C-terminal region of activation-induced cytidine deaminase (AID) is required for efficient class switch recombination and gene conversion

Activation-induced cytidine deaminase (AID) introduces single-strand breaks (SSBs) to initiate class switch recombination (CSR), gene conversion (GC), and somatic hypermutation (SHM). CSR is mediated by double-strand breaks (DSBs) at donor and acceptor switch (S) regions, followed by pairing of DSB ends in two S regions and their joining. Because AID mutations at its C-terminal region drastically impair CSR but retain its DNA cleavage and SHM activity, the C-terminal region of AID likely is required for the recombination step after the DNA cleavage. To test this hypothesis, we analyzed the recombination junctions generated by AID C-terminal mutants and found that 0- to 3-bp microhomology junctions are relatively less abundant, possibly reflecting the defects of the classical nonhomologous end joining (C-NHEJ). Consistently, the accumulation of C-NHEJ factors such as Ku80 and XRCC4 was decreased at the cleaved S region. In contrast, an SSB-binding protein, poly (ADP)-ribose polymerase1, was recruited more abundantly, suggesting a defect in conversion from SSB to DSB. In addition, recruitment of critical DNA synapse factors such as 53BP1, DNA PKcs, and UNG at the S region was reduced during CSR. Furthermore, the chromosome conformation capture assay revealed that DNA synapse formation is impaired drastically in the AID C-terminal mutants. Interestingly, these mutants showed relative reduction in GC compared with SHM in chicken DT40 cells. Collectively, our data indicate that the C-terminal region of AID is required for efficient generation of DSB in CSR and GC and thus for the subsequent pairing of cleaved DNA ends during recombination in CSR.Activation-induced cytidine deaminase (AID) is essential for three different genetic events: class switch recombination (CSR), gene conversion (GC), and somatic hypermutation (SHM), which contribute to Ig gene diversification (1–5). Although AID generates single-strand breaks (SSBs) in the Ig genes, subsequent repair steps for CSR and GC are similar to each other but are distinct from SHM in their mechanistic properties, i.e, in (i) generation of the double-strand breaks (DSBs), (ii) recombination, and (iii) the requirement for uracil-DNA-glycosylase (UNG) for the pairing of the DSB ends (6–10). Despite the similarities between GC and CSR, their repair mechanisms have distinct features: CSR recombination requires nonhomologous end joining (NHEJ), and GC depends on homologous recombination (HR). During CSR, DSB ends normally are joined by classical NHEJ (C-NHEJ), which requires specific repair proteins such as Ku80, XRCC4, or DNA ligase IV (11, 12). In the absence of C-NHEJ, a back-up end-joining pathway called “alternative end joining” (A-EJ), which is reported to be slower and also more error prone than C-NHEJ, joins the broken DSBs ends (13). On the other hand, HR, the most common form of homology-directed repair, requires long sequence homology between donor and acceptor DNA to complete the recombination step by recruiting a distinct set of repair proteins such as RAD54, RAD52, and RAD51 to the break sites (14, 15).Various studies on AID mutations in the N-terminal or C-terminal regions (4, 8, 9, 16–19) have shown that N-terminal AID mutants are compromised for CSR and are defective in SHM, indicating that the N-terminal region of AID is required for DNA cleavage (9, 16, 19). On the other hand, the C-terminal region of AID, which contains a nuclear-export signal and is responsible for AID’s shuttling activity between the nucleus and cytoplasm, is required for CSR-specific activity but not for DNA cleavage activity and SHM (8, 16). Among the series of AID C-terminal mutants examined, two mutants show characteristic features: P20, which has an insertion of 34 amino acids at residue 182 and normal nuclear-cytoplasmic shuttling activity, and JP8Bdel, which has a 16-amino acid truncation at residue 183, accumulates in the nucleus, and shows higher DNA break activity at the donor switch (S) region (16, 17). Although several reports suggest that the C-terminal region of AID is involved in protein stability (20, 21), C-terminal mutants of AID stabilized by fusing the hormone-binding domain of estrogen receptor (ER) also show similar CSR-defective phenotypes (8). Taken together, these data suggest that the DNA cleavage activity and CSR-specific activity depend on different regions of AID (8, 19). In addition, the C-terminal region of AID is essential for the interaction