Section of Immunobiology, Howard Hughes Medical Institute and Yale School of Medicine, New Haven, CT, USA
The first two authors contributed equally to this work
Correspondence to: D. G. Schatz; E-mail: david.schatz{at}yale.edu
Transmitting editor: M. J. Bevan
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Abstract |
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Keywords: activation-induced cytidine deaminase, B lymphocyte, class switch recombination, DNA double strand break
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Introduction |
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While a detailed molecular description of the mechanism underlying CSR remains elusive, recent discoveries have provided insight into some of the relevant molecular controls and revealed some striking similarities between the mechanisms of CSR and SHM. First, both processes are closely correlated with transcription across the region targeted for mutation or recombination, i.e. the V or the S region respectively (3). Second, SHM and CSR both require the presence of the activation-induced cytidine deaminase (AID), as the absence of this molecule abrogates these processes in vivo (4,5). AID shares sequence homology to APOBEC1, an RNA-editing enzyme, suggesting that AID might act to edit mRNA species that might then encode proteins involved in SHM and CSR. Recent biochemical studies, however, have suggested that AID acts as a DNA deaminase, specifically on single-stranded DNA, converting cytosine residues to uracils (68). Such single-stranded DNA could either be present transiently in the non-template strand during transcription of the V region (9), or in the case of class switch, in R-loops structures contained within transcribed switch repeats (10,11). How these lesions are recognized and resolved by specific or ubiquitous DNA repair machineries remains unknown. Finally, there is evidence suggesting that DNA breaks are involved in the initiation of SHM and CSR. Recent work has shown that blunt DNA double strand breaks (dsbs) are associated with SHM (1214), and that these breaks occurred in recombined IgH and IgL chain genes expressed by the RAMOS Burkitt lymphoma cell line, as well as in germinal center B cells following immunization. These dsbs were most abundant in the post-replicative (G2) phase of the cell cycle, and their location correlated with mutational hotspots. Furthermore, dsbs were detected at unequal frequency within mutation substrates, with an increased frequency of lesions observed at the 5' or upstream portions of recombined IgH and IgL chains. Initial reports described blunt SHM-associated dsbs as being introduced independently of AID (15,16); however, a more recent study suggests that the primary species of breaks involved in SHM is recessed, with their induction being dependent on AID (17).
There is little doubt that DNA dsbs are involved in the mechanism of CSR, as the intrachromosomal recombination results in excision of intervening genomic DNA and a circularization of this DNA fragment [reminiscent of the excision circles generated by cells undergoing V(D)J recombination]; however, direct evidence for such breaks is limited. An initial report described the detection of blunt DNA dsbs at a few positions in the S3 region of splenic B cells stimulated to switch to IgG3 using LPS (18). More recently,
-H2AX foci were found to co-localize with the IgH locus in ex vivo activated splenic B cells, suggesting DNA dsbs as intermediates in CSR (19). Association of these
-H2AX foci with IgH was also found to be dependent on AID (19). Another recent study reported that blunt, AID-dependent DNA dsbs could be detected by ligation-mediated PCR (LM-PCR) within the Sµ region of activated human B cells (20). While this evidence indicates that dsbs are associated with the mechanism of CSR, there is little information on the structure and kinetic regulation of these breaks. In particular, there has not yet been an attempt to detect staggered DNA dsbs in S regions, despite previous evidence suggesting that such lesions may be involved in CSR (21).
In this study we have used LM-PCR to detect and characterize CSR-associated DNA lesions in the genome of B cells stimulated to undergo CSR ex vivo. We report the specific induction of AID-dependent DNA dsbs within Sµ of switching B cells. These lesions were not observed in B cells activated to divide but not to undergo CSR. While blunt DNA lesions were observed, the predominant population of Sµ-associated dsbs were staggered. In addition, these breaks were detected prior to the expression of the newly switched isotypes and were present at approximately equal frequency at upstream and downstream regions of Sµ. These results provide direct evidence that staggered DNA dsbs are the predominant species of Sµ DNA lesions involved in CSR.
