DNA Acrobats of the Ig Class Switch
Clifford L. Wang and Matthias Wabl
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Copyright © 2004 by The American Association of
Immunologists All rights reserved.
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References
J Immunol 2004; 172:5815-5821; ;
doi: 10.4049/jimmunol.172.10.5815
http://www.jimmunol.org/content/172/10/5815
OF
THE
JOURNAL IMMUNOLOGY
BRIEF REVIEWS
DNA Acrobats of the Ig Class Switch1
Clifford L. Wang and Matthias Wabl 2
mmature B lymphocytes mature into small resting B lymphocytes with an Ag receptor of the IgM class. Without
prior interaction with Ag, B cells join gene segments to
create the Ag-binding Ig V region. Because numerous gene segments can be joined in numerous different combinations, the
naive B cells are able to create a sizable repertoire of Abs. Yet the
Abs of this primary repertoire are of a lesser quality; it seems
reasonable that in any large repertoire not aimed at a particular
Ag, low-affinity Abs should far outnumber high-affinity Abs.
However, the low affinity is in part offset by the higher avidity
of the IgM Abs that are secreted by the plasma cells derived from
the activated B lymphocytes. In the secreted IgM, the prototypical four-chain Ig structure is combined into pentamers or hexamers. In this case, the trade-off for higher avidity is a clumsy
structure that does not penetrate the entire organism; particularly, IgM does not pass the placenta and thus cannot protect
the fetus. Such protection depends on a switch from IgM to IgG
during the course of an immune response. In more general
terms, switching to other Ig classes serves to distribute a particular V region onto different effector functions and guide it to
different sites in the organism. Some 30 years ago, it was shown
I
that class switching occurs within a committed (i.e., surface Ag
receptor-expressing) lymphocyte (1–3), which in turn required
an explanation for how a given V region could be grafted onto
various C regions at the genetic level. The basic principles of
how this happens have been elucidated over the last three decades, and the biochemical details are now becoming clearer.
As mentioned above, the primary Ab repertoire is manifested
in IgM Abs, and cooperative binding by the 10 or 12 identical
Ag combining sites presumably counterbalance low affinity to
some extent. However, because the other secreted Ig classes
have only two (IgG, IgA, IgE) or four (IgA) combining sites,
this compensation is lost when the IgM H chain switches to the
other classes. Here, affinity maturation comes to rescue, and Ab
quality improves, often dramatically, upon exposure to Ag.
First, the clones of the primary repertoire with higher affinity to
the Ag are selected and induced to proliferate. During this proliferation, mutations arise in the Ab V regions at a rate that is a
million-fold higher than the normal, spontaneous mutation
rate (somatic hypermutation). If the mutations result in Abs of
even higher affinity, these clones are then selected, and they
proliferate further. The temporal connection between class
switching and hypermutation is also a mechanistic one, because
both are initiated by activation-induced cytidine deaminase
(AID)3 (4 – 6) and mediated, in part, by uracil-DNA glycosylase (UNG) (7, 8). Although this review will focus on class
switching, it will take some key observations from the hypermutation process to shed light on the mechanism of class
switching.
Basic requirements for the DNA acrobatics
Soon after it was recognized that the joining of V region gene
segments requires DNA deletion, it was discovered that the
genes encoding the H chain isotypes other than ␮ or ␦ are also
generated by joining and deletion (9). This deletion occurs between so-called switch (S) regions—repetitive sequences 5⬘ of
the constant (C) gene segments (10). These C gene segments are
arranged in a linkage group downstream of the V exon. During
switch recombination, the C␮ (which encodes the C region of the
H chain of IgM) is replaced by the C region of another isotype. A
priori, such a recombination process must be mediated by doublestrand breaks that are subsequently ligated (repaired).
Department of Microbiology and Immunology, University of California, San Francisco,
CA 94143
tional Institute of Allergy and Infectious Diseases Immunology Training Grant T32
AI07334.
