The Ig heavy chain intronic enhancer core region is necessary and sufficient to promote efficient class switch recombination
Eiko Sakai1,
Andrea Bottaro2,3 and
Frederick W. Alt1,2
1 Howard Hughes Medical Institute, The Children's Hospital, and
2 The Center for Blood Research and Department of Genetics, Harvard Medical School, Boston, MA 02115, USA
Correspondence to:
F. W. Alt, Howard Hughes Medical Institute, The Children's Hospital, 300 Longwood Avenue, Boston, MA 02115, USA
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Abstract
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The intronic IgH enhancer Eµ, which consists of the core enhancer (cEµ) flanked by 5' and 3' matrix attachment regions (MAR), has been implicated in the control of IgH locus recombination and transcription. Both cEµ and the MAR are required to enhance transcription of an IgH transgene. To elucidate the regulatory functions of cEµ versus its associated MAR in IgH class switch recombination (CSR), we have assayed ES cell lines which have targeted deletions of these elements, both individually and in combination, by the Rag-2-deficient blastocyst complementation method. Mutant B cells from chimeric mice were activated in culture and the influence of the mutations on CSR was assessed by analysis of B cell hybridomas. We find that the cEµ is necessary and sufficient for providing the functions of Eµ required for efficient CSR at the IgH locus. However, the 5' and 3' MAR sequences, as well as the known Iµ transcription start sites and the bulk of Iµ coding sequences, were dispensable for the process.
Keywords: Eµ enhancer, gene targeting, Ig class switching, matrix attachment region
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Introduction
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The IgH locus comprises a 5' region that harbors VH, DH and JH segments, and an ~120 kb 3' region that harbors the IgH constant regions genes (CµC
C
3C
1C
2bC
2aC
C
) (reviewed in 1). IgH variable region genes are assembled via V(D)J recombination at the pro-B cell stage, leading to the expression of µ IgH chains which contain constant region sequences encoded by the Cµ exons. Subsequent assembly and expression of Ig light chain variable region genes and Ig light chain expression leads to the generation of the IgM-expressing B cells (reviewed in 2). Activation of peripheral B cells in the context of various activators and lymphokines leads to the replacement of the Cµ exons with those of a downstream constant region gene by a recombination/ deletion mechanism. This class switch recombination (CSR) reaction, which occurs between repetitive switch (S) region sequences that lie upstream of the CH exons, leads to the production of Ig isotypes encoded by the downstream CH genes (e.g. IgG, IgE and IgA) (reviewed in 24).
CSR between S region sequences is preceded by and appears to require the induction of germline transcription from promoters which are located upstream of exons (termed I exons, e.g. Iµ, I
3, I
) which lie just 5' of the corresponding S regions (reviewed in 3,4). Primary germline transcripts are processed to a mature transcript in which the I exon is joined to the CH exons. Several lines of evidence, including gene-targeted mutational studies, have demonstrated the importance of germline transcription and of the elements upstream of the S regions (germline promoter and I exons) for CSR (reviewed in 3,4). It has been proposed that local transcription and/or local transcriptional regulatory elements play a direct role in regulating CSR activity at individual S regions (511). In addition, various gene-targeted mutational studies have implicated a long-range CSR control region downstream of the CH locus in regulation of the differential transcription of certain germline CH genes (3,1214).
Multiple enhancer elements have been identified within the IgH locus, including the Eµ intronic enhancer between JH and Cµ (1519; reviewed in 2022) and a series of enhancers (collectively referred to as the 3' IgH regulatory region) which lie downstream of C
(reviewed in 23). Eµ consists of a small 220 bp core element (hereafter referred as cEµ; Fig. 1A
) which is necessary and sufficient for transcriptional stimulation (reviewed in 24) and two flanking nuclear matrix attachment regions (MAR) which have been identified biochemically (25). These MAR have been demonstrated to contribute positively to Eµ function with respect to enhancement of transcription from a transgene promoter (2628). In addition to this particular function attributed to the Eµ MAR, a variety of general MAR functions have been proposed (review in 2934). For example, MAR often are found in close association with enhancers, promoters and putative replication origins, potentially serving to anchor these elements to specific nuclear matrix sites (25,3538). In addition, MAR have been implicated in constituting physical boundaries between genes by forming chromosome loops (35,36).

