Thymus-independent class switch recombination is affected by APRIL

The tumour necrosis factor (TNF) family member a proliferation-inducing ligand (APRIL) is implicated in various B-cell processes, such as class switch recombination, plasma cell differentiation and plasma cell survival. This was suggested from initial studies analysing B-cell responses in APRIL-deficient and transgenic mice, and mice deficient for the TNF receptors of APRIL, transmembrane activator and CAML interactor (TACI) and B-cell maturation antigen. Here, we present additional evidence for the importance of APRIL in thymus-independent (TI) B-cell responses, using APRIL-deficient and transgenic mice. APRIL-deficient mice show an impaired immunoglobulin A (IgA) response towards TI B-cell antigens, whereas APRIL transgenic mice show exaggerated TI B-cell responses. Moreover, antibody titres to TI antigens were sustained in APRIL transgenic mice for a long time and even increased up to 75 days in the case of IgA against 4-hydroxy-nitrophenacetyl-lipopolysaccharide.     

Evidence for the murine IgH mu locus acting as a hot spot for intrachromosomal homologous recombination

Homologous recombination accomplishes the exchange of genetic information between two similar or identical DNA duplexes. It can occur either by gene conversion, a process of unidirectional genetic exchange, or by reciprocal crossing over. Homologous recombination is well known for its role in generating genetic diversity in meiosis and, in mitosis, as a DNA repair mechanism. In the immune system, the evidence suggests a role for homologous recombination in Ig gene evolution and in the diversification of Ab function. Previously, we reported the occurrence of homologous recombination between repeated, donor and recipient alleles of the Ig H chain mu gene C (Cmu) region residing at the Ig mu locus in mouse hybridoma cells. In this study, we constructed mouse hybridoma cell lines bearing Cmu region heteroalleles to learn more about the intrachromosomal homologous recombination process. A high frequency of homologous recombination (gene conversion) was observed for markers spanning the entire recipient Cmu region, suggesting that recombination might initiate at random sites within the Cmu region. The Cmu region heteroalleles were equally proficient as either conversion donors or recipients. Remarkably, when the same Cmu heteroalleles were tested for recombination in ectopic genomic positions, the mean frequency of gene conversion was reduced by at least 65-fold. These results are consistent with the murine IgH mu locus behaving as a hot spot for intrachromosomal homologous recombination.     

The role of G-density in switch region repeats for immunoglobulin class switch recombination

The boundaries of R-loops are well-documented at immunoglobulin heavy chain loci in mammalian B cells. Within primary B cells or B cell lines, the upstream boundaries of R-loops typically begin early in the repetitive portion of the switch regions. Most R-loops terminate within the switch repetitive zone, but the remainder can extend a few hundred base pairs further, where G-density on the non-template DNA strand gradually drops to the genome average. Whether the G-density determines how far the R-loops extend is an important question. We previously studied the role of G-clusters in initiating R-loop formation, but we did not examine the role of G-density in permitting the elongation of the R-loop, after it had initiated. Here, we vary the G-density of different portions of the switch region in a murine B cell line. We find that both class switch recombination (CSR) and R-loop formation decrease significantly when the overall G-density is reduced from 46% to 29%. Short 50 bp insertions with low G-density within switch regions do not appear to affect either CSR or R-loop elongation, whereas a longer (150 bp) insertion impairs both. These results demonstrate that G-density is an important determinant of the length over which mammalian genomic R-loops extend.     

53BP1 is required for class switch recombination

53BP1 participates early in the DNA damage response and is involved in cell cycle checkpoint control. Moreover, the phenotype of mice and cells deficient in 53BP1 suggests a defect in DNA repair (Ward et al., 2003b). Therefore, we asked whether or not 53BP1 would be required for the efficient repair of DNA double strand breaks. Our data indicate that homologous recombination by gene conversion does not depend on 53BP1. Moreover, 53BP1-deficient mice support normal V(D)J recombination, indicating that 53BP1 is not required for "classic" nonhomologous end joining. However, class switch recombination is severely impaired in the absence of 53BP1, suggesting that 53BP1 facilitates DNA end joining in a way that is not required or redundant for the efficient closing of RAG-induced strand breaks. These findings are similar to those observed in mice or cells deficient in the tumor suppressors ATM and H2AX, further suggesting that the functions of ATM, H2AX, and 53BP1 are closely linked.     

