The contested role of uracil DNA glycosylase in immunoglobulin gene diversification

Class switch recombination (CSR) and somatic hypermutation (SHM) of immunoglobulin (Ig) genes are initiated by the activation-induced cytosine deaminase AID. The resulting uracils in Ig genes were believed to be removed by the uracil glycosylase (UNG) and the resulting abasic sites treated in an error-prone fashion, creating breaks in the Ig switch regions and mutations in the variable regions. A recent report suggests that UNG does not act as a glycosylase in CSR and SHM but rather has unknown activity subsequent to DNA breaks that were created by other mechanisms.     

Somatic hypermutation and class switch recombination in Msh6(-/-)Ung(-/-) double-knockout mice

Somatic hypermutation (SHM) and class switch recombination (CSR) are initiated by activation-induced cytosine deaminase (AID). The uracil, and potentially neighboring bases, are processed by error-prone base excision repair and mismatch repair. Deficiencies in Ung, Msh2, or Msh6 affect SHM and CSR. To determine whether Msh2/Msh6 complexes which recognize single-base mismatches and loops were the only mismatch-recognition complexes required for SHM and CSR, we analyzed these processes in Msh6(-/-)Ung(-/-) mice. SHM and CSR were affected in the same degree and fashion as in Msh2(-/-)Ung(-/-) mice; mutations were mostly C,G transitions and CSR was greatly reduced, making Msh2/Msh3 contributions unlikely. Inactivating Ung alone reduced mutations from A and T, suggesting that, depending on the DNA sequence, varying proportions of A,T mutations arise by error-prone long-patch base excision repair. Further, in Msh6(-/-)Ung(-/-) mice the 5' end and the 3' region of Ig genes was spared from mutations as in wild-type mice, confirming that AID does not act in these regions. Finally, because in the absence of both Ung and Msh6, transition mutations from C and G likely are "footprints" of AID, the data show that the activity of AID is restricted drastically in vivo compared with AID in cell-free assays.     

DNA polymerases β and λ do not directly affect Ig variable region somatic hypermutation although their absence reduces the frequency of mutations

During somatic hypermutation (SHM) of antibody variable (V) region genes, activation-induced cytidine deaminase (AID) converts dC to dU, and dUs can either be excised by uracil DNA glycosylase (UNG), by mismatch repair, or replicated over. If UNG excises the dU, the abasic site could be cleaved by AP-endonuclease (APE), introducing the single-strand DNA breaks (SSBs) required for generating mutations at A:T bp, which are known to depend upon mismatch repair and DNA Pol η. DNA Pol β or λ could instead repair the lesion correctly. To assess the involvement of Pols β and λ in SHM of antibody genes, we analyzed mutations in the VDJh4 3′ flanking region in Peyer's patch germinal center (GC) B cells from polβ^?/?polλ^?/?, polλ^?/?, and polβ^?/? mice. We find that deficiency of either or both polymerases results in a modest but significant decrease in V region SHM, with Pol β having a greater effect, but there is no effect on mutation specificity, suggesting they have no direct role in SHM. Instead, the effect on SHM appears to be due to a role for these enzymes in GC B cell proliferation or viability. The results suggest that the BER pathway is not important during V region SHM for generating mutations at A:T bp. Furthermore, this implies that most of the SSBs required for Pol η to enter and create A:T mutations are likely generated during replication instead. These results contrast with the inhibitory effect of Pol β on mutations at the Ig Sμ locus, Sμ DSBs and class switch recombination (CSR) reported previously. We show here that B cells deficient in Pol λ or both Pol β and λ proliferate normally in culture and undergo slightly elevated CSR, as shown previously for Pol β-deficient B cells.     

