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*
Departments of Medicine and Microbiology and Immunology, and Cancer Center, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642; and
Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 021139
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(1). Both in vivo and in vitro, class switch recombination (CSR) can be directed toward specific CH genes depending on the signals received by the B cell from activators and cytokines, and is preceded by transcriptional activation of the induced genes (1, 2). The transcripts generated at this stage (called CH germline, or "switch" transcripts) derive from promoter elements located directly upstream of the S regions targeted for rearrangement, and in their mature form contain a noncoding "I" exon spliced onto exon 1 of the CH gene. Targeted mutagenesis experiments have indicated that integrity of the germline transcription units (promoters and/or exons), rather than transcription through the S regions, is essential for CSR (3, 4, 5, 6, 7, 8, 9). More recently, splicing of germline transcript precursor has been shown to probably constitute an essential step in CSR activation (7, 10, 11). Thus, a 5'-truncated or spliced-out RNA species containing the S region may be an integral part of the CSR enzymatic machinery, or perhaps the same factors that effect RNA splicing may also be involved in DNA recombination pro- cesses. In support of the hypothesis of a functional connection between RNA splicing and CSR is the identification of proteins involved in RNA processing as components of switch region DNA-binding complexes (12, 13, 14) and the precedent of yeast factors involved in both splicing and meiotic recombination (15).
The IgH µ gene, like the other CSR-capable CH genes, harbors an I exon, Iµ, located immediately downstream of the IgH intronic enhancer (Eµ), which serves as its promoter (16, 17). Interestingly, the E4 box and the octamer elements within Eµ seem to be specifically required for Iµ transcription, rather than Eµ enhancer function (17), and ectopic expression of the E4-binding E47 transcription factor has been shown to be sufficient to induce Iµ transcripts in non-B lymphoid cells (18, 19). Thus, Iµ transcription appears to be a very specific and regulated aspect of Eµ activity, supporting the hypothesis of an important role for Iµ transcripts in B cell function. However, Iµ expression is quite constant throughout B cell development and does not increase upon CSR activation (16, 20, 21, 22). This observation, along with the finding that, in the majority of LPS-activated B cells, Sµ regions remain remarkably stable and show no signs of recombination activity (23, 24, 25), suggests that other layers of control, in addition to the production of Iµ transcripts, may be present for µ CSR.
Deletions encompassing Eµ and part of Iµ significantly reduce, but do not completely block, µ CSR (24, 25, 26). This is in contrast with the other CH genes, which are entirely dependent on the expression and splicing of their I region transcripts for efficient CSR (3, 4, 6, 7, 9). The analyzed mutations however involved only the 5'-most 500 bp of the 700-bp Iµ exon, leaving the splice donor site intact. Therefore, those experiments cannot rule out the possibility that transcripts generated from the more upstream VH or D region promoters could be aberrantly spliced at the remaining Iµ splice donor, allowing for at least some Iµ function.
To specifically test whether splicing of the Iµ exon is indeed necessary for µ CSR, we therefore generated a mutation that specifically deletes the Iµ splice donor.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The 
Iµ-s construct was generated by replacing the 236-bp
BglI-SpeI fragment spanning the Iµ splice donor
site with a LoxP-flanked pgk-neor gene (Fig. 1
). The two flanking arms are an 8.7-kb
EcoRI-BglI fragment at the 5', and a 632-bp
SpeI-HindIII fragment at the 3'; a pgk
promoter-driven tk gene was inserted at the 3' end of the
construct for double G418/gancyclovir selection of homologous
recombinants (27). Iµ-s transfected
G418/gancyclovir-resistant ES cells clones were screened for homologous
recombination by Southern blotting. Four targeted clones were injected
into C57BL/6 blastocysts, and resulting chimeric mice bred for germline
transmission with C57BL/6 mice. Germline heterozygous progeny of the
original chimeras were then bred with the adenovirus EIIA
promoter-driven Cre transgenic deleter mouse
(28) for LoxP-mediated deletion of the
neor gene, and deleted progeny were bred back
into C57BL/6 and to each other to obtain homozygotes.
