|
|||||||||
|
|
|
|
|
|
|
|
|
|
|
Blood, 15 May 2004, Vol. 103, No. 10, pp. 3828-3836. Prepublished online as a Blood First Edition Paper on February 24, 2004; DOI 10.1182/blood-2003-10-3470.
IMMUNOBIOLOGY Immunoglobulin class-switch recombination in mice devoid of any Sµ tandem repeatFrom the Laboratoire d'Immunologie, Unité mixte de recherche, Centre National de la Recherche Scientifique (CNRS UMR), Faculté de Médecine, Limoges, France; and Centre de Développement des Techniques Avancées pour l'Expérimentation Animale, Orléans, France.
Immunoglobulin heavy-chain class-switch recombination (CSR) occurs between highly repetitive switch sequences located upstream of the constant region genes. However, the role of these sequences remains unclear. Mutant mice were generated in which most of the Iµ-Cµ intron was deleted, including all the repeats. Late B-cell development was characterized by a severe impairment, but not a complete block, in class switching to all isotypes despite normal germ line transcription. Sequence analysis of the Iµ-Cµ intron in in vitro activated–mutant splenocytes did not reveal any significant increase in activation-induced cytidine deaminase (AID)–induced somatic mutations. Analysis of switch junctions showed that, in the absence of any Sµ repeat, the Iµ exon was readily used as a substrate for CSR. In contrast to the sequence alterations downstream of the switch junctions, very few, if any, mutations were found upstream of the junction sites. Our data suggest that the core Eµ enhancer could be the boundary for CSR-associated somatic mutations. We propose that the core Eµ enhancer plays a central role in the temporal dissociation of somatic hypermutation from class switching.
During B-cell development, the immunoglobulin (Ig) locus is the site of 2 types of rearrangements: V(D)J assembly that generates the variable (V) region exons at the heavy- and light-chain loci and class-switch recombination (CSR) at the heavy-chain (IgH) locus. Upon antigen challenge, mature B cells expressing IgM and/or IgD undergo diversification processes that affect both the V and the constant (C) genes. Point mutations and occasional insertions and deletions are introduced in the V regions during somatic hypermutation (SHM) and gene conversion eventually resulting in higheraffinity receptors. CSR specifically affects C genes through a deletional process whereby a downstream C-region gene is brought to proximity of a rearranged VDJ gene, allowing expression of one of the downstream isotypes (IgG, IgE, or IgA).1,2
CSR generally occurs between highly repetitive, G-rich switch (S) sequences located upstream of all the C genes except C CSR involves DNA breaks within partner S sequences followed by repair and ligation through a nonhomologous end-joining (NHEJ) mechanism with looped-out deletion of the intervening DNA. The final steps of CSR involve components of the general DNA repair machinery as well as mismatch-repair mechanisms. In contrast, the early steps requiring recognition and cleavage of S DNAs are still unclear. Both double-strand breaks and staggered single-strand breaks have been involved in the early steps of CSR.4,5 Frequent mutations have also been found in the vicinity of the breakpoints in the absence of any obvious consensus sequence or homology at the junction of the recombined S sequences.2,3,6 CSR is preceded by germ line transcription of target S region, which is directed by the upstream I promoter. Activation and targeting of CSR is correlated with the ability of certain mitogens and cytokines to induce or suppress germ line transcription of specific C genes. Cis-regulatory elements located upstream of the I promoters and downstream of the IgH locus, accurate splicing of germ line transcripts, and the polarity of transcription are critical for the efficiency of CSR.2,6-8 Germ line transcription has been suggested to be necessary for the accessibility of S sequences to putative recombinases effecting cleavage.2,6,9 Several studies also showed that germ line transcripts remain on the template DNA leading to RNA-DNA hybrids10-13 and long R-loops, which may serve as substrates for putative recombinase(s).14,15 Despite extensive efforts, the molecular bases of CSR, SHM, and gene conversion are not fully understood. These processes clearly require the activation-induced cytidine deaminase (AID), a member of the RNA-editing deaminase family, specifically expressed in germinal center B cells.16-21 However, neither the target(s) of AID nor its potential cofactor(s) are precisely known and the exact role of AID is still controversial.9,22-25 It has been suggested to act on one (or more) mRNA encoding putative endonuclease(s) that cleaves DNA in the V-region genes and S sequences.16,26 In contrast, indirect genetic evidence strongly suggests that V-region genes and S sequences are themselves the substrates of AID.27,28 In addition, recent biochemical and genetic studies provide good evidence that a target for AID could be the exposed single-stranded DNA during the transcription process.29-33 An increasing body of evidence shows that AID is responsible for the DNA cleavage that initiates CSR as well as the intraswitch rearrangements.34-36 In addition, AID seems to be the only B-cell–specific factor required for CSR, as ectopic expression of this enzyme is sufficient to induce CSR in fibroblasts.37 However, other studies strongly suggest that, in addition to AID, class-specific factors regulate isotype switching.38,39
Whatever the role of AID, the S sequences are clearly (but not exclusively) the sites for cleavage. Sequencing of switch junctions unambiguously identified the tandem repeats as the main site of recombination, though in the case of Sµ, breakpoints were also found outside the tandem repeats.3,40 How the different S regions are recognized by the CSR machinery is unclear, although increasing evidence points to a role of higher-order structures in targeting CSR.6,9 Deletion of the core Sµ from the mouse genome reduced the efficiency of CSR but preserved recombination events within the remaining intron sequences.41 In contrast, deletion of most of the I We thus sought to make a larger deletion of the Iµ-Cµ intron, including all the pentameric repeats and the sequences thought to play a regulatory role in µ gene expression.42-45 We show that, although severely impaired, CSR is not abolished, with a shift of switch junctions toward Iµ in the absence of proximal AID-induced mutations.
Gene targeting An Sµ targeting construct was generated using a plasmid containing an approximately 5 kilobase (kb) long 5' arm (StuI-SpeI fragment in which StuI was replaced by NotI site) tailored with a 1.3-kb ClaI-SalI fragment encompassing a neor gene flanked by loxP sites and an approximately 5-kb 3' arm XhoI fragment from phage MB8. An HSV tk gene was inserted in NotI site for negative selection. Embryonic stem (ES) cells (cell line CK35) were transfected by electroporation and selected using G418 (400 µg/mL) and ganciclovir (2 µM). Recombinant clones were identified by Southern blot analysis with an external probe (0.6 kb XhoI-XbaI fragment). Two ES clones showing homologous recombination were injected into C57Bl/6 blastocysts; the male chimeras were then mated with C57Bl/6 females. Germ line transmission of the mutation was checked by Southern blot. Homozygous mutant mice N/N were mated with EIIa-cre transgenic mice (a kind gift of Dr Heiner Westphal, used under a noncommercial research license agreement from Dupont Pharma, Wilmington, DE). The progeny was checked by Southern blot for recombinase (Cre)–mediated deletion. Northern blots
Total cellular RNA preparation and Northern blotting were carried out as described.46 Probes used for hybridization were as follows: for Cµ transcripts, a 1.2-kb XbaI-HindIII genomic fragment containing the murine Cµ1 to Cµ3 region; for RT-PCR analysis of germ line transcription Total RNA was prepared by the Tripure technique (Roche GmbH, Mannheim, Germany). One microgram of RNA was then retrotranscribed by addition of reverse transcriptase (RT; Invitrogen, Gröningen, The Netherlands). The oligonucleotide sequences, the polymerase chain reaction (PCR) conditions, and the expected sizes of amplified products corresponding to spliced germ line transcripts were described.46 Spleen cell cultures Splenocytes from 6- to 8-week-old mice were activated in vitro as described,46 except that we used cells at a density of 106 cells/mL. At days 0, 2, 4, and 5, aliquots of cells were removed in order to prepare RNA. Fluorescence-activated cell sorter (FACS) analysis Flow cytometry analysis of bone marrow cells. Bones from 6- to 8-week-old mice were flushed with 10% fetal calf