Ig Class Switch Recombination Research Articles

Comment on “Reassessment of the Role of Mut S Homolog 5 in Ig Class Switch Recombination Shows Lack of Involvement in cis- and trans-Switching”

We read with interest the recent article by Guikema et al. ([1][1]) where the authors concluded that Msh5 does not participate in Ig class switch recombination (CSR) in mice. This is clearly in contrast to our recent publication describing a role for Msh5 in CSR both in mice and humans ([2][2]), and

Response to Comment on “Reassessment of the Role of Mut S Homolog 5 in Ig Class Switch Recombination Shows Lack of Involvement in cis- and trans-Switching”

Response to Comment on “Reassessment of the Role of Mut S Homolog 5 in Ig Class Switch Recombination Shows Lack of Involvement in <i>cis</i>- and <i>trans</i>-Switching”

Reassessment of the Role of Mut S Homolog 5 in Ig Class Switch Recombination Shows Lack of Involvement in cis- and trans-Switching

When B cells are activated after immunization or infection, they exchange the gene encoding the Ig H chain C region by class switch recombination (CSR). CSR generally occurs by an intrachromosomal deletional recombination within switch (S) region sequences. However, approximately 10% of CSR events occur between chromosome homologs (trans- or interallele CSR), suggesting that the homologous chromosomes are aligned during CSR. Because the Mut S homolog 4 (Msh4) and Msh5 bind to Holliday junctions and are required for homologous recombination during meiosis in germ cells, we hypothesized these proteins might be involved in trans-chromosomal CSR (trans-CSR). Indeed, Msh4-Msh5 has recently been suggested to have a role in CSR. However, we find a large variety of alternative splice variants of Msh5 mRNA in splenic B cells rather than the full-length form found in testis. Most of these mRNAs are unlikely to be stable, suggesting that Msh5 might not be functional. Furthermore, we find that msh5 nullizygous B cells undergo CSR normally, have unaltered levels of trans-CSR, normal levels of DNA breaks in the Smu region, and normal S-S junctions. We also show that the S-S junctions from cis- and trans-CSR events have similar lengths of junctional microhomology, suggesting trans-CSR occurs by nonhomologous end joining as does intrachromosome (cis)-CSR. From these data, we conclude that Msh5 does not participate in CSR.

Collateral Damage from Antigen Receptor Gene Diversification

Chromosomal translocations that juxtapose antigen receptor genes and oncogenes are frequently associated with lymphoid malignancies. In this issue, Robbiani et al. (2008) show that activation-induced deaminase (AID), an enzyme involved in antigen receptor gene diversification, generates DNA double-strand breaks (DSBs) in oncogenes, and Tsai et al. (2008) propose that AID and the recombinase-activating gene (RAG) endonuclease may collaborate to generate off-target DSBs.

Analysis of intergenic transcription and histone modification across the human immunoglobulin heavy-chain locus

Ig class switch recombination (CSR) is initiated by activation-induced cytidine deaminase (AID) mediated deamination of the switch (S) regions; the resultant mismatch is processed to yield the DNA breaks required for recombination. Whereas many of the pathways involved in the mechanism of recombination have been identified, little is known about how CSR is regulated. AID action is known to require transcription of the Ig heavy-chain genes. However, it is not understood how AID is restricted to the Ig genes. Many aspects of gene expression are known to be regulated by modification of chromatin structure. In turn, chromatin is known to be regulated by several RNA-dependent activities. We have mapped the transcriptional and chromatin landscape of the human Ig heavy-chain locus to investigate the effect these activities have on CSR. We demonstrate that the Ig heavy-chain constant genes and 3'-regulatory regions are in an active chromatin conformation in unstimulated total human B cells: the locus undergoes both genic and intergenic transcription and possesses histone modifications associated with "active" chromatin (acetylated H3 and H4 and lysine 4 trimethylated H3). However, on cytokine stimulation, these modifications spread into the S regions, demonstrating a chromatin remodeling activity associated with switching. Surprisingly, after stimulation, the S regions also accumulate lysine 9 trimethylated H3, a modification previously associated with gene silencing. These data demonstrates that the Ig locus is maintained with a complex pattern of both positive and negative histone marks and suggest that some of these marks may have dual functions.