of AID with poly (A)⁺ RNA via a specific cofactor (22). Because CSR requires de novo protein synthesis, we proposed that after DNA cleavage the C-terminal region of AID may be involved in the regulation of the recombination step through generation of a new protein (8, 16, 22).DSBs induced by AID during CSR ultimately are joined by the efficient DNA repair pathway that requires C-NHEJ factors such as Ku70/80 (12, 23). However, in the absence of C-NHEJ, the A-EJ pathway that relies on microhomology can join the broken DNA ends, although this pathway is associated with chromosomal translocations (11, 24). Previously, we reported that JP8Bdel enhances aberrant c-myc/IgH translocations and that it fails to carry out the efficient recombination between donor and acceptor S regions in the IgH locus (8). Therefore, it is important to examine whether the AID C-terminal mutants affect the S–S joining in CSR.In the current work we examined whether the C-terminal region of AID is involved in DNA synapse formation and recombination during CSR in CH12F3-2 and spleen B cells. We also examined the effect of AID C-terminal mutations on GC in chicken DT40 cells, which depends on HR between pseudo V genes and the downstream IgVλ region. Using these CSR- and GC-monitoring systems, we demonstrate that efficient CSR and GC require the C-terminal region of AID for the formation of DSB from SSB and subsequent end synapse. Considering these findings together, we conclude that, in addition to DNA cleavage, AID has a unique function in the generation of DSBs, which is required for S–S synapse formation and joining in CSR and recombination in GC.     

Differential effect of aneuploidy on the X chromosome and genes with sex-biased expression in Drosophila

Global analysis of gene expression via RNA sequencing was conducted for trisomics for the left arm of chromosome 2 (2L) and compared with the normal genotype. The predominant response of genes on 2L was dosage compensation in that similar expression occurred in the trisomic compared with the diploid control. However, the male and female trisomic/normal expression ratio distributions for 2L genes differed in that females also showed a strong peak of genes with increased expression and males showed a peak of reduced expression relative to the opposite sex. For genes in other autosomal regions, the predominant response to trisomy was reduced expression to the inverse of the altered chromosomal dosage (2/3), but a minor peak of increased expression in females and further reduced expression in males were also found, illustrating a sexual dimorphism for the response to aneuploidy. Moreover, genes with sex-biased expression as revealed by comparing amounts in normal males and females showed responses of greater magnitude to trisomy 2L, suggesting that the genes involved in dosage-sensitive aneuploid effects also influence sex-biased expression. Each autosomal chromosome arm responded to 2L trisomy similarly, but the ratio distributions for X-linked genes were distinct in both sexes, illustrating an X chromosome-specific response to aneuploidy.Changes in chromosomal dosage have long been known to affect the phenotype or viability of an organism (1–4). Altering the dosage of individual chromosomes typically has a greater impact than varying the whole genome (5–7). This general rule led to the concept of “genomic balance” in that dosage changes of part of the genome produce a nonoptimal relationship of gene products. The interpretation afforded these observations was that genes on the aneuploid chromosome produce a dosage effect for the amount of gene product present in the cell (8).However, when gene expression studies were conducted on aneuploids, it became known that transacting modulations of gene product amounts were also more prevalent with aneuploidy than with whole-genome changes (9–14). Assays of enzyme activities, protein, and RNA levels revealed that any one chromosomal segment could modulate in trans the expression of genes throughout the genome (9–15). These modulations could be positively or negatively correlated with the changed chromosomal segment dosage, but inverse correlations were the most common (10–13). For genes on the varied segment, not only were dosage effects observed, but dosage compensation was also observed, which results from a cancelation of gene dosage effects by inverse effects operating simultaneously on the varied genes (9, 10, 14–18). This circumstance results in “autosomal” dosage compensation (14, 16–18). Studies of trisomic X chromosomes examining selected endogenous genes or global