Parp3 Negatively Regulates Immunoglobulin Class Switch Recombination

To generate highly specific and adapted immune responses, B cells diversify their antibody repertoire through mechanisms involving the generation of programmed DNA damage. Somatic hypermutation (SHM) and class switch recombination (CSR) are initiated by the recruitment of activation-induced cytidine deaminase (AID) to immunoglobulin loci and by the subsequent generation of DNA lesions, which are differentially processed to mutations during SHM or to double-stranded DNA break intermediates during CSR. The latter activate the DNA damage response and mobilize multiple DNA repair factors, including Parp1 and Parp2, to promote DNA repair and long-range recombination. We examined the contribution of Parp3 in CSR and SHM. We find that deficiency in Parp3 results in enhanced CSR, while SHM remains unaffected. Mechanistically, this is due to increased occupancy of AID at the donor (Sμ) switch region. We also find evidence of increased levels of DNA damage at switch region junctions and a bias towards alternative end joining in the absence of Parp3. We propose that Parp3 plays a CSR-specific role by controlling AID levels at switch regions during CSR.     

A role for Mlh3 in somatic hypermutation

Somatic hypermutation (SHM) and class switch recombination (CSR) allow B cells to make high affinity antibodies of various isotypes. Both processes are initiated by activation-induced cytidine deaminase (AID) to generate dG:dU mismatches in the immunoglobulin genes that are resolved differently in SHM and CSR to introduce point mutations and recombination, respectively. The MutL homolog MLH3 has been implicated in meiosis and DNA mismatch repair (MMR). Since it interacts with MLH1, which plays a role in SHM and CSR, we examined these processes in Mlh3-deficient mice. Although deficiencies in other MMR proteins result in defects in SHM, Mlh3(-/-) mice exhibited an increased frequency of mutations in their immunoglobulin variable regions, compared to wild type littermates. Alterations of mutation spectra were observed in the Jh4 flanking region in Mlh3(-/-) mice. Nevertheless, Mlh3(-/-) mice were able to switch to IgG3 or IgG1 with similar frequencies to control mice. This is the first instance where a loss of a DNA repair protein has a positive impact on the rate of SHM, suggesting that Mlh3 normally inhibits the accumulation of mutations in SHM.     

Assessing somatic hypermutation in Ramos B cells after overexpression or knockdown of specific genes

B cells start their life with low affinity antibodies generated by V(D)J recombination. However, upon detecting a pathogen, the variable (V) region of an immunoglobulin (Ig) gene is mutated approximately 100,000-fold more than the rest of the genome through somatic hypermutation (SHM), resulting in high affinity antibodies. In addition, class switch recombination (CSR) produces antibodies with different effector functions depending on the kind of immune response that is needed for a particular pathogen. Both CSR and SHM are initiated by activation-induced cytidine deaminase (AID), which deaminates cytosine residues in DNA to produce uracils. These uracils are processed by error-prone forms of repair pathways, eventually leading to mutations and recombination. Our current understanding of the molecular details of SHM and CSR come from a combination of studies in mice, primary cells, cell lines, and cell-free experiments. Mouse models remain the gold standard with genetic knockouts showing critical roles for many repair factors (e.g. Ung, Msh2, Msh6, Exo1, and polymerase η). However, not all genes are amenable for knockout studies. For example, knockouts of several double-strand break repair proteins are embryonically lethal or impair B-cell development. Moreover, sometimes the specific function of a protein in SHM or CSR may be masked by more global defects caused by the knockout. In addition, since experiments in mice can be lengthy, altering expression of individual genes in cell lines has become an increasingly popular first step to identifying and characterizing candidate genes. Ramos - a Burkitt lymphoma cell line that constitutively undergoes SHM - has been a popular cell-line model to study SHM. One advantage of Ramos cells is that they have a built-in convenient semi-quantitative measure of SHM. Wild type cells express IgM and, as they pick up mutations, some of the mutations knock out IgM expression. Therefore, assaying IgM loss by fluorescence-activated cell scanning (FACS) provides a quick read-out for the level of SHM. A more quantitative measurement of SHM can be obtained by directly sequencing the antibody genes. Since Ramos cells are difficult to transfect, we produce stable derivatives that have increased or lowered expression of an individual gene by infecting cells with retroviral or lentiviral constructs that contain either an overexpression cassette or a short hairpin RNA (shRNA), respectively. Here, we describe how we infect Ramos cells and then use these cells to investigate the role of specific genes on SHM (Figure 1).     

