DNA polymerase β is able to repair breaks in switch regions and plays an inhibitory role during immunoglobulin class switch recombination

Immunoglobulin (Ig) class switch recombination (CSR) is initiated by activation-induced cytidine deaminase (AID), which converts cytosines to uracils in switch (S) regions. Subsequent excision of dU by uracil DNA glycosylase (UNG) of the base excision repair (BER) pathway is required to obtain double-strand break (DSB) intermediates for CSR. Since UNG normally initiates faithful repair, it is unclear how the AID-instigated S region lesions are converted into DSBs rather than correctly repaired by BER. Normally, DNA polymerase β (Polβ) would replace the dC deaminated by AID, leading to correct repair of the single-strand break, thereby preventing CSR. We address the question of whether Polβ might be specifically down-regulated during CSR or inhibited from accessing the AID-instigated lesions, or whether the numerous AID-initiated S region lesions might simply overwhelm the BER capacity. We find that nuclear Polβ levels are induced upon activation of splenic B cells to undergo CSR. When Polβ−/− B cells are activated to switch in culture, they switch slightly better to IgG2a, IgG2b, and IgG3 and have more S region DSBs and mutations than wild-type controls. We conclude that Polβ attempts to faithfully repair S region lesions but fails to repair them all

Structure of germline immunoglobulin heavy-chain gamma 1 transcripts in interleukin 4 treated mouse spleen cells

Antibody class switching is mediated by a DNA recombination event that replaces the C mu gene with one of the other heavy (H) chain constant region (CH) genes located 3\u27 to the C mu gene. The regulation of this process is essential to the immune response because different CH regions provide different biological functions. Correlative evidence indicates that the isotype (class) specificity of the switch is determined by the accessibility of specific CH genes as indicated by hypomethylation and transcriptional activity. For example, RNAs transcribed from specific unrearranged CH genes are induced prior to switching under conditions that promote subsequent switching to these same CH genes. The function of transcription of these germline CH genes is unknown. In this report, we describe the structure of RNA transcribed from unrearranged gamma 1 genes in mouse spleen cells treated with LPS plus a HeLa cell supernatant containing recombinant interleukin 4. The germline gamma 1 RNA is initiated at multiple start sites 5\u27 to the tandem repeats of the gamma 1 switch (S gamma 1) region. As is true for analogous RNAs transcribed from unrearranged gamma 2b and alpha genes, the germline gamma 1 RNA has an I exon transcribed from the region 5\u27 to S gamma 1 sequences, which is spliced at a unique site to the C gamma gene. The germline gamma 1 RNA has an open-reading frame (ORF) that potentially encodes a small protein 48 amino acid in length

Characterization of an interleukin 4 (IL-4) responsive region in the immunoglobulin heavy chain germline epsilon promoter: regulation by NF-IL-4, a C/EBP family member and NF-kappa B/p50

A large body of data indicate that antibody class switching is directed by cytokines by inducing or repressing transcription from unrearranged, or germline, CH genes. Interleukin 4 (IL-4) induces transcription of the germline C epsilon genes in activated B cells and subsequently, cells in this population will undergo switch recombination to immunoglobulin E. Furthermore, the data suggest that transcription of germline C epsilon genes is required for class switching. In this paper we define DNA elements required for induction of transcription of the germline C epsilon genes by IL-4. To do this, segments of DNA from the 5\u27 flank of the initiation sites for germline epsilon RNA were ligated to a luciferase reporter gene and transfected into two mouse B cell lines, one of which can be induced to switch to IgE. By analysis of a series of 5\u27 deletion constructs and linker-scanning mutations, we demonstrate that a 46-bp segment (residing at -126/-79 relative to the first RNA initiation site) contains an IL-4 responsive region. By electrophoretic mobility shift assays, we find that this segment binds three transcription factors: the recently described NF-IL4, one or more members of the C/EBP family of transcription factors, and NF-kappa B/p50. Mutation of any of the binding sites for these three factors abolishes or reduces IL-4 inducibility of the epsilon promoter. A 27-bp segment within this IL-4 response region containing binding sites for NF-IL4 and a C/EBP factor is sufficient to transfer IL-4 inducibility to a minimal c-fos promoter

