RNA 2'-O-methylation promotes persistent R-loop formation and AID-mediated IgH class switch recombination.

BACKGROUND: RNA-DNA hybrids or R-loops are associated with deleterious genomic instability and protective immunoglobulin class switch recombination (CSR). However, the underlying phenomenon regulating the two contrasting functions of R-loops is unknown. Notably, the underlying mechanism that protects R-loops from classic RNase H-mediated digestion thereby promoting persistence of CSR-associated R-loops during CSR remains elusive. RESULTS: Here, we report that during CSR, R-loops formed at the immunoglobulin heavy (IgH) chain are modified by ribose 2'-O-methylation (2'-OMe). Moreover, we find that 2'-O-methyltransferase fibrillarin (FBL) interacts with activation-induced cytidine deaminase (AID) associated snoRNA aSNORD1C to facilitate the 2'-OMe. Moreover, deleting AID C-terminal tail impairs its association with aSNORD1C and FBL. Disrupting FBL, AID or aSNORD1C expression severely impairs 2'-OMe, R-loop stability and CSR. Surprisingly, FBL, AID's interaction partner and aSNORD1C promoted AID targeting to the IgH locus. CONCLUSION: Taken together, our results suggest that 2'-OMe stabilizes IgH-associated R-loops to enable productive CSR. These results would shed light on AID-mediated CSR and explain the mechanism of R-loop-associated genomic instability.

AID in non-Hodgkin B-cell lymphomas: The consequences of on- and off-target activity.

Activation induced cytidine deaminase (AID) is a key element of the adaptive immune system, required for immunoglobulin isotype switching and affinity maturation of B-cells as they undergo the germinal center (GC) reaction in peripheral lymphoid tissue. The inherent DNA damaging activity of this enzyme can also have off-target effects in B-cells, producing lymphomagenic chromosomal translocations that are characteristic features of various classes of non-Hodgkin B-cell lymphoma (B-NHL), and generating oncogenic mutations, so-called aberrant somatic hypermutation (aSHM). Additionally, AID has been found to affect gene expression through demethylation as well as altered interactions between gene regulatory elements. These changes have been most thoroughly studied in B-NHL arising from GC B-cells. Here, we describe the most common classes of GC-derived B-NHL and explore the consequences of on- and off-target AID activity in B and plasma cell neoplasms. The relationships between AID expression, including effects of infection and other exposures/agents, mutagenic activity and lymphoma biology are also discussed.

Heterologous expression of hAID in E. coli leads to the production of a splice isoform of AID: hAIDδC, a mystery to be explored.

Activation-induced cytidine deaminase (AID) is a key player that initiates antibody diversification in activated B-cell. AID mediates somatic hypermutation (SHM) and class switch recombination (CSR) via the deamination of cytosine to uracil at the Ig locus, resulting in the production of high-affinity antibodies. AID is predominantly restricted to Ig genes, whereas off-targeting of AID leads to lymphocyte-related malignancies. Interestingly, apart from FL-AID other splice isoforms of AID are highly expressed in the lymphocyte malignancies. In our study, we found that the heterologous expression of hAID-FL in E. coli cells produced two induced bands of hAID as demonstrated by SDS-PAGE and western blotting. Remarkably, peptide mapping data predicted that one band is hAID-FL and the other is its splice isoform, hAIDδE4a. To get an insight into why E. coli cells expressed hAID-FL and hAID variant, we mutated the 5' and 3' splice site of a putative intron of hAID, but it failed to produce only hAID-FL. Incidentally, hAID expressed with fusion partners also displayed two bands, and peptide mapping data strongly suggest that besides hAID-FL, the lower band showed a significant number of amino acids missing towards the C-terminal domain (named as hAIDδC). Our results are the first report to show that expression of recombinant hAID alone or irrespective of solubilization tags in E. coli cells produced hAID-FL and hAIDδC. It will be fascinating to explore the potential mechanism underlying the expression of hAIDδC from recombinant hAID plasmid in E. coli cells.

