(XRCC1) Deficiency Enhances Class Switch Recombination and Is

X-ray repair cross-complementing protein 1 (XRCC1) deﬁciency enhances class switch recombination and is permissive for alternative end joining

Li Han1, Weifeng Mao1,2, and Kefei Yu3

Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, MI 48824

Edited* by Frederick W. Alt, Howard Hughes Medical Institute, Harvard Medical School, Children’s Hospital, Immune Disease Institute, Boston, MA, and approved February 10, 2012 (received for review December 15, 2011) DNA double-strand breaks (DSBs) are essential intermediates in Ig the DNA end, the DNA-dependent protein kinase (DNA-PKcs) gene rearrangements: V(D)J and class switch recombination (CSR). that regulates end joining by phosphorylating other proteins (in- In contrast to V(D)J recombination, which is almost exclusively de- cluding itself), and the ligase complex containing XLF, XRCC4, pendent on nonhomologous end joining (NHEJ), CSR can occur in and DNA ligase 4. Also involved is a growing list of auxiliary NHEJ-deficient cells via a poorly understand backup pathway (or factors, including end processing nucleases (e.g., Artemis) and pathways) often termed alternative end joining (A-EJ). Recently, polymerases (μ and λ), polynucleotide kinases, 53BP1, and many several components of the single-strand DNA break (SSB) repair DNA damage response proteins (ATM, H2AX, Chk1, etc.). machinery, including XRCC1, have been implicated in A-EJ. To de- Although both V(D)J and class switch recombination rely on termine its role in A-EJ and CSR, Xrcc1 was deleted by targeted the generation and repair of DSBs, the dependence on NHEJ mutation in the CSR proficient mouse B-cell line, CH12F3. Here we is distinctively different between these two reactions. Whereas demonstrate that XRCC1 deficiency slightly increases the efficiency RAG-generated DSBs are almost exclusively joined by NHEJ, S of CSR. More importantly, Lig4 and XRCC1 double-deficient cells region breaks can be joined in NHEJ-deficient cells at a reduced switch as efficiently as Lig4-deficient cells, clearly indicating that but still considerable rate (4–6). DSB repair in the absence of an XRCC1 is dispensable for A-EJ in CH12F3 cells during CSR. intact NHEJ system has been collectively termed alternative end joining (A-EJ) (3, 4). A-EJ could be a component-substitution DNA double-strand break (DSB) is one of the most severe form of NHEJ or a distinct pathway (or pathways) (7–9). So far, Aforms of DNA damage and can result in chromosome loss or components of A-EJ have not been conclusively defined. A-EJ translocations. A variety of endogenous and exogenous sources has attracted much research attention recently because of its can induce DSBs, including ionizing radiation, reactive oxygen implication in chromosomal translocations that could lead to species, and some chemicals. On the other hand, physiological oncogenic transformations (10). Many translocation junctions processes during lymphocyte development such as V(D)J and have microhomology (DNA sequences that can be assigned to Ig class switch recombination (CSR) rely on DSBs to rearrange either of the two ends), which is characteristic of A-EJ. A-EJ is genetic information in somatic cells. sometimes called microhomology-mediated end joining (9). V(D)J recombination is a site-specific DNA recombination However, the presence of microhomology at the junction is not initiated by the RAG proteins, which are evolved from an an- a criterion to distinguish A-EJ from NHEJ, as NHEJ also prefers cient DNA transposase. The RAG complex recognizes specific short homology between the two ends (9, 11, 12). DNA sequences called recombination signal sequences (RSS) The final stage of DSB repair depends on DNA ligases. Ver- and cuts the DNA on one side of the RSS. The ensuing repair of tebrates have three ATP-dependent ligases (I, III, and IV) (13). the four DNA ends that are produced from a pair of cleavage Lig4 appears to be dedicated to NHEJ as no other function has events results in joining of subexonic coding fragments to form been described for Lig4 outside the NHEJ realm. XRCC4 an exon encoding the antigen-binding domain of a B- or T-cell complexes with and stabilizes Lig4. Deficiency of either Lig4 or receptor. In contrast, CSR in antigen-stimulated mature B cells XRCC4 essentially abolishes NHEJ. Conceivably, joining of S is a regionally specific recombination between two repetitive re- region breaks in Lig4-deficient cells must depend on Lig1 and/or gions [called switch (S) regions] that precede each of the con- Lig3. Normally, Lig1 complexes with proliferating cell nuclear stant regions (1). Looping out intervening sequences between antigen and is recruited to the replication fork to join Okazaki two S regions allows the expression of a new constant region that fragments during DNA replication (13). Lig3 complexes with was further downstream and results in a switch of Ig class (or XRCC1 and was generally considered the ligase involved in isotype) from IgM to IgG, IgE, or IgA (2). CSR is initiated by single-strand break (SSB) repair pathways. It has been shown activation-induced cytidine deaminase (AID) that converts DNA that cellular Lig3 activity is dependent on XRCC1 (14), which is cytosines into uracils at S regions. Through mechanisms that are a scaffold protein that interacts with many other DNA repair not yet fully understood, repair of AID-generated uracils in the S factors (e.g., Parp1, Pol β, APE1, PNKP, aprataxin, etc.) (13). region ultimately results in DSBs (2), which serve as critical inter- Although the traditional view of Lig3 in nuclear DNA repair has mediates in an overall cut-and-paste chromosomal deletion (2). In vertebrate cells, DSB repair mechanisms generally fall into two major categories: homologous recombination (HR) and Author contributions: K.Y. designed research; L.H. and W.M. performed research; L.H., nonhomologous end joining (NHEJ) (3). HR relies on the pres- W.M., and K.Y. analyzed data; and K.Y. wrote the paper. ence of another copy of DNA sequences that are highly similar to The authors declare no conflict of interest. the one harboring the DSB. Copying genetic information from the *This Direct Submission article had a prearranged editor. intact copy allows high-fidelity repair of the DSB. In complex 1L.H. and W.M. contributed equally to this work. genomes rich in repetitive DNA sequences, HR is restricted to S 2Present address: Department of Biotechnology, Dalian Medical University, Dalian and G2 phase of the cell cycle when sister chromatids are available 116044, China. as a source of homology. In contrast, NHEJ is the major DSB 3To whom correspondence should be addressed. E-mail: [email protected]. repair pathway that operates throughout the cell cycle. The core This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. NHEJ components include the Ku70/86 heterodimer that binds to 1073/pnas.1120743109/-/DCSupplemental.

