R

A

Aa

b

c

d

a

KABASCHSP

1

brIereaiTa[gcvhs

lde

H

1h

Seminars in Immunology 24 (2012) 246– 254

Contents lists available at SciVerse ScienceDirect

Seminars in Immunology

j ourna l ho me page: www.elsev ier .com/ locate /ysmim

eview

ctivation induced deaminase: How much and where?

lexandre Orthweina,b, Javier M. Di Noiaa,b,c,d,∗

Institut de Recherches Cliniques de Montréal, Montréal, Québec, H2W 1R7, CanadaDepartment of Microbiology and Immunology, Université de Montréal, Montréal, Québec, H3C 3J7, CanadaDepartment of Medicine, Faculty of Medicine, Université de Montréal, Montréal, Québec, H3C 3J7, CanadaDepartment of Medicine, McGill University, Montréal, Québec, Canada

r t i c l e i n f o

eywords:ctivation-induced deaminase (AID)-lymphocytentibody gene diversificationomatic hypermutationlass switch recombinationumoral immunityubcellular localization

a b s t r a c t

Activation induced deaminase (AID) plays a central role in adaptive immunity by initiating the processesof somatic hypermutation (SHM) and class switch recombination (CSR). On the other hand, AID also pre-disposes to lymphoma and plays a role in some autoimmune diseases, for which reasons AID expressionand activity are regulated at various levels. Post-translational mechanisms regulating the amount andsubcellular localization of AID are prominent in balancing AID physiological and pathological functionsin B cells. Mechanisms regulating AID protein levels include stabilizing chaperones in the cytoplasm andproteins efficiently targeting AID to the proteasome within the nucleus. Nuclear export and cytoplasmic

rotein stability retention contribute to limit the amount of AID accessing the genome. Additionally, a number of factorshave been implicated in AID active nuclear import. We review these intertwined mechanisms proposingtwo scenarios in which they could interact as a network or as a cycle for defining the optimal amount ofAID protein. We also comparatively review the expression levels of AID necessary for its function duringthe immune response, present in different cancers as well as in those tissues in which AID has been

emo

implicated in epigenetic r

. Antibody diversification in germinal center B cells

V(D)J recombination assembles the primary repertoire of anti-ody genes during B cell development. B cells that have successfullyearranged their immunoglobulin (Ig) genes express membranegM and/or IgD and migrate to the periphery where they will bexposed to foreign antigens. The first cognate antibody–antigenecognition of naïve B cells is usually not of high affinity. Thisndows the system with enough flexibility to interact with almostny possible antigen; however, high affinity antibody–antigennteractions are critical for neutralizing or disposing of antigens.hus, there are mechanisms to further change the antibodies. B cellsctivated by cognate antigen initiate the germinal center reaction1]. Germinal center B cells divide rapidly while diversifying theenes encoding for the heavy and light antibody chains. Diversifi-ation occurs through the introduction of point mutations in the

ariable regions of the IgH and IgL by the mechanism of somaticypermutation (SHM). Antibody variants produced by SHM areelected for improved antigen recognition by antigen presenting

Abbreviations: AID, activation induced deaminase; ALL, acute lymphoblasticeukemia; CML, chronic myeloid leukemia; CSR, class switch recombination; DLBCL,iffuse large B cell lymphoma; Ph+, Philadelphia chromosome-positive; NES, nuclearxport signal; NLS, nuclear localization signal; SHM, somatic hypermutation.∗ Corresponding author at: IRCM, 110 Avenue des Pins Ouest, Montréal, Québec,2W 1R7, Canada. Tel.: +1 514 987 5642; fax: +1 514 987 5528.

E-mail address: javier.di.noia@ircm.qc.ca (J.M. Di Noia).

044-5323/$ – see front matter © 2012 Elsevier Ltd. All rights reserved.ttp://dx.doi.org/10.1016/j.smim.2012.05.001

deling of the genome by demethylating DNA.© 2012 Elsevier Ltd. All rights reserved.

cells and T cells, which leads to affinity maturation of the antibodyresponse. At the same time, the constant exons of the IgH encod-ing for IgM and IgD are exchanged by exons encoding IgG, IgE, orIgA isotypes through the mechanism of class switch recombina-tion (CSR); thereby leading to the production of antibodies withconserved specificity but different biological properties. Both, SHMand CSR are mutagenic mechanisms that despite having differentend results (i.e. single point mutations versus a chromosomal dele-tion of several tens of kbp), share several molecular steps. The mostimportant step common to both SHM and CSR is their initiation bythe enzyme Activation induced deaminase (AID) [2,3].

AID is part of the AID/APOBEC family of cytosine deaminase-related enzymes, most of which have the unique capacity ofdeaminating deoxycytidine in single stranded DNA thus convertingit into deoxyuridine [4]. This is already a mutagenic lesion caus-ing a C:G to T:A base change after replication. Processing of theuracil by base excision and mismatch repair enzymes leads to thebroader spectrum of point mutations characterizing SHM, and toDNA double strand breaks, which are necessary intermediates inCSR (reviewed in [5–7]).

2. AID levels and disease

2.1. AID deficiency and haploinsufficiency

As expected from its central role in antibody diversification, lossof function mutations of AID cause an immunodeficiency syndrome

ars in

ctio[trilwdtPmtotossffhvaTA

2

bidwtmarAchsIscSatianobIbrqtmsbbA

A

A. Orthwein, J.M. Di Noia / Semin

haracterized by the absence of switched isotypes, recurrent infec-ions, and lymphoid tissue hyperplasia [3], which is recapitulatedn Aicda−/− mice [2]. Aicda+/− mice show an intermediate degreef lymphoid hyperplasia, between that of wt and Aicda−/− mice8,9]. AID haploinsufficient mice have ∼40% of AID mRNA and pro-ein levels compared to wt [9,10] and several groups have foundeduced CSR and SHM in Aicda+/− mice [8–11]. The decrease in CSRs roughly proportional to the decrease in AID mRNA and proteinevels although there are some discrepancies among the studies,

hich could originate from using different mouse strains and/orifferent in vitro experimental conditions [12,13]. There is consis-ency between studies in that SHM is reduced to ∼30% of the wt ineyer patches of Aicda+/− [8,9], with some more variability wheneasured elsewhere, ranging from 70% of wt in lymph nodes JH4 [8]

o only 15% of wt in S� region of B cells activated ex vivo [10]. Thesebservations suggest that AID is limiting for antibody diversifica-ion although it is not the only factor determining the efficiencyf CSR and SHM. In vivo, the effect is highly compensated for byelection [9,10] and it is unclear how compromised the immuneystem of an individual with reduced AID levels would be. Judgingrom the lack of clinical symptoms in humans carrying only oneunctional AID allele [3,14], this is probably not a major issue foruman health. However, it would have in all likelihood been disad-antageous during evolution. The previous considerations suggest

mechanism by which the minimal levels of AID were selected.he pathological side effects of AID probably set the upper limit ofID expression.

.2. AID levels in antibody diversification and cancer

Consistent with the view that AID levels are limiting for anti-ody diversification, higher levels of AID protein generally translate

nto more CSR and SHM, and are accompanied by an increased inci-ence of potentially transforming genomic lesions. Mouse modelsith modified AID expression levels nicely provided evidence for

his. First, several transgenic mice overexpressing AID have beenade, all of which show increased CSR and SHM [15–18]. There

re differences in the magnitude of the effect, which might beelated to the transgene design. The two transgenic lines in whichID was expressed from a ubiquitous promoter showed a signifi-ant but modest increase [15,16,18]. In these mice, B cells expressigh levels of AID throughout their development and some counter-election is possible, which could dampen the extent of the effect.n contrast, transgenic AID under the control of the Ig� enhancerhowed maximal expression in germinal center B cells and in thisase there was a very high increase in CSR and SHM levels [17].econdly, boosting AID expression in B cells by removing the neg-tive post-transcriptional regulation of AID by miR-155 also leadso higher levels of CSR [19,20]. Yet, none of these mice showedncreased levels of switched serum Ig, but this is probably regulatedt another level (selection, homeostatic proliferation), and does notecessarily reflect the intrinsic CSR capacity of the B cells. This lackf correlation between in vitro CSR and serum Ig levels, has alsoeen observed in MSH2-deficient and UNG-deficient mice [21–24].

nterestingly, mice in which higher levels of AID were achievedy removing the miR-155 binding sites from its mRNA showededuced affinity maturation, but no differences in the quantity oruality of SHM at the Ig variable regions [19]. This would suggesthat expression of AID above physiological levels could compro-

ise B cell viability, in line with the evidence that AID limits theize of the germinal center by causing B cell apoptosis [25]. It mighte that the upper limit for AID physiological expression levels could

e influenced by the increased apoptosis that occurs with elevatedID.