HomA and HomB, outer membrane proteins of Helicobacter pylori down-regulate activation-induced cytidine deaminase (AID) and Ig switch germline transcription and thereby affect class switch recombination (CSR) of Ig genes in human B-cells.

H. pylori is one of the major causes of chronic gastritis, peptic ulcer disease (PUD), gastric mucosa-associated lymphoid tissue lymphoma (MALT) and gastric carcinoma. H. pylori toxin VacA is responsible for host cell apoptosis, whereas CagA is known to aberrantly induce expression of activation-induced cytidine deaminase (AID) in gastric epithelial cells that causes mutations in oncogenes and tumour suppressor genes, leading to the transformation of normal cells into cancerous cells. Although, a significant amount of research has been conducted to understand the role of bacterial factors modulating deregulated host cell pathways, the interaction between H. pylori and immune cells of the marginal zone and its consequences are still not well understood. HomB and HomA, outer membrane proteins (OMPs) from H. pylori, which assist in the adhesion of bacteria to host cells, are found to be associated with H. pylori virulent strains and promote inflammation. Interestingly, we observed that the interaction of HomB/HomA OMPs with B-cells transiently downregulates AID expression and Ig switch germline transcription. Downregulation of AID leads to impairment of class switch recombination (CSR), resulting in significantly reduced switching to IgG and IgA antibodies. Besides, we examined the immune-suppressive response of B-cells and observed that the cells stimulated with HomA/B show upregulation in the levels of IL10, IL35, as well as PDL1, a T-cell inhibition marker. Our study suggests the potential role of OMPs in immune response modulation strategies used by the pathogen to evade the immune response. These results provide a better understanding of H. pylori pathogenesis and assist in identifying novel targets for therapy.

Unfolding the Role of Splicing Factors and RNA Debranching in AID Mediated Antibody Diversification.

Activated B-cells diversify their antibody repertoire via somatic hypermutation (SHM) and class switch recombination (CSR). SHM is restricted to the variable region, whereas, CSR is confined to the constant region of immunoglobulin (Ig) genes. Activation-induced cytidine deaminase (AID) is a crucial player in the diversification of antibodies in the activated B-cell. AID catalyzes the deamination of cytidine (C) into uracil (U) at Ig genes. Subsequently, low fidelity repair of U:G mismatches may lead to mutations. Transcription is essential for the AID action, as it provides a transient single-strand DNA substrate. Since splicing is a co-transcriptional event, various splicing factors or regulators influence the transcription. Numerous splicing factors are known to regulate the AID targeting, function, Ig transcription, and AID splicing, which eventually influence antibody diversification processes. Splicing regulator SRSF1-3, a splicing isoform of serine arginine-rich splicing factor (SRSF1), and CTNNBL1, a spliceosome interacting factor, interact with AID and play a critical role in SHM. Likewise, a splicing regulator polypyrimidine tract binding protein-2 (PTBP2) and the debranching enzyme (DBR1) debranches primary switch transcripts which later forms G-quadruplex structures, and the S region guide RNAs direct AID to S region DNA. Moreover, AID shows several alternate splicing isoforms, like AID devoid of exon-4 (AIDΔE4) that is expressed in various pathological conditions. Interestingly, RBM5, a splicing regulator, is responsible for the skipping of AID exon 4. In this review, we discuss the role and significance of splicing factors in the AID mediated antibody diversification.

Production of Herbicide-Sensitive Strain to Prevent Volunteer Rice Infestation Using a CRISPR-Cas9 Cytidine Deaminase Fusion.