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Serum from >6- to 16-wk-old homozygous and heterozygous mutant mice as well as normal littermates and C57BL/6 controls was analyzed by ELISA using mouse isotype-specific goat polyclonal Abs (Southern Biotechnology Associates, Birmingham, AL). Splenocytes from 6- to 16-wk-old mutants and controls were cultured for 5 days in complete RPMI 1640 medium with 10% FCS and 20 µg/ml LPS, 10 µg/ml dextran sulfate with or without 25 ng/ml recombinant mouse IL-4 (R&D Systems, Minneapolis, MN). At day 5, supernatants were collected and analyzed by ELISA, and B cell blasts were stained for surface Ig heavy chain isotype expression using fluorochrome-conjugated monoclonal rat Abs (PharMingen, San Diego, CA, and Southern Biotechnology). Stained cells were analyzed by flow cytometry on a Becton Dickinson FACScalibur instrument using CellQuest software (Mountain View, CA).
RNA analysis
Total RNA was extracted from splenocytes, LPS-activated blasts, and indicated cell lines using the Trizol reagent (Life Technologies, Rockville, MD). Northern blot analysis was performed after electrophoresis of 15 µg of total RNA on 2% formaldehyde and 1% agarose gels using random-primed 32P-labeled probes. Antisense probes for RNase protection assays (RPAs) were labeled with [32P]UTP using the T3/T7 Maxiscript kit (Ambion, Austin, TX). A total of 15 µg of total RNA/sample was hybridized to the radiolabeled probes and subjected to RPA using the RPAII kit (Ambion). The RPA products were separated by 6 M urea/PAGE and detected by autoradiography for 2–16 h.
5' rapid amplification of cDNA end (RACE) cloning of new µ-containing transcripts
Cµ-containing transcripts were cloned from total RNA from the Eµ-deleted hybridoma FSKO-LPS7 (25) using the Boehringer-Mannheim 5'/3' RACE kit (Indianapolis, IN) with the following nested Cµ antisense oligonucleotides: AS-Cµ1 (5'-TTCTGGTAGTTCCAGGTGAA-3'), 3'µCH1 (5'-ACCAGATTCTTATCAGACAG-3'), and Cµ1AS29–47 (5'-CTCTCGCAGGAGACGAGG-3'). The resulting mixture of PCR products was cloned into a PCR2.1 vector (Invitrogen, San Diego, CA), and individual recombinant plasmids were directly analyzed by sequencing.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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J1 ES cells were transfected with the Iµ-s construct and
grown under double G418/gancyclovir selection (27).
Resistant colonies were picked, expanded, and analyzed by Southern
blotting. In the germline configuration, the CµXH probe detects 2
bands on BamHI-digested DNA because of the presence of an
internal BamHI site (Fig. 1
A). The larger 12-kb
band corresponds to the 3' portion of the µ gene and its 3' flanking
sequences, which would not be affected by targeting of the Iµ exon.
The smaller 9.5-kb band spans the JH-Cµ intron, and upon Iµ-s
homologous recombination is reduced to about 7 kb. This novel pattern
was detected in 14 of 168 analyzed clones (Fig. 1B).
Homologous integration of the 5' arm of the construct was confirmed
using the 5' arm-internal Eµ probe (Fig. 1A; data not
shown). Four targeted clones were injected into C57BL/6 blastocysts,
chimeric mice were generated, and germline transmission of the mutation
was obtained from two independent clones (Fig. 1C). Mice
bearing the targeted mutation (Iµ-sneo/neo
mice) were then bred to the deleter transgenic mouse
(28), which induces LoxP-Cre-recombinase-mediated deletion
of the neor gene at the flanking LoxP sites. In
the Iµ-sneo mice, the Cre-deletion event causes the
replacement of the neor-containing 4.2-kb
BamHI band with a shorter 2.7-kb band
(Iµ-s-/- mice, Fig. 1C). The
Cre-deleted mice were then bred back in the C57BL/6 background; the
genotype of the line is therefore mixed (mostly C57BL/6, with FVB
contribution from the deleter strain and 129 from the
original ES cells).
