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Transcription-Dependent Somatic
Hypermutation Occurs at Similar Levels on
Functional and Nonfunctional Rearranged
IgH Alleles
Laurent Delpy, Christophe Sirac, Caroline Le Morvan and
Michel Cogné
J Immunol 2004; 173:1842-1848; ;
doi: 10.4049/jimmunol.173.3.1842
http://www.jimmunol.org/content/173/3/1842
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References
The Journal of Immunology
Transcription-Dependent Somatic Hypermutation Occurs at
Similar Levels on Functional and Nonfunctional Rearranged
IgH Alleles1
Laurent Delpy,2 Christophe Sirac,2 Caroline Le Morvan, and Michel Cogné3
G
eneration of the Ab repertoire in mammalians requires
several steps of gene remodeling which finally allow the
selection of cells producing a clonally restricted, single
sort of Ab. During establishment of the primary repertoire in pro-B
cells, DJ rearrangement first occurs on both alleles while by contrast, functional VDJ rearrangement on a single allele later terminates primary recombination of IgH loci. Secondary rearrangements occurring in immature B lymphocytes seem to mostly target
the functionally rearranged allele while accessibility of the excluded allele would be down-regulated (1). Evidence has even
been provided in the mouse that differential accessibility of both
IgH chain loci may be established before entry into the B cell
development pathway, as early as day 12 of the embryonic life (2).
Differences in the methylation status of alleles have been reported
and correlated with asymmetry of both their replication patterns
and their nuclear localization (2, 3). This asymmetry likely accounts for the lowered frequency of cells carrying rearrangements
on both alleles, which is below what would be expected if any
nonfunctional rearrangement on a first allele were indeed followed
with a rearrangement attempt on the second allele (4). By contrast
to several cytokine genes for which allelic exclusion was clearly
demonstrated at the transcriptional level, little is known about a
*Laboratoire d’Immunologie, Centre National de la Recherche Scientifique Unité
Mixte de Recherche 6101, Equipe labellisée “La Ligue,” Faculté de Médecine, Limoges, France
Received for publication August 12, 2003. Accepted for publication April 26, 2004.
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 grants from Réseau SWITCH, Ligue Nationale Contre
le Cancer, and Conseil Régional du Limousin.
2
potential asymmetry regarding transcriptional accessibility of Ig
alleles (5, 6). That nonfunctionally rearranged V sequences are
frequently cloned from genomic libraries but are virtually absent
from cDNA libraries or RT-PCR experiments could be taken as an
indication of a strong allelic exclusion at the transcriptional level.
However, Ig transcripts have been shown to be the targets of
mRNA surveillance control pathways that may result in a 4- to
100-fold accelerated degradation of nonfunctional transcripts (7).
Because of this efficient nonsense-mediated decay, steady-state
levels of mature Ig transcripts cannot provide any evaluation of the
transcription rate of the functional vs the nonfunctional allele in a
given B cell.
Following Ag encounter, the Ig genes in those B cells triggered
by the Ag are subjected to a second wave of diversification by
somatic hypermutation (SHM).4 This process leads to the introduction of multiple single nucleotide changes in and downstream
of the rearranged V(D)J segment, allowing the expression of a
secondary repertoire from which those B cells carrying mutated
Abs of improved Ag-binding affinity can be selected (reviewed in
Ref. 8). A first phase of SHM relies on the activation-induced
cytidine deaminase (AID) while a second phase may involve mismatch repair enzymes and translesion polymerases, but the molecular mechanisms selectively recruiting this machinery to Ig genes
are still unknown (9 –12). In vitro, transcription of DNA substrates
allows deoxycytidine deamination by AID (13, 14). In vivo, although not sufficient to ensure mutability, transcription appears as
necessary and is directly linked to SHM: the location of the mutation domain is determined by the position of the transcription
start site and recruitment of the mutational machinery is affected
by the cis-acting promoter and transcriptional enhancer elements
(15, 16). In mutable Ig transgenes integrated outside or within the
L.D. and C.S. contributed equally to this work.
3
Address correspondence and reprint requests to Prof. Michel Cogné, Centre National de la Recherche Unité Mixte de Recherche 6101, Laboratoire d’Immunologie,
2, rue du Dr. Marcland, 87025 Limoges, Cedex, France. E-mail address:
[email protected]
Copyright © 2004 by The American Association of Immunologists, Inc.
