The C-Terminal Domain of Cernunnos/XLF Is Dispensable for DNA Repair In Vivo

The core nonhomologous end-joining DNA repair pathway is composedof seven factors: Ku70, Ku80, DNA-PKcs, Artemis, XRCC4 (X4),DNA ligase IV (L4), and Cernunnos/XLF (Cernunnos). AlthoughCernunnos and X4 are structurally related and participate inthe same complex together with L4, they have distinct functionsduring DNA repair. L4 relies on X4 but not on Cernunnos forits stability, and L4 is required for optimal interaction ofCernunnos with X4. We demonstrate here, using in vitro-generatedCernunnos mutants and a series of functional assays in vivo,that the C-terminal region of Cernunnos is dispensable for itsactivity during DNA repair.

INTRODUCTION

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INTRODUCTION

Nonhomologous end joining (NHEJ) represents the main pathwayfor solving DNA double-strand breaks (DSB) in mammals. The coreof the NHEJ pathway is composed of seven proteins: Ku70, Ku80,DNA-dependent protein kinase catalytic subunit (DNA-PKcs), Artemis,XRCC4 (X4), DNA ligase IV (L4), and Cernunnos/XLF (Cernunnos)(reviewed in reference 18). Briefly, the Ku70-Ku80 heterodimerbound to broken DNA recruits the serine/threonine kinase DNA-PKcs.DNA-PK phosphorylates downstream effectors such as the nucleaseArtemis. The X4-L4 complex carries out the final joining ofsynapsed DNA ends in association with Cernunnos (2, 6). Cernunnoswas identified through cDNA functional complementation of afibroblast cell line obtained from a human patient with immunedeficiency and microcephaly (5). The same factor, called XLF,was identified through a yeast two-hybrid screen with X4 asa bait (2).

Cernunnos is structurally related to X4 and consists of a globularhead domain followed by a coiled-coil region and an unstructuredC-terminal domain (2, 6, 12). One major difference between thestructures of X4 and Cernunnos appears in the coiled-coil region.While this region is linear in X4, a hinge in the middle ofthe coiled-coil of Cernunnos folds back the end of the domaintoward the head (3, 14).

Cernunnos interacts with the X4-L4 complex in vivo and in vitro(2, 6). Cernunnos and X4 both appear to interact directly withL4, but the Cernunnos-L4 interaction seems to be very weak (7).In addition, purified Cernunnos associates with DNA in a sequence-independentmanner (20) but in a DNA length-dependent manner, like X4 (15).Although the X4-L4 complex can ligate DNA in vitro (10), Cernunnosfurther improves this activity (11, 15, 16, 20). Cernunnos seemsimportant, in particular, for the ligation of mismatched ornoncohesive DNA ends, but not for that of compatible DNA ends,in vitro (10, 20).

Cernunnos is therefore a "core" NHEJ component, but limitedinformation is available about its precise function during DNArepair in vivo. We show here that although X4 and Cernunnosshare sequence and structural homologies, their functions aredistinct. We also demonstrate that Cernunnos requires L4 forits association with X4. Lastly, the Cernunnos C terminus isdispensable for DNA repair following ionizing radiation (IR)and V(D)J recombination.

MATERIALS AND METHODS

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MATERIALS AND METHODS

Cells. Cernunnos-deficient cells (from patient P2 in reference 5) andOTel control cells, which are skin fibroblasts (17), are transformedwith simian virus 40 and immortalized with telomerase. X4 knockout(KO) and wild-type (WT) mouse embryonic fibroblasts (MEFs),generated from embryos at embryonic day 14.5, were immortalizedafter cell transfection with plasmid pLasWT expressing simianvirus 40 T antigen as described previously (23). The L4-defectiveN114P2 cells and the parental cell line Nalm6 were gifts fromM. R. Lieber (9).

Cloning. WT Cernunnos, deletion mutants (see Fig. 3A), and the WT X4Orf were PCR amplified from a cDNA library and cloned into thepcDNA3.1 vector (Invitrogen). Chimeras C1X2, X1C2 (see Fig.4A), and C1GFP (see Fig. S7A in the supplemental material) wereobtained using a double PCR amplification procedure and werecloned into the same vector. All constructs were subcloned intothe pMND-Myc-internal ribosome entry site (IRES)-green fluorescentprotein (GFP) retroviral vector by using the BD In-Fusion protocol(Clontech).

