Activation-induced cytidine deaminase (AID) and proteins involved in nonhomologous end joining are critical for CSR activity. AID deamination of deoxycytidine residues appears to initiate events that result in S region DNA cleavages (6, 7), whereas nonhomologous end joining proteins may be involved in the religation of broken S region DNA ends (8–10). CSR also involves the uracil DNA glycosylase protein, possibly for the generation of abasic sites from AID-generated dU–dG DNA mismatches (11). DNA mismatch repair (MMR) proteins are also important for CSR (12, 13), but their roles are unclear. Studies of S sites located within a portion of Sµ showed that the absence of the Msh2 MMR protein caused CSR to focus on GAGCT/GGGGT sequences (12). Furthermore, the presence of the SµTR region is critical for class switching in Msh2-deficient mice, suggesting that Msh2 affects the sequences that are targeted or selected for CSR (14). Msh2 is also important for the low levels of CSR found in uracil DNA glycosylase–deficient mice, suggesting an Msh2 role in some DNA cleavages that initiate CSR (15).

We have analyzed the locations of S sites in two mutant mouse strains that lack either the SµTR region or the Msh2 protein to determine how these mutations affect CSR targeting. S site locations in wild-type and SµTR^–/– mice show that CSR events occur within a 4–5-kb DNA region located downstream of the Iµ promoter and provide the first indication that CSR is targeted to a fixed-length "domain" that is linked to the location of a transcriptional promoter. Comparing S site locations in wild-type with Msh2^–/– mice shows that the absence of Msh2 focuses CSR sites on the SµTR region. This finding indicates that Msh2 has a role in targeting or selecting specific sequences for CSR within the accessible DNA domain.

	Results and Discussion

Top Abstract Results and Discussion Materials and Methods References

 
Upstream S sites are not affected by SµTR deletion
We used several digestion-circularization (DC)-PCR assays to analyze CSR sites in stimulated splenic B cells. To quantify Sµ/S1 CSR sites located upstream of the SµTR region in wild-type and SµTR^–/– mice, a BsrGI digestion was added to the standard EcoRI DC-PCR assay (16) before DNA circularization (Fig. 1 a). Only Sµ/S1 junctions upstream of the Sµ BsrGI site will lack the BsrGI site and generate EcoRI fragments that can be self-ligated and PCR amplified. Comparing band intensities in EcoRI DC-PCR with those in EcoRI–BsrGI DC-PCR assays allows the percentages of junctions located upstream or downstream of the Sµ BsrGI site to be calculated.

View larger version (33K): [in this window] [in a new window]   Figure 1. SµTR–/– and wild-type mice have similar numbers of upstream CSR events. (a) DC-PCR measures CSR events upstream of the SµTR region. A BsrGI site distinguished CSR junctions located upstream and downstream of the site. Only CSR sites in sequences upstream of BsrGI generate PCR signals. Total CSR events are measured by EcoRI DC-PCR (E, EcoRI; BGI, BsrGI). PCR primers are indicated by arrowheads. (b) Twofold dilutions of genomic DNAs were assayed by Sµ/S1 EcoRI DC-PCR and EcoRI–BsrGI DC-PCR. Samples were normalized using nAChR DC-PCR reactions. (c) CSR events in wild-type or SµTR–/– mice as percentages of total wild-type CSR events (set to 100%). Mean values and SEM are from six independent experiments with two mice per group.

Switch recombinations in SµTR^–/– mice shift to downstream JH-Cµ regions
To further locate S sites within the JH-Cµ intron, downstream CSR events measured in HindIII and XbaI DC-PCR assays were compared with the total number of switch events measured by the EcoRI DC-PCR assay (Fig. 2, a and b). Because the three DC-PCRs can have different efficiencies, we used a control plasmid (pSµ/S3) that contained Sµ and S3 sequences together with the three enzyme sites to normalize the DC-PCR assays. The frequencies of CSR sites within different segments of the JH-Cµ region were determined by comparison with standard curves generated using pSµ/S3.

View larger version (16K): [in this window] [in a new window]   Figure 2. SµTR–/– mice have increased levels of CSR events targeted to downstream sequences. (a) DC-PCR measuring CSR junction sites downstream of the SµTR region. HindIII and XbaI sites downstream of the SµTR region were used in DC-PCR assays to measure CSR junctions in sequences downstream of each site. Total CSR sites were determined by EcoRI DC-PCR (E, EcoRI; H1 and H2, HindIII; X, XbaI). Primer pairs used for the different DC-PCR assays are indicated by small arrows of different types. (b) Wild-type and SµTR–/– CSR events measured by EcoRI, HindIII, and XbaI real-time DC-PCR. Copy numbers for CSR events were derived from pSµ/S3 standard curves. Rag-1 PCR normalized CSR events relative to Rag-1 copy number. Mean values and SEM are from seven independent experiments with four mice per group. (c) Percentage of SµTR–/– switch events downstream of the H2 relative to events downstream of H1 (set to 100%). Mean values and SEM are from three independent experiments per group.