4
Abbreviations used in this paper: SHM, somatic hypermutation; FR, framework
region; PP, Peyer patch; PNA, peanut agglutinin; AID, activation-induced cytidine
deaminase.
0022-1767/04/$02.00
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Allelic exclusion of IgH chain expression is stringently established before or during early B cell maturation. It likely relies both
on cellular mechanisms, selecting those cells in which a single receptor allows the best possible Ag response, and on molecular
restrictions of gene accessibility to rearrangement. The extent to which transcriptional control may be involved is unclear.
Transcripts arising from nonfunctional alleles would undergo nonsense-mediated degradation and their virtual absence in mature
cells cannot ensure that transcription per se is down-regulated. By contrast, somatic hypermutation may provide an estimate of
primary transcription in Ag-activated cells since both processes are directly correlated. For coding regions, the rate and nature
of mutations also depend upon Ag binding constraints. By sequencing intronic sequence downstream mouse VDJ genes, we could
show in the absence of such constraints that somatic hypermutation intrinsically targets nonfunctional rearranged alleles at a
frequency approaching that of functional alleles, suggesting that transcription also proceeds on both alleles at a similar rate. By
contrast and confirming the strong dependency of somatic hypermutation upon transcription, we show that artificial blockade of
transcription on the nonfunctional allele by a knock-in neomycin resistance cassette keeps the VDJ region unmutated even when
its promoter is intact and when it is fully rearranged. The Journal of Immunology, 2004, 173: 1842–1848.
The Journal of Immunology
Materials and Methods
Mice
C57BL/6 wild-type mice (wt “b” allotype) were mated either with 129/Ola
wild-type mice (wt “a” allotype), providing the control wild-type a/b mice.
C57BL/6 mice were also mated with the B cell-deficient mouse line JH-NeoE␮, which carries a homozygous disrupted a allotype IgH locus. The JHNeo-E␮ mutation features the insertion of a neomycin resistance cassette at the
NaeI site immediately downstream of JH4 (Fig. 1A). This mutation is known
both to preserve the occurrence of V(D)J recombination and to result in a
complete transcriptional silencing of rearranged V(D)J segments (25). Heterozygous wt/JH-Neo-E␮ mice were selected, in which all B cells carried
a functional b allotype IgH locus and a nonfunctional a allotype IgH locus.
Cell cytometry and sorting procedures
Peyer patches (PP) were recovered by dissection from the small intestine.
Cell suspension was prepared by pressing PP through a nylon mesh and
cells were washed three times in cold DMEM containing 10% FCS (Invitrogen, Gaithersburg, MD). Cell suspensions were adjusted at 106
cells/ml after elimination of clumps of dead cells. Cells were incubated
with anti-B220-SPRD for 30 min at 4°C. After two washes with PBS
containing 10% FCS, FITC-conjugated peanut agglutinin (PNA) was
added and incubated on ice for an additional 30 min, before washing and
resuspension in medium. Purification of B220⫹PNAhigh and B220⫹PNAlow
cells was realized on a FACSVantage (BD Biosciences, Franklin Lakes,
NJ). The purification rates of these cell populations were constantly above
95% (Fig. 1B).
DNA extraction and amplification
Genomic DNA was extracted from PNAhigh B cells using the QIAamp
Tissue kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. DNA was amplified by PCR in 50 ␮l of Taq 1⫻ PCR buffer
containing 200 ␮M of each dNTP, 100 ng of each primer, and 1 U Taq
polymerase (Pharmacia, Piscataway, NJ), with the following program: 1
cycle at 94°C for 5 min, 35 cycles (94°C for 45 s, 58°C for 45 s, and 72°C
for 45 s), and 1 cycle at 72°C for 7 min. The following primers were used:
forward VH7183, 5⬘- CGGTACCAAGAASAMCCTGTWCCTGCAAATG
ASC-3⬘ (complementary to the third framework region of VH7183 family
genes) and various backward primers: 3⬘JH2, 5⬘-CAGTAAGCAAAC
CAGGCACA-3⬘, corresponding to an intronic sequence between the JH2
and JH3 segments; 3⬘ NaeI, 5⬘- TCCTAAAAGGCTCTGAGATCCC-3⬘,
FIGURE 1. Strategy for the analysis of mutations in PP lymphocytes. A,
PCR strategy for the analysis of V(D)J rearrangements involving VH7183
family gene segments and the JH1 or JH2 segment in wild-type mice
(VH7183-3⬘JH2 PCR) and involving the various JH segments in aJH-Neo-E␮/
bwt mice (VH7183-3⬘ NaeI PCR). B, Sorting of PP germinal center B cells
after staining with B220-SPRD and PNA-FITC. Cells from the B220⫹/
PNAhigh gate enriched in centroblasts were sorted and used for PCR cloning of V(D)J genes.