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Methods |
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B cell preparation and ex vivo culture
Small, resting B cells were prepared as detailed elsewhere (22). Briefly, spleens obtained from mice were homogenized and subject to red blood cell depletion and adherent cell depletion. T cells were removed by complement depletion (Cedarlane Laboratories, Ontario, Canada) following incubation with purified anti-mouse Thy-1.2, CD8 and CD4 antibodies (all from Pharmingen, San Diego, CA). Small, resting B cells were obtained by Percoll fractionation and were routinely >95% IgDhi and CD19+ as measured by FACS. B cells were cultured in B cell media (BCM) comprising RPMI (Invitrogen, Carlsbad, CA) supplemented with 2 mM L-glutamine, 0.1 mM non-essential amino acids, 10 mM HEPES (pH 7.4), 100 µg/ml streptomycin, 60 µg/ml penicillin, 5 x 105 M 2-mercaptoethanol (all from Sigma, St Louis, MO), 1 mM Na-pyruvate (Invitrogen), and 10% heat inactivated FCS (Gemini Bio-products, Woodland, CA).
B cells were stimulated at a density of 5 x 105 cells per ml with LPS (20 µg/ml) (Sigma) in the presence or absence of IL-4-containing supernatant (400 U/ml, a generous gift of Dr C. Janeway) or anti-mouse IgD (1.19, 10 µg/ml, a generous gift of Dr P. Lalor by way of Dr P. Hodgkin, WEHI, Melbourne) and IL-4. In certain experiments, B cells were labeled with CFSE (Molecular Probes Inc., Eugene, OR) prior to culture in order to monitor cell division by CFSE dilution. Details of CFSE labeling and quantitation and analysis of division profiles can be found elsewhere (23).
Flow cytometry and FACS analysis
To examine whether B cells had undergone CSR, B cells were harvested at various times and stained with antibodies against murine IgG1 (phycoerythrin) and IgG3 (biotin) (both from BD Pharmingen). The biotin-labeled primary mAb was counter-stained with Strepavidin-Tricolor (Caltag Laboratories, Burlingame, CA). Where possible, at least 104 live cell events were acquired using either FACScan or FACSCalibur (BD Biosciences). All FACS analysis was performed using CellQuest (BD Biosciences).
LM-PCR of DNA dsbs
Immediately following culture harvest, cells were washed twice in PBS/0.1% BSA and dead cells and cell fragments were removed by centrifugation over a Ficoll gradient (ICN Pharmaceutical, CA). Live B cells were immediately washed and embedded in low-melting temperature agarose (American Bioanalytical, MA; final concentration of 0.25%) that had been equilibrated to 37°C. Without exception, plugs were made from cells (usually 106 per plug) where post-Ficoll viability levels were >90% as measured by dye exclusion. In some instances, propidium iodide staining was also used to confirm viability levels (not shown). Cells in agarose plugs were subject to genomic DNA preparation and double-stranded DNA linker ligation as previously described (12). In some instances, plug DNA was treated with T4 DNA polymerase (New England Biolabs, MA) so as to blunt any staggered DNA lesions prior to linker ligation.
Linker-ligated DNA was subject to two rounds of LM-PCR: 10 (or 15) cycles with a 55°C annealing step followed by 28 cycles with a 62°C annealing step (2 µl of the 50 µl first LM-PCR was used to prime the second 25 µl reaction). PCRs were performed using Hotstart (Qiagen Inc., CA) or Jumpstart (Sigma) Taq polymerase and nested, gene-specific and linker-specific oligos (Table 1). PCR products were resolved on agarose gels, blotted onto nylon membranes and hybridized to gene-specific, 32P-labeled oligo probes. Input DNA control PCRs amplified genomic HPRT in a standard 30 cycle reaction.