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FIG. 3. Analysis of Cernunnos domains. (A) Schematic representation of WT Cernunnos and its mutants. (B) Mean results of three V(D)J recombination assays on chromosomal substrates integrated into Cernunnos-deficient cells (Cer-RSS), calculated as described in the legend to Fig. 1A. (C) Survival of Cernunnos-deficient cells, transduced with the pMND vector containing WT or mutant (1-230, 1-178, 1-167, or 1-141) Cernunnos or WT X4, 14 days after bleomycin treatment. Results are expressed as in Fig. 1C. (D) Percentage of IRIF-positive cells. Forty cells, transduced (GFP⁺) or not (GFP^–) and either left untreated or subjected to IR, were scored for DAPI, GFP, and ${gamma}$ H2AX foci 2 h and 24 h after IR (2 Gy). A cell was considered IRIF positive if it had more than 10 IRIFs. (E) Whole-cell lysates (WCL) from 293T cells transfected with V5-tagged WT or mutant (1-230, 1-178, or 1-167) Cernunnos were immunoprecipitated with irrelevant (IgG) and anti-V5 rabbit antibodies. (F) 293T cells were cotransfected with Myc-tagged WT Cernunnos and V5-tagged WT or mutant (1-230, 1-178, or 1-167) Cernunnos. For panels E and F, the immunoprecipitates were analyzed by WB as indicated.

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FIG. 4. Analysis of chimeras between Cernunnos and X4. (A) Summary of chimeras resulting from swapping of domains between WT Cernunnos and X4. (B) Mean results of three V(D)J recombination assays on chromosomal substrates integrated into Cernunnos-deficient cells (Cer-RSS). Results are expressed as described in the legend to Fig. 1A. (C) Survival of Cernunnos-deficient cells, transduced with the pMND-Myc-IRES-GFP vector containing WT Cernunnos, C1X2, X1C2, or WT X4, 14 days after bleomycin treatment. Results are expressed as in Fig. 1C. (D) Survival of Cernunnos-deficient cells transduced with an empty MND vector or with the vector carrying WT Cernunnos or C1X2, without any treatment. Cells were monitored for 21 days. J1, day 1; J21, day 21. Asterisks indicate statistical significance (P < 0.05). (E) 293T cells were transfected with V5-tagged WT Cernunnos, C1X2, X1C2, or WT X4. Whole-cell lysates (WCL) were immunoprecipitated with irrelevant (IgG) and anti-V5 rabbit antibodies. (F) 293T cells were cotransfected with Myc-tagged WT Cernunnos and V5-tagged WT Cernunnos, C1X2, or X1C2. For panels E and F, WCL were treated as for Fig. 3E.

V(D)J recombination assays. In-chromosome V(D)J recombination assays were performed as previouslydescribed (5, 17). For complementation analysis, 2.5 µgof the plasmid of interest was cotransfected during the V(D)Jassay. Extrachromosomal V(D)J recombination assays were performedwith pBlueRec (13) in MEFs or with the pRecCS substrates (5)in human fibroblasts.

WB and IP experiments. Cernunnos, X4, truncation mutants, and chimera-expressing constructswere used to transfect 293T cells. Immunoprecipitations (IP)were performed as described previously (6) on precleared lysatesusing polyclonal rabbit anti-immunoglobulin G (anti-IgG) (SantaCruz), anti-V5 tag (Abcam), anti-Myc tag (Sigma-Aldrich), anti-L4(also used for revelation; Abcam), and anti-X4 (Serotec) antibodies.Immunoprecipitates were analyzed by Western blotting (WB) withmurine antibodies directed against the V5 tag (Invitrogen),the Myc tag (Santa Cruz), and X4 (Genetex).

