Activation-induced Cytidine Deaminase Targeting Research Articles

The Mismatch Repair Protein Msh6 Influences the In Vivo AID Targeting to the Ig Locus

Somatic hypermutation (SHM) and class switch recombination (CSR) are initiated by activation-induced cytidine deaminase (AID), which preferentially deaminates deoxycytidines at WRC (W = A/T, R = A/G) motifs in vitro. The mechanisms responsible for targeting AID and for organizing the queue of enzymes involved in vivo have been elusive. Here, we examined point mutant knockin Msh6 mice (Msh6(TD/TD)), which lack the second phase of SHM but retain all the proteins involved, and found that AID was frequently targeted to non-WRC motifs. Unexpectedly, by comparing SHM and CSR in wild-type, Msh6(TD/TD), and age-matched Msh6(-/-) mice, we discovered that the presence of Msh6 protein influenced the AID targeting in phase one of SHM and mediated the proper targeting of recombination sites in CSR in vivo. Our data suggest that Msh6 plays a scaffolding role in the first phase of SHM, in addition to its enzymatic role in the second phase of SHM.

Shifts in targeting of class switch recombination sites in mice that lack μ switch region tandem repeats or Msh2

The mechanisms that target class switch recombination (CSR) to antibody gene switch (S) regions are unknown. Analyses of switch site locations in wild-type mice and in mice that lack the Sμ tandem repeats show shifts indicating that a 4–5-kb DNA domain (bounded upstream by the Iμ promoter) is accessible for switching independent of Sμ sequences. This CSR-accessible domain is reminiscent of the promoter-defined domains that target somatic hypermutation. Within the 4–5-kb CSR domain, the targeting of S site locations also depends on the Msh2 mismatch repair protein because Msh2-deficient mice show an increased focus of sites to the Sμ tandem repeat region. We propose that Msh2 affects S site location because sequences with few activation-induced cytidine deaminase targets generate mostly switch DNA cleavages that require Msh2-directed processing to allow CSR joining.

AID binds to transcription-induced structures in c-MYC that map to regions associated with translocation and hypermutation

Translocation and aberrant hypermutation of c-MYC are common in B-cell lymphomas. Activation-induced Cytidine Deaminase (AID) initiates switch recombination and somatic hypermutation in B cells by targeted deamination of transcribed genes. We show that transcription of the immunoglobulin S regions and c-MYC results in formation of similar DNA structures, 'G-loops', which contain a cotranscriptional RNA: DNA hybrid on the C-rich strand and single-stranded regions and G4 DNA on the G-rich strand. AID binds specifically to G-loops within transcribed S regions and c-MYC, and G-loops in c-MYC map to the regions associated with translocation breakpoints and aberrant hypermutation in B-cell lymphomas. Aberrant targeting of AID to DNA structures formed upon c-MYC transcription may therefore contribute to the genetic instability of c-MYC in B-cell malignancies.

VH replacement in rearranged immunoglobulin genes

Examples suggesting that all or part of the V(H) segment of a rearranged V(H)DJ(H) may be replaced by all or part of another V(H) have been appearing since the 1980s. Evidence has been presented of two rather different types of replacement. One of these has gained acceptance and has now been clearly demonstrated to occur. The other, proposed more recently, has not yet gained general acceptance because the same effect can be produced by polymerase chain reaction artefact. We review both types of replacement including a critical examination of evidence for the latter. The first type involves RAG proteins and recombination signal sequences (RSS) and occurs in immature B cells. The second was also thought to be brought about by RAG proteins and RSS. However, it has been reported in hypermutating cells which are not thought to express RAG proteins but in which activation-induced cytidine deaminase (AID) has recently been shown to initiate homologous recombination. Re-examination of the published sequences reveals AID target sites in V(H)-V(H) junction regions and examples that resemble gene conversion.

Hyper‐immunoglobulin M syndromes caused by intrinsic B‐lymphocyte defects

Hyper-immunoglobulin M (IgM) syndromes are primary immunodeficiencies characterized by normal or elevated serum IgM levels with the absence of other isotypes, pinpointing to a defect in the Ig class switch recombination (CSR). The delineation of hyper-IgM syndromes made it possible to better define the mechanisms underlying the two major events of antibody maturation in humans, CSR and introduction of somatic hypermutation (SHM) in the variable region of immunoglobulins. The description of the activation-induced cytidine deaminase (AID) deficiency, characterized by a defect in both CSR and SHM, demonstrated for the first time that this molecule acts as a master player in the antigen-induced Ig gene-modification events responsible for both CSR and SHM. However, deleterious mutations located in the C-terminus lead to a CSR defect without affecting SHM, providing evidence for a role of AID in CSR distinct from the cytidine deaminase activity, likely by binding to a specific CSR cofactor. Molecular causes of two other hyper-IgM conditions have not yet been defined. However, they may be caused by either a defect in AID targeting on S regions or a CSR-specific DNA-repair defect. The mechanism of action of AID remains somewhat debated, but the observation that uracil-DNA-glycosylase deficiency leads to a severe hyper-IgM syndrome strongly argues in favor of a DNA-editing activity of AID.