Received for publication March 8, 2004. Accepted for publication March 22, 2004.
2
Address correspondence and reprint requests to Dr. Matthias Wabl, Department of Microbiology and Immunology, University of California, San Francisco, CA 94143-0414.
E-mail address: [email protected]
The costs of publication of this article were defrayed in part by the payment of page charges.
This article must therefore be hereby marked advertisement in accordance with 18 U.S.C.
Section 1734 solely to indicate this fact.
1
This work was supported by National Institutes of Health Grant AG20684. C.L.W. was
supported by the Leukemia Research Foundation and National Institutes of Health/NaCopyright © 2004 by The American Association of Immunologists, Inc.
3
Abbreviations used in this paper: AID, activation-induced cytidine deaminase; UNG,
uracil-DNA glycosylase; APOBEC1, apolipoprotein B mRNA-editing catalytic polypeptide 1; AT, ataxia telangiectasia; S region, switch region.
0022-1767/04/$02.00
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Small resting B lymphocytes all start out producing IgM
Abs. Upon encountering Ag, the cells become activated
and make a switch from IgM to other Ig classes. This class
switch serves to distribute a particular V region to different Ig C regions. Each C region mediates a specialized effector function, and so, through switching, an organism
can guide its Abs to various sites. Creating the new H
chain requires loop-out and deletion of DNA between
switch regions. These DNA acrobatics require transcription of the switch regions, presumably so that necessary
factors can gain access to the DNA. These requisite switching factors include activation-induced cytidine deaminase
and components of general DNA repair, including base
excision repair, mismatch repair, and double-strand
break repair. Despite much recent progress, not all important factors have been discovered, especially those that may
guide recombination to a particular subclass. The Journal of Immunology, 2004, 172: 5815–5821.
5816
BRIEF REVIEWS: DNA ACROBATS OF THE Ig CLASS SWITCH
DNA loop-out
Holding together the S regions
Although the looping of the DNA between S regions could occur by the spontaneous, random motion of the DNA, we and
others have sought more active switching components. Here
the formation of the switch loop could be facilitated by components of a switch recombinase system. Such a system would
FIGURE 1. Three outcomes of the DNA loop-out,
cutting, and ligation mechanism of Ig class switch. The
DNA conformation is from the mouse Ig locus. Rectangles, V or C region exons; ovals, S regions. Yellow/
blue dual-tone ovals represent the ligation junction between two different S regions— here between S␮ and
S␥2b. Green circles represent factors that promote the
DNA loop-out. Inversion abolishes H chain expression
because of the reversed orientation. Only deletion allows
expression of the ␥2b chain. See also Ref. 11.
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When the DNA is cut at two places this leaves four new ends.
These ends may be rejoined in three different ways. One restores the original configuration. Another way leads to an inversion of the DNA between the cuts. The third way leads to
deletion; the intervening deleted sequence ligates its ends to
form a circle (switch circle), which is lost. The remaining ends
are rejoined, grafting a different C region with the extant V region. If the ends were free and not in proximity to each other
when the cuts are rejoined, then circular religation (a unimolecular reaction) and deletion should be highly favored over inversion (requiring bimolecular reaction). But because inversions are found almost as frequently as switch deletions (11),
this implicates more advanced DNA acrobatics. We and others
have predicted the existence of switch loops (11–14) where the
DNA ends are in proximity before cutting and rejoining (Fig.
1). Looping-out could occur after or before the first cut, but
needs to precede the second cut in order for the inversion scenario to occur. Because a switch recombination event requires
two breaks separated by long distances (up to 175 kb), a looping
mechanism likely would make the rejoining of the chromosomal ends efficient.
be able to recognize and bring together the S region of ␮ (S␮)
and the S region of the future isotype. Although the repetitive
sequences of the S regions share some homology, the presence
of inversions indicates that switch recombination is not mediated by homologous recombination. The fact that inversions
between the S regions can occur without homology at the joining sites (11, 15) further implicates nonhomologous
recombination.