When cultivated rice seed fall into fields, they may overwinter and spontaneously germinate the next spring. Such germinated plants are termed "volunteer rice." Volunteer grains originating from feed rice varieties may differ in certain traits, such as quality and taste, as compared with those of rice cultivated for human consumption, which may reduce the overall quality of the final harvested grain. Many rice varieties show resistance to benzobicyclon (BBC), a beta-triketone herbicide (bTH) that inhibits 4-hydroxyphenylpyruvate dioxygenase (HPPD). Recently, the rice gene HIS1 (HPPD INHIBITOR SENSITIVE 1) conferring resistance to BBC and other bTHs was identified. In this study, to suppress the occurrence of volunteer rice infestation, we attempted to generate a BBC-sensitive rice strain via the knockout of the HIS1 gene using genome editing techniques. The production of a his1 knockout line was carried out by the start-codon substitution or stop-codon creation using CRISPR-Cas9 cytidine deaminase fusion, which is useful as a novel amino acid sequence is not generated due to the shifting of the reading frame. The mutation frequencies of independent transgenic plants were 3.6, 13.5, 13.8, and 21.2% at four gRNAs for start-codon substitution and three stop-codon creations. The his1 knockout lines were conferred with sensitivity to BBC, re-confirming by genome editing that this is indeed the gene responsible for BBC resistance/sensitivity. The his1 knockout lines also exhibited a sensitive phenotype to other bTHs, including sulcotrione, mesotrione, tembotrione, and tefuryltrione, compared with the wild-type variety 'Nipponbare.' These results demonstrate the potential of herbicide-sensitive rice produced by genome editing technology as a material to control volunteer feed rice using pre-labeled herbicides for varieties consumed by humans.

SRSF1-3, a splicing and somatic hypermutation regulator, controls transcription of IgV genes via chromatin regulators SATB2, UBN1 and histone variant H3.3.

SRSF1, a member of the SR protein family, is an important splicing factor and regulator of splicing. Multiple splicing isoforms have been reported for this gene. SRSF1-3, a splicing isoform of SRSF1, is necessary for AID-dependent SHM of IgV genes. However, its precise role in SHM remains enigmatic. Transcriptomic analysis of SRSF1-3 reconstituted cells shows upregulation of transcription factor SATB2 and chromatin regulator UBN1. The increased SATB2 and UBN1 are strikingly enriched in the MAR and promoter regions of the IgL gene, respectively. Furthermore, UBN1 enrichment at the promoter region was coupled with a hundred-fold enhanced occupancy of the histone variant H3.3 at the IgL promoter, that is a hallmark of efficient SHM. The enhanced occupancy of SATB2 at the MAR, UBN1 and histone variant H3.3 at the IgL promoter leads to an increase in IgL transcription, revealing a role of SRSF1-3 in SHM. Thus, SRSF1-3 is likely involved in the regulation of SHM, via upregulation of a crucial transcription factor SATB2, as well as, by overexpression of a chromatin modulator of Ig genes, UBN1, which further assists in the recruitment of the histone variant H3.3. Furthermore, the splicing isoform SRSF1-3 regulates alternate splicing pattern of splicing isoforms for various crucial genes. The present study provides the first evidence that a splicing isoform of an SR protein can regulate the post-transcriptional processing of RNA in vivo.

AID preferentially targets the top strand in nucleosome sequences.

AID initiates both somatic hypermutation (SHM) and class switch recombination (CSR) in Ig genes. AID-induced mutations are linked with transcription initiation and elongation. Transcription occurs in the context of chromatin and thus RNA PolII and AID need to deal with nucleosomes. Both nucleosome stability and positioning significantly influence the accessibility of AID to Ig genes and the SHM pattern. Interestingly, in the nucleosome, SHM process seems to have a preference for the top strand. To know whether the preferential targeting of SHM to the top strand is due to a post-AID event, we expressed an inhibitor of Uracil DNA glycosylase (UNG), Ugi, into DT40 cells containing the nucleosome positioning sequence (MP2) and compared the SHM pattern. We observed a similar preference to the top strand for the high-affinity nucleosome positioning sequence in UNG inhibited cells. Furthermore, to understand whether the primary sequence of nucleosome sequence is influencing preferential targeting, we introduced two copies of MP2 sequence in the reverse orientation (MP2R) into a variable Ig gene. We observed that the MP2R cells also demonstrated preferential targeting of the non-transcribed strand in nucleosome as compared to the transcribed strand, confirming that in nucleosome sequences AID has better access to Cs on the top strand. The preferential targeting of AID on the top strand suggests that RNA Pol-II stalls while it transcribes the stable nucleosomes, thus giving ample opportunity for the transcribed strand to form R-loops with the nascent RNA, thereby gives limited access to AID on the bottom strand.