AID also contributes to the development of cancer, but whetherID levels correlate proportionally with the risk of developing

Immunology 24 (2012) 246– 254 247

cancer is unclear. AID oncogenicity is most likely a consequenceof its capacity to mutate and produce DNA breaks, thus initiatingchromosomal translocations affecting a number of loci in normal Bcells [26–30]. Increased levels of AID in B cells correlates with moreIgH-cMyc translocations [17,20] and increased mutations in somenon-Ig targets in vivo and in vitro [17,19,31]. However, this does notalways have oncogenic consequences [16,20] (see below).

Mice overexpressing AID have conclusively demonstrated theoncogenic capacity of AID. Ubiquitous transgenic overexpression ofAID leads to T cell lymphomas as well as lung adenomas and adeno-carcinomas, but not B cell malignancies [18]. B cells, in comparisonto other cell types that do not express AID, may have evolved pro-tective mechanisms against transformation, thus explaining thisobservation. Indeed, the apoptotic control that normally eliminatescells with AID-induced translocations [26], prevented B cell lym-phomas in an AID-Ig� transgenic model [17]. In the absence ofp53, AID-Ig� developed predominantly mature B cell lymphomas,even outpacing the T cell lymphomas that usually kill p53-deficientmice [17]. Curiously, germinal center B cells have reduced levelsof p53 to allow for the necessary DNA damage that accompaniesantibody gene diversification [32], which suggests the presence ofadditional mechanisms to prevent AID-initiated lymphomagenesis(for DNA repair regulation see Saribasak and Gearhart, this issue).For instance, off-target AID mutations are more frequent in theabsence of a number of DNA repair pathways, even with normalendogenous AID levels [31,33,34].

Endogenous levels of AID do initiate B cell transformation, albeitinfrequently. The etiological role of AID in B cell lymphomas orig-inating from germinal center B cells was demonstrated using theI�HABCL6 transgenic oncogene model, which deregulates BCL6 inB cells and results in frequent mature B cell lymphomas. Whencrossed with Aicda−/− mice, I�HABCL6 transgenic mice did notdevelop mature B cell lymphomas [35]. I�HABCL6 lymphomasare akin to human DLBCL (diffuse large B cell lymphoma), whichdisplays a high prevalence of AID expression [36–40], strongly sug-gesting an etiological role for endogenous AID in this lymphoma.In addition, endogenous AID levels contribute to chromosomaltranslocations of multiple partners with the Ig locus, such as theIgH-Ig� [41] or IgH-Pax5 [29,30], and even between two non-Iggenes [17,29,30]. Indeed, most of the translocations occurring ina p53-deficient background occur between c-Myc and miR-142[17], a molecular hallmark of acute prolymphocytic leukemia [42].Although a few DLBCL samples show higher than normal AID levels,in general DLBCL and follicular lymphoma samples show similar orlower AID levels than germinal center B cells [36–38,40]. Althoughlymphoma biopsies contain normal and non-B cells, where only AIDmRNA has been measured in many cases, it does not seem that AIDoverexpression is necessary for lymphomagenesis. The latter wasdemonstrated in the Balb/Bcl-xL mouse model of plasmacytoma.In this model, AID is required for most pristane-induced plasmacy-tomas, where it underpins the oncogenic IgH-cMyc translocation[34,39]. AID haploinsufficient mice show reduced incidence of lym-phoma compared to Aicda+/+, but still significantly higher than theAicda−/− [10]. Thus, significantly lower than normal levels of AIDare sufficient to cause plasmacytomas when they are combinedwith another predisposing condition. The IgH-cMyc translocation,which is caused by AID [26,28,43,44], is a hallmark of Burkitt’s lym-phoma in humans. AID is expressed at near normal levels in thistype of lymphoma [36]. Near to normal AID levels are also expressedin Ph+ ALL (Philadelphia chromosome-positive B cell acute lym-phoblastic leukemia) and CML (chronic myelogenous leukemia)[45–47]. In ALL and CML, AID can be a disease progression factor

by accelerating leukemia clonal evolution [46] and/or by mutat-ing the BCR-ABL1 oncogene, thus underpinning resistance to thetherapeutic drug imatinib [47]. Chronic lymphocytic leukemia isanother B cell malignancy expressing variable levels of AID due

2 ars in

tosiAtse

2

eaAdrB[mthltcabrai

3

svacrbtcriAvp

3

3

titbMwaneslHg

48 A. Orthwein, J.M. Di Noia / Semin

o population heterogeneity, where only a defined subpopulationf cells expresses most of AID mRNA [48,49]. The presence of thisubpopulation correlates with a worst prognosis, but AID mRNAn these cells is not higher than in normal B cells [48,49]. Finally,ID is also expressed in a number of tumor cell lines and primary

umors of non-lymphoid origin including breast [50], prostate [51],tomach [52], liver [53,54] and lung [55], where AID is normally notxpressed or expressed at much lower levels than in B cells [56].

.3. AID and autoimmunity

AID levels also influence antibody-mediated autoimmune dis-ases. AID is important in determining the severity of the pathology,s demonstrated in mouse models of lupus and arthritis in whichID deficiency, or even haploinsufficiency, results in a less severeisease [11,57–59]. In keeping with this, increased levels of AID cor-elate with higher levels of autoantibodies in the MRL/faslpr/lpr andXD2 mouse models of lupus and rheumatoid arthritis, respectively57,60–62]. Interestingly, AID-deficient mice also have autoim-

une disorders albeit of a different nature [63]. This fits well withhe predisposition to autoimmune disorders noted in AID-deficientuman patients [64]. The recent finding that low AID expression

evels during B cell development play a role in establishing B cellolerance could explain these findings [65,66]. Although, like in can-er, there are predisposing conditions beyond AID for developingutoimmunity, here again we find that AID levels are important inalancing an efficient immune response with disease. This equilib-ium may be altered by factors inducing AID, such as estrogens [67],nd could in part explain the higher susceptibility to autoimmunityn women.

. Regulating AID

The data summarized above illustrates how the optimal expres-ion levels of AID in germinal center B cells was selected duringertebrate evolution as a compromise between being able to mountn effective adaptive immune response and delaying the onset ofancer or autoimmune diseases. A strikingly complex network ofegulatory mechanisms exists to ensure that the optimal amount ofiologically active AID protein gets to the Ig locus. Any alteration inhe efficacy of these mechanisms could predispose to immunodefi-iency, autoimmunity or B cell lymphomas. Increased longevity andeduced selective pressure in humans have turned evolutionaryrrelevant side effects of AID into medical problems. UnderstandingID regulation and being able to manipulate its levels could pro-ide ways of targeting AID to prevent or ameliorate some of theseathologies.

.1. Aicda expression in and outside B cells

.1.1. Aicda in germinal center B cells and cancerAicda transcription is induced by cytokines and cell–cell interac-

ions in the context of antigen-triggered B cell activation during anmmune response (reviewed in [68] and see Vuong and Chaudhurihis issue). Thus, Aicda is highly transcribed in germinal center cellsut stringently repressed in plasma and memory B cells [69,70].easurements of AID mRNA in the B cell lineage agree well withestern blot and immunohistochemistry data, as well as with the

nalysis of an AID-GFP reporter mouse [40,69,71,72]. A combi-ation of promoter, enhancers and silencers located within fourvolutionary-conserved regulatory regions determines AID expres-

ion in germinal center B cells of the spleen and mucosal-associatedymphoid tissues (Peyer’s patches, tonsils, lymph nodes) [68,73].owever, the original belief of AID being exclusively expressed inerminal center B cells has been revised, as AID expression is also

Immunology 24 (2012) 246– 254

found in other tissues, not only in several pathological situationsbut also in normal tissues.

It is conceivable that the expression of AID in B lymphoid malig-nancies can be induced by similar stimuli than in mature B cells,or remains as a relict of the physiological counterpart of the neo-plasia. In other cases, such as leukemia originating from progenitorB cells (like in Ph+ ALL) or myeloid cells (like CML), where AID hasbeen convincingly detected [45,47], it is less clear but the BCR-ABL1oncogene itself could be involved in inducing Aicda [45]. It is alsounclear how AID expression is induced in malignant epithelial cellsfrom solid tumors. Ectopic AID expression in these tissues is likelyto be caused by the transcription factor Nf-�B, a critical transcrip-tion factor in orchestrating inflammatory and innate immunityresponses. In fact, Nf-�B participates in Aicda induction in nor-mal B cells [44,74] and was shown to mediate AID expression inhepatocytes [52]. Furthemore, epithelial cancers with AID expres-sion are often associated to chronic inflammation, sometimes withan underlying infection such as Hepatitis C virus [52,75], Heli-cobacter pylori [76,77] or human immunodeficiency virus [78,79].Also, other infectious agents like Epstein-Barr virus [78], Abelsonmurine leukemia virus [80] and Moloney murine leukemia virus[81], or stimulation of innate immune signaling through toll-likereceptors [44] can induce AID, probably through Nf-�B [81]. Insummary, it is difficult to generalize the reasons behind AID expres-sion in cancer cells, let alone its relevance for the pathology. It islikely that the clinical history and the selection acting in each caseinfluence the variations in AID expression levels we see betweenpatients.

