IgH Chain Class Switch Recombination: Mechanism and Regulation Janet Stavnezer and Carol E. Schrader This information is current as J Immunol 2014; 193:5370-5378; ; of September 26, 2021. doi: 10.4049/jimmunol.1401849 http://www.jimmunol.org/content/193/11/5370 Downloaded from References This article cites 133 articles, 66 of which you can access for free at: http://www.jimmunol.org/content/193/11/5370.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2014 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Th eJournal of Brief Reviews Immunology

IgH Chain Class Switch Recombination: Mechanism and Regulation Janet Stavnezer and Carol E. Schrader IgH class switching occurs rapidly after activation of with an S region farther downstream. S regions are G rich and mature naive B cells, resulting in a switch from expres- have a high density of WGCW (A/T-G-C-A/T) motifs, the sion of IgM and IgD to expression of IgG, IgE, or IgA; preferred target for activation-induced cytidine deaminase this switch improves the ability of Abs to remove the (AID), the that initiates CSR by deaminating cytosines pathogen that induces the humoral immune response. within S-region DNA, converting deoxycytidine to dU (2, 3). Class switching occurs by a deletional recombination of the base excision repair (BER) and mismatch repair between two switch regions, each of which is associated (MMR) pathways then convert the dUs to DNA DSBs, which with a H chain constant region gene. Class switch re- are required for CSR (4, 5) (Fig. 2). The DSBs are subse- Downloaded from combination (CSR) is instigated by activation-induced quently recombined by an end-joining type of DNA recom- cytidine deaminase, which converts cytosines in switch bination, predominantly by nonhomologous end joining regions to uracils. The uracils are subsequently removed (NHEJ). The use of NHEJ, rather than homologous recom- by two DNA-repair pathways, resulting in mutations, bination, is consistent with the facts that S-region DSBs are single-strand DNA breaks, and the double-strand breaks induced and recombined during the G1 phase (6–9) and that http://www.jimmunol.org/ required for CSR. We discuss several aspects of CSR, different S regions do not share long stretches of identity (1), which are required for homologous recombination. including how CSR is induced, CSR in B cell progen- CSR occurs very rapidly postinfection or immunization, itors, the roles of transcription and chromosomal loop- prior to formation of germinal centers, which generally form ing in CSR, and the roles of certain DNA-repair enzymes 7–10 d after exposure to Ag. For example, using mice in CSR. The Journal of Immunology, 2014, 193: expressing a transgenic BCR, both IgM+ and IgG2a+ cells 5370–5378. were detected in B cell follicles from days 2 to 4 after im- munization, but only IgG2a+ cells were detected in germinal

fter immunization or infection, activated naive centers, indicating that CSR occurred prior to germinal center by guest on September 26, 2021 BcellscanswitchfromexpressingIgMandIgDon formation (10). Also, CSR was detected in nontransgenic A their surface to expressing IgG, IgE, or IgA. This mice 4 d postinfection with Salmonella (11). However, CSR is isotype/class switch changes the effector function of the Ab also detected in germinal center B cells from human tonsils and improves its ability to eliminate the pathogen that in- (12), and IgA CSR occurs in Peyer’s patch germinal centers duced the response. Isotype switching involves replacement (13–15), in which B cells are constantly stimulated by the gut microbiota. CSR also occurs during T-independent responses, of the m and d HchainC(CH) regions of the expressed Ig which do not induce germinal centers (16). Thus, CSR starts with g, «,ora CH regions, and it occurs by a DNA re- combination event termed “class switch recombination” prior to somatic hypermutation (SHM) of V region [V(D)J] genes, an AID-dependent process that occurs mainly in ger- (CSR). Fig. 1 is a diagram (not to scale) of the CH genes and minal centers, and which, after selection, can result in Abs CSRinmice;humanCH genes are similarly arranged al- though not identical. with increased affinity for Ag. The data suggest that CSR may CSR is a deletional DNA recombination occurring between continue as long as B cells are undergoing activation. switch (S) regions, which are located upstream of all of the Induction of CSR CH genes, with the exception of Cd, and are 1–10 kb in length (1). Recombination occurs between dsDNA breaks (DSBs) Many studies of CSR have been performed using cultures of introduced into the donor m S(Sm) region and a downstream/ mouse splenic B cells, because these cells can be induced in acceptor S region located from ∼65–160 kb downstream; oc- culture to undergo robust CSR within ∼3 d by treatment with casionally, downstream S regions can subsequently recombine the B cell mitogen LPS, acting through the innate receptor

