Does antisense make sense of AID targeting?

Vertebrates make high-affinity antibodies to protect themselves against pathogenic organisms and their products. The generation of these antibodies requires the highly mutagenic enzyme activation-induced cytidine deaminase (AID) (1). AID deaminates dC to dU in ssDNA, and this mutation recruits additional error-prone repair to carry out somatic hypermutation (SHM) of the antibody variable (V) regions that encode the antigen-binding site, and class switch recombination (CSR), which allows those antigen binding sites to be expressed with different constant regions that mediate effector functions. However, it is still unclear how the genomic instability that is initiated by AID is restricted to only discrete parts of the Ig genes but spares the rest of the genome (2–4). In this issue of PNAS, Perlot et al. (5) provide a partial answer to this puzzle by identifying both sense and antisense transcripts of the V and switch (S) regions, both of which are targeted for mutation by AID; but they find only sense transcripts of the μ constant (Cμ) region, which is not targeted by AID (Fig. 1).

Fig. 1.

Sense and antisense transcription at the Ig locus. A scheme of the IgH locus is shown and the elements that are targeted by AID (orange) or spared (blue) are highlighted. As shown by Perlot et al. (5), both sense and antisense transcription is detected at the variable region (VDJ) and its immediate flanking sequences and in the switch (Sμ, Sγ1, and Sγ2b) regions, but only sense transcripts are detected at the μ constant (Cμ) region. NTS, upper, nontranscribed strand; TS, lower, transcribed strand. Mutation rate is shown only for the V(D)J region. AID is represented as a dimer.

The finding of antisense transcripts of the V and S regions in primary murine B cells is important because it addresses the unsolved problem of why both the lower (transcribed) and the upper (nontranscribed) strands of the V and S region DNA are highly mutated in mice and humans (6). This has been confusing because biochemical studies with semipurified AID (3, 4) and experiments in which AID was expressed in bacterial cells (7) have found AID-induced mutations mostly on the upper strand of the targeted DNA. This suggested that something like the large transcription apparatus was protecting the lower strand, whereas the upper strand was accessible to AID (Fig. 1). A number of explanations have been proposed for why both strands of the V and S regions are mutated in vivo, but not in vitro, such as differences between the mammalian and the bacterial RNA polymerases used in vitro (4), the accessibility of both strands in the supercoiled DNA at the edges of the transcription bubble (8), or ssDNA-binding replication protein A (RPA) participating with AID in mutation (4). Antisense transcription of the V and S regions provides an attractive, although not exclusive, alternative.

Antisense transcripts had previously been observed in the V region of a human Burkitt′s lymphoma cell line (9), in S regions within the context of chromosomal translocations (10–12), and in pro-B cells during V(D)J recombination (13), but Perlot et al. (5) are the first to identify such transcripts from a re arranged V region in primary B cells. In the S regions, transcription of the C rich lower strand results in R-loops, where the very G rich nascent RNA hybridizes the template strand and prevents AID action while leaving the upper strand single stranded and accessible to AID (14). However, the G:C content of the V region does not predispose it to R-loop formation, and R-loops have not been found there.

There is a considerable body of data that suggests that high rates of transcription are required for SHM and CSR (15–17). The fact that many highly transcribed genes in B cells expressing AID do not undergo high rates of mutation (2–4) has led to a number of models of how the targeting of AID to V and S regions is achieved. These include: (i) the participation of cis-acting DNA sequences that could recruit scaffolding proteins or form macromolecular DNA structures with different accessibility (3, 17); (ii) the existence of different AID cofactors for targeting to the V or S regions (18); and (iii) an increase in accessibility of the V and C regions due to high rates of transcription and associated epigenetic changes (3). The importance of transcription during hypermutation is also highlighted by the observation that the frequency of mutation rises sharply starting 100–200 bp downstream from the transcription start site, is highest over the coding exons of the V region, and falls off slowly till no mutations are detected 1–1.5 kb downstream (2) (Fig. 1). Although the sharp increase in mutation is often attributed to Pol II density or transition to elongation, the reasons for a decrease in the mutation rate as the distance from the promoter increases is not understood (2). If there were equal rates of sense and antisense transcription and if they played equal roles in targeting mutation, it would be expected that there would be a maximum rate of mutation within a few hundred base pairs of the antisense start site, and this would obscure the decrease in mutation 1 or 1.5 kb downstream from the start site of the sense transcript. However, this is not seen, suggesting a number of possibilities: it could support the idea that there is much less antisense than sense transcription; that the two transcripts play different roles in targeting mutation; or that this mutation pattern is not directed by transcription at all.

The identification of the promoter or start site for the antisense transcripts is critical to this analysis. Although the authors found multiple potential start sites in the J regions, they were unable to document promoter activity by using reporter assays. However, they did show that the production of antisense RNAs does not depend on the presence of the core intronic enhancer (cEμ in Fig. 1) by analyzing B cells from a mouse that lacks that regulatory element (5). Perlot et al. (5) also found both sense and antisense transcripts even at early stages of B cell differentiation when AID is not expressed and SHM and CSR are not occurring. This finding suggests that sense and antisense transcription are coordinately regulated and that antisense transcription may be initiated in preparation for SHM and CSR, but it could also mean that antisense transcription has no direct role in SHM or CSR. In addition, the authors report much lower steady-state levels of the antisense transcripts than of the spliced sense transcript. This was also observed in Ramos cells (9). However, when they compared the abundance of unspliced sense and antisense pre-mRNA, they were similar. Although this might suggest that the rates of transcription of both strands were similar, the authors point out that there are many possible explanations for the different abundance of the two transcripts, including more rapid degradation of the antisense transcripts (5).

Antisense transcription may be initiated in preparation for SHM and CSR.

Is there a role for transcription (sense and antisense) beyond providing accessibility? Other workers in the field have speculated about whether the sense transcript itself plays some role in targeting AID to hotspots for mutation within the V region. For example, it has been suggested that stem–loop-like structures in the nascent RNA could result in transcriptional pausing or affect the targeting of AID in some other way, increasing the likelihood of mutations at particular locations (19, 20), and this could equally apply to the antisense transcript. It is also possible that the two nascent RNAs could interact with each other (21). Furthermore, it might be worthwhile to consider what would happen if transcription apparati traveling in opposite directions, but on the same piece of DNA, approached and collided with each other.

In any case, the detection of sense and antisense transcription of the V and S regions, which undergo AID-induced mutation, and the absence of antisense transcription across the nonmutating μ constant region, are provocative findings. Whether antisense transcription plays a direct or indirect role in making the V and S regions susceptible to AID, or this bidirectional transcription is merely a reflection of some other property of those regions that makes them accessible to AID, the presence of antisense transcripts raises important questions that merit further study.