Mechanisms regulating the targeting and activity of activation induced cytidine deaminase

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ScienceDirect Mechanisms regulating the targeting and activity of activation induced cytidine deaminase David J Fear Activation induced cytidine deaminase (AID) plays a central role in the vertebrate adaptive immune response, initiating immunoglobulin (Ig) somatic hypermutation (SHM) and classswitch recombination (CSR). AID converts deoxycytosine (dC) in the DNA to deoxyuridine (dU), causing a DNA base-pairing mismatch. How this mismatch is recognised and resolved determines whether the site will undergo mutation, recombination or high-fidelity repair. Although AID action is essential for antibody diversification it is also known to act upon many non-Ig genes where it can cause tumourigenic mutations and translocations. Although much is known about the pathways of Ig diversification, there is still very little known about the mechanisms that target AID to its sites of action and regulate the different repair processes that can participate at these sites. Addresses MRC & Asthma UK Centre in Allergic Mechanisms of Asthma, Department of Asthma Allergy & Respiratory Science, King’s College London, 5th Floor, Tower Wing, Guy’s Hospital, St. Thomas Street, London SE1 9RT, United Kingdom

encoding the variable region, altering antigen specificity, a function also carried out by gene conversion in some animals (reviewed in [2]). Following affinity maturation (reviewed in [3]), these events raise the potential antibody repertoire to over 1011 different specificities, greater than the number of circulating B cells. As B cells interact with TH cells in the GC, Igs are further diversified by class-switch recombination (CSR); replacing one Ig heavy-chain constant (CH) region gene (initially Cm) with one of the downstream CH genes (either Cg, Ce or Ca in mammals), thereby altering the effector functions of the antibody. Surprisingly, each of these different secondary diversification events are initiated by the action of a single enzyme, activation induced cytidine deaminase (AID) [4–6]. Since the discovery of AID, almost 15 years ago, much has been learnt regarding the mechanics of these activities. However, tantalising questions remain regarding how AID is targeted to its sites of action and, perhaps more importantly, how the rest of the genome is protected from such a potentially dangerous activity.

Corresponding author: Fear, David J. ([email protected])

SHM Current Opinion in Immunology 2013, 25:619–628 This review comes from a themed issue on Immunogenetics and transplantation Edited by Miles Davenport and Deborah K Dunn-Walters For a complete overview see the Issue and the Editorial Available online 24th October 2013 0952-7915/$ – see front matter, # 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.coi.2013.05.017

Introduction Diversification of the immunoglobulin (Ig) repertoire is essential for the normal operation of the vertebrate adaptive immune system, allowing it to respond to an everchanging, and almost limitless, spectrum of pathogenic challenges. During B cell ontogeny the primary Ig repertoire is composed by the ordered recombination of Variable (V), Diversity (D) and Joining (J) gene segments. This process of V(D)J recombination constructs the variable portion of the Ig heavy and light chains (reviewed in [1]), producing approximately 108 different antibody specificities. Following antigen encounter, mature B cells undergo further diversification events in the germinal centres (GC) of the lymph nodes. Somatic hypermutation (SHM) introduces point mutations into the DNA www.sciencedirect.com

SHM introduces mutations into the variable region of the Ig gene at rate of up to 104–103 mutations per base per division, 10–100-fold higher than the ‘background’ rate of mutation across the genome [7]. AID initiates this process by converting deoxycytosine (dC) in the variable region DNA to deoxyuridine (dU) [8], resulting in a base-pairing mismatch. SHM occurs when this lesion is ‘repaired’ in an error-prone manner [9] (Figure 1). A first phase of mutation occurs at the site of the original dU lesion: When DNA replication occurs before resolution of this mismatch, uracil is used as a template and a C to T transition takes place in one daughter cell [8]. Alternatively, dU can be removed by uracil-N-glycosylase (UNG) [10], a component of the base excision repair (BER) pathway [11]. DNA replication over the resultant abasic site by Rev1 [12] and the translesion synthesis polymerase Pol u (POLQ [13]) then results in transitions and transversions at the site of AID action. A second phase of mutation occurs when the mismatch repair (MMR) pathway targets low-fidelity, ‘long patch’ repair. Here, bases surrounding the initial dU lesion are removed [14] and replaced in an error-prone manner through the action of DNA Pol h (POL H [15]) and Pol z (Pol Z or REV3L [14]), spreading mutations to the surrounding base pairs (reviewed in [13]). The reason why these activities stimulate low-fidelity repair at the Ig genes in B cells but highfidelity repair (through the action of Pol b (POL B [16]) elsewhere in the genome remains a crucial issue in our Current Opinion in Immunology 2013, 25:619–628

