of September 3 2014This information is current as

with Mutator ActivityBinding of AID to DNA Does Not Correlate

ChaudhuriAllysia J Matthews Solomon Husain and Jayanta

ol1400433httpwwwjimmunolorgcontentearly20140529jimmun

published online 30 May 2014J Immunol

MaterialSupplementary

DCSupplementalhtmlhttpwwwjimmunolorgcontentsuppl20140529content1400433

Subscriptionshttpjimmunolorgsubscriptions

is online at The Journal of ImmunologyInformation about subscribing to

PermissionshttpwwwaaiorgjicopyrighthtmlSubmit copyright permission requests at

Email AlertshttpjimmunolorgcgialertsetocReceive free email-alerts when new articles cite this article Sign up at

Print ISSN 0022-1767 Online ISSN 1550-6606 Immunologists Inc All rights reservedCopyright copy 2014 by The American Association of9650 Rockville Pike Bethesda MD 20814-3994The American Association of Immunologists Inc

is published twice each month byThe Journal of Immunology

at Northern Illinois U

niv Univ L

ibPeriodicals Dept on Septem

ber 3 2014httpw

ww

jimm

unolorgD

ownloaded from

at N

orthern Illinois Univ U

niv LibPeriodicals D

ept on September 3 2014

httpww

wjim

munolorg

Dow

nloaded from

The Journal of Immunology

Binding of AID to DNA Does Not Correlate with MutatorActivity

Allysia J Matthews Solomon Husain and Jayanta Chaudhuri

The DNA deaminase activation-induced cytidine deaminase (AID) initiates somatic hypermutation (SHM) and class switch recom-

bination (CSR) by deaminating cytidines to uridines at V region (V) genes and switch (S) regions The mechanism by which AID is

recruited to V genes and S region DNA is poorly understood In this study we used the CH12 B lymphoma line to demonstrate that

although S regions can efficiently recruit AID and undergo mutations and deletions AID neither binds to nor mutates the V gene

thus clearly demonstrating intraimmunoglobulin locus specificity Depletion of the RNA-binding protein polypyrimidine tract

binding protein-2 previously shown to promote recruitment of AID to S regions enables stable association of AID with the V

gene Surprisingly AID binding to the V gene does not induce SHM These results unmask a striking lack of correlation between

AID binding and its mutator activity providing evidence for the presence of factors required downstream of AID binding to effect

SHM Furthermore our findings suggest that S regions are preferred targets for AID and aided by polypyrimidine tract binding

protein-2 act as ldquosinksrdquo to sequester AID activity from other genomic regions The Journal of Immunology 2014 193 000ndash000

Activation-induced cytidine deaminase (AID) is essentialfor somatic hypermutation (SHM) and class switch re-combination (CSR) (1 2) During SHM AID deaminates

deoxycytidines to deoxyuridines at the V region exons (V gene) ofthe Ig H and L chains (3) Engagement of base excision repair andmismatch repair pathways along with DNA synthesis by error-prone DNA polymerases at the deoxycytidinedeoxyuridine mis-match mutates the V genes at a high rate (sim1022ndash1023 mutationsbpgeneration) leading to selection of B cells with increased Agaffinity (4) CSR exchanges the initially expressed Igh C regionCm for an alternative set of downstream CH exons (or genes) suchas Cg Claquo or Ca altering the B cell expression from IgM toa secondary Ab isotype (IgG IgE IgA) with distinct effectorfunction (5) CSR is a deletional-recombination reaction that isinitiated by AID-mediated deamination of transcribed repetitiveswitch (S) region DNA elements that precede each CH gene (5)End-joining of double strand breaks (DSBs) between two distinctS regions deletes the intervening DNA as an extrachromosomalcircle and juxtaposes a new CH gene downstream of the rearrangedVDJ segment Thus CSR allows for the generation of Ig mole-cules with the same affinity for Ag but with new effector functionAID is a generalmutator and canmutate and induceDSBs atmany

non-Ig genes (6ndash11) In fact aberrant AID activity at oncogenes is

a major contributing factor in the ontogeny of a large number ofmature B cell lymphomas (12) Despite the ability of AID to targetnon-Ig genes the V genes and S region DNA serve as major AIDtargets with the efficiency of AID association at the Ig loci beingseveral fold higher than at non-Ig genes (7 8) In addition to speci-ficity of AID for the Ig loci there is evidence for intra-Ig locusspecificity because B cells undergoing CSR in culture do not mutatetheir variable regions (13 14) Thus mechanisms must exist to ac-tively recruit AID to V genes and S regions during SHM and CSRrespectively Several factors including Spt5 polypyrimidine tractbinding protein-2 (Ptbp2) RNA exosome subunits and 14-3-3 adapterproteins have been implicated in the recruitment of AID to S regions(7 15ndash17) although the precise role of these proteins in CSR is yet tobe fully elucidated The mechanism by which AID is specificallyrecruited to V genes is even more enigmatic Unlike S regions that areunique in their GC richness and in their ability to form RNADNAhybrid structures (R-loops) upon transcription (18 19) V genes donot present a recognizable primary or predicted secondary structurethat could explain specificity for AID binding The RGYW (R = AGY = CT W = AT) tetranucleotide does serve as an SHM hot-spotmotif and E2A-transcription factor binding sites promote SHM (620) however the ubiquitous nature of these sequences at almost alltranscribed genes fails to explain AID specificityPreviously we identified Ptbp2 as an AID interactor (15) De-

pletion of Ptbp2 significantly impaired CSR as a result of a defectin the recruitment of AID to S regions In this study we used the Blymphoma cell line CH12 to show that when AID recruitment to Sregions is impaired through Ptbp2 depletion association of AIDwith the expressed V gene is remarkably promoted Surprisinglydespite the binding of AID to V genes SHM is not inducedTherefore AID binding does not correlate with mutation activitysuggesting that SHM requires specific factors andor subversion ofDNA repair pathways that operate downstream of AID binding

