The mu switch region tandem repeats are important, but not required, for antibody class switch recombination - PubMed (original) (raw)

The mu switch region tandem repeats are important, but not required, for antibody class switch recombination

T M Luby et al. J Exp Med. 2001.

Abstract

Class switch DNA recombinations change the constant (C) region of the antibody heavy (H) chain expressed by a B cell and thereby change the antibody effector function. Unusual tandemly repeated sequence elements located upstream of H chain gene exons have long been thought to be important in the targeting and/or mechanism of the switch recombination process. We have deleted the entire switch tandem repeat element (S(mu)) from the murine (mu) H chain gene. We find that the S(mu) tandem repeats are not required for class switching in the mouse immunoglobulin H-chain locus, although the efficiency of switching is clearly reduced. Our data demonstrate that sequences outside of the S(mu) tandem repeats must be capable of directing the class switch mechanism. The maintenance of the highly repeated S(mu) element during evolution appears to reflect selection for a highly efficient switching process rather than selection for a required sequence element.

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Figures

Figure 1

Generation of the ΔSμ mice. (A) Diagram of Sμ tandem repeats and map of the JH-Cμ intron. Eμ, intronic enhancer; Cμ, μ constant region exons; μmem, μ membrane exons; E, EcoRI; H, HindIII; B, BamHI; K, KpnI. In the expanded view of Sμ, each vertical line represents either a GAGCT or GGGGT sequence and the two HindIII sites represent the pair of sites flanking Sμ in the diagram of the wild-type (WT) allele. (B) Southern blot analyses of knockout mice. Genomic DNAs from mice with the indicated phenotypes were digested and hybridized. The left blot contains BamHI digests and was hybridized with pJ11 (reference 52), a 1.8-kb BamHI-EcoRI fragment containing JH3 and JH4. Sizes of wild-type (wt) and ΔSμ alleles are ∼8 and 5.3 kb, respectively. The right blot contains EcoRI digests and was hybridized sequentially with, first, a 900-bp KpnI-XbaI probe containing the μ membrane exons and, second, pM2-20 (reference 52), a 1.8-kb probe containing ∼1.4-kb of Sμ tandem repeat sequences. Sizes of the wild-type and ΔSμ alleles are ∼12 and 8.9 kb, respectively. Positions of λ HindIII DNA fragments are indicated. μmemb, μ membrane exons.

Figure 2

Analysis of IgM and IgG production in unimmunized ΔSμ mice and their littermate controls. (A) Animals were bled and total IgG titers were determined for three wild-type, six heterozygous, and eight mutant mice at weeks 6, 9, 12, and 15. (B) Isotype profile of serum from wild-type, heterozygous, and mutant mice were analyzed at week 15. (C) Serum IgM titers at week 15 were determined on a subset of wild-type, heterozygous, and mutant mice. (D) Surface IgM staining of wild-type and mutant mice was examined by flow cytometry using a FITC-conjugated monoclonal antibody reactive with mouse IgM.

Figure 3

Reduced, but easily detectable IgG expression on B cells from ΔSμ mice. IgG2b is more severely affected than IgG1 by the deletion of Sμ tandem repeats. Splenic B cells from wild-type or ΔSμ mice were cultured with either LPS plus IL-4 to induce IgG1 or with LPS plus dextran sulfate (DXS) to induce IgG2b. On day 4 the cells were stained for surface IgG and IgM and analyzed by flow cytometry. The percentage of cells in the upper gate (indicated in the top right corner of each panel) includes cells positive for IgG alone and for IgG and IgM together. The gates for IgG1 were set on wild-type cells treated with LPS alone, which does not induce switching to IgG1, and then were adjusted for the other isotypes to optimize separation of the populations. Gates for wild-type and ΔSμ cells were always the same for a given isotype.

Figure 4

DC-PCR demonstrates switch recombination at the DNA level is reduced in B cells from ΔSμ mice stimulated with LPS or LPS plus IL-4 (L+4). DNA template levels were adjusted by normalizing to the amount of AchR DC-PCR product and then twofold dilutions of adjusted levels of input or a plasmid standard were amplified for AchR, and for Sμ/Sγ1 and Sμ/Sγ2b recombination, with the incorporation of [α-32P]dCTP. The fold reductions relative to wild-type (WT) from two independent PCR reactions were: IgG1, 3.6 and 3.65; IgG2b, 9.7 and 8.5.