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Fig. 1. Targeted mutations of cEµ and MAR elements. (A) Schematic of the IgH locus showing DQ52, JH gene segments, intronic enhancer (Eµ), switch µ region (Sµ) and the µ constant region gene (Cµ). BamHI (B), HindIII (H), HinfI (Hf), SacI (S) and XbaI (X) restriction sites are shown. Also shown is probe A (a 0.9 kb XbaIBamH I fragment between Sµ and Cµ). The schematic is not drawn to scale. (B) Schematic diagram of targeted mutations of cEµ and MAR elements. Shaded and filled boxes represent MAR (5'MAR, a 350 bp XbaIHinfI fragment and 3'MAR, a 430 bp HinfIXbaI fragment ) and cEµ (a 220 bp HinfIHinfI fragment) elements respectively. Factor binding sites are indicated by open circles (E, µE15, µB, and Oct). The Iµ exon is shown by a bracket and the transcription start sites are shown over the 3'MAR by arrows. The loxP sites are indicated by open triangles. Mutant ES cell lines were employed in which the cEµ ( cEµ), the 5' MAR ( 5'MAR), the 3' MAR ( 3'MAR) or both MAR ( MARS) were replaced by single loxP sites (40).
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Several gene-targeted mutational studies in which relatively large regions of DNA sequence encompassing Eµ were removed suggested that this enhancer may be positively involved in regulating CSR (7,39). In one study, Sµ recombination activity was suppressed significantly, although not completely, by a 6 kb deletion of the region extending from DQ52 through cEµ (39). More recently, we have demonstrated that deletion of a more limited set of sequences spanning Eµ similarly impaired CSR (7). To specifically evaluate the function of Eµ in CSR and to elucidate potential individual roles of the 5'MAR, the cEµ, and the 3'MAR elements, we now have assayed effects of deletion of these components, either individually or in combination, on CSR activity in B lymphocytes.
To generate the mutant B cells, we utilized mutant F1 ES cell lines which harbored heterozygous deletion mutations in either cEµ (a 220 bp HinfIHinfI fragment), the 5'MAR (a 350 bp XbaIHinfI fragment), the 3'MAR (a 430 bp HinfIXbaI fragment) or both the 5'MAR and 3'MAR (Fig. 1B
) (40). These mutant ES cells were termed
cEµ,
5'MAR,
3'MAR or
MARS respectively. In the F1 ES cell line, the IgHa and IgHb alleles can be distinguished by polymorphisms for restriction endonuclease sites; in all of the mutant ES cell lines, deletions were targeted into the IgHa allele via the cre/loxP approach, resulting in the replacement of the individual Eµ/MAR elements by a single loxP site (7,39). The mutant ES cell lines were used to generate mutant B lymphocytes via the Rag-2-deficient blastocyst complementation method (41) as previously described (40). Mutant and control splenic B lymphocytes were activated for 5 days by culturing in the presence of bacterial lipopolysaccharide, at which time hybridomas were generated (7,40). IgG-expressing hybridomas thus generated have undergone CSR on their productive allele, and previous studies have shown that the majority of these hybridomas have also undergone CSR activation (either as complete SµSH switch events or as internal Sµ deletions) on the non-expressed allele (7,42,43). We therefore assessed the extent of CSR on the non-functional allele by Southern blot analysis of genomic DNA isolated from hybridoma lines using BamHI restriction and hybridization to a probe (probe A; Fig. 1A
) which can detect loss of Cµ (i.e. complete switch events) on the non-productive allele. In addition, SacI-digested DNA also was assayed for hybridization to probe A in order to detect internal deletions within Sµ, which are also indicative of CSR activity (39,43).