Materials and MethodsCell culture and protein analysis

CH12 cells (21) were stimulated at a density of 0253 106 cellsml for 96 hwith anti-CD40 Ab (CIT) (1 mgml HM40-3 eBioscience San DiegoCA) IL-4 (125 mgml 404-ML RampD Systems Minneapolis MN) andTGF-b1 (01 ngml 240-B RampD Systems) IgA+ cells were generated

Immunology Program Memorial Sloan-Kettering Cancer Center Gerstner Sloan-Kettering Graduate School New York NY 10065 and Immunology and MicrobialPathogenesis Program Weill-Cornell Medical School New York NY 10065

Received for publication February 18 2014 Accepted for publication April 25 2014

This work was supported by National Institutes of Health Grants 1R01AI072194 (toJC) and T32AI007621 and T32CA009149 (to AJM)

Address correspondence and reprint requests to Dr Jayanta Chaudhuri ImmunologyProgram Memorial Sloan-Kettering Cancer Center Gerstner Sloan-Kettering Grad-uate School New York NY 10065 E-mail address chaudhujmskccorg

The online version of this article contains supplemental material

Abbreviations used in this article AID activation-induced cytidine deaminase ChIPchromatin immunoprecipitation CIT anti-CD40 Ab IL-4 and TGF-b1 CSR classswitch recombination Ct cycle threshold DSB double strand break PKA proteinkinase A PKA-Ca catalytic subunit of protein kinase A pS38-AID AID phosphor-ylated at serine residue 38 Ptbp2 polypyrimidine tract binding protein-2 RPAreplication protein A S switch SHM somatic hypermutation shRNA short hairpinRNA

Copyright 2014 by The American Association of Immunologists Inc 0022-176714$1600

wwwjimmunolorgcgidoi104049jimmunol1400433

Published May 30 2014 doi104049jimmunol1400433 at N

orthern Illinois Univ U

niv LibPeriodicals D

ept on September 3 2014

httpww

wjim

munolorg

Dow

nloaded from

from CIT-stimulated CH12 cells by negative selection with anti-IgMMicroBeads (Miltenyi Biotec San Diego CA) followed by positive se-lection using anti-IgA biotin Ab (eBioscience) and Streptavidin MicroBeads(Miltenyi Biotec) The predominantly IgA+ cell population was subclonedby serial dilution

Flow cytometry and Western blotting

Cells were stained with IgMndashPE-Cy 7 (R6-602 BD Biosciences San JoseCA) and IgA-FITC (C10-3 BD Biosciences) and acquired on an LSR II(BD Biosciences) Data were analyzed using FlowJo software (TreeStarAshland OR) DAPI (Invitrogen Grand Island NY) was used for exclu-sion of dead cells Whole-cell lysates were prepared in Nonidet P-40 lysisbuffer (20 mM Tris [pH 75] 5 [vv] glycerol 150 mM NaCl 5 mM2-ME and 05 [vv] Nonidet P-40) The following primary Abs wereused anti-Ptbp2 (ab57619 Abcam Cambridge MA) anti-AID (22) andanti-Gapdh (loading control 6C5 Millipore Billerica MA)

Chromatin immunoprecipitation analysis

Knockdown in CH12 cells was described previously (15) Chromatinimmunoprecipitation (ChIP) assays were performed as described previ-ously (15 23) PCR amplification and detection were carried out on a Bio-Rad CFX96 system and threshold cycle (Ct) values were calculated usingCFX Manager software by setting the threshold within the linear phaseusing a log-amplification plot For detection of specific DNA sequences inChIP samples iQ SYBR Green Supermix (Bio-Rad Hercules CA) wasused for Sm and p53 amplifications whereas FastStart Universal ProbeMix was used for VDJ amplification along with FAM-labeled UniversalProbe 45 (both from Roche Indianapolis IN) Melt-curve analysis wasused for SYBR reactions to verify the presence of a single amplicon of thecorrect size ChIP ldquorelative unitsrdquo were calculated by first normalizing theCt value of each Ab sample by the Ct value of the input sample (CtAbCtinput) The inverse of this normalized Ab Ct value was then taken (1NormCtAb) The resulting value for the nonspecific IgG Ab sample wasthen subtracted from each of the specific Ab (SA) samples to obtain thefinal ldquorelative unitsrdquo [(1NormCtSA) ndash (1NormCtIgG)] Primer sequencesare listed in Supplemental Table II and primer positions within the Ighlocus are depicted in Supplemental Fig 1A