Department of Microbiology and Physiological Systems, University of Massachusetts Abbreviations used in this article: AID, activation-induced cytidine deaminase; AP, Medical School, Worcester, MA 01605 apyrimidinic/apurinic; APE, AP ; ATM, ataxia-telangiectasia mutant; BER, base excision repair; 3C, chromosome conformation capture; Cm, m C region; Received for publication July 21, 2014. Accepted for publication September 2, 2014. CH, H chain C; CSR, class switch recombination; DSB, dsDNA break; Em,Em intron This work was supported by National Institutes of Health Grants R01-AI23283 and enhancer; 39Ea,39 Ca enhancer; GLT, germline transcript; HEL, hen egg lysozyme; R21-AI99988 (both to J.S. and C.E.S.). MMR, mismatch repair; MRN, Mre11-Nbs1-Rad50; NHEJ, nonhomologous end join- ing; Pol, polymerase; Sm, m S; S, switch; SHM, somatic hypermutation; ss, single Address correspondence and reprint requests to Dr. Janet Stavnezer, Department of stranded; SSB, ssDNA break; UNG, uracil DNA glycosylase; WT, wild-type. Microbiology and Physiological Systems, University of Massachusetts Medical School, AS8-1053, 368 Plantation Street, Worcester, MA 01605. E-mail address: janet. Ó [email protected] Copyright 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1401849 The Journal of Immunology 5371

FIGURE 1. Diagram of the mouse IgH genes in naive mature B cells expressing IgM and IgD as well as CSR to IgG2b. During CSR to IgG2b, AID deaminates the Sm and Sg2b regions, insti- gating DSB formation. The Sm and Sg2b regions recombine by an intrachromosomal deletional recombination, which causes the expressed VDJ segment to become associated with the Cg2b gene. Splicing diagrams of the m and d mRNAs, the g2b GLTs, and g2b mRNA are indicated beneath the genes. Em and 39Ea are the two major enhancers that regulate expression of Ig H genes and CSR. Downloaded from

TLR4, or by signaling through CD40, the most important Likewise, when HEL is added to HEL-specific B cells acti- receptor for T cell help. LPS alone induces AID expression, vated with TLR7 or TLR9, CSR to IgG1 is also greatly in- but a cytokine, such as IL-4, must be added to induce AID creased (22). Unlike mouse B cells, human peripheral blood http://www.jimmunol.org/ when Ab to CD40 (anti-CD40) is used. These ligands also or tonsillar B cells switch poorly in culture to either CD40 or induce cell proliferation, another requirement for CSR (17, TLR signaling (29); perhaps activation through BCR signal- 18). Surprisingly, splenic B cells do not require a signal ing would help. provided by the BCR to switch in culture, because neither In conclusion, it is likely that CSR in vivo depends upon LPS nor anti-CD40 triggers signaling via the BCR. This activation of B cells via their BCR, in addition to secondary might be explained by the large amounts of LPS (usually 10– signals from CD40 and TLR signaling. It is unclear whether 50 mg/ml) used in these cultures, which would not normally the BCR signaling is required for inducing CSR or is only

