID from Japanese puffer and zebra fish for class-switching activity in mouse B cells. We find that fish AID catalyzes class switch recombination in mammalian B cells. Thus, AID had the potential to catalyze this reaction before the teleost and tetrapod lineages diverged, suggesting that the later appearance of a class-switching reaction was dependent on the evolution of switch regions and multiple constant regions in the IgH locus.

Figure 1.

m- and z-AID are catalytically active in E. coli and S. cerevisiae. (A) Mutator assay in E. coli. Expression of m-, z-, and f-AID was induced for 3 h in exponentially growing cultures that were then assayed for kanamycin resistance. Points represent the mutation frequency of individual starting colonies. The mean values and standard deviations from three experiments are given. (B) Mutator assay in ung ⁺ and ung ⁻ S. cerevisiae. Expression of m-, f-, and z-AID was induced by galactose for 48 h at 30°C, and cultures were assayed for ura3 auxotrophy in 5-FOA–containing medium. Points represent the mutation frequency in cultures from individual starting colonies. The mean values and standard deviations are given. ung ⁺ background data were from four experiments. One experiment in the ung⁻ background is shown.

Figure 2.

AID from bony fish induces class switch recombination in mouse AID^−/− splenocytes. (A) Representative flow cytometry profiles of IL-4 and LPS-stimulated B cells from AID^−/− mice infected with retroviruses encoding m-, f-, or z-AID or an empty vector control. The y axis shows GFP expression, and the x axis shows cell surface IgG1. The percentage of cells in each indicated quadrant is shown. (B) Percentage of switching to IgG1 by GFP⁺ cells. Points represent individual cultures. (C) Immunoblot analysis of protein extracts from AID^−/− B cell cultures infected with retroviruses encoding FLAG-tagged AID. GFP expression was used as a loading control.

Figure 3.

Normal Sμ–Sγ1 switch recombination junctions in z-AID–expressing mouse B cells. (A) Representative switch junctions cloned from mouse AID^−/− B cells infected with m- or z-AID–encoding retroviruses. Overlap was determined by identifying the longest region at the switch junction of perfect uninterrupted donor/acceptor identity. Sμ and Sγ1 germ line sequences are shown above and below each junction sequence, respectively. Bold letters indicate regions of microhomology and underlined letters indicate mutations. (B) Length of microhomologies at Sμ–Sγ1 junctions (n = 26 for both m- and z-AID–derived junctions). (C) Scatter analysis of the Sμ–Sγ1 breakpoints derived from in vitro–stimulated B cells. The axes indicate the position relative to Sμ and Sγ1 sequences (available from GenBank/EMBL/DDBJ under accession nos. J00440 and AJ851869, respectively). Closed circles denote breakpoints induced by m-AID, and open circles denote breakpoints induced by z-AID. Data are pooled from two independent experiments.