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Figure 1

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DNA repair pathways utilised in somatic hypermutation. The different DNA repair pathways utilised in the resolution of AID mediated DNA lesions during SHM are illustrated, depicting how these activities lead to transitions, transversions and mutation spreading (in red) or error free repair (black). SHM is initiated by AID-mediated DNA deamination of Ig variable regions, leading to a base-pairing mismatch. DNA replication before uracil removal results in C-T transitions in one daughter cell. Alternatively, if uracil is removed by UNG action (base excision repair — BER) transitions and transversions can occur following replication by one of the translesion synthesis polymerases (Rev1 or Pol u). Resolution of uracil mismatches can also occur via the mismatch repair (MMR) pathway, leading to the spreading of mutations away from the initial site of AID action, or through the high-fidelity homologus recombination (HR) pathway.

understanding not only SHM but also B cell oncogenesis. Recent evidence analysing AID-induced, UNG-dependent, mutations by next-generation sequencing revealed that the choice between high-fidelity and low-fidelity repair appears to be sequence-dependent [17,18] and may be targeted by the presence of mono-ubiquitylated proliferating cell nuclear antigen (PCNA) [19] possibly in combination with the abasic sites generated by exo-1 at the Ig loci [20]. However, it is also likely that the sheer number of sites of DNA damage induced at the Ig loci during SHM overwhelm the high-fidelity repair pathways, leaving some sites to be repaired through errorprone mechanisms [16].

CSR CSR takes place between two switch (S) regions, located upstream of each CH gene, excising the intervening DNA Current Opinion in Immunology 2013, 25:619–628

as a ‘switch circle’ and juxtaposing the variable region with the downstream CH gene (Figure 2). In the case of CSR, dU (resulting from AID action in the switch regions) is removed by the combined action of UNG and the apyrimidinic endonuclease (APE1) [10,21], leaving a singlestrand DNA break (SSB). Double-strand breaks (DSBs) are formed when SSBs are in close proximity on opposite DNA strands, or by ‘end processing’ through the action of the MMR pathway [22]. After the formation of DSBs in ‘donor’ and ‘target’ switch regions, synapsis takes place [23,24] and recombination occurs through the classical nonhomologous end-joining (C-NHEJ) [25] and alternative end-joining (A-EJ) pathways [26] (Figure 3). For both pathways, DNA breaks are detected by the Mre11/ Rad50/Nbs1 (MRN) [27] and ataxia telangiectasia mutated (ATM) [28] complexes. C-NHEJ appears to be the primary means by which CSR takes place [26]: Ku70 and Ku80 heterodimers and DNA-dependent protein kinase (DNAPKs) form a scaffold holding the non-homologous DNA ends together, modifying the DNA ends and recruiting DNA ligase IV to rejoin the DNA ends. A-EJ relies on micro-homologies within the DNA ends [26] (generated by MRN and CtIP action) and occurs independently of Ku and DNA ligase IV [29] possibly utilising PARP [30] and CtIP [31] to function as scaffolds for the additional factors required for DNA end processing and ligation via DNA ligase I or III [32]. Following break detection by MRN, the binding, and subsequent phosphorylation, of 53BP1 inhibits the A-EJ pathways through the exclusion of end resection enzymes [33]. Inhibition of A-EJ not only limits intra-switch region recombination but also alters the landscape of resultant translocations, possibly protecting cells from potentially tumourigenic events [34].

AID binds promiscuously to many ‘off target’ genes The choice of which pathways are used by the cell to resolve AID-induced DNA lesions to a great extent regulates the biological effect of AID activity. However, it is clear that AID activity is not just restricted to the Ig locus. AID has previously been shown to act at a number of ‘off target’, non-Ig genes, inducing mutation in CD79A, CD79B, FAS, PIM1, PAX5 and BCL6 [35,36] and causing chromosomal instability [37]. In recent years much effort has been focused on identifying the genomic distribution of AID and the extent of its activity. Schatz and colleagues [7] were one of the first groups to explore the extent of genomic AID activity; sequencing 150 B cell expressed genes. This study revealed widespread AID activity at approximately 40% of the genes studied. Even more surprisingly, the analysis of wild type versus Msh2/, UNG/ mice, demonstrated that only approximately half of these target genes were repaired through high-fidelity BER/MMR pathways [7]. More recently Hasham et al. demonstrated that XRCC2 deficiency leads to an increase in AID-induced DNA www.sciencedirect.com