Of the Sµ/S3 recombinations in SµTR^–/– mice located downstream of the H1 site, about half occurred downstream of the XbaI site located near the 5' end of the Cµ exons (Fig. 2 b). Comparisons of EcoRI DC-PCR with XbaI DC-PCR assays indicated that 30% of Sµ/S3 recombinations in SµTR^–/– mice occurred at sequences downstream of the XbaI site.

For wild-type and SµTR^–/– mice, we compared the locations of CSR sites determined by DC-PCR with the locations of CSR events in hybridoma panels (3). For wild-type mice, 23 hybridomas show five (22%) CSR sites upstream of the SµTR, 17 (78%) sites within the SµTR, and no sites downstream of the SµTR. DC-PCR analyses of wild-type CSR events show 17% upstream, 82% SµTR, and 1% downstream. For CSR sites in SµTR^–/– mice, 10 hybridomas show three (30%) upstream of the H1 site and seven (70%) downstream of H1, whereas DC-PCR shows 33% upstream and 67% downstream. Thus, although the hybridoma panels are small and will miss CSR events that result in nonfunctional Ig proteins, the comparisons show a good correlation between the distributions of CSR sites as determined by the two methods.

The CSR site frequency between the HindIII and XbaI sites might be slightly underestimated because of the H2 HindIII site in the Cµ segment (Fig. 2 a). For SµTR^–/– mice, CSR events occurring downstream of H2 were estimated using the HindIII-circularized B cell DNA samples. Among SµTR^–/– mice with CSR sites located downstream of H1, only 7% were located downstream of H2 (Fig. 2 c), assuming that both HindIII DC-PCR assays have equal efficiencies. An examination of the threshold cycles of the two HindIII PCR assays indicated that the efficiencies differ by less than a factor of two (not depicted). Thus, although the majority of switch recombinations in SµTR^–/– mice are skewed toward downstream JH-Cµ intron sequences, there are few junction sites downstream of the Cµ exons.

Lack of the MMR Msh2 protein shifts the targeting of CSR to focus on the SµTR region
The role of Msh2 in the CSR process is unclear. Previous analyses of SµTR^–/–:Msh2^–/– mice have shown that the SµTR region is critical for isotype switching when Msh2 is lacking (14). These studies suggested that Msh2 might be more important for switching in the sequences flanking the SµTR region than within the SµTR itself. To directly test this model, S site locations were determined in Msh2^–/– mice that retain the SµTR region.

CSR sites throughout the JH-Cµ region were analyzed by DC-PCR in Msh2^–/– mice. CSR sites upstream of the SµTR in Msh2^–/– mice were decreased about sixfold relative to the wild type, whereas switching within the SµTR was only reduced by a factor of 1.4 (Fig. 3, a and b). The fraction of CSR events located upstream in Msh2^–/– mice was significantly different from the wild type (two-sided P < 0.0001 by ² test; Fig. 3 b). Very few CSR events occurred downstream of the SµTR in both Msh2^–/– and wild-type mice (Fig. 3 C), and we cannot assess whether any specific reductions occur. Within the regions that exhibited the majority of CSR activity, however, our results show a shift of CSR site focus into the SµTR region in Msh2^–/– mice (Fig. 4) and support the model that Msh2 is more important for CSR in non-SµTR sequences and less important for CSR within the SµTR.

View larger version (16K): [in this window] [in a new window]   Figure 3. CSR within sequences upstream of the SµTR are reduced in Msh2–/– mice. (a) Twofold dilutions of genomic DNAs were assayed by Sµ/S1 EcoRI DC-PCR and EcoRI–BsrGI DC-PCR. Samples were normalized using nAChR DC-PCR. (b) CSR sites in wild-type or Msh2–/– mice measured relative to total wild-type CSR (set to 100%). Mean values and SEM of at least four independent experiments are presented. (c) CSR events in Msh2–/– mice measured by EcoRI, HindIII, and XbaI real-time DC-PCR and compared with wild-type mice. Mean values and SEM are from six independent experiments with two Msh2–/– mice and seven independent experiments with four wild-type mice.