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Results |
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We stimulated small, resting CFSE-labeled B cells purified from AID+/+ (C57BL/6) and AID/ mice with LPS and IL-4 or anti-IgD and IL-4. After 4 days of culture, LPS- and IL-4-stimulated AID+/+ B cells had divided up to 7 divisions and undergone CSR to IgG1. In response to anti-IgD and IL-4, B cells were able to proliferate (45 divisions; Fig. 1A) but failed to undergo CSR, in accordance with previously published data (25). In contrast, AID/ B cells divided but failed to undergo CSR in response to LPS and IL-4. Note that there was virtually no difference in the proliferative ability of AID+/+ and AID/ B cells in response to LPS and IL-4, with both types of cells having undergone a similar number of cell divisions by day 4. This result indicates that any defects in CSR-associated DNA breaks detected in AID/ B cells could not be attributed to defects in cellular replication.
After 4 days of culture, B cells were harvested and live cells were separated from dead cells or cell fragments by centrifugation over a Ficoll gradient. Removal of these dead cells was crucial, as the degradation of genomic DNA during apoptosis generates high levels of non-specific DNA dsbs. Such breaks could potentially mask the DNA lesions that are specifically induced during CSR. In all experiments, cells were taken from populations exhibiting viability levels of >90% (not shown). Genomic DNA from such cells was prepared and manipulated in agarose plugs to limit the generation of random DNA breaks due to physical manipulations prior to ligation.
To detect DNA dsbs in the Sµ switch region, we amplified the respective fragments from linker-ligated genomic DNA by PCR using Sµ and linker-specific primers. The products were separated on agarose gels, blotted and visualized by hybridization with a Sµ specific oligonucleotide. Note that the Sµ-specific PCR primers and probe lie upstream of the highly repetitive portion of Sµ (Fig. 3B). DNA dsbs were detected in the upstream region of Sµ in AID+/+ (C57BL/6) B cells stimulated with LPS+IL-4, whereas such breaks were not detectable in AID-deficient B cells stimulated under identical conditions (Fig. 1B). Treatment of genomic DNA with T4 DNA polymerase prior to linker ligation (to blunt 5' or 3' overhanging DNA ends) resulted in an increase in the level of detectable breaks (Fig. 1B), suggesting that a significant portion of the Sµ DNA lesions detectable by LM-PCR are staggered. A progressive 10-fold titration of linker-ligated template DNA indicated that dsbs in Sµ could be detected in as little as 500 cells activated with LPS and IL-4 when DNA ends in the agarose plug had been blunted prior to linker ligation. Note that no breaks were detectable in Cµ under any circumstances (Fig. 1B).
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CSR-associated DNA dsbs are predominantly staggered and are detected at equal frequency in 5' and 3' Sµ
Figure 1(B) indicated that there was an increased detection of Sµ dsbs in T4 DNA polymerase treated samples, suggesting that a substantial fraction of CSR-associated DNA breaks were staggered. To more accurately determine the frequencies of staggered and blunt Sµ dsbs, we performed LM-PCR on progressive 5-fold serial dilutions of T4 DNA polymerase treated or untreated genomic DNA prepared from AID+/ and AID/ B cells cultured with LPS and IL-4 for 4 days (Fig. 3A). In T4 DNA polymerase treated samples, dsbs could be amplified from AID+/ genomic DNA equivalent to as few as 80 cells, whereas no breaks could be detected in <2 x 103 cell equivalents of DNA from untreated samples, a difference of 2030-fold. These results provide further evidence that the predominant species of steady-state CSR-associated DNA lesions detectable by LM-PCR are staggered DNA dsbs. Note that there was a dramatic reduction in the level of dsbs in AID/ B cells, and there was no evidence of DNA breaks in Cµ in either AID+/+ or AID/ samples (data not shown).