Thus, switch recombination uses components of the nonhomologous recombination pathway for general DNA repair.
Clearly involved in the switch process is the histone H2AX (16).
In its phosphorylated form, it helps prevent aberrant repair of
both programmed and general DNA breakage by recruiting the
Mre11-Rad50-Nbs1 complex to the repair foci. Mice that lack
either one or both H2AX alleles in the concomitant absence of
p53 develop B lineage lymphomas with chromosome 12
(IgH)/15 (c-myc) translocations, with hallmarks of either aberrant V(D)J or class switch recombination (17) (however, H2AX
is not needed for hypermutation (16)). But, although B cells
lacking H2AX show impaired authentic class switch recombination, the frequency of intraswitch region deletions is normal
(16). This indicates that lesions and ligation are normal, but
long-distance joining is not. Thus, histone H2AX may facilitate
synapsis by chromatin remodeling.
Another candidate for attachment to the S regions and loop
formation are the Ku70 and Ku80 proteins of the nonhomologous end joining repair pathway, because both are required for
switch recombination (18, 19). Working together as a heterodimer (20), Ku70/Ku80 binds to ss- and dsDNA ends. It has
The Journal of Immunology
been suggested that Ku70/Ku80 can bind to some DNA sequences without open ends (21). Perhaps the (predicted only)
proclivity to form hairpin-like structures avails the S regions to
Ku70/Ku80 or other factors binding (21) before DNA cuts occur. These secondary structures would occur within a particular
S region; it is different from the looping out of dsDNA, which
is deleted as a result of a completed recombination process. So
we can imagine that one or more proteins, perhaps Ku70/
Ku80, bind to S␮ and to another S region and bring them into
close proximity. This might occur with (22) or without (23, 24)
the help of DNA-PKcs. In conventional (nonhomologous)
double-strand repair, DNA-PKcs is a DNA-dependent phosphokinase that is recruited by Ku70/80. For class switching,
however, there is no absolute requirement for DNA-PKcs (see
Ligating DNA ends).
Exposure of ssDNA by transcription
particular, switching requires synthesis of a germline transcript
that is composed of the I exon, the intron containing the S region, and the C region gene segments (Fig. 2A). When S␣ is
transcribed in the physiological direction in vitro, stable RNADNA structures could form, and the newly synthesized RNA
molecule would remain associated with the S region DNA template strand (30, 31). In vivo, the so-called R-loop (Fig. 2B) can
exceed 1 kb in length (32). This would expose the nontemplate
DNA strand for putative switch recombinase factors. However,
for class switching to occur, not only must there be transcription from the I exon promoter, but also the transcript must be
spliced (33, 34). Perhaps splicing factors need to be recruited to
the S regions (35). Alternatively, the spliced transcript could
hybridize to the S region DNA, allowing it to form a ssDNA
switch bubble (Fig. 2C). It is also possible that the spliced transcript interacts directly with AID. It would be somewhat surprising if a minimal R-loop model (i.e., a transcription bubble)
would explain the great efficiency of switching after B cell activation. If only a transcription bubble would be needed, perhaps
many other transcribed genes might recombine.
Introducing the cuts
The switch recombination breakpoints in frogs (36) and mice
(37) are usually at positions (microsites) where a ssDNA folding
program predicts the transition from a stem to a loop structure
FIGURE 2. Exposing ssDNA at the S region. A, DNA composed of V region, I promoter (arrow), I exon, S region, and C region. I promoter-transcribed RNA,
a colored line composed of the I and C exons (blue) and the large intron (orange). B, Stable R-loop model. GAGCTGAGCTG is a common repeated nucleotide
motif in the S region highly susceptible to AID-mediated DNA cutting. See also Ref. 32. C, Switch bubble model. Spliced mRNA transcript consisting of I and C
exons, blue line.
