An evolutionarily conserved target motif forimmunoglobulin class-switch recombination

Ali A Zarrin1,3, Frederick W Alt1, Jayanta Chaudhuri1, Nicole Stokes1, Dhruv Kaushal1,Louis Du Pasquier2 & Ming Tian1,3

Immunoglobulin H class-switch recombination (CSR) occurs between switch regions and requires transcription and activation-

induced cytidine deaminase (AID). Transcription through mammalian switch regions, because of their GC-rich composition,

generates stable R-loops, which provide single-stranded DNA substrates for AID. However, we show here that the Xenopus laevis

switch region Sl, which is rich in AT and not prone to form R-loops, can functionally replace a mouse switch region to mediate

CSR in vivo. X. laevis Sl–mediated CSR occurred mostly in a region of AGCT repeats targeted by the AID–replication protein A

complex when transcribed in vitro. We propose that AGCT is a primordial CSR motif that targets AID through a non-R-loop

mechanism involving an AID–replication protein A complex.

Immunoglobulin molecules are composed of heavy (IgH) and light(IgL) chains. Both IgH and IgL chains have an N-terminal variable (V)region, which is involved in antigen binding, and a C-terminalconstant region. There are several different IgH chain constant regions(for example, Cm, Cg and Ca), and these determine the class (IgM, IgGor IgA) and effector function of the immunoglobulin (antibody)molecule. The loci that encode IgH and IgL chains (Igh and Igl,respectively) undergo three distinct genetic alterations in B lineagecells. Early in development, Igh and Igl V-region exons are assembledfrom germline gene segments by V, diversity (D) and joining (J)recombination1. Productive Igh and Igl assembly generates matureB cells, which express surface IgM. After encountering cognateantigen, mature B cells further augment antibody specificity byintroducing point mutations into assembled Igh and Igl V-regionexons through somatic hypermutation (SHM). SHM permits selectionof B cells containing V-region mutations that encode higher-affinityantibody2,3. During an immune response, antibody class also can bealtered through IgH class-switch recombination (CSR). CSR replacesthe initially expressed Cm exons with one of several downstream sets ofCH exons (referred to as CH genes)4,5.

In the mouse, CH genes are arranged in the order 5¢-V(D)J-Cm-Cd-Cg3-Cg1-Cg2b-Cg2a-Ce-Ca-3¢. CSR occurs in switch (S) regions, whichare 1- to 12–kilobase (kb) repetitive DNA elements 5¢ of individual CH

genes. CSR results from recombination between the S region upstreamof Cm (Sm) and a downstream S region, accompanied by deletion ofintervening sequences4. Gene-targeted deletion of S regions greatlydiminishes or eliminates CSR, suggesting that S regions are specializedtargets for CSR6–8. Mammalian S regions are G rich on the non-template strand and are composed mainly of tandem repetitive units

in which certain motifs, such as TGGGG, GGGGT, GGGCT, GAGCTand AGCT, occur frequently4. As there are no consensus CSRrecombination sites in S regions or regions of extensive homology atCSR junctions, CSR is described as a region-specific recombinationprocess that is distinct from homologous recombination or site-specific V(D)J recombination9.

Even though IgH CSR and SHM result in very different outcomes(whole-scale deletions versus point mutations) and functionally targetdifferent sequences (S regions versus V-region exons), both processesrequire the B cell–specific activation-induced deaminase (AID)enzyme10,11. The function of AID in SHM and CSR has been debated.One theory proposes that AID works by RNA editing to deaminatespecific cytidines in unknown mRNAs and thereby allow them toencode CSR and SHM enzymes5. However, all evidence supportingthis model has been indirect12–14. In contrast, a wealth of experimentalevidence suggests that AID acts by deaminating cytidines in DNA15.Thus, AID is a single-stranded DNA (ssDNA)–specific cytidinedeaminase in vitro16–22. Moreover, genetic evidence indicates thatAID-mediated cytidine deamination of V regions or S regions gen-erates DNA lesions that are differentially processed to effect SHM andCSR23–25. Although one published report argued in favor of an RNA-editing mechanism based in part on an inability to find ectopicallyexpressed AID associated with S regions during CSR14, two otherstudies, one assaying ectopically expressed AID26 and another assayingendogenous AID27, demonstrated that AID associates with appro-priate S regions during CSR. Thus, the last two studies directly linkAID to DNA in a physiological setting.

Given that AID can deaminate ssDNA but not duplex DNA,elucidation of the mechanisms involved in the generation of ssDNA

Published online 7 November 2004; doi:10.1038/ni1137

1Howard Hughes Medical Institute, The Children’s Hospital, CBR Institute for Biomedical Research, and Department of Genetics, Harvard University Medical School,Boston, Massachusetts 02115, USA. 2Institute of Zoology, University of Basel, Basel, Switzerland. 3These authors contributed equally to this work. Correspondence shouldbe addressed to F.W.A. (alt@enders.tch.harvard.edu).

NATURE IMMUNOLOGY VOLUME 5 NUMBER 12 DECEMBER 2004 1 27 5

A R T I C L E S©

2004

Nat

ure

Pub

lishi

ng G

roup

ht

tp://

ww

w.n

atur

e.co

m/n

atur

eim

mun

olog

y

targets in chromosomal DNA has been an ongoing quest. Both CSRand SHM require transcription through the target sequences, and suchtranscription seems to be directly involved in generating the substratefor AID15. For mammalian CSR, transcription through S regions cangenerate ssDNA targets for AID activity in the form of R-loops, inwhich the S region transcript is hybridized to the template DNA andthe nontemplate DNA strand is displaced as a ssDNA loop28,29. Theability to generate R-loops is attributed to the characteristic basecomposition of mammalian S regions and, in particular, the G-richnontemplate strand4. Inversion of Sg1 inhibits the ability to form R-loops after transcription in vitro28 and correspondingly diminishes theability to support CSR in vivo7. However, inverted S regions, whichhave little or no R-loop formation, still support considerable CSR,suggesting that mechanisms in addition to R-loop formation maycontribute to the generation of AID target DNA during CSR7. Thisidea also derives support from the finding of low CSR in B cellslacking Sm tandem repeats6,8.

Point mutations that occur during SHM, although scatteredthroughout the V-region exon and immediate downstream flankingsequence, tend to occur at ‘hotspots’ composed of the DGYWtetramer (where D is A or G or T; Y is C or T; and W is A or T)30.Among DGYW variants, AGCT is the preferred sequence target30,31.Studies have suggested that AID is preferentially targeted to SHMhotspot motifs in association with replication protein A (RPA)27. RPAis a ssDNA-binding protein involved in DNA replication and repair32.In vitro studies have shown that the AID-RPA complex, but neitherprotein alone, preferentially binds and deaminates transcribed DNAthat contains SHM hotspots27. The AID-RPA complex, which dependson B cell–specific AID modifications, may access transcribed V-regiongenes in the context of transcription ‘bubbles’27. After deamination,RPA can remain bound to DNA, where it is proposed to function inthe recruitment of downstream repair pathways necessary to effectSHM. In vitro deamination of transcribed S regions occurs in theabsence of RPA because of the stable ssDNA substrate in R-loops.However, there still could be a function for the AID-RPA complex inCSR, both in the context of downstream factor recruitment and innon-R-loop-mediated CSR. Point mutations reminiscent of those seenin V genes after SHM also occur at DGYW sequences in S regions33–35.