Human PMS2 deficiency is associated with impaired immunoglobulin class switch recombination

Immunoglobulin (Ig) class switch recombination (CSR) deficiencies are rare primary immunodeficiencies characterized by the lack of switched isotype (IgG/IgA/IgE) production. In some cases, CSR deficiencies can be associated with abnormal somatic hypermutation. Analysis of CSR deficiencies has helped reveal the key functions of CSR-triggering molecules, i.e., CD40L, CD40, and effector molecules such as activation-induced cytidine deaminase and uracil N-glycosylase. We report a new form of B cell–intrinsic CSR deficiency found in three patients with deleterious, homozygous mutations in the gene encoding the PMS2 component of the mismatch repair machinery. CSR was found partially defective in vivo and markedly impaired in vitro. It is characterized by the defective occurrence of double-strand DNA breaks (DSBs) in switch regions and abnormal formation of switch junctions. This observation strongly suggests a role for PMS2 in CSR-induced DSB generation.

Single-strand DNA breaks in Ig class switch recombination that depend on UNG but not AID

B lymphocytes switch from secreting IgM to secreting IgG, IgA or IgE through a DNA recombination, class switch recombination (CSR), whose mechanism is incompletely understood. CSR is thought to be triggered by activation-induced deaminase (AID), which is believed to deaminate cytosines to uracil in single-strand regions of switch region DNA. Subsequent excision of uracils by uracil DNA glycosylase (UNG) (product of the UNG gene) generates abasic sites, which are targeted for DNA cleavage, producing DNA breaks that are critical intermediates in CSR. Consistent with this model, CSR-related double-strand breaks (DSBs)--detected by ligation-mediated PCR (LMPCR)--have been reported to be dramatically reduced in B cells from either AID(-/-) or UNG(-/-) mice. Here we examine single-strand breaks (SSBs) using LMPCR and report, surprisingly, that CSR-related anti-sense strand breaks in Sgamma regions are dependent only on UNG, and not AID, suggesting participation of a cytosine deaminase other than AID. This conclusion is supported by the sequences at these DNA breaks, which show a bias for a consensus sequence different from that reported for AID. The SSBs appear to be part of the normal CSR pathway since in B cells in which CSR is blocked by deletion of Smu, the content of Sgamma SSBs is elevated as though the breaks resolve inefficiently owing to the lack of a recombination partner for completing mu-to-gamma CSR. These results suggest a narrower role for AID in CSR than previously recognized and prompt a search for a putative alternative cytosine deaminase participating in CSR.

Fixing DNA breaks during class switch recombination

Immunoglobulin (Ig) class switch recombination (CSR) involves the breakage and subsequent repair of two DNA sequences, known as switch (S) regions, which flank IgH constant region exons. The resolution of CSR-associated breaks is thought to require the nonhomologous end-joining (NHEJ) DNA repair pathway, but the role of the NHEJ factor DNA-dependent protein kinase catalytic subunit (DNA-PKcs) in this process has been unclear. A new study, in which broken IgH-containing chromosomes in switching B cells were visualized directly, clearly demonstrated that DNA-PKcs and, unexpectedly, the nuclease Artemis are involved in the resolution of switch breaks.

Mechanisms for Decreased Function of B Cells in Aged Mice and Humans

The immune system has been known for some time to be compromised in aged individuals, e.g., both mice and humans, and in both humoral and cellular responses. Our studies have begun to elucidate intrinsic B lymphocyte defects in Ig class switch recombination, activation-induced cytidine deaminase, and E47 transcription factor expression. These defects occur in both mice and humans. Our studies have also shown that tristetraprolin is one of the key players in regulating the decreased E47 mRNA stability in aged B lymphocytes. These and current studies should lead to improvements in B lymphocyte function in aged populations.