RNA sequencing (RNA-seq) studies illustrate that the inverse effect can also account for sex chromosome dosage compensation in Drosophila (15, 19–21). In concert, autosomal genes are largely inversely affected by trisomy of the X chromosome (15, 19, 21).The dosage effects of aneuploidy can be reduced to the action of single genes whose functions tend to be involved in heterogeneous aspects of gene regulation but which have in common membership in macromolecular complexes (8, 22–24). This fact led to the hypothesis that genomic imbalance effects result from the altered stoichiometry of subunits that affects the function of the whole and that occurs from partial but not whole-genome dosage change (8, 22–25). Genomic balance also affects the evolutionary trajectory of duplicate genes differently based on whether the mode of duplication is partial or whole-genome (22, 23).Here we used RNA-seq to examine global patterns of gene expression in male and female larvae trisomic for the left arm of chromosome 2 (2L). The results demonstrate the strong prevalence of aneuploidy dosage compensation and of transacting inverse effects. Furthermore, because both trisomic males and females could be examined, a sexual dimorphism of the aneuploid response was discovered. Also, the response of the X chromosome to trisomy 2L was found to be distinct from that of the autosomes, illustrating an X chromosome-specific effect. Genes with sex-biased expression, as determined by comparing normal males and females, responded more strongly to trisomy 2L. Collectively, the results illustrate the prevalence of the inverse dosage effect in trisomic Drosophila and suggest that the X chromosome has evolved a distinct response to genomic imbalance as would be expected under the hypothesis that X chromosome dosage compensation uses the inverse dosage effect as part of its mechanism (15).     

Activation induced deaminase C-terminal domain links DNA breaks to end protection and repair during class switch recombination

Activation-induced deaminase (AID) triggers antibody class switch recombination (CSR) in B cells by initiating DNA double strand breaks that are repaired by nonhomologous end-joining pathways. A role for AID at the repair step is unclear. We show that specific inactivation of the C-terminal AID domain encoded by exon 5 (E5) allows very efficient deamination of the AID target regions but greatly impacts the efficiency and quality of subsequent DNA repair. Specifically eliminating E5 not only precludes CSR but also, causes an atypical, enzymatic activity-dependent dominant-negative effect on CSR. Moreover, the E5 domain is required for the formation of AID-dependent Igh-cMyc chromosomal translocations. DNA breaks at the Igh switch regions induced by AID lacking E5 display defective end joining, failing to recruit DNA damage response factors and undergoing extensive end resection. These defects lead to nonproductive resolutions, such as rearrangements and homologous recombination that can antagonize CSR. Our results can explain the autosomal dominant inheritance of AID variants with truncated E5 in patients with hyper-IgM syndrome 2 and establish that AID, through the E5 domain, provides a link between DNA damage and repair during CSR.Antibodies change during an immune response by increasing their affinity for cognate antigen and acquiring new biological properties that reside in the constant region of the heavy chain. These changes originate from modifications in the Ig genes. Somatic hypermutation (SHM) introduces single base pair mutations over the Ig variable exon (IgV), changing the antibody affinity (1, 2). Class switch recombination (CSR) exchanges the exons encoding for the constant region of the heavy chain that defines IgM for those exons defining IgG, IgA, or IgE. This process involves the stepwise generation and subsequent repair of DNA double strand breaks (DSBs) (3, 4).Activation-induced deaminase (AID) initiates both SHM and CSR by deaminating deoxycytidine to deoxyuridine at the Ig loci (2). During CSR, removal of AID-generated deoxyuridine from opposite DNA strands at two distant switch (S) regions by either the uracil DNA-glycosylase (UNG) or components of the mismatch repair pathway initiates DNA processing leading to DSBs. These DSBs at the Igh evoke a DNA damage response and are resolved by either classical nonhomologous end joining (C-NHEJ) requiring the DSBs end-binding heterodimer Ku70/80, the scaffold protein Xrcc4, and Ligase4 (4, 5) or an ill-defined alternative end-joining (A-EJ) pathway (6) for productive CSR. CSR requires the