Normal CSR in Iµ splice donor mutants
As a first approach to assessing the effects of deletion of the
Iµ splice donor on CSR, we tested serum Ig levels in
Iµ-s-/- mice and controls and found no
significant differences between the cohorts (Fig. 2A). Similarly, supernatants
from 5-day LPS/dextran splenocyte cultures with or without IL-4 had
similar levels of all tested Ig isotypes (IgM, IgG1, IgG3, and IgG2b)
in Iµ-s-/- and control mice (Fig. 2B). Homozygous mutant B cells also displayed a pattern of
expression of surface IgM, IgG1, IgG2b, IgG3 (Fig. 3), and IgE (data not shown)
indistinguishable from that of controls.
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1 exon (7, 10), does not affect
CSR. Lack of steady-state Iµ transcripts in Iµ splice donor mutants
To confirm that replacement of the Iµ splice donor site with a
LoxP site did not lead to the production of aberrantly spliced
Iµ-like transcripts, we performed Northern blot analysis of total RNA
from mutant and control LPS blasts using an Iµ-specific probe. Unlike
control samples, which display a prominent 2.5-kb Iµ-hybridizing
band, mutants show only weak hybridizing signals with this probe (Fig. 4A). These weak bands are
however most likely derived from cross-hybridizing transcripts, since
they are also observed in RNA from LPS blasts in which the entire
region corresponding to the Iµ probe
(EcoRI-HindIII fragment) has been replaced by a
neor gene (Fig. 4A)
(29). Thus, in the absence of the Iµ splice donor site,
there is no accumulation of steady-state Iµ transcripts. Although
this is presumably due to instability of the long unspliced precursor,
we cannot rule out the possibility that transcriptional regulatory
elements relevant for Iµ expression have been affected by the
Iµ-s mutation.
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If the widely accepted model of a role for I exon splicing in CSR
(1, 7) is correct, the results mentioned above raise the
question of how µ CSR is accomplished in the absence of detectable
Iµ splicing. We hypothesized that other previously unrecognized
transcripts from the µ locus could provide the required splicing
event. Normal cells express a number of different µ-containing
transcripts (20, 21) hampering the identification of
potential new transcripts. As a simplified model, we took
advantage of hybridoma lines expressing only a limited subset of µ
transcripts. The hybridomas were generated from B cells heterozygous
for a deletion extending from the Eµ enhancer to the 5' portion of
Iµ, up to 320 bp upstream of the splice donor (Eµ-del hybridomas)
(25). The Cµ gene has been lost on the normal allele of
these IgG-secreting hybridomas because of CSR, while it is retained on
the targeted allele. In addition, the JH region of the targeted locus
is in the germline configuration because of a significant defect in VDJ
recombination (29). Finally, because of the Eµ deletion,
these hybridomas cannot express any Iµ-initiated transcripts. Thus,
the Eµ-del- hybridomas represent a simplified system to look for
Eµ-independent transcripts in the µ locus. The only known Cµ
transcripts that can be expressed from the targeted loci are those
originating from the DQ52 promoter, which proceed through the JH region
and splice the JH1 segment onto the Cµ1 exon
(µo transcripts) (30, 31). These
transcripts can be detected with a µo-specific
probe mapping between DQ52 and JH1. Surprisingly, however, Northern
blot analysis showed that only a subset of Cµ-containing transcripts
from the hybridomas also hybridizes with the µo
probe (Fig. 4B). Initial RPA with Cµ1-specific probes
showed that these transcripts were in the sense orientation and
included unknown sequences spliced onto the Cµ1 acceptor site (data
not shown). These data show that a previously unrecognized class of
transcripts is expressed in the µ locus of Eµ-del-deleted
hybridomas, and that these transcripts include the Cµ gene spliced
onto novel sequences.
Cloning and characterization of two classes of µx transcripts
The detection of a novel type of transcripts in Eµ/Iµ mutants
strengthened our hypothesis that other transcripts may be responsible
for the CSR activity in Iµ splice donor mutants. If this is the case,
however, these transcripts should originate in a region relevant for
CSR (possibly upstream of Sµ), and they should be expressed in normal
cells, in particular in B cells undergoing CSR. We therefore utilized
5' RACE to identify Cµ containing transcripts from one of the
Eµ-del hybridomas described above. From the pool of clones obtained
by 5' RACE, we sequenced nine clones selected for hybridizing with a
Cµ-specific oligonucleotide but not the µo
probe. Two of these clones showed µ locus sequences spliced onto the
Cµ1 exon; both sequences mapped to the region between Iµ and Sµ.