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Figure 2

Mouse IgH locus Chromosome 12 VD J

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Organisation of the immunoglobulin heavy chain locus and mechanics of class-switch recombination. The murine and human immunoglobulin heavy chain loci are depicted, showing the location of the germline I exons (light coloured boxes), switch regions (ovals) and germline heavy chain genes (dark boxes), the rearranged variable region is illustrated in purple. CSR is preceded by germline gene transcription, initiated from I exons. The concerted action of AID, UNG and APE1 cause DNA breaks in the heavy chain switch regions, leading to recombination and juxtapositioning of the downstream heavy chain gene (in this case IgE) with the variable region. Intervening DNA sequences are excised as a switch circle and can yield ‘hybrid’ switch circle transcripts across the recombined switch region.

breaks, suggesting that the homologous recombination (HR) pathway also contributes to counteracting AIDinduced mutation [38]. Using truly ‘genome-wide’ techniques, independent studies from the Alt [39] and Nussenzweig [40] www.sciencedirect.com

laboratories investigated the distribution of AID-induced DNA translocations (high throughput genome-wide translocation sequencing, HTGTS, or translocation capture sequencing, TC-seq). These studies, together with one using 4C methodology [41], again highlighted the large number of AID-induced lesions that arise across the Current Opinion in Immunology 2013, 25:619–628

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Figure 3

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Recombination pathways utilised during class-switch recombination. The pathways and major components used by the classical non-homologus endjoining (NHEJ) and alternative end-joining (AEJ) pathways during CSR are shown. For both pathways, DNA breaks are first detected by the MRN complex, leading to a cascade of ATM mediated phosphorylation events. Pathways then diverge, possibly due to the presence or absence of 53BP1 regulating end processing activities. NHEJ utilises Ku and DNA-PK to join switch rejoins without homology. AEJ relies on CTIP and PARP to join switch regions with areas of micro-homology.

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genome, and implicated the transcriptional machinery as playing an important role in targeting AID to these sites. However, while many AID targets are certainly actively transcribed, studies investigating genome-wide DNA breaks (by ChIP-seq for Nbs1 [42]) and direct ChIPseq of AID distribution [43] have revealed that up to half of the sites of ‘off target’ AID binding and AID activity do not correspond to transcribed regions of the genome. In fact, AID ChIP-seq data generated in our laboratory suggest that AID may specifically move to more distant intergenic regions of the genome following stimulation of B cells to undergo GC reactions in vivo (D Fear, unpublished data). Together, these studies have highlighted the extent of AID’s promiscuity and the magnitude of the task that DNA repair mechanisms have in correcting this (on top of the background DNA damage accrued by all cells on a daily basis [44]). So far, most research into the mechanisms targeting AID has been focused on the transcribed regions of the genome. However, by targeting AID to intergenic regions of the genome, B cells may protect the more sensitive RNA coding or regulatory regions of the genome from dangerous AID action. This is an area of AID biology that warrants additional investigation. It is hard to understand why such a dangerous protein is distributed so widely across the genome. To answer this, it has been suggested that AID might have other functions that necessitate its widespread genomic distribution. Several studies have implicated AID as playing a role in the active removal of DNA methylation marks via thymine DNA glycosylase [45,46]. Although this remains a controversial topic it could explain why the cell is willing to ‘take the risk’ of potential widespread genomic damage.

Targeting AID activity For AID to function, it must first find its DNA targets. Through the use of next-generation sequencing technologies it has become clear that AID has far more genomic targets than initially suspected and much effort has been focused on understanding the mechanisms targeting AID to these sites. While a systematic review of all these factors is beyond the scope of this article (many have been discussed extensively in previous reviews [47–49]) several recent discoveries warrant further discussion (Figure 4). R-loops and WRC motifs

The DNA sequence of the Ig variable and switch regions are highly enriched for WRC motifs (A/T G/A C), described as being ‘hotspots’ for AID activity [50]. It has been suggested that these motifs might, at least locally, target AID to its sites of action [51]. In addition, an interaction with 14-3-3 adaptor proteins [52] has been proposed to target AID to conserved AGCT sequence motifs found www.sciencedirect.com

within the Ig switch regions [53]. Some of these sequence motifs have indeed been found to be enriched at sites of AID-induced mutation across the genome [42,43]. However, we have been unable to detect any enrichment of these sequences at regions bound by AID in our ChIP-seq study (D Fear, unpublished data). Although the density of these sequence motifs almost certainly affects AID activity at its sites of action it seems unlikely that they contribute to the targeting of AID except at the local level. Recruitment by transcription factors