Phylogenetic comparisons also suggest that CSR may occur inde-pendently of S-region R-loops. Evolutionarily, the earliest vertebrateto carry out CSR was the amphibian36. In contrast to the case formouse and human, Xenopus laevis Sm (XSm) would not be predictedto form R-loops, as it is devoid of G-rich pentamers (such as GGGGTand GGGCT) and is in fact AT rich37. However, XSm shares severalfeatures with its counterparts in mammals and birds38, most notably

repetitive sequences with a high frequency of AGCT, the SHM motifthat can target AID-RPA. To further elucidate potential mechanismsfor AID-targeting in CSR, we have tested the ability of XSm to mediateCSR in mice and to target the AID-RPA complex when transcribedin vitro.

RESULTS

XSl does not form an R-loop in vitro

To directly evaluate the ability of XSm to form R-loops, we cloned XSmunder control of the bacteriophage T7 RNA polymerase promoter in aplasmid and assayed R-loop formation with a P1 nuclease assay28. Inthe P1 assay, R-loops are demonstrated by high sensitivity of thenontemplate strand to P1 nuclease. As described28, mouse Sg1 showedsubstantial sensitivity to P1 (being almost completely degraded at aconcentration of 1 � 104 pg per reaction) when transcribed in thephysiological orientation but little sensitivity when transcribed in thenonphysiological orientation (being nearly intact at a concentration of1 � 105 pg per reaction; Fig. 1, lanes 2 and 7). In contrast, when XSmwas subjected to the same assay in the physiological orientation, itremained largely intact even after treatment with the highest concen-tration of P1 nuclease (Fig. 1, lane 13). When transcribed in the

Sγ1 (+) Sγ1 (–)

5.7 17.7

NT

T7

5.7 17.7

T7XSµ (+) XSµ (–)

5.7 9.8

T7

5.7 9.8

T7

10

8

6

5

23kb P1 (pg)

*NT* NT* NT*

Input

Input

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

105 104 103 102 101 105 104 103 102 101 105 104 103 102 101 105 104 103 102 101 0000

Cγ1

Iγ1

Sγ1a deleted locus (∆Sγ1

a)RV RVRI

XSµ+

XSµ+

XSµ–

Xpf +

Xpf +

Xpf –

neor

XSµ replacement construct

Wild-type Sγ1

Xpf intron replacement construct

Homologous recombination Cre-mediated recombination

Xpf + allele (physiological orientation)

Xpf – allele (inverted orientation)

XSµ+ allele (physiological orientation)

XSµ– allele (inverted orientation)

Sγ1b endogenous locus

P

tk

tk

HH

H

H

H

H

H

H

H

H

H

H

P

3′

H

H

neor

Figure 2 Targeted replacement of the Sg1a allele. Genomic structure of

mouse Sg1 loci on a mutated a allele (Igha) or wild-type b allele (Ighb).

EI, EcoRI; EV, EcoRV; H, HindIII, P, PstI. Triangles, loxP sites; tk, gene

encoding thymidine kinase; +, physiological transcription orientation; –,

inverted transcription orientation; rectangular black boxes, Ig1 and Cg1;

arrows, Ig1 promoter; ovals, S regions (wild-type or targeted).

Figure 1 R-loop formation of XSm versus mouse

Sg1. XSm and mouse Sg1 were transcribed with T7

RNA polymerase and then were treated with

increasing concentrations of P1 nuclease (1 � 10

to 1 � 105 pg per reaction). Reaction products

were denatured and were analyzed by Southern

blot with an oligonucleotide probe specific for the

nontemplate (NT) strand (*). Input, undigestedDNA; (+), physiological transcription orientation;

(–), inverted transcription orientation. Top,

transcription constructs; numbers below each

construct indicate distance (in kb) from the

5¢ end of the nontemplate strand.

1 27 6 VOLUME 5 NUMBER 12 DECEMBER 2004 NATURE IMMUNOLOGY

A R T I C L E S©

2004

Nat

ure

Pub

lishi

ng G

roup

ht

tp://

ww

w.n

atur

e.co

m/n

atur

eim

mun

olog

y

nonphysiological orientation, XSm was also largely resistant to P1,although there seemed to be modest sensitivity at the highestconcentration (Fig. 1, lane 19). We conclude that, in contrastto mouse Sg1, XSm did not show substantial R-loop-forming ability,a conclusion that we have validated by an independent method(discussed below).

Replacement of mouse Sc1 with XSlTo test the CSR activity of XSm in mice, we used an embryonic stemcell clone in which most of the intron containing Sg1 had been deletedon one allele7 (Fig. 2; DSg1). The embryonic stem cell clone used forthis targeting was generated from the 129-C57BL/6 F1 mouse7. In theF1 embryonic stem cell, the two Igh alleles belong to different IgHallotypes (IgHa from 129/Sv strain; IgHb from C57BL/6 strain) thatare distinguishable by polymorphisms. Because all of our targetingconstructs were from DNA from 129 mice, all mutations wereintroduced into the Igha allele, leaving the Ighb allele in wild-typeconfiguration. This configuration allowed us to assay the relative CSRof the mutated Igha allele versus that of the wild-type Ighb allele aftergenerating mature B cells from the mutant embryonic stem cell clones.Mature B cell clones from the DSg1 embryonic stem cell clone wereshown to fail to undergo CSR to Sg1 on the mutated allele7. Thus, byinsertion of a test sequence into the DSg1 allele, its capacity to mediateCSR can be evaluated7.

To test the ability of XSm to support CSR in mice, we first inserted a4.1-kb fragment of XSm in place of the 12-kb mouse Sg1. In mouseB cells, CSR depends on transcription through the S region from a‘germline’ promoter associated with a noncoding exon (called theI exon) upstream of the S region4 (Fig. 2). The targeting constructused, with insertion of the XSm sequence in the physiological orienta-tion relative to the germline Ig1 promoter, also contained a neomycin-resistance selectable marker gene (neor). Because neor can interferewith germline transcription from I promoters39,40, it was flanked byloxP sequences in the same orientation and was subsequently deletedby Cre recombination. The loxP sequences are 34–base pair (bp)recombination signal sequences for bacteriophage Cre recombinase41;exposure to Cre allows deletion of sequences that are flanked by loxPsites in the same orientation. To analyze effects of transcriptionalorientation, the inserted XSm was flanked by loxP sites lying inopposite orientation, one of which was generated by recombinationof the direct loxP sites flanking neor (Fig. 2). Cre-mediated recombi-nation between inverted loxP sites results in inversion of the inter-

vening DNA sequence. Thus, we were able to use Cre recombinase togenerate embryonic stem cells in which XSm was positioned in bothorientations relative to the Ig1 promoter (Fig. 2). Finally, as a control,we also generated embryonic stem cells in which we replaced Sg1 witha fragment of approximately 4 kb from intron 10 of the xerodermapigmentosum complementation group F (Xpf) gene (Fig. 2). Wedeleted neor and generated clones with both orientations of the Xpfintron by the loxP-Cre method as described above. We chose the Xpfintron because it contains no extensive repetitive sequence and its basecomposition (A, 29%; T, 26%; C, 20%; G, 25%) is representative ofthe genomic average in mouse.

