Sergio Roa, Fei Li Kuang, and Matthew D. Scharff* Department of Cell Biology, Albert Einstein College of Medicine, Bronx, NY 10461
V
ertebrates 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 constant (C) region, which is not targeted by AID (Fig. 1). 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 AIDinduced 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. www.pnas.org兾cgi兾doi兾10.1073兾pnas.0800935105
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 (S, S␥1, and S␥2b) regions, but only sense transcripts are detected at the constant (C) 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.
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 rearranged 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 Author contributions: S.R., F.L.K., and M.D.S. wrote the paper. The authors declare no conflict of interest. See companion article on page 3843. *To whom correspondence should be addressed. E-mail:
[email protected]. © 2008 by The National Academy of Sciences of the USA
PNAS 兩 March 11, 2008 兩 vol. 105 兩 no. 10 兩 3661–3662
COMMENTARY
Does antisense make sense of AID targeting?
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 (cE 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 1. Muramatsu M, et al. (1999) Specific expression of activation-induced cytidine deaminase (AID), a novel member of the RNA-editing deaminase family in germinal center B cells. J Biol Chem 274:18470 –18476. 2. Di Noia JM, Neuberger MS (2007) Molecular mechanisms of antibody somatic hypermutation. Annu Rev Biochem 76:1–22. 3. Goodman MF, Scharff MD, Romesberg FE (2007) AIDinitiated purposeful mutations in immunoglobulin genes. Adv Immunol 94:127–155. 4. Teng G, Papavasiliou FN (2007) Immunoglobulin somatic hypermutation. Annu Rev Genet 41:107–120. 5. Perlot T, Li G, Alt FW (2008) Antisense transcripts from immunoglobulin heavy chain locus V(D)J and switch regions. Proc Natl Acad Sci USA 105:3843–3848. 6. Milstein C, Neuberger MS, Staden R (1998) Both DNA strands of antibody genes are hypermutation targets. Proc Natl Acad Sci USA 95:8791– 8794. 7. Ramiro AR, Stavropoulos P, Jankovic M, Nussenzweig MC (2003) Transcription enhances AID-mediated cytidine deamination by exposing single-stranded DNA on the nontemplate strand. Nat Immunol 4:452– 456.
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
Antisense transcription may be initiated in preparation for SHM and CSR. 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). 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 8. Shen HM, Storb U (2004) Activation-induced cytidine deaminase (AID) can target both DNA strands when the DNA is supercoiled. Proc Natl Acad Sci USA 101:12997– 13002. 9. Ronai D, et al. (2007) Detection of chromatin-associated single-stranded DNA in regions targeted for somatic hypermutation. J Exp Med 204:181–190. 10. Julius MA, et al. (1988) Translocated c-myc genes produce chimeric transcripts containing antisense sequences of the immunoglobulin heavy chain locus in mouse plasmacytomas. Oncogene 2:469 – 476. 11. Apel TW, Mautner J, Polack A, Bornkamm GW, Eick D (1992) Two antisense promoters in the immunoglobulin mu-switch region drive expression of c-myc in the Burkitt’s lymphoma cell line BL67. Oncogene 7:1267–1271. 12. Morrison AM, et al. (1998) Deregulated PAX-5 transcription from a translocated IgH promoter in marginal zone lymphoma. Blood 92:3865–3878. 13. Bolland DJ, et al. (2004) Antisense intergenic transcription in V(D)J recombination. Nat Immunol 5:630 – 637. 14. Yu K, Roy D, Bayramyan M, Haworth IS, Lieber MR (2005) Fine-structure analysis of activation-induced deaminase accessibility to class switch region R-loops. Mol Cell Biol 25:1730 –1736.
3662 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0800935105
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 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. ACKNOWLEDGMENTS. S.R. is supported by Postdoctoral Fellowship EX-2006-0732 from the Spanish Ministry of Education and Science. F.L.K. is supported by the Medical Scientist Training Program T32 GM 007288. M.D.S. is supported by RO1CA72649 and R01CA102705 and by the Harry Eagle Chair provided by the National Women’s Division of the Albert Einstein College of Medicine.
15. Fukita Y, Jacobs H, Rajewsky K (1998) Somatic hypermutation in the heavy chain locus correlates with transcription. Immunity 9:105–114. 16. Jung S, Rajewsky K, Radbruch A (1993) Shutdown of class switch recombination by deletion of a switch region control element. Science 259:984 –987. 17. Storb U, et al. (2007) Targeting of AID to immunoglobulin genes. Adv Exp Med Biol 596:83–91. 18. Shinkura R, et al. (2004) Separate domains of AID are required for somatic hypermutation and class-switch recombination. Nat Immunol 5:707–712. 19. Storb U, et al. (1998) A hypermutable insert in an immunoglobulin transgene contains hotspots of somatic mutation and sequences predicting highly stable structures in the RNA transcript. J Exp Med 188:689 – 698. 20. Steele EJ, Lindley RA, Wen J, Weiller GF (2006) Computational analyses show A-to-G mutations correlate with nascent mRNA hairpins at somatic hypermutation hotspots. DNA Repair (Amst) 5:1346 –1363. 21. Giallourakis C, Alt FW, Bassing CH (2004) V(D)J recombinational accessibility-heading in the opposite direction? Nat Immunol 5:561–562.
Roa et al.