In particular, the two clones, µx18 and
µx31, correspond to almost contiguous stretches
located immediately upstream of the Sµ repeats (Fig. 5). A consensus splice donor is present
in both genomic sequences immediately 3' of the site of junction of the
novel exon with the Cµ1 exon, suggesting that the cloned products
resulted from a bona fide spliced RNA transcript rather than
artifactual PCR products. Both clones, although probably not complete
(see below), have multiple stop codons in all three reading frames, a
feature common to all I region germline transcripts (reviewed in Ref.
2).
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A). The latter probably
represent precursor RNAs or the same exons spliced onto other
sequences, such as the C genes expressed by the normal allele and
the fusion partner IgH loci.
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B)
as well as in Iµ-s-/- samples (Fig. 6C). Using RPA probes for µx and
Cµ exon sequences in combination, we could estimate the amount of
µx-Cµ transcripts to be about 1–5% of the
total µ transcripts in splenocytes and activated B cells (data not
shown). Thus, the steady-state level of µx
transcripts is rather low, although clearly detectable, and quite
constant in lines at different developmental stages, a pattern that is
very similar to that of Iµ. While our data suggest that, like Iµ,
the novel transcripts are not up-regulated by activation, specific
experiments comparing pure resting B lymphocytes (rather than total
splenocytes) and B cell blasts are required to conclusively establish
this point. However, the expression of µx
transcripts from loci bearing a deletion of the Eµ enhancer and Iµ
5' region (such as Eµ-del hybridomas) indicates that these novel
transcripts are under different regulatory elements than Iµ. One of the novel Iµ-like transcript likely initiates within the Iµ-Sµ region
An important aspect regarding the µx transcripts is whether they indeed initiate independently of other µ locus transcripts from specific promoter elements, or whether they represent alternatively spliced versions of VDJ- or Iµ-initiated transcripts.
As discussed above, the µx transcripts are expressed at very low levels, and they are nested within a large intron of two mRNAs, VDJ-Cµ and Iµ-Cµ, which are transcribed at a much higher rate. These two factors complicate the search for initiation sites using conventional techniques, generating high lane background in RPA, primer extension, and S1 nuclease protection experiments.
Nevertheless, RPA analysis using a genomic probe spanning the 5'
portion of the cloned µx18 sequence and its 5'
flanking regions revealed three predominant protected bands located
within a 80-bp range (Fig. 7A). These bands may represent
either bona fide initiation sites for the µx18
transcript or splice acceptor sites from a yet unidentified upstream
exon. However, no splice acceptor consensus sequences are detected in
the relevant regions by either the hspl or nnssp
splice site search programs available at the Baylor College of Medicine
Search Launcher web site
[http://dot.imgen.bcm.tmc.edu:9331/seq-search/gene-search.html)
(32)]. Moreover, the region immediately preceding the
putative initiation sites contains a number of potential transcription
factor DNA binding sites, including an octamer and an E47 consensus
binding site (Fig. 7B), both of which have been shown to be
critical for Iµ expression (17). A more detailed
structural and functional analysis of this putative promoter region to
establish whether elements in this region are involved in
µx18 transcription is in progress.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, C
, and C
genes is dependent on the expression of I region germline transcripts,
and that splicing of the germline transcript precursors is probably an
essential step for CSR activation (Refs. 7, 10, 11 ;
reviewed in Ref. 1). It is therefore rather surprising
that analogous experiments in the µ locus show that only 50–70% of
the CSR activity at the µ locus is eliminated by deletion of the 5'
half of the Iµ exon and its promoter elements located within the Eµ
intronic enhancer (24, 25, 26). However, µ locus
transcription is still clearly detectable in these mutants, and since
the Iµ splice donor site was not deleted by the mutation, this site
could still have served as donor for splicing events of large precursor
transcripts generated upstream of the JH segments. Therefore, those
experiments were not conclusive in terms of the role of Iµ splicing
in CSR. In the present report, we show that a specific mutation of the Iµ splice donor does not abolish CSR, even in the absence of detectable splicing of the residual Iµ sequences. We therefore reasoned that either the CSR/germline transcript "splicing model" had to be revised, at least for the µ locus, or that other transcripts were providing the necessary splicing event for µ CSR in these mutants. Indeed, we were able to show that two novel exon sequences, µx18 and µx31, mapping to the region between Iµ and Sµ, are spliced onto the Cµ1 exon in B cells from different stages of development, including switching LPS-activated B cell blasts. Although transcripts for both sequences are expressed from mutants lacking the Eµ enhancer, we were able to identify potential initiation sites in the Iµ-Sµ region only for µx18, which may therefore represent a bona fide second class of germline transcript. As discussed in Results, the low level of expression of the µx transcripts does not allow us to completely rule out the possibility that they represent alternative splicing products of larger µ locus precursor transcripts. Thus, a definitive proof of the origin of µx18 and µx31 transcripts will require additional experiments.