View larger version (20K): [in this window] [in a new window]   Figure 4. CSR junction sites and AID target sites within different segments of the JH-Cµ intron. Histograms of CSR site, WRC motif, and WGCW motif frequencies are shown for different segments of the JH-Cµ region. (a) Comparisons of wild-type with Msh2–/– mice that both have wild-type Sµ regions. (b) Comparisons of SµTR–/– mice with a shortened Sµ region. DNA segments are drawn in proportion on the x axis. Areas of boxes in the histogram are proportional to frequencies in each segment, resulting in box heights that indicate relative recombinational frequencies per nucleotide. Boxes for mutant mice are drawn to scale relative to wild-type mice. Switching reductions in SµTR–/– varied slightly in different DC-PCR assays and were averaged. Junction site distributions in each segment were assumed to be uniform. Wild-type CSR frequencies are set to 100%; other CSR frequencies are normalized to this total. Total WRC and WGCW motif frequencies were set to 100% for each mouse strain. The SµTR is only partially sequenced (available from GenBank/EMBL/DDBJ under accession no. AC073553). Densities of motifs are assumed conserved throughout the SµTR. The total numbers of each motif in the Iµ–Cµ region are indicated in the top right of each graph.

View larger version (15K): [in this window] [in a new window]   Figure 5. Model depicting the targeting domain for CSR in the JH-Cµ region for wild-type and SµTR–/– mice. The JH-Cµ intron region is shown with small circles cartooning WRC motifs (potential AID cleavage sites) in the JH-Cµ region. Open ovals represent domains accessible to CSR cleavage; regions not accessible to CSR are indicated by shaded ovals.

The CSR-accessible DNA domain could also involve a chromatin structure that regulates the switching machinery, similar to the ability of chromatin structure to regulate V(D)J recombination (20, 21). Studies of B cells undergoing somatic hypermutation indicate that mutating V(D)J segments are found in chromatin where the histones H3 and H4 are hyperacetylated relative to the C-region of the same gene (22). For switching, however, relationships between CSR and S region H3/H4 hyperacetylation are complex, suggesting that other modifications or protein factors might be needed for CSR targeting (23). In pro–B cells, dimethylation of histone 3 at lysine 4 in the JH-Cµ region peaks around Cµ with a rapid drop off within 2 kb of this central peak (21). Thus, the dimethylation of histone 3 at lysine 4 modification rises rapidly in the region just downstream of the SµTR that forms the 3' boundary of the switching domain and could be involved in limiting CSR accessibility.

In wild-type mice, CSR sites correlate with the distribution of WRC motifs (Fig. 4 a) that are known sites for AID deamination on single-stranded DNAs (24, 25). In SµTR^–/– mice, however, CSR sites do not correlate with WRC motifs (Fig. 4 b). The HindIII–XbaI focus of SµTR^–/– CSR sites could reflect the high concentration of nontranscribed G residues in this region (3). Such G-rich sequences promote RNA–DNA R-loops in S regions and might provide single-stranded DNA targets for AID activity (5, 26).

Mice with a larger Sµ-region deletion show more drastic CSR reductions and have Sµ sites mostly constrained to the Iµ exon region (4). Sequence elements important for the 4–5-kb CSR domain proposed in Fig. 5 may be disrupted by the larger Sµ deletion. Identifying sequences that establish the CSR domain might distinguish transcriptional and chromatin structure targeting models and lead to the identification of targeting factors. S regions clearly contain sequences important for CSR because replacing S region sequences with a random DNA sequence does not provide for CSR (27).

Msh2 may be important for processing "frayed" switch DNA cleavages
Our results show that S sites in Msh2^–/– mice are strongly focused into the SµTR region, indicating that CSR events within SµTR sequences are much less dependent on Msh2 than are events within non-SµTR sequences. The Msh2 activity that leads to this sequence-specific effect is not clear. Msh2 is known to recognize base pair mismatches, and recent studies suggest that Msh2 recognition of G–U might lead to S region DNA breaks (15). However, Msh2 G–U recognition appears to play a minor role in switch cleavage (15), and there is no apparent reason for this G–U recognition activity to affect switching outside of SµTR sequences to a greater extent than within the SµTR.

The shifts of CSR sites in Msh2^–/– mice are consistent with a model that we have previously proposed in which Msh2 processes DNA "flaps" found predominantly on cleavage ends generated from sequences outside of the SµTR (14). This model suggests a postcleavage end-processing function for Msh2 that converts staggered single-stranded breaks to the double-stranded breaks required for CSR, in addition to its role in initiating some switch cleavages.