3.1.2. Aicda transcription in other normal tissuesAID is expressed outside of the B cell compartment under nor-

mal conditions. The most notable example is the ovary, in whichbasal AID mRNA levels are ∼50–70% of those found in the spleen[56,82]. Furthermore, Aicda is induced by estrogen which increaseAID mRNA a few fold in spleen but by >20-fold in the ovary [67].Breast tissue also expresses AID when stimulated with estrogens[67], and several human breast cancer cells express AID mRNA[50], but we do not know the basal levels in normal breast tis-sue. Other examples of AID expression in normal non-lymphoidcells include prostate, heart and lung, although the estimates ofAID levels in these cases are less precise [51,56,82]. The presence ofAID would suggest a physiological role for it in these tissues, espe-cially in the ovary where levels are quite high, but it is unclear whatit is.

Evidence is accumulating to suggest that AID could be part ofa mechanism to demethylate 5meC at CpG sites in the genome,thereby influencing gene expression [56,83,84]. A role in epigeneticreprograming during development has been proposed, which couldexplain the presence of AID in cell types such as oocytes, spermato-cytes, primordial germ cells or embryonic stem cells [56,67,82–86].As mentioned, oocytes express AID mRNA to comparable levels ofB cells, although protein expression has not been tested [56,82].AID mRNA is very low in testis [56,69,82], which contrasts withthe fact that the protein has been detected by IF in spermatocytes[86]. There is discrepancy about the timing when AID is expressedin primordial germ cells although all reports find mRNA levels sub-stantially lower than in B cells (5–10%) [56,84,85]. Similarly AIDmRNA levels in stem cells are ∼5–10% of those found in B cells[56,83]. Thus, the amount of AID required for its proposed role inepigenetic reprogramming seems to be considerably lower thanthat required for antibody diversification. Even lower AID mRNAlevels than in stem cells have been measured during B cell develop-

ment [65,66], so low that it cannot be detected by using an AID-GFPreporter gene [69]. And yet, convincing evidence suggests that it isenough to play a role in B cell tolerance [65,66]. The possibility ofquite low levels of AID, which would be ineffective for antibody

ars in

deu

3

3

biatld[ei[

tdifnsrcHhAtpTAdokiptoinraCttnptwtcwrlon[lttcf

A. Orthwein, J.M. Di Noia / Semin

iversification in a normal immune response, being functional isven more striking when one considers the post-translational reg-latory mechanisms restraining AID in activated B cells.

.2. Post-translational regulation of AID in B cells

.2.1. AID subcellular localizationAID is predominantly cytoplasmic (see Häsler, Rada, and Neu-

erger, this issue) in steady-state as first reported for AID-GFPn Ramos B cells [87], and confirmed by subcellular fractionationnd IHC in primary B cells [40,71,88]. The deletion or mutation ofhe C-terminal 10 amino acids of AID led to its nuclear accumu-ation, identifying this region as a nuclear export signal (NES) andemonstrating that AID is a nuclear-cytoplasmic shuttling protein89–91]. This leucine-rich NES is a typical recognition motif for thexportin CRM1, as was demonstrated by using the CRM1-specificnhibitor leptomycin B [90,91] and by coimmunoprecipitation92,93].

AID gains access to the nucleus by active, energy-dependentransport [93]. Due to its small size (24 kDa), AID could in principleiffuse passively through the nuclear pores. However, it is actively

mported into the nucleus, as demonstrated by its capacity to con-er nuclear localization to large proteins that are well above theuclear pore passive diffusion cut-off [93]. The nuclear localizationignal (NLS) of AID has not been completely defined. AID N-terminalegion (roughly residues 5–50) contains multiple basic residues, aharacteristic of many NLS, and it is clearly a major part of it [90,93].owever, by itself it is not sufficient to mediate nuclear import ofeterologous proteins, which requires the first 181 out of the 198ID amino acids [93]. There are other residues elsewhere in the pro-

ein that form part of and/or are critical in displaying the NLS, whichrompted the suggestion that AID has a conformational NLS [93].he factors mediating AID nuclear import are also not well defined.ID binds in vitro to several karyopherin-� importins, which areedicated nuclear import factors, but their functionality has beennly indirectly inferred because oxidative stress, which inhibitsaryopherin-�-mediated nuclear import [94,95], also inhibits AIDmport [93]. However, AID also interacts with CTNNBL1, a nuclearrotein presumed to work in mRNA splicing, which binds to NLShrough an armadillo-like domain [96,97]. Nuclear accumulationf an AID variant with a mutated NES is partially compromisedn DT40 CTNNBL1−/− cells, suggesting a role for CTNNBL1 in AIDuclear import [97]. Still, CH12 CTNNBL1−/− cells did not show anyeduction in CSR demonstrating that it is at least redundant withnother mechanism for importing AID into the nucleus [98]. SinceTNNBL1 interacts with importin-�s [97], they could be part ofhe same pathway. GANP, a protein associated with the RNA shut-ling machinery [99], has also been proposed to mediate AID activeuclear import, largely based on the observation that its overex-ression increases the nuclear fraction of AID [100]. Regardless ofhe pathway, it is clear that AID is actively imported into the nucleushile its small size would allow it to passively diffuse, which raises

he question of why this would be necessary. The explanationould be that there is a need to counteract cytoplasmic retention,hich prevents its diffusion [93]. Indeed, a motif in AID C-terminal

egion, overlapping with, but being distinct from the NES, is able toimit the passive diffusion of GFP into the nucleus [93]. Separationf function mutations exist to corroborate that these two sig-als are distinct and mediate different protein–protein interactions93]. The translation factor eEF1� could participate in retention, ateast in part, since it is stoichiometrically associated with AID in

he cytoplasm and mutating the proposed AID cytoplasmic reten-ion motif can disrupt this interaction [93,101]. However, geneticonfirmation has not been possible since eEF1� is an essentialactor.

Immunology 24 (2012) 246– 254 249

AID subcellular localization is dynamic and reflects the equi-librium between nuclear export and cytoplasmic retention, andthe opposing active nuclear import. The fraction of steady statenuclear AID resulting from this equilibrium has been consistentlyestimated to be around <10% [40,88,91]. Whether this equilibriumis regulated (such as to increase diversification after some signal-ing event, at a particular cell cycle stage [102], etc.), or whether theproportion of AID in the nucleus is constant at all times [68,103], isunresolved. Drastic alterations or truncation of the AID NES abolishCSR, most likely because this region also contains a domain medi-ating interactions required for CSR [90,91,104,105]. Nevertheless,the relocalization of AID into the nucleus by mutating or delet-ing the NES increases SHM and immunoglobulin gene conversionin overexpression studies in vitro [90,104,105]. Similarly, mutatingthe C-terminal cytoplasmic retention signal of AID leads to fasternuclear import and higher SHM and CSR [93,105].

The interpretation of this data at face value, indicates thatalterations in the mechanisms of AID subcellular localization haveconsequences on antibody diversification and that nuclear exclu-sion limits the biological activity of AID. However, there are caveatsin this interpretation as most AID nuclear variants tend to showhigher catalytic activity [91,104,106] and lower expression lev-els [91,105,107]. The contribution of these characteristics to theobserved effect on antibody diversification is unknown and wecould be over or underestimating the magnitude of the effect.

3.2.2. AID stabilityThe stability of AID is directly linked to its subcellular localiza-

tion with cytoplasmic AID being much more stable than nuclearAID [107]. The ∼8 h half-life of AID in B cells represents in fact arough average of its ∼2.5 h nuclear and 18–20 h cytoplasmic half-life [107]. Indeed, inhibition of AID nuclear export with leptomycinB and mutation or deletion of the NES, as well as disruption of AIDcytoplasmic retention, increase the nuclear fraction of AID and cor-relate with a decrease in its half-life [93,101,105,107,108]. On theother hand, restricting AID to the cytoplasm resulted in increasedAID half-life [93,107]. There are some mechanistic explanationsfor this difference in stability. HSP90 interacts with AID in thecytoplasm and prevents its polyubiquitination and therefore itsproteasomal degradation [108]. The first step in the HSP90 molec-ular chaperoning pathway and stabilization is the interaction ofAID with the HSP40 and HSP70 system [109]. In particular, thereis a specific dependence on the HSP40 DnaJa1, since its deficiencyresults in decreased stability and reduced levels of AID, accompa-nied by loss of biological activity [109]. Simultaneous inhibition ofHSP90 and the proteasome, but not inhibiting only the proteasome,results in massive accumulation of polyubiquitinated AID [108].Thus, there seems to be very little turnover of cytoplasmic AID,stabilization being the default pathway. HSP90 is critical for thisstabilization, most likely just after AID protein synthesis, but doesnot exclude the possibility that later complexes with eEF1� [101] orimportins [93], could also be stabilizing. It has also been proposedthat some maturation step such as post-translational modificationor oligomerization could stabilize AID [103,108].