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The intrastrand secondary structure by itself may not be sufficient for the S region to be a target of the recombinase. As with
hypermutation, switching requires transcription (Fig. 2) (Refs.
25–27; reviewed in Ref. 28). This is in line with the activity of
AID, which apparently requires ssDNA exposed by transcription or by specific binding proteins. This requirement was also
demonstrated by the transcription-dependent recombination
of an exogenous substrate in AID-producing fibroblasts (29). In
5817
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BRIEF REVIEWS: DNA ACROBATS OF THE Ig CLASS SWITCH
FIGURE 3. AID-mediated DNA cutting at the S region. See also Ref. 96.
shown that AID introduces C-to-U mutations at the nontemplate strand only (47). This means that nicks would be introduced in the upper (reading) strand only, and no double-strand
breaks could be formed without spontaneous nicking on the
template strand. Also, exonuclease activity around the nick
would not help under those circumstances—the other strand
would simply not be nicked. However, it is possible that repair
is subverted in that it works on the nonmutated rather than on
the mutated strand, which would introduce the lesions on the
other strand. A similar role of mismatch repair has been suggested to contribute to hypermutation (58). At any rate, the
preferred AID target of a G on the reading (upper, nontemplate) strand embedded in the RGYW in hypermutation is not
consistent with C being targeted in the nontemplate strand.
Nor is the fact that RGYW apparently can be targeted on both
strands in cultured cells (59 – 61) and in the whole animal (62).
But what if AID edits RNA?
It is also known that the requirements for AID structure on class
switching are greater than on hypermutation. If mutated, the
C-terminal end of AID still can mediate hypermutation, but
not class switching (63, 64). Apparently, the C-terminal region
needs to bind another molecule, which may be another protein,
but also DNA, or RNA. There are also cell lines that express
endogenous AID, but do not switch extrachromosomal switch
substrates (65); and there is another one that does not switch or
hypermutate the endogenous ␮ gene.4 Finally, mitogen-stimulated B cells switch but do not hypermutate their Ig genes (66).
In the work of Ito et al. (67), it has been proposed that AID,
in addition to working on DNA, also edits a specific RNA. At
the moment, however, there is no experimental evidence that
AID can in fact act on RNA, let alone any evidence that it does
act on RNA physiologically. But it is quite interesting that AID
4
F. J. X. Spillmann and M. Wabl. Endogenous AID expression in cell line WEHI-231.
Submitted for publication.
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within the S region. It seems clear that AID binds to the chromatin at the S region (38) and, in conjunction with UNG (7, 8,
39) and yet-to-be-identified apyrimidinic endonuclease(s), introduces nicks into DNA (Fig. 3). This model suggests how
AID cuts DNA—many nicks in close proximity would lead to
the eventual double-stranded break (40, 41), which are staggered (42). Indeed, AID is required for DNA cleavage in switch
recombination (43), although it has also been concluded that
AID functions in a postcleavage step, at least in the somatic hypermutation process (44). Specifically, AID may (4) or may not
(45) deaminate free cytidine, but it converts also deoxycytidine
into deoxyuridine in DNA (41, 46 – 49). The noncanonical
DNA base deoxyuridine, in turn, may be removed by UNG (7,
8), thereby creating an abasic site on one DNA strand. This site
then may be attacked by an apurinic apyrimidinic endonuclease, which creates the ssDNA breaks.