Figure 5

(A) ΔSμ and wild-type (WT) recombination junctions in the JH –Cμ intron. The wild-type junctions pictured are all from μ − γ1 recombinations and were derived from published sequences. Junctions nos. 1 and 2 are from reference 53, nos. 3 and 4 from reference 7, nos. 5–7 from reference 54, no. 8 from reference 21, nos. 9–19 from reference 23, nos. 20 and 21 from reference 55, no. 22 from reference 56, and no. 23 from reference 57. ΔSμ recombination junctions were generated by PCR from IgG1-producing hybridomas. Sites were from hybridomas 16B3 (A), 18A4 (B), 8B5 (C), 8A2 (D), 18B6 (E), 18B2 (F), 9A6 (G), 8C4 (H), 4D5 (I), and 16B5 (J). Only the relevant restriction sites are shown on the map. E, EcoRI; H, HindIII; X, XbaI. Location of the PCR primer used to generate ΔSμ junctions is represented by small arrowheads. (B) ΔSμ and wild-type (WT) switch junctions upstream of Cγ1. The wild-type junctions are all μ −γ1 recombinations and are derived from (reference 55). The wild-type junctions shown here are a subset of the group shown in A because we could not accurately place all the breakpoints within the γ1 tandem repeats. ΔSμ junctions were generated by PCR from IgG1-producing hybridomas; the small arrowheads represents the location of the PCR primer used.

Figure 5

Figure 6

Sequence analysis of ΔSμ IgG1 hybridoma switch recombination junctions. Approximately 80 bp of each clone is shown, 40 bp upstream and 40 bp downstream of each breakpoint. The sequence on top is the corresponding germline μ sequence, and the sequence on the bottom is the germline γ1 sequence. A small vertical line is drawn between bases if they are identical. The switch junctions are shown as either a large vertical line if there are not any shared base pairs, or as a box if there are shared base pairs. Junctions are assigned according to the following criteria: if there is a base change close to the potential breakpoint it must be followed by at least one base that is the same as germline; if there are two or more base changes they must be followed by at least the same number of germline bases. Base changes are marked with an asterisk, insertions have no corresponding germline sequence, and deletions have the corresponding germline base placed below the germline sequence.

Figure 7

Southern blots of ΔSμ IgG1 hybridomas. DNAs from 32 hybridomas were digested with EcoRI and hybridized. The blots on the left were hybridized with a 700-bp EcoRI-HindIII fragment located just 3′ of the Eμ enhancer (pJ14c; reference 53). On the right, the same blots were probed with a 10-kb EcoRI fragment containing the Sγ1 S region (pγ1EH10.0; reference 58). The germline bands are marked with an arrowhead and the sizes are ∼8.9 kb for the μ intron and 16 kb for the γ1 intron. Positions of Hind III fragments of λ phage DNA are indicated for each gel.

Figure 8

Analysis of the frequency of G nucleotides in the ΔSμ JH-Cμ intron. The top three graphs wrap around to form a continuous sequence from JH to the beginning of Cμ in the ΔSμ mouse. The bottom graph represents a portion of the sequence that has been removed in the ΔSμ mouse; however, ∼2.5 kb of tandem repeats is not included in this graph because the sequence is not known. The analysis was done by counting the number of G residues for nucleotide nos. 1–10 (numbers not shown), and plotting the value on the graph. This was repeated for nucleotide nos. 2–11, nos. 3–12, etc. Essentially, a peak in the graph indicates a stretch of DNA with numerous G residues. A random sequence would have 2.5 G residues per 10 nucleotides. An oval on the top line of the graph represents runs of G that are three nucleotides or greater. The positions of the recombination sites in the ΔSμ IgG1 hybridomas are indicated by a horizontal line between the 7th and 8th line of the graph. Relevant restriction sites and features of the intron are indicated.

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The mu switch region tandem repeats are important, but not required, for antibody class switch recombination - PubMed (original) (raw)