A Novel DT40 Antibody Library for the Generation of Monoclonal Antibodies.

Early etiological diagnosis is very important for the control of sudden viral infections, and requires antibodies with both high sensitivity and high specificity. Traditional antibody preparation methods have limitations, such as a long and arduous cycle, complicated operation, and high expenses. A chicken lymphoma cell line, DT40, is known to produce IgM-type antibodies and undergo gene conversion and somatic mutation in the variable region of the immunoglobulin gene during culture. Here, the DT40 cell line was developed to produce antibody libraries and prepare antibody rapidly in vitro. Since hypermutation in DT40 cells was regulated by the activation-induced cytidine deaminase (AID) gene, AID expression needs to be controlled to either fix the Ig sequence by stopping mutation or improve affinity by resuming mutation after the antibodies have been selected. In this study, we generated a novel AID-inducible DT40 cell line (DT40-H7), in which the endogenous AID gene was knocked out using the CRISPR/Cas9 genome editing system, and an inducible AID gene, based on the Tet-Off expression system, was stably transfected. AID expression was controlled in DT40-H7 cells in a simple and efficient manner; gene conversion and point mutations were observed only when AID was expressed. Using the antibody library generated from this cell line, we successfully obtained monoclonal antibodies against the NS1 protein of Zika virus. The DT40-H7 cell line represents a useful tool for the selection and evolution of antibodies and may also be a powerful tool for the rapid selection and generation of diagnostic antibodies for emerging infectious diseases.

HIV-1 Tat protein induces aberrant activation of AICDA in human B-lymphocytes from peripheral blood.

Individuals infected with human immunodeficiency virus (HIV) are at increased risk for Burkitt lymphoma, a B-cell malignancy which occurs after a chromosomal translocation rearranging the MYC oncogene with an immunoglobulin gene locus, usually the IGH heavy chain gene locus. We have previously reported that the HIV protein Tat which circulates in all HIV-positive individuals whatever their immune status caused an increased rate of colocalization between IGH and MYC in B-cells nuclei. We here present in vitro evidence that Tat activates the expression of the AICDA gene that encodes the activation-induced cytidine deaminase whose physiological function is to create double-strand breaks for immunoglobulin gene maturation. In the presence of Tat, DNA damage was observed concomitantly in both MYC and IGH, followed by DNA repair by nonhomologous end joining. AICDA was further found overexpressed in vivo in peripheral blood B-cells from HIV-infected individuals. Thus, the capacity of Tat to spontaneously penetrate B-cells could be sufficient to favor the occurrence of MYC-IGH oncogenic rearrangements during erroneous repair, a plausible cause for the increased incidence of Burkitt lymphoma in the HIV-infected population.

Targeted Base Editing with CRISPR-Deaminase in Tomato.

The Target-AID system, consisting of a complex of cytidine deaminase and deficient CRISPR/Cas9, enables highly specific genomic nucleotide substitutions without the need for template DNA. The Cas9-fused cytidine deaminase is guided by sgRNAs and catalyzes the conversion of cytosine to uracil. The resulting U-G DNA mismatches trigger nucleotide substitutions (C to T or G to A) through DNA replication and repair pathways. Target-AID also retains the benefits of conventional CRISPR/Cas9 including robustness in various organisms, high targeting efficiency, and multiplex simultaneous gene editing. Our research group recently developed plant-optimized Target-AID system and demonstrated targeted base editing in tomato and rice. In this chapter, we introduce methods for Target-AID application in tomato.