Induction of immunoglobulin isotype switching in cultured I.29 B lymphoma cells. Characterization of the accompanying rearrangements of heavy chain genes

The murine B cell lymphoma I.29 contains cells expressing surface IgM or IgA with identical heavy chain variable regions (9, 25, and D. Klein and J. Stavnezer, unpublished data). Purified IgM+ cells from the lymphoma have been adapted to culture and induced to switch to IgA, IgE, or IgG2 by treatment with lipopolysaccharide (LPS) or by treatment with a monoclonal anti-I.29 antiidiotype plus LPS. Clones of IgM+ cells have been obtained and induced to switch. Under optimal conditions, 30% of the cells in the culture expressed IgA 8 d after the inducers were added, and by 15 d 90% of the cells were IgA+. In actively switching cultures, up to 50% of the cells whose cytoplasm stained positively with anti-IgA stained simultaneously with anti-IgM, which indicates that the appearance of IgA+ cells in the cultures was due to isotype switching and not to clonal outgrowth. Examination by Southern blotting experiments of the Ig heavy chain genes in I.29 cells before and after switching revealed that isotype switching was accompanied by DNA recombinations that occurred within or immediately 5\u27 to the tandemly repeated switch sequences. Within 3 d after the addition of inducers of switching, the nonexpressed chromosome underwent a variety of deletions or expansions within the S mu region, and a portion of the S alpha regions had undergone a 0.9-kb deletion. In cultures that contained at least 12% IgA+ cells, rearranged, expressed alpha genes, produced by recombination between the S mu region within the expressed mu gene and the S alpha region, were detected

Mlh1 Can Function in Antibody Class Switch Recombination Independently of Msh2

Mismatch repair proteins participate in antibody class switch recombination, although their roles are unknown. Previous nucleotide sequence analyses of switch recombination junctions indicated that the roles of Msh2 and the MutL homologues, Mlh1 and Pms2, differ. We now asked if Msh2 and Mlh1 function in the same pathway during switch recombination. Splenic B cells from mice deficient in both these proteins were induced to undergo switching in culture. The frequency of switching is reduced, similarly to that of B cells singly deficient in Msh2 or Mlh1. However, the nucleotide sequences of the Sμ-Sγ3 junctions resemble junctions from Mlh1- but not from Msh2-deficient cells, suggesting Mlh1 functions either independently of or before Msh2. The substitution mutations within S regions that are known to accompany switch recombination are increased in Msh2- and Mlh1 single-deficient cells and further increased in the double-deficient cells, again suggesting these proteins function independently in class switch recombination. The finding that MMR functions to reduce mutations in switch regions is unexpected since MMR proteins have been shown to contribute to somatic hypermutation of antibody variable region genes

Role for Mismatch Repair Proteins Msh2, Mlh1, and Pms2 in Immunoglobulin Class Switching Shown by Sequence Analysis of Recombination Junctions

B cells from mice deficient in mismatch repair (MMR) proteins show decreased ability to undergo class switch recombination in vitro and in vivo. The deficit is not accompanied by any reduction in cell viability or alterations in the cell cycle in B cells cultured in vitro. To assess the role of MMR in switching we examined the nucleotide sequences of Sμ-Sγ3 recombination junctions in splenic B cells induced in culture to switch to IgG3. The data demonstrate clear differences in the sequences of switch junctions in wild-type B cells in comparison with Msh2-, Mlh1-, and Pms2-deficient B cells. Sequences of switch junctions from Msh2-deficient cells showed decreased lengths of microhomology between Sμ and Sγ3 relative to junctions from wild-type cells and an increase in insertions, i.e., nucleotides which do not appear to be derived from either the Sμ or Sγ3 parental sequence. By contrast, 23% of junctions from Mlh1- and Pms2-deficient cells occurred at unusually long stretches of microhomology. The data indicate that MMR proteins are directly involved in class switching and that the role of Msh2 differs from that of Mlh1 and Pms2

