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• W2127111507 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. 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. The classical nonhomologous end-joining (C-NHEJ) pathway is critical for repair of general DNA double strand breaks (DSBs) in mammalian cells and for repair of programmed DSBs during lymphocyte development. There are six well-characterized C-NHEJ factors (Dudley et al., 2005Dudley D.D. Chaudhuri J. Bassing C.H. Alt F.W. Mechanism and control of V(D)J recombination versus class switch recombination: similarities and differences.Adv. Immunol. 2005; 86: 43-112Crossref PubMed Scopus (207) Google Scholar), and another, XLF/Cernunnos (XLF), that was discovered more recently (Ahnesorg et al., 2006Ahnesorg P. Smith P. Jackson S.P. XLF interacts with the XRCC4-DNA ligase IV complex to promote DNA nonhomologous end-joining.Cell. 2006; 124: 301-313Abstract Full Text Full Text PDF PubMed Scopus (557) Google Scholar, Buck et al., 2006Buck D. Malivert L. de Chasseval R. Barraud A. Fondaneche M.C. Sanal O. Plebani A. Stephan J.L. Hufnagel M. le Deist F. et al.Cernunnos, a novel nonhomologous end-joining factor, is mutated in human immunodeficiency with microcephaly.Cell. 2006; 124: 287-299Abstract Full Text Full Text PDF PubMed Scopus (553) Google Scholar). The Ku80/Ku70 heterodimer (Ku) binds DSBs where, among other functions, it recruits downstream C-NHEJ factors. DNA bound Ku forms a complex with and activates the DNA-dependent protein kinase catalytic subunit (DNA-PKcs), which activates endonuclease activity of Artemis (Goodarzi et al., 2006Goodarzi A.A. Yu Y. Riballo E. Douglas P. Walker S.A. Ye R. Harer C. Marchetti C. Morrice N. Jeggo P.A. Lees-Miller S.P. DNA-PK autophosphorylation facilitates Artemis endonuclease activity.EMBO J. 2006; 25: 3880-3889Crossref PubMed Scopus (216) Google Scholar, Ma et al., 2002Ma Y. Pannicke U. Schwarz K. Lieber M.R. Hairpin opening and overhang processing by an Artemis/DNA-dependent protein kinase complex in nonhomologous end joining and V(D)J recombination.Cell. 2002; 108: 781-794Abstract Full Text Full Text PDF PubMed Scopus (792) Google Scholar, Moshous et al., 2001Moshous D. Callebaut I. de Chasseval R. Corneo B. Cavazzana-Calvo M. le Deist F. Tezcan I. Sanal O. Bertrand Y. Philippe N. et al.Artemis, a novel DNA double-strand break repair/V(D)J recombination protein, is mutated in human severe combined immune deficiency.Cell. 2001; 105: 177-186Abstract Full Text Full Text PDF PubMed Scopus (683) Google Scholar). The Artemis endonuclease processes a subset of DNA ends to prepare them for end joining (Rooney et al., 2002Rooney S. Sekiguchi J. Zhu C. Cheng H.L. Manis J. Whitlow S. DeVido J. Foy D. Chaudhuri J. Lombard D. Alt F.W. Leaky Scid phenotype associated with defective V(D)J coding end processing in Artemis-deficient mice.Mol. Cell. 2002; 10: 1379-1390Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar). Finally, DNA Ligase IV (Lig4), in association with XRCC4, performs end ligation. Ku70, Ku80, XRCC4, and Lig4 are considered “core” C-NHEJ factors because they are conserved in evolution and are required for all known C-NHEJ reactions, whereas, DNA-PKcs and Artemis evolved more recently and are particularly important for joining ends that require processing (Dudley et al., 2005Dudley D.D. Chaudhuri J. Bassing C.H. Alt F.W. Mechanism and control of V(D)J recombination versus class switch recombination: similarities and differences.Adv. Immunol. 