Bleomycin sensitivity assay. Virus supernatants, produced from pMND-Myc-IRES-GFP constructs,containing Cernunnos (WT or 1-230, 1-178, 1-167, or 1-141 mutant[where numbers represent the positions of the amino acids ineach mutant]) cDNA or WT or chimeric (C1X2 or X1C2) X4 cDNA,were used to transduce Cernunnos-deficient cells or X4KO orWT MEFs as described previously (5) in order to generate stablecell lines expressing C-terminal Myc-tagged proteins and GFP.Established cell lines were incubated at 37°C with mediumalone or with medium containing increasing doses of bleomycinfor 1 h, after which the medium was replaced by complete medium.Cells were split regularly and analyzed for GFP expression byfluorescence-activated cell sorting over 14 days (Becton DickinsonFACScan flow cytometer). The ratio of GFP^– (untransduced)to GFP⁺ (transduced) cells at the initiation of culture wasset to 1.

Immunofluorescence detection of IR-induced foci (IRIFs). Cernunnos-deficient fibroblasts were transduced with the emptypMND-Myc-IRES-GFP retroviral vector or the pMND-Myc-IRES-GFPvector containing Cernunnos (WT or the 1-230 deletion mutant),seeded on cover slides, and gamma-irradiated (2 Gy). DNA repairfoci were labeled either with a monoclonal mouse anti- ${gamma}$ H2AX antibodyor a polyclonal rabbit anti-53BP1 antibody, revealed by an AlexaFluor 546-coupled goat anti-mouse or anti-rabbit secondary antibody(Molecular Probes), 2 h and 24 h after IR, as described previously(5). Slides were counterstained with 0.1 µg/ml of DAPI(4',6'-diamidino-2-phenylindole), mounted in Fluorsave (Calbiochem),and viewed by epifluorescence microscopy.

RESULTS AND DISCUSSION

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RESULTS AND DISCUSSION

Separate functions of Cernunnos and X4. We analyzed the respective roles of X4 and Cernunnos duringV(D)J recombination by using an in-chromosome V(D)J recombinationassay (5). The Cernunnos-deficient cell line carrying an intrachromosomalsubstrate (Cer-RSS) rearranges the substrate upon transfectionwith RAG1 and RAG2 and ectopic expression of WT Cernunnos (setas 100% efficiency), as expected, but not upon overexpressionof X4 (Fig. 1A). Conversely, X4 but not Cernunnos complementsthe V(D)J defect in X4KO MEFs in a V(D)J extrachromosomal assay(Fig. 1B). During general DNA repair, a selective advantageis conferred by ectopic expression of Cernunnos only in Cernunnos-deficienthuman fibroblasts and by ectopic expression of X4 only in X4KOMEFs upon treatment with the radiomimetic drug bleomycin, asmanifested by the dose-response increase in the GFP ratio followingtreatment (Fig. 1C and D).

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FIG. 1. Evidence for distinct functions of Cernunnos and X4. (A) Mean results of three V(D)J recombination assays on chromosomal substrates integrated into Cernunnos-deficient fibroblasts (Cer-RSS) and WT OTel fibroblasts (OTel-RSS). Relative V(D)J recombination is calculated based on the recombination frequency obtained by transfection with RAG1 and RAG2 for OTel-RSS and with RAG1, RAG2, and Cernunnos for Cer-RSS cells. ns, nonsignificant difference (P > 0.005); *, statistically significant difference (P < 0.005); ***, highly statistically significant difference (P < 0.001). (Inset) Expression of V5-tagged Cernunnos and X4 constructs in 293T cells. (B) V(D)J recombination assay in X4KO and WT MEFs using the pBlueRec system. Recombination of the substrate was analyzed by PCR. (C, D, and E) Survival of Cernunnos-deficient cells (C) and X4KO (D) and WT (E) MEFs transduced with the pMND retrovirus containing Cernunnos or X4 14 days after bleomycin treatment.

We conclude that Cernunnos and X4, although structurally related,exert specific and not interchangeable functions during DNADSB repair. This is in accord with the lack of redundancy ofthese two factors in vivo, both in KO mice and in humans. Attestingto their specific functions during DNA repair, their crystalstructures reveal important differences (3, 14). Notably, X4has a linear coiled-coil domain with which L4 interacts, whereasthe Cernunnos coiled-coil forms a hinge that folds back theC terminus of the coiled-coil toward the head domain. The functionalconsequence of this major difference is not clearly establishedyet, but at least, it would mask the sequence known in X4 tocorrespond to the L4-interacting region.