MyD88 Negatively Controls Hypergammaglobulinemia with Autoantibody Production during Bacterial Infection

A large body of evidence has convincingly shown that Toll-like receptors are necessary sensors for infections with pathogens, but their activation was also suggested to generate autoimmunity. During experimental infections, the lack of these sensors or of their signaling molecules should lead to a deficient immune response. We found out that MyD88, the major adaptor of the Toll/interleukin-1 (Toll/IL-1) receptor signaling pathway, can actually act as a negative regulator of B-cell function in some settings. MyD88-deficient mice infected by Borrelia burgdorferi developed extreme hypergammaglobulinemia compared to wild-type animals, with high levels of immunoglobulin M (IgM) autoantibodies. In vivo, cell transfer experiments and cell blocking assays showed that this phenotype was not linked to the absence of MyD88 in B cells but rather to CD4 T-cell and likely dendritic cell dysfunctions leading to a Th1-to-Th2 cytokine switch. In addition, our results suggest a relative defect in the Ig class switch recombination process, since MyD88 knockout mice developed mostly IgM antibodies. Collectively, these data emphasize the complex role of the Toll/IL-1 receptor pathway in tuning the immune response against infection and avoiding autoimmunity.

Alternative endings.

The repair of chromosome breaks is essential to maintain genome integrity. Both in mammalian cells and in budding yeast, repair in the G1 phase of the cell cycle is particularly dependent on nonhomologous end-joining (NHEJ) to repair broken chromosomes. NHEJ is important in repairing double-strand breaks (DSBs) that arise during both V(D)J recombination and class switch recombination (CSR) events in the immune system. End-joining also is responsible for generating chromosomal translocations that are associated with various cancers. Work by Guirouilh-Barbat et al. (1) published in a recent issue of PNAS and by several other laboratories (2–4) has revealed that–in addition to the “classical” NHEJ pathway–there is a robust alternative pathway that also contributes to the generation of translocations. Our current understanding of the mechanisms of NHEJ results from a dynamic interplay between genetics and biochemistry both in mammals and in budding yeast (for reviews, see refs. 5–7). A major motivation to identify the genes responsible for NHEJ has been the study of end-joinings in both V(D)J recombination and Ig CSR in mammals, driven by the identification of immune-compromised humans. A second impetus has been the identification of genes conferring x-ray resistance. These studies led to the identification of DNA-protein kinase catalytic subunit (PKcs) and the Ku70 and Ku80 proteins, which play a central role both in immune cell recombination and in resistance to ionizing radiation. Further work led to the identification of a number of XRCC genes conferring radio-resistance. Subsequent studies in budding yeast confirmed that Ku proteins were also essential for NHEJ (there is no DNA-PKcs). Research using budding yeast identified other NHEJ components, notably DNA ligase 4, its partner Lif1 (whose mammalian homolog is XRCC4), and Nej1 (whose mammalian homolog is XLF/Cernunnos) † . Ku-deficient mice are viable but severely immunocompromised, whereas mice … *E-mail: haber{at}brandeis.edu

Brca1 in immunoglobulin gene conversion and somatic hypermutation

Defects in Brca1 confer susceptibility to breast cancer and genomic instability indicative of aberrant repair of DNA breaks. Brca1 was previously implicated in the homologous recombination pathway via effects on the assembly of recombinase Rad51. Activation-induced cytidine deaminase (AID) deaminates C to U in B lymphocyte immunoglobulin ( Ig) DNA to initiate programmed DNA breaks. Subsequent uracil-glycosylase mediated U removal, and perhaps further processing, leads to four known classes of mutation: Ig class switch recombination that results in a region-specific genomic deletion, Ig somatic hypermutation that introduces point mutations in Ig V-regions, Ig gene conversion in vertebrates that possess Ig pseudo- V genes, and translocations common to B cell lymphomas. We tested the involvement of Brca1 in AID-dependent Ig diversification in chicken DT40 cells. The DT40 cell line diversifies Ig Vλ mainly by gene conversion, and less so by point mutation. Brca1-deficiency caused a shift in Vλ diversification, significantly reducing the proportion of gene conversions relative to point mutations. Thus, Brca1 regulates AID-dependent DNA lesion repair. Interestingly, while Brca1 is required to recruit ubiquitinated FancD2 to DNA damage, the phenotype of Brca1-deficient DT40 differs from the one of FancD2-deficient DT40, in which both gene conversion and non-templated mutations are impaired.