Inheritance of co-edited genes by CRISPR-based targeted nucleotide substitutions in rice.

The CRISPR/Cas9 system is a revolutionary genome-editing tool for directed gene editing in various organisms. Cas9 variants can be applied as molecular homing devices when combined with various functional effectors such as transcriptional activators or DNA modification enzymes. Target-AID is a synthetic complex of nuclease deficient Cas9 fused to an activation-induced cytidine deaminase (AID) that enables targeted nucleotide substitution (C to T or G to A). We previously demonstrated that the introduction of desired point mutations into target genes by Target-AID confers herbicide tolerance to rice callus. Inheritance of the introduced mutations, as well as the removal of transgenes, are key issues that must be addressed in order to fully develop Target-AID as a plant breeding technique. Here we report the transmission of such mutations from the callus to regenerants and their progenies, leading to a generation of selectable marker-free (SMF) herbicide tolerant rice plants with simultaneous multiplex nucleotide substitutions. These findings demonstrate that Target-AID can be developed into novel plant breeding technology which enables improvement of multiplex traits at one time in combination with sophisticated targeted base editing with the simplicity and versatility of CRISPR/Cas9 system.

Activation-Induced Cytidine Deaminase Aided In Vitro Antibody Evolution.

Activation-induced cytidine deaminase (AID) initiates somatic hypermutation (SHM) by converting deoxycytidines (dC) to deoxyuracils (dU) which then can induce other mutations, and plays a central role in introducing diversification of the antibody repertoire in B cells. Ectopic expression of AID in bacteria and non-B cells can also lead to frequent mutations in highly expressed genes. Taking advantage of this feature of AID, in recent years, systems coupling in vitro somatic hypermutation and mammalian cell surface display have been developed, with unique benefits in antibody discovery and optimization in vitro. Here, we provide a protocol for AID mediated in vitro protein evolution. A CHO cell clone bearing a single gene expression cassette has been constructed. The gene of an interested protein for in vitro evolution can be easily inserted into the cassette by dual recombinase-mediated cassette exchange (RMCE) and constantly expressed at high levels. Here, we matured an anti-TNFα antibody as an example. Firstly, we obtained a CHO cell clone highly displaying the antibody by dual RMCE. Then, the plasmid expressing AID is transfected into the CHO cells. After a few rounds of cell sorting-cell proliferation, mutant antibodies with improved features can be generated. This protocol can be applied for improving protein features based on displaying levels on cell surface and protein-protein interaction, and thus is able to enhance affinity, specificity, and stability besides others.

Enhancers Improve the AID-Induced Hypermutation in Episomal Vector for Antibody Affinity Maturation in Mammalian Cell Display.

The induction of somatic hypermutation (SHM) in various cell lines by activation-induced cytidine deaminase (AID) has been used in protein-directed selection, especially in antibody affinity maturation. Several antibody affinity maturation systems based on mammalian cells have been developed in recent years, i.e., 293T, H1299, Raji and CHO cells. However, the efficiency of in vitro AID-induced hypermutation is low, restricting the application of such systems. In this study, we examined the role of Ig and Ek enhancers in enhancing SHM in the episomal vector pCEP4 that expresses an anti-high mobility group box 1 (HMGB1) full-length antibody. The plasmid containing the two enhancers exhibited two-fold improvement of mutation rate over pCEP4 in an AID expression H1299 cell line (H1299-AID). With the engineered episomal vector, we improved the affinity of this antibody in H1299-AID cells by 20-fold.

AID Biology: A pathological and clinical perspective.