Interactions within the ligation complex. Cernunnos, X4, and L4 are part of the same complex (2, 6, 21).It is also known that in the absence of X4, L4 is not stable(4). A defect in Cernunnos protein does not result in L4 destabilization(6) (Fig. 2B), and the overexpression of Cernunnos in X4KO MEFscould not rescue L4 stability (Fig. 2A). The ectopic expressionof Cernunnos or X4 has no effect on L4 expression in Cernunnos-deficientcells (Fig. 2B). We conclude that L4 does not rely on Cernunnosfor its stability in vivo. This is in accord with a weak Cernunnos-L4interaction (3, 15), even though surface plasmon resonance studiesusing purified proteins in vitro have demonstrated such an interaction(14).

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FIG. 2. The Cernunnos-X4-L4 complex. (A) Whole-cell lysates (WCL) from X4KO MEFs, either left untransfected or transfected with a V5-tagged Cernunnos or X4 expression construct, and from L4KO and WT MEFs were analyzed by WB with the indicated antibodies. (B) WCL from WT fibroblasts and Cernunnos-deficient cells transfected with a V5-tagged Cernunnos or X4 expression construct as for panel A. (C) WCL from N114P2 (L4KO) or Nalm6 (parental) cells, transduced (+) or not (–) with pMND-Cernunnos-Myc-IRES-GFP, were immunoprecipitated with irrelevant antibodies (IgG) or anti-Myc antibodies and then blotted and revealed as indicated. *, endogenous X4; **, V5-tagged X4; ***, IgG heavy chain.

To further understand their interdependence within the X4-L4-Cernunnoscomplex, we analyzed the behavior of X4 and Cernunnos in theabsence of L4 by using the N114P2 cell line, an L4-defectivederivative of the human Nalm6 pre-B-cell tumor (9). IP of exogenousCernunnos protein from WT Nalm6 cells coprecipitated both X4and L4 (Fig. 2C), as previously described (2, 6). In contrast,IP of Cernunnos from the L4-deficient N114P2 cells failed topull down endogenous X4 to a detectable level. Knockdown ofL4 by short hairpin RNA has been shown to reduce the recruitmentof Cernunnos to the site of DNA damage (21). The opposite isnot true, since both X4 and L4 are recruited at DNA DSB in theabsence of Cernunnos.

We conclude from these experiments that the formation of a completeX4-L4-Cernunnos complex relies both on X4 for the stabilizationof L4 and on L4 for the inclusion of Cernunnos within the complex.Given the degradation of L4 in X4-deficient cells (4), it isnot possible to analyze directly the relation of L4 and Cernunnosin the absence of X4.

Important domains of Cernunnos. Given the evolutionary relation between X4 and Cernunnos andtheir specific functions during DNA repair, we aimed at identifyingtheir various functional domains by generating Cernunnos deletionmutants and X4-Cernunnos chimeras through domain swapping. Inall cases, the resulting mutants were analyzed for their stability,their participation in the formation of X4-L4-Cernunnos complexes,their activity during V(D)J recombination, and their capacityto ensure general DNA DSB repair in vivo.

We previously divided the Cernunnos protein into three structurallydistinct domains based on analysis of its sequence (6): thehead (amino acids [aa] 1 to 141), a coiled-coil central region(aa 142 to 230), and a nonstructured C-terminal region (aa 231to 299) (Fig. 3A). Only the full-length and C-terminus-deletedforms of Cernunnos complemented the V(D)J defect in Cer-RSScells (Fig. 3B) in transient transfection assays; all the otherdeletion mutants, extending within the coiled-coil central region,demonstrated sharp decreases in their activities. This was particularlythe case for the 1-178 and 1-167 mutants, which recapitulatemutations identified in Cernunnos patients (5). Likewise, theCernunnos head-only construct (1-141) was inefficient in V(D)Jrecombination, but its mirror form (the 142-299 construct) couldnot be tested due to poor stability (data not shown).

