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V(D)J recombination process and the Pre-B to immature B-cells transition are altered in Fanca − / − mice

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ABSTRACT

B-lymphocytes in the bone marrow (BM) must generate a functional B-cell receptor and overcome the negative selection induced by reactivity with autoantigens. Two rounds of DNA recombination are required for the production of functional immunoglobulin heavy (Ig-HCs) and light (LCs) chains necessary for the continuation of B-lymphocyte development in the BM. Both rounds depend on the joint action of recombination activating gene-1 (RAG-1) and RAG-2 endonucleases with the DNA non-homologous end-joining pathway. Loss of the FANC gene leads to the chromosome breakage and cancer predisposition syndrome Fanconi anemia. Because the FANC proteins are involved in certain aspects of the recombination process, we sought to determine the impact of the FANC pathway on the Ig diversification process using Fanca−/− mice. In this work we demonstrated that Fanca−/− animals have a mild B-cell differentiation defect characterized by a specific alteration of the IgM− to IgM+ transition of the B220low B-cell population. Pre-B cells from Fanca−/− mice show evidence of impaired kLC rearrangement at the level of the Vk-Jk junction. Furthermore, Fanca−/− mice showed a skewed Vκ gene usage during formation of the LCs Vk-Jk junctions. Therefore, the Fanca protein appears as a yet unidentified factor involved in the primary diversification of Ig.

No MeSH data available.


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Normal heavy chain rearrangement in Fanca−/− mice.(A) Distribution of CDR3 lengths of VHDJH4 rearrangements amplified from genomic DNA that was isolated from BM IgM− B cells of Fanca−/− and WT mice. The average values are listed in Table 1. (B) Average numbers of added N-nucleotides per sequence in VHDJH4 rearrangements of Fanca−/− and WT mice are plotted either for VH to DJH and D to JH junctions (“Total”) or for VH to DJH (“VHD”) alone or D to JH (“DJH”) junctions alone. The results are displayed as the mean ± SEM. (C) The proportion of P-nucleotide additions at either the VH to DJH4 (“VHD”) or the D to JH4 (“DJH”) junctions. The data for HC rearrangements are from three independent pools of three mice per genotype (the numbers of sequences analysed are indicated in Table 1).
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f2: Normal heavy chain rearrangement in Fanca−/− mice.(A) Distribution of CDR3 lengths of VHDJH4 rearrangements amplified from genomic DNA that was isolated from BM IgM− B cells of Fanca−/− and WT mice. The average values are listed in Table 1. (B) Average numbers of added N-nucleotides per sequence in VHDJH4 rearrangements of Fanca−/− and WT mice are plotted either for VH to DJH and D to JH junctions (“Total”) or for VH to DJH (“VHD”) alone or D to JH (“DJH”) junctions alone. The results are displayed as the mean ± SEM. (C) The proportion of P-nucleotide additions at either the VH to DJH4 (“VHD”) or the D to JH4 (“DJH”) junctions. The data for HC rearrangements are from three independent pools of three mice per genotype (the numbers of sequences analysed are indicated in Table 1).

Mentions: To obtain a better characterization of the transition from the pro-B (the phase where HC rearrangement occurs) to the pre-B stage in Fanca deficient cells, we addressed V(D)J rearrangement efficiency by a PCR-based assay using VH and JH4 consensus primers to amplify HC-rearranged junctions in DNA isolated from Fanca+/+ and Fanca−/− BM B220+ IgM− cells. The rearranged junctions were analysed for length, size range and junctional diversity of CDR3. Our data showed that the average length, size range and length distribution of CDR3 of rearranged VDJH4 genes as well as the proportion of in-frame (vs. out-of–frame sequences were similar between Fanca−/− and WT mice (Table 1 and Fig. 2A). A determination of the length of each V, D, and J segment contributing to the CDR3 region showed no considerable difference between WT and the Fanca−/− mice (Table S2). Additionally, we found that the average number of N-additions, whether estimated for both the VH-D and D-JH junctions (total) or for either the VH-D or the D-JH junction alone appeared to be similar between the two groups of mice (Fig. 2B). On the contrary, P-additions at the VH-D or D-JH junctions differ significantly between Fanca−/− and WT mice (Fig. 2C). Surprisingly, whereas the ratio of P-additions at D-JH vs VH-D junctions was similar in WT cells (0.86+/−0.2), a significant disequilibrium was observed in Fanca−/− cells (ratio of 3.11+/−0.8). In other words, in Fanca−/− B cells, we observed 3 times more sequences with P-additions at D-JH junctions than at VH-D.


