AID binds cooperatively with UNG and Msh2-Msh6 to Ig switch regions dependent upon the AID C terminus

Activation-induced cytidine deaminase (AID) is induced in B cells during an immune response and is essential for both class-switch recombination (CSR) and somatic hypermutation of Ab genes. The C-terminal 10 aa of AID are required for CSR but not for somatic hypermutation, although their role in CSR is unknown. Using retroviral transduction into mouse splenic B cells, we show that the C terminus is not required for switch (S) region double-strand breaks (DSBs) and therefore functions downstream of DSBs. Using chromatin immunoprecipitation, we show that AID binds cooperatively with UNG and the mismatch repair proteins Msh2-Msh6 to Ig Sμ and Sγ3 regions, and this depends on the C terminus and the deaminase activity of AID. We also show that mismatch repair does not contribute to the efficiency of CSR in the absence of the AID C terminus. Although it has been demonstrated that both UNG and Msh2-Msh6 are important for introduction of S region DSBs, our data suggest that the ability of AID to recruit these proteins is important for DSB resolution, perhaps by directing the S region DSBs toward accurate and efficient CSR via nonhomologous end joining.     

High resolution analysis of the chromatin landscape of the IgE switch region in human B cells

Antibodies are assembled by a highly orchestrated series of recombination events during B cell development. One of these events, class switch recombination, is required to produce the IgG, IgE and IgA antibody isotypes characteristic of a secondary immune response. The action of the enzyme activation induced cytidine deaminase is now known to be essential for the initiation of this recombination event. Previous studies have demonstrated that the immunoglobulin switch regions acquire distinct histone modifications prior to recombination. We now present a high resolution analysis of these histone modifications across the IgE switch region prior to the initiation of class switch recombination in primary human B cells and the human CL-01 B cell line. These data show that upon stimulation with IL-4 and an anti-CD40 antibody that mimics T cell help, the nucleosomes of the switch regions are highly modified on histone H3, accumulating acetylation marks and tri-methylation of lysine 4. Distinct peaks of modified histones are found across the switch region, most notably at the 5' splice donor site of the germline (I) exon, which also accumulates AID. These data suggest that acetylation and K4 tri-methylation of histone H3 may represent marks of recombinationally active chromatin and further implicates splicing in the regulation of AID action.     

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.     

Mismatch repair converts AID-instigated nicks to double-strand breaks for antibody class-switch recombination

Mismatch repair (MMR) proteins are important for antibody class-switch recombination (CSR), but their roles are unknown. We propose a model for the function of MMR in CSR in which MMR proteins convert single-strand nicks instigated by activation-induced cytidine deaminase (AID) into the double-strand breaks (DSBs) that are required for CSR. This model does not invoke any novel functions for MMR but simply posits that, owing to numerous single-strand nicks in the switch (S) regions of both DNA strands, when MMR proteins are recruited by U:G mismatches, they excise one strand of DNA and soon reach a nick on the opposite strand. This halts excision activity and creates a DSB. This model explains why B cells that lack either S mu and MSH2 or UNG and MSH2 cannot undergo CSR.     

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.     

Structural phylogenetic analysis of activation-induced deaminase function

In mammals, activation-induced deaminase (AID) initiates somatic hypermutation (SHM) and class switch recombination (CSR) of Ig genes. SHM and CSR activities require separate regions within AID. A chromosome region maintenance 1 (CRM1)-dependent nuclear export signal (NES) at the AID C terminus is necessary for CSR, and has been suggested to associate with CSR-specific cofactors. CSR appeared late in AID evolution, during the emergence of land vertebrates from bony fish, which only display SHM. Here, we show that AID from African clawed frog (Xenopus laevis), but not pufferfish (Takifugu rubripes), can induce CSR in AID-deficient mouse B cells, although both are catalytically active in bacteria and mammalian cell systems, albeit at decreased level. Like mammalian AID, Takifugu AID is actively exported from the cell nucleus by CRM1, and the Takifugu NES can substitute for the equivalent region in human AID, indicating that all the CSR-essential NES motif functions evolutionarily predated CSR activity. We also show that fusion of the Takifugu AID catalytic domain to the entire human noncatalytic domain restores activity in mammalian cells, suggesting that AID features mapping within the noncatalytic domain, but outside the NES, influence its function.     

Nbs1 ChIP-Seq Identifies Off-Target DNA Double-Strand Breaks Induced by AID in Activated Splenic B Cells

Activation-induced cytidine deaminase (AID) is required for initiation of Ig class switch recombination (CSR) and somatic hypermutation (SHM) of antibody genes during immune responses. AID has also been shown to induce chromosomal translocations, mutations, and DNA double-strand breaks (DSBs) involving non-Ig genes in activated B cells. To determine what makes a DNA site a target for AID-induced DSBs, we identify off-target DSBs induced by AID by performing chromatin immunoprecipitation (ChIP) for Nbs1, a protein that binds DSBs, followed by deep sequencing (ChIP-Seq). We detect and characterize hundreds of off-target AID-dependent DSBs. Two types of tandem repeats are highly enriched within the Nbs1-binding sites: long CA repeats, which can form Z-DNA, and tandem pentamers containing the AID target hotspot WGCW. These tandem repeats are not nearly as enriched at AID-independent DSBs, which we also identified. Msh2, a component of the mismatch repair pathway and important for genome stability, increases off-target DSBs, similar to its effect on Ig switch region DSBs, which are required intermediates during CSR. Most of the off-target DSBs are two-ended, consistent with generation during G1 phase, similar to DSBs in Ig switch regions. However, a minority are one-ended, presumably due to conversion of single-strand breaks to DSBs during replication. One-ended DSBs are repaired by processes involving homologous recombination, including break-induced replication repair, which can lead to genome instability. Off-target DSBs, especially those present during S phase, can lead to chromosomal translocations, deletions and gene amplifications, resulting in the high frequency of B cell lymphomas derived from cells that express or have expressed AID.     

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.     

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.     

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.     

AID is required for c-myc/IgH chromosome translocations in vivo

Chromosome translocations between c-myc and immunoglobulin (Ig) are associated with Burkitt's lymphoma in humans and with pristane- and IL6-induced plasmacytomas in mice. These translocations frequently involve Ig switch regions, suggesting that they might be the result of aberrant Ig class switch recombination (CSR). However, a direct link between CSR and chromosome translocations has not been established. We have examined c-myc/IgH translocations in IL6 transgenic mice that are mutant for activation induced cytidine deaminase (AID), the enzyme that initiates CSR. Here we report that AID is essential for the c-myc/IgH chromosome translocations induced by IL6.