Loss of IL-7 Receptor α on CD4+ T Cells Defines Terminally Differentiated B Cell-Helping Effector T Cells in a B Cell-Rich Lymphoid Tissue

IL-7 plays important roles in development and homeostatic proliferation of lymphocytes. IL-7 uses a receptor composed of IL-7Ralpha (CD127) and the common gamma-chain (CD132) to transmit its signal. It has been unknown how CD127 is regulated during Th cell differentiation to the B cell-helping T cell lineage. In this study, we report that loss of CD127 defines terminally differentiated B cell-helping effector T cells in human tonsils. Although naive CD4(+) T cells uniformly express CD127, the memory/effector (non-FOXP3(+)) CD4(+) T cells are divided into CD127(+) and CD127(-) cells. The CD127(-) T cells are exclusively localized within the germinal centers where B cells become plasma and memory B cells, whereas CD127(+) T cells are found in T cell areas and the area surrounding B cell follicles. Consistently, the CD127(-) T cells highly express the B cell zone homing receptor CXCR5 with concomitant loss of CCR7. Compared with CD127(+) memory T cells, CD127(-) T cells have considerably shorter telomeres, do not proliferate in response to IL-7, and are prone to cell death. The CD127(-) T cells produce a large amount of the B cell follicle-forming chemokine CXCL13 upon stimulation with B cells and Ags. Most importantly, they are highly efficient in helping B cells produce Igs of all isotypes in a manner dependent on CD40L and ICOS and inducing activation-induced cytidine deaminase and Ig class switch recombination. The selective loss of CD127 on the B cell-helping effector T cells would have implications in regulation and termination of Ig responses.

AID-deficient Bcl-xL transgenic mice develop delayed atypical plasma cell tumors with unusual Ig/Myc chromosomal rearrangements

Activation-induced cytidine deaminase (AID) is required for immunoglobulin (Ig) class switch recombination and somatic hypermutation, and has also been implicated in translocations between Ig switch regions and c-Myc in plasma cell tumors in mice. We asked if AID is required for accelerated tumor development in pristane-treated Bcl-xL transgenic BALB/c mice deficient in AID (pBxAicda−/−). pBxAicda −/− mice developed tumors with a lower frequency (24 vs. 62%) and a longer mean latency (108 vs. 36 d) than AID-sufficient mice. The tumors appeared in oil granuloma tissue and did not form ascites. By interphase fluorescence in situ hybridization, six out of nine pBxAicda −/− primary tumors had T(12;15) and one had T(6;15) chromosomal translocations. Two tumors were transplantable and established as stable cell lines. Molecular and cytogenetic analyses showed that one had an unusual unbalanced T(12;15) translocation, with IgH Cμ and Pvt-1 oriented head to tail at the breakpoint, resulting in an elevated expression of c-Myc. In contrast, the second was T(12;15) negative, but had an elevated N-Myc expression caused by a paracentric inversion of chromosome 12. Thus, novel mechanisms juxtapose Ig and Myc-family genes in AID-deficient plasma cell tumors.

Immunodeficiency and Autoimmune Phenomena in Female Hyper‐IgM Syndrome

We report an unusual case that highlights the clinical problems associated with autoimmune phenomena. A female (born 1972) was referred to our hospital with systemic lupus erythematosus (SLE) diagnosis. During the follow-up (7 years), we observed the appearance and the disappearance of a lot of autoantibodies detected. The history of recurrent bacterial sinopulmonary infections since puberty and enlargement of lymphonodes, elevated IgM, very low IgA and normal IgG levels, and the variable autoantibody profile oriented toward a "defective Ig class switch recombination" disorder: the hyper-IgM syndrome. Immunodeficiency and autoimmune phenomena may occur concomitantly in the same individual and sometimes the differential diagnosis is difficult.