2005; 86: 43-112Crossref PubMed Scopus (207) Google Scholar). Developing B and T lymphocytes assemble immunoglobulin (Ig) and T cell receptor (TCR) variable region exons from variable (V), diversity (D), and joining (J) gene segments via V(D)J recombination. V(D)J recombination is initiated by the recombination activating gene (RAG) 1 and 2 endonuclease, which introduces DSBs between participating V, D, or J coding sequences and flanking recombination signal (RS) sequences. RAG-mediated cleavage produces two blunt 5′ phosphorylated RS ends and two covalently sealed (hairpin) coding ends. Subsequently, C-NHEJ fuses the RS ends and the coding ends to form RS and coding joins, respectively (Dudley et al., 2005Dudley D.D. Chaudhuri J. Bassing C.H. Alt F.W. Mechanism and control of V(D)J recombination versus class switch recombination: similarities and differences.Adv. Immunol. 2005; 86: 43-112Crossref PubMed Scopus (207) Google Scholar). Core C-NHEJ factors are required for both coding and RS joins, while DNA-PKcs and Artemis are usually not required for blunt ligation of RS ends, but are instead required for coding join formation due to their role in opening and processing hairpin intermediates (Dudley et al., 2005Dudley D.D. Chaudhuri J. Bassing C.H. Alt F.W. Mechanism and control of V(D)J recombination versus class switch recombination: similarities and differences.Adv. Immunol. 2005; 86: 43-112Crossref PubMed Scopus (207) Google Scholar). Mature B cells undergo Ig heavy-chain (IgH) class switch recombination (CSR), a process that replaces the Cμ IgH constant region (CH) exons with downstream CH exons. Long switch (S) regions that precede CH exons are targets for activation-induced cytidine deaminase (AID), an enzyme that introduces DNA lesions that lead to DSBs (Stavnezer et al., 2008Stavnezer J. Guikema J.E. Schrader C.E. Mechanism and regulation of class switch recombination.Annu. Rev. Immunol. 2008; 26: 261-292Crossref PubMed Scopus (666) Google Scholar). DSBs in the donor S region upstream of Cμ (Sμ) are joined to DSBs in a downstream acceptor S region to complete CSR. A substantial fraction of normal CSR joins are generated by C-NHEJ, which joins ends that lack homology to form “direct” joins and also joins ends with several base pair homologies to form microhomology (MH) joins (Yan et al., 2007Yan C. Souza E.K. Franco S. Hickernell T. Boboila C. Gumaste S. Geyer M. Alimzhanov M. Manis J. Rajewsky K. Alt F.W. IgH Class switching and translocations Use a robust non-classical end-joining pathway.Nature. 2007; 449: 478-482Crossref PubMed Scopus (446) Google Scholar). Thus, C-NHEJ-deficient B lymphocytes activated for CSR accumulate IgH locus chromosomal breaks and translocations due to inability to join broken S regions (Yan et al., 2007Yan C. Souza E.K. Franco S. Hickernell T. Boboila C. Gumaste S. Geyer M. Alimzhanov M. Manis J. Rajewsky K. Alt F.W. IgH Class switching and translocations Use a robust non-classical end-joining pathway.Nature. 2007; 449: 478-482Crossref PubMed Scopus (446) Google Scholar). However, while XRCC4 and Lig4 are absolutely required for V(D)J recombination, substantial CSR (up to 50% of normal levels) occurs in their absence due to an alternative end-joining (A-EJ) pathway that strongly prefers to join ends with MH (Yan et al., 2007Yan C. Souza E.K. Franco S. Hickernell T. Boboila C. Gumaste S. Geyer M. Alimzhanov M. Manis J. Rajewsky K. Alt F.W. IgH Class switching and translocations Use a robust non-classical end-joining pathway.Nature. 