be provided to B cells in vivo. Also, much more continuous required for initial activation of the B cells, and the T cell by guest on September 26, 2021 CD40 signaling is provided in these cultures than normally signals are responsible for inducing CSR. In addition, it would be available in vivo. In vivo, CD40 signaling would be appears likely that TLR signaling is important for both T- delivered to Ag-specific B cells during contacts with Ag- independent and T-dependent responses in vivo. It is possible specific Th cells. CD40 signaling is very important for CSR that primary and multiple secondary signals given together in vivo, as demonstrated by the lack of B cell proliferation and increase the robustness of the CSR response. CSR in response to T-dependent Ags in mice and humans deficient in CD40 signaling (19, 20). Also, TLR signaling is Regulation of isotype specificity by transcription of unrearranged CH important for in vivo immune responses and CSR in response genes to viruses (21). Naive B cells have the potential to switch to any isotype. Although Ag is not necessary to obtain CSR in culture, Isotype specificity is directed by induction of transcription addition of low levels (1–100 ng/ml) of soluble hen egg ly- across S regions, because AID-induced and CSR sozyme (HEL) Ag to LPS- or anti-CD40–stimulated mouse are restricted to S regions that are undergoing transcription splenic B cells expressing a HEL-specific BCR increases CSR (5, 30–33). Located upstream of each acceptor S region are (22). Increasing the amount of Ag to 1 mg/ml inhibited CSR. transcription promoters, which are activated by cytokines that Also, low levels (nanogram amounts) of anti-IgD dextran induce CSR to that specific isotype. Transcription from these added to LPS-activated cultures of splenic B cells increases promoters produces germline transcripts (GLTs), so-called CSR (23–25). The increase in CSR is not due to increased cell because they are transcribed from unrearranged genes. GLTs proliferation (24, 26). In contrast, addition of large concen- are spliced, as diagrammed in Fig. 1 for g2b GLT. Because of trations (10 mg/ml) of anti-IgM to LPS cultures or anti- their unusually high G content, S-region transcripts form CD40 cultures reduces CSR, although it does not inhibit cell R-loops (RNA-DNA hybrids) with the bottom strand DNA, proliferation (27, 28). The inhibitory-signaling pathway has rendering the nontranscribed (top strand) single stranded (ss) been studied, and it appears to be a feedback response to (34–36). The substrate for AID is ssDNA; thus, R-loops extensive BCR cross-linking. cause the top strand to become an extensive AID substrate. BCR signaling appears to be important for CSR in culture However, it is known that AID attacks both the top and under conditions when signals provided by accessory/secondary bottom strands at S regions nearly equally (37). Thus, the signals are limiting. Ligands for the innate receptors TLR1/ R-loop must be removed during CSR to expose the bottom 2, TLR7, and TLR9 were shown to induce little or no CSR strand to AID. in culture. When anti-IgD dextran is added to these cultures, The R-loop is thought to cause RNA polymerase (Pol) II to CSR to several isotypes is increased synergistically (24). stall during transcription of S regions, resulting in accumu- 5372 BRIEF REVIEWS: Ab CLASS SWITCH RECOMBINATION

ubiquitinate Pol II, leading to degradation of Pol II (42). Pol II degradation leads to transcription termination, creating substrates for the exosome. Interestingly, AID is found associated with polyubiquitinated Pol II, and Nedd4 ubiquitination activity was shown to promote interaction of AID with Spt5 andwiththeRNAexosome,aswellastopromotebinding of the RNA exosome to transcribed S regions. Nedd4 also reduces the level of g1 GLTs, presumably due to degrada- tion by the exosome. Thus, Nedd4 activity appears to in- crease the amount of ss bottom strand DNA and to help recruit AID to S regions. This model has been supported by results indicating that Pol II pausing leads to transcription termination and increased SHM in IgH V genes, dependent on the RNA exosome (43, 44). GLTs must be spliced to support CSR, although the reason for this is unknown (33, 45–49). Because splicing is cotran- scriptional, it is possible that splicing factors are involved

in recruiting AID to S regions (50) and/or that splicing is Downloaded from required for R-loops to form, perhaps because the RNA must thread back into the DNA to form an R-loop (36). The splicing regulator PTBP2, a protein that binds AID and S- region transcripts, is important for efficient CSR. Knockdown of this protein reduces the association of AID with S regions

