It was observed several years ago that mutations are often found close to the sites of switch recombination, and some of these mutations could be the consequence of the resolution of nucleotide mismatches arising in heteroduplexes formed during the switching process (19
). However, it has more recently become clear that mutations can also be detected in Sμ far upstream of the site of switch recombination, that such “preswitch” mutations are AID dependent and can occur even on IgH alleles that have not undergone evident switching recombination (21
). The distribution of those AID-dependent mutations that are not generated as a consequence of the switch recombination itself could give insight into the nature of AID targeting.
Although mutations in the preswitch region have largely been described in immortalized B cell lines or in B cells that have been activated with LPS in vitro, they can also be detected in germinal center B cells sorted from mouse Peyer's patches (25
). Although the mutation loads in the preswitch region are often quite light, a considerably higher mutation load accumulates in germinal center B cells obtained from msh2−/− ung−/−
mice (). The absence of both uracil-DNA glycosylase (UNG) and MSH2 in these animals means that the AID-generated U:G lesions are not recognized for processing into switch recombination and are also probably less likely to be repaired (25
). Instead, the cells seem simply to replicate across the U:G lesions and, consequently, accumulate large numbers of C→T and G→A transition mutations upstream of Sμ. The msh2−/− ung−/−
mice can therefore be used to obtain large databases of switch-associated mutations with no contribution to the database arising from mutations generated as a result of the switch recombination itself. Furthermore, in contrast to what is observed in normal mice, there is no mutagenic patch repair of the U:G lesion in msh2−/− ung−/−
mice, and all the observed mutations are likely to directly mark the sites of AID-catalyzed deamination events. Thus, the observed mutations are entirely restricted to transitions at C:G pairs with no substitutions accumulating at A:T pairs () (25
). In fact, this restriction to transitions at C:G pairs means that it is possible to check that individual databases are relatively free from contamination by PCR error, a considerable concern in lightly mutated databases, especially with highly repetitive target sequences.
Figure 1. Distribution of Sμ-associated mutations. (A) Comparison of switch-associated mutations in wild-type and msh2−/− ung−/− mice. Mutations in Sμ-associated regions m.1 and m.2 and in Sα-associated region (more ...)
The accumulation of mutations across a region extending from JH4 through to Cμ4 was analyzed in germinal center Peyer's patch B cells obtained from a pair of msh2−/− ung−/− mice. Primers were designed so as to amplify the whole region in sections of ~1,500 base pairs. The results are shown in , excluding the highly repetitive part of Sμ, because we were unable to amplify this region using the same PCR strategy as was used for the rest of the region.
The two mice showed similar distributions of mutations. As expected for mutations attributable to error-free replication across the U:G lesion, >99% of the 1,399 mutations identified in the 130 Sμ sequences analyzed were transitions at C:G pairs (). Consistent with the target site preference of AID, if all these mutations are computed as C deaminations, 69% of the mutations are found to fall within a WRC consensus. This is comparable with the 24.3% that would be anticipated if the mutations were randomly targeted to different C residues within the target (a calculation that takes account of the sequence of the Sμ target that is being analyzed).
Mutations in the vicinity of the IgH S regions
The Sμ mutation domain begins ~150 nt downstream of the start sites of the Sμ germline transcripts and extends over 5 kb, tapering at a low level into Cμ exons. Interestingly, the Eμ enhancer is largely spared of mutations, apparently being located just downstream of the VHDJH mutation domain and upstream of the Sμ mutation domain ().
To identify possible target sites of AID-catalyzed deamination in the acceptor S regions, we amplified and sequenced regions in and around the repetitive regions of the Sγs, S
, and Sα from the same Peyer's patch germinal center B cell DNA samples that had been used for analysis of Sμ-associated mutations ( and ). Scarcely any mutations were observed associated with S
, which might possibly reflect little or no switching to IgE in Peyer's patches. However, with all four Sγs, the results were similar to those obtained with Sμ, although the mutation loads were somewhat lower. Thus, mutations were observed both up- and downstream of the repetitive portions of the Sγs with the major mutation domains appearing to begin just downstream of the Iγ start sites. The mutations were all transitions at C:G pairs, focused on a WRC consensus (where W = A/T and R = A/G). With regard to Sα, although mutations were readily detected in the 3′ flanking region, we detected no mutations in the vicinity of the Iα exon. We do not know the reason for this, but one possibility is that there could be alternative start sites for the Sα transcripts in Peyer's patch germinal centers.