corresponding to an intronic sequence ⬃300 bp downstream of JH4 on a
wild-type allele (but 2 kb downstream of JH4 on a JH-Neo-E␮ knock-in
allele); and N3, 5⬘-TCTTGACGAGTTCTTCGAGGGGATCGGCA- 3⬘, a
primer homologous to the inserted neo gene 3⬘ end, proximal to the JH4
element, thus able to specifically amplify those rearrangements occurring
on the knock-in allele.
Cloning and sequencing
The PCR products were separated on a 1.2% agarose gel and cloned into
the pCRII-TOPO vector (Invitrogen) according to the manufacturer’s instructions. Plasmids were isolated using a standard Triton X-100/lysozyme
method and inserted sequences were sequenced from both ends using the
M13Reverse and M13(⫺20) forward primers and an automated laser fluorescent DNA ABI PRISM 3100 sequencer (PerkinElmer, Branchburg,
NJ). Mutations were analyzed on a limited interval including the rearranged JH segment and the following 180 bp of intronic sequence, a region
known to be strongly targeted by SHM on expressed genes (8, 26). Sequences from wild-type a/b mice and from heterozygous awt/bJH-Neo-E␮
animals were checked for mutation by comparison with the known germline sequences of the JH region from the a allotype (26) or the b allotype
(GenBank accession number AC073553).
Results
Functionally and nonfunctionally rearranged alleles accumulate
mutations at close rates in B cells from wild-type mice
PP PNAhigh B cells from wt a/b mice were sorted and checked for
hypermutation. To appreciate SHM in these cells regardless of
their Ag specificity, intronic sequences downstream of rearranged
VDJ junctions were amplified using a forward VH7183 family
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IgH locus, a linear correlation between expression and mutability
has been reported (17–19).
V(D)J rearrangement and presence of a V promoter are known
as mandatory for high-level SHM (18, 20). Although unrearranged
V segments and incomplete DJ rearrangements are transcribed at
low levels in immature B cells, V to DJ approximation boosts
transcription by positioning one VH promoter under the control of
the E␮ enhancer (reviewed in Ref. 21). Regarding allelic exclusion, the relative transcription rate of Ig loci in wild-type B cells
carrying two VDJ rearrangements has not been studied; by contrast, the presence of somatic mutations within out-of-frame rearranged V genes has been documented (22, 23). Since functional
and nonfunctional Ig sequences are strongly related and cannot be
individually studied by the means of hybridization techniques, we
thus decided to use the accumulation of mutations for the quantitative evaluation of the relative transcription rates of both alleles in
primary B cells that carry double VDJ rearrangements. Mutations
are known to freely accumulate in the framework regions of nonfunctional genes (23), while CDR mutations of the expressed alleles
are selected by the Ag-driven cell selection (8). We thus restricted our
analysis to intronic sequences located immediately downstream of
rearranged J segments, for which the accumulation of mutations only
reflected activity of the hypermutation machinery, with no selection
for the production of any translated gene product. These experiments
were conducted both in wild-type B cells and in B cells carrying an
heterozygous targeted disruption that was previously demonstrated to
abolish VDJ transcription while preserving all cis-regulatory elements
of the IgH locus and still allowing VDJ rearrangement on this nonfunctional allele (24).
1843
1844
HYPERMUTATION OF ALLELICALLY EXCLUDED V GENES
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FIGURE 2. Mutations in functional vs nonfunctional rearrangements from wild-type and from aJH-Neo-E␮/bwt mice. A, Intron sequences directly flanking the
JH segment of VHDJH genes cloned from wild-type B cells were classified into two groups depending on whether they came from genes with a functional or a
nonfunctional V(D)J junction. B, Intron sequences flanking the JH segment of rearranged VHDJH genes cloned from aJH-Neo-E␮/bwt B cells in which sequences from
the wild-type allele are functional while sequences from the knock-in allele are allelically excluded. JH1GL, JH2GL, and JH3GL represent the germline unmutated
sequences; exon sequences are in bold.