The µx18 transcripts share a number of similarities with the Iµ transcripts. Like Iµ, the µx18 exon is spliced onto the Cµ1 exon and does not possess any significant open reading frame, although, like Iµ, it could potentially be translated starting at a Cµ1 ATG codon (33). Also similar to Iµ, µx18 is expressed at relatively constant levels throughout B cell development and possibly through activation. Finally, if our RPA initiation site data are correct, µx18 may initiate in proximity to putative binding sites for Oct and E47, which are the principle effectors of Iµ transcription (17). It should be noted, however, that µx18 and µx31 represent only a small fraction (a few percent) of the total µ-containing transcripts, in contrast to the high levels of expression of Iµ, whose levels are comparable to those of VDJ-µ transcripts (16, 21).
Detection of µx31-Cµ- and µx18-Cµ-containing transcripts in Eµ-del hybridomas strongly suggests that their expression is independent of Eµ. Transgenic studies from Sigurdadottir et al. (34) have shown that a potential novel enhancer element involved in IgH transcription in activated B cells may map to the region between Iµ and Sµ. In addition, DNase-hypersensitive sites have been detected in the µx exon regions in human B cells, and at least in T cells in the mouse (35, 36). It is intriguing to speculate that such potential enhancer element(s) may be involved in µx transcript expression, just as Eµ acts as the Iµ promoter.
In mice bearing the JHT mutation (24), which spans the entire DQ52-JH-Eµ region but not the putative Sigurdadottir enhancer (34), only traces of µ-containing transcripts are detected. It would therefore be of interest to establish whether these remaining µ transcripts in JHT mutants are indeed the µx18 and µx31 transcripts at their normal steady-state rate (which as we discussed is much lower than other µ transcripts), or whether the presence of the larger deletion does in fact affect their expression, either by deleting some unknown regulatory element or by significantly altering the structure of the locus. Since the JHT heterozygotes, like the other Eµ mutants, are still able to switch at low levels, we would predict that they would express µx18 and µx31 transcripts.
Also notable is the fact that DNase I-hypersensitive sites were
recently identified about 1–2 kb 3' of the I1 exon
(37), raising the possibility that other germline
transcription units may possess a structure similar to the
Iµ/µx region. However, whether additional
transcripts are indeed generated from the 1 region remains to be
shown.
Finally, why should the µ locus present with such a complex, apparently redundant transcriptional activity? One obvious but teleological argument is that multiple transcription units may protect against the effects of mutations eliminating one or the other. Since Iµ mutations could be predicted to block CSR altogether, they would result in extremely severe immunodeficiency comparable to that observed in diseases such as hyper-IgM syndrome or common variable immunodeficiency.
A more likely explanation is that duplication of the Iµ/µx transcription units may have resulted from the inherent instability of S regions, which often undergo germline deletion/duplication events (38); this would explain the presence of octamer and E47 binding sites upstream of both Iµ and µx18 as remnants of the original duplicated unit. We should however point out that both the E47 and Oct sites in the µx18 putative initiation region, although conforming to published consensus sequences, are not closely related to those in the Eµ enhancer. Finally, as discussed above, an inherent promoter activity may be associated with the enhancer element identified by Sigurdadottir et al. (34).
