53BP1 facilitates long-range DNA end-joining during V(D)J recombination

Abstract

Variable, diversity and joining (V(D)J) recombination and class-switch recombination use overlapping but distinct non-homologous end joining pathways to repair DNA double-strand-break intermediates. 53BP1 is a DNA-damage-response protein that is rapidly recruited to sites of chromosomal double-strand breaks, where it seems to function in a subset of ataxia telangiectasia mutated (ATM) kinase-, H2A histone family member X (H2AX, also known as H2AFX)- and mediator of DNA damage checkpoint 1 (MDC1)-dependent events. A 53BP1-dependent end-joining pathway has been described that is dispensable for V(D)J recombination but essential for class-switch recombination. Here we report a previously unrecognized defect in the joining phase of V(D)J recombination in 53BP1-deficient lymphocytes that is distinct from that found in classical non-homologous-end-joining-, H2ax-, Mdc1- and Atm-deficient mice. Absence of 53BP1 leads to impairment of distal V-DJ joining with extensive degradation of unrepaired coding ends and episomal signal joint reintegration at V(D)J junctions. This results in apoptosis, loss of T-cell receptor alpha locus integrity and lymphopenia. Further impairment of the apoptotic checkpoint causes propagation of lymphocytes that have antigen receptor breaks. These data suggest a more general role for 53BP1 in maintaining genomic stability during long-range joining of DNA breaks.

Figure 1. Antigen-receptor-associated aberrations in 53BP1^−/− lymphocytes

a, Upper panel: schematic of the TCRα-TCRδ locus with positions of the BACs used for generation of DNA-FISH probes indicated. Middle panel: Representative examples of projections of confocal sections, analyzed by three-dimensional FISH on freshly isolated thymocytes. Bottom panel: frequency at which TCRcα or (TCRvα+ TCRcα) signal is lost from one allele in WT, 53BP1^−/−, p53^−/−, 53BP1^−/−p53^−/−, 53BP1^−/−Nbs1^tr735 and Atm^−/− thymocytes (>200 cells analyzed per genotype in each of two experiments;error bars, s.d.). b, TCRα associated chromosomal aberrations in lymph node T cells determined by FISH using a TCRα locus spanning BAC (red signal, right panel), a chromosome 14 paint (green signal, right panel) and a telomere repeat specific probe (white signal, right panel). T cells were stimulated with anti-TCR/CD28 antibodies for 48 hours. (error bars, s.d., n≥3).c, Frequency of IgH-associated abnormalities in metaphase spreads from bone marrow and splenic B cells from WT, 53BP1^−/−, 53BP1^−/−p53^−/− and 53BP1^−/−Nbs1^tr735 mice (error bars, s.d., n≥3). B220⁺ bone marrow cells were cultured on irradiated S17 stromal cells in the presence of IL7 for 5 days. Splenic CD43-negative B cells were cultured for 1 day with RP105, which induces proliferation but not CSR. FISH was performed using probes specific for the IgHcα locus (red signal, right panel), chromosome 12 (green signal, right panel) and telomeric repeats (white signal, right panel).

Figure 2. Processing of Jα coding ends in 53BP1 deficient thymocytes

a, Southern blot analysis of total thymocyte and kidney DNA digested with StuI and hybridized with a CαI probe. The same blot was stripped and re-probed with a RAG2 probe as a loading control. The fragments corresponding to the germline TCRα^SJ allele (*) allele, the Jα56 coding ends, as well as the molecular mass markers (in kb) are indicated. b, Strategy for PCR amplification of free Jα coding ends captured by TdT-mediated poly-adenylation. c, Southern blot analysis of coding ends from TCRα^SJ/SJ:WT and TCRα^SJ/SJ:53BP1^−/− thymocytes. Serial five-fold dilutions and a mock-polyadenylated control are shown. IL-2 gene PCR templated with serial five-fold dilutions of the genomic DNA used for polyadenylation is shown as a loading control. d, Examples of sequences of coding joints formed by V(D)J recombination. Germline coding sequences (bold face lowercase letters) are indicated on the top. Nucleotide insertions (P/N) are indicated by capital letters. Sequences of inserts^a–d are listed in Supplementary Table 2.

Figure 3. Decreased efficiency of long-range VDJ recombination and TCRα locus contraction in 53BP1^−/− thymocytes

a, Schematic of the mouse TCRα/δ locus is shown in the top panel with the four different TCRδ rearrangements depicted individually below. Frequency of TCRδ locus rearrangements in total thymocytes from 53BP1^−/− and WT littermates. Quantitative assessment of genomic DNA rearrangements of Dδ1 to Dδ2, Dδ2 to Jδ1, and Vδ4 and Vδ5 to (D)Jδ1 genes were performed by quantitative PCR and normalized to the signal of the non-rearranging DNA 3’ of Jδ2. Results are averaged from 4 53BP1^−/− and 2 WT mice with duplicate measurements, and standard errors of the mean are shown. (WT vs. 53BP1^−/−: D2-J1, p=0.851; D1–D2, p=0.001;V4-DJ1, p=0.003; V5-DJ1, p<0.001). b, Distances separating the TCRVα and TCRCα loci in freshly isolated CD4/CD8 double negative (DN) or double positive (DP) thymocytes. The data were obtained by three-dimensional DNA-FISH using the TCRα BAC probes as indicated (top panel). As control, the probe set RP23-309A8 and RP24-336F10 (1Mb distance) on mouse chromosome 1D was used. Light grey bars: 0 to 0.3 µm; dark grey bars: 0.3–0.5 µm and black bars: 0.5 to 2 µm. Distance distribution histograms are shown in Supplementary Fig. 4, and Supplementary Table 3 lists sample sizes and average distances. (error bars, s.d.; *****: p<0.0001).

Figure 4. Model for 53BP1’s role in promoting and/or maintaining synapsis during V(D)J recombination

Before recombination, 53BP1 is loosely associated with chromatin (Step 1). RSS’s that are at a close physical distance (a) spend more time near the RAG1/2- bound RSS than distant RSS’s (b), and have a high probability of synapsis whether or not 53BP1 is present. 53BP1 homo-oligomerization increases the effective local concentration of distant RSSs thereby promoting their pairing. When RAG1/2 associates with and cleaves a single RSS, 53BP1 accumulates at the lesion and binds tightly to flanking chromatin in a manner dependent on H2AX and MDC1 (Steps 2 and 3). After RAG1/2 mediated DSB formation (step 4), 53BP1 stabilizes the post-cleavage complex, thereby increasing the efficiency of non-homologous end-joining (Step 5).