Sequence analysis of blunt Sµ LM-PCR products from switching AID+/ B cells indicated that the breakpoints were distributed throughout the S region as well as in the 5' flanking region (Supplementary fig. 1). Although these breakpoints do not necessarily reflect the location of the initial DNA lesion(s), they suggest that CSR-associated DNA breaks occur throughout the entire Sµ region. Consequently, we were interested in determining whether CSR-associated breaks could be detected in both the 5' and 3' regions of Sµ. This issue has not previously been addressed, and is of some mechanistic importance because of the finding that there is a strong upstreamdownstream asymmetry for blunt dsbs associated with SHM (12,13). Using LM-PCR, we detected upstream ends of dsbs at the 5' end of Sµ and downstream ends at the 3' end of Sµ in AID+/+ B cells at approximately equal frequency (Fig. 3B). There was no evidence of breaks within Cµ (not shown).
Note that in Fig. 3(B), Sµ dsbs could be detected in AID-deficient B cells, albeit at lower levels compared to AID+/+ cells. An increased primary LM-PCR cycle number [15 cycles in Fig. 3(B) compared with 10 cycles for the experiments in Figs 1, 2, 3(A) and 4] is the most likely explanation. As the increased cycle number significantly enhanced the detection of dsbs in AID+/+ cells, it is possible that the dsbs seen in Sµ of AID/ cells are bona fide, and reflect a low level of residual break induction in the absence of AID. Alternatively, the Sµ dsbs observed in AID/ cells could result from increased genomic instability in highly transcribed switch regions. It is also possible that the presence of low levels of dsbs in AID/ B cells may represent background generated by the LM-PCR procedure itself, or breaks amplified from genomic DNA of a contaminating population of dead cells. Irrespective, it is clear that the DNA lesions in AID-deficient B cells are present at a much reduced level compared to AID+/+ and that staggered dsbs are the predominant population of DNA lesions present in both the upstream and downstream portions of Sµ.
Specific induction of Sµ-associated dsbs occurs prior to switched isotype expression
Our data support the conclusion that Sµ dsbs are specifically introduced into Sµ of B cells stimulated to undergo CSR. If these DNA lesions are intermediates in CSR, it would be expected that they would be introduced prior to expression of downstream isotypes. Figure 4(A) indicates that Sµ dsbs could be detected in B cells activated with LPS for 2 days, despite the absence of any detectable surface IgG3. This result clearly indicated that the induction of Sµ dsbs precedes surface expression of the downstream, switched IgH C genes and is consistent with the idea that break induction occurs prior to CSR. B cells from anti-IgD and IL-4 control cultures failed to switch and did not show evidence of Sµ dsbs. Analysis of cell division by CFSE dilution indicated that very little proliferation had occurred in B cells from either culture (Fig. 4B), further suggesting that the absence of Sµ dsbs in anti-IgD and IL-4 cultures could not be attributed to reduced division in comparison to LPS and IL-4 cultures (Fig. 1A). Note that no dsbs were observed in Cµ under either culture condition (not shown).
