AID is essential for immunoglobulin V gene conversion in a cultured B cell line

Following productive V gene rearrangement, the functional immunoglobulin genes in the B lymphocytes of man and mouse are subjected to two further types of genetic modification. Class-switch recombination, a region-specific but largely nonhomologous recombination process, leads to a change in constant region of the expressed antibody. Somatic hypermutation introduces multiple single nucleotide substitutions in and around the rearranged V gene segments and underpins affinity maturation. However, in chicken and rabbits (but not man or mouse), an additional mechanism, gene conversion, is a major contributor to V gene diversification. It has been demonstrated recently that both switch recombination and hypermutation are ablated in mice and humans lacking AID, a B cell-specific protein of unknown molecular activity. Here we show that disruption of AID in the DT40 chicken B cell lymphoma leads to a failure to perform immunoglobulin V gene conversion. Thus, AID is required for all three immunoglobulin gene modification programs (gene conversion, hypermutation, and switch recombination) and acts in the initiation or execution of these processes rather than in bringing the B cell to an appropriate stage of differentiation.     

AID and Igh switch region-Myc chromosomal translocations

Chromosomal translocations involving Ig heavy chain switch regions and an oncogene, like Myc, represent early initiating events in the development of many B cell malignancies. These translocations are widely believed to result from aberrant class switch recombination (CSR). Recent reports have produced conflicting models for the role of activation-induced cytidine deaminase (AID) in this process. Here, we discuss possible roles of AID, CSR, and somatic hypermutation in generating chromosomal translocations and in tumor progression.     

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.     

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.     

Transcription-coupled mutagenesis by the DNA deaminase AID

Activation-induced deaminase (AID) initiates switch recombination and somatic hypermutation of immunoglobulin genes in activated B cells. Compelling evidence now shows that AID travels with RNA polymerase II to deaminate actively transcribed DNA.     

Stimulation of V(D)J recombination by histone acetylation

V(D)J recombination assembles functional immunoglobulin and T cell receptor genes from individual gene segments [1]. A common recombination mechanism, initiated by the proteins RAG1 and RAG2 at conserved recombination signal sequences (RSSs), operates at all rearranging loci [2] [3]. It has been proposed that the key regulator of the reaction is 'accessibility' of the RSS within chromatin [4]. Recently, the packaging of RSSs into nucleosomes was shown to inhibit initiation of V(D)J recombination [5] [6]. Nevertheless, the tight tissue specificity of regulation cannot be explained by nucleosome-mediated repression alone because a significant fraction of RSSs would be predicted to lie in linker regions between nucleosomes. Therefore, some aspect of the regulation of the recombination reaction must rely on the disruption of higher-order chromatin structure. Here, we report that histone acetylation directly stimulates the recombination reaction in vivo in the correct cell- and stage-specific manner. Neither expression of RAG genes nor activity of RAG proteins was increased by acetylation. Furthermore, histone acetylation failed to overcome nucleosome-mediated repression of RSS recognition and cleavage in vitro. Our data suggest a role for histone acetylation in stimulating recombination in vivo through disruption of higher-order chromatin structures.