Activation-induced cytidine deaminase induces reproducible DNA breaks at many non-Ig Loci in activated B cells

After immunization or infection, activation-induced cytidine deaminase (AID) initiates diversification of immunoglobulin (Ig) genes in B cells, introducing mutations within the antigen-binding V regions (somatic hypermutation, SHM) and double-strand DNA breaks (DSBs) into switch (S) regions, leading to antibody class switch recombination (CSR). We asked if, during B cell activation, AID also induces DNA breaks at genes other than IgH genes. Using a nonbiased genome-wide approach, we have identified hundreds of reproducible, AID-dependent DSBs in mouse splenic B cells shortly after induction of CSR in culture. Most interestingly, AID induces DSBs at sites syntenic with sites of translocations, deletions, and amplifications found in human B cell lymphomas, including within the oncogene B cell lymphoma11a (bcl11a)/evi9. Unlike AID-induced DSBs in Ig genes, genome-wide AID-dependent DSBs are not restricted to transcribed regions and frequently occur within repeated sequence elements, including CA repeats, non-CA tandem repeats, and SINEs.     

Alkyladenine DNA glycosylase (Aag) in somatic hypermutation and class switch recombination

Somatic hypermutation (SHM) and class switch recombination (CSR) of immunoglobulin (Ig) genes require the cytosine deaminase AID, which deaminates cytosine to uracil in Ig gene DNA. Paradoxically, proteins involved normally in error-free base excision repair and mismatch repair, seem to be co-opted to facilitate SHM and CSR, by recruiting error-prone translesion polymerases to DNA sequences containing deoxy-uracils created by AID. Major evidence supports at least one mechanism whereby the uracil glycosylase Ung removes AID-generated uracils creating abasic sites which may be used either as uninformative templates for DNA synthesis, or processed to nicks and gaps that prime error-prone DNA synthesis. We investigated the possibility that deamination at adenines also initiates SHM. Adenosine deamination would generate hypoxanthine (Hx), a substrate for the alkyladenine DNA glycosylase (Aag). Aag would generate abasic sites which then are subject to error-prone repair as above for AID-deaminated cytosine processed by Ung. If the action of an adenosine deaminase followed by Aag were responsible for significant numbers of mutations at A, we would find a preponderance of A:T>G:C transition mutations during SHM in an Aag deleted background. However, this was not observed and we found that the frequencies of SHM and CSR were not significantly altered in Aag-/- mice. Paradoxically, we found that Aag is expressed in B lymphocytes undergoing SHM and CSR and that its activity is upregulated in activated B cells. Moreover, we did find a statistically significant, albeit low increase of T:A>C:G transition mutations in Aag-/- animals, suggesting that Aag may be involved in creating the SHM A>T bias seen in wild type mice.     

Spontaneous class switch recombination in B cell lymphopoiesis generates aberrant switch junctions and is increased after VDJ rearrangement

Mature B cells replace the mu constant region of the H chain with a downstream isotype in a process of class switch recombination (CSR). Studies suggest that CSR induction is limited to activated mature B cells in the periphery. Recently, we have shown that CSR spontaneously occur in B lymphopoiesis. However, the mechanism and regulation of it have not been defined. In this study, we show that spontaneous CSR occurs at all stages of B cell development and generates aberrant joining of the switch junctions as revealed by: 1) increased load of somatic mutations around the CSR break points, 2) reduced sequence overlaps at the junctions, and 3) excessive switch region deletion. In addition, we found that incidence of spontaneous CSR is increased in cells carrying VDJ rearrangements. Our results reveal major differences between spontaneous CSR in developing B cells and CSR induced in mature B cells upon activation. These differences can be explained by deregulated expression or function of activation-induced cytidine deaminase early in B cell development.     