2007; 449: 478-482Crossref PubMed Scopus (446) Google Scholar). DNA-PKcs deficiency results in variably decreased CSR, and Artemis deficiency has little effect on CSR, likely in part because many broken S region ends do not require processing (Dudley et al., 2005Dudley D.D. Chaudhuri J. Bassing C.H. Alt F.W. Mechanism and control of V(D)J recombination versus class switch recombination: similarities and differences.Adv. Immunol. 2005; 86: 43-112Crossref PubMed Scopus (207) Google Scholar). However, both factors are required for some CSR joins, because their absence leads to IgH locus breaks in activated B cells (Franco et al., 2008Franco S. Murphy M.M. Li G. Borjeson T. Boboila C. Alt F.W. DNA-PKcs and Artemis function in the end-joining phase of immunoglobulin heavy chain class switch recombination.J. Exp. Med. 2008; 205: 557-564Crossref PubMed Scopus (67) Google Scholar). Deficiency in mice for any of the six well-characterized C-NHEJ factors results in severe combined immunodeficiency (SCID) owing to inability to complete V(D)J recombination (Dudley et al., 2005Dudley D.D. Chaudhuri J. Bassing C.H. Alt F.W. Mechanism and control of V(D)J recombination versus class switch recombination: similarities and differences.Adv. Immunol. 2005; 86: 43-112Crossref PubMed Scopus (207) Google Scholar). In addition, deficiency for these C-NHEJ factors results in increased cellular ionizing radiation (IR) sensitivity and genomic instability. Deficiency for XRCC4, Lig4, and Ku also leads to neuronal apoptosis, which, in the case of XRCC4 and Lig4 deficiency, is so severe that it causes late embryonic lethality (Lee and McKinnon, 2007Lee Y. McKinnon P.J. Responding to DNA double strand breaks in the nervous system.Neuroscience. 2007; 145 (Published online August 23, 2006): 1365-1374Crossref PubMed Scopus (79) Google Scholar). V(D)J recombination is not completely blocked in Ku70- or Artemis-deficient mice, leading to a “leaky” SCID phenotype with very small populations of mature T cells (Dudley et al., 2005Dudley D.D. Chaudhuri J. Bassing C.H. Alt F.W. Mechanism and control of V(D)J recombination versus class switch recombination: similarities and differences.Adv. Immunol. 2005; 86: 43-112Crossref PubMed Scopus (207) Google Scholar). Mice that are doubly deficient for any of the six characterized C-NHEJ factors plus the p53 checkpoint protein succumb to pro-B cell lymphomas, which routinely harbor RAG-dependent translocations between IgH and chromosomal regions near the c-Myc or N-Myc oncogenes (Dudley et al., 2005Dudley D.D. Chaudhuri J. Bassing C.H. Alt F.W. Mechanism and control of V(D)J recombination versus class switch recombination: similarities and differences.Adv. Immunol. 2005; 86: 43-112Crossref PubMed Scopus (207) Google Scholar). Although they routinely die from pro-B lymphoma, C-NHEJ/p53-deficient mice also develop medulloblastomas in situ (Lee and McKinnon, 2002Lee Y. McKinnon P.J. DNA ligase IV suppresses medulloblastoma formation.Cancer Res. 2002; 62: 6395-6399PubMed Google Scholar), and specific deletion of XRCC4 in p53-deficient developing neurons leads to aggressive medulloblastomas with recurrent chromosomal translocations, reflecting the neuronal DSB repair defect (Yan et al., 2006Yan C.T. Kaushal D. Murphy M. Zhang Y. Datta A. Chen C. Monroe B. Mostoslavsky G. Coakley K. Gao Y. et al.XRCC4 suppresses medulloblastomas with