Reduced Isotype Switching in Splenic B Cells from Mice Deficient in Mismatch Repair Enzymes

Mice deficient in various mismatch repair (MMR) enzymes were examined to determine whether this repair pathway is involved in antibody class switch recombination. Splenic B cells from mice deficient in Msh2, Mlh1, Pms2, or Mlh1 and Pms2 were stimulated in culture with lipopolysaccharide (LPS) to induce immunoglobulin (Ig)G2b and IgG3, LPS and interleukin (IL)-4 to induce IgG1, or LPS, anti–δ-dextran, IL-4, IL-5, and transforming growth factor (TGF)-β1 to induce IgA. After 4 d in culture, cells were surface stained for IgM and non-IgM isotypes and analyzed by FACS®. B cells from MMR-deficient mice show a 35–75% reduction in isotype switching, depending on the isotype and on the particular MMR enzyme missing. IgG2b is the most affected, reduced by 75% in Mlh1-deficient animals. The switching defect is not due to a lack of maturation of the B cells, as purified IgM+IgD+ B cells show the same reduction. MMR deficiency had no effect on cell proliferation, viability, or apoptosis, as detected by [3H]thymidine incorporation and by propidium iodide staining. The reduction in isotype switching was demonstrated to be at the level of DNA recombination by digestion-circularization polymerase chain reaction (DC-PCR). A model of the potential role for MMR enzymes in class switch recombination is presented

The μ Switch Region Tandem Repeats Are Important, but Not Required, for Antibody Class Switch Recombination

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 (Sμ) from the murine μ H chain gene. We find that the Sμ 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 Sμ tandem repeats must be capable of directing the class switch mechanism. The maintenance of the highly repeated Sμ element during evolution appears to reflect selection for a highly efficient switching process rather than selection for a required sequence element

Evidence for Class-Specific Factors in Immunoglobulin Isotype Switching

Immunoglobulin class switch recombination (SR) occurs by a B cell–specific, intrachromosomal deletional process between switch regions. We have developed a plasmid-based transient transfection assay for SR to test for the presence of transacting switch activities. The plasmids are novel in that they lack a eukaryotic origin of DNA replication. The recombination activity of these switch substrates is restricted to a subset of B cell lines that support isotype switching on their endogenous loci and to mitogen-activated normal splenic B cells. The factors required for extrachromosomal plasmid recombination are constitutively expressed in proliferating splenic B cells and in B cell lines capable of inducibly undergoing immunoglobulin SR on their chromosomal genes. These studies suggest that mitogens that induce switching on the chromosome induce accessibility rather than switch recombinase activity. Finally, we provide evidence for two distinct switching activities which independently mediate μ→α and μ→γ3 SR

Shifts in targeting of class switch recombination sites in mice that lack mu switch region tandem repeats or Msh2

The mechanisms that target class switch recombination (CSR) to antibody gene switch (S) regions are unknown. Analyses of switch site locations in wild-type mice and in mice that lack the Smu tandem repeats show shifts indicating that a 4-5-kb DNA domain (bounded upstream by the Imu promoter) is accessible for switching independent of Smu sequences. This CSR-accessible domain is reminiscent of the promoter-defined domains that target somatic hypermutation. Within the 4-5-kb CSR domain, the targeting of S site locations also depends on the Msh2 mismatch repair protein because Msh2-deficient mice show an increased focus of sites to the Smu tandem repeat region. We propose that Msh2 affects S site location because sequences with few activation-induced cytidine deaminase targets generate mostly switch DNA cleavages that require Msh2-directed processing to allow CSR joining