Alternative end-joining catalyzes robust IgH locus deletions and translocations in the combined absence of ligase 4 and Ku70

Abstract

Class switch recombination (CSR) in B lymphocytes is initiated by introduction of multiple DNA double-strand breaks (DSBs) into switch (S) regions that flank immunoglobulin heavy chain (IgH) constant region exons. CSR is completed by joining a DSB in the donor S mu to a DSB in a downstream acceptor S region (e.g., S gamma1) by end-joining. In normal cells, many CSR junctions are mediated by classical nonhomologous end-joining (C-NHEJ), which employs the Ku70/80 complex for DSB recognition and XRCC4/DNA ligase 4 for ligation. Alternative end-joining (A-EJ) mediates CSR, at reduced levels, in the absence of C-NHEJ, even in combined absence of Ku70 and ligase 4, demonstrating an A-EJ pathway totally distinct from C-NHEJ. Multiple DSBs are introduced into S mu during CSR, with some being rejoined or joined to each other to generate internal switch deletions (ISDs). In addition, S-region DSBs can be joined to other chromosomes to generate translocations, the level of which is increased by absence of a single C-NHEJ component (e.g., XRCC4). We asked whether ISD and S-region translocations occur in the complete absence of C-NHEJ (e.g., in Ku70/ligase 4 double-deficient B cells). We found, unexpectedly, that B-cell activation for CSR generates substantial ISD in both S mu and S gamma1 and that ISD in both is greatly increased by the absence of C-NHEJ. IgH chromosomal translocations to the c-myc oncogene also are augmented in the combined absence of Ku70 and ligase 4. We discuss the implications of these findings for A-EJ in normal and abnormal DSB repair.

Fig. 1.

Robust ISDs occur in Ku70- and Ku70/Lig4-deficient IgM⁺ B-cell hybridomas. (A) Schematic of the IgH locus with genomic probes and restriction sites used for Southern blotting. Sμ ISD events were detected with a Cμ probe and confirmed with an Iμ probe (Fig. S2). Sγ1 ISD events were detected with an Iγ1 probe and confirmed with an Sγ1 probe (Fig. S2). (B) Ku70-deficient hybridomas display frequent ISDs as shown in representative panels of IgM⁺ Ku70^−/−H/+L⁺ and WT hybridomas probed with the indicated Southern probes. K, kidney DNA (indicates size of germline Sμ and Sγ1 regions); NS1, genomic DNA from NS1 hybridoma fusion partner. (C) Quantification of Sμ and Sγ1 ISD frequency in Ku70^−/−HL, Ku70^−/−/Lig4^−/−HL, and WT HL IgM⁺ hybridomas.

Fig. 2.

Examples of junctions from WT HL, Ku70HL, Ku70/Lig4HL, and Lig4HL primary B cells. GenBank annotated sequences for Sμ (MUSIGD07), Sγ1 (MUSIGHANB), and Sε (MUSIGHANX) were used for alignment. For each individual alignment, the annotated GenBank sequence is given at the top. The number of the nucleotides involved in the the junction is indicated on the left (top and bottom) for each alignment; between them the ISD junction sequence is shown. Junctional MHs are shaded in gray; the junctional insertion is underlined. The length of junctional MHs and insertions for each junction is indicated on the right.

Fig. 3.

Frequent IgH breaks and translocations in Ku70- and Lig4-deficient primary B cells stimulated for CSR. (A) Diagram with the 3′IgH BAC probe (green) and 5′IgH BAC probe (red) used for IgH FISH and examples of normal IgH loci, IgH breaks, and IgH translocations. Normal IgH loci retain both green and red locus-flanking signals. An IgH break results in split green and red signals. Translocations involving the IgH locus join the proximal region of chromosome12 (red signal) to other chromosomal fragments. (B) Percentage of metaphases with general breaks (Left) and IgH breaks (Right). (C) Percentage of IgH translocations.

Fig. 4.

Ku70-, Ku70/Lig4- and Lig4-deficient B cells activated for CSR display increased IgH–c-myc translocations. (A) Schematic of the IgH and c-myc loci with the location of the primers used for PCR amplification indicated. Gray primers: Der15 amplification; black primers: Der12 amplification (37). (B) Examples of Der12 IgH–c-myc translocations from αCD40 plus IL-4–stimulated Ku70^−/−HL, Ku70^−/−/Lig4^−/−HL, and control (WT HL) B cells. (C) Quantification of IgH–c-myc translocation frequency. Average and SD, genotype, and number of mice analyzed are indicated for each bar graph.