Once inside the nucleus, AID is much less stable, either becauseit looses stabilizing interactions and/or it is actively destabilized.Unlike cytoplasmic AID, nuclear AID seems to be constantly tar-geted to the proteasome by ubiquitin-dependent and -independentpathways [107,110]. Proteasomal inhibition is sufficient for sub-stantial accumulation of polyubiquitinated nuclear AID [107]. TheE3 ubiquitin ligases modifying nuclear AID are unknown. MDM2interacts with AID and could be one of them [111]. However, DT40

cells deficient in, or overexpressing, MDM2 show a very modestincrease and decrease in Ig gene conversion, respectively [111].Thus, MDM2 is either redundant with some other ubiquitin lig-ase or not relevant for AID. Interestingly, AID with no internal

2 ars in

lgpibA

cDDltrtois[oa

doebaAeit

3

pscTsta[c

a[ueipaiciowi

4

4e

pb

50 A. Orthwein, J.M. Di Noia / Semin

ysine residues is still significantly polyubiquitinated [107], sug-esting either N-terminal ligation or a non-canonical pathway ofolyubiquitination for AID. A proportion of AID interacts with, and

s targeted for degradation by, the nuclear protein REG-�, whichrings proteins for proteasomal degradation in an ubiquitin- andTP-independent fashion [110] (see below).

The cytoplasmic stabilization by the DnaJa1–HSP90 pathway isritical for, and limiting in, producing AID. Inhibiting HSP90, ornaJa1 farnesylation (which prevents binding to AID), as well asnaJa1 knockdown or knockout, all result in lower endogenous AID

evels and a nearly proportional decrease in antibody diversifica-ion by SHM and CSR [108,109]. This data is in keeping with theesults from AID haploinsufficient mice [9,10]; supporting the viewhat AID protein is limiting. Also consistent with this view, DnaJa1verexpression increases AID levels and antibody diversification

n vitro [109]. Similarly, REG-� deficiency results in higher AIDteady-state levels, increased nuclear AID stability and CSR in mice110]. Thus, modulating AID stability seems to be one more wayf limiting AID expression and yet another mechanism restrainingntibody diversification.

In the absence of AID-specific repressors, HSP90 inhibitorserived from geldanamycin, which are already in clinical trials,r farnesyltransferase inhibitors to inactivate DnaJa1, could bexploited to indirectly target AID [108,109]. They offer the possi-ility to treat some of the pathologies associated with exacerbatedntibody diversification, like autoimmune diseases, or derived fromID side effects such as imatinib resistance in CML or tumor clonalvolution. The challenge here is to identify a disease where AIDmplication and onset can be predicted to use these inhibitors athe right time.

.2.3. AID phosphorylationIn addition to AID protein levels and subcellular localization,

hosphorylation regulates AID activity. Five evolutionary con-erved AID residues, have been found to be phosphorylated in Bells: Ser3, Thr27, Ser38, Thr140 and Tyr184 [8,112–116]. Despiteyr184 being very close to the NES and the cytoplasmic retentionignal, and Ser3, Thr27 and Ser38 being close to or immersed inhe NLS, neither phosphonull nor phosphomimicking mutationst any of these sites seem to affect AID subcellular localization103,115,117]. Also, it is not known whether phosphorylation mayontribute to AID stability.

Phosphorylation of Thr27 inhibits AID-induced deaminationnd CSR in vitro, suggesting it modulates AID specific activity112,113,117], but whether this is used in vivo to regulate AID isnknown. Phosphorylation at Ser3 reduces AID biological activityx vivo but does not impact its catalytic activity, and its role andmportance in vivo are unknown [114]. On the other hand, phos-horylation of Ser38 and Thr140 are not essential for AID catalyticctivity in vitro, but both significantly increase AID biological activ-ty in vivo [8,112,115,118,119]. Ser38 is phosphorylated by PKA onhromatin, which allows AID to interact with RPA and greatly facil-tates CSR and SHM [8,112,113,115,119,120]. However, the effectf mutating Ser38 and Thr140 in vivo was much more pronouncedhen combined with AID haploinsufficiency [8,118], thus suggest-

ng that these modifications are not limiting for AID activity.

. Conclusion and questions

.1. Regulation of AID in cells with high versus low levels of AIDxpression

The post-translational regulation of AID (by stabilization, com-artmentalization, and phosphorylation) greatly contributes toalancing antibody gene diversification and widespread DNA

Immunology 24 (2012) 246– 254

damage in germinal center B cells. The expected (and in some casesknown) contribution of AID to cancer is by the same mechanisms ituses for antibody diversification: induction of mutations and DNAbreaks. Where the information is available, the AID levels in lym-phoid malignancies and solid tumors from human patients tendto be within the same range as those in B cells. In many of thesecancers, AID post-translational regulation mechanisms seem to be,for the most part, functional. Indeed, many B cell lymphoma celllines expressing AID have been used to study compartmentaliza-tion, stability and phosphorylation of AID, and these findings werethen confirmed in primary cells. In almost every cell line tested,AID is cytoplasmic and responds to leptomycin B; is sensitive toHSP90 inhibition, and is phosphorylated. However, there could bequantitative or qualitative differences that go unnoticed becausenot much work has been done on actual activated germinal center Bcells. For instance, there could be differences in the relative strengthof the different mechanisms regulating AID subcellular localizationand/or stability, which may shift the balance of AID regulation andpromote DNA damage. This could partly explain why certain tis-sues can be more sensitive to transformation than B cells in AIDtransgenic mice (i.e. thymus, lung or testis) [18,121].

Tissues expressing very low AID levels are quite a different cat-egory to consider. First there is genetic evidence suggesting thatthe expression of AID during early B cell development in the bonemarrow plays a physiological role by eliminating autoreactive Bcells [65,66]. It was suggested that AID expression during B celldevelopment mutates the IgV region [122,123]. This is reminis-cent of what is observed in sheep, rabbits and cattle where SHMor Ig gene conversion contributes to the diversity of the primaryantibody repertoire [124–126]. In humans, AID expression duringearly B cell development could be an evolutionary relic or may con-tribute to tolerance by mutating autoreactive clones, thus changingtheir specificity. However, given the almost linear proportionalitybetween AID protein levels and SHM in germinal center B cells, it isdifficult to imagine that the 1000-fold lower levels of AID expressedin immature B cells when compared to germinal center B cells [66],could do much SHM. The mechanism of AID-mediated B cell tol-erance is unknown but one wonders whether the 1000-fold lessmRNA linearly translates into 1000-fold less AID, or whether only10% of this AID is in the nucleus and how unstable is it. Simi-lar questions can be asked about cells where AID is proposed tounderpin epigenetic reprogramming. One might need to invoke amuch more efficient (and error free) mechanism for AID-mediateddemethylation than for SHM.

4.2. A network or a cycle for AID protein regulation?

It is likely that multiple mechanisms were selected during evo-lution to restrain the activity of AID. The alternatives, widespreadgenomic damage or immunodeficiency, are highly deleterioustraits. Nevertheless, the redundancy of mechanisms with, appar-ently, the same function is striking. The total AID levels expressedin B cells are much higher than the amount of it that can be foundin the nucleus at any time, and there are multiple mechanisms toenforce this nuclear exclusion. These mechanisms could be seenas a cycle or as a network (Fig. 1). The first model envisions thatthese mechanisms actually form a circuit (f.i. release from HSP90 isalways followed by AID associated to cytoplasmic retention, whichprecedes nuclear import, etc.). This could allow mechanisms withapparently the same effect to play distinct roles. Cytoplasmic reten-tion could contribute more to AID nuclear exclusion than nuclearexport (as the largely cytoplasmic distribution of endogenous AID

in Ramos cells treated with leptomycin B could suggest [93]).Nuclear export could contribute to the recycling of AID from thenucleus in keeping with the decay observed when nuclear exportis inhibited. An alternative model could be that there are several

A. Orthwein, J.M. Di Noia / Seminars in Immunology 24 (2012) 246– 254 251

AHA-1

Networ k Pharmacologi cal d est abilizat ion

Proteasome

Hsp90 inhibito rs FTI

HSC70ATP

ADP

CYTOPLASMICRETENTION

CYTOPLASMICRETENTION

NUCLEARIMPORT

NUCLEARIMPORT

NUCLEAREXPORT

NUCLEARDEGRADATION

NUCLEAREXPORT

CYTOPLASMICDEGRADATION

NUCLEARDEGRADATION

TARGETING

TARGETING

STABILIZATION

STABILIZATION

Oligomerization ?PTM ?