4604–4608 | PNAS | March 20, 2012 | vol. 109 | no. 12 www.pnas.org/cgi/doi/10.1073/pnas.1120743109 Downloaded by guest on September 26, 2021 been recently challenged (15, 16), the importance of XRCC1 in from XRCC1+/+ cells, consistent with the lack of effect hap- SSB repair has been well established. lodeficiency has on XRCC1 expression levels in these cells. Because A-EJ relies on microhomology and because both Lig1 and Lig3 are SSB ligases, we considered the possibility that A-EJ Slightly Increased CSR in XRCC1-Deficient Cells. To determine results from a pair of XRCC1-dependent SSB ligations at DNA whether XRCC1 is involved in CSR, cell growth and CSR efficiency were compared between XRCC1 proficient (+/Δ) and ends containing long nucleotide overlaps. In addition, XRCC1 Δ Δ deficient (Δ/Δ) cells. XRCC1 / cells grow more slowly than wild- has recently been implicated in A-EJ (17, 18), along with other +/Δ SSB repair factors (19). To assess the role of XRCC1 in CSR and type or XRCC1 cells, as assessed by live cell counting, re- fi gardless of the presence or absence of cytokine stimulation (Fig. A-EJ, we deleted Xrcc1 in wild-type and Lig4-de cient CH12F3 Δ/Δ cells by targeted mutation. Here we demonstrate that XRCC1 2A). We considered that the apparent slow growth of XRCC1 fi fi cells might be attributable to increased cell death because of the de ciency alone slightly enhances the ef ciency of CSR. More Δ/Δ importantly, deletion of both Lig4 and Xrcc1 genesdoesnot inability of XRCC1 cells to repair oxidation-associated DNA fi damage. However, shifting cells from 20% oxygen to 3% only abolish CSR. In fact, cells de cient in both XRCC1 and Lig4 Δ/Δ switch as efficiently, or slightly better than, Lig4-deficient cells. partially improve the growth of XRCC1 cells. Although the mechanism underlying the slow growth These data demonstrate unequivocally that XRCC1 is dis- Δ Δ of XRCC1 / cells has not been determined, it is unlikely that pensable for A-EJ during CSR. Δ Δ XRCC1 / cells have an intrinsic proliferation defect because Δ Δ XRCC1 / cells undergo robust CSR (Fig. 2B), which is known to Results and Discussion Δ Δ be cell-proliferation dependent. In fact, XRCC1 / cells switch Gene Targeting of XRCC1. XRCC1 is a DNA repair factor that has Δ more efficiently than XRCC1+/ cells (Figs. 2B and 3D). The in- recently been implicated as a component of A-EJ (17, 18). Be- crease in CSR efficiency in the absence of XRCC1 is small cause XRCC1 is essential for mouse embryonic development (∼19%), but consistent (P = 0.014). The mechanism by which (20), there is a lack of direct genetic evidence for its involvement XRCC1 deficiency promotes CSR efficiency is unknown. One fi Δ Δ in CSR and A-EJ. XRCC1-de cient Chinese hamster ovary cell possible explanation is that XRCC / cells accumulate more SSBs lines are viable (21), suggesting that XRCC1 is dispensable for in S regions that enhance the chance of DSB formation. In this somatic cell growth. We therefore attempted gene targeting of regard, our data are also consistent with previous reports regarding Xrcc1 in a mouse B-cell line (CH12F3) that is capable of robust fl – an inhibitory effect on CSR by several SSB repair factors (22, 23). cytokine-dependent CSR. DNA sequences anking exons 4 6of To determine whether XRCC1 deficiency affects switch fi Δ Xrcc1 were ampli ed from CH12F3 genomic DNA and used as junction microhomology, postswitched clones of XRCC1+/ and Δ Δ homology blocks for gene targeting (Fig. 1A). In successfully XRCC1 / cells were isolated and each junction was amplified – fl targeted clones, exons 4 6 are replaced with a oxed puromycin- individually (Fig. S1). The average nucleotide overlap is 2 bp resistant gene expression cassette that can be excised by Cre- regardless of the genotype, and there is no obvious difference LoxP reaction (Fig. 1 A and B). Removal of exons 4–6 results in with regard to switch junction microhomology (Fig. 4). This a frameshift of all downstream exons and yields a null allele as observation is consistent with a recent report from Boboila et al. evidenced by the complete lack of XRCC1 as shown by Western (24), but is at odds with another study that showed a slight de- blot analysis (Fig. 1C). In contrast to XRCC1 haplodeficient crease of nucleotide overlaps from 2.5 bp in wild-type to 1.7 bp in mouse primary B cells (18), deletion of one copy of Xrcc1 in XRCC1 haplodeficient cells (18). Δ Δ CH12F3 cells does not alter XRCC1 protein levels. Although In this study, XRCC1 / junctions have more insertions; this Δ Δ there is a moderate reduction of Lig3 protein in XRCC1 / cells modest effect must be interpreted cautiously. In these analyses, it (consistent with a role of XRCC1 in stabilizing Lig3) (14), Lig3 is difficult to differentiate a 1-bp insertion from a point mutation. Δ Δ Δ protein levels in XRCC1+/ cells (Fig. 1C) are indistinguishable There are six 1-bp insertions in the XRCC1 / group compared IMMUNOLOGY

