Functional overlaps between XLF and the ATM-dependent DNA double strand break response

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DNA Repair 16 (2014) 11–22

Contents lists available at ScienceDirect

DNA Repair

j ourna l ho me pa ge: www.elsev ier .com/ locate /dnarepai r

unctional overlaps between XLF and the ATM-dependent DNAouble strand break response�

ipul Kumar1, Frederick W. Alt ∗,1, Valentyn Oksenych ∗,1

oward Hughes Medical Institute, Program in Cellular and Molecular Medicine, Boston Children’s Hospital, Department of Genetics,arvard Medical School, Boston, MA 02115, United States

r t i c l e i n f o

rticle history:eceived 22 November 2013eceived in revised form 14 January 2014ccepted 24 January 2014vailable online 20 February 2014

eywords:ernunnosTM

a b s t r a c t

Developing B and T lymphocytes generate programmed DNA double strand breaks (DSBs) during theV(D)J recombination process that assembles exons that encode the antigen-binding variable regions ofantibodies. In addition, mature B lymphocytes generate programmed DSBs during the immunoglobulinheavy chain (IgH) class switch recombination (CSR) process that allows expression of different antibodyheavy chain constant regions that provide different effector functions. During both V(D)J recombinationand CSR, DSB intermediates are sensed by the ATM-dependent DSB response (DSBR) pathway, which alsocontributes to their joining via classical non-homologous end-joining (C-NHEJ). The precise nature of theinterplay between the DSBR and C-NHEJ pathways in the context of DSB repair via C-NHEJ remains under

NA-PKcsHEJ(D)J recombination3BP1

investigation. Recent studies have shown that the XLF C-NHEJ factor has functional redundancy withseveral members of the ATM-dependent DSBR pathway in C-NHEJ, highlighting unappreciated majorroles for both XLF as well as the DSBR in V(D)J recombination, CSR and C-NHEJ in general. In this review,we discuss current knowledge of the mechanisms that contribute to the repair of DSBs generated duringB lymphocyte development and activation with a focus on potential functionally redundant roles of XLFand ATM-dependent DSBR factors.

. Repair of programmed DSBs during lymphocyteevelopment and activation

Classical non-homologous end-joining (C-NHEJ) is a majorammalian double-strand break (DSB) repair pathway that is

ctive throughout the cell cycle but which has particular impor-ance in the G1 cell cycle phase. There are two programmedNA recombination processes in lymphocytes that employ C-NHEJ:(D)J recombination in developing B and T lymphocytes, and IgHlass switch recombination (CSR) in activated mature B lympho-ytes [1–4]. In addition to C-NHEJ, homologous recombination (HR)s the other known major DSB repair pathway in mammalian cellsnd predominates in the S/G2 phases of the cell cycle. Moreover,

ne or more alternative end-joining (A-EJ) DSB repair mechanismsave been observed to fuse at least certain classes of DSBs in the

� This is an open-access article distributed under the terms of the Creative Com-ons Attribution-NonCommercial-No Derivative Works License, which permits

on-commercial use, distribution, and reproduction in any medium, provided theriginal author and source are credited.∗ Corresponding authors.

E-mail addresses: [email protected] (F.W. Alt),[email protected] (V. Oksenych).1 All authors contributed equally to this work.

568-7864/$ – see front matter © 2014 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.dnarep.2014.01.010

© 2014 Elsevier B.V. All rights reserved.

absence of C-NHEJ; however, the overall functional significance ofA-EJ in the presence of C-NHEJ is yet to be determined [1,2].

In developing B and T cell lymphocytes, variable (V), diversity(D) and joining (J) gene segments are assembled to form V(D)J exonsthat encode the N-terminal “variable” portion of immunoglob-ulin (Ig) or T cell receptor (TCR) proteins that provide antigenbinding specificity. Together with downstream exons that encodeC-terminal “constant” region portions of Ig or TCR proteins, theyform complete Ig or TCR genes [5]. V(D)J recombination is initi-ated by the Recombination activating gene 1 and 2 proteins, whichtogether form an endonuclease (RAG) that recognizes short recom-bination signal (RS) sequences that flank V, D, or J coding sequences[6] and then introduces DSBs between an appropriate set of RSs andV, D, or J coding gene segments [7,8]. RAG generates DSBs at thesesequences in the form of blunt 5′-phosphorylated signal RS ends(“SEs”) and hairpin-sealed coding ends (“CEs”) [9]. The RAG com-plex holds cleaved V(D)J CEs and SEs in a post-cleavage synapticcomplex and, in a yet to be determined manner, directs joining oftwo appropriate CEs to each other to form V(D)J coding joins (“CJs”)and joining of the two SEs to each other to form signal joins (“SJs”),by the C-NHEJ pathway [10] (Fig. 1).

V(D)J recombination end-joining occurs exclusively by C-NHEJand does not occur at all in the absence of core C-NHEJ factors[11]. In this regard, RAG2, through unknown mechanisms, func-tions to exclude potential A-EJ repair pathways [12]. Consistent

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Fig. 1. V(D)J recombination in developing lymphocytes requires the RAG endonuclease and C-NHEJ factors. This figure depicts an example of a V(D)J recombination eventoccurring between a V gene segment and a rearranged DJ gene segment to form a complete V-D-J exon. (A) The RAG1/2 endonuclease (green ovals) is recruited to recombinationsignal (RS) sequences flanking a “V” gene segment and a “DJ” gene segment. The white triangle represents a 23-RSS, and the black triangle represents a 12-RSS. (B) AfterDSB induction, RAG holds hairpin-sealed coding ends and blunt signal ends in a post-cleavage synaptic complex. (C) The Ku70/Ku80 heterodimer (denoted by the light bluesemicircle and dark blue semicircle, respectively) recognizes and binds DNA ends. Hairpin-sealed coding ends require DNA-PKcs (light blue oval) and the nuclease Artemis( 4, yelK lex bs

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red circle) to open and process the hairpins before ligation. (D) DNA ligase 4 (Ligu-dependent manner. XLF (orange circle) is also recruited to the DNA repair compignal joins (left) and coding joins (right), respectively.

ith V(D)J joining occurring only by C-NHEJ, the entire V(D)Jecombination reaction occurs only in the G1 cell cycle phase,

restriction solidified by the degradation of RAG2 at the G1/Sransition [13,14]. Overall, the “V(D)J recombinase” consists of theymphocyte-specific RAG cleavage component and lymphocyte-pecific TdT diversification component (see below), along withore generally expressed C-NHEJ proteins that comprise the join-

ng component. As discussed below, recent studies also implicateTM-dependent DSBR factors as potential joining components of

he V(D)J recombinase. It is notable that during V(D)J recombina-ion, RAG must alter the normal process of C-NHEJ from simplyejoining the two ends of a given DSB to direct joins of CEs to CEs andEs to SEs from two separate DSBs, leading to an inversional or dele-ional outcome depending on the relative chromosomal orientationf the initiating RSs to each other [13,15,16].