joining of two simultaneous DSBs located far apart and deletion of the intervening chromosomal segment (3). As a side effect, CSR can also produce chromosomal translocations involving the Ig loci (7). The recombination of variable diversity joining (VDJ) gene fragments is also a long-range intrachromosomal joining, but in that case, the initiating recombination-activating gene (RAG)1/2 endonuclease protects the DNA ends and promotes C-NHEJ to prevent aberrant joining (8, 9). No analogous role of AID on DNA repair during CSR has been shown so far, although AID has been suggested to stabilize inter–S-region synapsis (10). The C terminus of AID is necessary for CSR but not SHM for unknown reasons (11, 12). This requirement might reflect a role of this domain in repair, given that C-terminally truncated AID variants still produce DSBs at the S regions in B cells (13–15). However, the fact that AID can be replaced by the yeast endonuclease I-SceI for efficient CSR in engineered mice seems to argue against its need for repair (16). Thus, it is still unclear whether AID contributes to the repair steps of CSR.AID deficiency causes a hyper-IgM immunodeficiency syndrome (HIGM2) in humans. Most HIGM2 patients carry deleterious mutations in AICDA (the AID gene), which are inherited as autosomal recessive (AR) traits (17, 18). These patients lack SHM and CSR, are susceptible to infections, and develop lymphadenopathies because of germinal center hyperplasia (17, 18). Intriguingly, a small proportion of HIGM2 patients carries only one mutated AICDA allele. There is no explanation as to why these alleles are autosomal dominant (AD), but in every case, the AD allele encodes for an AID protein missing the last 8 or 12 aa (12, 15). Because this region is necessary for CSR and because AD HIGM2 patients show normal SHM, the simplest explanation would be that AD AID variants behave as dominant negatives specifically for CSR, as suggested by the families’ pedigrees (15). We hypothesized that studying this proposed dominant-negative effect could also shed light on the role of AID C terminus and show a role of AID in late steps of CSR.     

Functional requirements of AID’s higher order structures and their interaction with RNA-binding proteins

Activation-induced cytidine deaminase (AID) is essential for the somatic hypermutation (SHM) and class-switch recombination (CSR) of Ig genes. Although both the N and C termini of AID have unique functions in DNA cleavage and recombination, respectively, during SHM and CSR, their molecular mechanisms are poorly understood. Using a bimolecular fluorescence complementation (BiFC) assay combined with glycerol gradient fractionation, we revealed that the AID C terminus is required for a stable dimer formation. Furthermore, AID monomers and dimers form complexes with distinct heterogeneous nuclear ribonucleoproteins (hnRNPs). AID monomers associate with DNA cleavage cofactor hnRNP K whereas AID dimers associate with recombination cofactors hnRNP L, hnRNP U, and Serpine mRNA-binding protein 1. All of these AID/ribonucleoprotein associations are RNA-dependent. We propose that AID’s structure-specific cofactor complex formations differentially contribute to its DNA-cleavage and recombination functions.Activation-induced cytidine deaminase (AID), which is expressed in antigen-stimulated mature B cells, is essential for Ig somatic hypermutation (SHM) and class-switch recombination (CSR) (1, 2). AID induces DNA breaks at the variable (V) and switch (S) regions during SHM and CSR, respectively (3, 4). Although both processes are initiated by AID-induced DNA cleavage, point mutations at the V region are executed mostly by error-prone DNA repair whereas CSR is accomplished by recombination of cleaved ends at donor and acceptor S regions (5, 6). However, the detailed mechanisms by which AID carries out the two mechanistically distinct functions for SHM and CSR have yet to be uncovered (7). Studies on AID mutants revealed that AID’s N- and C-terminal domains are distinctly required for its DNA-cleavage and recombination functions, respectively (8–10). Mutations at the N terminus of AID impair SHM as well as CSR whereas those at the C terminus abrogate CSR only and show increased SHM activity. Recent studies demonstrated that the CSR process after DNA cleavage, including the synapsis formation between cleaved ends, is impaired with the C-terminally defective AID, indicating that AID’s C terminus confers a CSR-specific recombination function, independent of AID’s DNA cleavage function, by an unknown mechanism (11, 12).AID belongs to the