Fig. 1. Gene targeting of Xrcc1.(A) Genomic organization of the wild-type and targeted Xrcc1 loci. Gray boxes indicate exons. Open block arrow (Puro) indicates expression cassette of puromycin-resistant gene. Shaded block arrow (DTA) indicates diphtheria toxin A chain. BamH I restriction sites are indicated by “B.” Probes used in Southern blot analysis are depicted at the Top. Plus indicates wild-type allele. P and Δ indicate targeted allele with or without the puro cassette, respectively. (B) Southern blot analysis of BamH I-digested genomic DNA from wild-type and targeted cells. Genotypes, sizes of bands, and probes are indicated. (C) Western blot analysis of protein expression in wild type (WT), XRCC1 haplodeﬁcient (+/Δ), and XRCC1-deﬁcient (Δ/Δ) cells. The same blot was stripped and reprobed with different monoclonal antibodies as indicated.

Han et al. PNAS | March 20, 2012 | vol. 109 | no. 12 | 4605 Downloaded by guest on September 26, 2021 Fig. 2. Proliferation and CSR of XRCC1-proficient and -deficient cells. (A) Propagation of live cells (trypan exclusion) over a span of 3 d in unstimulated (−CIT) Δ Δ Δ or stimulated (+CIT) cultures for wild-type (WT), XRCC1-proficient (XRCC1+/ ), and -deficient (XRCC1 / ) cells, respectively. Error bars indicate SD of three independent experiments. (B) Representative FACS analysis of CSR by surface staining of IgA after 72 h of growth in the absence or presence of cytokines (CIT). Numbers in boxed areas indicate percentages.