Mature B cells are generated through the productive assembly ofn IgH V(D)J exon and an Ig light chain (IgL) VJ exon; which, respec-ively, lead to production of IgH and IgL proteins that together formhe B cell antigen receptor (“BCR”). In the peripheral immune sys-em (e.g. spleen and lymph nodes), mature B cells may encounterntigens that bind to their BCR, causing them to become activatednd to undergo additional genomic alterations including IgH classwitch recombination (CSR). Through CSR, the set of exons encod-

ng the initially expressed IgH constant region (C�) are replaced

ith one of several sets of downstream CH exons (C�, C�, C�), lead-ng to IgH class switching from IgM to IgG, IgA, or IgE. CSR resultsn secretion of specific antibodies that are endowed with optimal

low oval) and XRCC4 (orange oval) are recruited to the DNA repair complex in ay Ku. (E) The XRCC4/Lig4 complex ligates signal ends and coding ends, resulting in

effector functions for elimination of particular pathogens. CSR is ini-tiated by the Activation-induced cytidine deaminase (AID), whichdeaminates C’s to U’s within long (i.e. up to 10 kb), highly repet-itive switch (“S”) regions that lie just upstream of each set of CHexons, thereby triggering downstream mechanisms that generateS region DSBs that are requisite intermediates for CSR [1,2]. Tocomplete CSR, DSBs in a donor S region upstream of C� (S�) anda downstream acceptor S region (e.g. S�, S�, S�) are fused by C-NHEJ. However, in the complete absence of C-NHEJ, CSR, unlikeV(D)J recombination, can still be completed, albeit at somewhatreduced efficiency, by A-EJ [2,17–19].

2. C-NHEJ in the repair of DSB during V(D)J recombinationand other processes

The Ku70, Ku80, XRCC4 and DNA Ligase 4 (Lig4) factors are oftendescribed as the evolutionarily conserved “core” C-NHEJ factors[1,2,20,21]. Ku70 and Ku80 form a dimer (“Ku”) that recognizesand binds to DSBs [22]. XRCC4 and Lig4 form a complex that isrequisite for the ligation phase of C-NHEJ [1,20,21]; these proteinsapparently are recruited to DSB ends by Ku [23,24]. Due to theirrecognition and joining functions, respectively, Ku and XRCC4/Lig4are required for all known forms of C-NHEJ; for example, during

V(D)J recombination they are required for joining of blunt SEs andfor joining of hairpin-sealed CEs [1,2]. Accordingly, deficiency forany of the core C-NHEJ factors (Ku70, Ku80, XRCC4 or Lig4) in miceleads to inability to join CEs or SEs during V(D)J recombination,

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esulting in a complete block in B and T cell development and aevere combined immunodeficiency (SCID), that is essentially asevere as that of RAG-deficient mice where V(D)J recombinationannot be initiated [2,25,26].

The DNA-dependent protein kinase catalytic subunit (DNA-Kcs) and Artemis, which is a DNA-PKcs-activated endonuclease,re C-NHEJ factors that have specialized functions in C-NHEJ thatre required for joining DNA ends that must be processed beforeoining. For example, joining of hairpin-sealed CEs is nearly abro-ated in the absence of DNA-PKcs or Artemis due to their inability toe opened in the absence of DNA-PKcs-activated Artemis endonu-lease activity [27]. In accord with the requirement for DNA-PKcsnd Artemis to generate V(D)J recombination CJs, complete defi-iency for DNA-PKcs or Artemis in mice also results in a block in Bnd T cell development and a SCID due to inability to join V(D)JEs and thereby form functional Ig and TCR genes required forevelopment beyond progenitor stages [2,25]. Processing of DSBsor C-NHEJ also may involve various DNA polymerases, such asNA polymerase � and DNA polymerase � [20]. In addition, theeveloping lymphocyte specific terminal deoxynucleotidyl trans-erase enzyme (TdT), the first recognized component of the V(D)Jecombinase [28], adds non-templated nucleotides to V(D)J junc-ions prior to their ligation, thereby greatly increasing junctionaliversity and vastly expanding the diversity of antibody and TCRepertoires [28–30]. Once DSB ends are processed, if necessary, theRCC4/Lig4 complex ligates them to complete the reaction.

DNA-PKcs clearly has functions in C-NHEJ beyond activatingrtemis. In this regard, ligation of blunt SEs occurs normally in thebsence of Artemis; but SE joining is variably impaired, depend-ng on cell type, both with respect to frequency and fidelity inhe absence of DNA-PKcs. Thus, SE joining occurs normally inNA-PKcs-deficient embryonic stem (ES) cells but shows vari-ble impairment in developing lymphocytes and somatic cell lines31–38]. Among other possibilities, such variability in SE joiningmong different DNA-PKcs-deficient cell types might be explainedy differential expression in different cell types of factors that areunctionally redundant with DNA-PKcs C-NHEJ functions beyondhose associated with Artemis activation. As discussed below, twonown factors, XLF and ATM, can, at least partially, compensate forNA-PKcs in SE joining. Non-Artemis-related C-NHEJ functions ofNA-PKcs revealed by defective SE joining during V(D)J recombi-ation in DNA-PKcs-deficient cells have been suggested to includeNA end-bridging functions based on biochemical studies [39–41].hile DNA-PKcs may also play such end-bridging functions in C-

HEJ more generally, such a role is difficult to study in the contextf V(D)J recombination CE joining due to requisite function of DNA-Kcs in the opening of hairpin-sealed CEs prior to joining.