serum (FCS)–containing RPMI 1640. After disaggregation and washing, cells were stained with spectral red–conjugated anti-B220 and phycoerythrin-conjugated anti–c-kit, anti-CD43, anti-CD25, anti-IgM, or anti-IgD (Southern Biotechnology, Birmingham, AL). Data were obtained on 1.5 x 104 viable cells by using a Coulter XL apparatus (Beckman Coulter, Fullerton, CA). Flow cytometry analysis of spleen cells. At day 5 of stimulation, splenocytes (5 x 105 cells/assay) were stained with spectral red–conjugated anti-B220 and phycoerythrin-conjugated anti-IgM, anti-IgG3, or anti-IgG2b or fluorescein isothiocyanate (FITC)–conjugated anti-IgG1, anti-IgG2a, or anti-IgA (Southern Biotechnology). Data were obtained as described in the previous paragraph. ELISA assays Sera or supernatants from spleen cell cultures (harvested after 5 days of stimulation) were analyzed for the presence of the various Ig classes and subclasses by enzyme-linked immunosorbent assay (ELISA) as described.46 PCR and sequencing For AID-induced mutations, in vitro stimulation of splenocytes, high– molecular weight DNA preparation, and PCR conditions were as described,36 except that we used 20 µg/mL lipopolysaccharide (LPS; Sigma, St Louis, MO) supplemented or not with 1 ng/mL of mouse recombinant interleukin 4 (IL4; PeproTech, Rocky Hill, NJ) and the cultures were stopped at day 5. PCR products were cloned in Topo I vector (Invitrogen), transfected in TG1 bacterial strain, and plated without preculture at 37°C to avoid generation of sister clones. Sequences were determined on both strands by using M13 universal primers and aligned by using the Clustalw program (Institut Pasteur, Paris, France). Oligonucleotides Amplification of germ line transcripts. Primers Imf, Cmr, Ig2af, Cg2ar, Ig2bf, Cg2br, Iaf, Car, Acti-4, and Acti-5 have been described.46 The primers were as follows: cEµf, 5'-GAGGATTCAGCCGAAACTGGAG-3';5'NaeI, 5'-TCAGGTAAGAATGGCCTCTCC-3'. Amplification of Iµ-Cµ region. The primers were as follows: 3'Eµf, 5'-CATTCTGGTCAAAACGGCTTCACAAATC-3'; 5'Sµb, 5'-GGTCTCTATTCTTTCTCAATTC-3'; 3'Sµf, 5'-GCCAGTGTCTCAGAGGGAAGCC-3'; H5', 5'-GTAAGGAGGGACCCAGGCTAAG-3'; H3', 5'-CAGTCCAGTGTAGGCAGTAGA-3'. Amplification of JH4-Eµ enhancer region. The primers were as follows: VH7183, 5'-CAACCATTAGTAGTGGTGGTAG-3'; 5'Eµb, 5'-ATACACATACTTCTGTGTTCCTTTG-3'.
Amplification of switch breakpoints. Splenocytes from mutant homozygous (
Generation of mice carrying a large deletion of the Iµ-Cµ intron
In order to delete a large portion of the Iµ-Cµ intron, a targeting vector was designed to remove all the scattered pentamers beyond the core Sµ (defined as a
Early B-cell development in Iµ-Cµ intron–deleted mice
The early B-cell compartment in mutant mice was analyzed by flow cytometry using a set of specific surface markers. Bone marrow cells were labeled with anti-B220 and anti–c-kit to check for pro-B cells. Normal pro-B-cell population was found in These results indicate that deletion of the Iµ-Cµ intron leads to a slight increase in the pre–B-cell compartment but has no effect on the other developmental stages. Serum antibodies and in vitro immunoglobulin production by mutant splenocytes
In order to analyze the sera, homozygous
A totally different picture emerged from the analysis of Ig production in vitro. Splenocytes from littermates were stimulated with LPS supplemented or not with IL4 for 5 days, and supernatants were analyzed by ELISA. Whereas no difference was detected for IgM production between wt (+/+), heterozygous ( Altered cell surface immunoglobulin expression in Iµ-Cµ intron–deleted mice
We also checked alteration of CSR in the mutant mice by studying cell surface expression of the different isotypes on activated splenic B cells. Splenocytes from wt, heterozygous, or homozygous mutant mice were activated with LPS alone to induce switching to IgG3 and IgG2b, with LPS+IL4 to induce switching to IgG1, with LPS+IFN
Altered expression of class-switched genes but normal germ line transcription in stimulated mutant splenocytes
We then asked if the pattern of class-switched gene expression would mirror that found for cell surface expression. To this end, total RNA from unstimulated, LPS-, or LPS+IL4–stimulated splenocytes was hybridized with a Cµ probe and with a C