1845
The Journal of Immunology
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FIGURE 2. (Continued)
1846
Untranscribed rearranged VDJ gene segments do not undergo
SHM on the targeted allele of heterozygous wt/ JH-Neo-E␮
germinal center B cells
Although preserving V(D)J recombination to JH1, JH2, and JH3,
the JH-Neo-E␮ mutation blocks transcription of both germline JH
and rearranged DJH or VDJH segments (24). Although B cell development is arrested in homozygous JH-Neo-E␮ mutant mice
(24), using heterozygous mice with a single knock-in IgH allele
allows normal B cell maturation and warrants that all functionally
expressed sequences originate from the wild-type allele. Since the
JH-Neo-E␮ knock-in features the insertion of a neomycin resistance cassette at a NaeI site located ⬃100 bp downstream of JH4,
it could be used as a “tag” allowing to easily distinguish sequences
FIGURE 3. Comparison of hypermutation in functional and nonfonctional IgH alleles from wild-type
(upper panel) and from aJH-Neo-E␮/bwt mice (lower
panel). The data are presented as pie charts and indicate the proportion of sequences that contain 0, 1, 2,
etc., mutations in the rearranged JH segment and the
succeeding intronic 180-bp sequence.
from the knock-in allele or from the functional allele with a wildtype NaeI site.
As for wild-type mice, PP PNAhigh B cells from heterozygous
wt/JH-Neo-E␮ mice were sorted and checked for hypermutation.
Although the disrupted IgH locus was from the a allotype, heterozygous mice obtained after breeding with C57/B6 mice provided a wild-type b allotype IgH locus. By force, B cells of such
mice all carry a functionally rearranged b allotype JH region and an
allelically excluded a allotype locus. Since a number of B cells
carry double complete VDJ rearrangements, it was readily possible
from these cells, to amplify rearranged sequences originating from
both alleles. To that goal, a unique VH7183 consensus forward
primer was used along with a backward primer either specific for
the knock-in neo gene downstream of JH4 or specific for the wildtype JH4 3⬘ flanking intron. In addition, the use of heterozygous
IgH a/b allotype mice in which known allelic differences are located in the JH germline region provided a further control of the
origin of cloned sequences. The respective mutation frequencies
on both a functional b wt allele and a rearranged but untranscribed
a allele carrying the JH-Neo-E␮ mutation could finally be appreciated in identical conditions and in the very same PNAhigh B cell
samples (Fig. 2B).
A complete lack of somatic mutation of rearranged JH sequences was noticed on the untranscribed neo knock-in allele,
reaching 1.1 mutations/1000 bp (i.e., close to the range of mutations introduced by the Taq polymerase enzyme in PCR experiments) instead of a simultaneous 30 mutations/1000 bp on the
wild-type b control allele (Table I). The JH-Neo-E␮ knock-in thus
significantly blocked SHM of nonfunctional alleles carrying VDJ
rearrangements (Student’s t test, p ⬍ 0.02).
Discussion
In transgenic models, the hypermutation mechanism is known to
act only on gene copies that are demethylated as well as transcribed (15–19, 26). Although endogenous alleles of Ig loci may
show some asymmetry with regard to accessibility, DNA methylation, and pattern of early replication, it has not been clearly ascertained whether the processes of primary transcription and of
SHM preferentially target the actively expressed allele. Mutations
were previously observed in FRs of nonfunctional rearranged V␭
genes but their frequency could not be compared with that of functional genes in which FR mutations are negatively selected (22).
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framework region (FR) 3 consensus primer along with a backward
primer located in the intron downstream of JH2
Sequence analysis focused on the rearranged JH segment and the
following intronic 180-bp sequence, a region known to be strongly
targeted by the SHM process (8). This sequencing strategy also
avoided ambiguities for the assignment of V sequences to their
germline counterparts. Mutations were counted either only in the
intronic sequence or in the complete sequence including the JHencoded fourth FR (FR4). Sequencing also allowed us to check
VDJ junction sequences for functionality and to classify them into
two groups: functional sequences featured an in-frame rearrangement with no stop codon created at the VD or DJ junction and
nonfunctional sequences either carried out-of-frame junctions or
included premature termination codons within the D segment.
