Overlapping activation-induced cytidine deaminase hotspot motifs in Ig class-switch recombination

Abstract

Ig class-switch recombination (CSR) is directed by the long and repetitive switch regions and requires activation-induced cytidine deaminase (AID). One of the conserved switch-region sequence motifs (AGCT) is a preferred site for AID-mediated DNA-cytosine deamination. By using somatic gene targeting and recombinase-mediated cassette exchange, we established a cell line-based CSR assay that allows manipulation of switch sequences at the endogenous locus. We show that AGCT is only one of a family of four WGCW motifs in the switch region that can facilitate CSR. We go on to show that it is the overlap of AID hotspots at WGCW sites on the top and bottom strands that is critical. This finding leads to a much clearer model for the difference between CSR and somatic hypermutation.

Fig. 1.

Targeting of RMCE cassette into Sα locus in CH12F3 cells. (A) Genomic organization of the germ-line and targeted Sα locus. The map is drawn to scale. Exons are indicated by filled squares. L2 and 3L are wild-type and mutant loxP sites, respectively. PGK, phosphoglycerate kinase promoter; Puro, puromycin-resistant gene; ΔTK, truncated thymidine kinase gene; DTA, diphtheria toxin A chain. Restriction sites: B, BamHI; K, KpnI; S, SbfI. (B) Southern blot of BamHI-digested genomic DNA from wild-type and targeted cells. The 5′-probe detects only the productive allele and the 3′-probe detects both alleles. (C) RMCE. Exchange plasmid containing floxed mutant sequence and Cre expression plasmid are contransfected into Sα-targeted CH12F3 cells. Successful exchange events are screened by counter selection against TK gene using GANC.

Fig. 2.

Validation of RMCE-based CSR assay in CH12F3 cells. (A) EcoRI-digested genomic DNA from GANC-resistant clones was analyzed by Southern blot with the indicated probe. The expected sizes of the hybridized bands are listed in the diagram. Gray and black triangles indicate wild-type and mutant loxP sites, respectively. Block arrows indicate the orientations of the exchanged fragments. “E” indicates EcoRI site. SaFL indicates full-length Sα. Lig4In indicates an intronic sequence from murine DNA ligase IV gene (AC138397: 142058–144068). Plus and minus indicate physiological and nonphysiological orientations, respectively. (B) RMCE-mediated sequence knock-in at the Sα locus. Exchange efficiency is defined as the percentage of correctly exchanged clones against the total GANC-resistant clones. (C) Class-switch assay. Each dot represents an independent clone generated by RMCE in 1F7 cells. One of at least three independent experiments was shown.

Fig. 3.

Construction of synthetic switch region. (A) Structure of the 80-bp linker (monomer) reflecting the consensus Sα repeat described in ref. . (B) Resolution of ligated concatemers on a 1.2% agarose gel. Lane 1, 1-kb DNA ladder (Invitrogen). Lane 2, ligation products (concatemers). The number of repeats in each band was shown to the right side of the gel. (C) Comparison of class-switch efficiency between the size-matched synthetic and native core Sα region. Each dot represents an independent clone. One of at least three independent experiments was shown. (D) The level of GLTs in stimulated cells. Error bars represent SEM of at least three independent experiments. Each experiment contains at least two independent clones from each construct.

Fig. 4.

Mutagenesis of AGCT motif in Sα region. (A) The sequence of the consensus Sα repeat. The six AGCT sites are underlined. (B) Class-switch assay of Sα mutations. The name of each mutant reflects the sequence change from the AGCT. Each dot represents an independent clone. One of at least three independent experiments was shown. (C) The level of GLTs in stimulated cells. (D) AGCC-2 mutant. Two point mutations (indicated by arrows) were introduced into the AGCC mutant to disrupt the proximity of AID hotspots (boxed area). Cytosine residues in AID hotspots are labeled red. (E and F) Class-switch (E) and germ-line (F) transcript level in AGCC-2 mutant. Error bars represent SEM of at least three independent experiments.

Fig. 5.

CSR efficiency correlates with switch region WGCW density. (A) CSR assay of Sα replacement constructs. Sγ1–2.2B, 2,172 bp BamHI-BamHI fragment of mouse Sγ1 (D78344: 5824–8000). Sγ3–1.2HSc, 1,181 bp of HindIII-SacI fragment of mouse Sγ3 (48). Sα-2.1HX, 2,076 bp HindIII-XbaI fragment of mouse Sα (AC160982: 124768–122693). Sγ2b-2.0Sp, 2,051 bp SpeI-SpeI fragment of mouse Sγ2b (D78344: 29538–31593). Sα-1.1Con, 1,120 bp of Sα made by ligation of 14 repeats of consensus 80 mer (Fig. 3). (B) The level of GLTs in stimulated cells. Error bars represent SEM of at least three independent experiments. Each experiment contains at least two independent clones from each construct. (C) CSR correlates with S region WGCW density. Error bars represent SD of three independent experiments.