Lymphocyte-specific compensation for XLF/cernunnos end-joining functions in V(D)J recombination

Abstract

Mutations in XLF/Cernunnos (XLF) cause lymphocytopenia in humans, and various studies suggest an XLF role in classical nonhomologous end joining (C-NHEJ). We now find that XLF-deficient mouse embryonic fibroblasts are ionizing radiation (IR) sensitive and severely impaired for ability to support V(D)J recombination. Yet mature lymphocyte numbers in XLF-deficient mice are only modestly decreased. Moreover, XLF-deficient pro-B lines, while IR-sensitive, perform V(D)J recombination at nearly wild-type levels. Correspondingly, XLF/p53-double-deficient mice are not markedly prone to the pro-B lymphomas that occur in previously characterized C-NHEJ/p53-deficient mice; however, like other C-NHEJ/p53-deficient mice, they still develop medulloblastomas. Despite nearly normal V(D)J recombination in developing B cells, XLF-deficient mature B cells are moderately defective for immunoglobulin heavy-chain class switch recombination. Together, our results implicate XLF as a C-NHEJ factor but also indicate that developing mouse lymphocytes harbor cell-type-specific factors/pathways that compensate for the absence of XLF function during V(D)J recombination.

Figure 1. Characterization of XLF^Δ/Δ ES cells

(A) Western blotting assay for XLF in WT, XLF^Δ/Δ and XLF^Δ/Δ ES cells that over-express truncated XLF. The number in the brackets indicates the cDNA construct copy number in each line. "XLF" denotes the mobility of authentic XLF while "ΔXLF" denotes the mobility of XLF^Δα4/5. NS1, 2 and 3 indicate mobility of commonly observed non-specific bands observed in WT and XLF^Δ/Δ extracts. The * and ** symbols denote XLF^Δα4/5 bands from 4 and 6 copy transfectants, respectively (B) IR sensitivity of WT, XLF^Δ/Δ ES cells and XLF^Δ/Δ ES over-expressing XLF^Δα4/5 from a pGK-promoter based expression vector. (C) Summary of transient V(D)J recombination results on WT, XLF^Δ/Δ and XLF^Δ/Δ ES cells that over-expressed XLF^Δα4/5 Values reflect two independent experiments from two independent XLF^Δα4/5 over-expressing ES lines (6 and 4 copies; see Table S1 for details).

Figure 2. Characterization of XLF^Δ/Δ MEFs

(A) Proliferation of two independent sets (set a from 129/Sv background and set b from 129/BL6 mixed background) of primary WT and XLF^Δ/Δ MEFs. (B) IR sensitivity of the two independent sets of WT and XLF^Δ/Δ MEFs. (C) T- FISH analyses of spontaneous cytogenetic abnormalities in passage one WT and XLF^Δ/Δ MEF lines (n=3 for each genotype). Chromosome and chromatid breaks were scored as descried (Franco et al., 2008). (D) Transient V(D)J recombination results from immortalized WT and XLF^Δ/Δ MEFs. Values were obtained from multiple independent experiments from two independent XLF^Δ/Δ MEF lines (see Sup. Fig. 3 and Table S2 for details).

Figure 3. Lymphocyte Development in XLF^Δ/Δ Mice

(A) Western blotting analysis of XLF cross-reactive protein expression in extracts from indicated lymphoid organs of littermate-matched WT and XLF^Δ/Δ mice (see Sup. Fig. 1 for more examples). Symbols are as described in Fig. 1 legend. (B) Representative FACS analyses of lymphocyte development in four week old WT, XLF^Δ/Δ and RAG2^−/− mice. (C) Percentage and number of developing and mature lymphocytes in bone marrow, thymus and spleen from WT and XLF^Δ/Δ mice. Cell numbers in different fractions indicated in panel C were calculated by determining total cell numbers in each organ and then determining the percentage in each fraction (indicated by boxes on FACS plots in panel B).

Figure 4. Characterization of XLF^Δ/Δ A-MuLV Transformed Pro-B cells

(A) Western blotting for XLF expression in WT and XLF^Δ/Δ A-MuLV transformed pro-B cells and XLF^Δ/Δ A-MuLV transformant that over-expresses WT XLF from an integrated SV40- promoter based vector containing a puromycin resistant gene (denoted as XLF^Δ/Δ +XLF). See Sup. Figure 1 for more examples. (B) IR sensitivity of WT, XLF^Δ/Δ and XLF^Δ/Δ +XLF A-MuLV transformed pro-B cells. (C) Repair of IR induced DNA DSBs by WT, XLF^Δ/Δ and XLF^Δ/Δ +XLF A-MuLV transformed pro-B cells. The average and standard deviation of three experiments are plotted. (D) Summary of the transient V(D)J recombination assays on WT, XLF^Δ/Δ and XLF^Δ/Δ +XLF A-MuLV transformed pro-B cells (see Sup. Fig. 7 and Table S3 for details).

Figure 5. Chromosomal V(D)J Recombination in WT and XLF^Δ/Δ A-MuLV Transformants

(A) The pMX-INV V(D)J recombination substrate (adapted from Bredemeyer et al., 2006). The pMX-INV vector has a single pair of recombination RSs that flank an inverted green fluorescent protein (GFP) cassette. Normal V(D)J recombination allows for GFP expression. Also shown is a schematic representation of non-rearranged (NR), cleavage intermediates (CE and SE) and joining products (CJ and SJ) of pMX-INV. The long terminal repeats (LTR), packaging sequence, GFP cDNA, IRES-hCD4 cDNA (I-hCD4), 5’ 12-recombination signal (12-RS) and 3’ 23-RS (filled and open triangles, respectively), EcoRV (V) site, NcoI (N) site, and C4 probe are indicated. (B) Clones of WT, XRCC4^−/− and three independent XLF^Δ/Δ (a, b, and c) pro-B cell lines were treated with STI571 for 0, 48 and 96 hours and rearrangement was assayed for GFP expression. (C) Southern blot analyses of the rearrangement status of an inversional V(D)J recombination substrate (on right). For the upper gel, DNA was digested with EcoRV and NcoI and probed with the indicated C4 probe. The NR (non-rearranged) and 3’CE are both 2.2kb and the rearranged product is 4kb. For the lower gel, DNA was digested with EcoRV and probed with the same C4 probe. The NR and CJ are 5 kb bands and the 3’CE is a 2.2 kb band.

Figure 6. Class Switch Recombination in XLF^Δ/Δ Mice

(A) Proliferation of purified splenic B cells from WT and XLF^Δ/Δ mice following αCD40/IL-4 stimulation. (B) Representative FACS analysis of CD43⁻ splenocytes from WT, AID^−/− and XLF^Δ/Δ mice after a four-days of indicated stimulations. (C) Percentage of IgH locus breaks measured by IgH locus-specific FISH in WT, XLF^Δ/Δ AID^−/− and XLF^Δ/Δ AID^−/− B metaphases after cells were stimulated for 4 days with αCD40/IL-4. Data represent the average and standard deviation from multiple experiments.