?

CRM1

DnaJa1

HSP90

AID eEF1α

CTNNBL1GANP

?

Poly Ub REG-γ

Cycle

Proteasome

E3?

TranscriptionRNA processing factorsPKA phosphorylation

AID

ATP

ADP

DnaJa1HSP90

A

CB

E3 (?)

P

Importin α

Fig. 1. Major post-translational regulation steps affecting AID levels. (A) Schematic representation of steps participating in stability and subcellular localization regulationrepresented within a cycle. Only selected AID interacting factors are shown. AID is synthesized in the cytoplasm, where unfolded AID is met by the HSP40-HSC70 system, thespecific action of the HSP40 DnaJa1 allows transferring AID into the HSP90 molecular chaperoning stabilization cycle. After some undefined maturation step, or conformationalchange, AID is passed onto eEF1� and/or other cytoplasmic retention factors before active nuclear import. A number of factors could be implicated, alternatively or jointlyin AID nuclear import. Inside the nucleus AID is either exported by CRM1 or targeted to the Ig loci by interacting with a number of RNA processing factors, where itis phosphorylated by PKA. AID is (subsequently) degraded in the nucleus either through ubiquitin- or REG�-dependent proteasomal degradation. (B) Simplified schematicrepresentation of the same steps as in A, but in the form of a network in which most pools of AID are interconnected (see text). (C) Schematic representation of AID cytoplasmicdegradation following inhibition of the HSP90 molecular chaperoning pathway. HSP90 inhibitors prevent the ATP hydrolysis cycle of the chaperone. FTI, farnesyltransferasei bilizato

otbAtbp

nhibitors, prevent farnesylation of DnaJa1, which is required for binding to and staf cytoplasmic AID.

ptions after each regulatory point, with as many possible destina-ions competing for AID (f.i. release from HSP90 could be followedy cytoplasmic retention or nuclear import or degradation; nuclear

ID could have a similar probability of being exported, targeted to

he Ig locus or degraded, etc.). Of course, a third possibility woulde a mixture of both models, but this simplification could help inostulating testable hypothesis.

ion of AID. Both inhibitors lead to polyubiquitination and proteasomal degradation

Much experimental work is still needed. The germinal center isprobably the only normal tissue in which the presence of AID pro-tein has been accurately measured. Detection elsewhere has mostly

relied on RT-PCR [56,67,82–84] or non-quantitative immunode-tection by IHC or IF. Comparing the relative levels of AID proteinbetween different tissues; developing sensitive methods to fol-low AID in precursor B or stem cells, comparing the regulatory

2 ars in

mcs

A

iIioT

R

52 A. Orthwein, J.M. Di Noia / Semin

echanisms of AID between germinal center and transformed Bells or non B cell expression sites, would all contribute to under-tanding AID.

cknowledgments

We thank Stephen Methot for reading the manuscript. The workn our laboratory was funded by operating grants from the Canadiannstitutes of health research and the Cancer Research Society and annfrastructure grant from the Canadian Fund for Innovation, leaderspportunity fund. JMDN is supported by a Canada Research Chairier 2 and AO by a Cole Foundation doctoral fellowship.

eferences

[1] Klein U, Dalla-Favera R. Germinal centres: role in B-cell physiology and malig-nancy. Nature Reviews Immunology 2008;8:22–33.

[2] Muramatsu M, Kinoshita K, Fagarasan S, Yamada S, Shinkai Y, Honjo T. Classswitch recombination and hypermutation require activation-induced cyti-dine deaminase (AID), a potential RNA editing enzyme. Cell 2000;102:553–63.

[3] Revy P, Muto T, Levy Y, Geissmann F, Plebani A, Sanal O, et al. Activation-induced cytidine deaminase (AID) deficiency causes the autosomal recessiveform of the hyper-IgM syndrome (HIGM2). Cell 2000;102:565–75.

[4] Conticello SG. The AID/APOBEC family of nucleic acid mutators. Genome Biol-ogy 2008;9:229.

[5] Stavnezer J, Guikema JE, Schrader CE. Mechanism and regulation of classswitch recombination. Annual Review of Immunology 2008;26:261–92.

[6] Peled JU, Kuang FL, Iglesias-Ussel MD, Roa S, Kalis SL, Goodman MF, et al.The biochemistry of somatic hypermutation. Annual Review of Immunology2008;26:481–511.

[7] Di Noia JM, Neuberger MS. Molecular mechanisms of antibody somatic hyper-mutation. Annual Review of Biochemistry 2007;76:1–22.

[8] McBride KM, Gazumyan A, Woo EM, Schwickert TA, Chait BT, NussenzweigMC. Regulation of class switch recombination and somatic mutation by AIDphosphorylation. The Journal of Experimental Medicine 2008;205:2585–94.

[9] Sernandez IV, de Yebenes VG, Dorsett Y, Ramiro AR. Haploinsufficiency ofactivation-induced deaminase for antibody diversification and chromosometranslocations both in vitro and in vivo. PLoS One 2008;3:e3927.

[10] Takizawa M, Tolarova H, Li Z, Dubois W, Lim S, Callen E, et al. AID expressionlevels determine the extent of cMyc oncogenic translocations and the inci-dence of B cell tumor development. The Journal of Experimental Medicine2008;205:1949–57.

[11] Jiang C, Zhao ML, Diaz M. Activation-induced deaminase heterozygousMRL/lpr mice are delayed in the production of high-affinity pathogenicantibodies and in the development of lupus nephritis. Immunology2009;126:102–13.

[12] Wu X, Stavnezer J. DNA polymerase beta is able to repair breaks in switchregions and plays an inhibitory role during immunoglobulin class switchrecombination. The Journal of Experimental Medicine 2007;204:1677–89.

[13] Kaminski DA, Stavnezer J. Antibody class switching differs among SJL, C57BL/6and 129 mice. International Immunology 2007;19:545–56.

[14] Durandy A, Peron S, Taubenheim N, Fischer A. Activation-induced cytidinedeaminase: structure–function relationship as based on the study of mutants.Human Mutation 2006;27:1185–91.

[15] Rush JS, Liu M, Odegard VH, Unniraman S, Schatz DG. Expression ofactivation-induced cytidine deaminase is regulated by cell division, providinga mechanistic basis for division-linked class switch recombination. Proceed-ings of the National Academy of Sciences of the United States of America2005;102:13242–7.

[16] Muto T, Okazaki IM, Yamada S, Tanaka Y, Kinoshita K, Muramatsu M, et al.Negative regulation of activation-induced cytidine deaminase in B cells. Pro-ceedings of the National Academy of Sciences of the United States of America2006;103:2752–7.

[17] Robbiani DF, Bunting S, Feldhahn N, Bothmer A, Camps J, Deroubaix S, et al.AID produces DNA double-strand breaks in non-Ig genes and mature Bcell lymphomas with reciprocal chromosome translocations. Molecular Cell2009;36:631–41.

[18] Okazaki IM, Hiai H, Kakazu N, Yamada S, Muramatsu M, Kinoshita K, et al.Constitutive expression of AID leads to tumorigenesis. The Journal of Experi-mental Medicine 2003;197:1173–81.

[19] Teng G, Hakimpour P, Landgraf P, Rice A, Tuschl T, Casellas R, et al.MicroRNA-155 is a negative regulator of activation-induced cytidine deami-nase. Immunity 2008;28:621–9.

[20] Dorsett Y, McBride KM, Jankovic M, Gazumyan A, Thai TH, Robbiani DF, et al.

MicroRNA-155 suppresses activation-induced cytidine deaminase-mediatedMyc-Igh translocation. Immunity 2008;28:630–8.

[21] Bardwell PD, Woo CJ, Wei K, Li Z, Martin A, Sack SZ, et al. Altered somatichypermutation and reduced class-switch recombination in exonuclease 1-mutant mice. Nature Immunology 2004;5:224–9.

Immunology 24 (2012) 246– 254

[22] Rada C, Di Noia JM, Neuberger MS. Mismatch recognition and uracil excisionprovide complementary paths to both Ig switching and the A/T-focused phaseof somatic mutation. Molecular Cell 2004;16:163–71.

[23] Ehrenstein MR, Neuberger MS. Deficiency in Msh2 affects the efficiency andlocal sequence specificity of immunoglobulin class-switch recombination:parallels with somatic hypermutation. EMBO Journal 1999;18:3484–90.