We generated chimeric mice by injecting the various mutantembryonic stem clones into blastocysts from recombination activatinggene 2 (RAG2)–deficient mice. In chimeric mice derived using thisRAG2-deficient blastocyst complementation approach, all mature Band T cells were derived from the injected embryonic stem cells. Tomeasure relative ability of the mutant and wild-type Sg1 alleles tomediate CSR, we stimulated splenocytes from chimeric mice withantibody to CD40 (anti-CD40) plus interleukin-4 (IL-4), a treatmentthat preferentially targets CSR to the Sg1 region. To confirm that theS-region replacements did not affect germline transcription from theIg1 promoter, we used an established RT-PCR assay to comparegermline transcript abundance from the wild-type (nontargeted)and targeted Sg1 alleles7. In this assay we found no obvious differencesin the relative expression of the DSg1, XSm or Xpf replacement (Igha)alleles versus the wild-type (Ighb) allele (Fig. 3).

129/

SV

F1 C57BL/

6

∆S γ1XS µ

+

XS µ –

147

242

123

217

Size (bp)

Mar

ker

γ1a

γ1b

Xpf +

Xpf –

F1

Figure 3 RT-PCR analysis of the Ighg1 germline transcripts. A fragment

of the cDNA of the germline transcript between Ig1 and Cg1 was amplified

by RT-PCR, then the cDNA from the two alleles was distinguished by

MboI digestion, which demonstrates a polymorphic MboI site in the

Ighb allele7. Representative data from at least two independent experiments

are presented.

0

20

40

60

80

100

120

F1 ∆Sγ1 XSµ+ XSµ

– Xpf + Xpf –

IgG

1a /Ig

G1

tota

l (%

)

Figure 4 ELISA of IgG1 in the supernatants of splenocyte cultures. The ratioof IgG1a/IgG1 total of the wild-type mice is defined arbitrarily as 100% CSR

efficiency for the Igha allele. Each dot represents the average of two to three

measurements of one chimera and error bars represent standard deviation of

the mean (triangles).

Table 1 IgG1a-producing hybridomas/IgG1b-producing hybridomas

Genotype IgG1a/IgG1b CSR (%)

F1 63/42 100.0

DSg1 0/160 0.0

XSm+ 43/115 24.9

XSm� 27/110 16.4

Xpf+ 2/52 2.6

Xpf� 6/147 2.7

Numbers of IgG1a- and IgG1b-producing hybridomas for each genotype. Relative CSRfrequency is defined by the IgG1a-producing hybridoma/IgG1b-producing hybridomaratio and is arbitrarily set as 100% for F1 cells. P ¼ 0.000024, efficiency of CSR forXSm

� versus Xpf� (Chi-square).

NATURE IMMUNOLOGY VOLUME 5 NUMBER 12 DECEMBER 2004 1 27 7

A R T I C L E S©

2004

Nat

ure

Pub

lishi

ng G

roup

ht

tp://

ww

w.n

atur

e.co

m/n

atur

eim

mun

olog

y

XSl mediates efficient CSR in mice

In B cells derived from F1 embryonic stem cells with replacement ofSg1, CSR to IgGa and IgG1b must be mediated through XSm (or theXpf intron) and endogenous mouse Sg1

b, respectively, with the relativetiters of IgG1a versus IgG1b reflecting CSR efficiency on the two alleles.After stimulating cells with anti-CD40 plus IL-4, we used enzyme-linked immunosorbent assay (ELISA) to measure supernatant IgG1a

concentrations and, as an IgG1b-specific antibody is not available,total IgG. With these values, we then calculated the IgG1a/IgG1 ratio.We arbitrarily defined the IgG1a/IgG1 ratio for wild-type F1 cells as100% CSR efficiency for the wild-type Ighg1

a allele (Fig. 4). In B cellswith deletion of Sg1

a, IgG1a was undetectable in the culture super-natant and served as the background for this assay (Fig. 4). Insertionof XSm in either orientation restored supernatant IgG1a to approxi-mately 60% of wild-type (Fig. 4). As an index of the specificity, theXpf intron in either orientation supported the generation of IgG1a

only slightly above the background noted with the DSg1 allele (Fig. 4).As an independent assay for CSR efficiency, we also measured the

IgG1a/IgG1b expression ratio in hybridomas generated from thevarious chimeric mice. Because of allelic exclusion, only one Igh allelein a given B cell is functionally expressed1. Thus, in a population of F1B cells, approximately half express IgMa, while the other half produceIgMb. In IgG1-producing hybridomas derived from wild-type F1 Bcells, we consistently obtained slightly more IgG1a hybridomas thanIgG1b hybridomas7 (Table 1). The reason for this over-representationof the IgG1a producers is not clear. Thus, for comparison, wedesignated the IgG1a/IgG1b ratio of F1 cells as 100% CSR efficiencyon the Igha allele. In cells with replacement of XSm, this ratio wasapproximately 20% that of F1 cells in either transcriptional orienta-tion, whereas the Xpf intron generated a ratio of only 2–3% in eithertranscriptional orientation, which although very low was detectably

above the DSg1 background (Table 1). Although the results of both theELISA and hybridoma methods led to the same conclusions, therelative CSR efficiency of XSm seemed somewhat different whenmeasured by ELISA (60%) versus hybridoma analysis (20%). Asdiscussed before7, we would argue that the hybridoma analyses aremore accurate, as the IgG1a/IgG1b ratio reflects a direct comparison ofthe CSR frequency at the two alleles. The decreased frequency of CSRsupported by XSm compared with that supported by the endogenousSg1 may be attributed in part to the shorter length of XSm (4.1 kbversus 12 kb for Sg1). In support of this, preliminary experimentshave shown that CSR efficiency is dependent on S-region length(A.A.Z. and F.W.A., data not shown). Thus, based on both theELISA and hybridoma analyses, we conclude that XSm but not anunrelated sequence of similar length (the Xpf intron) supports