In the hypermutation process, when one analyzes DNA sequences, it appears as if AID preferentially attacks a G on the
reading strand, embedded in an RGYW motif (with R representing a purine; G, deoxyguanidine; Y, a purine; and W, either
A or T) (50, 51). In fact, we know that AID attacks a C in
ssDNA (41, 46 – 49); therefore, the G in the short motif marks
the site of attack of the C on the other DNA strand. Because S
regions have many repeats of AGCT—a palindromic RGYW
motif—there will be many mutations (16), and, as a consequence, many nicks introduced into the S region DNA. Moreover, the G䡠U mismatch may be dealt with by mismatch repair,
which removes longer stretches of ssDNA around the mismatch
by an exonuclease (52) (Fig. 3); if this stretch happens to have a
single nick on the other strand, a double-strand break would
also result. In fact, components of the mismatch repair system
are involved in the switch process (53–57). Without them,
there is less switching; and the recombination breakpoints are
more likely to occur in consensus motifs.
The sequence of events described above do not quite fit all of
the characteristics ascribed to AID. Some experiments have
The Journal of Immunology
Ligating DNA ends
After the appropriate cuts are made, the open DNA ends need
to be ligated, i.e., the lesions need to be repaired. Indicative of
an involvement of the Mre11-Rad50-Nbs1 complex, B cells
from patients with Mre11 deficiency (ataxia telangiectasia
(AT)-like disorder) switch less efficiently, and the switch recombination junctions are aberrant (70). In the main pathway
for nonhomologous end joining, the phosphokinase DNAPKcs, after it has been recruited by Ku70/80, phosphorylates
itself and Ku70/80, and colocalizes at DNA damage foci with
other repair proteins such as H2AX and 53BP1 (71). The Ku/
DNA-PKcs complex also recruits and phosphorylates XRCC4
and ligase IV (reviewed in Ref. 20). The requirement for DNAPKcs in switch recombination is not absolutely clear. On the
one hand, it seems natural that, with Ku70/80 being strictly
needed, DNA-PKcs would follow. In cultured cells, such a requirement was reported (22). Yet, in the mouse, this requirement is not strict (23), particularly not for the IgG1 isotype
(24). Perhaps ATM (reviewed in Ref. 72), the protein mutated
in AT, can replace DNA-PKcs (73). ATM is another important
protein kinase in the double-strand repair pathway; and AT patients suffer from immunodeficiency. Whatever the exact requirements for kinases, in the end, ligase(s) need to join the
open ends to generate the complete switched gene, in which the
V exon is now in close proximity to the C region gene segment
of the new isotype. To date, the identity of the ligases required
are not known, although there are indications that ligase IV is
among them (74).
Guiding the switch recombinase
At the cellular level, B cells need input from Th cells in order for
them to switch their Ig isotype (reviewed in Ref. 75). There is a
large body of evidence of how different lymphokines instruct
the switch recombinase system to a particular isotype (reviewed
in Ref. 28). The lymphokines are synthesized by the Th cells,
which in turn have been directed to differentiate into Th1 or
Th2 cells by lymphokines synthesized by professional APCs.
Different types of pathogen are handled by different APCs,
which in turn synthesize different lymphokines. This is why in
the end, the pathogen instructs the isotype of the Ab. However,
the question remains as to how the lymphokines accomplish
this. Obviously, as they bind to the appropriate receptor on the
surface of the cell that is to switch Ig isotype, a signaling cascade
is initiated that ultimately leads to transcription off the I region
promoter (reviewed in Ref. 28). But is this and AID all that is
needed to set off the switch process? In other words, are there no
S region-specific factors needed (76)? S regions do differ among
each other (77), and they differ more from one S region to another than between the same S region of different species (78,
79). This may indicate other requirements (65, 80), in addition
to transcription and subsequent splicing. Various factors that
bind to S regions have been reported; however, none of these
factors are expressed specifically in switching B lymphocytes.