In summary, our results highlight a surprisingly complex and unique structure for the µ locus germline transcript region, with two, possibly three Iµ-like exons. These findings suggest a likely explanation for the puzzling result of Eµ/Iµ-independent switching, and most importantly provide a new framework for the characterization of the elements involved in the control of CSR at the µ locus.
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Acknowledgments |
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Iµ-s mutant mouse strain,
and to Dick Insel, John Manis, Kathy Seidl, and Eiko Sakai for careful
review of this manuscript. A.B. also thanks Bortolo Nardini for
inspiration. |
Footnotes |
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2 Address correspondence and reprint requests to Dr. Andrea Bottaro, Immunology Unit, University of Rochester Medical Center, Box 695, 601 Elmwood Avenue, Rochester, NY 14642. E-mail address:
3 Abbreviations used in this paper: S, switch; CH, heavy chain constant region; CSR, class switch recombination; ES, embryonic stem; RPA, RNase protection assay; RACE, rapid amplification of cDNA end.
Received for publication July 2, 1999. Accepted for publication November 19, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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2b promoter and exon. EMBO J. 12:3529.[Medline]
promoter by gene targeting alters control of immunoglobulin heavy chain class switching. Proc. Natl. Acad. Sci. USA 90:3705. exon-deficient mice: role of germline transcription in class switch recombination. J. Clin. Invest. 97:477.[Medline]
2b exon for class switching. Int. Immunol. 10:1683. exon-replacement mice synthesize a spliced HPRT-C transcript which may explain their ability to switch to IgA: inhibition of switching to IgG in these mice. Int. Immunol. 11:37. hybrid germline transcripts subsequent to immunoglobulin heavy chain class switching. Int. Immunol. 6:491.1. Int. Immunol. 10:527.This article has been cited by other articles:
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  Z. Oruc, A. Boumediene, M. Le Bert, and A. A. Khamlichi Replacement of I{gamma}3 germ-line promoter by I{gamma}1 inhibits class-switch recombination to IgG3 PNAS, December 18, 2007; 104(51): 20484 - 20489. [Abstract] [Full Text] [PDF]
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 M. Samara, Z. Oruc, H.-L. Dougier, T. Essawi, M. Cogne, and A. A. Khamlichi Germ line transcription in mice bearing neor gene downstream of I{gamma}3 exon in the Ig heavy chain locus Int. Immunol., April 1, 2006; 18(4): 581 - 589. [Abstract] [Full Text] [PDF]
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 B. Tirosh, N. N. Iwakoshi, L. H. Glimcher, and H. L. Ploegh XBP-1 specifically promotes IgM synthesis and secretion, but is dispensable for degradation of glycoproteins in primary B cells J. Exp. Med., August 15, 2005; 202(4): 505 - 516. [Abstract] [Full Text] [PDF]
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 H. Liu, J. Wang, and E. M. Epner Cyclin D1 activation in B-cell malignancy: association with changes in histone acetylation, DNA methylation, and RNA polymerase II binding to both promoter and distal sequences Blood, October 15, 2004; 104(8): 2505 - 2513. [Abstract] [Full Text] [PDF]
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A. A. Khamlichi, F. Glaudet, Z. Oruc, V. Denis, M. Le Bert, and M. Cogne Immunoglobulin class-switch recombination in mice devoid of any S{micro} tandem repeat Blood, May 15, 2004; 103(10): 3828 - 3836. [Abstract] [Full Text] [PDF]
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
)-flanked pgk-neor
gene replaces a 236-bp BglI-SpeI fragment
spanning the Iµ splice donor site. Bottom, Structure
of the targeted locus after Cre-mediated deletion. B,
Southern blot analysis of 
µx18) and the approximate position of
the putative major initiation sites (*) are indicated. Underlined
sequences mark some potential binding sites for various transcription
factors, identified from the TRANSFAC database of transcription factor
binding sites (