recurrent translocations in p53-deficient mice.Proc. Natl. Acad. Sci. USA. 2006; 103: 7378-7383Crossref PubMed Scopus (96) Google Scholar). Artemis mutations in humans lead to SCID (Moshous et al., 2001Moshous D. Callebaut I. de Chasseval R. Corneo B. Cavazzana-Calvo M. le Deist F. Tezcan I. Sanal O. Bertrand Y. Philippe N. et al.Artemis, a novel DNA double-strand break repair/V(D)J recombination protein, is mutated in human severe combined immune deficiency.Cell. 2001; 105: 177-186Abstract Full Text Full Text PDF PubMed Scopus (683) Google Scholar). In contrast, XLF mutations in humans lead to microcephaly and a combined immunodeficiency that is less severe than that associated with Artemis mutations (Ahnesorg et al., 2006Ahnesorg P. Smith P. Jackson S.P. XLF interacts with the XRCC4-DNA ligase IV complex to promote DNA nonhomologous end-joining.Cell. 2006; 124: 301-313Abstract Full Text Full Text PDF PubMed Scopus (557) Google Scholar, Buck et al., 2006Buck D. Malivert L. de Chasseval R. Barraud A. Fondaneche M.C. Sanal O. Plebani A. Stephan J.L. Hufnagel M. le Deist F. et al.Cernunnos, a novel nonhomologous end-joining factor, is mutated in human immunodeficiency with microcephaly.Cell. 2006; 124: 287-299Abstract Full Text Full Text PDF PubMed Scopus (553) Google Scholar, Dai et al., 2003Dai Y. Kysela B. Hanakahi L.A. Manolis K. Riballo E. Stumm M. Harville T.O. West S.C. Oettinger M.A. Jeggo P.A. Nonhomologous end joining and V(D)J recombination require an additional factor.Proc. Natl. Acad. Sci. USA. 2003; 100: 2462-2467Crossref PubMed Scopus (147) Google Scholar). XLF-deficient human fibroblasts (Buck et al., 2006Buck D. Malivert L. de Chasseval R. Barraud A. Fondaneche M.C. Sanal O. Plebani A. Stephan J.L. Hufnagel M. le Deist F. et al.Cernunnos, a novel nonhomologous end-joining factor, is mutated in human immunodeficiency with microcephaly.Cell. 2006; 124: 287-299Abstract Full Text Full Text PDF PubMed Scopus (553) Google Scholar, Dai et al., 2003Dai Y. Kysela B. Hanakahi L.A. Manolis K. Riballo E. Stumm M. Harville T.O. West S.C. Oettinger M.A. Jeggo P.A. Nonhomologous end joining and V(D)J recombination require an additional factor.Proc. Natl. Acad. Sci. USA. 2003; 100: 2462-2467Crossref PubMed Scopus (147) Google Scholar) and mouse ES cells (Zha et al., 2007Zha S. Alt F.W. Cheng H.L. Brush J.W. Li G. Defective DNA repair and increased genomic instability in Cernunnos-XLF-deficient murine ES cells.Proc. Natl. Acad. Sci. USA. 2007; 104: 4518-4523Crossref PubMed Scopus (87) Google Scholar) are IR-sensitive and have DSB repair defects, including severely impaired V(D)J coding and RS joining on episomal substrates. XLF shares structural features with XRCC4, including an N-terminal head domain and a C-terminal coiled-coil domain required for homodimer formation (Ahnesorg et al., 2006Ahnesorg P. Smith P. Jackson S.P. XLF interacts with the XRCC4-DNA ligase IV complex to promote DNA nonhomologous end-joining.Cell. 2006; 124: 301-313Abstract Full Text Full Text PDF PubMed Scopus (557) Google Scholar, Andres et al., 2007Andres S.N. Modesti M. Tsai C.J. Chu G. Junop M.S. Crystal structure of human XLF: a twist in nonhomologous DNA end-joining.Mol. Cell. 2007; 28: 1093-1101Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, Li et al., 2008Li Y. Chirgadze D.Y. Bolanos-Garcia V.M. Sibanda B.L. Davies O.R. Ahnesorg P. Jackson S.P. Blundell T.L. Crystal structure of human XLF/Cernunnos reveals unexpected differences from