APOBEC (apolipoprotein B mRNA-editing enzyme catalytic polypeptide) family of cytidine deaminases (CDDs) and shows high sequence homology with APOBEC1 (A1) (1, 13, 14), which edits apolipoprotein B (APOB) mRNA. The APOB mRNA editing ability of A1 is highly dependent on its cofactors, A1CF/ACF (15, 16) and RBM47 (17), both of which belong to the heterogeneous nuclear ribonucleoprotein (hnRNP) family. Recently, two A1CF-like hnRNPs, hnRNP K and hnRNP L, were identified as the cofactors of AID and found to be involved in the cleavage and recombination of DNA, respectively (18). Because the N and C termini of AID differentially regulate two functions of AID—cleavage and recombination, respectively—we speculated that the AID termini would be critical for function-coupled cofactor association. For instance, the N or C terminus of AID may function as a molecular switch that induces an AID–AID interaction, enabling AID to exert distinct physiological functions through its association with cofactors. Regrettably, however, there is little structural information available that can explain any of AID’s regulatory modes of action, including its cofactor association mechanisms, in the context of its physiological functions.Although a significant amount of structural information is available for a number of APOBEC family members, the 3D structures of A1 and AID are yet to be resolved (19, 20). The CDD family of enzymes exists in nature in a variety of structural forms, including monomeric, dimeric, and tetrameric forms, and comparative structural modeling using the yeast CDD structure predicts a dimeric structure for both A1 and AID (21, 22). On the other hand, homology modeling with the APOBEC2 (A2) crystal structure, which seems to be a tetramer composed of two head-to-head interacting dimers, predicts that AID forms a tetramer (23). Notably, A2 was later reported to exist as a monomer in solution (24). Similarly, an atomic force microscopic (AFM) study found that AID exists in the cell predominantly as a monomer associated with a single-strand DNA substrate (25). However, the same AFM dataset was interpreted differently by another group of investigators, who concluded that AID probably forms an A2-like tetramer in solution (26). The modeling of AID’s catalytic pocket in reference to eight APOBEC family members suggested that most of the AID–DNA complex remains in an inactive state due to occlusion by the substrate DNA, which may explain its weak catalytic activity for cleaving DNA in vitro (27).One of the limitations of the computational modeling of AID’s structure is that AID’s N-and C-terminal sequences are substantially different from those of other APOBEC members and thus reside outside the modeling template. Although the structural outcome of a protein can differ by a variety of reasons, including the methods applied (28), none of the AID studies mentioned above explain why the C-terminal deletion of AID leads to the loss of CSR function only. Therefore, model-based computational simulation may not explain the physiological structure–function relationship of AID in B cells.Here, we explored AID’s structure–function relationship using a bimolecular fluorescence complementation (BiFC) assay, which detects homo- or heteromeric protein–protein interactions in live cells (29, 30). For the homomeric interaction assay, the target protein is fused to two nonfluorescent halves of a green or red fluorescent protein. An interaction between two of the target proteins brings the two nonfluorescent halves of the fluorescent protein into close proximity, reconstituting the fluorescence. The BiFC assay thus allows a rapid analysis of the dimerization of a protein of interest in live cells.By combining this assay with other biochemical approaches, such as coimmunoprecipitation (co-IP) and glycerol gradient sedimentation, we revealed the presence of both monomeric and dimeric forms of AID in analyzed cells. Intriguingly, C-terminal AID mutants that lost CSR function showed a severe dimerization defect, suggesting that AID’s C terminus is required to stabilize the dimeric structure that is required for CSR. We also showed that the AID monomer and dimer associate with different RNA-binding proteins (RBPs) to form ribonucleoprotein (RNP) complexes. Based on these findings, we propose that the monomeric AID–RNP complex includes hnRNP K (18) and contributes to the DNA cleavage function of AID whereas the dimeric AID–RNP complexes include hnRNP L (18), hnRNP U (31), or Serpine mRNA-binding protein 1 (SERBP1) (32) and contribute to the recombination step of CSR.     