Of the hybridomas generated from wild-type cells, only 5% (two of 35) retained Sµ/Cµ in the germline configuration on the non-productive allele; of the remainder, 24% had undergone internal Sµ deletions and 70% had deleted Cµ (Table 1
). Of 21 IgG-expressing hybridomas with a
cEµ mutation, nine (43%) retained the targeted, non-expressed allele in germline configuration, a highly significant increase compared to normal hybridomas (Yates' corrected
2 = 9.91, P = 0.0016) (Table 1
). This is a phenotype comparable to that observed in
Eµ mutant B cells (in which 53% non-expressed alleles in the germline configuration) (7). These data demonstrate that cEµ is involved in effecting CSR. In
3'MAR or
MARS B cell hybridomas, the mutated and non-mutated alleles underwent equally efficient CSR. In the
5'MAR hybridomas, a slightly higher number of germline
5'MAR alleles versus normal alleles was observed (two of 94 normal versus 10 of 94
5'MAR) (Table 1
); this increase is of borderline significance (Yates' corrected
2 = 4.36, P = 0.037). This small increase might indicate a minor effect of the 5'MAR on CSR regulation; although the finding of normal CSR in hybridomas with mutation of both MAR suggests that this may just be a sampling anomaly.
A potential explanation for the observed phenotype of
cEµ mutants is that the decrease in CSR activity in these allele represents an indirect effect of the mutation.
cEµ alleles differ from normal alleles in that they undergo inefficient V to DJ rearrangement (40); therefore, most of the
cEµ alleles in the hybridomas are in DJ status (40). Thus, potential VH-proximal regulatory elements are absent from
cEµ loci, and may affect their expression and/or ability to switch.
To eliminate this potential bias, we analyzed CSR frequency specifically in the subset of hybridomas bearing only DJ rearrangements on their normal or targeted alleles (Table 2
). In the three different
MAR mutants, only two out of a total 37 DJ-rearranged normal alleles had germline Sµ regions (5.4%). Therefore, normal DJ alleles are able to undergo CSR at high frequency. Similarly, DJ alleles bearing
5'MAR,
3'MAR or
MARS mutations do not significantly differ from their normal counterparts (Table 2
). On the other hand, DJ-rearranged
cEµ alleles display the same significant decrease in CSR activity (eight of 15 germline Sµ regions or 53%) that was observed in the total sample (Table 1
). Therefore, we conclude that V to DJ rearrangement does not affect the rate of CSR in cis and that the inhibition of CSR observed in
cEµ mutants is not due to secondary effects of impaired VDJ rearrangement. Taken together, the CSR data in the various mutant hybridomas demonstrates that cEµ is important for regulating IgH CSR and can function in this regard in the absence of its associated MAR.
Our current studies show that the cEµ is both necessary and sufficient for Eµ function in CSR (although not absolutely required for residual activity). In this context, the core is known to act as a promoter for germline Iµ transcripts (44), whose start sites are within the 3'MAR (Fig. 1A
). Furthermore, the 3'MAR constitutes ~70% of the Iµ exon sequence (25,45). Therefore, our results with the
3'MAR mutants clearly indicate, somewhat surprisingly, that neither the transcription start sites nor the presence of the bulk of the Iµ exon are essential for CSR, ruling out a specific role for this conserved exon sequence per se in promoting CSR (Table 1
). Finally, the data from the
MARS mutation also rule out an essential role of the MAR in CSR via chromosome organization and nuclear matrix interaction. There are several potential explanations for the CSR-promoting activity of cEµ in the absence of MAR. First, even in the absence of the classical Iµ initiation sites, cEµ may be able to generate some form of germline transcript that employs the RNA splice donor site retained in the sequence at the 3' end of the Iµ exon. Indeed, some novel transcription start sites were observed in a transgene lacking the MAR (27). Second, cEµ can bind a number of nuclear factors which could be involved in recruiting the CSR machinery, or in facilitating the interaction between the µ locus and downstream CH genes. Finally, cEµ may drive the initiation of DNA replication as Eµ is embedded in a replication initiation region (38) and DNA replication has been implicated in the CSR mechanism itself (46).
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Acknowledgments
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We thank to Laurie Davidson and Roger Ferrini for technical assistance with mouse work, and the members of the Alt lab for helpful discussions. This work was supported by AI35714 and AI20047 (to F. W. A.). E. S. was supported in part by the Japan Society for Promotion of Science.
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Abbreviations
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C | constant |
CSR | class switch recombination |
H | heavy chain |
MAR | matrix attachment region |
S | switch |
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Notes
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3 Present address: Immunology Unit, Department of Medicine, University of Rochester Medical Center, Rochester, NY 14642, USA 
Transmitting editor: D. Kitamura
Received 27 March 1999,
accepted 17 June 1999.
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