Figure 2. Distribution of Sγ-, -, and α-associated mutations. The distribution of transition mutations at C:G pairs around Sγ, , and α in the Peyer's patch germinal center B cells of msh2−/− ung (more ...)
Several immunoglobulin S regions have been shown to be able to adopt R loop conformations during transcription (27
), which could expose a single-stranded DNA substrate for AID action. We were therefore interested in ascertaining whether AID displayed any preference for deaminating the DNA strand that is not used as the template for transcription (the nontemplate strand) at the S regions in vivo. This, presumably, could be detected as a strand asymmetry in the pattern of the C→T transitions. No such asymmetry is evident from the m.1, m.2, or m.3 regions ( and ). Although most of these sequences derive from regions that flank (rather than lie within) the highly repetitive portions of the S regions, no evidence of preferential deamination of the nontemplate DNA strand is observed even if attention is focused on sequences derived from parts of Sγ3 or Sγ2b that have been shown to be able to form R loops in vivo ().
Figure 3. Absence of major strand polarity in Sμ-associated mutations. (A) Transition mutations at C:G pairs in regions m.1to m.4 were categorized as mutations at C on either the transcription template or nontemplate strand (TS or NTS, respectively) and (more ...)
We extended the analysis to the repetitive portion of Sμ. Although we had failed to amplify this highly repetitive DNA in a single-stage PCR reaction using a variety of different primers, we did achieve success with a nested PCR approach. The Sμ clones obtained in this way were heavily mutated, forming part of a region that can form R loops in vitro (28
), but certainly showed no evidence in favor of preferential deamination of the nontemplate DNA strand ().
We wished to learn whether the cytokines that stimulate isotype-specific switching also play a role in recruitment of AID-catalyzed deamination. We therefore screened for S region mutations in B cells that had been activated with LPS in vitro in the presence of either IL-4 (to induce switching to IgG1) or IFN-γ (to induce switching to IgG2a). In all cases, mutations accumulated in the Sμ 5′ flank with mutations in Sγ1 being specifically enhanced in the IL-4 cultures and mutations in Sγ2a enhanced in the IFN-γ cultures (). No mutations were detected in Sγ1 or Sγ2a in the absence of the relevant cytokine.
Figure 4. Cytokine induction of switch-associated mutations in LPS-cultured B cells. (A) In vitro class switching of splenic B cells from a wild-type (control) C57BL/6 mouse analyzed by flow cytometry after a 7-d culture with LPS plus either IL-4 or IFN-γ. (more ...)
Analysis of individual mutated S region sequences from LPS-activated B cells from msh2−/− ung−/− mice reveals that several harbor clusters of linked C→T transition mutations on either the top or bottom DNA strand (). The abundance of contiguous same-strand deamination mutations is significantly higher than would be anticipated if each deamination mutation occurred as an independent event; the deviation from random is significant at the 97% level if the pool of sequences in is analyzed using a χ2 test. These clusters do not appear to reflect any strand asymmetry of the DNA target in respect of the location of intrinsic mutational hotspot motifs. Thus, examples can be seen where, in a specific segment of the S region, the top strand has been targeted for deamination in one clone, whereas it is the bottom strand of the same segment that has been targeted in another ().
Figure 5. DNA strand polarity of switch-associated mutations in LPS-cultured B cells. (A) DNA strand polarity of C→T transition mutations in individual S region sequences amplified from splenic B cells from msh2−/− ung−/− (more ...)
The results suggest that, at least occasionally in B cells from msh2−/− ung−/− mice, AID can introduce clustered deaminations on a single DNA strand, possibly through processive action. One might then expect to see clones with clustered deaminations even in sequences obtained from B cells that have been cultured for shorter periods with LPS. Indeed, although the overall mutation load is lower in Sμ m.2 sequences isolated from B cells on day 5 as opposed to day 7 of culture, the abundance of sequences with clustered same-strand deamination mutations appears similar ().
Although same-strand clustering is also discernable among some of the sequences from Peyer's patch B cells that cover the repetitive region of Sμ (), the heavy mutation loads evident in these sequences obtained from in vivo–activated B cells (probably reflecting an increased number of rounds of mutation) may mask the evidence of apparently processive deamination.