Of 50 functional and 23 nonfunctional sequences analyzed from
PP PNAhigh B cells, somatic mutations were readily observed on
both classes of rearranged alleles in wild-type animals (Figs. 2A
and 3). SHM reached the level of 13.1 mutations/1000 bp for functionally rearranged genes and 11.9 mutations/1000 bp for nonfunctionally rearranged alleles (Table I). This difference was not statistically significant. Since our sequencing interval included the JH
region encoding the V domain FR (FR4) for which substitutions
might be counterselected in expressed functional genes, we also restricted the comparison to intronic sequences flanking the rearranged
JH. This comparison again indicated similar mutation frequencies of
13.6 and 11.1%, respectively, for the functional and the nonfunctional
sequences. In both cases, the frequency was not significantly lower for
nonfunctional genes (Student’s t test, p ⬎ 0.05).
HYPERMUTATION OF ALLELICALLY EXCLUDED V GENES
The Journal of Immunology
1847
Table I. Comparison of mutation frequencies observed in the functional or nonfunctional IgH alleles from wild-type mice or from aJH-Neo-E␮/bwt
micea
JH-neo-E␮/wt Mice
Normal Mice
No. of junctions (and total bp) analyzed
Mutation frequency (/1000 bp)
Significant difference between functional vs nonfunctional
a
Functional
V(D)J Genes
Nonfunctional
V(D)J Genes
50 (9085 bp)
13.6%
23 (4171 bp)
11.15%
NS
Functional
V(D)J Genes
Nonfunctional
V(D)J Genes
20 (3615 bp)
20 (3640 bp)
30.0%
1.1%
p ⬍ 0.02
Sequences located immediately downstream rearranged V(D)J genes were determined and compared to their germline counterparts.
rangement of one allele. Both our data and those from dual BCRexpressing mice with a double knock-in of functionally rearranged
genes (28) clearly indicate that for IgH genes, double rearrangements result in allelic inclusion at the transcriptional level and that
allelic asymmetry is either unable to completely block transcription or that this blockade is relieved by VDJ rearrangement. Importantly and further indicating transcriptional competence of both
IgH alleles in mature B cells, germline transcripts from constant
genes that precede class switching were also shown to arise from
both the expressed and the “allelically excluded” IgH locus (29).
The major conclusion of the present work is that whether they
are functional or not, rearranged VDJ genes undergo SHM at
roughly similar rates. By contrast, preventing transcription of an
allelically excluded VDJ blocks its accessibility to SHM. It can be
inferred from these observations that allelic exclusion of IgH expression in normal B cells is not effected at the transcriptional level
for the numerous mature cells carrying double VDJ rearrangements. The early asymmetry in the replication pattern or nuclear
localization observed for Ig loci could thus have limited effects,
acting only by down-regulating V to DJ rearrangement in pro-B
cells. Later on and in a number of selected B cells, allelic asymmetry could also be strongly reinforced secondarily to VDJ rearrangement, as a consequence of the asymmetric activation of a
single VH promoter in those cells with a single V to DJ rearrangement. Both our data and those showing allelic inclusion in mice
with double knock-in of functional VDJ segments favor the hypothesis that allelic exclusion is mostly effected by the stochastic
process of VDJ rearrangement. Since in-frame rearrangements encoding a functional H chain able to pair with its L chain partner
constitute rare events, they are highly unlikely to occur on both
alleles and to be simultaneously expressed at the protein level. Our
findings that nonfunctionally rearranged alleles are not silenced at
the level of VDJ transcription further demonstrates that, by contrast to cytokine genes, transcriptional asymmetry plays little role
in the allelic exclusion of Ig gene expression. Downstream of transcription, these findings underline the importance of RNA surveillance pathways that degrade nonfunctional mature Ig mRNAs, thus
preventing the “wastage” that would result from their translation
into truncated Ig chains. They also confirm that in the course of B
cell malignancies, both Ig loci may be accessible to mutations,
recombinations, and translocations.
Acknowledgments
We thank Vincent Denis and Claire Carrion for their kind technical
assistance.
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HYPERMUTATION OF ALLELICALLY EXCLUDED V GENES