Δ − − with one in the XRCC1+/ group. In addition, junction 10 in the (IgM IgA ) cells (Fig. 3C). These cells are likely the ones that are Δ Δ XRCC1 / group also has this ambiguity (insertion vs. mutation), undergoing CSR because they are more prominent in Lig4-defi- and junction 19 could have an insertion or a small deletion in the cienct cells (Fig. 3C), consistent with our previous observations Sα nearby the junction. These ambiguities are intrinsic in ana- that CSR in Lig4-deficient cells is kinetically slower than in wild- lyzing switch junction; this is even more problematic when ana- type cells. This double-negative population is even more exag- Δ Δ Δ Δ lyzing junctions between highly homologous S regions, as is the gerated in XRCC1 / Lig4 / cells, which raises the possibility case in our study. Although XRCC1 deficiency might minimally that some of these cells harbor large deletions due to the excessive alter the frequency of mutations (18) or small deletions/inser- DNA damage in the absence of XRCC1 and insufficient end tions because of its role in single-strand break repair, these data joining in the absence of Lig4. This possibility might explain our suggest that this is not a major affect. difficulty in obtaining switch junctions from this genotype. To rule out the possibility that the observed effects were due CSR in XRCC1 and Lig4 Double-Deficient Cells. XRCC1 was recently to a clonal variation, genetic complementation experiments were Δ Δ implicated as a component of A-EJ (17, 18). If that were the case, carried out to transduce Lig4 or Xrcc1 cDNA into XRCC1 / Δ Δ Δ Δ deletion of Xrcc1 in NHEJ-deficient cells (e.g., Lig4 / ) should Lig4 / cells by recombinant retroviruses. Expression of Lig4 or inhibit CSR. To test this hypothesis, we targeted Xrcc1 in Lig4- XRCC1 from transduced cDNA was confirmed by Western blot Δ Δ deficient cells (Lig4 / )(5).LackofbothLig4andXRCC1protein analysis (Fig. 5A). As expected, complementation by Lig4 cDNA Δ Δ Δ Δ in XRCC1 / Lig4 / cells was confirmed by Western blot analysis restored robust CSR (Fig. 5B). In contrast, transduction with Δ Δ Δ Δ (Fig. 3A). XRCC1 / Lig4 / cultures grow considerably slower Xrcc1 cDNA resulted in slightly diminished CSR efficiency Δ Δ than XRCC1 / cultures based on live cell counting (Fig. 3B). compared with an empty virus control (Fig. 5B), consistent with Δ Δ Consistent with previous studies (4, 5), CSR in Lig4 / cells is re- our earlier observations. duced to 25–40% compared with wild-type cells after 3 d of cyto- A recent study implied that XRCC1 might be involved in A-EJ Δ Δ kine stimulation. As can be seen, deletion of Xrcc1 in Lig4 / cells during CSR. The implication was based on a small decrease of does not further reduce CSR efficiency (Fig. 3 C and D); we con- switch junction microhomology between wild-type and XRCC1 cluded that XRCC1 is dispensable for A-EJ. In fact, CSR efficiency haplodeficient primary B cells (18); however, CSR efficiency was Δ Δ Δ Δ in XRCC1 / Lig4 / cells is slightly higher than in cells deficient in not affected by XRCC1 haplodeficiency. Our study reaches only Lig4 (Fig. 3 C and D), consistent with our earlier observation a different conclusion. Here, we have introduced targeted dele- that XRCC1 deficiency modestly promotes CSR. Staining for both tions of Xrcc1 on both alleles in CH12F3 cells; we find (un- IgM and IgA isotypes revealed a population of double-negative equivocally) that XRCC1 is not required for CSR efficiency and

4606 | www.pnas.org/cgi/doi/10.1073/pnas.1120743109 Han et al. Downloaded by guest on September 26, 2021 Fig. 3. CSR in cells deficient in both XRCC1 and Lig4. (A) Western blot analysis of protein expression. (Lane 1) Wild type; (lane 2) XRCC1Δ/Δ;(lane3)Lig4Δ/Δ;(lane4) Lig4Δ/Δ XRCC1Δ/Δ. The same blot was stripped and reprobed with different antibodies as indicated. (B) Propagation of live cells (trypan exclusion) over a span of 3 d in unstimulated (−CIT) or stimulated (+CIT) cultures. Error bars indicate SD of three independent experiments. (C) Representative FACS analysis of CSR by surface staining of IgM (PE labeled) and IgA (FITC labeled) after 72 h growth in the absence or presence of cytokines (CIT). Numbers in boxed areas indicate percentages. (D) Relative CSR efficiency of various genotypes. Error bars indicated SD of at least three independent experiments (wild-type CSR efficiency set to 100%). P value Δ Δ Δ was calculated by two-tailed paired t test. Comparison between XRCC1+/ and XRCC1 / is based on five independent experiments.