Many factors that act upon DNA and chromatin are recruited by interacting with specific transcription factors. E2A (Ig enhancer-binding factors E12/E47) recognition motifs, E-box elements, have been heavily implicated in targeting AID to its sites of action: E-box motifs have been shown to be sufficient to target SHM to transcribed genes in transgenic cell lines [54,55] and are enriched in the promoters of genes shown to be mutated by AID [7], an analysis that has recently been refined to include YY1 and C/EBP binding sites [56]. While these motifs certainly play an important role in driving the expression of AID target genes, their direct role in targeting AID to these genes is less clear. To date, other genome-wide studies investigating sites of DNA breaks [42] and direct AID binding [43] have failed to show an enrichment of these motifs, while no biochemical studies have been able to demonstrate an association. Targeting by histone modifications

Specific patterns of histone modifications are frequently used by cells to direct factors to their genomic targets, an idea almost certainly oversimplified as the ‘histone-code hypothesis’ [57]. Following stimulation of CSR, Ig switch regions are enriched for nucleosomes containing histone H3 modified by tri-methylation of specific lysine residues located within the N-terminal tail (lysines 4 and 9 termed H3K4me3 and H3K9me3 respectively) [58,59]. The first direct evidence that these modifications may be involved in regulating AID activity came when a knockout of PTIP, a component of the H3K4 methyltransferase complex MML3–MML4, resulted in reduced H3K4me3 at Ig switch regions and impaired CSR to downstream IGH genes [60]. Similarly, knockdown of SSRP1, Spt16 [61] and spt6 [62], components of the FAcilitates Chromatin Transcription (FACT) complex have also shown to result in reduced Ig switch region H3K4 tri-methylation and reduced CSR. Although a strong correlation between H3K4me3 and AID localisation clearly exists [43,63], AID does not contain any domains known to recognise methylated histones and no factors have so far been described that might mediate this interaction. Conversely, AID has been shown to form a complex with KAP1 and HP1, which in turn interacts with H3K9me3 histone modifications, while knockdown of KAP1 leads to reduced AID deposition at switch regions and reduced Current Opinion in Immunology 2013, 25:619–628

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Figure 4

(a) Recruitment of AID by sequence specific factors

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Mechanisms involved in targeting AID. Three of the potential mechanisms targeting AID to its sites of action are illustrated. In all figures histone octamers are represented by grey circles and AID is depicted as being complexed with RNA polymerase (RNAP) within a ‘transcription bubble’ with the nascent RNA chain in red. AID action is depicted by red Us. (a) AID may be targeted to its sites of action by an interaction with the transcription factors C/EBP, YY1 and E2A (although no complex has as yet been detected), or via 14-3-3 adaptor proteins recognising ACGT motifs within the switch regions. (b) While it has been shown that H3K4 tri-methylation (H3K4me) is mediated by PTIP and FACT it is not known how AID interprets this modification. Conversely, AID may be recruited to sites of H3K9 tri-methylation (H3K9me3) via KAP yet the enzyme that mediates this modification is not certain. Recruitment of AID to the transcription complex (c) appears to occur following stalling of transcription; upon release of RNAP, AID can function processively, helped by the stabilisation of single-stranded DNA by the binding of RPA and formation of R-loops. Finally, it has been hypothesised that degradation of the R-loop by the RNA exosome may allow deamination of the template strand. Current Opinion in Immunology 2013, 25:619–628

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mutation [64]. Although the enzyme complexes that mediate this modification at AID target sites have not been positively identified, a H3K9 methyltransferase, Suv39h1, has been previously shown to regulate CSR to IgA [65], compelling evidence for a role of these modifications in the targeting of AID.

of inflammatory cell signalling events as well as posttranscriptional control by miRNAs, nuclear localisation and phosphorylation (reviewed in [76]). Understanding the concerted action of all these mechanisms will be required to fully understand how AID functions in normal B cells and its contribution to tumourigenesis.