ba

TAGGGTGAGCTGAGCTGGGTGAGC TAGAGTGAGCTGAGCTGTGCATTACAGTCGCGACCAAGCTGTGCATTA

GTAGACTGTAATGAACTGGAATGA GTAGACT.TAG.AATATACTGAATCGGTTTAGAAACAATATACTGAAT

GCTAAACTAGGCTGGCTTAACCGA GCTAAACTAGGCAGTTCCGTACACTTGAACAGAACTAGTTCCGTACAC

AGCTGGGCCGCTAAGCTAAACTAG AGCTGGGCCGCTACTGCAAGCTATAGCTTTTGCAGGACTGCAAGCTAT

TCCTGGGATTCTGGAAGAAAAGAT TCCTGGGATTCTTTAATGCAGTATGTTTTTACACAATTAATGCAGTAT

GGAAGCTAATTTAGAATCAAGTAA GGAAGCTAAAGGTCAGCTTGGTCTCTCGGTTTAGAATCAGCTTGGTCT

CTGTCTCTACTTCAGTTATACATG CTGTCTCTACTTGCTGAGCATTGTTTTCCAGCTCAAGCTGAGCATTGT

AGATAAAATGGATACCTCAGTGGT SµAGATAAAATGAGGGGCTGGAACTG 140 TTAGCTTGGTCAGGGCTGGAACTG XSµ+

Sµ288XSµ–

Sµ19XSµ–

Sµ71XSµ–

Sµ441XSµ–

Sµ35XSµ–

Sµ99XSµ–

Sµ459XSµ–

Sµ18XSµ–

Sµ49XSµ–

Sµ54XSµ+

Sµ109XSµ+

Sµ56XSµ+

Sµ324 XSµ+

Sµ21 XSµ+

Sµ199XSµ+

Sµ14XSµ+

Sµ220XSµ+

TGAGCTGGGGTGAGCTCAGCTATG TGAGCTGGGGTGATTCAATGCATATGTATAGAATAAATTCAATGCATA

GCTGAGCTGAGCTGGGGTGAGCTG GCTGAGCTGAGATATATAGTGAATCTGAATTCATTCTATATAAAGCTC

TGGCTGAGCTGAGATGGGTGGGCT TGGCTGAGCTGAGAGCATTATACACCAAAACAAGCGGAGCATTATACA

GGTGAGCTGAGCTGAGCTTGACTG GGTGAGCTGAGGTTGCAGGACTGCAGTGCACAGCTTTTGCAGGACTGC

ATGGCTGAGCTGAGATGGGTGGGC ATGGCTGAGCTGATGAATTCAGTAGCTTTATATAGAATGAATTCAGTA

ATGCGCTAAACTGAGGTGATTACT ATGCGCTAAACTAATTATTCTGTACTGCAGCTATGAAATTATTATTCT

ATTTTAGAAGCT------------ ATTTTAGAAGCTCCACTGTACAGCTGAGCAAGCTATGCACTGTACAGC

ATGCGCTAAACTGAGGTGATTACT ATGCGCTAAACTAATTATTATTTTCTGCAGCTATGAAATTATTATTTT

GAGCTGGGGTGA------------ GAGCTGGGGTGATCCAAAAAGCTGTAGTGTATTGCTTCCAAAAAGCTG

GGGCCGCTAAGCTAAACTAGGCTG GGGCCGCTAAGCTACAGCTTGCTTTTCTGTACATTGTACAGCTTGCTT

CTACGCGTGTTGGGGTGAGCTGATCTGAAATGAGCTACTCTG CTACGCGTGTTGGGGTGAGCTCCTGGAACAGGAGAAGGCCCTAGCTGCTAGCTTTGTGTGGGGCCTGGAACAGGAGAAGGCCCT

GCTGAGCTGGGGTGAGCTGAGCTGAGCTGGGGTGAGCTGAGC SµGCTGAGCTGGGGTGAGCTGAAGCTTTGTGTGGGCCTGGAACA 215AAGTGACTCGGAAAGCTGCTAGCTTTGTGTGGGCCTGGAACA Xpf+

Sµ103Xpf+

Sµ15Xpf–

Sµ3Xpf–

ATGGGGTGAGCTGAGCTGGGCTGAGCTGGACTGAGCTGAGCT ATGGGGTGAGCTGAGCTGGGTTTTCTCTCATGGACTATACTTAGCTCCTTAGTAGTGTAGCGAATTTCCTCATGGACTATACTT

AGCTGAGCTAGGGTGAGCTGAGCTGGGTGAGCTGAGCTGAGC AGCTGAGCTAGGGTGAGCTGATTTGATAATAGACTGCTGCTGATCACGTATATACCACTTCCATTTGATAATAGACTGCTGCTG

d

cSµ to XSµ+ Sµ to XSµ

–Sµ to Xpf+

Sµ to Xpf–

Figure 5 Sequence analysis of Sm to XSm+, XSm

�, Xpf+ and

Xpf� hybridoma junctions. Nucleotide sequences surrounding

CSR junction: XSm+ (a) and XSm

� (b). Dashed line, the core

mouse Sm region that has not been fully sequenced. (c) Xpf+.

(d) Xpf�. The AGCT nucleotides of XSm and Xpf are in italics;

underlining indicates insertions.

XSµ+

XSµ–

Cγ1

Cγ1

Iγ1

Iγ1

1.0

2.0 3.0 4.0

AGCT

kb

AGCT

1.0

2.03.04.0kb

Figure 6 Sm to XSm switch junctions. Recombination junctions were amplified

from individual hybridomas using PCR. Each breakpoint is indicated as a

filled circle. Top, locations of AGCT motifs and breakpoints in XSm+; bottom,

XSm�. Vertical lines in XSm, location of AGCT motifs (Fig. 7).

1 27 8 VOLUME 5 NUMBER 12 DECEMBER 2004 NATURE IMMUNOLOGY

A R T I C L E S©

2004

Nat

ure

Pub

lishi

ng G

roup

ht

tp://

ww

w.n

atur

e.co

m/n

atur

eim

mun

olog

y

relatively efficient CSR in mice. In addition, we conclude that unlikemouse Sg1 (ref. 7), XSm mediates CSR with equal efficiency in bothtranscriptional orientations.