[24] Rada C, Williams GT, Nilsen H, Barnes DE, Lindahl T, Neuberger MS.Immunoglobulin isotype switching is inhibited and somatic hyper-mutation perturbed in UNG-deficient mice. Current Biology 2002;12:1748–55.

[25] Zaheen A, Boulianne B, Parsa JY, Ramachandran S, Gommerman JL, MartinA. AID constrains germinal center size by rendering B cells susceptible toapoptosis. Blood 2009;114:547–54.

[26] Ramiro AR, Jankovic M, Callen E, Difilippantonio S, Chen HT, McBride KM, et al.Role of genomic instability and p53 in AID-induced c-myc-Igh translocations.Nature 2006;440:105–9.

[27] Robbiani DF, Bothmer A, Callen E, Reina-San-Martin B, Dorsett Y, Difilippan-tonio S, et al. AID is required for the chromosomal breaks in c-myc that leadto c-myc/IgH translocations. Cell 2008;135:1028–38.

[28] Dorsett Y, Robbiani DF, Jankovic M, Reina-San-Martin B, Eisenreich TR,Nussenzweig MC. A role for AID in chromosome translocations betweenc-myc and the IgH variable region. The Journal of Experimental Medicine2007;204:2225–32.

[29] Chiarle R, Zhang Y, Frock RL, Lewis SM, Molinie B, Ho YJ, et al. Genome-wide translocation sequencing reveals mechanisms of chromosome breaksand rearrangements in B cells. Cell 2011;147:107–19.

[30] Klein IA, Resch W, Jankovic M, Oliveira T, Yamane A, Nakahashi H, et al.Translocation-capture sequencing reveals the extent and nature of chromo-somal rearrangements in B lymphocytes. Cell 2011;147:95–106.

[31] Yamane A, Resch W, Kuo N, Kuchen S, Li Z, Sun HW, et al. Deep-sequencingidentification of the genomic targets of the cytidine deaminase AID and itscofactor RPA in B lymphocytes. Nature Immunology 2011;12:62–9.

[32] Phan RT, Dalla-Favera R. The BCL6 proto-oncogene suppresses p53 expressionin germinal-centre B cells. Nature 2004;432:635–9.

[33] Liu M, Duke JL, Richter DJ, Vinuesa CG, Goodnow CC, Kleinstein SH, et al. Twolevels of protection for the B cell genome during somatic hypermutation.Nature 2008;451:841–5.

[34] Hasham MG, Donghia NM, Coffey E, Maynard J, Snow KJ, Ames J, et al.Widespread genomic breaks generated by activation-induced cytidine deam-inase are prevented by homologous recombination. Nature Immunology2010;11:820–6.

[35] Pasqualucci L, Bhagat G, Jankovic M, Compagno M, Smith P, Muramatsu M,et al. AID is required for germinal center-derived lymphomagenesis. NatureGenetics 2008;40:108–12.

[36] Smit LA, Bende RJ, Aten J, Guikema JE, Aarts WM, van Noesel CJ.Expression of activation-induced cytidine deaminase is confined to B-cellnon-Hodgkin’s lymphomas of germinal-center phenotype. Cancer Research2003;63:3894–8.

[37] Lossos IS, Levy R, Alizadeh AA. AID is expressed in germinal center B-cell-likeand activated B-cell-like diffuse large-cell lymphomas and is not correlatedwith intraclonal heterogeneity. Leukemia 2004;18:1775–9.

[38] Bodor C, Bognar A, Reiniger L, Szepesi A, Toth E, Kopper L, et al. Aber-rant somatic hypermutation and expression of activation-induced cytidinedeaminase mRNA in mediastinal large B-cell lymphoma. British Journal ofHaematology 2005;129:373–6.

[39] Deutsch AJ, Aigelsreiter A, Staber PB, Beham A, Linkesch W, Guelly C, et al.MALT lymphoma and extranodal diffuse large B-cell lymphoma are targetedby aberrant somatic hypermutation. Blood 2007;109:3500–4.

[40] Pasqualucci L, Guglielmino R, Houldsworth J, Mohr J, Aoufouchi S, PolakiewiczR, et al. Expression of the AID protein in normal and neoplastic B cells. Blood2004;104:3318–25.

[41] Jankovic M, Robbiani DF, Dorsett Y, Eisenreich T, Xu Y, Tarakhovsky A, et al.Role of the translocation partner in protection against AID-dependent chro-mosomal translocations. Proceedings of the National Academy of Sciences ofthe United States of America 2010;107:187–92.

[42] Gauwerky CE, Huebner K, Isobe M, Nowell PC, Croce CM. Activation of MYC ina masked t(8;17) translocation results in an aggressive B-cell leukemia. Pro-ceedings of the National Academy of Sciences of the United States of America1989;86:8867–71.

[43] Kovalchuk AL, duBois W, Mushinski E, McNeil NE, Hirt C, Qi CF, et al. AID-deficient Bcl-xL transgenic mice develop delayed atypical plasma cell tumorswith unusual Ig/Myc chromosomal rearrangements. The Journal of Experi-mental Medicine 2007;204:2989–3001.

[44] Ramiro AR, Jankovic M, Eisenreich T, Difilippantonio S, Chen-Kiang S, Mura-matsu M, et al. AID is required for c-myc/IgH chromosome translocations invivo. Cell 2004;118:431–8.

[45] Feldhahn N, Henke N, Melchior K, Duy C, Soh BN, Klein F, et al. Activation-induced cytidine deaminase acts as a mutator in BCR-ABL1-transformedacute lymphoblastic leukemia cells. The Journal of Experimental Medicine2007;204:1157–66.

[46] Gruber TA, Chang MS, Sposto R, Muschen M. Activation-induced cytidine

deaminase accelerates clonal evolution in BCR-ABL1-driven B-cell lineageacute lymphoblastic leukemia. Cancer Research 2010;70:7411–20.

[47] Klemm L, Duy C, Iacobucci I, Kuchen S, von Levetzow G, Feldhahn N, et al. TheB cell mutator AID promotes B lymphoid blast crisis and drug resistance inchronic myeloid leukemia. Cancer Cell 2009;16:232–45.

ars in

A. Orthwein, J.M. Di Noia / Semin

[48] McCarthy H, Wierda WG, Barron LL, Cromwell CC, Wang J, Coombes KR,et al. High expression of activation-induced cytidine deaminase (AID) andsplice variants is a distinctive feature of poor-prognosis chronic lymphocyticleukemia. Blood 2003;101:4903–8.

[49] Palacios F, Moreno P, Morande P, Abreu C, Correa A, Porro V, et al. Highexpression of AID and active class switch recombination might account fora more aggressive disease in unmutated CLL patients: link with an activatedmicroenvironment in CLL disease. Blood 2010;115:4488–96.

[50] Babbage G. Immunoglobulin heavy chain locus events and expression ofactivation-induced cytidine deaminase in epithelial breast cancer cell lines.Cancer Research 2006;66:3996–4000.

[51] Lin C, Yang L, Tanasa B, Hutt K, Ju BG, Ohgi K, et al. Nuclear receptor-inducedchromosomal proximity and DNA breaks underlie specific translocations incancer. Cell 2009;139:1069–83.

[52] Endo Y, Marusawa H, Kinoshita K, Morisawa T, Sakurai T, Okazaki IM, et al.Expression of activation-induced cytidine deaminase in human hepatocytesvia NF-kappaB signaling. Oncogene 2007;26:5587–95.

[53] Komori J, Marusawa H, Machimoto T, Endo Y, Kinoshita K, Kou T, et al.Activation-induced cytidine deaminase links bile duct inflammation tohuman cholangiocarcinoma. Hepatology 2008;47:888–96.

[54] Kou T, Marusawa H, Kinoshita K, Endo Y, Okazaki I-m Ueda Y, et al. Expres-sion of activation-induced cytidine deaminase in human hepatocytes duringhepatocarcinogenesis. International Journal of Cancer 2006;120:469–76.

[55] Shinmura K, Igarashi H, Goto M, Tao H, Yamada H, Matsuura S, et al. Aber-rant expression and mutation-inducing activity of AID in human lung cancer.Annals of Surgical Oncology: The Official Journal of the Society of SurgicalOncology 2011.

[56] Morgan HD, Dean W, Coker HA, Reik W, Petersen-Mahrt SK. Activation-induced cytidine deaminase deaminates 5-methylcytosine in DNA and isexpressed in pluripotent tissues: implications for epigenetic reprogramming.Journal of Biological Chemistry 2004;279:52353–60.

[57] Hsu HC, Yang P, Wu Q, Wang JH, Job G, Guentert T, et al. Inhibition of thecatalytic function of activation-induced cytidine deaminase promotes apo-ptosis of germinal center B cells in BXD2 mice. Arthritis and Rheumatism2011;63:2038–48.