Some of these factors are well characterized in processes other
than switch recombination. They include Pax-5/B cell-specific
activating protein (reviewed in Ref. 81), LR1 (82), NF-␬B/p50
(83, 84), switch nuclear A-site protein (includes E47) (85, 86),
and S␮BP-2 (87). Among them, NF-B/p50, Pax-5, and E47
have also been shown to affect levels of Ig switching, with the E
proteins directly regulating expression of AID (88). Recently, a
ubiquitously expressed, DNA-binding, late SV40 factor has
been reported to bind S regions and to repress class switching to
IgA (89). However, in general, the direct role of these factors in
the switch recombination process has yet to be integrated. But,
clearly, there may be more factors needed than the well-studied
ones. Indeed, there are hyper-IgM syndrome patients with a selective defect that has not been attributed to any of the known
genetic defects (90). In some of these patients, the defect might
lie in survival signals delivered to switched B cells, but in others,
switching might be impaired.
Concluding remarks
The S regions seem to be particularly good targets for recombination because of their highly repeated WRCY (RGYW on the
complementary strand) sequence motif, where an embedded C
can be a target of AID. Apart from transcription, subsequent
splicing of the germline transcript is necessary in order for the
switch recombination to proceed. No such specific transcript
has been identified for hypermutation of the V region—a process that shares with class switching the requirement for AID,
UNG, and mismatch repair proteins. It almost seems as if hypermutation at the V region is a byproduct of the class switching; with affinity maturation as a welcome afterthought when
the switch process leads to the loss of avidity. Although hypermutation results in a much higher frequency of mutations, the
intrinsic rate, with 10⫺3 to 10⫺4 per base pair per cell generation (91–93), pales in comparison to another mutation process
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is a nucleocytoplasmic shuttling protein with a bipartite nuclear
localization signal and a nuclear export signal (67), although it
is predominantly found in the cytoplasm when tagged by green
fluorescent protein (68). It has been suggested that AID may
have mechanisms similar to those of apolipoprotein B mRNAediting catalytic polypeptide 1 (APOBEC1) (67). According to
this hypothesis, edited mRNA (encoding an unknown polypeptide) in the AID-cofactor complex is transported to the cytoplasm for translation. The amino acid sequence of AID is somewhat similar to APOBEC1, and when AID was initially
discovered, it was suggested that AID would also be an mRNAediting enzyme. If so, it could prove wrong most of the DNA
cutting pathway proposed earlier in this review. With the RNAediting mechanism of APOBEC1, this cytidine deaminase introduces a premature stop codon into apolipoprotein B100
mRNA. The majority of these edited mRNA molecules is exported
to the cytoplasm as a complex associated with APOBEC1 and a
cofactor, APOBEC1 complementation factor, to avoid nonsensemediated decay. Nonsense-mediated decay affects mRNAs with
premature stop codons, presumably to avoid translation of short
polypeptides that would act as dominant-negative factors. The
similarity between AID and APOBEC1 activity might hinge on
the inhibition of nonsense-mediated decay, which works quite
efficiently in B lymphocytes (69). The I exon of the germline
transcripts contains stop codons in all three frames. Without protection from nonsense-mediated decay, there would not be a sufficient quantity of transcripts to form the switch bubble. Perhaps
AID and the putative cofactor need to bind to the spliced germline
transcript. Interestingly, when synthesized in insect cells, AID is
bound to RNA, which needs to be removed by RNase for AID to
exhibit its enzymatic activity (48). Presumably, nonspecific insect
mRNA blocks binding of specific nucleic acid. However, the fact
that spliced germline transcripts cannot be supplied in trans (33,
34) would speak against the course of events outlined above.
5819
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BRIEF REVIEWS: DNA ACROBATS OF THE Ig CLASS SWITCH
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abundance of RGYW target motifs in the S regions.
The interest in the basic mechanism of Ig class switching is
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relatively common. Even more important is IgE as the primary
culprit in immediate type of hypersensitivity. IgE-secreting
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to generate the gene encoding the ⑀-chain of IgE may provide
the basis for a simple screening test, in which chemicals are assayed for their potential to bind to the factor and, thereby, to
inhibit its function. Chemicals that inhibit the factor will inhibit the generation of IgE, and thus have the potential to prevent an allergic reaction to any substance.
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