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Comparative Study
. 2004 Nov 1;200(9):1103-10.
doi: 10.1084/jem.20041162.

ATM is required for efficient recombination between immunoglobulin switch regions

Affiliations
Comparative Study

ATM is required for efficient recombination between immunoglobulin switch regions

Bernardo Reina-San-Martin et al. J Exp Med. .

Abstract

Ataxia telangiectasia mutated (ATM) kinase is critical for initiating the signaling pathways that lead to cell cycle checkpoints and DNA double strand break repair. In the absence of ATM, humans and mice show a primary immunodeficiency that includes low serum antibody titers, but the role of ATM in antigen-driven immunoglobulin gene diversification has not been defined. Here, we show that although ATM is dispensable for somatic hypermutation, it is required for efficient class switch recombination (CSR). The defect in CSR is not due to alterations in switch region transcription, accessibility, DNA damage checkpoint protein recruitment, or short-range intra-switch region recombination. Only long-range inter-switch recombination is defective, indicating an unexpected role for ATM in switch region synapsis during CSR.

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Figures

Figure 1.
Figure 1.
ATM is required for efficient CSR, but not for SHM. Flow cytometry analysis of WT and ATM−/− B cells stimulated with LPS plus IL-4 for 4 d (A) or LPS alone (B and C) for 5 d. Cell division as measured by CFSE dye dilution is shown on the top. The percentage of cells expressing (A) IgG1, (B) IgG2b, and (C) IgG3 after a specific number of cell divisions is indicated on the bottom. Cells were stained with Topro-3 before acquisition and analysis was performed on live B cells (Topro-3). (D) Mutation analysis in the JH4 intron (reference 46) of WT and ATM−/− germinal center B cells (B220+ Fas+ GL-7+) obtained from the lymph nodes of immunized mice. Segment sizes in the pie charts are proportional to the number of sequences carrying the number of mutations indicated in the periphery of the charts. The frequency of mutations per basepair sequenced and the total number of independent sequences analyzed is indicated underneath and in the center of each chart, respectively. Statistical significance was determined by a two-tailed t test assuming unequal variance and comparing to WT (P = 0.914). Percent nucleotide substitutions adjusted for base composition is shown to the right of each pie chart. Percentage of mutations within hotspot motifs (references –83) is indicated underneath each panel. The total number of mutations analyzed was as follows: WT, 88 mutations/28,120 bp; ATM−/−, 63 mutations/20,920 bp.
Figure 2.
Figure 2.
Switch region accessibility in the absence of ATM. (A) Real-time RT-PCR for μ sterile transcript (μ ST), γ1 sterile transcript (γ1 ST), and post-switch γ1 circle transcript (γ1 CT) in WT (closed bars) and ATM−/− (open bars) B cells stimulated with LPS and IL-4 for 3 d. Mean results from four independent cultures are expressed as fold induction relative to WT. (B) Mutations in Sμ determined in WT and ATM−/− B cells sorted for five cell divisions and expressing IgM. The number of mutations was as follows: WT, 31 mutations/83,773 bp; ATM−/−, 14 mutations/55,069 bp. Pie charts are as in Fig. 1. Statistical significance was determined by a two-tailed t test assuming unequal variance and comparing to background (resting B cells from WT mice) or WT. P-values are indicated below each pie chart.
Figure 3.
Figure 3.
CSR junctions in the absence of ATM. (A) Histogram depicting the percentage of sequences with the indicated length of microhomologies at Sμ/Sγ1 junctions in WT (closed bars) and ATM−/− (open bars) B cells. Overlap was determined by identifying the longest region at the switch junction of perfect uninterrupted donor/acceptor identity. (B) Mutations in the vicinity of the junctions obtained from WT and ATM−/− B cells. Pie charts are as in Fig. 1. Statistical significance was determined by a two-tailed t test assuming unequal variance and comparing to WT. P-values are indicated below each pie chart.
Figure 4.
Figure 4.
Intra-switch region recombination in the absence of ATM. Southern blot analysis of (A) Sμ and (B) Sγ1 regions in IgM-secreting hybridomas derived from ATM−/− B cells. Restriction enzymes and probes used are indicated in the top panels. Δ, deletions. Control digests performed on tail DNA (ATM−/−) and the SP2/0Ag-14 (SP2) fusion partner were loaded on lanes 1 and 2. The SP2 cell line has a deletion in Cμ and no hybridization is observed. The same deletions in Sμ were found using an Eμ probe (not depicted). Number of deletions over hybridomas screened is indicated below each panel. Molecular weight markers in kilobase pairs are indicated on the left side of each panel.
Figure 5.
Figure 5.
Activation of 53BP1 in response to CSR-associated DSBs. Distribution of 53BP1 in activated B cells from WT, ATM−/−, Ku80−/−, and H2AX−/− mice. B cells were stained with anti-53BP1 antibodies followed by DNA FISH detection of the CH region. Fluorescent images represent a single optical section.

References

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