(51, 52). PTBP2 is known to regulate alternative splicing and http://www.jimmunol.org/ many aspects of RNA metabolism (53), although its specific role(s) in CSR is not defined. Also, CTNNBL1, a component of the spliceosome, interacts with AID and is important for both CSR and SHM (50). These interesting studies suggest avenues that might lead to an understanding of why splicing of GLTs is important for CSR, although at this time the role of splicing is not understood. by guest on September 26, 2021 Regulation of CSR by chromosome looping Sm and the acceptor S regions must be in contact to re- combine. Chromosome conformation capture (3C) experi- ments showed that, in mature naive splenic B cells prior to CSR, the Em intron enhancer (Em)andthe39 Ca enhancer FIGURE 2. Models for the generation of DNA DSBs during CSR. (A) (39Ea)/regulatory region, located ∼220 kb apart in the ge- Diagram of how the BER pathway converts AID-induced dUs to DNA nome, are positioned sufficiently near each other in the B breaks. ( ) Model of how the MMR pathway converts SSBs produced by nucleus to be cross-linked by formaldehyde treatment via UNG and APE activity to DSBs appropriate for NHEJ. See text for more information. proteins bound to them (54–57). Gene targeting experi- ments showed that segments of the 39Ea are essential for CSRtoallisotypes(58).ThisEm–39Ea interaction causes lation of Pol II in S regions, although some other feature of S Sm and the downstream S regions to be located within the regions might be responsible (38, 39). Interestingly, AID was same chromosomal loop (Fig. 3A). In cells activated to demonstrated to be in a complex with Spt5, a protein that switch, but that have not yet switched to IgG1 by treatment associates with Pol II when it is stalled on the DNA template, with LPS plus IL-4, the Em-Sm-Cm (m Cregion)andSg1- suggesting that Pol II stalling recruits AID to S regions (40). Cg1 loci are found in 3C experiments to be positioned near In addition, stalled Pol II can lead to transcription termina- each other and the 39Ea segment (Fig. 3B). In cells treated tion, generating 39 ends of S-region transcripts that become with LPS alone, which induces CSR to IgG3, the Sg3-Cg3 substrates for the RNA exosome, a complex that degrades locus is associated with Em-Sm-Cm and with 39Ea instead of RNA from the 39 end. The RNA exosome was shown in with Sg1-Cg1 (54, 55). Because LPS plus IL-4 induces g1 biochemical (in vitro) experiments to allow AID to target the GLTs, these results suggest that factors that induce tran- bottom strand in transcribed duplex DNA (41), which is scription from the GLT promoter and/or the GLTs them- otherwise mostly inaccessible to AID when associated with selves are involved in recruiting the Sg1-Cg1 locus to the newly transcribed RNA. This finding provides an explanation positions of the Em-Sm-Cm locus and 39Ea. Also, it is pos- for how AID targets the bottom strand. sible that looping allows transcriptional activators that bind These in vitro experiments have been bolstered by in vivo the enhancers to gain access to GLT promoters; thus, asso- experiments in which CSR was reduced by 50% in cells lack- ciation of a S-CH acceptor locus with the Em-Sm-Cm locus ing Nedd4, an E3 ubiquitin ligase. Nedd4 monoubiquitinates and with 39Ea might contribute to transcription or even be paused Pol II, causing an unknown ubiquitin ligase to poly- required for germline transcription. The Journal of Immunology 5373