C-NHEJ is involved in repair of DSBs more generally. Defi-iency for core C-NHEJ factors in lymphoid or non-lymphoid cellseads to IR sensitivity and increased genomic instability, associ-ted defects in repair of DSBs ranging from DSBs introduced byhe I-SceI enzyme in reporter constructs [42,43] to DSBs intro-uced during CSR in activated B cells [17–19,42] and to DSBsesulting from unknown factors during neuronal development. Inhe latter context, deficiency for XRCC4 or Lig4 results in severend widespread p53-mediated apoptotic death of newly generatedeurons in response to DSBs [44–48]. This severe neuronal death

s associated with late embryonic lethality of XRCC4- and Lig4-eficient mice, which can be rescued by p53 deficiency [45,46]. Kueficiency in mice also leads to increased p53-mediated death ofewly generated neurons [49], but not as severely as that seen inRCC4 or Lig4 deficiency and, correspondingly, Ku deficiency does

ot lead to embryonic lethality [50,51]. Why Ku deficiency is lessevere in this context is unknown but one hypothesis, based onhe finding that Ku-deficiency can rescue the embryonic lethalityf Lig4 deficiency [52,53], is that Ku binding may block access of

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ends to alternative DSB repair pathways in XRCC4- or Lig4-deficientcells [1]. Deficiency for non-core C-NHEJ factors has a more vari-able and less severe effect for C-NHEJ events beyond the joining ofCEs during V(D)J recombination. DNA-PKcs deficiency also leadsto increased IR-sensitivity, genomic instability and reduced CSRbut to a much lesser degree than observed for core C-NHEJ fac-tor deficiencies [31,32,54–56]. Artemis deficiency also may variablyimpact these processes but to an even lesser extent than DNA-PKcsdeficiency [32,54,57,58]. Correspondingly, neither DNA-PKcs- norArtemis-deficient mice have growth defects or neuronal defects[31,50,58].

C-NHEJ-deficient mice are not highly disposed to developmentof lymphoid or other cancers even though their developing and acti-vated lymphocyte Ig and TCR loci translocate due to mis-joiningof persistent RAG-initiated DSBs. Lack of tumor development inC-NHEJ-deficient mice is thought to be due to elimination ofcells with persistent DSBs or oncogenic translocations via thep53-dependent G1/S checkpoint. Correspondingly, all tested C-NHEJ/p53 double-deficient mice, except XLF-deficient mice (seebelow), rapidly develop RAG-dependent pro-B lymphomas withIgH locus translocations that lead to c-myc or, in the case ofArtemis deficiency, N-myc oncogene amplification [45,46,59–62].Core C-NHEJ-deficient mice that are also p53-deficient consistentlydevelop medulloblastoma brain tumors, consistent with an impor-tant, but unknown, role of C-NHEJ in development of the nervoussystem [44,48,63,64].

The XRCC4-like factor (XLF) [65,66] has also been implicated injoining of DSBs, although its requirement for C-NHEJ appears vari-able and, in that regard, it is not required for robust developmentalV(D)J recombination in mice [67,68], due to a functional redun-dancy between XLF and various DSBR factors in C-NHEJ ([69–71];discussed below) and a functional redundancy with DNA-PKcs in SEjoining [32]. Correspondingly, germline deficiency for XLF in micedoes not lead to any major impacts on survival or development,including that of lymphocytes. In the latter context, while there aremodest effects on B and T cell development, these largely may bedue to impacts on repair of DSBs other than those involved in V(D)Jrecombination [67,68]. Also, consistent with functionally redun-dant factors that could compensate for XLF in end-joining, thereis no obvious impact of XLF deficiency on neuronal developmentin mice. Due to the compensatory functions of XLF and the ATM-dependent DSBR, we will discuss XLF in more detail later in thereview.

3. ATM-dependent DNA double-strand break responseproteins

The Ataxia telangiectasia (AT) mutated (ATM) protein kinaseis a key upstream member of the ATM-dependent DSBR pathway[72]. ATM belongs to the phosphoinositide 3-kinase related proteinkinase (PIKK) family that includes DNA-PKcs and Ataxia telangiec-tasia and Rad3-related protein (ATR) [73]. After DSB generationin G1, ATM activates several downstream factors including p53.Activation of p53 mediates the p53-dependent G1/S checkpoint toarrest cells with unrepaired DSBs to facilitate proper DSB repairor to cause apoptosis of cells with persistent DSBs [74–76]. TheDSBR also participates directly in repair of DSBs, including thoseinvolved in V(D)J recombination [77,78] and those involved in CSR[79]. Following activation via DSBs, ATM phosphorylates a set ofproteins that includes histone H2AX, mediator of DNA damagecheckpoint 1 (MDC1) and the p53-binding protein 1 (53BP1), which

generate large foci in chromatin flanking DSBs [73]. In this regard,phosphorylated histone H2AX (“�-H2AX”) promotes recruitmentof MDC1 [80], which contributes to the generation of a positivefeedback loop that promotes spreading of phosphorylated H2AX

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ver hundreds of kilobases (kb) within chromatin on either side ofhe DSB [81–84]. MDC1 also recruits ubiquitin ligases RNF8 andNF168, the latter of which modifies H2A family histones (H2And H2AX) to promote stable 53BP1 association within these foci85–88]. Beyond potential roles in checkpoint signaling, forma-ion of these ATM-dependent foci have been proposed to tetherSB ends for re-joining via C-NHEJ [89]. DSBR factors downstreamf ATM also have been implicated in directing repair into C-NHEJersus HR or A-EJ, for example by preventing end resection [90–94].