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Discussion |
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Our current model of CSR implies that two double-strand DNA cleavage events within targeted S regions are essential. However, there is limited direct evidence for DNA lesions associated with CSR, and we have little idea of how these lesions are generated and processed, or of their structural properties. The first evidence for CSR-associated DNA breaks identified blunt DNA lesions within S3 of splenic B cells stimulated to switch to IgG3 (18); however there was no data on the upstream recombination partner Sµ. Further support for the existence of DNA breaks came from fluorescence in situ hybridization experiments that showed the presence of phosphorylated
-H2AX foci within the IgH locus in primary B cells treated to undergo CSR (19). Such foci are formed at DNA lesions, but the limited resolution of this technique did not allow determination of the precise location of the breaks, nor did it provide information regarding the structural characteristics of these lesions. More recent evidence suggests that blunt, AID-dependent dsbs are associated with Sµ in activated human B cells (20), and our data confirm this finding; however, it remains to be determined whether such blunt breaks are the principal DNA lesions in CSR. First, analysis of both recombinant junctions in substrates designed to undergo CSR by inversion revealed frequent short deletions and duplications, suggesting staggered DNA dsbs as an intermediate in CSR (21). Second, in SHM, blunt dsbs within the endogenous rearranged Ig genes are AID-independent (15,16), whereas staggered dsbs are AID-dependent (17). In addition, staggered breaks would arguably be the most plausible initial product if AID acts to convert cytosines to uracils at different positions in the top and bottom strands of target DNA. The subsequent generation of a nick by the action of the ubiquitous base excision repair machinery at two such cytosines would result in a staggered dsb. The involvement of base excision repair in CSR is supported by the finding that the frequency of switching in uracil-N-glycosylase (UNG)-deficient B cells is dramatically reduced (26). In addition, UNG deficiency results in a profound reduction in the level of blunt Sµ-associated dsbs in human B cells activated to undergo CSR (27), and in a shift of mutation patterns at G/C bases (26,27). Thus, given the parallels between SHM and CSR and the findings reported here, staggered dsbs may play a central role in both processes. Nevertheless, existing data do not rule out the participation of blunt dsbs in SHM or CSR, and it is possible that blunt ends are the precursors of staggered ends in SHM (17), and the products of processing of staggered ends in CSR.
In this study we provide evidence that DNA dsbs in the Sµ region are associated with CSR. Several findings support this claim. First, under identical culture conditions, no dsbs are found in the Cµ region, which is located immediately downstream of Sµ. Second, the DNA dsbs are specifically generated when B cells are stimulated to undergo CSR, whereas they are not detectable in B cells stimulated to divide but not to isotype switch. Third, the induction of DNA dsbs precedes the presence of Ig of switched isotype on the cell surface, suggesting that DNA cleavage is a requirement for the generation of switched isotypes. Finally, both blunt and staggered DNA breaks are largely or exclusively created in an AID-dependent manner, and AID is essential for CSR to occur (4).
Further characterization of Sµ-associated breaks suggested that the primary steady-state species of DNA lesions in CSR are staggered dsbs. Titration of genomic DNA indicated that these staggered lesions could be detected at an 25-fold higher frequency than blunt dsbs. This finding raises the issues of how these staggered breaks are generated, and why they are present in such a large excess as compared to blunt dsbs. There are at least three possibilities: (1) blunt and staggered DNA breaks are generated and/or resolved with unequal efficiencies; (2) DNA breaks are initially blunt, and then are either rapidly rejoined or processed to generate staggered ends that persist for a longer time; (3) DNA breaks are initially staggered and some fraction are made blunt before end-joining occurs. Since our experimental approach only determines the steady-state population of DNA dsbs, we cannot distinguish between these possibilities.
Our results provide strong support for the model of staggered DNA dsb intermediates in the switch repeat region during CSR (3,21). As CSR targets (at least) two S regions for recombination, and as efficient recombination is thought to require close juxtaposition of a pair of S regions, it remains important to determine whether breaks in the two targeted S regions are generated synchronously (analogous to V(D)J recombination and bacterial transposition events), or, if instead, one break (i.e. in Sµ) occurs first, perhaps even prior to switch region synapsis. Furthermore, it would be interesting to obtain a more detailed analysis of the kinetics of dsb induction and possible changes in the structure of such lesions over time. Placing the CSR-associated DNA breaks into the well-defined sequence of events that includes the induction of germline transcripts, expression of AID and the generation of post-switch transcripts, would advance our understanding of the molecular mechanism of CSR.
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Supplementary data |
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Acknowledgements |
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Abbreviations |
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BCMB cell media
CFSE5(and 6) carboxyfluorescein diacetate succinimydl ester
CSRclass switch recombination
dsbdouble-strand break
LM_PCRligation-mediated PCR
MFImean fluorescence intensity
SHMsomatic hypermutation
UNGuracil-N-glycolase
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References |
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