We then sought to analyze germ line transcription, which is generally a prerequisite of CSR. Total RNA was prepared from appropriately stimulated splenocytes and was subjected to an RT-PCR in semiquantitative conditions, using a set of specific primers for germ line promoters and C exons. We found no obvious alteration in Iµ-Cµ and I Altogether, these results show that deletion of most of the Iµ-Cµ intron has no effect on germ line transcription but drastically affects class-switched gene expression and hybrid germ line transcription. Lack of AID-induced somatic mutations in Iµ-Cµ intron–deleted splenocytes
Recent work34,36 has shown that AID introduces inducible DNA lesions in the Sµ region that are thought to initiate CSR. We sought to determine the mutation frequency in the Iµ-Cµ intron from homozygous mutant mice. We first amplified 868 bp between the 3' matrix attachment region (MAR) and Cµ1 from unstimulated, LPS-, or LPS+IL4–activated
One possibility to account for the lack of increased mutation frequency in the altered Iµ-Cµ intron could be that the remaining sequences are not prone to a high frequency of mutation even in the wt locus; we therefore sought to analyze splenocytes from wt (+/+) mice. This was achieved by sequencing the regions corresponding to those remaining in the mutated intron (3' to Iµ and 5' to Cµ1 exons); the length of the regions to be sequenced in the wt alleles was extended to include an equivalent number of nucleotides to that brought by the targeting vector (loxP site and cloning sites) in the mutant allele. A slight accumulation of mutations was found in the 5' region (539 bp) as well as in the 3' region (399 bp; 7 and 2 mutations, respectively) from LPS-activated splenocytes, although a higher "background" was found in the 5' region from unstimulated splenocytes (4 mutations; Table 1 and Figure 5B). This could be due to some amplified sequences from preactivated splenocytes, from memory B cells, or from some rare class-switched splenocytes.
Altogether, these results suggest that the remaining sequences in the mutated Iµ-Cµ intron are poor targets of AID and that the deleted sequences offer better substrates for AID. We therefore asked if in heterozygous ( These data strongly suggest that AID specifically targets the Sµ sequences on the wt allele and has no obvious bystander effect on the mutated allele. Iµ is readily used as a substrate for CSR in the absence of mutations upstream of switch junctions in Iµ-Cµ intron–deleted mice
Having shown that deletion of the major portion of the Iµ-Cµ intron leads to a severe impairment but not a complete abrogation of CSR and that this decrease is likely to be due to a decrease in CSR initiation as reflected by the lack of AID-induced mutations, we sought to analyze the junction sites and the flanking mutations on class-switched alleles. We sequenced 27 independent clones, the sequence of the recombined S
Within the limits of our data set, neither a "hot spot" for CSR nor a clear-cut bias toward G-rich sequences in Iµ was found. No obvious microhomology was found within the 10 nucleotides flanking the switch junction. Interestingly, the most proximal switch donor site within Iµ was found in the 3' MAR (clone TS23; Figure S3). When we looked at mutations upstream of switch junctions, very few mutations were found. In group A, only one mutation (clone TS19) and in group B, 3 mutations all clustered in a single clone (clone TS17) were found in the proximal Iµ sequences. In contrast, the usual alterations associated with CSR (point mutations, deletions, duplications) were observed downstream of S Previous work strongly suggests that splicing of germ line transcripts is critical for CSR efficiency.50 In our case, all clones from group A have deleted the canonical Iµ splice donor site, which suggests that cryptic splice sites may have been activated. We adopted an RT-PCR approach on unstimulated and LPS-activated splenocyte's RNA. PCR and sequence analysis of spliced products mostly revealed normal use of the canonical splice sites (not shown), whereas a shorter and lesser abundant amplified product revealed an upstream cryptic splice site correctly spliced to the canonical Cµ1 acceptor site in LPS-activated splenocyte's RNA (Figure 6B). These results suggest that in addition to or in the absence of the canonical Iµ splice donor site, an upstream cryptic splice site on Iµ transcripts occasionally enables the splicing of Iµ-Cµ transcripts.