Circular DNA is excised by immunoglobulin class switch recombination

We have purified extrachromosomal circular DNAs from adult mouse spleen cells, and cloned into a phage vector the BamHl fragments hybridizing with C mu and S gamma 1 probes. We obtained 52 S mu+S gamma 1+ clones by screening 1.4 million phage clones derived from spleen cells stimulated with bacterial lipopolysaccharide and interleukin 4. We have identified the breakpoints of six clones that contain S gamma 1 and S mu sequences fused in the 5' to 3' orientation. All these switch recombination sites were assigned to the central repetitive sequences of the S mu and S gamma 1 regions. Since the common S mu-S gamma 1 sequences at the recombination sites are at most 2 bases long, typical homologous recombination cannot account for their joining. These findings provide direct evidence that mu-gamma 1 class switching can occur by the looping out and excision of chromosomal DNA, with formation of a circle.     

Directed Ig class switch recombination in activated murine B cells

Immunoglobulin class switch recombination occurs at frequencies of up to 10%/cell/generation in activated murine B-lymphocytes. We analysed cH gene rearrangements and switch recombinations from active and inactive IgH loci of B-cells activated in various ways and immortalized by cell fusion. Although about half of the IgM+ cells show rearrangement of c mu genes, the deletion of c mu is a rare event. Half of the IgG3+ and IgG1+ cells show rearrangement of c mu genes on the inactive IgH locus and the other half of the IgG+ cells have deleted c mu from both IgH loci by switch recombination. This recombination is directed to the same switch regions on both IgH loci in 60-80% of all cases. Interleukin 4 may play a critical role in programming murine B-lymphocytes for specific switch recombination.     

Nucleic acid structures and enzymes in the immunoglobulin class switch recombination mechanism

Class switch recombination is the gene rearrangement process by which our B lymphocytes change from IgM production to IgG, IgA, or IgE. Unlike the well-characterized V(D)J recombination, the mechanism of class switch recombination has been largely enigmatic until very recent progress has begun to shed light on this gene rearrangement process. Progress has been made on the enzymes involved in leading to the DNA cleavage events and on identifying the unusual DNA structures that those enzymes recognize.     

Connection between induction of DNA lesions and DNA recombination/repair during Ig class switch recombination

Comment on: Kracker S, et al. Proc Natl Acad Sci USA 2010; 107:22225-30.     

Borrelia burgdorferi-induced inflammation facilitates spirochete adaptation and variable major protein-like sequence locus recombination.

Spirochete adaptation in vivo is associated with preferential Borrelia burgdorferi gene expression. In this paper, we show that the administration of B. burgdorferi-immune sera to IFN-gammaR-deficient mice that have been infected with B. burgdorferi N40 for 4 days causes spirochete clearance. In contrast, immune sera-mediated clearance of B. burgdorferi N40 is not apparent in immunocompetent mice, suggesting a role for IFN-gamma-mediated responses in B. burgdorferi N40 host adaptation. B. burgdorferi-immune sera also induces clearance of B. burgdorferi N40 that have been passaged in vitro 75 times (B. burgdorferi N40-75), a derivative of B. burgdorferi N40 that does not rapidly adapt in vivo in immunocompetent mice. B. burgdorferi N40-75 produce lower levels of IFN-gamma and IL-12 in mice than does B. burgdorferi N40, and the administration of these cytokines to B. burgdorferi N40-75-infected mice results in an increased spirochetal burden, further indicating that IFN-gamma-mediated events promote B. burgdorferi survival. Differential immunoscreening and RT-PCR demonstrate that IFN-gamma-mediated signals facilitate spirochete recombination at the variable major protein like sequence locus, a site for early antigenic variation in vivo, and that recombination rates by B. burgdorferi N40 are lower in IFN-gammaR-deficient mice than in control animals. These results suggest that the murine immune response can promote the in vivo adaptation of B. burgdorferi.     

Requirement of non-canonical activity of uracil DNA glycosylase for class switch recombination

Activation-induced cytidine deaminase (AID) and uracil DNA glycosylase (UNG) are required for class switch recombination (CSR). AID is involved in the DNA cleavage step of CSR, but the precise role of UNG is not yet understood. Mutations and deletions are footprints of abortive DNA cleavage in the immunoglobulin switch region in splenic B cells stimulated to undergo CSR. However, a UNG deficiency did not reduce the number of such footprints, indicating UNG is dispensable for the DNA cleavage step. Mutagenesis experiments revealed that the role of UNG in CSR depends on its WXXF motif. This motif is also essential for the interaction of UNG with the HIV viral peptide Vpr, which recruits UNG to the HIV particle. Furthermore, exogenous Vpr had a dominant-negative effect on CSR. These results suggest that UNG is recruited to the CSR machinery through its WXXF motif by a Vpr-like host factor and plays a novel non-canonical role in a CSR step that follows DNA cleavage.