The human AT syndrome includes progressive ataxia, immun-deficiency, radio-sensitivity, genomic instability, increased Ig andCR locus translocations in normal lymphocytes, and B and Tell lymphomas [95,96]. The phenotype of ATM-deficient miceverlaps with that of AT patients and includes general cellularadio-sensitivity and genomic instability (as determined cytoge-etically), modest immunodeficiency, IgH CSR defects (30-50% oformal), and susceptibility to T cell lymphomas that all carryecurrent chromosomal translocations involving the TCR� locus97,98]. Cytogenetic studies showed that most chromosomal aber-ations in ATM-deficient cells, similar to those of C-NHEJ deficientells [79,99–101], occur in the form of chromosomal breaks andranslocations, supporting the notion that ATM plays a most crit-cal role during DSB repair in pre-replicative (e.g. G1) cells. Whilehromosomal translocations have not been well-characterized inmmature ATM-deficient human T cell acute lymphocyte leukemias“T-ALLS”), it seems likely that, as has recently been reported foruman T-ALLs more generally, TCR� locus translocations will alsoe a major feature of immature ATM-deficient human T-ALLs [102].he CSR defects in ATM-deficient mice are associated with substan-ially impaired end-joining during CSR, which leads to unrepairedID-initiated S region DSBs in activated B cells. These breaksrogress at high frequency to chromosomal breaks and translo-ations observed by metaphase fluorescence in situ hybridizationFISH), supporting the notion that the DSBR contributes to tethering

region DSBs for end-joining during CSR [79].While there is clearly a substantial level of normal V(D)J recom-

ination in AT patients and ATM-deficient mice, the frequent Ig orCR locus translocations observed in normal T and B cells of ATatients and ATM-deficient mice were noted to be consistent with

V(D)J recombination joining defect [95]. With respect to roles ofTM in V(D)J recombination, studies that employed ATM-deficientouse pro-B cell lines demonstrated that ATM deficiency or inhi-

ition of ATM kinase activity leads to release of some CEs fromAG-held V(D)J recombination post-cleavage complexes, leadingo unrepaired RAG-generated DSBs that can progress to chromoso-

al breaks and translocations [78]. Such “free” CEs are also joinedt increased frequency to cleaved SEs to generate hybrid or, poten-ially, open and shut joins instead of normal CJs [78]. Thus, ATMontributes to promoting proper C-NHEJ during V(D)J recombi-ation by stabilizing RAG-mediated DSB post-cleavage complexesuring V(D)J recombination [78]. Furthermore, ATM also has aole in SE joining that is revealed in the absence of DNA-PKcs33,103], that could reflect overlapping functions in phosphorylat-ng common DNA repair substrates and/or in more direct roles inNA end-tethering. With respect to downstream substrates, defi-iencies for H2AX, MDC1 and 53BP1 DSBR factors all also havelear-cut impacts on general C-NHEJ as best illustrated by effectsn CSR; however, deficiencies for these factors have quite mod-st impacts on V(D)J recombination and lymphocyte development81,104,105].

H2AX- and MDC1-deficient mice appear relatively normal,lthough their cells have increased IR-sensitivity and cytogenetic

enomic instability [79,81,104]. Correspondingly, activated H2AX-eficient or MDC1-deficient B cells have a modest reduction inSR associated with substantial levels of IgH locus chromosomalreaks, indicating roles for both in the end-joining phase of CSR.

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Cytogenetic studies revealed that H2AX and MDC1 likely func-tion in DSB repair both during pre-replicative and post-replicativecell cycle phases [79,81,101], as evidenced by an increase in bothchromosomal and chromatid breaks in H2AX-deficient or MDC1-deficient cells [79,104,106]. Neither H2AX deficiency nor MDC1deficiency had any readily detectable impact on V(D)J recombina-tion in vivo or on lymphocyte development. Yet, in a p53-deficientbackground, H2AX deficiency, or haplo-insufficiency, predisposedto thymic lymphomas and to pro-B and B cell lymphomas [77].Notably, H2AX/p53 double-deficient pro-B lymphomas harboredoncogenic IgH locus translocations with junctions that involveV(D)J recombination-associated DSBs, a finding that led to theproposal that H2AX may function in suppressing occasional gen-eration of unrepaired DSBs during V(D)J recombination [77,107].In this regard, H2AX was subsequently shown to protect per-sistent RAG-initiated DSBs in C-NHEJ-deficient (e.g. Artemis- orLig4-deficient) pro-B lines from undergoing aberrant resection [90](also see below).

53BP1-deficient mice also appear relatively normal but againtheir cells have increased IR sensitivity [105,108]. Yet, other thanCSR-activated B cells, 53BP1-deficient cells have only modest,if any, cytogenetic instability. However, cytogenetic analyses ofthose cells that do show genomic instability indicate that 53BP1deficiency mainly leads to chromosomal breaks consistent witha major 53BP1 role in genome stability maintenance occurringin pre-replicative cells as observed for ATM [70,79,99,108,109].53BP1-deficient mice have modestly reduced lymphocyte num-bers and their differentiating T lymphocytes have very modestV(D)J recombination defects [105,110,111]. Strikingly, though, CSRis nearly abrogated in the absence of 53BP1 [105,110]. In this con-text, CSR-activated 53BP1-deficient B cells have much greater levelsof cytogenetic instability than other 53BP1-deficient cell types butnearly all of it is associated with AID-dependent IgH locus breaksand translocations.

The more dramatic CSR deficiency and associated IgH locusinstability identified in 53BP1-deficient B cells compared to otherDSBR-deficient B cells indicates a more specialized role for 53BP1in CSR, beyond that of the ATM-dependent DSBR [79]. Roles of53BP1 that might contribute to CSR defects include protection ofDSB ends from resection and potentially promoting their joiningin the context of the DSBR [2,79,91–93,105,110,111], although theoverall mechanisms by which 53BP1 plays an especially crucialrole in CSR compared to other DSBR factors remains to be eluci-dated. Finally, while 53BP1-deficient mice are not cancer prone,53BP1-deficient mice that are also p53-deficient develop thymic orB cell lymphomas [109,112], and 53BP1-deficient mice that havederegulated AID expression may also develop B cell lymphomas[113].