Mutation analysis

Genomic DNAwas isolated from 15ndash3 3 106 CH12 cells cultured with orwithout CIT for 96 h Amplification of sequences for mutation analysis wasdone using Phusion DNA polymerase (New England Biolabs Ipswich MA)PCR products were cloned into a Zero Blunt TOPO cloning kit (Invitrogen)Individual clones were sequenced with vector-derived M13 primers andmutation frequency was calculated by dividing the total number of uniquemutations by the total number of bases sequenced Mutation data are plottedas a pie chart showing the portion of total sequences containing 0 1 or 2mutations The number of individual clones sequenced is indicated at thecenter of each chart and the mutation frequency (unique mutationsbasessequenced) and the number of sequences containing deletions if applicableare indicated below The VDJ sequence expressed by CH12 cells is Gen-Bank M345811 Primer sequences are listed in Supplemental Table II andprimer positions within the Igh locus are depicted in Supplemental Fig 1A

Statistical analysis

A two-tailed paired Student t test was used to assess the significance ofChIP data p 005 was considered significant A z-test for comparingproportions of two independent groups was used to assess the significanceof mutation frequencies p 005 was considered significant

ResultsAID binds to S regions but not to V gene segments in CH12cells

Upon stimulation with CIT the mouse B lymphoma line CH12expresses AID and undergoes robust CSR to IgA (21 24) (Fig 1A1B) Given that CSR in CH12 cells is dependent on several of theknown factors implicated in CSR of primary B lymphocytes (715 25) these cells serve as a bona fide model system to elucidateCSR Despite the high frequency of CSR CIT-stimulated CH12cells do not undergo endogenous SHM (26)To test whether the failure to undergo SHM is due to preferred

recruitment of AID to the S regions as opposed to the V gene wecarried out ChIP experiments in CH12 cells with AID Ab using

histone H3 Ab as a positive control We analyzed the DNAndashproteincomplexes for the presence of the Sm Igh V gene (VDJ) andTrp53 (which encodes p53 non-Ig control) genomic sequence usingsequence-specific primers (Supplemental Fig 1A SupplementalTable II) AID did not associate with Igh regions in unstimulatedcells or with p53 under any condition (Supplemental Fig 1B 1C 1F)As expected AID binding to Sm was readily observed in stimulatedcells however no AID was detected at the Igh V gene segment(Fig 1C Supplemental Fig 1F)One plausible explanation for the observed specificity is that S

regions being rich in RGYW sequence motifs and having the abilityto form ssDNA in the context of R-loops are significantly better thanV genes at recruiting AID (18 19 27 28) Therefore we hypoth-esized that in ldquoterminally switchedrdquo IgA+ CH12 cells which cannotundergo further CSR the Igh V gene would have a better chance ofcompeting favorably against the smaller recombined S region forAID binding To test this notion we purified IgA+ cells from CIT-stimulated CH12 cells and subcloned an IgA+ clone by serial dilu-tion (Fig 1A) When grown in the absence of cytokines the IgA+

clone expressed no detectable AID whereas robust AID expressionwas induced upon stimulation with CIT (Fig 1B) As in CH12 cellsAID did not associate with Igh regions in unstimulated IgA+ cells orwith the Trp53 (p53 non-Ig control) genomic sequence under anycondition (Supplemental Fig 1B 1C 1F) However contrary to ourpredictions AID binding to the Igh V gene segment was notdetected in CIT-stimulated IgA+ cells (Fig 1C Supplemental Fig1F) Instead AID was found associated with the recombined SmndashSaDNA (Fig 1C Supplemental Fig 1F) Thus AID exhibits intra-Ighlocus specificity with a bias for binding to S regions not only in bulkCH12 cells undergoing CSR but also in IgA+ cells in which the IghV gene might be expected to have a better chance of competingfavorably against the remaining S region for AID binding

Activated CH12 cells do not undergo SHM

To test the possibility that the failure to detect AID at the V gene isnot due to the transient nature of the interaction we isolated ge-nomic DNA from IgA+ cells and sequenced the expressed Igh Vgene using sequence-specific primers (Supplemental Fig 1ASupplemental Table II) The rate of mutation of the Igh V gene(VDJ) in CIT-stimulated IgA+ cells was similar to that observedfor unstimulated IgA+ cells suggesting that SHM was not induced(Fig 2A Supplemental Table IA) In contrast the recombinedSmndashSa sequence in CIT-stimulated IgA+ cells had significantlevels of mutations (Fig 2B Supplemental Table IB) Further-more the original recombined SmndashSa junction present in the un-stimulated IgA+ cell line underwent deletions to create novel Sjunctions (Fig 2B 2C) Thus although AID in IgA+ cells boundto and mutated the remaining S region it neither associated withnor induced SHM at the Igh V gene

Localization of AID at the Igh locus is altered upon Ptbp2depletion

The RNA-binding protein Ptbp2 interacts with AID and facilitatesthe recruitment of AID to S regions (15) We investigated thepossibility that in Ptbp2-depleted cells AID that fails to bind to Sregions can be ldquoretargetedrdquo to the V gene segment Using a shorthairpin RNA (shRNA) directed against the 39 untranslated regionof Ptbp2 mRNA we knocked down Ptbp2 expression in CH12cells with ldquoscrambledrdquo shRNA serving as a control (Fig 3A)As expected (15) AID binding to S regions was significantly re-duced (sim2-fold p = 0003) and CSR was impaired (Fig 3B 3CSupplemental Fig 1G) Strikingly the reduction in AID at Sregions was accompanied by a significant increase (6-fold p =001) in AID binding specifically to the Igh V gene of stimulated