IL-4-induced AID expression and its relevance to IgA class switch recombination

Activation-induced cytidine deaminase (AID) is an inducible gene that plays a critical role in Ig class switch recombination and somatic hypermutation in B cells. We explored the mechanisms by which IL-4 induces AID expression in mouse B cells. IL-4 increased AID expression and over-expression of Stat6 further augmented IL-4-induced promoter activity. The involvement of Stat6 in the promoter activity was confirmed using ChIP assays and site-directed mutagenesis. Treatment with H89, a PKA inhibitor, markedly decreased IL-4-induced AID expression, and over-expression of CREB enhanced it. These results indicate that Stat6 and PKA/CREB are involved in IL-4-induced AID expression. The relevance of these signal transducing molecules was verified using the TGFβ1-induced IgA isotype switching model. Our results indicate that IL-4, through Stat6 and PKA/CREB, induces AID expression leading to Ig isotype switching event.

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.

Abrogation of Lupus Nephritis in Activation-Induced Deaminase-Deficient MRL/lpr Mice

We generated MRL/lpr mice deficient in activation-induced deaminase (AID). Because AID is required for Ig hypermutation and class switch recombination, these mice lack hypermutated IgG Abs. Unlike their AID wild-type littermates, AID-deficient MRL/lpr mice not only lacked autoreactive IgG Abs but also experienced a dramatic increase in the levels of autoreactive IgM. This phenotype in AID-deficient mice translated into a significant reduction in glomerulonephritis, minimal mononuclear cell infiltration in the kidney, and a dramatic increase in survival to levels comparable to those previously reported for MRL/lpr mice completely lacking B cells and well below those of mice lacking secreted Abs. Therefore, this study wherein littermates with either high levels of autoreactive IgM or autoreactive IgG were directly examined proves that autoreactive IgM Abs alone are not sufficient to promote kidney disease in MRL/lpr mice. In addition, the substantial decrease in mortality combined with a dramatic increase in autoreactive IgM Abs in AID-deficient MRL/lpr mice suggest that autoreactive IgM Abs might not only fail to promote nephritis but may also provide a protective role in MRL/lpr mice. This novel mouse model containing high levels of autoreactive, unmutated IgM Abs will help delineate the contribution of autoreactive IgM to autoimmunity.

Activation-induced cytidine deaminase action is strongly stimulated by mutations of the THO complex.

Activation-induced cytidine deaminase (AID) is a B cell enzyme essential for Ig somatic hypermutation and class switch recombination. AID acts on ssDNA, and switch regions of Ig genes, a target of AID, form R-loops that contain ssDNA. Nevertheless, how AID action is specifically targeted to particular DNA sequences is not clear. Because mutations altering cotranscriptional messenger ribonucleoprotein (mRNP) formation such as those in THO/TREX in yeast promote R-loops, we investigated whether the cotranscriptional assembly of mRNPs could affect AID targeting. Here we show that AID action is transcription-dependent in yeast and that strong and transcription-dependent hypermutation and hyperrecombination are induced by AID if cells are deprived of THO. In these strains AID-induced mutations occurred preferentially at WRC motifs in the nontranscribed DNA strand. We propose that a suboptimal cotranscriptional mRNP assembly at particular DNA regions could play an important role in Ig diversification and genome dynamics.

A primary immunodeficiency characterized by defective immunoglobulin class switch recombination and impaired DNA repair

Immunoglobulin class switch recombination (CSR) deficiencies are rare primary immunodeficiencies, characterized by a lack of switched isotype (IgG, IgA, or IgE) production, variably associated with abnormal somatic hypermutation (SHM). Deficiencies in CD40 ligand, CD40, activation-induced cytidine deaminase, and uracil-N-glycosylase may account for this syndrome. We previously described another Ig CSR deficiency condition, characterized by a defect in CSR downstream of the generation of double-stranded DNA breaks in switch (S) μ regions. Further analysis performed with the cells of five affected patients showed that the Ig CSR deficiency was associated with an abnormal formation of the S junctions characterized by microhomology and with increased cell radiosensitivity. In addition, SHM was skewed toward transitions at G/C residues. Overall, these findings suggest that a unique Ig CSR deficiency phenotype could be related to an as-yet-uncharacterized defect in a DNA repair pathway involved in both CSR and SHM events.