A role for the MutL mismatch repair Mlh3 protein in immunoglobulin class switch DNA recombination and somatic hypermutation

Class switch DNA recombination (CSR) and somatic hypermutation (SHM) are central to the maturation of the Ab response. Both processes involve DNA mismatch repair (MMR). MMR proteins are recruited to dU:dG mispairs generated by activation-induced cytidine deaminase-mediated deamination of dC residues, thereby promoting S-S region synapses and introduction of mismatches (mutations). The MutL homolog Mlh3 is the last complement of the mammalian set of MMR proteins. It is highly conserved in evolution and is essential to meiosis and microsatellite stability. We used the recently generated knockout mlh3(-/-) mice to address the role of Mlh3 in CSR and SHM. We found that Mlh3 deficiency alters both CSR and SHM. mlh3(-/-) B cells switched in vitro to IgG and IgA but displayed preferential targeting of the RGYW/WRCY (R = A or G, Y = C or T, W = A or T) motif by Sgamma1 and Sgamma3 breakpoints and introduced more insertions and fewer donor/acceptor microhomologies in Smu-Sgamma1 and Smu-Sgamma3 DNA junctions, as compared with mlh3(+/+) B cells. mlh3(-/-) mice showed only a slight decrease in the frequency of mutations in the intronic DNA downstream of the rearranged J(H)4 gene. However, the residual mutations were altered in spectrum. They comprised a decreased proportion of mutations at dA/dT and showed preferential RGYW/WRCY targeting by mutations at dC/dG. Thus, the MMR Mlh3 protein plays a role in both CSR and SHM.     

Individual Substitution Mutations in the AID C Terminus That Ablate IgH Class Switch Recombination

Activation-induced cytidine deaminase (AID) is essential for class switch recombination (CSR) and somatic hypermutation (SHM) of Ig genes. The C terminus of AID is required for CSR but not for SHM, but the reason for this is not entirely clear. By retroviral transduction of mutant AID proteins into aid ^-/- mouse splenic B cells, we show that 4 amino acids within the C terminus of mouse AID, when individually mutated to specific amino acids (R190K, A192K, L196S, F198S), reduce CSR about as much or more than deletion of the entire C terminal 10 amino acids. Similar to ΔAID, the substitutions reduce binding of UNG to Ig Sμ regions and some reduce binding of Msh2, both of which are important for introducing S region DNA breaks. Junctions between the IgH donor switch (S)μ and acceptor Sα regions from cells expressing ΔAID or the L196S mutant show increased microhomology compared to junctions in cells expressing wild-type AID, consistent with problems during CSR and the use of alternative end-joining, rather than non-homologous end-joining (NHEJ). Unlike deletion of the AID C terminus, 3 of the substitution mutants reduce DNA double-strand breaks (DSBs) detected within the Sμ region in splenic B cells undergoing CSR. Cells expressing these 3 substitution mutants also have greatly reduced mutations within unrearranged Sμ regions, and they decrease with time after activation. These results might be explained by increased error-free repair, but as the C terminus has been shown to be important for recruitment of NHEJ proteins, this appears unlikely. We hypothesize that Sμ DNA breaks in cells expressing these C terminus substitution mutants are poorly repaired, resulting in destruction of Sμ segments that are deaminated by these mutants. This could explain why these mutants cannot undergo CSR.     

An evolutionary view of the mechanism for immune and genome diversity

An ortholog of activation-induced cytidine deaminase (AID) was, evolutionarily, the first enzyme to generate acquired immune diversity by catalyzing gene conversion and probably somatic hypermutation (SHM). AID began to mediate class switch recombination (CSR) only after the evolution of frogs. Recent studies revealed that the mechanisms for generating immune and genetic diversity share several critical features. Meiotic recombination, V(D)J recombination, CSR, and SHM all require H3K4 trimethyl histone modification to specify the target DNA. Genetic instability related to dinucleotide or triplet repeats depends on DNA cleavage by topoisomerase 1, which also initiates DNA cleavage in both SHM and CSR. These similarities suggest that AID hijacked the basic mechanism for genome instability when AID evolved in jawless fish. Thus, the risk of introducing genome instability into nonimmunoglobulin loci is unavoidable but tolerable compared with the advantage conferred on the host of being protected against pathogens by the enormous Ig diversification.     