2 INTRALOCUS SPECIFICITY OF AID

at Northern Illinois U

niv Univ L

ibPeriodicals Dept on Septem

ber 3 2014httpw

ww

jimm

unolorgD

ownloaded from

Ptbp2-depleted cells (Fig 3C Supplemental Fig 1D 1G) AIDwas not substantially enriched at other regions of the Igh locusincluding 1 kb upstream and downstream of the rearranged VDJ(59Vh1-53 and 39Jh2 respectively) Cm and the Im promoter(Fig 3D) Surprisingly AID was not detected at the expressedVJk L chain locus (Fig 3D) This could be due to occlusion of theAb-binding epitope while AID is bound to the L chain locus ormore interestingly a suggestion that the level of Ptbp2 preferen-tially effects AID relocalization within the Igh locus during SHMthus only the binding of AID to the S region and Igh V geneexons is altered upon Ptbp2 depletion Finally AID was not found

to be associated with control genomic sequences such as Trp53(p53) or other non-Ig genes that were shown to be upregulated inB cells undergoing CSR (23) (Fig 3E) Thus when the associationof AID with S regions is impaired its interaction with the expressedIgh V gene segment is specifically and significantly promoted

AID bound to the Igh V gene in PTBP2-depleted cells isphosphorylated

AID is phosphorylated at serine 38 (S38) and mutation of S38to alanine impairs the ability of AID to mediate SHM and CSR

(23 29ndash33) To determine whether AID bound to the V gene was

phosphorylated at S38 (pS38-AID) we carried out ChIP experi-

ments using pS38-AIDndashspecific Ab (23) In accordance with AID

bound to Sm the amount of pS38-AID localized to Sm was re-

duced in Ptbp2-depleted CH12 cells (Fig 4A) This reduction was

accompanied by a significant increase (sim7-fold p = 005) in pS38-

AID levels specifically associated with the Igh V gene (Fig 4A)

Localization of pS38-AID to the Trp53 (p53 non-Ig control)

genomic sequence was not amplified from pS38-AID samples

(Supplemental Fig 1E) Thus AID is not only specifically tar-

geted to the V gene segment in Ptbp2-depleted CH12 cells it is

phosphorylated at S38AID phosphorylated at S38 interacts with the ssDNA-binding

protein replication protein A (RPA) (29 30) To test whether RPAlocalization also is altered in Ptbp2-depleted cells we carried outChIP analyses using an Ab specific for the 32-kDa subunit of RPARPA levels at Sm were significantly reduced (sim25-fold p = 003) inPtbp2-depleted CH12 cells (Fig 4A) In contrast there was a sig-nificant increase (sim3-fold p = 001) in RPA localization specificallyto the Igh V gene segment (Fig 4A Supplemental Fig 1E)Phosphorylation of AID at S38 is mediated by protein kinase A

(PKA) (23 30 31 34) and it is believed that AID is phosphory-lated by S regionndashbound PKA to activate the CSR cascade (23 35)In keeping with AID-independent recruitment of PKA to S regions(23) the catalytic subunit of PKA (PKA-Ca) was detected at Sm inCIT-stimulated Ptbp2-depleted cells (Fig 4B) However PKA-Cawas not detected at the Igh V gene segment (Fig 4B) even thoughphosphorylated AID was readily detectable (Fig 4A) Thus not allproteins known to bind S regions are retargeted to the V gene in theabsence of Ptbp2 thereby ruling out the possibility that the bindingof AID to the Igh V gene is due to general deregulation of proteinndashDNA associations in Ptbp2-depleted CH12 cellsOur findings that PKA-Ca is associated with S regions but not

with the Igh V gene regardless of Ptbp2 expression lends credence

FIGURE 1 AID expressed in IgA+ cells does not bind the expressed Igh V gene (A) Flow cytometry analysis of IgM (left panel) or IgA (right panel)

expression on unstimulated (U) or CIT-stimulated (S) CH12 or IgA+ cells (B) Western blot analysis of whole-cell extracts using AID and Gapdh (loading

control) Abs (C) ChIP analysis to detect binding of AID or histone H3 to Sm and Igh V gene (VDJ) in stimulated CH12 or IgA+ cells Two independent

experiments are shown ldquoChIP (Relative Units)rdquo is defined as the reciprocal of the quotient of the crossing threshold (Ct) value of specific Ab immu-

noprecipitation signal and the Ct value of input signal with nonspecific IgG immunoprecipitation signal (background) subtracted from this value

FIGURE 2 AID does not induce SHM in CH12 cells (A) Mutation fre-

quency of the Igh V region (VDJ) Unstimulated (U) IgA+ cells accumulated

one mutation in 66297 bases and stimulated (S) IgA+ cells had one mutation

in 68593 bases (B) Mutation frequency of SmndashSa junction Unstimulated

(U) IgA+ cells accumulated 2 mutations in 65515 bases and 1 sequence with

a deletion and CIT-stimulated (S) IgA+ cells had 20 unique mutations in

64568 bases and 17 sequences with a deletion Data were obtained from two

independent samples The p values were calculated using a z-test (C) The

generation of deletions at the SmndashSa junction in CIT-stimulated IgA+ cells

DSBs generated in Sm and Sa upon initial stimulation of an IgM+ CH12 cell

are synapsed to form an initial SmndashSa junction Stimulation of an IgA+ cell

generates a new set of DSBs and results in the formation of a new SmndashSa

junction deleting the intervening switch sequence

The Journal of Immunology 3

at Northern Illinois U

niv Univ L

ibPeriodicals Dept on Septem

ber 3 2014httpw

ww

jimm

unolorgD

ownloaded from

to the proposal (23) that AID phosphorylation has a different role inSHM than in CSR It is generally believed that transcribed S regionsform R-loops allowing AID to access S regions independent of its

