Interplay between Target Sequences and Repair Pathways Determines Distinct Outcomes of AID-Initiated Lesions

Abstract

Activation-induced deaminase (AID) functions by deaminating cytosines and causing U:G mismatches, a rate-limiting step of Ab gene diversification. However, precise mechanisms regulating AID deamination frequency remain incompletely understood. Moreover, it is not known whether different sequence contexts influence the preferential access of mismatch repair or uracil glycosylase (UNG) to AID-initiated U:G mismatches. In this study, we employed two knock-in models to directly compare the mutability of core Sμ and VDJ exon sequences and their ability to regulate AID deamination and subsequent repair process. We find that the switch (S) region is a much more efficient AID deamination target than the V region. Igh locus AID-initiated lesions are processed by error-free and error-prone repair. S region U:G mismatches are preferentially accessed by UNG, leading to more UNG-dependent deletions, enhanced by mismatch repair deficiency. V region mutation hotspots are largely determined by AID deamination. Recurrent and conserved S region motifs potentially function as spacers between AID deamination hotspots. We conclude that the pattern of mutation hotspots and DNA break generation is influenced by sequence-intrinsic properties, which regulate AID deamination and affect the preferential access of downstream repair. Our studies reveal an evolutionarily conserved role for substrate sequences in regulating Ab gene diversity and AID targeting specificity.

FIGURE 1.

Gene targeting of 5′cSμ region into endogenous IgH V region locus. (A) Targeting strategy of 5′cSμ region. Restriction endonuclease map of the endogenous Igh locus is shown. The closed circle represents the Igh intronic enhancer (iEμ), and the closed boxes represent DQ52 and J_H1–4 elements. Targeting construct was used to introduce the modified 5′cSμ allele (V-5′cSμ) into wt 129 ES cells. The XhoI–EcoRI fragment containing the DQ52 and J_H1–4 elements was replaced by the V-5′cSμ cassette and the floxed neomycin gene. Closed triangles represent the loxP sites. Arrow indicates the V_H186.2 promoter. An asterisk indicates the stop codon in the leader sequence. Striped box indicates 5′cSμ region. (B) Southern blot analysis of targeted ES cells. Left panel, EcoRI-digested ES cell DNA was hybridized with 3′ probe (J_H probe). Right panel, HindIII-digested DNA before and after deletion of the neomycin gene was hybridized with 5′ probe A (DQ52 probe). Germline (GL) and targeted bands (in kb) are indicated by arrows. (C) Schematic of VB1–8 productive and V-cSμ passenger alleles. Top panel: Configuration of VB1–8 allele. The pattern-filled box indicates the VB1–8 exon sequence. The open box (L) indicates the leader of the VB1–8 exon. The V pro indicates the V_H186.2 promoter. The oval box indicates Eμ. Bottom panel, Configuration of V-5cSμ allele. A 760-bp of the cSμ region replaced a large portion of the VB1–8 exon sequence with 21 bp of the V region exon and 19-bp J_H2 exon left flanking the 5′cSμ region, and a stop codon (*) was introduced into the leader sequence (L). (D) Semiquantitative RT-PCR analysis of V-Cμ transcripts in unstimulated or stimulated B cells from VB1–8 or V-cSμ KI mice. The cDNA samples were prepared from unstimulated or stimulated B cells as described in Materials and Methods, and diluted in 1:5 serials for actin (1:5, 1:25, and 1:125) or 1:3 serials for V-Cμ transcripts (no dilution, 1:3 and 1:9). Representative data are shown from three independent experiments.

FIGURE 2.

Mutation frequency of VB1–8 and V-cSμ alleles in wt or repair factor–deficient backgrounds. (A) Mutation frequency of VB1–8 allele in wt (n = 4), Ung^−/− (n = 5), Msh2^−/− (n = 3), and DKO (n = 4) mice (see details in Supplemental Table I). (B) Mutation frequency of the V-cSμ allele in wt (n = 5), Ung^−/− (n = 3), Msh2^−/− (n = 3), and DKO (n = 4) mice (see details in Supplemental Table II). (C) Statistical significance was calculated with a Student t test between different genetic backgrounds (two tailed, two samples with equal variance).

