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. 2012 Mar 6;109(10):3903-8.
doi: 10.1073/pnas.1120160109. Epub 2012 Feb 21.

Overlapping functions between XLF repair protein and 53BP1 DNA damage response factor in end joining and lymphocyte development

Xiangyu Liu  1 , Wenxia Jiang, Richard L Dubois, Kenta Yamamoto, Zachary Wolner, Shan Zha
Affiliations

Overlapping functions between XLF repair protein and 53BP1 DNA damage response factor in end joining and lymphocyte development

Xiangyu Liu et al. Proc Natl Acad Sci U S A. .

Abstract

Nonhomologous end joining (NHEJ), a major pathway of DNA double-strand break (DSB) repair, is required during lymphocyte development to resolve the programmed DSBs generated during Variable, Diverse, and Joining [V(D)J] recombination. XRCC4-like factor (XLF) (also called Cernunnos or NHEJ1) is a unique component of the NHEJ pathway. Although germ-line mutations of other NHEJ factors abrogate lymphocyte development and lead to severe combined immunodeficiency (SCID), XLF mutations cause a progressive lymphocytopenia that is generally less severe than SCID. Accordingly, XLF-deficient murine lymphocytes show no measurable defects in V(D)J recombination. We reported earlier that ATM kinase and its substrate histone H2AX are both essential for V(D)J recombination in XLF-deficient lymphocytes, despite moderate role in V(D)J recombination in WT cells. p53-binding protein 1 (53BP1) is another substrate of ATM. 53BP1 deficiency led to small reduction of peripheral lymphocyte number by compromising both synapse and end-joining at modest level during V(D)J recombination. Here, we report that 53BP1/XLF double deficiency blocks lymphocyte development at early progenitor stages, owing to severe defects in end joining during chromosomal V(D)J recombination. The unrepaired DNA ends are rapidly degraded in 53BP1(-/-)XLF(-/-) cells, as reported for H2AX(-/-)XLF(-/-) cells, revealing an end protection role for 53BP1 reminiscent of H2AX. In contrast to the early embryonic lethality of H2AX(-/-)XLF(-/-) mice, 53BP1(-/-)XLF(-/-) mice are born alive and develop thymic lymphomas with translocations involving the T-cell receptor loci. Together, our findings identify a unique function for 53BP1 in end-joining and tumor suppression.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
XLF and 53BP1 have redundant functions in lymphocyte development. (A) Representative flow cytometric analyses of T cells in thymus and spleen from WT, XLF−/−, 53BP1−/−, 53BP1−/−XLF−/−, and DNA-PKcs−/− mice (see Experimental Procedures for further description of the mouse lines). The histogram represents the total live thymocytes. The dashed line in the histogram provides a visual reference for the relative level of surface CD3ε staining. DN, CD4−CD8− double negative. For the “Thymus DN” staining, the total population is thymic DN cells, not total thymocytes. (B) Representative flow cytometric analyses of B cells in bone marrow, spleen, and lymph node from WT, XLF−/−, 53BP1−/−, and 53BP1−/−XLF−/− mice. The numbers on each blot (A and B) represent the percentage of live cells represented by a given population.
Fig. 2.
Fig. 2.
Lymphocyte development defects in 53BP1−/−XLF−/− mice are due to impaired V(D)J recombination. (A) Representative flow cytometric analyses of bone marrow and spleen from WT, XLF−/−53BP1−/−, 53BP1−/−XLF−/−HL, and 53BP1−/−XLF−/−EμBcl2+ mice. Numbers on the plot are percentage of total live cells (determined by forward and side scatter) represented by indicated population. (B) The number of total DN and single-positive (SP) thymocytes and IgM+ splenic B cells were calculated based on total organ cellularity and the percentage of the given cell type (by flow cytometric analyses). Each value listed represents the average ± SD from at least three mice of each genotype between 4 and 12 wk of age. The P values are calculated between corresponding groups by using two-tailed Student's t test. The original data are provided in Fig. S2.
Fig. 3.
Fig. 3.
53BP1−/−XLF−/− B cells have end joining during chromosomal V(D)J recombination. (A and D) Schematic of the pMX-INV (A) and pMX -SJ (B) retroviral V(D)J recombination substrates (13). The diagrams illustrate the unrearranged substrate (UR), CE/SE intermediates, and CJ/SJ products. The RSS (open and filled triangles), GFP, IRES-truncated hCD4 (ihCD4), and the LTRs are marked. The positions of EcoRV (E) sites and NcoI (N) sites are shown. hCD4 probe is located within ihCD4. (B and C) Southern blot analysis with hCD4 probe of EcoRV+NcoI (Upper) and EcoRV (Lower) digested genomic DNA from the indicated v-abl–transformed B cells containing pMX-INV substrates treated with STI571 for 2 or 4 d with or without ATM kinase inhibitor (15 μM). (E) Southern blot analysis with hCD4 probe of EcoRV digested genomic DNA from the indicated lines containing integrated pMX-SJ substrate The results shown in B and E were obtained from pools of B-cell lines with diverse integration sites of given substrate. C showed the result of cell line with single clonal integration of pMX-INV and the corresponding clones with ectopic expression of XLF.
Fig. 4.
Fig. 4.
End-joining in XLF−/− B cells requires the Tudor domain, but not the BRCT domain, of 53BP1. 53BP1−/−XLF−/− B cells with single clonal integration of pMX-INV (Inv 26) cells were infected with pBMN-ihCD2-53BP1WT, pBMN-ihCD2-53BP1ΔBRCT, or pBMN-ihCD2-53BP1ΔBRCT. (A) Flow cytometry analysis for GFP expression in different cells with identical substrate integration treated with STI for 0, 2, or 4 d with or without ATM inhibitor. (B) Southern blot analysis with hCD4 probe of EcoRV+NcoI (Upper) and EcoRV (Lower) digested genomic DNA from the indicated v-abl–transformed B cells treated with STI571 with or without ATM inhibitor as indicated.
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