4. XLF is a C-NHEJ factor but is not required for normalV(D)J recombination in mice

The XRCC4-like factor (XLF, also known as Cernunnos, or Nhej1)was identified as a potential C-NHEJ factor through both cDNAcomplementation of cells derived from an IR-sensitive humanimmunodeficiency patient and through a yeast two-hybrid screenfor XRCC4-interacting proteins [65,66]. In humans, inactivat-ing mutations of XLF result in an autosomal recessive disordercharacterized by immunodeficiency that may become progres-sively more severe; but which, in general, is not as severe asthe complete SCID phenotypes associated with human Artemis

or DNA-PKcs deficiency [66,114–116]. Similar to patients withhypomorphic mutations in Lig4, human XLF deficiency alsois associated with microcephaly and radio-sensitivity [66,116].Moreover, XLF-deficient human fibroblasts that ectopically

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xpressed RAG are substantially impaired for ability to undergo(D)J recombination in the context of episomal and intrachromo-omal V(D)J recombination substrates, consistent with a role forLF in C-NHEJ [66,117].

Two independent XLF-deficient mouse lines were both rela-ively normal overall; but XLF-deficient MEFs and ES cells, similaro cells from XLF-deficient patients, had significant V(D)J recom-ination defects [67,68]. Yet, XLF-deficient mice have only veryildly reduced peripheral T and B cell numbers. Moreover, dis-

ributions of developing B and T cells in the bone marrow andhymuses of XLF-deficient mice are comparable to those of WTounterparts [67,68], in striking contrast to the block in lympho-yte differentiation at the progenitor stage of core C-NHEJ-deficientice and DNA-PKcs- or Artemis-deficient mice [2,25]. Abelsonurine Leukemia virus transformed pro-B cells (Abl pro-B cells)

reated with the Abl kinase inhibitor Gleevec (STI571) undergo1 cell cycle arrest, induce RAG expression, and carry out V(D)J

ecombination at the endogenous Ig� light chain locus, as well as inhe context of transient or chromosomally-integrated V(D)J recom-ination substrates [78]. XLF-deficient Abl pro-B lines performed(D)J recombination on such substrates similarly to WT Abl pro-

lines, despite the fact that these lines were mildly IR-sensitive,uggesting a potential differential effect of XLF deficiency on DSBepair during V(D)J recombination versus more general DSB repairn these cells [32,67,70,71].

Despite the lack of a V(D)J recombination defect in developingymphocytes, other types of XLF-deficient cells show mani-estations of C-NHEJ deficiency. Both XLF-deficient MEFs andLF-deficient ES cells were IR-sensitive and impaired for joining(D)J CEs and SEs within transient V(D)J recombination substrates

67,68,118], but in neither case was the impact as severe as isbserved for XRCC4-deficient cells. Cytogenetic genomic insta-ility is present in XLF-deficient MEFs and is primarily in theorm of chromosomal breaks, implicating a role for XLF in repairf pre-replicative DSBs [32,67,70,118]. Also consistent with anffect of XLF deficiency on general C-NHEJ, XLF-deficient mature

cells have reduced CSR coupled with increased unrepaired IgHhromosome breaks [67]. XLF/p53 double-deficient mice, unlikether C-NHEJ/p53 double-deficient mice, rarely die of pro-B cellymphomas; but rather die of T cell lymphomas characteristic of53 deficiency [67]. As C-NHEJ/p53 double-deficient pro-B cell

ymphomas routinely contain oncogenic translocations involvingAG-generated IgH locus DSBs, the rarity of this type of tumor

n XLF/p53 double-deficient mice is consistent with normal V(D)Jecombination in developing XLF-deficient lymphocytes. Notably,owever, many XLF/p53 double-deficient mice also develop medul-

oblastomas (MBs), as seen in other C-NHEJ/p53 double-deficientice [17,119,120]. The occurrence of recurrent MBs in XLF/p53

ouble-deficient mice is consistent with a general C-NHEJ defectn MB progenitors and warrants deeper investigation into under-ying mechanisms. As XLF clearly functions as a C-NHEJ factor in

broad variety of cell types, the relatively normal V(D)J recom-ination in XLF-deficient progenitor lymphocytes and Abl pro-Bell lines coupled with the normal lymphocyte development inLF-deficient mice [67,68], suggested the existence of factors thatompensate for XLF function in C-NHEJ, specifically in the contextf V(D)J recombination in developing lymphocytes [67].

Most current models for XLF function derive from biochemicalnd structural assays, which suggest that XLF largely works in con-ert with the structurally similar XRCC4 factor. Both XLF and XRCC4ontain a globular head domain, an alpha-helical stalk domain,nd an unstructured C-terminal domain [65,121]. In this regard,

LF forms heterodimers with XRCC4 through interactions of theirespective globular head domains [65,121]. Known functions ofRCC4 include stabilization of Lig4 through direct interaction [122]nd stimulation of Lig4 activity [123]. XLF/XRCC4 heterodimers

ir 16 (2014) 11–22 15

form protein filaments [121,124–127] that tether DNA ends in vitroand which have been speculated to enhance XRCC4-dependentrecruitment of Lig4 to DSBs [124,126]. In biochemical assays, XLFalso enhances efficiency of the XRCC4/Lig4 complex to ligate lin-earized plasmids in vitro [128]. Notably, non-lymphoid cells thatexpress mutant forms of XRCC4 that are unable to interact withXLF carry out joining of SEs, but not CEs, in the context of extra-chromosomal V(D)J recombination substrates [129], suggestingpotential functional compensation for XLF-XRCC4 end-bridging byRAG or other factors in the post-cleavage synaptic complex forjoining of SEs [121,129,130].