XRCC4 with implications for NHEJ.EMBO J. 2008; 27 (Published online November 29, 2007): 290-300Crossref PubMed Scopus (92) Google Scholar). XLF interacts with XRCC4 (Ahnesorg et al., 2006Ahnesorg P. Smith P. Jackson S.P. XLF interacts with the XRCC4-DNA ligase IV complex to promote DNA nonhomologous end-joining.Cell. 2006; 124: 301-313Abstract Full Text Full Text PDF PubMed Scopus (557) Google Scholar) and boosts end ligation efficiency of the XRCC4/Lig4 complex in vitro (Gu et al., 2007Gu J. Lu H. Tsai A.G. Schwarz K. Lieber M.R. Single-stranded DNA ligation and XLF-stimulated incompatible DNA end ligation by the XRCC4-DNA ligase IV complex: influence of terminal DNA sequence.Nucleic Acids Res. 2007; 35: 5755-5762Crossref PubMed Scopus (94) Google Scholar, Lu et al., 2007Lu H. Pannicke U. Schwarz K. Lieber M.R. Length-dependent binding of human XLF to DNA and stimulation of XRCC4.DNA ligase IV activity.J. Biol. Chem. 2007; 282: 11155-11162Crossref PubMed Scopus (88) Google Scholar). XLF also stimulates end joining of mismatched noncohesive ends and blunt ends, but has little impact on ligation of cohesive ends (Tsai et al., 2007Tsai C.J. Kim S.A. Chu G. Cernunnos/XLF promotes the ligation of mismatched and noncohesive DNA ends.Proc. Natl. Acad. Sci. USA. 2007; 104: 7851-7856Crossref PubMed Scopus (138) Google Scholar). XLF has an apparent yeast homolog, known as Nej1 (Callebaut et al., 2006Callebaut I. Malivert L. Fischer A. Mornon J.P. Revy P. de Villartay J.P. Cernunnos interacts with the XRCC4 x DNA-ligase IV complex and is homologous to the yeast nonhomologous end-joining factor Nej1.J. Biol. Chem. 2006; 281: 13857-13860Crossref PubMed Scopus (100) Google Scholar, Hentges et al., 2006Hentges P. Ahnesorg P. Pitcher R.S. Bruce C.K. Kysela B. Green A.J. Bianchi J. Wilson T.E. Jackson S.P. Doherty A.J. Evolutionary and functional conservation of the DNA non-homologous end-joining protein, XLF/Cernunnos.J. Biol. Chem. 2006; 281: 37517-37526Crossref PubMed Scopus (67) Google Scholar), which interacts with Lif1p, the yeast homolog of XRCC4 (Teo and Jackson, 2000Teo S.H. Jackson S.P. Lif1p targets the DNA ligase Lig4p to sites of DNA double-strand breaks.Curr. Biol. 2000; 10: 165-168Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar), suggesting that XLF is a “core” C-NHEJ factor. Although a role of XLF in C-NHEJ might explain the lymphocytopenia of human XLF mutant patients, the reason for their mildly impaired lymphocyte development is not clear. Also, one XLF mutant patient was diagnosed with hyper IgM syndrome, suggesting an XLF role in CSR (Buck et al., 2006Buck D. Malivert L. de Chasseval R. Barraud A. Fondaneche M.C. Sanal O. Plebani A. Stephan J.L. Hufnagel M. le Deist F. et al.Cernunnos, a novel nonhomologous end-joining factor, is mutated in human immunodeficiency with microcephaly.Cell. 2006; 124: 287-299Abstract Full Text Full Text PDF PubMed Scopus (553) Google Scholar). To further elucidate XLF function, we generated and characterized XLF mutant mice. To characterize XLF function in mouse cells, we employed gene-targeted mutation to delete exons 4 and 5 from both copies of the XLF gene in ES cells (Zha et al., 2007Zha S. Alt F.W. Cheng H.L. Brush J.W. Li G. Defective DNA repair and increased genomic instability in Cernunnos-XLF-deficient murine ES cells.Proc. Natl. Acad. Sci. USA. 2007; 104: 4518-4523Crossref PubMed Scopus (87) Google Scholar). The resulting XLFΔ/Δ ES cells were severely impaired for