Targeting by RNA biosynthesis

Acknowledgements

By far the most extensively studied aspect of AID targeting has been the link with RNA polymerase (RNAP) and RNA biosynthesis (including transcription, splicing and degradation). Before CSR, the participating CH genes are transcribed from germline (I) exons located upstream of the switch regions, producing ‘sterile’ germline transcripts (Figure 2). These transcripts appear to have two functions: Firstly, they produce the regions of singlestranded DNA, stabilised by the binding of Replication Protein A (RPA [67]), required for AID action [66]. Secondly, transcription may directly contribute to AID targeting and processivity through the formation of a complex between AID and RNAP [68]. More recently this picture has been further refined. AID binding is enriched specifically at regions containing stalled RNAP [43], while several factors associated with stalled RNAP including spt5 [69] and the Polymerase-Associated Factor (PAF) complex [70] have been shown to directly interact with AID. Additionally, PTBP2 [71], SRSF1 [72] and CTNNBL1 [73], components of the splicing machinery and components of the RNA exosome [74] have been shown to either interact with AID or regulate CSR and SHM. The association between AID and such a bewildering array of different parts of the RNA biosynthesis machinery may simply reflect the fact that these factors frequently reside within the large, and heterogeneous, RNAP holoenzyme complex. However, these associations may also reflect the complex secondary RNA/ DNA structures that are formed at Ig switch regions (and other AID targets), and their role in ‘fine tuning’ AID action to genes with distinct sequence characteristics. Thus, AID may be tethered to RNAP via spt5/PAF complex when RNAP stalls, possibly upon reaching regions of secondary structure such as R-loops or spliceosome activity. Finally, RNA degradation of the RNA:DNA R-loop structure (formed upon transcription of the G rich switch regions [75]) by the RNA exosome might be required for facilitating AID activity on both the template and non-template DNA strands.

Research in D.F.’s laboratory is supported by the Biotechnology and Biological Science Research Council, BB/H019634/1, and by King’s Health Partners R&D Challenge Fund, R120505. The author also acknowledges financial support from the Department of Health via the National Institute for Health Research (NIHR) comprehensive Biomedical Research Centre award to Guy’s & St Thomas’ NHS Foundation Trust in partnership with King’s College London and King’s College Hospital NHS Foundation Trust; and support of the MRC and AUK Centre in Allergic Mechanisms of Asthma by the Medical Research Council and Asthma UK.

Conclusion It is becoming ever more apparent that AID is subject to multiple layers of regulation, as befits an enzyme whose dysregulated activity has such dire consequences for genome integrity. The aspects of AID regulation discussed above primarily deal with how AID finds its targets, and how the resultant DNA lesions are processed. However, AID is also subject to transcriptional control, being expressed specifically following receipt www.sciencedirect.com

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21. Schrader CE, Linehan EK, Mochegova SN, Woodland RT, Stavnezer J: Inducible DNA breaks in Ig S regions are dependent on AID and UNG. J Exp Med 2005, 202:561-568.

37. Ramiro AR, Jankovic M, Callen E, Difilippantonio S, Chen HT, McBride KM, Eisenreich TR, Chen J, Dickins RA, Lowe SW et al.: Role of genomic instability and p53 in AID-induced c-myc-Igh translocations. Nature 2006, 440:105-109.

22. Ehrenstein MR, Neuberger MS: Deficiency in Msh2 affects the efficiency and local sequence specificity of immunoglobulin class-switch recombination: parallels with somatic hypermutation. EMBO J 1999, 18:3484-3490. 23. Larson ED, Duquette ML, Cummings WJ, Streiff RJ, Maizels N: MutSalpha binds to and promotes synapsis of transcriptionally activated immunoglobulin switch regions. Curr Biol 2005, 15:470-474. 24. Wuerffel R, Wang L, Grigera F, Manis J, Selsing E, Perlot T, Alt FW, Cogne M, Pinaud E, Kenter AL: S–S synapsis during class switch recombination is promoted by distantly located transcriptional elements and activation-induced deaminase. Immunity 2007, 5:711-722. 25. Casellas R, Nussenzweig A, Wuerffel R, Pelanda R, Reichlin A, Suh H, Qin XF, Besmer E, Kenter A, Rajewsky K et al.: Ku80 is required for immunoglobulin isotype switching. EMBO J 1998, 17:2404-2411. 26. Yan CT, Boboila C, Souza EK, Franco S, Hickernell TR, Murphy M, Gumaste S, Geyer M, Zarrin AA, Manis JP et al.: IgH class switching and translocations use a robust non-classical endjoining pathway. Nature 2007, 449:478-482.