switching occurs in cells treated with LPS plus CD40L plus IL-4 (67). GLT expression correlates with CSR (i.e., g2b and « GLTs, but not g3org1 GLTs, are detected in pro-B cells, activated without or with IL-4, respectively). The explanation for this restricted choice of isotypes appears to be that pro- B cells have a chromosomal loop between the Cg3 and Cg1 loci that sequesters these genes away from the Em and 39Ea enhancers (67). This loop was not detected in mature splenic B cells. Likewise, ex vivo bone marrow pre-B cells from C57BL/6 mice switch to g2b but not to g3; surprisingly, however, BALB/c pre-B cells switch to g2b and g3 (66). The pre-B cells in both mouse strains also switched to a, consis- tent with results indicating that mice that cannot express IgM FIGURE 3. Diagrams of models of chromosome looping within the CH can undergo CSR to IgA (69). Two mature mouse B cell lines A gene locus obtained from 3C assays. ( ) In mature naive B cells, the Em and (CH12F3 and I.29m) can be induced to switch in culture, but 39Ea enhancers are interacting. (B) A model for the interactions predicted from 3C assays for splenic B cells induced to switch to IgG1, showing the switching is restricted to IgA, or rarely, in I.29m, to IgE or interactions between the two enhancers and the Em-Sm-Cm and Sg1-Cg1 IgG2a. Perhaps these cell lines have a chromosomal loop that loci. The S regions are not diagrammed, and the sites of interaction are not sequesters the other CH loci from the enhancers. Much more Downloaded from precisely known, so the diagram only indicates approximate locations. See text research is needed to understand how specific chromosomal for further explanation. loops are regulated and the roles of chromosomal loops in regulating CSR. It is important to identify the proteins regulating loop formation between the Em and 39Ea enhancers and CH genes Introduction of DSBs in S regions by AID involves the BER and MMR in B cells before and during CSR. YY-1, a protein that binds pathways http://www.jimmunol.org/ both the 39Ea and Em enhancers is a candidate (59, 60). Although AID is rapidly induced after B cell activation, the Better evidence is available for PTIP, which was shown to enzymes and proteins that convert the AID-induced dUs to interact with the B cell–specific protein Pax5 and to be im- DSBs are constitutively expressed, because the lesions that portant for the interaction between the 39Ea and the g1 GLT these proteins repair occur in all types of cells. Uracil, whether promoter (61). Mice deficient in PTIP have reduced g3, g2b, as a result of AID activity or caused by spontaneous hydrolysis and g1 GLTs and reduced CSR to these isotypes (62), but it of cytosine, can be excised by the BER enzyme uracil DNA is unknown how much of this effect is due to reduced in- glycosylase (UNG), which leaves an abasic (apyrimidinic/ teraction between the CH loci and the 39Ea and how much apurinic [AP]) site (Fig. 2A). Although cells express four by guest on September 26, 2021 is due to the fact that PTIP is important for recruiting the uracil DNA glycosylases (UNG, Smug1, MBD4, and TDG), histone methyl transferase MLL, which produces the acti- deficiency in UNG alone results in a 95–99% reduction in vating histone modification H3K4me3. It is possible that CSR in mice and humans (4, 70, 71) and reduces S-region MLL and/or H3K4me3 are important for the looping. DSBs detected during CSR in cultured splenic B cells nearly 2 2 CTCF and cohesin, two proteins involved in contraction/ to levels observed in aid / B cells (72). Recently, it was 2/2 looping of the VH and Vk gene loci (63), do not appear to found that, in ung mice, Smug1 can partially substitute 2/2 2/2 bind within the 180-kb region encoding the CH genes nor to (i.e., in smug1 ung splenic B cells, CSR is reduced the 39Ea.However,cohesinwasshowntoberequiredfor another 5-fold) (71). Smug1 deficiency by itself has no effect optimum CSR, but the mechanism of its contribution is on CSR, probably as a result of its low abundance (73) and unknown (64, 65). low activity on dU in ssDNA, whereas UNG is abundant and more active on ssDNA than on dsDNA (74). Because AID CSR in B cell progenitors only has activity on ssDNA, it is possible that UNG excises Pre-B cells purified from mouse bone marrow express low most dUs prior to reformation of the DNA duplex. amounts of AID and were shown to have ongoing H chain Abasic (AP) sites are subsequently cut by AP endonuclease switch recombination (m . g2b), despite the fact that they do (APE)1 and/or APE2, creating an ssDNA break (SSB) not express L chains and IgM (66). This was determined by (Fig. 2A). If SSBs are sufficiently near each other on both two molecular assays for switch recombination events at the DNA strands, DSBs are produced. APE1 is essential for 2 2 DNA level, detection of RNA transcripts from the excised viability; although apex1 / mice have not been produced, 2 DNA circles (circle transcripts), and postswitch transcripts apex1+/ mice have DNA repair defects. Both APE1 and produced by transcription of the recombined genes. In a dif- APE2 contribute to CSR in splenic B cells induced to ferent study, pro-B cells from Rag1-deficient mice could be switch in culture (75). S-region DSBs are greatly reduced in 2 2 2 induced in culture to switch at the DNA level from m . g2b apex1+/ apex2 / splenic B cells but are only marginally 2 2 2 and from m . «, although these cells do not express either reduced in apex1+/ or apex2 / cells, indicating that m-chains or L chains (67). The physiological role of this H these enzymes are partially redundant. However, deletion chain switching is unknown, although AID activity in pro- of these enzymes in CH12F3 B lymphoma cells gave dif- and pre-B cells might contribute to autoimmunity (68). ferent results. APE1-deficient cells have an 80% reduction in Interestingly, and unlike CSR regulation in mature B cells, CSR, whereas APE2 deficiency has no effect on CSR (76). m . g2b, but not m . g3, switching occurs in pro-B cells The different results in splenic B cells and CH12F3 cells activated with LPS plus CD40L, and m . «, but not m . g1, remains a puzzle. 5374 BRIEF REVIEWS: Ab CLASS SWITCH RECOMBINATION