ability to form V(D)J coding and RS joins in transient assays and were IR-sensitive (Zha et al., 2007Zha S. Alt F.W. Cheng H.L. Brush J.W. Li G. Defective DNA repair and increased genomic instability in Cernunnos-XLF-deficient murine ES cells.Proc. Natl. Acad. Sci. USA. 2007; 104: 4518-4523Crossref PubMed Scopus (87) Google Scholar). Expression of a wild-type (WT) XLF complementary DNA (cDNA) rescued IR sensitivity and defective V(D)J recombination in XLFΔ/Δ ES cells, confirming the specificity of the XLF targeting (Zha et al., 2007Zha S. Alt F.W. Cheng H.L. Brush J.W. Li G. Defective DNA repair and increased genomic instability in Cernunnos-XLF-deficient murine ES cells.Proc. Natl. Acad. Sci. USA. 2007; 104: 4518-4523Crossref PubMed Scopus (87) Google Scholar). Our targeting strategy allowed in-frame splicing from XLF exon 3 to 6 to generate a truncated XLF transcript that was readily detectable (Zha et al., 2007Zha S. Alt F.W. Cheng H.L. Brush J.W. Li G. Defective DNA repair and increased genomic instability in Cernunnos-XLF-deficient murine ES cells.Proc. Natl. Acad. Sci. USA. 2007; 104: 4518-4523Crossref PubMed Scopus (87) Google Scholar). Despite truncated XLF transcripts, a cross-reactive XLF protein was not detected in XLFΔ/Δ ES cell extracts (Zha et al., 2007Zha S. Alt F.W. Cheng H.L. Brush J.W. Li G. Defective DNA repair and increased genomic instability in Cernunnos-XLF-deficient murine ES cells.Proc. Natl. Acad. Sci. USA. 2007; 104: 4518-4523Crossref PubMed Scopus (87) Google Scholar). However, when large amounts of extract from XLFΔ/Δ cells were assayed, we sometimes detected very low levels of an XLF cross-reactive band of the approximate size predicted for truncated XLF protein (see below). To estimate maximal levels of the putative XLF protein fragment in XLFΔ/Δ ES cells relative to those of authentic XLF in WT ES cells, we performed serial dilutions of WT extracts. Based on such experiments, maximal levels of truncated XLF in XLFΔ/Δ ES cells, if present at all, generally appeared to be less than 1% of WT XLF levels (Figure 1A; see Figure S1 available online). The crystal structure of XLF (Andres et al., 2007Andres S.N. Modesti M. Tsai C.J. Chu G. Junop M.S. Crystal structure of human XLF: a twist in nonhomologous DNA end-joining.Mol. Cell. 2007; 28: 1093-1101Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, Li et al., 2008Li Y. Chirgadze D.Y. Bolanos-Garcia V.M. Sibanda B.L. Davies O.R. Ahnesorg P. Jackson S.P. Blundell T.L. Crystal structure of human XLF/Cernunnos reveals unexpected differences from XRCC4 with implications for NHEJ.EMBO J. 2008; 27 (Published online November 29, 2007): 290-300Crossref PubMed Scopus (92) Google Scholar) reveals that the potential truncated XLF protein generated by our targeting (lacking aa 131 to 196) would lack the α4 (aa 128 to 171) and α5 (aa 171 to 185) regions, which form the coiled-coil domain required for XLF homodimerization and, indirectly, for XRCC4 interaction (Andres et al., 2007Andres S.N. Modesti M. Tsai C.J. Chu G. Junop M.S. Crystal structure of human XLF: a twist in nonhomologous DNA end-joining.Mol. Cell. 2007; 28: 1093-1101Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, Li et al., 2008Li Y. Chirgadze D.Y. Bolanos-Garcia V.M. Sibanda B.L. Davies O.R. Ahnesorg P. Jackson S.P. Blundell T.L. Crystal structure of human XLF/Cernunnos reveals unexpected differences from XRCC4 with implications for NHEJ.EMBO J. 2008; 27 (Published online November 