38. Hasham MG, Donghia NM, Coffey E, Maynard J, Snow KJ, Ames J, Wilpan RY, He Y, King BL, Mills KD: Widespread genomic breaks generated by activation-induced cytidine deaminase are prevented by homologous recombination. Nat Immunol 2010, 11:820-826. 39. Chiarle R, Zhang Y, Frock RL, Lewis SM, Molinie B, Ho YJ,  Myers DR, Choi VW, Compagno M, Malkin DJ et al.: Genomewide translocation sequencing reveals mechanisms of chromosome breaks and rearrangements in B cells. Cell 2011, 147:107-119. A seminal paper that (together with Klein et al. [40]) is the first to use nextgeneration sequencing to explore the true extent of AID mediated translocations across the genome. Together, these studies reveal important aspects of the targeting mechanisms of AID and allow for crucial comparisons to studies investigating AID distribution and AID mediated mutation. 40. Klein IA, Resch W, Jankovic M, Oliveira T, Yamane A, Nakahashi H,  Di Virgilio M, Bothmer A, Nussenzweig A, Robbiani DF et al.: Translocation-capture sequencing reveals the extent and nature of chromosomal rearrangements in B lymphocytes. Cell 2011, 147:95-106. See annotation to [39].

28. Reina-San-Martin B, Chen HT, Nussenzweig A, Nussenzweig MC: ATM is required for efficient recombination between immunoglobulin switch regions. J Exp Med 2004, 200:1103-1110.

41. Hakim O, Resch W, Yamane A, Klein I, Kieffer-Kwon KR,  Jankovic M, Oliveira T, Bothmer A, Voss TC, Ansarah-Sobrinho C et al.: DNA damage defines sites of recurrent chromosomal translocations in B lymphocytes. Nature 2012, 484:69-74. An important study that uses 4C technology to investigate intra-chromosomal contacts involving IGH and myc (targets of AID mediated translocation) across the genome. This study combines 4C, TC-seq and ChIP-seq for RPA to determine that DNA damage is determined by both intra-chromosomal contact frequency and DNA damage frequency.

29. Boboila C, Yan C, Wesemann DR, Jankovic M, Wang JH, Manis J, Nussenzweig A, Nussenzweig M, Alt FW: Alternative end-joining catalyzes class switch recombination in the absence of both Ku70 and DNA ligase 4. J Exp Med 2010, 207:417-427.

42. Staszewski O, Baker RE, Ucher AJ, Martier R, Stavnezer J,  Guikema JE: Activation-induced cytidine deaminase induces reproducible DNA breaks at many non-Ig Loci in activated B cells. Mol Cell 2011, 41:232-242.

27. Petersen S, Casellas R, Reina-San-Martin B, Chen HT, Difilippantonio MJ, Wilson PC, Hanitsch L, Celeste A, Muramatsu M, Pilch DR et al.: AID is required to initiate Nbs1/ gamma-H2AX focus formation and mutations at sites of class switching. Nature 2001, 414:660-665.

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Regulation of AID Fear 627

An important study into the genomic distribution of AID mediated DNA breaks (identified by ChIP-seq for Nbs1). A comprehensive bioinformatic analysis of the sites of DNA damage sheds light on some of the mechanisms responsible for targeting AID activity. 43. Yamane A, Resch W, Kuo N, Kuchen S, Li Z, Sun HW, Robbiani DF,  McBride K, Nussenzweig MC, Casellas R: Deep-sequencing identification of the genomic targets of the cytidine deaminase AID and its cofactor RPA in B lymphocytes. Nat Immunol 2011, 12:62-69. The first study to comprehensively investigate AID distribution across the genome by ChIP-seq. This not only confirms how widespread AID is across the genome but also identifies the role polymerase stalling may play in AID targetting. 44. Spry M, Scott T, Pierce H, D’Orazio JA: DNA repair pathways and hereditary cancer susceptibility syndromes. Front Biosci 2007, 12:4191-4207. 45. Guo JU, Su Y, Zhong C, Ming GL, Song H: Hydroxylation of 5 methylcytosine by TET1 promotes active DNA demethylation in the adult brain. Cell 2011, 145:423-434. Together with Cortellino et al. [46] this paper was one of the first to demonstrate a potential role for AID outside of immunoglobulin diversification. These studies could explain why such widespread AID activity is tolerated by the cell despite it dangerous side effects. 46. Cortellino S, Xu J, Sannai M, Moore R, Caretti E, Cigliano A, Le  Coz M, Devarajan K, Wessels A, Soprano D et al.: Thymine DNA glycosylase is essential for active DNA demethylation by linked deamination-base excision repair. Cell 2011, 146:67-79. See annotation to [45]. 47. Storck S, Aoufouchi S, Weill JC, Reynaud CA: AID and partners: for better and (not) for worse. Curr Opin Immunol 2011, 23:337-344. 48. Kothapalli NR, Fugmann SD: Targeting of AID-mediated sequence diversification to immunoglobulin genes. Curr Opin Immunol 2011, 23:184-189. 49. Pavri R, Nussenzweig MC: AID targeting in antibody diversity. Adv Immunol 2011, 110:1-26. 50. Pham P, Bransteitter R, Petruska J, Goodman MF: Processive AID-catalysed cytosine deamination on single-stranded DNA simulates somatic hypermutation. Nature 2003, 424:103-107. 51. Kohli RM, Maul RW, Guminski AF, McClure RL, Gajula KS, Saribasak H, McMahon MA, Siliciano RF, Gearhart PJ, Stivers JT: Local sequence targeting in the AID/APOBEC family differentially impacts retroviral restriction and antibody diversification. J Biol Chem 2010, 285:40956-40964.