Association of CSR junctions with AGCT

To determine the sites in XSm that are used for CSR in mouse B cells,we determined the nucleotide sequence of Sm to XSm or Xpf junctionsgenerated from individual hybridomas (Fig. 5). Nucleotide junctionsfor both XSm and Xpf carried insertions, deletions and mutationstypical of S junctions. For XSm, 12 of 20 junctions were at or near (lessthan 7 bp away from) AGCT sites (Fig. 5a,b). In addition, three of fourSm-Xpf junctions occurred in the vicinity of AGCT sites (Fig. 5c,d).CSR breakpoints were preferentially localized in a region of XSmapproximately 1.5 kb in size proximal to the Ig1 promoter whentranscribed in the physiological transcription (Fig. 6). Notably, CSRbreakpoints in X. laevis B cells also localize in this region37. This areaof XSm regions had a particularly high density of AGCT motifs (Fig. 6).The apparent association of CSR junctions with this AGCT-rich regionwas confirmed in experiments in which the orientation of the XSm wasinverted. In this configuration, the AGCT-rich region was moved distalto the promoter and the CSR recombination sites were correspond-ingly focused in this distal region (Fig. 6). The correlation between theCSR breakpoints and the region rich in AGCT motifs was statisticallysignificant in both the physiological (r ¼ 0.75, P o 0.05) and invertedXSm (r o 0.86, P ¼ 0.05) orientations (Supplementary Fig. 1 online).Thus, these findings suggest that the AGCT motif, or perhaps morelikely a high density of this motif, serves as an optimal target for CSR.The 4-kb Xpf intron sequence contained only 30 AGCT motifs,compared with 226 in the XSm of similar size, which could explainits inability to support efficient CSR.

The observation that the non-R-loop forming XSm region served asan efficient substrate for CSR in vivo prompted us to test if this AGCT-rich region is preferentially deaminated by the AID-RPA complexwhen transcribed in vitro. For this, we used a cleavage assay in whichdeamination is demonstrated both by diminution of the template andby the appearance of lower-molecular-weight cleavage products27.Although we noted only very modest deamination of XSm in thepresence of AID alone, there was robust deamination of the XSmregion in either transcriptional orientation in the presence of bothRPA and AID (Fig. 7a). For XSm, deamination was demonstrated bothby the loss of the substrate band and by the appearance of discretecleavage products (Fig. 7a). The presence of discrete cleavage productsin these assays suggests that certain AGCT sites are better targets thanothers, at least in the T7 polymerase deamination assay. The ability ofAID and RPA together, but not AID alone, to deaminate transcribedXSm is reminiscent of what has been noted with transcribed substratesthat do not form R-loops but which are rich in SHM motifs27. Thus,the lack of deamination noted on transcribed XSm with AID alone also

provides additional evidence that XSm does not form R-loops27

(Fig. 7). In keeping with previous studies27, deamination occurredmainly on the nontemplate strand, whereas the template strandunderwent only modest deamination (data not shown). Moreover,in notable recapitulation of the in vivo S-region breakpoints, thepattern of deamination for either substrate correlated well with thelocation of the AGCT-rich sequence in the DNA and with respect toCSR breakpoints in the context of transcriptional orientation (Figs. 6versus 7a). Finally, the Xpf intron (in the reverse orientation) was avery poor substrate relative to XSm for the AID-RPA complex in thisassay; whereas modest deamination was noted in the form of cleavageproducts, there was no readily detectable diminution of the substrateband in the same conditions in which this was noted for XSm (Fig. 7b).Based on these findings, we conclude that the AID-RPA complexprobably targets XSm for CSR in mice.

DISCUSSION

We have taken an evolutionary approach to elucidate aspects of themechanism of CSR. Here we have shown that XSm, which is verydivergent in overall sequence content from mammalian S regions,functions in place of mouse Sg1 to support efficient CSR in mice.Previous work with CSR substrates led to the conclusion that XSmsupports CSR only at greatly reduced efficiency compared with mouseS regions42. The discrepancy between the findings of that previouswork and our study here suggests that such artificial constructs maynot accurately mimic CSR at the endogenous Igh locus. Several studiesof mouse S regions have indicated involvement of transcription-derived S-region R-loops in providing the ssDNA substrate onwhich AID initiates CSR. The ability of mouse S regions to form R-loops derives from their GC-rich structure and the fact that they are Grich on the nontemplate strand. In contrast, XSm, consistent with itsAT-rich content, does not have R-loop-forming ability in vitro, asassessed by both the P1 nuclease and AID-RPA deamination assays.The lack of R-loop-forming ability of XSm but the retention of itsability to support efficient CSR in mice supports the suggestion thatCSR in mammals may also occur through a non-R-loop mechanism7.

As would be predicted on the basis on a non-R-loop mechanism fortargeting CSR, XSm supported CSR similarly in either orientationrelative to transcription from the Ig1 promoter. Moreover, in bothorientations, CSR junctions occurred mainly in a region of XSm thatwas rich in AGCT motifs. Notably, alignment of various S regionsamong amphibians, birds and mammals showed conservation of theAGCT motif, which may well serve as a primordial target sequence forCSR38. As in SHM, AGCT may not be the only functional CSR motif.Unlike the XSm region, the xenopus Sx region, which is downstream ofthe XSm region and mediates CSR to the xenopus Igx, does not containabundant AGCT motifs37. Instead, tetramers such as AGCA andTGCA are well represented37. Additional motifs have also been

Substrate

CleavedDNA

CleavedDNA

AID +

RPA

AID +

RPA

AID +

RPA +

T7

AID +

RPA +

T7

RPA + T

7

AID +

T7

RPA + T

7

AID +

T7

AID +

RPA

AID +

RPA +T7

RPA + T

7

AID +

T7

XSµ+

T7 T7

RPA + T

7

AID +

T7

AID +

RPA +

T7

4.03.0

0.50

2.0

0.751.01.5

kb

T7

XSµ

T7

Xpf–

Substrate4.03.0

0.50

2.0

0.751.01.5

kb

XSµ–

a bFigure 7 The AID-RPA complex deaminates the XSm region in vitro.

(a) Deamination pattern of XSm when transcribed in the physiological

orientation or inverted orientation. The XSm transcription plasmid was

linearized at the 3¢ end of the S region and then transcribed with T7 RNA

polymerase, and was subsequently subjected to DNA deamination in thepresence of RPA and increasing amounts of AID (wedges above lanes).

Reaction products were analyzed by Southern blot with an oligonucleotide

probe (black box) specific for the nontemplate strand of the plasmid.

Data are from one representative of three independent experiments.

The main areas of deamination approximately reside in the AGCT-rich

sequences of XSm (Fig. 6). (b) Deamination assays of XSm and Xpf�

analyzed as described above. Data are from one representative of

two experiments.

NATURE IMMUNOLOGY VOLUME 5 NUMBER 12 DECEMBER 2004 1 27 9

A R T I C L E S©

2004

Nat

ure

Pub

lishi

ng G

roup

ht

tp://

ww

w.n

atur

e.co

m/n

atur

eim

mun

olog

y

identified in chicken38. All of these identified motifs correspond to theaccepted DGYW hotspot sequence for SHM30. Thus, it is possible thatthe DGYW motif, of which AGCT is a variant, may represent theoverall preferred substrate for AID deamination and thus may func-tion in both SHM and CSR. Finally, we note that many of the CSRjunctions, in both XSm and the Xpf intron, were at or very near AGCTmotifs. Therefore, it is possible that deamination of deoxycytidines inthe context of this motif may be a major event in the initiation of CSR,similar to its proposed function in the initiation of SHM15. However,many junctions were also outside the AGCT motif, potentiallyreflecting deamination of deoxycytidine residues outside the AGCTsequence. Still, the finding of junctions outside the AGCT motifs couldbe consistent with AID-mediated cytidine deamination within AGCT(and related motifs) as a major initiating event for CSR. It has beenproposed that processing of AID-initiated lesions by repair pathwayscould extend the lesion and ultimately result in a double-strand DNAbreak at a more distant site43.