Activation-induced cytidine deaminase (AID), primarily expressed in activated mature B lymphocytes in germinal centers, is the key factor in adaptive immune response against foreign antigens. AID is responsible for producing high-affinity and high-specificity antibodies against an infectious agent, through the physiological DNA alteration processes of antibody genes by somatic hypermutation (SHM) and class-switch recombination (CSR) and functions by deaminating deoxycytidines (dC) to deoxyuridines (dU), thereby introducing point mutations and double-stranded chromosomal breaks (DSBs). The beneficial physiological role of AID in antibody diversification is outweighed by its detrimental role in the genesis of several chronic immune diseases, under non-physiological conditions. This review offers a comprehensive and better understanding of AID biology and its pathological aspects, as well as addresses the challenges involved in AID-related cancer therapeutics, based on various recent advances and evidence available in the literature till date. In this article, we discuss ways through which our interpretation of AID biology may reflect upon novel clinical insights, which could be successfully translated into designing clinical trials and improving patient prognosis and disease management.

The Complex Interplay between DNA Injury and Repair in Enzymatically Induced Mutagenesis and DNA Damage in B Lymphocytes.

Lymphocytes are endowed with unique and specialized enzymatic mutagenic properties that allow them to diversify their antigen receptors, which are crucial sensors for pathogens and mediators of adaptive immunity. During lymphocyte development, the antigen receptors expressed by B and T lymphocytes are assembled in an antigen-independent fashion by ordered variable gene segment recombinations (V(D)J recombination), which is a highly ordered and regulated process that requires the recombination activating gene products 1 & 2 (RAG1, RAG2). Upon activation by antigen, B lymphocytes undergo additional diversifications of their immunoglobulin B-cell receptors. Enzymatically induced somatic hypermutation (SHM) and immunoglobulin class switch recombination (CSR) improves the affinity for antigen and shape the effector function of the humoral immune response, respectively. The activation-induced cytidine deaminase (AID) enzyme is crucial for both SHM and CSR. These processes have evolved to both utilize as well as evade different DNA repair and DNA damage response pathways. The delicate balance between enzymatic mutagenesis and DNA repair is crucial for effective immune responses and the maintenance of genomic integrity. Not surprisingly, disturbances in this balance are at the basis of lymphoid malignancies by provoking the formation of oncogenic mutations and chromosomal aberrations. In this review, we discuss recent mechanistic insight into the regulation of RAG1/2 and AID expression and activity in lymphocytes and the complex interplay between these mutagenic enzymes and DNA repair and DNA damage response pathways, focusing on the base excision repair and mismatch repair pathways. We discuss how disturbances of this interplay induce genomic instability and contribute to oncogenesis.

Epstein-Barr virus (EBV)-associated epithelial and non-epithelial lesions of the oral cavity.

Epstein-Barr virus (EBV) is known to be associated with the development of malignant lymphoma and lymphoproliferative disorders (LPDs) in immunocompromised patients. EBV, a B-lymphotropic gamma-herpesvirus, causes infectious mononucleosis and oral hairy leukoplakia, as well as various pathological types of lymphoid malignancy. Furthermore, EBV is associated with epithelial malignancies such as nasopharyngeal carcinoma (NPC), salivary gland tumor, gastric carcinoma and breast carcinoma. In terms of oral disease, there have been several reports of EBV-related oral squamous cell carcinoma (OSCC) worldwide. However, the role of EBV in tumorigenesis of human oral epithelial or lymphoid tissue is unclear. This review summarizes EBV-related epithelial and non-epithelial tumors or tumor-like lesions of the oral cavity. In addition, we describe EBV latent genes and their expression in normal epithelium, inflamed gingiva, epithelial dysplasia and SCC, as well as considering LPDs (MTX- and age-related) and DLBCLs of the oral cavity.

Coupling recombinase-mediated cassette exchange with somatic hypermutation for antibody affinity maturation in CHO cells.