APE2 is a very inefficient endonuclease, about 1000-fold less forming a platform for Artemis and PALF, as well as active than APE1. APE2 has stronger exonuclease activity than for DNA polymerases, and greatly stimulating the ligation APE1; thus, one could envision APE1 producing the SSB and activity of DNA ligase IV-XRCC4-XLF (reviewed in Refs. APE2 creating a gap at the SSB, which could increase the 87–89). The Mre11-Nbs1-Rad50 (MRN) complex also rap- probability that an SSB might become a DSB. However, evidence idly binds DSBs, but it is unclear whether KU and MRN suggesting that APE2 acts as an endonuclease at AID-induced AP compete or cooperate at S-region DSBs. Rad50 has a long sites in splenic B cells was obtained by determining the locations coiled-coil domain with a hook at the end by which MRN of S-region DSBs in APE-deficient mice compared with wild- complexes bound at different DSBs can interact and tether the type (WT) mice. APE2 deficiency reduced their preference DSBs to each other (90). MRN recruits additional factors, in- for G:C bp, so that the proportion of breaks at G:C bp were not cluding the kinase ataxia-telangiectasia mutant (ATM), which significantly different from their proportion in the sequence activates and coordinates the cellular response to DSBs. KU and 2 itself, whereas DSBs in apex1+/ cells were similar to WT cells MRN were both shown to bind S-region DSBs (6, 91–93) and (75). It is not known why APE2 is important for CSR, especially contribute to CSR (94–98). because APE1 is a much more efficient endonuclease. There is a great deal of mechanistic flexibility in NHEJ, A major question in the field is why AID-induced lesions are which is necessary because DSBs can differ greatly, having 39 not accurately repaired, despite the fact that BER is a highly or 59 ss tails of different lengths, and possibly even having active and error-free repair pathway. In most cells, DNA Pol b hairpins, in addition to being blunt. Generally, the junctions accurately replaces the excised , ligase III–XRCC1 formed between two S regions have 0 or 1 bp of micro- Downloaded from seals the phosphodiester backbone, and DSBs are not gener- homology between Sm and the acceptor S region (i.e., one ated (Fig. 2A). In fact, DNA Pol b modestly inhibits S-region cannot discern whether the 1 bp came from Sm or the ac- DSBs and CSR, suggesting that it competes with DSB for- ceptor S region, although this microhomology can increase up mation but is overwhelmed by the numbers of AID-induced to 6 bp or more). In cells deficient in an NHEJ protein (e.g., SSBs (77). Another study suggested an additional possibility. KU or ligase IV) or deficient in Mlh1 or Pms2, S–S junctions

In this study, SHM of Ab V region genes was examined in show increased lengths of microhomology, especially if Sm– http://www.jimmunol.org/ germinal center B cells from Peyer’s patches (78). These cells Sa junctions are analyzed, because these S regions have the are undergoing constant stimulation by gut microbes and most homology with each other (99). To explain the increased undergo robust SHM. The results suggest that the use of lengths of junctional microhomology, investigators have APE2, instead of APE1, in repair of AID-induced lesions in thought that an alternate end-joining pathway, or micro- germinal centers converts BER to an error-prone repair pathway homology-mediated end joining, substitutes for NHEJ (100– (78). Germinal center B cells express very low levels of APE1 102). However, it is not clear whether microhomology- protein and mRNA compared with APE2. APE2 was found to mediated end joining is really a defined pathway or, instead, +/2