29, 2007): 290-300Crossref PubMed Scopus (92) Google Scholar). Thus, this truncated XLF protein (referred to as XLFΔα4/5) potentially would be nonfunctional. To test for XLFΔα4/5 activity, we asked whether highly overexpressing XLFΔα4/5 cDNA in XLFΔ/Δ ES cells complemented IR sensitivity or defective V(D)J recombination. For this purpose, we introduced a PGK promoter-driven XLFΔα4/5 cDNA expression vector into XLFΔ/Δ ES cells and isolated clones with different copy numbers. Enforced XLFΔα4/5 cDNA expression generated an XLF cross-reactive protein of the size predicted for XLFΔα4/5, with the highest copy number lines expressing XLFΔα4/5 at levels up to 30%–40% those of XLF in WT ES cells (Figure 1A) and which were at least 50- to 100-fold higher than those of XLFΔα4/5 potentially expressed in XLFΔ/Δ cells (Figures 1A and S1; see below). However, even such high level overexpression of XLFΔα4/5 did not detectably rescue IR sensitivity (Figure 1B) or V(D)J recombination defects of XLFΔ/Δ ES cells (Figure 1C; Table S1). Therefore, we conclude that XLFΔα4/5 only accumulates to very low levels in XLFΔ/Δ ES; in any case, it is not functional when highly overexpressed. Thus, by available criteria, our targeting appears to generate a functionally null allele. Three independent XLF+/Δ ES cell clones were injected for germline transmission and resulting XLF+/Δ mice were bred to generate XLFΔ/Δ mice in a 129/Sv background. XLFΔ/Δ mice were fertile, born at Mendelian ratios, and were normal in size (data not shown). Western blotting analyses confirmed undetectable or very low level expression of an anti-XLF cross-reactive band of the size predicted for XLFΔα4 in various tissues of XLFΔ/Δ mice (see below). Although the human XLF mutation is associated with microcephaly and Ku, XRCC4, and Lig4 deficiency with increased apoptosis of developing neurons in mice, we did not observe excessive neuronal cell death or a significant reduction of brain weight in embryonic (E17.5; n = 3) or adult (P30; n = 3) XLFΔ/Δ mice (Figure S2). We also did not observe overt malignancies including thymic lymphomas in a cohort (n = 18) of XLFΔ/Δ-deficient mice followed for 18 months (data not shown). XLF+/Δ cells and mice were indistinguishable from WT, excluding dominant negative effects of the XLFΔ allele (data not shown). We generated mouse embryonic fibroblasts (MEFs) from day 13.5 XLFΔ/Δ and littermate control (WT) embryos. Proliferation of XLFΔ/Δ MEFs was indistinguishable from that of WT MEFs derived from both inbred (pure 129/Sv, set a) and mixed (129/BL6 mixed, set b) backgrounds (Figure 2A). Consistent with findings on XLFΔ/Δ ES cells, XLFΔ/Δ MEFs exhibited substantially increased IR sensitivity compared with WT controls (Figure 2B). Furthermore, telomere fluorescence in situ hybridization (T-FISH) revealed that 13% ± 3% of metaphases from XLFΔ/Δ MEFs had at least one chromosomal break, in comparison to 1% ± 2% in metaphases from WT MEFs (Figure 2C, left panel). Almost all chromosomal breaks in XLFΔ/Δ MEFs were chromosome breaks, as opposed to chromatid breaks (Figure 2C, right panel), similar to the distribution of chromosomal abnormalities associated with deficiency for various C-NHEJ factors (Franco et al., 2008Franco S. Murphy M.M. Li G. Borjeson T. Boboila C. Alt F.W. DNA-PKcs and Artemis function in the end-joining phase of immunoglobulin heavy chain class switch recombination.J. Exp. Med. 2008; 205: 557-564Crossref PubMed Scopus (67) Google Scholar, Mills et al., 2004Mills K.D. Ferguson D.O. Essers J. Eckersdorff M. Kanaar R. Alt F.W. Rad54 and DNA Ligase IV cooperate to maintain mammalian chromatid stability.Genes Dev. 2004; 18: 1283-1292Crossref PubMed Scopus (107) Google Scholar, Yan et al., 2007Yan C. Souza E.K. Franco S. Hickernell T. Boboila C. Gumaste S. Geyer M. Alimzhanov M. Manis J. Rajewsky K. Alt F.W. IgH Class switching and translocations Use a robust non-classical end-joining pathway.Nature. 