phosphorylation status (36) Phosphorylation of AID at S38 is stillrequired to promote formation of DSBs at S regions through inter-action with APE1 (35) as well as for the repair of DSBs throughrecruitment of RPA to S regions (8 23) In contrast transcribed Vgenes do not readily reveal ssDNA in the context of R-loops and it islikely that AID only binds V genes in the context of a pS38ndashAIDndashRPA complex because of the ability of RPA to bind and stabilizessDNA within transcription bubbles (29) Our findings that pS38-AID and RPA but not PKA-Ca are detected at the Igh V genesupport the notion that AID binding to V genes requires priorphosphorylation However we cannot exclude the possibilities thatPKA-Ca was not detected at the V gene segment as a result of thetransient nature of the interaction or that other proteins bound to theV gene segment mask the Ab binding site of V genendashbound PKA-Ca Additionally we cannot rule out the possibility that AID boundto the V region exons is not phosphorylated by PKA but is insteadmodified by an unidentified kinase

AID localization to the V gene does not induce SHM

To determine whether AID binding induced SHM we sequencedthe Igh V gene (VDJ) Surprisingly the mutation frequency inPtbp2-depleted cells stimulated for 96 h was similar to that inscrambled cells (Fig 5A Supplemental Table IC) MoreoverPtbp2-depleted cells stimulated continuously for 3 wk did notaccumulate additional mutations (Supplemental Table IC) Al-though the mutation frequency was higher than in unstimulatedcells (Supplemental Table IC) it was still considerably lower thantypical SHM (32 33 37) The absence of SHM activity in Ptbp2-depleted cells cannot be attributed to a deficiency in Igh V gene(VDJ) transcription because transcription through this region wasnot markedly altered (2-fold change) upon Ptbp2 depletion(Supplemental Fig 2A) Most importantly there was no correlation

FIGURE 3 AID is retargeted from Sm to the Igh V gene in Ptbp2-depleted CH12 cells (A) Western blot of Ptbp2 AID and Gapdh in whole-cell extracts

of unstimulated (U) or CIT-stimulated (S) CH12 cells expressing scrambled (Scr) or Ptbp2 shRNA constructs Data are representative of six independent

experiments (B) Quantification of CSR in stimulated cells CSR frequency in Ptbp2-depleted cells was expressed as a percentage of CSR in scrambled

shRNA cells which was arbitrarily set to 100 (n = 6) p = 00004 paired two-tailed Student t test (C) ChIP analyses of histone H3 (positive control) and

AID bound to Sm and Igh V gene (VDJ) DNA sequence in stimulated cells expressing either scrambled or Ptbp2 shRNA (n = 4) p = 0003 p = 001

paired two-tailed Student t test (D) AID or histone H3 binding to region 59 of VDJ (59Vh1-53) Cm region 39 of VDJ (39Jh2) Im promoter (Im prom) or Ig

L chain V gene (VJk) in CIT-stimulated scrambled or Ptbp2-depleted (Ptbp2) CH12 cells (n = 4) (E) AID or histone H3 binding to Cdc45l Dusp1 Fabp5

Melk and p53 gene regions in CIT-stimulated scrambled and Ptbp2-depleted (Ptbp2) CH12 cells (n = 4) Data represent the mean and error bars show SD

FIGURE 4 pS38-AID and RPA but not PKA-Ca are retargeted from

Sm to the Igh V gene in Ptbp2-depleted CH12 cells ChIP analyses of

histone H3 (positive control) and pS38-AID and RPA (A) or PKA-Ca

bound to Sm and Igh V gene (VDJ) DNA sequence (B) in stimulated cells

expressing either scrambled or Ptbp2 shRNA constructs (n = 3) Data

represent the mean and error bars show SD p = 005 p = 003 p =

001 paired two-tailed Student t test ns not significant

4 INTRALOCUS SPECIFICITY OF AID

at Northern Illinois U

niv Univ L

ibPeriodicals Dept on Septem

ber 3 2014httpw

ww

jimm

unolorgD

ownloaded from

between the levels of AID bound to the V gene and SHM fre-quency (Figs 3C 5A) In contrast the region 59 of the Sm-re-petitive region (34) was highly mutated (Fig 5B SupplementalTable ID) A majority of the mutations in this region were tran-sition mutations and all mutations occurred at CG bp (Fig 5C)This spectrum suggests that mutations in CH12 cells arise mainlyfrom replication across deaminated residues with a small portionrepaired via base-excision repair engagement The lack of muta-tion at AT bases could indicate that the mismatch repair pathwayis either not active or is not recruited to the Igh locus in CH12 cells(38) Additionally there was a marked lack of G to T mutations inscrambled cells whereas Ptbp2-depleted cells showed both G to Aand G to T mutations The reason behind this intriguing observationis not clear but it is possible that alterations in AID andor Ptbp2at S regions could modulate the engagement of error-prone poly-merases during CSRMany of the mutations identified in the region 59 of the Sm-