be important for mutations at A:T bp, whereas apex1 Bcells that NHEJ has partially redundant components. For example, by guest on September 26, 2021 have unperturbed SHM (78). Because an SSB is required as an in the absence of ligase IV, ligase I and ligase III can perform entry point for the error-prone translesion polymerase DNA end joining, but not as efficiently, and the other components Pol h to introduce mutations at A:T bp, this suggests that of NHEJ can still participate (88, 103). Deficiencies in an APE2 is indeed acting as an endonuclease during SHM, cre- NHEJ component might expose the DSBs to end-resection ating SSBs. It is possible that the use of APE2 might inhibit activities; also, the less efficient recombination might allow error-free repair of AID-induced lesions. Interestingly, APE2, time for increased end resection prior to ligation and result in but not APE1, interacts with PCNA, a protein that recruits increased use of microhomology, which can help to stabilize DNA Pol h to DNA (79). the junctions. In situations in which fewer S-region DSBs are When the AID-UNG-APE pathway induces SSBs that are too induced [e.g., in human patients heterozygous for AID with 2 2 2 2 far apart on opposite DNA strands to produce DSBs, MMR can a C-terminal deletion (104) or in mlh1 / or pms2 / mouse convert the SSBs to a DSB (5) (Fig. 2B). The Msh2-Msh6 and human B cells (7, 99, 105)], junctional microhomology is heterodimer binds to U:G mismatches in duplex DNA, as well also increased. Perhaps this occurs because DSBs are limiting, as recruits Mlh1-Pms2 and exonuclease 1, which initiates re- thus delaying recombination. Note that although Msh2- section from a SSB located 59 to the mismatch (80, 81). Pms2 deficient cells have reduced DSBs (7), they do not have also has endonuclease activity and can create additional SSBs increased junctional microhomology, indicating that reduced on the previously nicked strand, providing additional entry sites DSB frequency does not always result in increased use of for exonuclease 1 or other nucleases (82, 83). As diagrammed microhomology. The explanation for this difference from in Fig. 2B, this resection should result in creation of a DSB Mlh1- and Pms2-deficient cells is unknown. with a long ss tail, which, when filled in by DNA Pol, creates a blunt or nearly blunt DSB appropriate for NHEJ (5). Several The AID C terminus is essential for CSR but not for SHM types of data support this model, including the facts that The fact that AID lacking the C terminal 8–17 aa (DAID) S-region DSBs and CSR are reduced by 2–7-fold in MMR- cannot support CSR but appears to support normal SHM of deficient B cells (7, 84, 85) and that, in the absence of Sm V(D)J segments has been known for several years, but the ex- tandem repeats, with their numerous AID target hotspots, CSR planation is still unclear (106, 107). Recruitment of UNG and is absolutely dependent upon Msh2 (86). Msh2-Msh6 to Sm is impaired in cells expressing DAID, likely decreasing the introduction of DSBs. However, the numbers of Recombination of S-region DSBs by NHEJ Sm and Sg DSBs detected are normal, suggesting that DSBs S–S recombination occurs by NHEJ, which involves binding accumulate due to defective repair and recombination (93, 108, of the abundant toroidal heterodimer KU70/80 to each DSB, 109). In agreement, there is reduced recruitment of NHEJ The Journal of Immunology 5375 proteins to Sm regions and increased lengths of S–S junctional verting it to gH2AX, which, in turn, interacts with 53BP1 microhomology in cells expressing DAID(93,104,110,111). (122), although additional kinases, other enzymes, and his- However, how the C terminus recruits NHEJ proteins, and tone modifications also recruit and activate 53BP1 (123, whether this is important for the greatly reduced CSR, is still 124). Resection at S-region DSBs during the G1 phase nor- unknown. mally is inhibited by 53BP1 and its effector protein Rif1, two proteins very important for CSR that function epistatically The role of ATM during CSR and phosphorylation of AID and whose activities favor recombination by NHEJ (120, 123, 2 2 ATM kinase is a major regulator of the DNA damage response. 125–131). Surprisingly, S–S junctions in 53bp1 / cells do ATM is rapidly activated by the binding of MRN to DSBs; in not have increased microhomology (126). Another activity of turn, ATM phosphorylates MRN and several downstream 53BP1 that is essential for CSR is its ability to oligomerize effectors of DSB repair (112). Humans with ATM mutations (127, 132). 53BP1 likely binds DSBs in both Sm and down- have numerous problems due to poor DNA repair functions. stream S regions, and, most likely, the oligomerization of Mouse splenic B cells lacking ATM have reduced CSR (30% 53BP1 helps to hold two different S regions together in the of WT B cells) (113, 114) and impaired V(D)J recombination proper conformation. This might explain why inverted S- (115). In response to S-region DSBs, ATM induces phos- region sequences and a segment from the IgH Em enhancer have been detected between the recombining S-region seg- phorylation of AID at S38 by an undefined pathway, and this 2 2 2 2 increases the ability of AID to induce S-region DSBs and to ments in 53bp1 / cells (126). In addition, in 53bp1 / cells bind to APE1 via an unknown protein (116). APE2 has not induced to undergo CSR there are numerous deletions within Downloaded from been tested. Thus, AID is activated by a feed-forward Sm, suggesting that DSBs within Sm failed to find an acceptor mechanism involving ATM. Phosphorylation of AID at S38 S-region recombination partner. Thus, 53BP1 is essential for is not necessary for its deaminase activity in cell-free experi- CSR, and it appears to have multiple roles, although they are ments, although activated cultured splenic B cells expressing still not entirely understood. AIDS38A have greatly reduced Sm DSBs and CSR (116–