Two new isotype-specific switching activities detected for Ig class switching

Ig class switch recombination (CSR) occurs by an intrachromosomal deletional process between switch (S) regions in B cells. To facilitate the study of CSR, we derived a new B cell line, 1.B4.B6, which is uniquely capable of mu --> gamma3, mu --> epsilon, and mu --> alpha, but not mu --> gamma1 CSR at its endogenous loci. The 1.B4.B6 cell line was used in combination with plasmid-based isotype-specific S substrates in transient transfection assays to test for the presence of trans-acting switching activities. The 1.B4.B6 cell line supports mu --> gamma3, but not mu --> gamma1 recombination, on S substrates. In contrast, normal splenic B cells activated with LPS and IL-4 are capable of plasmid-based mu --> gamma1 CSR and demonstrate that this S plasmid is active. Activation-induced deaminase (AID) was used as a marker to identify existing B cell lines as possible candidates for supporting CSR. The M12 and A20 cell lines were identified as AID positive and, following activation with CD40L and other activators, were found to differentially support mu --> epsilon and mu --> alpha plasmid-based CSR. These studies provide evidence for two new switching activities for mu --> gamma1 and mu --> epsilon CSR, which are distinct from mu --> gamma3 and mu --> alpha switching activities previously described. AID is expressed in all the B cell lines capable of CSR, but cannot account for the isotype specificity defined by the S plasmid assay. These results are consistent with a model in which isotype-specific switching factors are either isotype-specific recombinases or DNA binding proteins with sequence specificity for S DNA.     

Human MSH6 deficiency is associated with impaired antibody maturation

Ig class-switch recombination (Ig-CSR) deficiencies are rare primary immunodeficiencies characterized by defective switched isotype (IgG/IgA/IgE) production. Depending on the molecular defect, defective Ig-CSR may also be associated with impaired somatic hypermutation (SHM) of the Ig V regions. Although the mechanisms underlying Ig-CSR and SHM in humans have been revealed (at least in part) by studying natural mutants, the role of mismatch repair in this process has not been fully elucidated. We studied in vivo and in vitro Ab maturation in eight MSH6-deficient patients. The skewed SHM pattern strongly suggests that MSH6 is involved in the human SHM process. Ig-CSR was found to be partially defective in vivo and markedly impaired in vitro. The resolution of γH2AX foci following irradiation of MSH6-deficient B cell lines was also found to be impaired. These data suggest that in human CSR, MSH6 is involved in both the induction and repair of DNA double-strand breaks in switch regions.     

MSH6- or PMS2-deficiency causes re-replication in DT40 B cells,but it has little effect on immunoglobulin gene conversion or on repair of AID-generated uracils

The mammalian antibody repertoire is shaped by somatic hypermutation (SHM) and class switch recombination (CSR) of the immunoglobulin (Ig) loci of B lymphocytes. SHM and CSR are triggered by non-canonical, error-prone processing of G/U mismatches generated by activation-induced deaminase (AID). In birds, AID does not trigger SHM, but it triggers Ig gene conversion (GC), a ‘homeologous’ recombination process involving the Ig variable region and proximal pseudogenes. Because recombination fidelity is controlled by the mismatch repair (MMR) system, we investigated whether MMR affects GC in the chicken B cell line DT40. We show here that Msh6^−/− and Pms2^−/− DT40 cells display cell cycle defects, including genomic re-replication. However, although IgVλ GC tracts in MMR-deficient cells were slightly longer than in normal cells, Ig GC frequency, donor choice or the number of mutations per sequence remained unaltered. The finding that the avian MMR system, unlike that of mammals, does not seem to contribute towards the processing of G/U mismatches in vitro could explain why MMR is unable to initiate Ig GC in this species, despite initiating SHM and CSR in mammalian cells. Moreover, as MMR does not counteract or govern Ig GC, we report a rare example of ‘homeologous’ recombination insensitive to MMR.