RAD51 paralogs promote homology-directed repair at diversifying immunoglobulin V regions

Abstract

Background: Gene conversion depends upon the same factors that carry out more general process of homologous recombination, including homologous gene targeting and recombinational repair. Among these are the RAD51 paralogs, conserved factors related to the key recombination factor, RAD51. In chicken and other fowl, gene conversion (templated mutation) diversifies immunoglobulin variable region sequences. This allows gene conversion and recombinational repair to be studied using the chicken DT40 B cell line, which carries out constitutive gene conversion and provides a robust and physiological model for homology-directed repair in vertebrate cells.

Results: We show that DT40 contains constitutive nuclear foci of the repair factors RAD51D and XRCC2, consistent with activated homologous recombination. Single-cell imaging of a DT40 derivative in which the rearranged and diversifying immunoglobulin lambdaR light chain gene is tagged with polymerized lactose operator, DT40 PolyLacO-lambdaR, showed that RAD51D and XRCC2 localize to the diversifying lambdaR gene. Colocalizations correlate both functionally and physically with active immunoglobulin gene conversion. Ectopic expression of either RAD51D or XRCC2 accelerated the clonal rate of gene conversion, and conversion tracts were significantly longer in RAD51D than XRCC2 transfectants.

Conclusion: These results demonstrate direct functions of RAD51D and XRCC2 in immunoglobulin gene conversion, and also suggest that modulation of levels of repair factors may be a useful strategy to promote gene correction in other cell types.

Figure 1

Gene conversion diversifies chicken Ig genes. Gene conversion at the chicken Igλ locus. The rearranged λ gene variable (VJ) and constant (C) region is transcribed to encode the Igλ light chain polypeptide (at right); upstream pseudo-V (ψV) donors are templates for sequence transfer (above). Tracts of templated mutation are evident in the diversified V region and the encoded protein (below). Gene conversion proceeds by a pathway in which the target V region is cleaved and then undergoes homology-directed repair templated by the ψV regions (boxed inset).

Figure 2

RAD51, RAD51D-GFP and XRCC2-GFP form nuclear foci in DT40 B cells. Representative immunofluorescent images of normally proliferating DT40 stained with anti-RAD51 (left) or anti-GFP antibodies (center left); and of DT40 RAD51D-GFP or DT40 XRCC2-GFP cells stained with anti-GFP antibodies (center right, right). Merged DAPI image, below; bar, 10 μm.

Figure 3

RAD51, RAD51D-GFP and XRCC2-GFP foci localize to the diversifying λ_R gene in DT40 PolyLacO-λ_R cells. (A) Igλ locus tagged with polymerized lactose operator (PolyLacO) in DT40 PolyLacO-λ_R. The ψVλ array, PolyLacO, VJ and Cλ regions are indicated. (B) Immunofluorescent images of colocalizations of λ_R gene with RAD51 in single DT40 PolyLacO-λ_R RFP-LacI cells. Images of two representative cells are shown. λ_R, red signal from RFP-LacI binding at Igλ_R; α-RAD51, green; merge, λ_R and RAD51 signal; DAPI, nuclear DNA. Bar, 10 μm. (C) Immunofluorescent images of colocalizations of the tagged λ_R gene with RAD51D-GFP in stable DT40 PolyLacO-λ_R RFP-LacI RAD51D-GFP transfectants. λ_R, red signal from RFP-LacI binding at Igλ_R; α-GFP, green; merge, λ_R and GFP signal; DAPI, nuclear DNA. Bar, 10 μm. (D) Immunofluorescent images of colocalizations of the tagged λ_R gene with XRCC2-GFP in stable DT40 PolyLacO-λ_R RFP-LacI XRCC2-GFP transfectants. Notations as in panel C.

Figure 4

Representative three-dimensional images of RAD51D-GFP and XRCC2-GFP colocalizations with the λ_R gene. Serial images of a single nucleus were taken in 0.2 μm sections, spanning a total field depth of 1.0 μm. Above, λ_R/RAD51D-GFP colocalizations in stable DT40 PolyLacO-λ_R RFP-LacI RAD51D-GFP transfectants. Below, λ_R/XRCC2-GFP colocalizations in stable DT40 PolyLacO-λ_R RFP-LacI XRCC2-GFP transfectants. λ_R, red signal from RFP-LacI binding at Igλ_R; RAD51D-GFP or XRCC2-GFP, green; merge, λ_R and RAD51D-GFP or XRCC2-GFP signals; DAPI, nuclear DNA. Arrows indicate colocalizations.

Figure 5

λ_R/RAD51D-GFP colocalization depends upon AID-initiated DNA damage and reflects enrichment of RAD51D-GFP at λ_R. (A) Comparison of the fraction of cells exhibiting λ_R/RAD51D-GFP colocalizations in asynchronous cultures of DT40 PolyLacO-λ_R RFP-LacI RAD51D-GFP cells expressing or not expressing Ugi. (B) Products of PCR amplification from fixed DT40 chromatin after immunoprecipitation with anti-GFP or polyspecific IgG (IgG) antibodies. Results shown are representative of results of three separate experiments, with two different chromatin preparations. Amplicons derived from the rearranged (Vλ_R) or unrearranged (Vλ_U) Vλ regions, or the ovalbumin gene (Ova). Amplification was carried out at two-fold dilutions of template (triangles). Enrichment was calculated as the ratio of the Vλ and Ova amplicons, relative to the IgG control; standard deviations shown.

Figure 6

RAD51D or XRCC2 expression accelerates the clonal rate of Ig gene diversification. Fraction of sIgM loss variants (sIgM^- cells) in DT40 RAD51D, DT40 RAD51D-GFP, DT40 XRCC2 and DT40 XRCC2-GFP transfectants, normalized to DT40 GFP transfectants. Data are from two independent experiments; average fold increase is shown below.

Figure 7

Conversion tracts in DT40 RAD51D and XRCC2 transfectants. (A) Schematic diagram of mutations in 31 Vλ regions from DT40 RAD51D-GFP transfectants, aligned with the germline Vλ region (top). Complementarity-determining regions (CDRs) which make major contacts with antigen are indicated. Each Vλ sequence is represented as a horizontal line; bars identify gene conversion tracts (black, 2 or more nt differences with germline; gray, one nt difference); and lollipops identify point mutations with no match in the germline sequence. Potential ψV donors for each converted tract are identified above the bars. Range and average length of conversion tracts indicated below. (B) Schematic diagram of mutations in 27 Vλ regions from DT40 XRCC2-GFP transfectants, aligned with the germline Vλ region (top line). Notations as in panel A. (C) Pie chart showing relative fractions of gene conversion events resulting in changes at 1 nt or 2 or more nt, in Vλ genes from DT40 RAD51D-GFP and DT40 XRCC2-GFP transfectants.