histone modification across the human immunoglobulin heavychain locus. Proc Natl Acad Sci U S A 2008, 105:15872-15877. 59. Kuang FL, Luo Z, Scharff MD: H3 trimethyl K9 and H3 acetyl K9 chromatin modifications are associated with class switch recombination. Proc Natl Acad Sci U S A 2009, 106:5288-5293. 60. Daniel JA, Santos MA, Wang Z, Zang C, Schwab KR, Jankovic M, Filsuf D, Chen HT, Gazumyan A, Yamane A et al.: PTIP promotes chromatin changes critical for immunoglobulin class switch recombination. Science 2010, 329:917-923. 61. Stanlie A, Aida M, Muramatsu M, Honjo T, Begum NA: Histone3 lysine4 trimethylation regulated by the facilitates chromatin transcription complex is critical for DNA cleavage in class switch recombination. Proc Natl Acad Sci U S A 2010, 107:22190-22195. 62. Okazaki IM, Okawa K, Kobayashi M, Yoshikawa K, Kawamoto S, Nagaoka H, Shinkura R, Kitawaki Y, Taniguchi H, Natsume T et al.: Histone chaperone Spt6 is required for class switch recombination but not somatic hypermutation. Proc Natl Acad Sci U S A 2011, 108:7920-7925. 63. Kato L, Begum NA, Burroughs AM, Doi T, Kawai J, Daub CO, Kawaguchi T, Matsuda F, Hayashizaki Y, Honjo T: Nonimmunoglobulin target loci of activation-induced cytidine deaminase (AID) share unique features with immunoglobulin genes. Proc Natl Acad Sci U S A 2012, 109:2479-2484. 64. Jeevan-Raj BP, Robert I, Heyer V, Page A, Wang JH, Cammas F,  Alt FW, Losson R, Reina-San-Martin B: Epigenetic tethering of AID to the donor switch region during immunoglobulin class switch recombination. J Exp Med 2011, 208:1649-1660. The authors undertake a mass spectroscopy analysis of AID interacting proteins, identifying the H3K9me3 binding protein KAP1 by MS and co-IP experiments. A comprehensive analysis of KAP1 function in knockout cells reveals that it appears to be involved in targeting AID to switch regions decorated with this modification. This is the first factor to be identified that could directly target AID to a specific histone modification. 65. Bradley SP, Kaminski DA, Peters AH, Jenuwein T, Stavnezer J: The histone methyltransferase Suv39h1 increases class switch recombination specifically to IgA. J Immunol 2006, 177:1179-1188. 66. Chaudhuri J, Tian M, Khuong C, Chua K, Pinaud E, Alt FW: Transcription-targeted DNA deamination by the AID antibody diversification enzyme. Nature 2003, 422:726-730. 67. Chaudhuri J, Khuong C, Alt FW: Replication protein A interacts with AID to promote deamination of somatic hypermutation targets. Nature 2004, 430:992-998.

52. Xu Z, Fulop Z, Wu G, Pone EJ, Zhang J, Mai T, Thomas LM, AlQahtani A, White CA, Park SR et al.: 14-3-3 adaptor proteins recruit AID to 50 -AGCT-30 -rich switch regions for class switch recombination. Nat Struct Mol Biol 2010, 17:1124-1135.

68. Nambu Y, Sugai M, Gonda H, Lee CG, Katakai T, Agata Y, Yokota Y, Shimizu A: Transcription-coupled events associating with immunoglobulin switch region chromatin. Science 2003, 302:2137-2140.