V(D)J recombination process and the Pre-B to immature B-cells transition are altered in Fanca − / − mice
Normal heavy chain rearrangement in Fanca−/− mice.(A) Distribution of CDR3 lengths of VHDJH4 rearrangements amplified from genomic DNA that was isolated from BM IgM− B cells of Fanca−/− and WT mice. The average values are listed in Table 1. (B) Average numbers of added N-nucleotides per sequence in VHDJH4 rearrangements of Fanca−/− and WT mice are plotted either for VH to DJH and D to JH junctions (“Total”) or for VH to DJH (“VHD”) alone or D to JH (“DJH”) junctions alone. The results are displayed as the mean ± SEM. (C) The proportion of P-nucleotide additions at either the VH to DJH4 (“VHD”) or the D to JH4 (“DJH”) junctions. The data for HC rearrangements are from three independent pools of three mice per genotype (the numbers of sequences analysed are indicated in Table 1).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC5121645&req=5

f2: Normal heavy chain rearrangement in Fanca−/− mice.(A) Distribution of CDR3 lengths of VHDJH4 rearrangements amplified from genomic DNA that was isolated from BM IgM− B cells of Fanca−/− and WT mice. The average values are listed in Table 1. (B) Average numbers of added N-nucleotides per sequence in VHDJH4 rearrangements of Fanca−/− and WT mice are plotted either for VH to DJH and D to JH junctions (“Total”) or for VH to DJH (“VHD”) alone or D to JH (“DJH”) junctions alone. The results are displayed as the mean ± SEM. (C) The proportion of P-nucleotide additions at either the VH to DJH4 (“VHD”) or the D to JH4 (“DJH”) junctions. The data for HC rearrangements are from three independent pools of three mice per genotype (the numbers of sequences analysed are indicated in Table 1).
Mentions: To obtain a better characterization of the transition from the pro-B (the phase where HC rearrangement occurs) to the pre-B stage in Fanca deficient cells, we addressed V(D)J rearrangement efficiency by a PCR-based assay using VH and JH4 consensus primers to amplify HC-rearranged junctions in DNA isolated from Fanca+/+ and Fanca−/− BM B220+ IgM− cells. The rearranged junctions were analysed for length, size range and junctional diversity of CDR3. Our data showed that the average length, size range and length distribution of CDR3 of rearranged VDJH4 genes as well as the proportion of in-frame (vs. out-of–frame sequences were similar between Fanca−/− and WT mice (Table 1 and Fig. 2A). A determination of the length of each V, D, and J segment contributing to the CDR3 region showed no considerable difference between WT and the Fanca−/− mice (Table S2). Additionally, we found that the average number of N-additions, whether estimated for both the VH-D and D-JH junctions (total) or for either the VH-D or the D-JH junction alone appeared to be similar between the two groups of mice (Fig. 2B). On the contrary, P-additions at the VH-D or D-JH junctions differ significantly between Fanca−/− and WT mice (Fig. 2C). Surprisingly, whereas the ratio of P-additions at D-JH vs VH-D junctions was similar in WT cells (0.86+/−0.2), a significant disequilibrium was observed in Fanca−/− cells (ratio of 3.11+/−0.8). In other words, in Fanca−/− B cells, we observed 3 times more sequences with P-additions at D-JH junctions than at VH-D.

View Article: PubMed Central - PubMed

ABSTRACT

B-lymphocytes in the bone marrow (BM) must generate a functional B-cell receptor and overcome the negative selection induced by reactivity with autoantigens. Two rounds of DNA recombination are required for the production of functional immunoglobulin heavy (Ig-HCs) and light (LCs) chains necessary for the continuation of B-lymphocyte development in the BM. Both rounds depend on the joint action of recombination activating gene-1 (RAG-1) and RAG-2 endonucleases with the DNA non-homologous end-joining pathway. Loss of the FANC gene leads to the chromosome breakage and cancer predisposition syndrome Fanconi anemia. Because the FANC proteins are involved in certain aspects of the recombination process, we sought to determine the impact of the FANC pathway on the Ig diversification process using Fanca−/− mice. In this work we demonstrated that Fanca−/− animals have a mild B-cell differentiation defect characterized by a specific alteration of the IgM− to IgM+ transition of the B220low B-cell population. Pre-B cells from Fanca−/− mice show evidence of impaired kLC rearrangement at the level of the Vk-Jk junction. Furthermore, Fanca−/− mice showed a skewed Vκ gene usage during formation of the LCs Vk-Jk junctions. Therefore, the Fanca protein appears as a yet unidentified factor involved in the primary diversification of Ig.

No MeSH data available.


Related in: MedlinePlus