repetitive region and V gene of stimulated scrambled and Ptbp2-depleted cells were in RGYW hot-spot motifs (Supplemental Fig 2BSupplemental Table IE) Of the total bases sequenced for mutationanalysis the amount of available RGYW motifs in the Igh V gene(VDJ) is sim61more than the amount available in the region 59 of theSm-repetitive region (based on numbers listed in SupplementalTable ICndashE) however the percentage of RGYW motifs mutated inthe region 59 of the Sm-repetitive region is much higher than in theIgh V gene (VDJ) (Supplemental Fig 2B Supplemental Table IE)Consequently if the RGYW motifs found in the Igh V gene werebeing mutated at the same frequency as the RGYW motifs in theregion 59 of the Sm-repetitive region the number of RGYW motifsexpected to be mutated at the Igh V gene would be significantlyhigher (Supplemental Fig 2C Supplemental Table IE) Thus the lack

of detectable SHM in Ptbp2-depleted CH12 cells is not due to a de-ficiency in the amount of available hot-spot motifs in this regionrather it is likely due to the absence in expression of required repairmediators or uncharacterized SHM-specific factors in CH12 cells(Fig 6) In conclusion although known mediators of SHM namelyAID and RPA were abundant at the Igh V gene and the bound AIDwas phosphorylated at S38 SHM was not induced

DiscussionOur results clearly demonstrate that S regions are preferred targetsfor AID binding Even in cells that have undergone CSR to the lastCH gene AID is efficiently recruited to the remaining S region inlieu of the transcribed Igh V gene This strong preference for Sregions might represent a physiological mechanism for AID reg-ulation wherein S regions act as a ldquosinkrdquo to prevent AID frominteracting with non-Ig genomic sequences Being noncoding Sregions could sustain mutations and deletions without havingany deleterious effect on cell viability This is evident from theabundant mutations and deletions observed at SmndashSa junctionsupon re-expression of AID in IgA+ cells Thus active recruitmentof AID to S regions probably functions as a default pathway tosequester AID activity away from other genomic targetsIf AID expressed in a B cell is indeed actively recruited to the S

regions the question that follows is how AID is targeted to the Vgenes to initiate SHM Our studies clearly demonstrate that re-duction in Ptbp2 levels promotes binding of AID to Igh V genesegments Thus in a B cell undergoing SHM modulating Ptbp2expression or perturbing the interaction between AID and Ptbp2through posttranslational modifications of AID andor Ptbp2 couldincrease the amount of non-S regionndashbound AID (Fig 6) The re-lative expression of Ptbp2 in germinal center B cells undergoingSHM versus CSR will shed further light onto the differential role ofthis protein during an immune responseThe most striking finding is that despite binding to the Igh V

gene AID cannot induce SHM Over the past few years severalgenome-wide association analyses of AID reported that it has thepotential to bind to other genomic regions (7 11 39) Our findingsclearly enforce the notion that binding of AID is not synonymouswith mutations For SHM to take place at least in the context ofCIT-stimulated CH12 cells additional steps need to occur beyondAID binding It is feasible that CH12 cells lack a factor(s) re-quired for SHM andor that CIT induction is not sufficient to inducethe expression of all required proteins Another likely possibilityis that AID targeted to the Igh V gene in Ptbp2-depleted cellsis indeed actively deaminating but high-fidelity repair (6) as

FIGURE 5 AID binding to the Igh V gene does not induce SHM (A)

Mutation analysis of Igh V gene (VDJ) segment in CIT-stimulated CH12 cells

expressing either scrambled or Ptbp2 shRNA constructs Scrambled cells

accumulated three mutations in 65436 bases sequenced and Ptbp2 cells had

two mutations in 65436 bases sequenced (B) Mutation analysis of 59 Sm

region Scrambled cells accumulated 14 mutations in 62160 bases sequenced

and Ptbp2 cells had 9 mutations in 62720 bases sequenced Data represent

four independent samples (C) Characterization of mutations found in 59 Sm

region Percentage nucleotide substitutions from residue ldquoxrdquo (down left side)

to residue ldquoyrdquo (along top) with total percentage substitution for each residue

listed on the right Percentage transitions or transversions are indicated below

the figures The p values were calculated using the z-test

FIGURE 6 Model for AID binding to Igh V gene upon Ptbp2 depletion

Ptbp2 interacts with AID and actively recruits it to S regions When the

interaction is dampened through an unknown mechanism AID can then

bind to the V region exons AID bound to the Igh V gene is phosphorylated

and is bound to RPA however SHM is not induced Thus additional

factorsprocesses absent or inactive in CH12 cells are required for SHM

The Journal of Immunology 5

at Northern Illinois U

niv Univ L

ibPeriodicals Dept on Septem

ber 3 2014httpw

ww

jimm

unolorgD

ownloaded from

opposed to error-prone repair required for SHM ldquofixesrdquo thelesions in a nonmutagenic fashion Under physiological con-ditions SHM is associated with germinal center structures foundin secondary lymphoid organs where the interaction of B cellswith CD4+ Th cells (40) could induce SHM-promoting factors orsubversion of high-fidelity repair pathways that are absent fromthe in vitro culture system described in this studyIn conclusion the data presented in this article intriguingly

suggest that targeting of AID to S regions is the default mechanismin a CSR-sufficient B cell line and may indicate a role for S regionsas an AID ldquosinkrdquo sequestering AID away from the rest of thegenome Our findings identify Ptbp2 as a crucial mediator be-tween S region and Igh V gene AID targeting and show that AIDlocalization does not determine AID deamination activity or theinduction of mutation Additionally we describe a cell linendashbasedmodel system that will be invaluable in further elucidating factorsconditions required for SHM versus CSR

