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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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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.
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fig4: 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.

Mentions: AID activity generates multiple DSBs within a given S region, and CSR joins the most upstream DSB with the most downstream DSB from two different S regions, which can be separated by as much as 200 kb of DNA (Fig. 4 A). The intervening double-stranded DNA fragments are then degraded or joined to form excision circles. However, some rejoined sequences could be involved in unusual insertions of DNA fragments internal to an S region. This can result from local rejoining or ISR and can lead to the presence of Sμ-Sμ-Sγ1 or Sμ-Sγ1-Sγ1 sequence junctions, as opposed to the canonical Sμ-Sγ1 sequence (Reina-San-Martin et al., 2007). Remarkably, we found that the frequency of ISR was significantly higher in Fanca−/− than in WT B cells (12.6 vs. 3.8%; Fig. 4 B). The levels of Sμ ISR in Fanca−/− B cells (8.1%) were much higher than in WT B cells (1.2%), whereas the levels of Sγ1 ISR in Fanca−/− B cells were twofold greater than in WT B cells (4.7 vs. 2.5%; Fig. 4 C). These findings suggest that ISR at the Sμ region may occur more frequently than at the Sγ1 region, consistent with previously published data showing that Sμ appears to be intrinsically preferred by AID compared with other downstream S regions during CSR (Alt et al., 2013). Considering the small number of ISR junctions we obtained as well as the fact that the levels of Sμ ISR appeared to be higher than those for Sγ1 in WT mice, we cannot rule out the possibility that Fanca specifically inhibits ISR at Sμ to a greater extent than at downstream S regions. Interestingly, we also observed increased insertions of long sequences (>10 bp) at junctions from siFANCA-transfected cells bearing an intrachromosomal NHEJ reporter system (Fig. 3 D). Strikingly, with the exception of one junction that showed a large insertion from another region of the construct, these long inserted sequences originated from the 50-bp I-SceI–excised sequence. Again, this finding is consistent with our observations of ISR during the CSR process in Fanca−/− mice.


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)

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

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fig4: 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.
Mentions: AID activity generates multiple DSBs within a given S region, and CSR joins the most upstream DSB with the most downstream DSB from two different S regions, which can be separated by as much as 200 kb of DNA (Fig. 4 A). The intervening double-stranded DNA fragments are then degraded or joined to form excision circles. However, some rejoined sequences could be involved in unusual insertions of DNA fragments internal to an S region. This can result from local rejoining or ISR and can lead to the presence of Sμ-Sμ-Sγ1 or Sμ-Sγ1-Sγ1 sequence junctions, as opposed to the canonical Sμ-Sγ1 sequence (Reina-San-Martin et al., 2007). Remarkably, we found that the frequency of ISR was significantly higher in Fanca−/− than in WT B cells (12.6 vs. 3.8%; Fig. 4 B). The levels of Sμ ISR in Fanca−/− B cells (8.1%) were much higher than in WT B cells (1.2%), whereas the levels of Sγ1 ISR in Fanca−/− B cells were twofold greater than in WT B cells (4.7 vs. 2.5%; Fig. 4 C). These findings suggest that ISR at the Sμ region may occur more frequently than at the Sγ1 region, consistent with previously published data showing that Sμ appears to be intrinsically preferred by AID compared with other downstream S regions during CSR (Alt et al., 2013). Considering the small number of ISR junctions we obtained as well as the fact that the levels of Sμ ISR appeared to be higher than those for Sγ1 in WT mice, we cannot rule out the possibility that Fanca specifically inhibits ISR at Sμ to a greater extent than at downstream S regions. Interestingly, we also observed increased insertions of long sequences (>10 bp) at junctions from siFANCA-transfected cells bearing an intrachromosomal NHEJ reporter system (Fig. 3 D). Strikingly, with the exception of one junction that showed a large insertion from another region of the construct, these long inserted sequences originated from the 50-bp I-SceI–excised sequence. Again, this finding is consistent with our observations of ISR during the CSR process in Fanca−/− mice.

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.

Show MeSH
Related in: MedlinePlus