We generated mice in which most of the Iµ-Cµ intron was deleted, leaving intact just the necessary sequences for correct splicing of µ transcripts. In addition to the core Sµ, the deletion removed all the remaining scattered pentamers involved in CSR41 as well as intronic sequences thought to play a regulatory role in Ig gene expression.42-45 We found no evidence for such a regulatory role in activated B cells. With regard to early B-cell development, no obvious abnormality could be detected in pro-B- and immature B-cell compartments. In contrast, we did find a 2-fold increase in the pre–B-cell compartment by FACS analysis of bone marrow, which could indicate a delayed or decreased pre–B-cell–receptor (pre-BCR) expression. The reasons for such an alteration are unclear but are unlikely to involve the absence of NFSµ-U1 and BSAP binding sites, since their deletion in the mouse genome had apparently no effect on pre-B cells.45 Late B-cell development was characterized by an impairment, but not a complete block, in CSR to all isotypes. In the serum, the decrease was in the range of 2- to 10-fold for IgG subclasses, whereas the IgA titer was unaffected, which is reminiscent of the core Sµ–deleted mice.41
In contrast, in vitro LPS- or LPS/IL4–activated Ig production was more severely decreased (10- to 100-fold reduction for IgG3, IgG2b, and IgG1), pointing to an autonomous defect in the ability to effect CSR by mutant splenocytes. Accordingly, whereas µ transcripts were equally abundant in
Interestingly, germ line transcription was normal, be it from Iµ or from downstream I promoters reflecting their unaltered accessibility. In contrast, hybrid germ line transcripts in the form of Iµ-Cx (Cx being any C region gene apart from µ and In mice devoid of the core Sµ, CSR was decreased but not abrogated with an apparent bias toward the region downstream of the deletion.41 In our study, the bias was rather toward the region upstream of the deletion with a clear shift toward Iµ. The reason for this discrepancy may be due to the lack of any Sµ remnants in our mice.
Although PCR may induce some cloning bias, our sequences are likely representative of the spectrum of S junctions occurring in
Based on the analysis of a large number of switch junctions from a cell line induced to switch to C
Requirement for a canonical splicing of Iµ may also be less stringent than expected. Thus, cryptic sites downstream of the Iµ exon were activated in cells with a mutation of the usual I donor site.51 In Iµ-Cµ intron–deleted mice, we identified by an RT-PCR approach a cryptic splice site just downstream 3 of the Iµ transcription initiation sites.52,53 This proximal site may be rarely used and it may have a physiologic meaning in that it lies upstream of all the S junctions we found in our Altogether, these data strongly suggest that the 3' end of Iµ is not the 5' border for CSR as suggested.40 Rather, we favor the view that it is the core Eµ enhancer itself that is the actual border (below).
That CSR may, in some extreme situations, be rescued by elements outside the core Sµ or even within Iµ suggests that CSR may be physiologically vital to the cells committed to this pathway. It may indicate that, upon appropriate stimulation and even in the absence of the specific motifs, the cleavage/repair machinery may somehow find its way to the cEµ-Cµ region. In that view, Sµ repeats would provide the optimal substrate for efficient recombination, while other components of the machinery would target CSR to that peculiar region. Whether this is due to the fact that this region is the initial and crucial switch donor site or whether it is a general feature of the sequences preceding C genes can now be grasped by comparing our Sµ deletion with a similar deletion of S This suggests that beyond structural (S sequences, I promoters, etc) and functional (germ line transcription, splicing, etc) similarities, there are likely other levels of regulation to account for such differential effects.