We analyzed the JH-Cµ sequence to determine whether motifs associated with CSR might account for the S site distribution in Msh2^–/– mice. WRC motifs are preferred sites for AID (24, 25), and the WGCW motif is a site where AID activity on opposing strands could provide a double-stranded cleavage with a single base overhang (24). WRC motifs within the JH-Cµ intron correlate well with distributions of CSR sites in the 4–5-kb CSR domain for wild-type mice (Fig. 4 a). For Msh2^–/– mice, on the other hand, CSR sites correlate better with WGCW motifs (Fig. 4 a). CSR downstream of the SµTR is rare in both wild-type and Msh2^–/– mice, so that Msh2 effects are not discernable. However, SµTR^–/– mice show numerous S sites in this downstream region and previous analyses have shown that SµTR^–/–:Msh2^–/– mice show large decreases in all CSR events (14). Thus, Msh2 is also important when CSR occurs downstream of the SµTR, consistent with the paucity of WGCW sites within these sequences (Fig. 4 a). CSR sites in SµTR^–/–:Msh2^–/– mice also show a significant focus on WGCW motifs (two-tailed P = 0.0043 by Fisher's exact test) relative to CSR sites in SµTR^–/– mice (14), emphasizing the correlation between WGCW motifs and CSR sites when Msh2 is not present. Furthermore, the number of WGCW motifs in the wild-type Sµ region is about half the number of WRC motifs in the same region (Fig. 4 a). Perhaps the twofold reduction in CSR observed between wild-type and Msh2^–/– mice correlates roughly with the fraction of cleavage sites that cannot complete CSR because of frayed ends.

The involvement of Msh2 in processing S region breaks would suggest that Msh2 alters switch targeting by affecting the selection of switch breaks that can undergo CSR. Thus, Msh2 has an important role to broaden the types of DNA sequences that can successfully participate in CSR. Analyses of SµTR^–/– mice crossed with mice deficient in other MMR proteins, such as ExoI, Mlh1, and Pms2, should indicate whether these proteins are involved in the same processes of the CSR mechanism that are affected by Msh2.

	Materials and Methods

Top Abstract Results and Discussion Materials and Methods References

 
Mice and cell culture
SµTR^–/– and Msh2^–/– mice were used in these studies. Animal experiments were approved by Tufts University Institutional Animal Care and Use Committee. Splenic B cells were cultured with 50 µg/ml LPS (Sigma-Aldrich) with or without 0.3 ng/ml anti–-dextran, or with LPS and IL-4 (800 U/ml) as described previously (14).

DC-PCR
DC-PCR was performed as previously described (3, 14). Circularized DNAs were diluted twofold, and PCR was amplified as follows. For switch events located upstream of the SµTR, DNAs were digested with BsrGI and EcoRI before circularization. Sµ/S1 DC-PCR products were amplified and phosphorimager quantification was normalized using the nicotinic acetylcholine receptor (nAChR) gene (16). BsrGI digestion was monitored by PCR of the BsrGI site using primers Sµ_out (14) and BsrGI_bk (5'-TCATTACTGTGGCTGGAGAG-3'). Switching upstream was quantitated by comparing EcoRI–BsrGI DC-PCR with EcoRI DC-PCR products. For switch events located downstream of the SµTR, genomic DNAs were digested with EcoRI, HindIII, or XbaI, and circularized for real-time PCR using fluorescent SYBR green dye (Applied Biosystems). The pSµ/S3 control plasmid provided standard curves for each real-time DC-PCR. Rag-1 copy numbers were measured by real-time PCR using Rag-1 plasmid standards (Rag-1 plasmid provided by E. Marchlik, Tufts University, Boston, MA). Gel electrophoresis of PCR products and melting temperature analyses were used to confirm the specificity of real-time PCR amplifications. Total Sµ/S3 junctions were measured in an EcoRI DC-PCR assay using published primer pairs DC-µ.2 and DC-3.2 (28).

Switch events downstream of the H1 site (Fig. 2) were measured by HindIII DC-PCR using primers H3-IM (5'-CCTACACCAGATCATCCAGTACAGCT-3') and H3-G3F (5'-GATAGGACAGATGGAGCAGTTACAGA-3'). XbaI DC-PCR measured switch events downstream of the XbaI site located 5' of the Cµ exon (Fig. 2 a) using primer pairs XBA-IM (5'-GAACGTGACTTTGGAAGCCTTCA-3') and X1-G3F (5'-GCTGAGAGGAGACCTGGGTTG-3'). HindIII DC-PCR measured S junctions downstream of the H2 site (Fig. 2 c) using primers CMH3-R (5'-GTGGAGGGACCTGCAGTCAAG-3') and H3-G3F.

Statistics
² tests were used to assess the significance of Msh2^–/– upstream CSR differences. Fisher's exact test was used to assess the significance of differences in CSR at WGCW motifs between SµTR^–/–:Msh2^–/– and SµTR^–/– mice.