All direct experimental evidence suggests that both CSR andSHM are initiated by cytidine deamination in target DNA byAID16–21,24,27,44. In association with RPA, AID deaminates DNA in atranscription-dependent way in vitro, focusing on SHM hotspots27.Combining those observations, we propose that the AGCT motif orother variations of the DGYW tetramer serve(s) as optimal target sitesof the AID-RPA complex. SHM evolved earlier than CSR, as it hasbeen detected in all vertebrates including fish, whereas CSR is limitedto amphibians, birds and mammals36,45. Yet SHM and CSR shareseveral similar features, including the requirement for transcriptionand AID, and we have now provided evidence that the same sequencemotif may function to target AID in both processes. Point mutationsreminiscent of those noted in V genes after SHM are also found atDGYW sequences in S regions, consistent with the idea of a commonAID-targeting mechanism for CSR and SHM33–35. Therefore, CSRmay have evolved from SHM. In more primitive species such asamphibians, S regions consist simply of multimers of SHM motifssuch as AGCT37. In mammals, S regions seem to have further divergedby incorporating additional features, such as an R-loop-formingability, which may serve to increase efficiency of CSR.

The findings of our experiments in which we replaced Sg1 with asimilarly transcribed Xpf intron have several potentially importantimplications. The Xpf intron, which has only a low density of AGCTmotifs, supports only very low CSR relative to the xenopus S region ofsimilar size. Therefore, we can conclude that the ability of a sequenceto function efficiently in CSR is not simply a function of its location ortranscription amount. However, the Xpf intron does support a verylow frequency of CSR. Mutations with characteristics similar toSHM have been detected in several genes outside the Igh locus46,47.In addition, AID-dependent translocations between the c-Myc onco-gene and S-region sequences have been noted48. As the Xpf introndoes contain a low density of AGCT motifs, it is conceivable that thecombination of transcription with sparse hotspot motifs might besufficient to cause infrequent strand breaks through AID in activated Bcells, which could contribute to tumorigenic translocations if theyoccur in regions adjacent to oncogenes such as Bcl6 or Myc.

METHODSGeneration of mutant mice. A 6-kb EcoRI-PstI 5¢ homology arm from an Igh-

containing bacterial artificial chromosome genomic clone was cloned into an

XhoI site of the pLNtk targeting vector. A 4.5-kb EcoRV fragment containing

Cg1 was used as the 3¢ homology arm. The replacement construct for the Xpf

intron was generated by insertion of a 4-kb HindIII-XhoI fragment (accession

number AC004155) of intron 10 upstream of the 3¢ homology arm. The Xpf

intron 10 and flanking sequences were cloned from a l-phage library49. The

replacement construct for XSm was generated by insertion of a 4.1-kb EcoRV

fragment (positions 2,848 to 6,958 of accession number AF0012166) upstream

of the 3¢ homology arm37. These constructs were transfected into an embryonic

stem clone in which Sg1 was deleted7. Correctly targeted clones were detected by

Southern blot with a probe that hybridized downstream of the 3¢ homology

arm. The configuration of the targeted allele was further confirmed by

additional Southern blots with probes that hybridized to the Ig1 and neor

regions. For the deletion of neor through two flanking loxP sites, embryonic

stem clones were infected with recombinant adenovirus that expressed Cre

recombinase. Inversions of the XSm or Xpf sequences were achieved with a third

inverted loxP site. These embryonic stem cell clones were used to generate

chimeric mice through the RAG2-deficient blastocyst complementation system.

Mouse work was approved by the Institutional Animal Care and Use Commit-

tee of Children’s Hospital (Boston, Massachusetts).

Assays for class switching. We used protocols similar to those described

before7. Spleens were from chimeric mice 5–6 weeks old. Splenocytes

were cultured at a density of 1.5 � 106 cells/ml (approximately 0.5 � 106 B

lymphocytes/ml) in RPMI media supplemented with 10% FBS, 2 mM

glutamine, 100 units/ml of penicillin-streptomycin, 100 mM b-mercaptoetha-

nol, 1 mg/ml of anti–mouse CD40 (HM40-3; PharMingen) and 25 ng/ml of

recombinant mouse IL-4 (R&D Systems). Then, 6 d after stimulation, culture

supernatants were collected. The concentration of IgG1a and total IgG1 in the

culture supernatant was measured by ELISA. The allotype-specific monoclonal

antibody Igh-4a (PharMingen) was used to capture IgG1a ; total IgG1 was

captured with goat polyclonal anti-mouse IgG1 (Southern Biotechnology

Associates). In both cases, alkaline phosphatase–conjugated goat anti-mouse

IgG1 (Southern Biotechnology Associates) was used as the visualization anti-

body. Purified mouse IgG1a (PharMingen) was used as the standard.

Hybridomas were generated from splenocytes that had been stimulated for

4 d in vitro with anti-CD40 and IL-4. For each fusion, 20 � 106 splenocytes and

4 � 106 NS1 myeloma cells were used with 50% PEG 1500 (Roche) and

subsequently were grown in 15% FBS, 2 mM glutamine, 100 units/ml of

penicillin-streptomycin, 100 mM b-mercaptoethanol and hypoxanthine-

aminopterin-thymidine (Sigma). Clones that secreted IgG1 were identified by

ELISA. IgG1 allotype was determined by ELISA. As an antibody specific for

IgG1b is not available, hybridomas that produced only IgG1 and not IgG1a

were considered to produce IgG1b. The relative CSR efficiency was calculated

as the ratio of IgG1a-producing hybridomas to IgG1b-producing hybridomas

as described7.

Analyses of CSR junctions. CSR junctions were amplified from hybridomas by

nested PCR. Nested mouse Sm primers were 5¢-CTCTGGCCCTGCTTATTG-

TTG-3¢ followed by 5¢-AGACCTGGGAATGTATGGTT-3¢. The reverse nested

primers were located in exon 1 of Cg1: 5¢-CAATTTTCTTGTCCACCTTGGTG-

CTG-3¢ followed by 5¢-GTGTGCACACCGCTGGACAGG-3¢. PCR products

were sequenced directly or after cloning as described50. Switch junctions were

analyzed with the SeqMan program of DNASTAR Lasergene and the Mega-

BLAST program of the National Center for Biotechnology Information.