does not affect the fine structure of joined switch regions, strongly The moderate increase in CSR efficiency in the absence of arguing that XRCC1 is also dispensable for A-EJ. Moreover, these XRCC1 suggests that accumulation of unrepaired SSBs in S re- conclusions are in excellent agreement with those in a recent study gions promotes CSR, perhaps by increasing the chance of DSBs. from Alt and colleagues who also disrupted both alleles of Xrcc1 in Our data do not rule out a possible role of Lig3 in A-EJ and CSR. However, given the recent findings regarding the nonessential

switching B cells (24). IMMUNOLOGY role of Lig3 in nuclear DNA repair (15, 16) and the dispensability of its cofactor XRCC1 in A-EJ shown in this study, it seems more likely that Lig1 is the ligase that operates in A-EJ in the absence of intact NHEJ. Alternatively, both Lig1 and Lig3 may participate in A-EJ, and elimination of either one alone may not reveal a defect. Materials and Methods Reagents. Lig4 antibody was kindly provided by David Schatz (Yale University, New Haven, CT). Lig3 antibody was purchased from BD Biosciences (611876). XRCC1 antibody was purchased from Abcam (ab1838). Antibody against mouse β-actin was purchased from Santa Cruz (sc-47778).

Cell Culture and CSR Assay. CH12F3 cells were cultured in RPMI1640 medium supplemented with 10% (vol/vol) FBS and 50 μMofβ-mercaptoethanol. For CSR assay, healthy CH12F3 cells were seeded at 5 × 104 cells/mL in the presence of 1 μg/mL anti-CD40 antibody (eBioscience; 16–0402-86), 5 ng/mL of IL-4 Fig. 4. Length of microhomology in switch junctions. Percentage of switch (R&D Systems; 404-ML), and 0.5 ng/mL TGF-β1 (R&D Systems; 240-B) and junctions with the indicated nucleotide overlap (excluding nucleotide additions). grown for 72 h. Cells were stained with a FITC-conjugated antimouse IgA

Han et al. PNAS | March 20, 2012 | vol. 109 | no. 12 | 4607 Downloaded by guest on September 26, 2021 Δ Δ Δ Δ Fig. 5. Genetic complementation of Lig4 / XRCC1 / cells. (A) Western blot analysis of protein expression in wild-type (WT) cells and Lig4Δ/Δ XRCC1Δ/Δ cells infected with retroviruses car- rying Lig4, XRCC1, or no cDNA, as indicated. The same blot was stripped and reprobed with different antibodies as indicated. (B) Representative FACS analysis of CSR by surface staining of IgM (PE labeled) and IgA (FITC labeled) after 72 h growth in the absence or presence of cytokines (CIT). Numbers in boxed areas indicate percentages.

antibody (BD Biosciences; 559354) and analyzed on a ﬂow cytometer (LSR II; supernatants were harvested 48 h after transfection and used to infect BD Biosciences). CSR efﬁciency was determined as the percentage of XRCC1Δ/Δ Lig4Δ/Δ cells. Infected cells were selected by puromycin and sub- IgA+ cells. jected to CSR assays.

XRCC1 Gene Targeting. Two homology blocks (2.1 and 5.8 kb) were PCR Switch Junction Analysis. Individual IgA+ clones were isolated by limiting ampliﬁed from CH12F3 genomic DNA and used as homology blocks for dilutions of cytokine-stimulated cultures in 96-well plates. Switch junctions gene targeting. Gene targeting procedures have been reported previously were ampliﬁed with primers KY761 (5′-AACTCTCCAGCCACAGTAATGACC-3′) (5, 25, 26). and KY743 (5′-GAGCTCGTGGGAGTGTCAGTG-3′). PCR products were se- quenced at the genomic core facility at Michigan State University. Genetic Complementation. Lig4 or XRCC1 coding region sequences were cloned into the retroviral transfer vector pMSCV-puro (Clontech Laborato- ACKNOWLEDGMENTS. This work was supported by National Institutes of ries). Recombinant retroviruses were packaged in the Phoenix cell line. Viral Health Grant R01 AI081817 (to K.Y.).

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