2007; 449: 478-482Crossref PubMed Scopus (446) Google Scholar). SV40 Large T-antigen immortalized XLFΔ/Δ MEFs had substantially reduced ability to generate V(D)J coding or RS joins (5%–20% of WT levels), as measured by three independent transient V(D)J assays on two sets of XLFΔ/Δ and WT littermate MEFs (Figure 2D; Table S2). Notably, while residual coding joins recovered from transient assays of other C-NHEJ-deficient cells are mostly aberrant (e.g., large deletions), residual coding joins recovered from XLFΔ/Δ MEFs, like those from XLFΔ/Δ ES cells, resembled those of WT MEFs in terms of length and junctional modifications (Figure S3). Similarly, 80% of the residual RS joins recovered from XLFΔ/Δ MEFs were precise (Figure 2D). We conclude that XLFΔ/Δ MEFs generally resemble XLFΔ/Δ ES cells with respect to IR sensitivity, defective V(D)J recombination in transient assays, and increased genomic instability. Western blot analyses of extracts from total thymus, spleen, and bone marrow of XLFΔ/Δ mice showed that expression of putative XLFΔα4/5 protein occurred at levels ranging from undetectable (in some experiments titrating to less than 0.2% of authentic XLF levels in WT extracts) to very weakly detectable (less than 1% of levels of WT XLF) (Figures 3A and S1). Total cell numbers in the thymus and spleens of 4-week-old XLFΔ/Δ mice were, on average, about 40%–50% those of WT (Figure 3C). However, flow cytometry revealed that distribution of pro-B (CD43+B220+IgM−), pre-B (CD43−B220+IgM−), newly generated B (IgM+B220low) and recirculating B (IgM+B220hi) cells in XLFΔ/Δ bone marrow were similar to those of WT and XLF+/Δ littermates (Figures 3B and 3C). In addition, XLFΔ/Δ mice contained splenic IgM+ B cells at numbers approximately 30%–50% those of WT (Figures 3B and 3C). Similarly, despite the mild reduction in total thymocyte numbers, the distribution of CD4−CD8− (double negative), CD4+/CD8+ (double positive), and CD4+/CD8− or CD8+/CD4− (single positive) XLFΔ/Δ thymocytes was comparable to those of WT littermates (Figures 3B and 3C). Reduced levels of surface CD3 and TCRβ expression in ATM−/− thymocytes (Borghesani et al., 2000Borghesani P.R. Alt F.W. Bottaro A. Davidson L. Aksoy S. Rathbun G.A. Roberts T.M. Swat W. Segal R.A. Gu Y. Abnormal development of Purkinje cells and lymphocytes in Atm mutant mice.Proc. Natl. Acad. Sci. USA. 2000; 97: 3336-3341Crossref PubMed Scopus (156) Google Scholar) might reflect a modestly impaired V(D)J recombination (Huang et al., 2007Huang C.Y. Sharma G.G. Walker L.M. Bassing C.H. Pandita T.K. Sleckman B.P. Defects in coding joint formation in vivo in developing ATM-deficient B and T lymphocytes.J. Exp. Med. 2007; 204: 1371-1381Crossref PubMed Scopus (45) Google Scholar). In this regard, XLFΔ/Δ and WT thymocytes express similar surface CD3 and TCRβ levels (Figure S4). XLFΔ/Δ mice also contained normal populations of splenic single positive (CD4+CD8− and CD4−CD8+" @default.
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