R-loop assay. XSm was cloned into the same vector used before to show R-loop

formation in mouse S regions28. In vitro transcription and P1 nuclease assays

were done as described28. In vitro transcription reactions were carried out at

37 1C for 15 min in 40 mM Tris HCl, pH 8.0, 6 mM MgCl2, 10 mM DTT,

4 mM spermidine, 10 mM NaCl, 1 mM of each rNTP, 15 units of T7 RNA

polymerase and 100 ng of linearized plasmid in a volume of 20 ml. After phenol

extraction and precipitation, nuclease P1 digestion was carried out in 50 mM

sodium acetate, pH 5.5, 200 mM NaCl, 1 mM ZnSO4 and 25 ng of DNA. For

each 20-ml reaction, 1 � 10 to 1 � 105 pg P1 nuclease was used. The P1

reactions were incubated at 37 1C for 15 min. After RNase treatment and

precipitation, the DNA was incubated at 50 1C for 1 h in 50% dimethyl

sulfoxide, 1 M glyoxal, 10 mM sodium phosphate, pH 7.0, 0.5 mM EDTA and

1 mg sonicated salmon sperm DNA. Samples were separated by electrophoresis

through 1% agarose gels containing 10 mM sodium phosphate, pH 7.0, and

0.5 mM EDTA. Subsequently, the Southern blot was probed with template-

specific or nontemplate-specific digonucleotides.

1 28 0 VOLUME 5 NUMBER 12 DECEMBER 2004 NATURE IMMUNOLOGY

A R T I C L E S©

2004

Nat

ure

Pub

lishi

ng G

roup

ht

tp://

ww

w.n

atur

e.co

m/n

atur

eim

mun

olog

y

DNA deamination assay. DNA deamination was done on substrates that

contained a T7 RNA polymerase promoter upstream of the target DNA as

desribed27. DNA was transcribed with T7 RNA polymerase (Promega) in the

presence (or absence) of purified activated B cell AID (50–150 ng) and

recombinant RPA (200 ng) in a buffer containing 20 mM Tris HCl, pH 8.0,

100 mM NaCl, 1 mM dithiothreitol and 10 mM ZnCl2. After incubation at

30 1C for 1 h, samples were incubated with recombinant uracil glycosylase

(New England Biolabs) for 30 min at 37 1C and then treated with 0.1 M NaOH

at 90 1C for 10 min. Reaction products were denatured in the presence of 50%

dimethyl sulfoxide and 1 M glyoxal, separated by electrophoresis through 0.8%

agarose gels containing 10 mM sodium phosphate, pH 7.0, and analyzed by

Southern blot with an oligonucleotide complementary to T7 RNA polymerase

promoter as the probe.

Note: Supplementary information is available on the Nature Immunology website.

ACKNOWLEDGMENTSWe thank Y. Fujiwara, T. Borjeson and A. Williams for mouse work, andJ. Manis, R. Shinkura, C. Giallourakis, E. Pinaud, J. Wang and S. Ranganath fordiscussions. Supported by National Institutes of Health (AI31541 to F.W.A. andAI07512 to M.T.), National Cancer Institute of Canada (A.A.Z.) and HowardHughes Medical Institute (F.W.A.).

COMPETING INTERESTS STATEMENTThe authors declare that they have no competing financial interests.

Received 20 September; accepted 21 October 2004

Published online at http://www.nature.com/natureimmunology/

1. Jung, D. & Alt, F.W. Unraveling V(D)J recombination; insights into gene regulation.Cell 116, 299–311 (2004).

2. Martin, A. & Scharff, M.D. AID and mismatch repair in antibody diversification.Nat. Rev. Immunol. 2, 605–614 (2002).

3. Reynaud, C.A., Aoufouchi, S., Faili, A. & Weill, J.C. What role for AID: mutator, orassembler of the immunoglobulin mutasome? Nat. Immunol. 4, 631–638 (2003).

4. Manis, J.P., Tian, M. & Alt, F.W. Mechanism and control of class-switch recombination.Trends Immunol. 23, 31–39 (2002).

5. Honjo, T., Muramatsu, M. & Fagarasan, S. AID: how does it aid antibody diversity?Immunity 20, 659–668 (2004).

6. Luby, T.M., Schrader, C.E., Stavnezer, J. & Selsing, E. The m switch region tandemrepeats are important, but not required, for antibody class switch recombination.J. Exp. Med. 193, 159–168 (2001).

7. Shinkura, R. et al. The influence of transcriptional orientation on endogenous switchregion function. Nat. Immunol. 4, 435–441 (2003).

8. Khamlichi, A.A. et al. Immunoglobulin class-switch recombination in mice devoid ofany S m tandem repeat. Blood 103, 3828–3836 (2004).

9. Dunnick, W., Hertz, G.Z., Scappino, L. & Gritzmacher, C. DNA sequences atimmunoglobulin switch region recombination sites. Nucleic Acids Res. 21, 365–372(1993).

10. Revy, P. et al. Activation-induced cytidine deaminase (AID) deficiency causes theautosomal recessive form of the hyper-IgM syndrome (HIGM2). Cell 102, 565–575(2000).

11. Muramatsu, M. et al. Class switch recombination and hypermutation require activ-ation-induced cytidine deaminase (AID), a potential RNA editing enzyme. Cell 102,553–563 (2000).

12. Doi, T., Kinoshita, K., Ikegawa, M., Muramatsu, M. & Honjo, T. De novo proteinsynthesis is required for the activation-induced cytidine deaminase function in class-switch recombination. Proc. Natl. Acad. Sci. USA 100, 2634–2638 (2003).

13. Begum, N.A. et al. De novo protein synthesis is required for activation-induced cytidinedeaminase-dependent DNA cleavage in immunoglobulin class switch recombination.Proc. Natl. Acad. Sci. USA 101, 13003–13007 (2004).

14. Begum, N.A. et al. Uracil DNA glycosylase activity is dispensable for immunoglobulinclass switch. Science 305, 1160–1163 (2004).

15. Chaudhuri, J. & Alt, F.W. Class-switch recombination: interplay of transcription, DNAdeamination and DNA repair. Nat. Rev. Immunol. 4, 541–552 (2004).

16. Petersen-Mahrt, S.K., Harris, R.S. & Neuberger, M.S. AID mutates E. coli suggestinga DNA deamination mechanism for antibody diversification. Nature 418, 99–103(2002).

17. Bransteitter, R., Pham, P., Scharff, M.D. & Goodman, M.F. Activation-induced cytidinedeaminase deaminates deoxycytidine on single-stranded DNA but requires the actionof RNase. Proc. Natl. Acad. Sci. USA 100, 4102–4107 (2003).

18. Sohail, A., Klapacz, J., Samaranayake, M., Ullah, A. & Bhagwat, A.S. Humanactivation-induced cytidine deaminase causes transcription-dependent, strand-biasedC to U deaminations. Nucleic Acids Res. 31, 2990–2994 (2003).

19. Chaudhuri, J. et al. Transcription-targeted DNA deamination by the AID antibodydiversification enzyme. Nature 422, 726–730 (2003).