[58] Jiang C, Zhao ML, Scearce RM, Diaz M. Activation-induced deaminase-deficient MRL/lpr mice secrete high levels of protective antibodies againstlupus nephritis. Arthritis and Rheumatism 2011;63:1086–96.

[59] Jiang C, Foley J, Clayton N, Kissling G, Jokinen M, Herbert R, et al. Abrogationof lupus nephritis in activation-induced deaminase-deficient MRL/lpr mice.Journal of Immunology 2007;178:7422–31.

[60] Zan H, Zhang J, Ardeshna S, Xu Z, Park SR, Casali P. Lupus-prone MRL/faslpr/lprmice display increased AID expression and extensive DNA lesions, com-prising deletions and insertions, in the immunoglobulin locus: concurrentupregulation of somatic hypermutation and class switch DNA recombination.Autoimmunity 2009;42:89–103.

[61] White CA, Seth Hawkins J, Pone EJ, Yu ES, Al-Qahtani A, Mai T, et al. AIDdysregulation in lupus-prone MRL/Fas(lpr/lpr) mice increases class switchDNA recombination and promotes interchromosomal c-Myc/IgH loci translo-cations: modulation by HoxC4. Autoimmunity 2011;44:585–98.

[62] Xu X, Hsu HC, Chen J, Grizzle WE, Chatham WW, Stockard CR, et al. Increasedexpression of activation-induced cytidine deaminase is associated with anti-CCP and rheumatoid factor in rheumatoid arthritis. Scandinavian Journal ofImmunology 2009;70:309–16.

[63] Hase K, Takahashi D, Ebisawa M, Kawano S, Itoh K, Ohno H. Activation-inducedcytidine deaminase deficiency causes organ-specific autoimmune disease.PLoS One 2008;3:e3033.

[64] Quartier P, Bustamante J, Sanal O, Plebani A, Debre M, Deville A, et al. Clinical,immunologic and genetic analysis of 29 patients with autosomal recessivehyper-IgM syndrome due to activation-induced cytidine deaminase defi-ciency. Clinical immunology (Orlando, FL) 2004;110:22–9.

[65] Meyers G, Ng YS, Bannock JM, Lavoie A, Walter JE, Notarangelo LD, et al.Activation-induced cytidine deaminase (AID) is required for B-cell tolerancein humans. Proceedings of the National Academy of Sciences of the UnitedStates of America 2011;108:11554–9.

[66] Kuraoka M, Holl TM, Liao D, Womble M, Cain DW, Reynolds AE, et al.Activation-induced cytidine deaminase mediates central tolerance in B cells.Proceedings of the National Academy of Sciences of the United States ofAmerica 2011;108:11560–5.

[67] Pauklin S, Sernandez IV, Bachmann G, Ramiro AR, Petersen-Mahrt SK.Estrogen directly activates AID transcription and function. The Journal ofExperimental Medicine 2009;206:99–111.

[68] Ramiro A, Di Noia J. Regulatory mechanisms of AID function. In: DNA deami-nation and the immune system. Imperial College Press; 2010.

[69] Crouch EE, Li Z, Takizawa M, Fichtner-Feigl S, Gourzi P, Montano C, et al. Regu-lation of AID expression in the immune response. The Journal of ExperimentalMedicine 2007;204:1145–56.

[70] Shaffer AL, Lin KI, Kuo TC, Yu X, Hurt EM, Rosenwald A, et al. Blimp-1 orches-trates plasma cell differentiation by extinguishing the mature B cell geneexpression program. Immunity 2002;17:51–62.

[71] Cattoretti G, Büttner M, Shaknovich R, Kremmer E, Alobeid B, Niedobitek G.

Nuclear and cytoplasmic AID in extrafollicular and germinal center B cells.Blood 2006;107:3967–75.

[72] Muramatsu M, Sankaranand VS, Anant S, Sugai M, Kinoshita K, Davidson NO,et al. Specific expression of activation-induced cytidine deaminase (AID), a

Immunology 24 (2012) 246– 254 253

novel member of the RNA-editing deaminase family in germinal center Bcells. Journal of Biological Chemistry 1999;274:18470–6.

[73] Tran TH, Nakata M, Suzuki K, Begum NA, Shinkura R, Fagarasan S, et al. Bcell-specific and stimulation-responsive enhancers derepress aicda by over-coming the effects of silencers. Nature Immunology 2010;11:148–54.

[74] Dedeoglu F, Horwitz B, Chaudhuri J, Alt FW, Geha RS. Induction ofactivation-induced cytidine deaminase gene expression by IL-4 and CD40ligation is dependent on STAT6 and NFkappaB. International Immunology2004;16:395–404.

[75] Machida K, Cheng KT, Sung VM, Shimodaira S, Lindsay KL, Levine AM, et al.Hepatitis C virus induces a mutator phenotype: enhanced mutations ofimmunoglobulin and protooncogenes. Proceedings of the National Academyof Sciences of the United States of America 2004;101:4262–7.

[76] Matsumoto Y, Marusawa H, Kinoshita K, Endo Y, Kou T, Morisawa T, et al. Heli-cobacter pylori infection triggers aberrant expression of activation-inducedcytidine deaminase in gastric epithelium. Nature Medicine 2007;13:470–6.

[77] Matsumoto Y, Marusawa H, Kinoshita K, Niwa Y, Sakai Y, Chiba T.Up-regulation of activation-induced cytidine deaminase causes geneticaberrations at the CDKN2b-CDKN2a in gastric cancer. Gastroenterology2010;139:1984–94.

[78] Epeldegui M, Breen EC, Hung YP, Boscardin WJ, Detels R, Martinez-Maza O. Elevated expression of activation induced cytidine deaminase inperipheral blood mononuclear cells precedes AIDS-NHL diagnosis. AIDS2007;21:2265–70.

[79] Epeldegui M, Thapa DR, De la Cruz J, Kitchen S, Zack JA, Martinez-MazaO. CD40 ligand (CD154) incorporated into HIV virions induces activation-induced cytidine deaminase (AID) expression in human B lymphocytes. PLoSOne 2010;5:e11448.

[80] Gourzi P, Leonova T, Papavasiliou FN. A role for activation-induced cytidinedeaminase in the host response against a transforming retrovirus. Immunity2006;24:779–86.

[81] Gourzi P, Leonova T, Papavasiliou FN. Viral induction of AID is independentof the interferon and the toll-like receptor signaling pathways but requiresNF-kappaB. The Journal of Experimental Medicine 2007;204:259–65.

[82] MacDuff DA, Demorest ZL, Harris RS. AID can restrict L1 retrotransposi-tion suggesting a dual role in innate and adaptive immunity. Nucleic AcidsResearch 2009;37:1854–67.

[83] Bhutani N, Brady JJ, Damian M, Sacco A, Corbel SY, Blau HM. Reprogrammingtowards pluripotency requires AID-dependent DNA demethylation. Nature2010;463:1042–7.

[84] Popp C, Dean W, Feng S, Cokus SJ, Andrews S, Pellegrini M, et al. Genome-wideerasure of DNA methylation in mouse primordial germ cells is affected by AIDdeficiency. Nature 2010;463:1101–5.

[85] Hajkova P, Jeffries SJ, Lee C, Miller N, Jackson SP, Surani MA. Genome-widereprogramming in the mouse germ line entails the base excision repair path-way. Science 2010;329:78–82.

[86] Schreck S, Buettner M, Kremmer E, Bogdan M, Herbst H, Niedobitek G.Activation-induced cytidine deaminase (AID) is expressed in normal sper-matogenesis but only infrequently in testicular germ cell tumours. The Journalof Pathology 2006;210:26–31.

[87] Rada C, Jarvis JM, Milstein C. AID-GFP chimeric protein increases hyper-mutation of Ig genes with no evidence of nuclear localization. Proceedingsof the National Academy of Sciences of the United States of America2002;99:7003–8.

[88] Schrader CE, Linehan EK, Mochegova SN, Woodland RT, Stavnezer J. InducibleDNA breaks in Ig S regions are dependent on AID and UNG. The Journal ofExperimental Medicine 2005;202:561–8.

[89] Brar SS, Watson M, Diaz M. Activation-induced cytosine deaminase (AID) isactively exported out of the nucleus but retained by the induction of DNAbreaks. The Journal of Biological Chemistry 2004;279:26395–401.

[90] Ito S, Nagaoka H, Shinkura R, Begum NA, Muramatsu M, Nakata M,et al. Activation-induced cytidine deaminase shuttles between nucleus andcytoplasm like apolipoprotein B mRNA editing catalytic polypeptide 1. Pro-ceedings of the National Academy of Sciences of the United States of America2004;101:1975–80.