Heterologous expression of activation-induced cytidine deaminase (AID) can induce somatic hypermutation (SHM) for genes of interest in various cells, and several research groups (including ours) have successfully improved antibody affinity in mammalian or chicken cells using AID-induced SHM. These affinity maturation systems are time-consuming and inefficient. In this study, we developed an antibody affinity maturation platform in Chinese hamster ovary (CHO) cells by coupling recombinase-mediated cassette exchange (RMCE) with SHM. Stable CHO cell clones containing a single copy puromycin resistance gene (PuroR) expression cassette flanked by recombination target sequences (FRT and loxP) being able to highly express a gene of interest placed in the cassette were developed. The PuroR gene was replaced with an antibody gene by RMCE, and the antibody was displayed on the cell surface. Cells displaying antibodies on their membrane were transfected with the AID gene, and mutations of the antibody gene were accumulated by AID-mediated hypermutation during cell proliferation followed by flow cytometric cell sorting for cells bearing antibody mutants with improved affinity. Affinity improvements were detected after only one round of cell sorting and proliferation, mutant clones with 15-fold affinity improvement were isolated within five rounds of maturation (within 2 months). CHO cells are fast growing, stress-resistant and produce antibody with glycosylations suitable for therapy. Our antibody-evolution platform based on CHO cells makes antibody-affinity maturation more efficient and is especially convenient for therapeutic antibody affinity improvement.

Long-term arsenite exposure induces premature senescence in B cell lymphoma A20 cells.

Chronic arsenite exposure induces immunosuppression, but the precise mechanisms remain elusive. Our previous studies demonstrated that arsenite exposure for 24 h induces G0/G1 arrest in mouse B lymphoma A20 cells and the arrest is caused through induction of cyclin-dependent kinase inhibitor p16(INK4a) followed by accumulation of an Rb family protein, p130. In this study, we further investigated the consequences of long-term arsenite exposure of A20 cells. The results demonstrated that exposure to 10 µM sodium arsenite up to 14 days induces a great increase in G0/G1 arrest, irreversible cell growth suppression, cellular morphological changes and positive staining for senescence-associated ß-galactosidase. The long-term arsenite exposure also induced up-regulation of p16(INK4a) followed by robust accumulation of p130 and activation of the p53 pathway. Knockdown experiments with siRNA showed that p130 accumulation is essential for cell cycle arrest by long-term arsenite exposure. Since p16(INK4a) and the p53 pathway are known to be activated by DNA damage, we investigated the involvement of DNA damage formation by long-term arsenite exposure. We found that a variety of DNA repair-related genes were significantly down-regulated from 24 h of arsenite exposure and activation-induced cytidine deaminase was greatly up-regulated after long-term arsenite exposure. Consistent with these findings, long-term arsenite exposure increased a DNA double-strand break marker, γ-H2AX and increased mutation frequency in a Bcl6 gene region. These results revealed that long-term arsenite exposure induces premature senescence through DNA damage increase and p130 accumulation in lymphoid cells.

AID expression in B-cell lymphomas causes accumulation of genomic uracil and a distinct AID mutational signature.

The most common mutations in cancer are C to T transitions, but their origin has remained elusive. Recently, mutational signatures of APOBEC-family cytosine deaminases were identified in many common cancers, suggesting off-target deamination of cytosine to uracil as a common mutagenic mechanism. Here we present evidence from mass spectrometric quantitation of deoxyuridine in DNA that shows significantly higher genomic uracil content in B-cell lymphoma cell lines compared to non-lymphoma cancer cell lines and normal circulating lymphocytes. The genomic uracil levels were highly correlated with AID mRNA and protein expression, but not with expression of other APOBECs. Accordingly, AID knockdown significantly reduced genomic uracil content. B-cells stimulated to express endogenous AID and undergo class switch recombination displayed a several-fold increase in total genomic uracil, indicating that B cells may undergo widespread cytosine deamination after stimulation. In line with this, we found that clustered mutations (kataegis) in lymphoma and chronic lymphocytic leukemia predominantly carry AID-hotspot mutational signatures. Moreover, we observed an inverse correlation of genomic uracil with uracil excision activity and expression of the uracil-DNA glycosylases UNG and SMUG1. In conclusion, AID-induced mutagenic U:G mismatches in DNA may be a fundamental and common cause of mutations in B-cell malignancies.