53. Zarrin AA, Alt FW, Chaudhuri J, Stokes N, Kaushal D, Du PL, Tian M: An evolutionarily conserved target motif for immunoglobulin class-switch recombination. Nat Immunol 2004, 5:1275-1281.

69. Pavri R, Gazumyan A, Jankovic M, Di Virgilio M, Klein I, AnsarahSobrinho C, Resch W, Yamane A, Reina San-Martin B, Barreto V et al.: Activation-induced cytidine deaminase targets DNA at sites of RNA polymerase II stalling by interaction with Spt5. Cell 2010, 143:122-133.

54. Tanaka A, Shen HM, Ratnam S, Kodgire P, Storb U: Attracting AID to targets of somatic hypermutation. J Exp Med 2010, 207:405-415. 55. Michael N, Shen HM, Longerich S, Kim N, Longacre A, Storb U: The E box motif CAGGTG enhances somatic hypermutation without enhancing transcription. Immunity 2003, 19:235-242. 56. Duke JL, Liu M, Yaari G, Khalil AM, Tomayko MM, Shlomchik MJ,  Schatz DG, Kleinstein SH: Multiple transcription factor binding sites predict AID targeting in non-Ig genes. J Immunol 2013, 190:3878-3888. An excellent in-depth bioinformatic analysis of genes mutated by AID reveals that a conserved triumviret of transcription factor binding sites (Ebox, YY1 and C/EBP) correlates with mutational activity. 57. Gardner KE, Allis CD, Strahl BD: Operating on chromatin, a colorful language where context matters. J Mol Biol 2011, 409:36-46. 58. Chowdhury M, Forouhi O, Dayal S, McCloskey N, Gould HJ, Felsenfeld G, Fear DJ: Analysis of intergenic transcription and www.sciencedirect.com

70. Willmann KL, Milosevic S, Pauklin S, Schmitz KM, Rangam G,  Simon MT, Maslen S, Skehel M, Robert I, Heyer V et al.: A role for the RNA pol II-associated PAF complex in AID-induced immune diversification. J Exp Med 2012, 209:2099-2111. One of the very few studies that has investigated AID binding partners using a system based on endogenous AID, endogenous AID was epitope tagged in vivo (see also Refs. [71,74]). This study identified a large number of related proteins from two RNAP-associated complexes, PAF and FACT, highlighting the importance of the association between AID and the RNA biosynthesis pathway. 71. Nowak U, Matthews AJ, Zheng S, Chaudhuri J: The splicing  regulator PTBP2 interacts with the cytidine deaminase AID and promotes binding of AID to switch-region DNA. Nat Immunol 2011, 12:160-166. Another one of the studies that has investigated AID binding partners using in vivo tagged edogenous AID. This study identified PTBP2, a component of the RNA Splicing machinery, again shedding more light on the role of the association between AID and the RNA biosynthesis pathways. Current Opinion in Immunology 2013, 25:619–628

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72. Kanehiro Y, Todo K, Negishi M, Fukuoka J, Gan W, Hikasa T, Kaga Y, Takemoto M, Magari M, Li X et al.: Activationinduced cytidine deaminase (AID)-dependent somatic hypermutation requires a splice isoform of the serine/ arginine-rich (SR) protein SRSF1. Proc Natl Acad Sci U S A 2012, 109:1216-1221.

exosome targets the AID cytidine deaminase to both strands of transcribed duplex DNA substrates. Cell 2011, 144:353-363. An alternative approach to isolate and characterise endogenous AID complexes using in vitro transcribed templates identifies the RNA exosome as playing an important role in AID mediated deamination of transcribed DNA.

73. Conticello SG, Ganesh K, Xue K, Lu M, Rada C, Neuberger MS: Interaction between antibody-diversification enzyme AID and spliceosome-associated factor CTNNBL1. Mol Cell 2008, 31:474-484.

75. Yu K, Chedin F, Hsieh CL, Wilson TE, Lieber MR: R-loops at immunoglobulin class switch regions in the chromosomes of stimulated B cells. Nat Immunol 2003, 4:442-451.

74. Basu U, Meng FL, Keim C, Grinstein V, Pefanis E, Eccleston J,  Zhang T, Myers D, Wasserman CR, Wesemann DR et al.: The RNA

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76. Xu Z, Zan H, Pone EJ, Mai T, Casali P: Immunoglobulin classswitch DNA recombination: induction, targeting and beyond. Nat Rev Immunol 2012, 12:517-531.

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