Internal IgH class switch region deletions are position-independent and enhanced by AID expression

Abstract

Ig heavy chain class switch recombination (CSR) involves a recombination/deletion mechanism that exchanges the expressed C(H) gene with a downstream C(H) gene. CSR is mediated by highly repetitive switch (S) region sequences and requires the activation-induced deaminase (AID). The S region 5' of the C mu gene (S mu) can undergo high-frequency internal deletions in normal B cells and B cell lines activated for CSR, although the relationship of these deletions and CSR has not been elucidated. In this study, we introduced constitutively transcribed S mu or S gamma 2b regions into a pro-B cell line that can be activated for AID expression, CSR, and endogenous S mu deletions. We find that randomly integrated S region transcription units in these cells also undergo increased levels of internal rearrangements after cellular activation, indicating that the deletion process is independent of location within the Ig heavy chain locus and potentially AID-promoted. To test the latter issue, we generated hybridomas from wild-type and AID-deficient activated B cells and assayed them for internal S mu deletions and S region mutations. These studies demonstrated that efficient intra-S region recombination depends on AID expression and that internal S region deletions are accompanied by frequent mutations, indicating that most S region deletions occur by the same mechanism as CSR.

Figure 1

Induction of intra-S region deletions detected in transfected ISR substrates. (A) Design of the Dγ2b and μ-GFP constructs showing the position of S region sequences in relation to restriction sites and hybridization probes (hatched boxes) used for Southern analysis. See text for details. E, EcoR1. (B) (Left) AID transcripts are detectable by Northern blotting in the Dγ2b-containing subclone 18.4.1 and increase after activation. Dγ2b transcripts as detected by the β-globin probe display constitutive expression from the substrate even after activation. Rac-1 levels were used to determine RNA loading (33). (Right) AID transcripts are induced in μ-GFP subclone 26.4 after activation with LPS. (Middle) A Northern blot with GFP probe showing constitutive expression of the μ-GFP construct in subclone 26.4. (Bottom) A Northern blot of the same RNA hybridized with a Rac-1 control probe to indicate RNA loading. (C) Deletions within the Sμ region of the μ-GFP construct occur after activation of 18–81 A20 cells with LPS. Southern blotting analysis on DNA from 18–81 A20 (lane 15), 26.4 (lane 16), and 26.4 subclones (lanes 1–14). μ-GFP transfected (26.4) 18–81 A20 cells were grown in LPS or media alone for 2 days and then subcloned at limiting dilution. Genomic DNA was isolated and digested with EcoR1 and screened with the GFP-specific probe shown in A. Rearrangements are denoted by *.

Figure 2

Decreased frequency of Sμ deletions in hybridomas derived from AID^−/− B cells compared with WT, IgM-secreting hybridomas. (A) Genomic organization of the J_H-Cμ region of the IgH locus, including the location of the intronic IgH enhancer (Eμ), μS region (Sμ), and μ constant region exons (Cμ). B, BamH1; E, EcoR1; H, HindIII; S, Sac1; X, XbaI. Two-sided arrows indicate germ-line EcoR1/BamH1 and Xba1 restriction fragment sizes. Hatched boxes represent hybridization probes used for Southern blotting analyses. (B) Panel of hybridomas showing internal deletions within Sμ. Hybridoma DNA was doubly digested with BamH1/EcoR1 and Southern analysis was performed by using the 5′ Cμ−specific probe shown in A. W1 and W2 are two representative WT IgM-secreting hybridomas; A1-A5 are AID^−/− hybridomas. NS-1 is the hybridoma fusion partner. Kidney DNA (WT) indicates germ-line configuration. (C) To confirm that the deletions were between the Eμ and Cμ regions, Southern blotting analysis was done on Xba1-digested DNA and probed with the Iμ probe shown in A. Results on all Sμ-deleting hybridomas are summarized in Table 2. Rearrangements are denoted by *.

Figure 3

Mutations accumulate downstream of Sμ in WT IgM-secreting hybridomas. (A) The genomic configuration of the Sμ region is shown. PCR primers were designed 300 bp apart at the 3′ end of the Sμ core and used to amplify this sequence from hybridoma DNA. (B) (Left) The number of mutations found on each of 17 alleles derived from nine WT hybridomas, 12 of which contain Sμ deletions. The mutations were distributed as follows: four alleles with 1 bp, two with 2 bp, and one with 3 bp altered. (Right) Sequences derived from six WT hybridomas with 12 alleles revealed two alleles each with a single bp mutation.