To differentiate between the qualitative and quantitative consequencesof these various mutations, we performed a pilot experimentto determine the minimal amount of WT Cernunnos protein requiredto fully complement Cer-RSS cells for V(D)J recombination anddetection of the protein by WB in 293T cells (see Fig. S1 inthe supplemental material). Several situations can be noted:full complementation is obtained by overexpression (4 µgto 2.5 µg of plasmid), and the level of complementationthen decreases slowly to 60% efficiency when the amount of plasmidis decreased to 1 µg. By use of an amount of plasmid withwhich the protein is no longer detectable by WB (1 µgto 0.05 µg), the activity remains constant at 60%. Lastly,below 0.01 µg of plasmid, the V(D)J activity is no longercomplemented. In summary, one can still achieve statisticallysignificant complementation of the V(D)J recombination in Cer-RSScells with an amount of Cernunnos plasmid that does not leadto detectable expression of the protein in 293T cells. Sincethe deletion mutants for which test results are shown in Fig.3 are detected comparably to the WT form in 293T cells, theirloss of activity is most likely qualitative rather than dueto the modulation of their expression level.

To gain further insight into the functionality of these truncationmutants, we analyzed their propensity to confer a selectivegrowth advantage on Cernunnos-deficient cells upon treatmentwith increasing doses of bleomycin (Fig. 3C). A selective advantage,as judged by an increase in the frequency of GFP⁺ transducedcells, was obtained with the WT and the 1-230 construct, whilethe GFP ratio did not change with the other mutants. Lastly,transduction of Cernunnos-deficient cells with Cernunnos-IRES-GFPresulted in the disappearance of ${gamma}$ H2AX IRIFs (a hallmark of NHEJdeficiency [5]) in GFP⁺ transduced cells 24 h after IR (Fig.3D; see also Fig. S2 in the supplemental material), attestingto efficient DNA repair in these cells. Likewise, the efficientDNA repair obtained with the Cernunnos 1-230 mutant was attestedto by the disappearance of ${gamma}$ H2AX foci 24 h following IR.

We determined the level of Cernunnos expression upon retroviraltransduction in fibroblasts by performing quantitative WB analysis(see Fig. S3 in the supplemental material). After normalizationto the level of endogenous Ku70 expression, it appears thatthe exogenous Cernunnos-Myc level in transduced Cernunnos-deficientcells is only about 1.17-fold higher than the endogenous Cernunnoslevel in WT cells. Complementation by the Cernunnos 1-230 constructis therefore not caused by strong overexpression.

Altogether, these experiments establish that the C-terminalregion (aa 231 to 299) of Cernunnos is dispensable for its fullDNA repair activity in vivo. In contrast to that of the 1-230mutant, the activities of the other truncated forms could notreally be tested (Fig. 3B, C, and D) due to protein instability(Fig. 3E and F). Although they were expressed to detectablelevels in 293T cells, they were undetectable by either WB orimmunohistochemistry (see Fig. S4A and B in the supplementalmaterial) in Cernunnos-deficient cells. This could explain thelack of complementation in these cells. IP studies (Fig. 3E and F)demonstrated that the Cernunnos 1-230 mutant can integrate theX4-L4 complex (Fig. 3E) and is capable of heterodimerizationwith full-length Cernunnos (Fig. 3F). This is in accord withthe results of interaction studies using purified proteins invitro (3) but contrasts with those of studies with Saccharomycescerevisiae, which suggested that the C terminus of Nej1, theyeast ortholog of Cernunnos (6, 8), is required for interactionwith Lif1p, the X4 ortholog (19).

The unstructured 68-aa C-terminal region confers DNA bindingactivity on Cernunnos in vitro (3). Moreover, this region isthe site of the DNA-PK- and ATM-dependent phosphorylation ofseveral serine residues (S245 and S251 being the major phosphorylationsites) (22). Yet alanine substitution at these phosphorylationsites (22) or the deletion of the entire region (our study)has no detrimental impact on NHEJ in vivo, suggesting that thisin vitro DNA binding activity may not be relevant during NHEJ.Indeed, the X4-L4 complex is even targeted at DNA breaks inthe absence of Cernunnos (21). This strikingly contrasts withNHEJ in yeast, where it was shown that deletion of the C-terminalregion of Nej1 severely compromised NHEJ activity (1). Lastly,the C-terminal region of Cernunnos does not participate in theoverall three-dimensional structure of the protein, as evidencedby the fact that crystals were obtained with C-terminus-deletedproteins (3, 14). The role of the C-terminal region in otherputative functions of Cernunnos will await further studies.

