Kazuo Kinoshita

Constitutive Expression of AID Leads to Tumorigenesis

Genome stability is regulated by the balance between efficiencies of the repair machinery and genetic alterations such as mutations and chromosomal rearrangements. It has been postulated that deregulation of class switch recombination (CSR) and somatic hypermutation (SHM), which modify the immunoglobulin (Ig) genes in activated B cells, may be responsible for aberrant chromosomal translocations and mutations of non-Ig genes that lead to lymphocyte malignancy. However, the molecular basis for these genetic instabilities is not clearly understood. Activation-induced cytidine deaminase (AID) is shown to be essential and sufficient to induce both CSR and SHM in artificial substrates in fibroblasts as well as B cells. Here we show that constitutive and ubiquitous expression of AID in transgenic mice caused both T cell lymphomas and dysgenetic lesions of epithelium of respiratory bronchioles (micro-adenomas) in all individual mice. Point mutations, but not translocations, were massively introduced in expressed T cell receptor (TCR) and c-myc genes in T lymphoma cells. The results indicate that AID can mutate non-Ig genes including oncogenes, implying that aberrant AID expression could be a cause of human malignancy.

In situ class switching and differentiation to IgA-producing cells in the gut lamina propria

One of the front lines of the immune defence is the gut mucosa, where immunoglobulin-α (IgA) is continuously produced to react with commensal bacteria and dietary antigens. It is generally accepted that, after antigenic stimulation in the Peyers patches, IgA+ lymphoblasts (B220+IgA+) migrate through the lymph and blood circulation, and eventually home to the lamina propria of the intestine. Mice that lack activation-induced cytidine deaminase (AID) are defective in class switch recombination (CSR) and somatic hypermutation. CSR changes the immunoglobulin heavy chain constant region (CH) gene being expressed from Cµ to other CH genes, resulting in a switch of the immunoglobulin isotype from IgM to IgG, IgE or IgA. AID-/- mice also secrete large amounts of immunoglobulin-µ (IgM) into faeces, and accumulate B220-IgM+ plasma cells as well as B220+IgM+ cells in the gut. Here we show that lamina propria B220+IgA+ cells have just completed CSR, as they still express both AID and transcripts from circular DNA that has been ‘looped-out’ during CSR. Lamina propria IgM+ B cells seem to be pre-committed to switching to IgA+ in vitro as well as in vivo. Culturing lamina propria IgM+ B cells together with lamina propria stromal cells enhances preferential switching and differentiation of B cells to IgA+ plasma cells. We conclude that IgA+ cells in the gut lamina propria are generated in situ from B220+IgM+ lymphocytes.

AID mutant analyses indicate requirement for class-switch-specific cofactors

Activation-induced cytidine deaminase (AID) is the essential and sole B cell–specific factor required for class-switch recombination (CSR) and somatic hypermutation (SHM). However, it is not known how AID differentially regulates these two independent events. Involvement of several cofactors interacting with AID has been indicated by scattered distribution of loss-of-function point mutations and evolutionary conservation of the entire 198-amino-acid protein. Here, we report that human AID mutant proteins with insertions, replacements or truncations in the C-terminal region retained strong SHM activity but almost completely lost CSR activity. These results indicate that AID requires interaction with a cofactor(s) specific to CSR.

The AID enzyme induces class switch recombination in fibroblasts.

The switch of the immunoglobulin isotype from IgM to IgG, IgE or IgA is mediated by class switch recombination (CSR). CSR changes the immunoglobulin heavy chain constant region (CH) gene from Cμ to one of the other CH genes. Somatic hypermutation introduces massive numbers of point mutations in the immunoglobulin variable (V) region gene, giving rise to immunoglobulin with higher affinity. Activation-induced cytidine deaminase (AID), a putative RNA-editing cytidine deaminase, is expressed strictly in activated B cells and is indispensable in both CSR and somatic hypermutation. But the exact function of AID is unknown. Here we show that ectopic expression of AID induces CSR in an artificial switch construct in fibroblasts at a level comparable to that in stimulated B cells. Sequences around recombination junctions in the artificial substrate have features similar to endogenous CSR junctions. Furthermore, AID-induced CSR in fibroblasts is dependent on transcription of the target S region, as shown in endogenous CSR in B cells. The results show that AID is the only B-cell-specific factor required for initiation of the CSR reaction in the activated locus.