AcknowledgmentsWe thank Tasaku Honjo (Kyoto University Kyoto Japan) for providing

the CH12 cell line and David Schatz (Yale University New Haven CT) and

members of the Chaudhuri laboratory for helpful discussions and suggestions

DisclosuresThe authors have no financial conflicts of interest

References1 Muramatsu M K Kinoshita S Fagarasan S Yamada Y Shinkai and

T Honjo 2000 Class switch recombination and hypermutation requireactivation-induced cytidine deaminase (AID) a potential RNA editing enzymeCell 102 553ndash563

2 Revy P T Muto Y Levy F Geissmann A Plebani O Sanal N CatalanM Forveille R Dufourcq-Labelouse A Gennery et al 2000 Activation-induced cytidine deaminase (AID) deficiency causes the autosomal recessiveform of the Hyper-IgM syndrome (HIGM2) Cell 102 565ndash575

3 Petersen-Mahrt S K R S Harris and M S Neuberger 2002 AID mutatesE coli suggesting a DNA deamination mechanism for antibody diversificationNature 418 99ndash103

4 Papavasiliou F N and D G Schatz 2002 Somatic hypermutation of immu-noglobulin genes merging mechanisms for genetic diversity Cell 109(Suppl)S35ndashS44

5 Stavnezer J J E Guikema and C E Schrader 2008 Mechanism and regu-lation of class switch recombination Annu Rev Immunol 26 261ndash292

6 Liu M J L Duke D J Richter C G Vinuesa C C GoodnowS H Kleinstein and D G Schatz 2008 Two levels of protection for the B cellgenome during somatic hypermutation Nature 451 841ndash845

7 Pavri R A Gazumyan M Jankovic M Di Virgilio I Klein C Ansarah-Sobrinho W Resch A Yamane B Reina San-Martin V Barreto et al 2010Activation-induced cytidine deaminase targets DNA at sites of RNA polymeraseII stalling by interaction with Spt5 Cell 143 122ndash133

8 Yamane A W Resch N Kuo S Kuchen Z Li H W Sun D F RobbianiK McBride M C Nussenzweig and R Casellas 2011 Deep-sequencingidentification of the genomic targets of the cytidine deaminase AID and its co-factor RPA in B lymphocytes Nat Immunol 12 62ndash69

9 Robbiani D F S Bunting N Feldhahn A Bothmer J Camps S DeroubaixK M McBride I A Klein G Stone T R Eisenreich et al 2009 AID pro-duces DNA double-strand breaks in non-Ig genes and mature B cell lymphomaswith reciprocal chromosome translocations Mol Cell 36 631ndash641

10 Chiarle R Y Zhang R L Frock S M Lewis B Molinie Y J HoD R Myers V W Choi M Compagno D J Malkin et al 2011 Genome-widetranslocation sequencing reveals mechanisms of chromosome breaks and rear-rangements in B cells Cell 147 107ndash119

11 Staszewski O R E Baker A J Ucher R Martier J Stavnezer andJ E Guikema 2011 Activation-induced cytidine deaminase induces reproducibleDNA breaks at many non-Ig loci in activated B cells Mol Cell 41 232ndash242

12 Pasqualucci L G Bhagat M Jankovic M Compagno P SmithM Muramatsu T Honjo H C Morse III M C Nussenzweig and R Dalla-Favera 2008 AID is required for germinal center-derived lymphomagenesisNat Genet 40 108ndash112

13 Manser T 1987 Mitogen-driven B cell proliferation and differentiation are notaccompanied by hypermutation of immunoglobulin variable region genes JImmunol 139 234ndash238

14 Nagaoka H M Muramatsu N Yamamura K Kinoshita and T Honjo 2002Activation-induced deaminase (AID)-directed hypermutation in the immuno-globulin Smu region implication of AID involvement in a common step of classswitch recombination and somatic hypermutation J Exp Med 195 529ndash534

15 Nowak U A J Matthews S Zheng and J Chaudhuri 2011 The splicingregulator PTBP2 interacts with the cytidine deaminase AID and promotesbinding of AID to switch-region DNA Nat Immunol 12 160ndash166

16 Basu U F L Meng C Keim V Grinstein E Pefanis J Eccleston T ZhangD Myers C R Wasserman D R Wesemann et al 2011 The RNA exosometargets the AID cytidine deaminase to both strands of transcribed duplex DNAsubstrates Cell 144 353ndash363

17 Xu Z Z Fulop G Wu E J Pone J Zhang T Mai L M ThomasA Al-Qahtani C A White S R Park et al 2010 14-3-3 adaptor proteinsrecruit AID to 59-AGCT-39-rich switch regions for class switch recombinationNat Struct Mol Biol 17 1124ndash1135

18 Tian M and F W Alt 2000 Transcription-induced cleavage of immuno-globulin switch regions by nucleotide excision repair nucleases in vitro J BiolChem 275 24163ndash24172