Swapping between Cernunnos and XRCC4. We performed domain-swapping experiments to define the regionsof X4 and Cernunnos critical for their functions in DNA repair.In the C1X2 chimera (Fig. 4A), we associated the head (aa 1to 141) of Cernunnos with the coiled-coil and C-terminal domainof X4, while in X1C2, the head of Cernunnos was replaced withthat of X4. The C1X2 chimera complemented the V(D)J recombinationdefect of Cer-RSS cells to about 60% of the WT level (Fig. 4B),whereas the X1C2 chimera was inefficient. However, none of thechimeras were proficient at complementing the DNA repair defectfollowing bleomycin treatment, as judged by the lack of anyincrease in the GFP index in mixed-population studies (Fig.4C). This suggests that the "head" of Cernunnos hooked to the"body" of X4 has enough activity to deal with a few DNA lesions[V(D)J recombination] but is not active enough to manage themany bleomycin-induced DNA DSB.

Since the Cernunnos head is not stable, we verified that theactivity of the C1X2 chimera was not solely the consequenceof protein stabilization by the X4 coiled-coil by replacingthis region with that of GFP (C1GFP) (see Fig. S5 in the supplementalmaterial). The C1GFP protein failed to complement either V(D)Jrecombination activity or DNA repair (tested by the persistenceof IRIFs); this failure was not due to a mislocalization ordestabilization of this protein.

Accordingly, C1X2 conferred a slight spontaneous selective advantageon Cernunnos-deficient cells (in the absence of an exogenousDNA DSB inducer), comparable to that obtained with WT Cernunnos(Fig. 4D). The absence of X1C2 activity in Cernunnos-deficientcells could be caused either by the very low stability of X1C2compared to those of C1X2 and WT Cernunnos (Fig. 4E and F) orby its lack of an important region present in the X4 coiled-coildomains (such as the L4 interaction region) or the head of Cernunnos.Surprisingly, while the C1X2 chimera could associate with endogenousL4 to some extent (Fig. 4E), it did not heterodimerize withWT Cernunnos (Fig. 4F), arguing for a role of the coiled-coilregion in Cernunnos dimerization. This lack of heterodimerizationcould participate in the low functional activity of this chimera.While the C1X2 chimera was partly functional in Cernunnos-deficientcells, it was completely inefficient in complementing the absenceof X4 in MEFs from X4KO mice. C1X2 also failed to complementV(D)J recombination activity in X4KO MEFs (see Fig. S6 in thesupplemental material) and to confer a selective advantage followingbleomycin treatment (see Fig. S7 in the supplemental material),in contrast to the phenotype reversion obtained with WT X4 inboth cases.

Altogether, these findings demonstrate that the heads and coiled-coilregions of Cernunnos and X4 are not interchangeable, and theysuggest specific roles for each in NHEJ. While a particularfunction of the coiled-coil region of X4 has been decipheredin the binding of L4, the precise functions of the heads haveyet to be determined, possibly through the identification ofadditional partners.

ACKNOWLEDGMENTS

This work was supported by institutional grants from INSERMas well as by grants from the Ligue National contre le Cancer(Equipe labelisée La Ligue), the INCa/CancéropôleIdF, the Agence Nationale pour la Recherche (ANR'06), and theCommissariat à l'Energie Atomique (LRC-CEA 40V). L.M.is supported by MENRT and P.R.-M. by the Association pour laRecherche sur le Cancer. P.R. is a scientist from the CentreNational de la Recherche Scientifique.

FOOTNOTES

* Corresponding author. Mailing address: INSERM U768, Hopital Necker-Enfants Malades, 149, rue de Sevres, 75015 Paris, France. Phone: 33 (0) 1 44 49 50 81. Fax: 33 (0) 1 42 73 06 40. E-mail: devillar{at}necker.fr

Published ahead of print on 22 December 2008.

Supplemental material for this article may be found at http://mcb.asm.org/.

REFERENCES

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