Activation-induced Deaminase (AID)-directed Hypermutation in the Immunoglobulin Sμ Region Implication of AID Involvement in a Common Step of Class Switch Recombination and Somatic Hypermutation

Somatic hypermutation (SHM) and class switch recombination (CSR) cause distinct genetic alterations at different regions of immunoglobulin genes in B lymphocytes: point mutations in variable regions and large deletions in S regions, respectively. Yet both depend on activation-induced deaminase (AID), the function of which in the two reactions has been an enigma. Here we report that B cell stimulation which induces CSR but not SHM, leads to AID-dependent accumulation of SHM-like point mutations in the switch μ region, uncoupled with CSR. These findings strongly suggest that AID itself or a single molecule generated by RNA editing function of AID may mediate a common step of SHM and CSR, which is likely to be involved in DNA cleavage.

A hallmark of active class switch recombination: Transcripts directed by I promoters on looped-out circular DNAs

To specify when and where Ig class switch recombination (CSR) takes place, a good molecular marker closely associated with active CSR is required. CSR is accompanied by deletion of circular DNA from the Ig heavy chain locus. The circular DNA contains a DNA segment between Sμ and a target S region including its I promoter, which is driven by specific cytokine stimulation before CSR. We found that the specific I promoter is still active in looped-out circular DNA and directs production of I-Cμ transcripts termed “circle transcripts.” Reverse transcription–PCR demonstrated transient induction of specific circle transcripts upon CSR in a murine lymphoma cell line, CH12F3-2A, as well as spleen B cells. Production of the circle transcripts appeared to depend on expression of activation-induced cytidine deaminase (AID), an essential factor for CSR. A comparison of kinetics between circle transcripts and circular DNA showed more rapid disappearance of circle transcripts. Thus, circle transcripts may serve as a hallmark for active CSR in vitro and in vivo.

DNA double-strand breaks: prior to but not sufficient in targeting hypermutation.

The activation-induced cytidine deaminase (AID) is required for somatic hypermutation (SHM) and class-switch recombination (CSR) of immunoglobulin (Ig) genes, both of which are associated with DNA double-strand breaks (DSBs). As AID is capable of deaminating deoxy-cytidine (dC) to deoxy-uracil (dU), it might induce nicks (single strand DNA breaks) and also DNA DSBs via a U-DNA glycosylase-mediated base excision repair pathway (‘DNA-substrate model’). Alternatively, AID functions like its closest homologue Apobec1 as a catalytic subunit of a RNA editing holoenzyme (‘RNA-substrate model’). Although rearranged Vλ genes are preferred targets of SHM we found that germinal center (GC) B cells of AID-proficient and -deficient Vλ1-expressing GC B cells display a similar frequency, distribution, and sequence preference of DSBs in rearranged and also in germline Vλ1 genes. The possible roles of DSBs in relation to AID function and SHM are discussed.

Linking class-switch recombination with somatic hypermutation

The recent discovery of a molecular link between two apparently different genetic alteration events — class-switch recombination and somatic hypermutation — has led to the idea that the recognition and cleavage of target DNA in these two events might be mediated by similar or identical molecules to those involved in RNA editing. This could mean that the complexity of mammalian genetic information may be enriched by an interplay between RNA editing and DNA modification.

De novo protein synthesis is required for the activation-induced cytidine deaminase function in class-switch recombination

Activation-induced cytidine deaminase (AID) is required for class-switch recombination (CSR), somatic hypermutation, and gene conversion of Ig genes. Although AID has sequence similarity to an RNA-editing enzyme Apobec-1, how AID functions in CSR and somatic hypermutation is unknown. Because involvement of RNA-editing but not DNA-editing in CSR requires de novo protein synthesis after AID expression, we examined whether protein synthesis inhibitors could block CSR in the presence of the AID activity. For this purpose we constructed AID fused with the hormone-binding domain of the estrogen receptor (AID-ER), which was introduced into AID-deficient spleen B cells. When such transfectants were treated with an estrogen analogue, 4-hydroxytamoxifen (OHT), CSR was induced within 1 h. Cycloheximide or puromycin drastically suppressed OHT-induced CSR in AID-ER expressing AID−/− B cells when added 1 h before OHT but not after OHT, suggesting that de novo protein synthesis is required for an event downstream to AID expression in CSR. The results lend the weight to RNA-editing hypothesis for the function of AID.

Variable deletion and duplication at recombination junction ends: implication for staggered double-strand cleavage in class-switch recombination.

Immunoglobulin class-switch recombination (CSR) gives rise to looped-out circular DNA of a cleaved S segment, which is lost eventually after cell divisions. To understand the molecular mechanism of S region cleavage during CSR, we constructed artificial CSR substrates in which inversion-type CSR takes place to retain the cleaved S segment. Sequencing analyses of recombinant clones of these substrates revealed that varying degrees of deletions and duplications exist at CSR breakpoints, suggesting the involvement of staggered cleavage of the S region in CSR. In addition, mutations frequently found near junctions showed a similar profile of base replacement to Ig somatic hypermutation. These findings suggest that single-strand tails of staggered cleavage may be repaired by error-prone DNA synthesis.