2 Pitfalls in developing cancer chemoprevention agents

The antibody gene mutator activation-induced cytidine deaminase (AID) promiscuously damages oncogenes, leading to chromosomal translocations and tumorigenesis. Why nonimmunoglobulin loci are susceptible to AID activity is unknown. Here, we study AID-mediated lesions in the context of nuclear architecture and the B cell regulome. We show that AID targets are not randomly distributed across the genome but are predominantly grouped within super-enhancers and regulatory clusters. Unexpectedly, in these domains, AID deaminates active promoters and eRNA+ enhancers interconnected in some instances over megabases of linear chromatin. Using genome editing, we demonstrate that 3D-linked targets cooperate to recruit AID-mediated breaks. Furthermore, a comparison of hypermutation in mouse B cells, AID-induced kataegis in human lymphomas, and translocations in MEFs reveals that AID damages different genes in different cell types. Yet, in all cases, the targets are predominantly associated with topological complex, highly transcribed super-enhancers, demonstrating that these compartments are key mediators of AID recruitment.

5 Towards a high throughput platform for quantitative serum protein profiling

The antibody gene mutator activation-induced cytidine deaminase (AID) promiscuously damages oncogenes, leading to chromosomal translocations and tumorigenesis. Why nonimmunoglobulin loci are susceptible to AID activity is unknown. Here, we study AID-mediated lesions in the context of nuclear architecture and the B cell regulome. We show that AID targets are not randomly distributed across the genome but are predominantly grouped within super-enhancers and regulatory clusters. Unexpectedly, in these domains, AID deaminates active promoters and eRNA+ enhancers interconnected in some instances over megabases of linear chromatin. Using genome editing, we demonstrate that 3D-linked targets cooperate to recruit AID-mediated breaks. Furthermore, a comparison of hypermutation in mouse B cells, AID-induced kataegis in human lymphomas, and translocations in MEFs reveals that AID damages different genes in different cell types. Yet, in all cases, the targets are predominantly associated with topological complex, highly transcribed super-enhancers, demonstrating that these compartments are key mediators of AID recruitment.

Activation-induced cytidine deaminase (AID) can target both DNA strands when the DNA is supercoiled.

The activation-induced cytidine deaminase (AID) is required for somatic hypermutation (SHM) and class-switch recombination of Ig genes. It has been shown that in vitro, AID protein deaminates C in single-stranded DNA or the coding-strand DNA that is being transcribed but not in double-stranded DNA. However, in vivo, both DNA strands are mutated equally during SHM. We show that AID efficiently deaminates C on both DNA strands of a supercoiled plasmid, acting preferentially on SHM hotspot motifs. However, this DNA is not targeted by AID when it is relaxed after treatment with topoisomerase I, and thus, supercoiling plays a crucial role for AID targeting to this DNA. Most of the mutations are in negatively supercoiled regions, suggesting a mechanism of AID targeting in vivo. During transcription the DNA sequences upstream of the elongating RNA polymerase are negatively supercoiled, and this transient change in DNA topology may allow AID to access both DNA strands.

Replication protein A interacts with AID to promote deamination of somatic hypermutation targets.

Activation-induced cytidine deaminase (AID) is a single-stranded (ss) DNA deaminase required for somatic hypermutation (SHM) and class switch recombination of immunoglobulin genes. Class switch recombination involves transcription through switch regions, which generates ssDNA within R loops. However, although transcription through immunoglobulin variable region exons is required for SHM, it does not generate stable ssDNA, which leaves the mechanism of AID targeting unresolved. Here we characterize the mechanism of AID targeting to in-vitro-transcribed substrates harbouring SHM motifs. We show that the targeting activity of AID is due to replication protein A (RPA), a ssDNA-binding protein involved in replication, recombination and repair. The 32-kDa subunit of RPA interacts specifically with AID from activated B cells in a manner that seems to be dependent on post-translational AID modification. Thus, our study implicates RPA as a novel factor involved in immunoglobulin diversification. We propose that B-cell-specific AID-RPA complexes preferentially bind to ssDNA of small transcription bubbles at SHM 'hotspots', leading to AID-mediated deamination and RPA-mediated recruitment of DNA repair proteins.

Human activation-induced cytidine deaminase causes transcription-dependent, strand-biased C to U deaminations.

Activation-induced cytidine deaminase (AID) is required for the maturation of antibodies in higher vertebrates, where it promotes somatic hypermutation (SHM), class switch recombination and gene conversion. While it is known that SHM requires high levels of transcription of the target genes, it is unclear whether this is because AID targets transcribed genes. We show here that the human AID promotes C to T mutations in Escherichia coli which are stimulated by transcription. The mutations are strand-biased and occur preferentially in the non-transcribed strand of the target gene. Human AID purified from E.coli is active without prior treatment with a ribonuclease and deaminates cytosines in plasmid DNA in vitro. Further, the action of this enzyme is greatly stimulated by the transcription of the target gene in a strand-dependent fashion. These results confirm the prediction that AID may act directly on DNA and show that it can act on transcribing DNA in the absence of specialized DNA structures such as R-loops. It suggests that AID may be recruited to variable genes through transcription without the assistance of other proteins and that the strand bias in SHM may be caused by the preference of AID for the non-transcribed strand.