118). It is hypothesized that this feed-forward mechanism Questions remaining http://www.jimmunol.org/ increases AID activity at localized regions that have DSBs, There are many more interesting issues to discuss about such as at the IgH S regions, helping to explain why AID is so CSR than can even be discussed briefly in this article be- much more active at S regions than at off-target sites (91, 119, cause of space limitations. We recommend several recent 120). 2 2 reviews of CSR that include additional topics (33, 49, 103, Interestingly, atm / B cells induced to switch show de- 133–135). creased DSBs at Sg regions but have increased DSBs at Sm Although much is now known about CSR, there are still (9). These data have been interpreted to suggest that the feed- many questions left unanswered, some of which have been forward activation of AID by ATM is very important for AID mentioned in this review. Although AID deaminates off-target by guest on September 26, 2021 activity at acceptor S regions and that, when DSBs are poorly genomic sites, as well as induces DSBs at off-target sites, most induced in the acceptor S region, Sm DSBs accumulate due to of its activity is directed toward Ig loci. How AID is targeted to lack of a downstream partner. These data also suggest that S regions is mostly unknown. We know that transcription, and acceptor S-region DSBs are limiting for CSR. It was found probably RNA stalling and RNA splicing, are important; of previously that AID is more active at Sm regions than at Sg course, there are numerous transcribed genes with spliced regions (37, 121), suggesting that AID attacks the Sm region transcripts and stalled Pol II. What causes the stalling of Pol II first (121). Taken together, these data lead to the hypothesis at S regions? S regions and S-region transcripts are likely to that ATM is activated by Sm DSBs, resulting in phosphory- bind specific proteins, such as 14-3-3 (136) and PTBP2 that lation of AID, which increases its activity at the downstream S help to recruit AID, but are there others? How is the looping region that is colocalized due to chromosome looping. The of the CH gene loci and enhancers regulated? What causes studies of chromosomal looping described above suggest that AID-induced S-region DSBs to be restricted to the G1 phase? Sm and acceptor S regions are located near each other prior to Is AID activity restricted to the G1 phase, as is UNG activity? induction of AID and DSBs, because the looping might co- What signaling mechanism causes the S-region DSBs to be incide with expression of GLTs. Thus, when AID is activated repaired prior to the S phase, even in the absence of ATM or by phosphorylation, the acceptor S region might be localized p53 (9)? What is the role of APE2 in S-region DSBs, and why sufficiently near to be attacked. Close localization of the two S isn’t APE1 sufficient? Although APE1 is expressed as well as regions also decreases the likelihood of aberrant recombina- APE2 in splenic B cells induced to switch in culture, it is tion events. Surprisingly, ATM deficiency has no effect on cell poorly expressed in germinal center B cells. Does the low cycle regulation of S-region DSBs, because they are restricted expression of APE1 reduce CSR in germinal center cells? Is 2/2 to the G1 phase in atm cells, just as in WT cells, indicating APE1 well expressed during CSR in vivo in nongerminal that Sm DSBs only accumulate during the G1 phase and are center cells? How does phosphorylation of AID at S38 cause repaired before the S phase, even if the DSBs do not undergo increased S-region DSBs? Is this specific for S-region DSBs? Sm–Sx recombination (9). What does the C terminus of AID do during CSR? Although it might be involved in recruitment of NHEJ proteins, its Function of 53BP1 in CSR deletion has a much greater effect on CSR than loss of NHEJ 53BP1 is one of the DNA repair proteins that binds DSBs in functions. What is the importance and what are the roles of response to the phosphorylation activities of ATM. CSR is histone modifications of IgH genes for CSR? What are the reduced by 90% in cells deficient in 53BP1, but its roles in functions of AID expression in pro- and pre-B cells? Is this CSR are still not clear. ATM phosphorylates H2AX, con- advantageous or only deleterious? 5376 BRIEF REVIEWS: Ab CLASS SWITCH RECOMBINATION

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