The findings that XLF is not required for V(D)J recombination innormal developing lymphocytes but is required for ectopic V(D)Jrecombination in non-lymphoid cells led to the hypothesis thatother factors may functionally compensate for XLF to promoteV(D)J recombination in developing lymphocytes [67]. As men-tioned above, one such factor might be RAG, which holds CEsand SEs in a post-cleavage synaptic complex and directs C-NHEJ-mediated joining of two CEs and two SEs, respectively, to each other[131,132]. In this context, SEs are held more tightly than CEs inthe RAG post-cleavage synaptic complex [133]; which in the con-text of proposed XLF synapsis functions could explain the lack ofimpact on SE joining versus CE joining when XRCC4-XLF inter-action is disrupted [129]. If RAG does function redundantly withXLF during V(D)J recombination in developing lymphocytes, butnot in non-lymphoid cells, it is conceivable that normal physio-logical V(D)J recombination may have evolved to optimize abilityof RAG, for example via post-translational modifications, to holdCEs and/or SEs in post-cleavage synaptic complexes for properend-joining and/or to contribute to C-NHEJ factor recruitment[12,131,132,134]. However, additional studies, described below,demonstrated that factors other than RAG provide functionalredundancy with XLF in V(D)J recombination and C-NHEJ moregenerally.

5. XLF has functional redundancy with ATM

The ATM-dependent DSBR is activated in response to endoge-nous RAG-initiated DSBs at antigen receptor loci [135,136], anddeficiencies for ATM or several ATM downstream factors (H2AXand 53BP1) impair end-joining during V(D)J recombination, withimpacts ranging from moderate for ATM [78] to very modest forH2AX [79,104,106]. Such studies led to the proposal that the ATM-dependent DSBR, beyond activating checkpoints, may contributeto C-NHEJ through end-tethering during V(D)J recombination,CSR, and more generally [1,2,79,89,137]. Indeed, ATM was foundto stabilize RAG-mediated V(D)J breaks in the context of thepost-cleavage complex [78]. Together, these findings led to theevaluation of ATM and downstream DSBR factors as candidates forhaving functional redundancy with XLF, potentially through rolesin end-tethering. Correspondingly, XLF/ATM double-deficient micewere observed to have a severe block in B and T cell developmentat the progenitor stage when V(D)J recombination occurs, veryreminiscent of the SCID phenotype of C-NHEJ-deficient mice [71].In the XLF/ATM double-deficient background, the B cell develop-mental block was substantially rescued by introduction of germlinealleles containing pre-assembled IgH and IgL variable region exons(“HL alleles”), consistent with impairment resulting largely froma V(D)J recombination defect [71]. Correspondingly, XLF/ATMdouble-deficient Abl pro-B cells arrested in G1 to activate V(D)J

recombination accumulated unrepaired breaks in the endogenousIg� locus [71], and were severely impaired in ability to join CEs andSEs of RAG-initiated DSBs within chromosomally integrated V(D)Jrecombination substrates. Thus, combined deficiency for ATM and

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16 V. Kumar et al. / DNA Repair 16 (2014) 11–22

Fig. 2. XLF has functional redundancy with ATM and ATM substrates (H2AX, 53BP1) during C-NHEJ. (A) Factors of the DSBR pathway (ATM, �H2AX, 53BP1; pink oval)accumulate around a DSB. Ku70/Ku80 (light blue and dark blue semicircles) bind DNA ends. DNA-PKcs (light blue oval), XRCC4/Lig4 (orange and yellow ovals) and XLF(orange circle) are recruited to the C-NHEJ DNA repair complex by Ku. (B) and (C) In the absence of ATM (or H2AX, or 53BP1), C-NHEJ is functional. In the absence of XLF,C ATM, CX -NHEb

Xd

dArhtuaIsdadioi

srtcd

-NHEJ is also largely functional. (D) In cells with combined deficiency for XLF and

LF and 53BP1, C-NHEJ is dramatically reduced. It remains unclear which stage of Cy the question marks).

LF essentially abrogates chromosomal V(D)J recombination ineveloping lymphocytes (Fig. 2).

The studies of developing lymphocytes or pro-B cell lines clearlyemonstrate that XLF-deficient B lineage cells require ATM andTM-deficient B cells require XLF to perform C-NHEJ during V(D)Jecombination [71]. XLF/ATM double-deficient mouse fibroblastsave substantially increased cytogenetic instability as comparedo fibroblasts deficient for either factor alone (Oksenych and Alt,npublished), which among other possibilities, suggests that ATMnd XLF may have functional redundancy in C-NHEJ more generally.ndeed, studies of CSR in the XLF/ATM double-deficient backgroundtrongly support this notion. Thus, CSR to IgG1 in mature XLF/ATMouble-deficient B cells generated in a background containing HLlleles was reduced to the residual levels observed in core C-NHEJ-eficient cells (Fig. 2). As residual CSR in C-NHEJ-deficient B cells

s known to occur via A-EJ, these findings also suggested that thebserved functional redundancy for ATM and XLF primarily occursn the context of C-NHEJ and not for A-EJ [71].

Assays for V(D)J recombination within extra-chromosomal sub-trates introduced into XLF/ATM double-deficient Abl pro-B cells

evealed that both CE and SE joining occurred at levels similar tohose of WT Abl pro-B cells, in striking contrast to the essentiallyomplete block in chromosomal V(D)J recombination in XLF/ATMouble-deficient lines [71]. This finding suggested that XLF and

-NHEJ is nearly abrogated. In cells with combined deficiency for XLF and H2AX, orJ is defective in the combined absence of XLF and ATM or its substrates (as denoted

ATM functional redundancy may be more specific to chromoso-mal V(D)J recombination in the context of chromatin [71]. In thisregard, inhibition of ATM kinase activity in XLF-deficient Abl pro-B cells also led to a block in joining of RAG-cleaved CEs and SEswithin chromosomally integrated V(D)J recombination substrates,suggesting that XLF-redundant functions of ATM in chromosomalV(D)J recombination joining may be mediated, at least in part, bydownstream ATM substrates in the context of chromatin [71]. Thesefindings led to additional studies that implicated H2AX and 53BP1as factors that have functional redundancy with XLF in C-NHEJ dur-ing V(D)J recombination.

