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

Nguyen TV, Riou L, Aoufouchi S, Rosselli F - J. Exp. Med. (2014)

Bottom Line: 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.

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Affiliation: Centre National de la Recherche Scientifique UMR 8200, Institut Gustave Roussy, 94805 Villejuif, France Université Paris Sud, 91400 Orsay, France Programme Equipe Labellisées, Ligue Contre le Cancer, 75013 Paris, France.

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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.
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fig2: 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.

Mentions: To determine whether loss of Fanca affects Ig class switching, we determined the levels of IgM, IgG subclasses, and IgA in the serum from Fanca−/− and WT mice at 8–12 wk of age. All Ig isotype titers were similar between WT and Fanca−/− mice, indicating that Fanca−/− B cells are proficient for Ig secretion (Fig. 2 A). We next examined the intrinsic ability of Fanca−/− B cells to undergo CSR in vitro. Splenic B cells from Fanca−/− and WT mice were stimulated with either IL-4 plus αCD40 to induce switching from IgM to IgG1 or with LPS to induce switching to IgG3 and IgG2b. Stimulated B cells were cultured for 4 d and analyzed by flow cytometry for surface expression of IgG1, IgG3, and IgG2b. Fanca−/− B cells were able to switch to all isotypes examined (Fig. 2 B). We also examined the kinetics of in vitro switching to IgG1 in WT and Fanca−/− purified splenic B cells stimulated with αCD40 plus IL-4 for 2–5 d, and we observed that Fanca−/− and WT B cells switched to IgG1 at similar frequencies (Fig. 2 C). Building on previous observations made in Fancg−/− mice (Krijger et al., 2010), these data indicate that loss of the FANC pathway does not impair secondary Ig diversification and secretion.


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

Nguyen TV, Riou L, Aoufouchi S, Rosselli F - J. Exp. Med. (2014)

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.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4042646&req=5

fig2: 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.
Mentions: To determine whether loss of Fanca affects Ig class switching, we determined the levels of IgM, IgG subclasses, and IgA in the serum from Fanca−/− and WT mice at 8–12 wk of age. All Ig isotype titers were similar between WT and Fanca−/− mice, indicating that Fanca−/− B cells are proficient for Ig secretion (Fig. 2 A). We next examined the intrinsic ability of Fanca−/− B cells to undergo CSR in vitro. Splenic B cells from Fanca−/− and WT mice were stimulated with either IL-4 plus αCD40 to induce switching from IgM to IgG1 or with LPS to induce switching to IgG3 and IgG2b. Stimulated B cells were cultured for 4 d and analyzed by flow cytometry for surface expression of IgG1, IgG3, and IgG2b. Fanca−/− B cells were able to switch to all isotypes examined (Fig. 2 B). We also examined the kinetics of in vitro switching to IgG1 in WT and Fanca−/− purified splenic B cells stimulated with αCD40 plus IL-4 for 2–5 d, and we observed that Fanca−/− and WT B cells switched to IgG1 at similar frequencies (Fig. 2 C). Building on previous observations made in Fancg−/− mice (Krijger et al., 2010), these data indicate that loss of the FANC pathway does not impair secondary Ig diversification and secretion.

Bottom Line: 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Centre National de la Recherche Scientifique UMR 8200, Institut Gustave Roussy, 94805 Villejuif, France Université Paris Sud, 91400 Orsay, France Programme Equipe Labellisées, Ligue Contre le Cancer, 75013 Paris, France.

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Related in: MedlinePlus