[91] McBride KM, Barreto V, Ramiro AR, Stavropoulos P, Nussenzweig MC.Somatic hypermutation is limited by CRM1-dependent nuclear exportof activation-induced deaminase. The Journal of Experimental Medicine2004;199:1235–44.

[92] Ellyard JI, Benk AS, Taylor B, Rada C, Neuberger MS. The dependence of Ig class-switching on the nuclear export sequence of AID likely reflects interactionwith factors additional to Crm1 exportin. European Journal of Immunology2011;41:485–90.

[93] Patenaude AM, Orthwein A, Hu Y, Campo VA, Kavli B, Buschiazzo A, et al. Activenuclear import and cytoplasmic retention of activation-induced deaminase.Nature Structural & Molecular Biology 2009;16:517–27.

[94] Kodiha M, Chu A, Matusiewicz N, Stochaj U. Multiple mechanisms promotethe inhibition of classical nuclear import upon exposure to severe oxidativestress. Cell Death and Differentiation 2004;11:862–74.

[95] Miyamoto Y, Saiwaki T, Yamashita J, Yasuda Y, Kotera I, Shibata S, et al. Cellularstresses induce the nuclear accumulation of importin alpha and cause a con-

ventional nuclear import block. The Journal of Cell Biology 2004;165:617–23.

[96] Conticello SG, Ganesh K, Xue K, Lu M, Rada C, Neuberger MS. Interactionbetween antibody-diversification enzyme AID and spliceosome-associatedfactor CTNNBL1. Molecular Cell 2008;31:474–84.

2 ars in

[

[

[

[

[

[

[

[

[

[

[

[

54 A. Orthwein, J.M. Di Noia / Semin

[97] Ganesh K, Adam S, Taylor B, Simpson P, Rada C, Neuberger MS. CTNNBL1Is a novel nuclear localization sequence-binding protein that recognizesRNA-splicing factors CDC5L and Prp31. The Journal of Biological Chemistry2011;286:17091–102.

[98] Han L, Masani S, Yu K. Cutting edge: CTNNBL1 is dispensable for Ig class switchrecombination. Journal of Immunology 2010;185:1379–81.

[99] Wickramasinghe VO, McMurtrie PI, Mills AD, Takei Y, Penrhyn-Lowe S, Ama-gase Y, et al. mRNA export from mammalian cell nuclei is dependent on GANP.Current Biology 2010;20:25–31.

100] Maeda K, Singh SK, Eda K, Kitabatake M, Pham P, Goodman MF, et al. GANP-mediated recruitment of activation-induced cytidine deaminase to cell nucleiand to immunoglobulin variable region DNA. The Journal of Biological Chem-istry 2010;285:23945–53.

101] Häsler J, Rada C, Neuberger MS. Cytoplasmic activation-induced cytidinedeaminase (AID) exists in stoichiometric complex with translation elonga-tion factor 1� (eEF1A). Proceedings of the National Academy of Sciences ofthe United States of America 2011;108:18366–71.

102] Ordinario EC, Yabuki M, Larson RP, Maizels N. Temporal regulation of Iggene diversification revealed by single-cell imaging. Journal of Immunology2009;183:4545–53.

103] Patenaude AM, Di Noia JM. The mechanisms regulating the subcellular local-ization of AID. Nucleus 2010;1:325–31.

104] Barreto VM, Reina San-Martin BR, Ramiro AR, McBride KM, NussenzweigMC. C-terminal deletion of AID uncouples class switch recombination fromsomatic hypermutation and gene conversion. Molecular Cell 2003;12:501–8.

105] Geisberger R, Rada C, Neuberger MS. The stability of AID and its function inclass-switching are critically sensitive to the identity of its nuclear-exportsequence. Proceedings of the National Academy of Sciences of the UnitedStates of America 2009;106:6736–41.

106] Kohli RM, Abrams SR, Gajula KS, Maul RW, Gearhart PJ, Stivers JT. A portablehot spot recognition loop transfers sequence preferences from APOBEC familymembers to activation-induced cytidine deaminase. The Journal of BiologicalChemistry 2009;284:22898–904.

107] Aoufouchi S, Faili A, Zober C, D’Orlando O, Weller S, Weill J-C, et al. Pro-teasomal degradation restricts the nuclear lifespan of AID. The Journal ofExperimental Medicine 2008;205:1357–68.

108] Orthwein A, Patenaude A-M, Affar EB, Lamarre A, Young JC, Di NoiaJM. Regulation of activation-induced deaminase stability and antibodygene diversification by Hsp90. The Journal of Experimental Medicine2010;207:2751–65.

109] Orthwein A, Zahn A, Methot SP, Godin D, Conticello SG, Terada K, et al. Opti-mal functional levels of activation-induced deaminase specifically require theHsp40 DnaJa1. EMBO Journal 2011.

110] Uchimura Y, Barton LF, Rada C, Neuberger MS. REG-gamma associates with

and modulates the abundance of nuclear activation-induced deaminase. TheJournal of Experimental Medicine 2011;208:2385–91.

111] MacDuff DA, Neuberger MS, Harris RS. MDM2 can interact with the C-terminus of AID but it is inessential for antibody diversification in DT40 Bcells. Molecular Immunology 2006;43:1099–108.

Immunology 24 (2012) 246– 254

[112] Basu U, Chaudhuri J, Alpert C, Dutt S, Ranganath S, Li G, et al. The AID anti-body diversification enzyme is regulated by protein kinase A phosphorylation.Nature 2005;438:508–11.

[113] Chatterji M, Unniraman S, McBride KM, Schatz DG. Role of activation-induceddeaminase protein kinase A phosphorylation sites in Ig gene conversion andsomatic hypermutation. Journal of Immunology 2007;179:5274–80.

[114] Gazumyan A, Timachova K, Yuen G, Siden E, Di Virgilio M, Woo EM,et al. Amino-terminal phosphorylation of activation-induced cytidine deam-inase suppresses c-myc/IgH translocation. Molecular and Cellular Biology2011;31:442–9.

[115] McBride KM, Gazumyan A, Woo EM, Barreto VM, Robbiani DF, Chait BT,et al. Regulation of hypermutation by activation-induced cytidine deami-nase phosphorylation. Proceedings of the National Academy of Sciences ofthe United States of America 2006;103:8798–803.

[116] Pasqualucci L, Kitaura Y, Gu H, Dalla-Favera R. PKA-mediated phosphorylationregulates the function of activation-induced deaminase (AID) in B cells. Pro-ceedings of the National Academy of Sciences of the United States of America2006;103:395–400.

[117] Demorest ZL, Li M, Harris RS. Phosphorylation directly regulates the intrin-sic DNA cytidine deaminase activity of activation-induced deaminase andAPOBEC3G protein. The Journal of Biological Chemistry 2011;286:26568–75.

[118] Cheng HL, Vuong BQ, Basu U, Franklin A, Schwer B, Astarita J, et al. Integrityof the AID serine-38 phosphorylation site is critical for class switch recom-bination and somatic hypermutation in mice. Proceedings of the NationalAcademy of Sciences of the United States of America 2009;106:2717–22.

[119] Vuong BQ, Lee M, Kabir S, Irimia C, Macchiarulo S, McKnight GS, et al. Specificrecruitment of protein kinase A to the immunoglobulin locus regulates class-switch recombination. Nature Immunology 2009;10:420–6.

[120] Chaudhuri J, Khuong C, Alt FW. Replication protein A interacts withAID to promote deamination of somatic hypermutation targets. Nature2004;430:992–8.

[121] Takai A, Toyoshima T, Uemura M, Kitawaki Y, Marusawa H, Hiai H, et al. A novelmouse model of hepatocarcinogenesis triggered by AID causing deleteriousp53 mutations. Oncogene 2009;28:469–78.

[122] Han JH, Akira S, Calame K, Beutler B, Selsing E, Imanishi-Kari T. Class switchrecombination and somatic hypermutation in early mouse B cells are medi-ated by B cell and toll-like receptors. Immunity 2007;27:64–75.

[123] Mao C, Jiang L, Melo-Jorge M, Puthenveetil M, Zhang X, Carroll MC, et al. Tcell-independent somatic hypermutation in murine B cells with an immaturephenotype. Immunity 2004;20:133–44.

[124] Lucier MR, Thompson RE, Waire J, Lin AW, Osborne BA, Goldsby RA. Mul-tiple sites of V lambda diversification in cattle. Journal of Immunology1998;161:5438–44.

[125] Reynaud CA, Garcia C, Hein WR, Weill JC. Hypermutation generating the

sheep immunoglobulin repertoire is an antigen-independent process. Cell1995;80:115–25.

[126] Weinstein PD, Anderson AO, Mage RG. Rabbit IgH sequences in appendix ger-minal centers: VH diversification by gene conversion-like and hypermutationmechanisms. Immunity 1994;1:647–59.