20. Dickerson, S.K., Market, E., Besmer, E. & Papavasiliou, F.N. AID mediates hypermuta-tion by deaminating single stranded DNA. J. Exp. Med. 197, 1291–1296 (2003).

21. Ramiro, A.R., Stavropoulos, P., Jankovic, M. & Nussenzweig, M.C. Transcriptionenhances AID-mediated cytidine deamination by exposing single-stranded DNA onthe nontemplate strand. Nat. Immunol. 4, 452–456 (2003).

22. Yu, K., Huang, F.T. & Lieber, M.R. DNA substrate length and surrounding sequenceaffect the activation-induced deaminase activity at cytidine. J. Biol. Chem. 279,6496–6500 (2004).

23. Di Noia, J. & Neuberger, M.S. Altering the pathway of immunoglobulin hypermutationby inhibiting uracil-DNA glycosylase. Nature 419, 43–48 (2002).

24. Rada, C. et al. Immunoglobulin isotype switching is inhibited and somatic hypermuta-tion perturbed in UNG-deficient mice. Curr. Biol. 12, 1748–1755 (2002).

25. Imai, K. et al. Human uracil-DNA glycosylase deficiency associated with profoundlyimpaired immunoglobulin class-switch recombination. Nat. Immunol. 4, 1023–1028(2003).

26. Nambu, Y. et al. Transcription-coupled events associating with immunoglobulin switchregion chromatin. Science 302, 2137–2140 (2003).

27. Chaudhuri, J., Khuong, C. & Alt, F.W. Replication protein A interacts with AID topromote deamination of somatic hypermutation targets. Nature 430, 992–998 (2004).

28. Tian, M. & Alt, F.W. Transcription-induced cleavage of immunoglobulin switch regionsby nucleotide excision repair nucleases in vitro. J. Biol. Chem. 275, 24163–24172(2000).

29. Yu, K., Chedin, F., Hsieh, C.L., Wilson, T.E. & Lieber, M.R. R-loops at immunoglobulinclass switch regions in the chromosomes of stimulated B cells. Nat. Immunol. 4,442–451 (2003).

30. Rogozin, I.B. & Diaz, M. Cutting edge: DGYW/WRCH is a better predictor of mutabilityat G:C bases in Ig hypermutation than the widely accepted RGYW/WRCY motif andprobably reflects a two-step activation-induced cytidine deaminase-triggered process.J. Immunol. 172, 3382–3384 (2004).

31. Betz, A.G., Neuberger, M.S. & Milstein, C. Discriminating intrinsic and antigen-selected mutational hotspots in immunoglobulin V genes. Immunol. Today 14, 405–411 (1993).

32. Binz, S.K., Sheehan, A.M. & Wold, M.S. Replication protein A phosphorylation and thecellular response to DNA damage. DNA Repair (Amst) 3, 1015–1024 (2004).

33. Petersen, S. et al. AID is required to initiate Nbs1/g-H2AX focus formation andmutations at sites of class switching. Nature 414, 660–665 (2001).

34. Nagaoka, H., Muramatsu, M., Yamamura, N., Kinoshita, K. & Honjo, T. Activation-induced deaminase (AID)-directed hypermutation in the immunoglobulin Sm region:implication of AID involvement in a common step of class switch recombination andsomatic hypermutation. J. Exp. Med. 195, 529–534 (2002).

35. Zeng, X., Negrete, G.A., Kasmer, C., Yang, W.W. & Gearhart, P.J. Absence of DNApolymerase {eta} reveals targeting of C mutations on the nontranscribed strand inimmunoglobulin switch regions. J Exp. Med. 199, 917–924 (2004).

36. Du Pasquier, L. The immune system of invertebrates and vertebrates. Comp. Biochem.Physiol. B Biochem. Mol. Biol. 129, 1–15 (2001).

37. Mussmann, R., Courtet, M., Schwager, J. & Du Pasquier, L. Microsites for immunoglo-bulin switch recombination breakpoints from Xenopus to mammals. Eur. J. Immunol.27, 2610–2619 (1997).

38. Kitao, H., Arakawa, H., Yamagishi, H. & Shimizu, A. Chicken immunoglobulin m-chaingene: germline organization and tandem repeats characteristic of class switch recom-bination. Immunol. Lett. 52, 99–104 (1996).

39. Cogne, M. et al. A class switch control region at the 3¢ end of the immunoglobulin heavychain locus. Cell 77, 737–747 (1994).

40. Seidl, K.J. et al. An expressed neor cassette provides required functions of the 1g2bexon for class switching. Int. Immunol. 10, 1683–1692 (1998).

41. Sternberg, N. & Hoess, R. The molecular genetics of bacteriophage P1. Annu. Rev.Genet. 17, 123–154 (1983).

42. Tashiro, J., Kinoshita, K. & Honjo, T. Palindromic but not G-rich sequences are targetsof class switch recombination. Int. Immunol. 13, 495–505 (2001).

43. Bardwell, P.D. et al. Altered somatic hypermutation and reduced class-switch recom-bination in exonuclease 1-mutant mice. Nat. Immunol. 5, 224–229 (2004).

44. Petersen-Mahrt, S.K. & Neuberger, M.S. In vitro deamination of cytosine to uracil insingle-stranded DNA by apolipoprotein B editing complex catalytic subunit 1 (APO-BEC1). J. Biol. Chem. 278, 19583–19586 (2003).

45. Flajnik, M.F. Comparative analyses of immunoglobulin genes: surprises and portents.Nat. Rev. Immunol. 2, 688–698 (2002).

46. Pasqualucci, L., Neri, A., Baldini, L., Dalla-Favera, R. & Migliazza, A. BCL-6 mutationsare associated with immunoglobulin variable heavy chain mutations in B-cell chroniclymphocytic leukemia. Cancer Res. 60, 5644–5648 (2000).

47. Kuppers, R. & Dalla-Favera, R. Mechanisms of chromosomal translocations in B celllymphomas. Oncogene 20, 5580–5594 (2001).

48. Ramiro, A.R. et al. AID is required for c-myc/IgH chromosome translocations in vivo.Cell 118, 431–438 (2004).

49. Tian, M., Shinkura, R., Shinkura, N. & Alt, F.W. Growth retardation, early death, andDNA repair defects in mice deficient for the nucleotide excision repair enzyme XPF.Mol. Cell Biol. 24, 1200–1205 (2004).

50. Dudley, D.D. et al. Internal IgH class switch region deletions are position-independentand enhanced by AID expression. Proc. Natl. Acad. Sci. USA 99, 9984–9989 (2002).

NATURE IMMUNOLOGY VOLUME 5 NUMBER 12 DECEMBER 2004 1 28 1

A R T I C L E S©

2004

Nat

ure

Pub

lishi

ng G

roup

ht

tp://

ww

w.n

atur

e.co

m/n

atur

eim

mun

olog

y