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

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

Fanca−/− mice displayed a skewed Vκ gene usage in in-frame Vκ-Jκ1 rearrangements in BM IgM- B cells.Analysis of Vκ gene family usage of total (A) and in-frame (B) Vκ-Jκ1 rearrangements amplified from genomic DNA isolated from BM IgM− B cells of Fanca−/− and WT mice (*p < 0.05 with Fisher’s exact test). Vκ families are displayed according to chromosomal order relative to the Jκ genes cluster. The data are from four independent pools of four mice per genotype (numbers of analysed sequences are indicated in Table 1).
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f5: Fanca−/− mice displayed a skewed Vκ gene usage in in-frame Vκ-Jκ1 rearrangements in BM IgM- B cells.Analysis of Vκ gene family usage of total (A) and in-frame (B) Vκ-Jκ1 rearrangements amplified from genomic DNA isolated from BM IgM− B cells of Fanca−/− and WT mice (*p < 0.05 with Fisher’s exact test). Vκ families are displayed according to chromosomal order relative to the Jκ genes cluster. The data are from four independent pools of four mice per genotype (numbers of analysed sequences are indicated in Table 1).

Mentions: Next, we compared the Vκ repertoire usage in Vκ-Jκ1 and Vκ-Jκ4 rearrangements in BM IgM− B cells from Fanca−/− and WT mice. Regarding the total Vκ-Jκ1 rearranged junctions, Fanca−/− and WT mice shared a similar Vκ family usage profile, with Vκ1, Vκ4/5 and Vκ9/10 used more often (>50%), and the single-members Vκ11, Vκ22, VκRF, and Vκdv36 used rarely, as previously reported262728 (Fig. 5A). Remarkably however, with respect to in-frame Vκ-Jκ1 rearrangements, we noticed the presence of a higher than expected proportion of Vκ1, 2, 8, 21 and 23-family junction in Fanca−/− mice. Furthermore, the Vκ1 family was the most significantly increased in Fanca−/− mice. Interestingly the Vκ8 Vκ21 Vκ23 gene families are located less than 1.0 Mb from Jκ1, whereas Vκ1 is located more than 2 Mb away (Fig. 5C). A similar analysis of Vκ gene usage in Vκ-Jκ4 rearrangements failed to show differences with respect to both distance and Vκ family usage between WT and Fanca−/− mice (Figure S3) further supporting the specificity of the previous observation.


V(D)J recombination process and the Pre-B to immature B-cells transition are altered in Fanca − / − mice
Fanca−/− mice displayed a skewed Vκ gene usage in in-frame Vκ-Jκ1 rearrangements in BM IgM- B cells.Analysis of Vκ gene family usage of total (A) and in-frame (B) Vκ-Jκ1 rearrangements amplified from genomic DNA isolated from BM IgM− B cells of Fanca−/− and WT mice (*p < 0.05 with Fisher’s exact test). Vκ families are displayed according to chromosomal order relative to the Jκ genes cluster. The data are from four independent pools of four mice per genotype (numbers of analysed sequences are indicated in Table 1).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f5: Fanca−/− mice displayed a skewed Vκ gene usage in in-frame Vκ-Jκ1 rearrangements in BM IgM- B cells.Analysis of Vκ gene family usage of total (A) and in-frame (B) Vκ-Jκ1 rearrangements amplified from genomic DNA isolated from BM IgM− B cells of Fanca−/− and WT mice (*p < 0.05 with Fisher’s exact test). Vκ families are displayed according to chromosomal order relative to the Jκ genes cluster. The data are from four independent pools of four mice per genotype (numbers of analysed sequences are indicated in Table 1).
Mentions: Next, we compared the Vκ repertoire usage in Vκ-Jκ1 and Vκ-Jκ4 rearrangements in BM IgM− B cells from Fanca−/− and WT mice. Regarding the total Vκ-Jκ1 rearranged junctions, Fanca−/− and WT mice shared a similar Vκ family usage profile, with Vκ1, Vκ4/5 and Vκ9/10 used more often (>50%), and the single-members Vκ11, Vκ22, VκRF, and Vκdv36 used rarely, as previously reported262728 (Fig. 5A). Remarkably however, with respect to in-frame Vκ-Jκ1 rearrangements, we noticed the presence of a higher than expected proportion of Vκ1, 2, 8, 21 and 23-family junction in Fanca−/− mice. Furthermore, the Vκ1 family was the most significantly increased in Fanca−/− mice. Interestingly the Vκ8 Vκ21 Vκ23 gene families are located less than 1.0 Mb from Jκ1, whereas Vκ1 is located more than 2 Mb away (Fig. 5C). A similar analysis of Vκ gene usage in Vκ-Jκ4 rearrangements failed to show differences with respect to both distance and Vκ family usage between WT and Fanca−/− mice (Figure S3) further supporting the specificity of the previous observation.

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&minus;/&minus; mice. In this work we demonstrated that Fanca&minus;/&minus; animals have a mild B-cell differentiation defect characterized by a specific alteration of the IgM&minus; to IgM+ transition of the B220low B-cell population. Pre-B cells from Fanca&minus;/&minus; mice show evidence of impaired kLC rearrangement at the level of the Vk-Jk junction. Furthermore, Fanca&minus;/&minus; mice showed a skewed V&kappa; 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