FIGURE 3.

Frequency of deletions and insertions (indels) of VB1–8 and V-cSμ alleles. (A) Frequency of indels in VB1–8 allele in wt (n = 4), Ung^−/− (n = 5), Msh2^−/− (n = 3), and DKO (n = 4) mice (see details in Supplemental Table I). (B) The frequency of indels in the V-cSμ allele in wt (n = 5), Ung^−/− (n = 3), Msh2^−/− (n = 3), and DKO (n = 4) mice (see details in Supplemental Table II). (C) Statistical significance was calculated with a Student t test between different genetic backgrounds (two tailed, two samples with equal variance). (D) The percentage of indels that occurred in different regions of the KI cSμ sequence in V-cSμ/wt samples (n = 5). Data represent means ± SEM. (E) The percentage of indels that occurred in different regions of the KI cSμ sequence in V-cSμ/Msh2^−/− samples (n = 3). Data represent means ± SEM. Nucleotide position in KI cSμ region: AGCT sparse region, 99–599 bp; intermediate region, 600–717 bp; dense region, 718–1065 bp.

FIGURE 4.

Mutation spectrum in different repair factor–deficient backgrounds. (A) Percentage of A/T and C/G mutations in VB1–8 allele. (B) Percentage of A/T and C/G mutations in VcSμ allele. (C) Mutation spectrum of J_H4 intron, VB1–8 and VcSμ alleles in wt mice. (D) Mutation spectrum of VB1–8 and VcSμ alleles in UNG or MSH2-deficient mice.

FIGURE 5.

Mutation hotspots in VB1–8 allele. (A) Point mutations from all VB1–8/wt samples were compiled and plotted against base pair position. Mutation hotspots show a strong correlation with the CDR1, CDR2, and CDR3 regions (see details in Table I). Additional mutation hotspots correlated with the position of AGCT motifs on the VB1–8 allele [displayed in (C)]. (B) Point mutations from all VB1–8/DKO samples were compiled and plotted against base pair position. The overall mutation frequency of VB1–8/DKO samples was higher than that of the VB1–8/wt samples (A). The correlation between mutation hotspots and CDR regions or AGCT motifs was observed. (C) Position of AGCT motifs along the VB1–8 KI allele (1–1284 bp).

FIGURE 6.

Mutation hotspots in V-cSμ allele. (A) The point mutations from all V-cSμ/wt samples were compiled and plotted against base pair position. Most highly targeted mutation hotspots occur in the AGCT intermediate or dense regions at the position of an AGCT motif (see details in Table II). The AGCT sparse region also contains a relatively high amount of point mutations. (B) The point mutations from all VB1–8/DKO samples were compiled and plotted against base pair position. The position of mutation hotspots also correlated with the position of AGCT motifs [displayed in (C)]. The overall mutation frequency of V-cSμ/DKO samples was higher than that of the V-cSμ/wt samples (A). (C) Position of AGCT motifs along the V-cSμ KI allele (1–1352 bp).

FIGURE 7.

Correlation between AGCT density and mutation frequency. (A and B) Correlation plots for mutation frequency and AGCT density. The mutation frequency of the AGCT sparse, intermediate, and dense regions were correlated with the AGCT motif density (frequency of AGCT motifs per 100 bp) in the V-cSμ/wt (A) or V-cSμ/DKO (B) samples. The frequency of mutations did not correlate with the density of the AGCT motif proportionally. (C) A detailed analysis of the highly targeted hotspots in the AGCT dense region of the V-cSμ KI allele in DKO mice. The highly targeted hotspots (orange rectangles) almost exclusively occur at C/G base pairs within the AGCT motifs, reflecting the footprint of AID deamination in this region. A highly targeted hotspot was defined as any nucleotide with >15 substitutions, which ranks roughly in the 73rd percentile for all mutations.