19 Yu K F Chedin C L Hsieh T E Wilson and M R Lieber 2003 R-loops atimmunoglobulin class switch regions in the chromosomes of stimulated B cellsNat Immunol 4 442ndash451

20 Michael N H M Shen S Longerich N Kim A Longacre and U Storb2003 The E box motif CAGGTG enhances somatic hypermutation withoutenhancing transcription Immunity 19 235ndash242

21 Nakamura M S Kondo M Sugai M Nazarea S Imamura and T Honjo1996 High frequency class switching of an IgM+ B lymphoma clone CH12F3 toIgA+ cells Int Immunol 8 193ndash201

22 Chaudhuri J M Tian C Khuong K Chua E Pinaud and F W Alt 2003Transcription-targeted DNA deamination by the AID antibody diversificationenzyme Nature 422 726ndash730

23 Vuong B Q M Lee S Kabir C Irimia S Macchiarulo G S McKnight andJ Chaudhuri 2009 Specific recruitment of protein kinase A to the immuno-globulin locus regulates class-switch recombination Nat Immunol 10 420ndash426

24 Muramatsu M V S Sankaranand S Anant M Sugai K KinoshitaN O Davidson and T Honjo 1999 Specific expression of activation-inducedcytidine deaminase (AID) a novel member of the RNA-editing deaminasefamily in germinal center B cells J Biol Chem 274 18470ndash18476

25 Han L and K Yu 2008 Altered kinetics of nonhomologous end joining andclass switch recombination in ligase IV-deficient B cells J Exp Med 2052745ndash2753

26 Eto T K Kinoshita K Yoshikawa M Muramatsu and T Honjo 2003 RNA-editing cytidine deaminase Apobec-1 is unable to induce somatic hypermutationin mammalian cells Proc Natl Acad Sci USA 100 12895ndash12898

27 Misaghi S C S Garris Y Sun A Nguyen J Zhang A Sebrell K SengerD Yan M N Lorenzo S Heldens et al 2010 Increased targeting of donorswitch region and IgE in Sgamma1-deficient B cells J Immunol 185 166ndash173

28 Zhang T A Franklin C Boboila A McQuay M P Gallagher J P ManisA A Khamlichi and F W Alt 2010 Downstream class switching leads to IgEantibody production by B lymphocytes lacking IgM switch regions Proc NatlAcad Sci USA 107 3040ndash3045

29 Chaudhuri J C Khuong and F W Alt 2004 Replication protein A interactswith AID to promote deamination of somatic hypermutation targets Nature 430992ndash998

30 Basu U J Chaudhuri C Alpert S Dutt S Ranganath G Li J P SchrumJ P Manis and F W Alt 2005 The AID antibody diversification enzyme isregulated by protein kinase A phosphorylation Nature 438 508ndash511

31 Pasqualucci L Y Kitaura H Gu and R Dalla-Favera 2006 PKA-mediatedphosphorylation regulates the function of activation-induced deaminase (AID) inB cells Proc Natl Acad Sci USA 103 395ndash400

32 McBride K M A Gazumyan E M Woo T A Schwickert B T Chait andM C Nussenzweig 2008 Regulation of class switch recombination and somaticmutation by AID phosphorylation J Exp Med 205 2585ndash2594

33 Cheng H L B Q Vuong U Basu A Franklin B Schwer J AstaritaR T Phan A Datta J Manis F W Alt and J Chaudhuri 2009 Integrity of theAID serine-38 phosphorylation site is critical for class switch recombination andsomatic hypermutation in mice Proc Natl Acad Sci USA 106 2717ndash2722

34 McBride K M A Gazumyan E M Woo V M Barreto D F RobbianiB T Chait and M C Nussenzweig 2006 Regulation of hypermutation byactivation-induced cytidine deaminase phosphorylation Proc Natl Acad SciUSA 103 8798ndash8803

35 Vuong B Q K Herrick-Reynolds B Vaidyanathan J N Pucella A J UcherN M Donghia X Gu L Nicolas U Nowak N Rahman et al 2013 A DNAbreak- and phosphorylation-dependent positive feedback loop promotes immu-noglobulin class-switch recombination Nat Immunol 14 1183ndash1189

36 Matthews A J S Zheng L J DiMenna and J Chaudhuri 2014 Regulation ofimmunoglobulin class-switch recombination choreography of noncoding tran-scription targeted DNA deamination and long-range DNA repair Adv Immunol122 1ndash57

37 McKean D K Huppi M Bell L Staudt W Gerhard and M Weigert1984 Generation of antibody diversity in the immune response of BALBcmice to influenza virus hemagglutinin Proc Natl Acad Sci USA 813180ndash3184

38 Chahwan R W Edelmann M D Scharff and S Roa 2012 AIDing antibodydiversity by error-prone mismatch repair Semin Immunol 24 293ndash300

39 Klein I A W Resch M Jankovic T Oliveira A Yamane H NakahashiM Di Virgilio A Bothmer A Nussenzweig D F Robbiani et al 2011Translocation-capture sequencing reveals the extent and nature of chromosomalrearrangements in B lymphocytes Cell 147 95ndash106

40 Hamel K M V M Liarski and M R Clark 2012 Germinal center B-cellsAutoimmunity 45 333ndash347

6 INTRALOCUS SPECIFICITY OF AID

at Northern Illinois U

niv Univ L

ibPeriodicals Dept on Septem

ber 3 2014httpw

ww

jimm

unolorgD

ownloaded from