Fanca deficiency reduces A/T transitions in somatic hypermutation and alters class switch recombination junctions in mouse B cells

Abstract

Fanconi anemia is a rare genetic disorder that can lead to bone marrow failure, congenital abnormalities, and increased risk for leukemia and cancer. Cells with loss-of-function mutations in the FANC pathway are characterized by chromosome fragility, altered mutability, and abnormal regulation of the nonhomologous end-joining (NHEJ) pathway. Somatic hypermutation (SHM) and immunoglobulin (Ig) class switch recombination (CSR) enable B cells to produce high-affinity antibodies of various isotypes. Both processes are initiated after the generation of dG:dU mismatches by activation-induced cytidine deaminase. Whereas SHM involves an error-prone repair process that introduces novel point mutations into the Ig gene, the mismatches generated during CSR are processed to create double-stranded breaks (DSBs) in DNA, which are then repaired by the NHEJ pathway. As several lines of evidence suggest a possible role for the FANC pathway in SHM and CSR, we analyzed both processes in B cells derived from Fanca(-/-) mice. Here we show that Fanca is required for the induction of transition mutations at A/T residues during SHM and that despite globally normal CSR function in splenic B cells, Fanca is required during CSR to stabilize duplexes between pairs of short microhomology regions, thereby impeding short-range recombination downstream of DSB formation.

Figure 1.

Reduced A/T transitions during SHM in Fanca^−/− mice. (A) Distribution of mutations in the J_H4 intronic region (506 bp) that was amplified from Peyer’s patch PNA^high B cells isolated from WT and Fanca^−/− mice. (B) Proportion of sequences with numbers of mutations per clone (the central circle shows the total number of analyzed sequences) from WT (n = 5) and Fanca^−/− mice (n = 5). (C) The spectrum of base substitutions is expressed as a percentage of the total number of mutations (left), and the frequency of mutation (right) was corrected for base composition. Gray boxes denote a significant decrease in mutation frequency compared with WT (the χ² test was applied according to the SHMTool algorithm; *, P < 0.05; **, P < 10⁻³; ***, P < 10⁻⁴). Data for the SHM are from five independent experiments. (D) Differential expression of Polη in FANCA- and FANCG-deficient cells. Whole cell extracts were prepared from the indicated human lymphoblast cell lines and analyzed by immunoblotting for the expression of Polη, MSH2, FANCA, FANCG, and Vinculin (asterisks indicate nonspecific bands). Representative data from two independent experiments are shown.

Figure 2.

Robust CSR in Fanca^−/− mice. (A) Serum from Fanca^−/− (n = 5) and WT mice (n = 7) was collected and analyzed by ELISA for the indicated IgM, IgG subclasses, and IgA. The results are displayed as mean ± SEM values for the Fanca^−/− titer as a percentage of WT. Data are representative of two independent experiments with at least five mice per group. (B) Splenic B cells were isolated from Fanca^−/− and WT mice and stimulated in vitro with IL-4 and anti-CD40 or LPS. The expression of IgG1, IgG3, and IgG2b was determined 4 d later by flow cytometry. Bar graphs show the mean percentages of cells expressing the indicated IgG ± SEM. Data are representative of three independent experiments with at least three mice per group. (C) Splenic B cells were isolated from Fanca^−/− and WT mice and stimulated in vitro with IL-4 and anti-CD40. Surface IgG1 expression was determined by flow cytometry on days 2, 3, 4, and 5 after stimulation. Bar graphs show the mean percentages of IgG1⁺ cells ± SEM. Data are representative of five independent experiments with at least five mice per group.

Figure 3.

Altered CSR junctions in Fanca^−/− mice. (A) Splenic B cells from WT (n = 6) and Fanca^−/− (n = 6) mice were stimulated with anti-CD40 and IL-4, and 4 d later, Sμ–Sγ1 junctions were amplified by PCR from genomic DNA and then sequenced. Percentage of sequences with blunt joins (0), indicated MH, and junctional insertions (Ins) are shown. Data from six independent experiments were pooled and analyzed by the χ² test (**, P < 10⁻³). (B) A plot representing the relative frequency of sequences with blunt joins, MH, or insertions for WT and Fanca^−/− B cells from A. (C) A schematic representation of the I-SceI chromosomal reporter. (D) The GCV6 cells containing the I-SceI substrate described in C were transfected with siCT or siFANCA for 48 h and subsequently with I-SceI–expressing plasmid. 3 d later, I-SceI junctions were amplified from genomic DNA and then sequenced. Percentage of sequences with blunt joins (0), indicated MH, or insertions at the I-SceI–induced NHEJ junction are reported. Analyzed sequences were obtained from two independent experiments (*, P < 10⁻²).

Figure 4.

Increased ISR in Fanca^−/− mice. (A) A model for the joining of AID-generated DSBs between S regions during switch to IgG1. AID generates multiple DSBs within a given S region, and CSR joins two DSBs from two different S regions, producing canonical Sµ-Sγ1 junctions. The intervening double-stranded DNA fragments are then degraded or joined to form excision circles; occasionally some rejoined sequences exhibiting unusual junctions (e.g., Sµ-Sµ-Sγ1 or Sµ-Sγ1-Sγ1) resulting from local rejoining or ISR are observed. (B) Frequency of ISR in WT and Fanca^−/− B cells induced to switch to IgG1 by anti-CD40 and IL-4 for 4 d. ISR was examined by analysis of Sμ-Sγ1 junctions. Horizontal bars indicate the means (*, P < 0.05 with the two-tailed Student’s t test). (C) Bar graph shows the percentage of Sμ and Sγ1 ISR in WT and Fanca^−/− B cells from B. (D) Percentage of sequences exhibiting blunt joins (0), indicated MH, or insertions at the ISR junction. Data for the ISR analysis are based on five mice per genotype from five independent experiments.