Analysis of switch junctions did not reveal any obvious microhomology indicating that the breaks were normally resolved by the NHEJ repair pathway. Strikingly, whereas the usual alterations (point mutations, deletions, duplications) were found downstream of the S
The very rare mutations upstream of the junction sites, the lack of obvious internal rearrangements in the mutant allele in preliminary experiments by Southern blot, together with the absence of a significant increase in AID-induced mutations in the Iµ-Cµ intron from mutant splenocytes or in the I Computer search for secondary structures suggests that many stem-loop structures49 may form in the mutated Iµ-Cµ region. However, only about half the breakpoints were found within these structures indicating that the stem loops are not the main targets of CSR machinery. Sequence analysis of the Iµ-Cµ intron in activated heterozygous splenocytes revealed an exquisite specificity of AID and a lack of a bystander effect on the mutated intron. This could be due to the removal, by our deletion, of DNA motifs recognized by AID or a putative cleaving enzyme or cofactor(s). Indeed, several proteins binding Sµ have been identified2 whose role is still unclear. Bearing in mind the biallelic nature of germ line transcription,8,54 this indicates that, although required, transcription per se is not sufficient for AID-induced mutations as suggested33 but needs the simultaneous formation of higher-order structures common to all S sequences, one likely candidate being the R-loop.14,15 We would like to propose the following model to account for the decreased initial cleaving activity involved in CSR in our mice. Upon appropriate stimulation, the cleaving enzyme(s) preferentially binds to the transcriptional machinery that initiates from germ line promoters similarly to what has been proposed for SHM.55,56 As transcription proceeds, the transcriptional complexes may be slowed down by the secondary (or higher) structures of S sequences generated by the first complexes, increasing the probability that the endonuclease(s) introduces mutations. In the absence of such structures, the complexes will proceed without pausing and the probability of introducing mutations will decrease. Conversely, in the absence of germ line promoter,57-59 the endonuclease(s) may still cleave the DNA, albeit at lower frequency. In the case of µ gene, cEµ acts as both a germ line promoter and an enhancer of transcription initiating from the promoters of the variable regions (PVHs). An important implication of our model is that the endonuclease(s) will be targeted to the VH region (SHM) or to the Sµ region (CSR) depending on the commitment of cEµ. Therefore, cEµ may well be the key component of an activation-induced molecular switch that controls the temporal dissociation of SHM from CSR.
We are indebted to Marianne Brüggemann and Michael S. Neuberger for the kind gift of C  phages and to Chantal Kress for kindly providing ES cells. We would like to thank an unknown reviewer for very helpful comments.
Submitted October 10, 2003; accepted December 31, 2003.
Prepublished online as Blood First Edition Paper, February 24, 2004; DOI 10.1182/blood-2003-10-3470.
Supported by grants from Association pour la Recherche sur le Cancer (grant no. 4403), Ligue Nationale Contre le Cancer, the Switch Network, and Conseil Régional du Limousin.
F.G. and Z.O. contributed equally to this work.
The online version of the article contains a data supplement.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: A. A. Khamlichi, CNRS UMR 6101, Laboratoire d'Immunologie, Faculté de Médecine, 2 rue du Dr Marcland, F-87025 Limoges cedex, France; e-mail: khamlich{at}unilim.fr.
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Top
Abstract
Introduction
, and S
are composed of pentameric tandem repeats such as GGGGT, GGGCT, and GAGCT, while S
sequences, which also contain these elements, consist of repeats of a 49–base pair (bp) sequence.
/
2.9-kb HindIII fragment). Among 480 resistant clones, 2 clones showed a recombinant band with the expected size and both allowed germ line transmission. A total of approximately 4.6 kb were thus replaced by a neor cassette flanked by 2 loxP sites (
(transforming growth factor 
) comprises the switch donor sites within Iµ and group B (
) those within the mutated Iµ-Cµ intron. The relative position of cEµf primer is indicated. cEµ indicates the core Eµ enhancer; H, HindIII; R, EcoRI; and X, XbaI. (B) Putative cryptic splice site in Iµ exon. (Top) Relative position of the primers used in RT-PCR. (Bottom) The first line shows the localization of the putative cryptic donor splice site (underlined) downstream of the octamer motif of the core Eµ enhancer. Three transcription initiation sites