Somatic hypermutation of the AID transgene in B and non-B cells

Abstract

Somatic hypermutation (SHM) of the Ig genes is required for affinity maturation of the humoral response to foreign antigens. Activation-induced cytidine deaminase (AID), which is specifically expressed in germinal center centroblasts, is indispensable for this process. Expression of AID is sufficient to activate SHM in hybridomas, non-B cells, and Escherichia coli, suggesting that it initiates the mutational process by deaminating DNA. However, the cis-acting sequences that are responsible for targeting AID activity to the variable (V) region of Ig genes are unknown. Here we show that expression of AID in B cell lines (i.e., Burkitt's lymphoma Ramos and hybridoma P1-5) not only causes hypermutation of Ig sequences, but also of the AID transgene itself. Because it is possible that B cell-specific transacting factors bind to and recruit the "mutator" to the AID transgene, we tested whether the AID transgene can mutate in non-B cells. Indeed, we show that expression of AID in Chinese hamster ovary cells causes SHM of both the Ig and AID transgenes. These data suggest that high transcription rates alone may predispose any gene to mutation by AID but do not preclude that there are specific B cell factors that account for the extremely high rate of V mutation in vivo and its targeting to the V region.

Fig 1.

Mutations in the AID transgene from Ramos, hybridoma P1–5, and CHO are shown. Mutations located within the AID transgene from the Burkitt's lymphoma Ramos (uppercase), hybridoma P1–5 (lowercase), and CHO cells (uppercase bolded) are shown. Duplicate mutations from each clone were counted once in Table 1. Hotspot motifs (RGYW and WRCY) are bolded.

Fig 2.

AID induces SHM in CHO cells. (A) Murine Vn/E_CMV γ2a construct transfected into CHO cells to study SHM. As described in ref. , this heavy chain Ig construct has replaced the intronic μ enhancer with a CMV enhancer and contains a TAG nonsense codon within an RGYW hotspot motif at codon 38. (B Left) CHO clone CHO-LC18 (see Materials and Methods) stably transfected with heavy and light chain Ig genes was transfected with empty vector (○) or the hAID construct (•). Depending on expression of AID (see below), data were distributed into AID-negative (AID⁻) and AID-positive (AID⁺) columns. Frequency of nonsense revertants, as detected with the ELISA spot assay, is plotted. (Right) Ten subclones of CHO clones A.3 and A.9 were further analyzed by using the ELISA spot assay to calculate mutation rates by fluctuation analysis. (C) Northern blots for hAID and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in CHO transfectants. Clone numbers correspond to numbers in B Left.