6. XLF has redundant functions with ATM substrates H2AXand 53BP1

Combined deficiency for XLF and histone H2AX results in earlyembryonic lethality. The cause of such embryonic lethality remainsto be determined. One proposed model is that embryonic lethalitymay be caused by XLF and H2AX functional redundancy in DNArepair processes beyond those shared with ATM alone. Such func-

tions, for example, might be related to potential ATM-independentpost-replicative DNA repair roles for H2AX as suggested by theincreased levels of chromatid breaks in H2AX-deficient versusATM-deficient cells [71,79,81,101]. In this context, H2AX can be

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ctivated independently of ATM by ATR, DNA-PKcs [71,138–141]nd, conceivably, by other kinases. If this model is correct, oneight also expect MDC1 deficiency, which also leads to defects

n pre-replicative or post-replicative DSB repair defects, to lead tombryonic lethality when combined with XLF deficiency. Another,on-mutually exclusive model, in analogy to p53 rescue of embry-nic lethality of Lig4- or XRCC4-deficient mice [46], would be thatheckpoint defects associated with ATM deficiency, but not H2AXeficiency, rescue potential embryonic lethal DNA repair defects ofLF/ATM deficient mice [71]. If correct, p53 deficiency might res-ue the embryonic lethality of XLF/H2AX deficiency. However, it islso notable that XLF/H2AX embryonic lethality occurs much ear-ier than the late embryonic lethality of Lig4 or XRCC4 deficiency,oth of which are associated with dramatic neuronal apoptosis45–47,71].

It was not possible to assess potential defects in lympho-yte development or DNA repair in the context of XLF/H2AXouble-deficient mice due to early embryonic lethality. However,onditional inactivation of H2AX in XLF-deficient Abl pro-B linesad no impact on cellular viability. In these lines, combined H2AXnd XLF deficiency also results in severely impaired joining of RAG-enerated CEs and SEs; albeit not quite to the same extent as seenith dual ATM- and XLF-deficiency [71] (Fig. 2). However, while inLF/ATM double-deficient Abl pro-B lines, un-joined CEs and SEsppeared largely intact, in XLF/H2AX double-deficient Abl pro-Bines most un-joined CEs and SEs were highly resected, consistent

ith findings that H2AX protects such ends from resection in-NHEJ-deficient backgrounds [90]. At first glance, this differential

mpact of ATM and H2AX deficiency on the fate of un-joined CEs andEs in an XLF-deficient background might not seem consistent withuch H2AX functions lying downstream of ATM. However, ATMeficiency has a dual impact on resection of un-joined CEs and SEsuring V(D)J recombination, both protecting these ends from resec-ion via generation of phosphorylated �H2AX and also promotingheir resection by activating CtIP [90]. Thus, the defect in CE and SEoining per se in the combined absence of H2AX and XLF may reflect2AX functioning downstream of ATM activation at a DSB, both toromote joining, for example via an end-tethering function, andlso to specifically prevent end resection by ATM-activated CtIP.orrespondingly, inhibition of ATM kinase activity in XLF/H2AXouble-deficient Abl pro-B cell lines substantially restored accu-ulation of un-joined CEs and SEs that were not markedly resected.XLF and 53BP1 double-deficient mice are live born, but

ompared to XLF-deficient or 53BP1-deficient mice, are growthetarded, their fibroblasts have increased cytogenetic genomicnstability manifested primarily as chromosome breaks, and theyre more prone to thymic lymphoma [69,70]. Moreover, likeLF/ATM double-deficient mice, XLF/53BP1 double-deficient miceave a SCID phenotype with lymphocyte development essen-ially blocked at the progenitor B and T cell stages, consistentith impaired V(D)J recombination [69,70]. Indeed, analyses ofLF/53BP1 double-deficient Abl pro-B cell lines revealed a com-ined impact on ability to join RAG-generated CEs and SEs during(D)J recombination that was strikingly similar to the combined

mpact of dual XLF/H2AX deficiency (Fig. 2), including severelympaired joining of both CEs and SEs and dramatically increasedesection of the un-joined CEs and SEs that was rescued by treat-ent of cells with an ATM inhibitor [69,70]. The end protection role

f 53BP1 is consistent with such roles in other contexts includingSR [91–94]. While the V(D)J recombination defects of XLF/53BP1ouble-deficient Abl pro-B lines was substantial, as seen with dualLF/H2AX deficiency, some residual joining remained [69,70]. In

his context, the essentially complete SCID phenotype of XLF/53BP1ouble-deficient mice may reflect both greatly impaired V(D)Jecombination, as well as an impact on developing or more matureymphocytes due to effects on general C-NHEJ as suggested by

ir 16 (2014) 11–22 17

the increased genomic instability of XLF/53BP1 double-deficientfibroblasts.

Overall, the impact of H2AX or 53BP1 deficiency on V(D)J recom-bination and C-NHEJ in an XLF-deficient background is consistentwith both DSBR proteins having critical functions in both end-joining per se (e.g. by end-tethering roles) and end protection fromaberrant resection in the context of the ATM-dependent DSBR. As53BP1 may also have functions at DSBs independent of ATM, as itlikely does during CSR, a functional redundancy with XLF in thiscontext cannot be ruled out. XLF, on its own, does not appear tohave a major role in protecting un-joined RAG-initiated CEs and SEsfrom resection. Thus, these ends are highly resected in Lig4/H2AXdouble-deficient or Artemis/H2AX double-deficient Abl pro-B cells[90]. Likewise, in Artemis-, DNA-PKcs-, or XRCC4-deficient Ablpro-B cells that are also XLF-deficient, persistent, unrepaired CEsgenerated during attempted V(D)J recombination do not undergoincreased resection [32].