Genomic donor cassette sharing during VLRA and VLRC assembly in jawless vertebrates

Lampreys possess two T-like lymphocyte lineages that express either variable lymphocyte receptor (VLR) A or VLRC antigen receptors. VLRA⁺ and VLRC⁺ lymphocytes share many similarities with the two principal T-cell lineages of jawed vertebrates expressing the αβ and γδ T-cell receptors (TCRs). During the assembly of VLR genes, several types of genomic cassettes are inserted, in step-wise fashion, into incomplete germ-line genes to generate the mature forms of antigen receptor genes. Unexpectedly, the structurally variable components of VLRA and VLRC receptors often possess partially identical sequences; this phenomenon of module sharing between these two VLR isotypes occurs in both lampreys and hagfishes. By contrast, VLRA and VLRC molecules typically do not share their building blocks with the structurally analogous VLRB receptors that are expressed by B-like lymphocytes. Our studies reveal that VLRA and VLRC germ-line genes are situated in close proximity to each other in the lamprey genome and indicate the interspersed arrangement of isotype-specific and shared genomic donor cassettes; these features may facilitate the shared cassette use. The genomic structure of the VLRA/VLRC locus in lampreys is reminiscent of the interspersed nature of the TCRA/TCRD locus in jawed vertebrates that also allows the sharing of some variable gene segments during the recombinatorial assembly of TCR genes.The only two extant taxa of jawless vertebrates (agnatha), lampreys and hagfishes, occupy a unique position in chordate phylogeny and thus are a focal point for studies in comparative immunology. Although jawless vertebrates were shown to reject skin allografts and to produce serum agglutinins to mammalian red blood cells and bacteria (1, 2), the cellular and molecular bases for these adaptive responses remained elusive until the recent identification of their alternative adaptive immune system (3). In contrast to the antigen receptors of jawed vertebrates, whose structural framework is the Ig-domain, the basic building block of agnathan antigen receptors is the leucine-rich repeat (LRR) (4–6). In analogy to the situation in jawed vertebrates, mature genes of so-called variable lymphocyte receptors (VLRs) are combinatorially assembled from different types of genomic donor LRR cassettes; their sequences are inserted into incomplete germ-line VLR genes (4–6). A gene conversion-like process is postulated to underlie the VLR gene assembly (7, 8), through the activity of orthologs of mammalian activation-induced cytidine deaminase (AID), termed cytidine deaminases 1 and 2 (CDA1 and CDA2) (7, 9). As is the case for T-cell receptors (TCRs) and B-cell receptors (BCRs) of jawed vertebrates, combinatorial VLR assembly generates vast repertoires of diverse anticipatory receptors (4–6).Three VLR genes, VLRA, VLRB and VLRC, have been identified in lampreys and hagfishes (3, 9–12). The three VLR isotypes are differentially expressed by three distinct populations of lymphocytes in lampreys (9, 13). The two types of T-like cells of lamprey, VLRA⁺ and VLRC⁺ lymphocytes, are generated in the thymoid, a lymphoepithelial tissue equivalent to the thymus (13–15); this situation is analogous to the development in the thymus of the two distinct αβ and γδ T-cell lineages in jawed vertebrates. The VLRB⁺ cells appear to be generated in hematopoietic tissues outside of the thymoid (14), much like B cells in jawed vertebrates. These findings suggest that these basic pathways of lymphocyte differentiation already existed in a common ancestor of jawed and jawless vertebrates (4–6, 16).The close developmental relationship of the two principal lineages of T lymphocytes in jawed vertebrates is reflected in the unique genomic organization of the TCR genes that encode the four chains of the two different heterodimeric αβ and γδ TCRs (17). Here, we examine the sequence diversity and genomic organization of VLRA and VLRC receptor genes to gain insight into their functional and evolutionary relationship.