7. XLF and DNA-PKcs have functional overlaps in V(D)Jrecombination and C-NHEJ

DNA-PKcs and ATM are related PIKKs with shared substrates[140], are functionally redundant with respect to SE joiningduring V(D)J recombination [33,103], and have both been sug-gested to potentially play a role in tethering DSB ends duringC-NHEJ [39–41,78]. Correspondingly, DNA-PKcs also appears tohave functional overlap with XLF in C-NHEJ. Thus, XLF/DNA-PKcsdouble-deficient fibroblasts have increased levels of chromosomalbreaks compared to that of DNA-PKcs- or XLF-deficient fibroblasts[32]. Moreover, combined deficiency for XLF and DNA-PKcs abro-gates joining of RAG-initiated SEs within chromosomally integratedV(D)J recombination substrates in Abl pro-B cells [32]. Given thatXLF/Artemis double-deficient Abl pro-B cells did not display suchan impact on SE joining, the dramatically impaired SE joining inXLF/DNA-PKcs double-deficient Abl pro-B cells involves Artemis-independent DNA-PKcs functions. In this context, XLF/DNA-PKcsdouble-deficient mice are live born but die shortly after birth forunknown reasons; whereas XLF/Artemis double-deficient mice donot exhibit early post-natal lethality, consistent with XLF/DNA-PKcs functional redundancy separate from DNA-PKcs function inArtemis activation [32]. Finally, the apparent DNA-PKcs redundantfunction with XLF is mediated by its kinase activity, as DNA-PKcskinase inhibition in XLF-deficient Abl pro-B cells or mature B cellsabrogates SE joining and reduces CSR levels, respectively [32]. Thelatter finding also is consistent with a broader functional redun-dancy between XLF and DNA-PKcs in C-NHEJ in general.

8. Perspectives

The finding that the ATM-dependent DNA damage responseresults in the formation of large foci in chromatin surrounding DSBs[142,143] raised the possibility of a direct role for the DSBR in DSBrepair, and, in particular, in C-NHEJ. Likewise, the discovery thatXLF is required for protection from ionizing radiation and also forjoining of RAG-initiated DSBs in patient fibroblasts implicated thisXRCC4-related factor as a potential C-NHEJ component [66,144].A confounding issue, however, in considering potential roles ofATM-dependent DSBR factors and the XLF protein as C-NHEJfactors was the relative dispensability of these factors for C-NHEJduring V(D)J recombination. However, the finding that XLF sharesredundant functions with ATM and several of its downstream

DSBR factors with respect to C-NHEJ during V(D)J recombinationin developing lymphocytes greatly clarified the critical roles ofXLF and the DSBR in this process. Thus, in the absence of XLF,developing B and T lymphocytes are totally reliant on ATM and

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18 V. Kumar et al. / DNA Repair 16 (2014) 11–22

F ) Botha M ande

ArfVompdf

ra

Fs

ig. 3. XLF and ATM (and DSBR factors) have redundant functions during C-NHEJ. (And/or stabilize the XRCC4/Lig4 (orange and yellow ovals) complex at a DSB. (B) ATnds in close proximity before ligation.

TM downstream factors to carry out normal chromosomal V(D)Jecombination; likewise, in the absence of ATM or downstreamactors, developing B and T cells require XLF for C-NHEJ during(D)J recombination. Indeed, while XLF deficiency has little impactn lymphocyte development, and ATM deficiency has only aodest impact, combined ATM and XLF deficiency results in a SCID

henotype reminiscent of that observed in the context of C-NHEJeficiency [71]. A further surprising recent finding is the additional

unctional redundancy between XLF and DNA-PKcs in C-NHEJ [32].

The ongoing challenge is to elucidate the nature of theedundant functions of XLF and DSBR factors (and DNA-PKcs)nd whether they have overlapping roles in the same general

ig. 4. XLF and ATM (and DSBR factors) have complementary functions during C-NHEJtimulates XRCC4/Lig4 (orange and yellow ovals) activity. (B) ATM may stimulate XRCC4/

ATM (pink oval) and XLF (orange circle) may stimulate Lig4 (yellow oval) activity, its downstream substrates, combined with XLF/XRCC4 filaments, may tether DNA

function (e.g. end-tethering) (Fig. 3) or whether they representroles in different processes that provide different functions (e.g.end-tethering versus C-NHEJ factor recruitment) that are com-pensatory for each other in C-NHEJ [71]. A potential example ofcompensatory but different functions might include DNA end-tethering and C-NHEJ factor recruitment (or activation) (Fig. 4).Thus, efficient tethering of DSBs may keep them together longenough for ligation even at reduced repair efficiency due to

impaired C-NHEJ factor recruitment; conversely, efficient C-NHEJrecruitment might rapidly repair ends before they separate due toreduced tethering activity (Fig. 4). With respect to such potentialroles in C-NHEJ, DNA-PKcs and XLF have been directly implicated

. (A) ATM (pink oval) may function to hold DNA ends, while XLF (orange circle)Lig4, while XLF/XRCC4 filaments hold DNA ends.

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n DNA end-tethering [39–41,121,124–127], while ATM and itsownstream DSBR factors have been suggested to play sucholes either directly or indirectly [78,79,89,105,111]. With respecto C-NHEJ factor recruitment or activation, XLF was originallyescribed as a part of the XLF/XRCC4/Lig4 complex with potentialoles in the ligation phase of the reaction [65,128,145], and XLFas been found to stimulate Lig4 activity [128,145].

It is notable that single deficiencies for XLF or individual DSBRactors generally have a less pronounced effect on V(D)J recombina-ion than on other forms of C-NHEJ-mediated DSB repair includingSR. In theory, the lower V(D)J recombination impact might reflect

contribution by RAG in holding CE and SE in synaptic complexesnd/or promoting their joining by C-NHEJ [12,131,132]. In this con-ext, ATM or DNA-PKcs phosphorylation of RAG is not required forts function in normal cells [146]; but whether such an activityould contribute to the impact of ATM or DNA-PKcs deficiency on(D)J recombination in the absence of XLF remains to be tested.inally, functional redundancies between XLF and DSBR factorsr RAG might also contribute to the phenotypic diversity of defi-iencies for these factors in human patients or between humansnd mice. For example, the marked variation in degree of lym-hopenia observed in XLF-deficient patients [66,116] might reflectariations in the expression of XLF compensatory proteins. Con-ersely, the impact of ATM deficiency in humans can vary amongatients [95,147,148], and ATM-deficient mice do not exhibit thevert neurological defects observed in humans [95,98]. Potentially,uch variations could also reflect, at least in part, varying degreesf XLF compensation for ATM function in these different settings.

onflict of interest statement

The authors declare no conflict of interest.

cknowledgements

V.K. is supported by an NIH T32 training grant GM007226-38..W.A. is supported by NIH grant AI076210. F.W.A. is an Investigatorf the Howard Hughes Medical Institute.

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