Activation-induced Cytidine Deaminase Overexpression Research Articles

“Snapshotting” Somatic Hypermutation in Single Follicular Lymphoma Cells

IntroductionFollicular Lymphoma (FL) is one of the most prevalent B-cell neoplasms and despite recent advances remains incurable in most cases. FL cells are malignant counterparts of normal germinal center B-cells. At molecular level FL are characterized by the t(14;18) which results in the overexpression of the BCL-2. These apoptosis resistant cells accumulate somatic mutations associated with tumorigenesis and progression. Whereas several mutagenic mechanisms shape the FL genomic landscape, up to 22% of the mutations may be attributed to activation-induced cytidine deaminase (AID) activity.Under physiological conditions AID is responsible for somatic hypermutation (SHM) in immunoglobulin genes (IG) of germinal center B-cells. In fact, it is accepted that ongoing SHM still occurs at relatively high rates in FL cells as compared with other germinal center lymphoid neoplasms. Moreover, we recently demonstrated the direct effect of AID overexpression inducing somatic mutations driving murine and human B-cell neoplasm progression in vivo. Therefore, unveiling the molecular basis of AID activity in lymphoma cells remains essential to understand lymphomagenesis and to develop novel targeted therapies.The advent of single-cell high-throughput sequencing has enabled the analysis of molecular events at an unprecedented resolution. Therefore, we designed a study to capture AID-induced somatic hypermutation at the single cell level in FL.MethodsTumor samples derived from 14 FL patients were analyzed, as controls, we chose a B-cell malignancy with lower SHM rates and included 5 samples derived from chronic lymphocytic leukemia (CLL) and 2 from monoclonal B lymphocytosis (MBL). Single cell whole cDNA libraries were obtained by 10X Genomics. Immunoglobulin gene single cell libraries were prepared by enrichment with seminested amplification using 3 ′ constant domain primers followed by 10X Genomics prep. Both single-cell libraries were sequenced in paired-end mode (2 × 150 bp) on an Illumina Hiseq platform. We developed an immunoglobulin alignment tool to enable the analysis of highly mutated IG sequences derived from FL. A consensus sequence was obtained by transcript associated with a unique molecular identifier (UMI) for every cell. Then, the presence of variants in the IG by position in a particular cell was annotated. These observations were filtered using quality parameters (read depth => 25 , frequency of the event => 20%, UMIs supporting every variant => 5).ResultsWe analyzed an average of 926 cells per case. Our data confirms at the single cell level previous reports on high clonal heterogeneity and hypermutation rate (mean 18.7%) in FL. When analyzing intracellular heterogeneity we observed single FL cells expressing simultaneously transcripts derived from the same immunoglobulin VDJ rearrangement but displaying high confidence single nucleotide variants. After applying strict filtering strategies to account for potential technical issues we defined the occurrence of “SHM snapshot events” when a single cell displayed a set of transcripts from a particular VDJ rearrangement with and without the occurring single nucleotide variant. Such events were detected in 8 of the 14 FL analyzed samples. In those 8 samples, 114 events were observed in which two different transcripts of one specific IG were found within a single cell. AID-related motifs (WRCY, WA and RCG) were found in 45% of the SHM snapshot events. SHM snapshot events were undetectable in CLL (5 samples) and 3 SHM snapshot events were detected in MBL (2 samples). In addition, the occurrence of SHM snapshot events was significantly associated with AID expression (Fisher exact test, p-value < 0.001). On the other hand, AID expression was not detectable on CLL and MBL samples.ConclusionHere we report for the first time the occurrence of ongoing somatic hypermutation at a single cell level in FL. The simultaneous detection of both the pre and post mutation IG mRNA transcripts within a single cell may be indicative of SHM occurring more recently than the lifespan of mRNA transcript. The detection of this phenomenon in 57% of FL samples and its association with AID expression suggests that AID-induced mutagenesis may be acting at a much higher rate than expected. This work highlights the role of AID in shaping the tumor heterogeneity in FL and the need to further understand the role of this enzyme in lymphomagenesis and tumor progression. DisclosuresNo relevant conflicts of interest to declare.

Over-Expression of Activation-Induced Cytidine Deaminase in TCL1 Mice Leads to the Development of IGHV-Mutated CLL Clones That Associate with More Aggressive Disease

Introduction. Activation-induced cytidine deaminase (AID) mediates somatic hypermutation (SHM) and class switch recombination (CSR) of immunoglobulin genes (IG s), both of which are necessary for an effective immune response in normal B cells. An unwanted consequence of AID's role is its contribution to cellular transformation and tumor progression by aberrantly mutagenizing genes outside the IG locus (off-target mutations). In chronic lymphocytic leukemia (CLL), higher levels of IGHV clonal mutations in IGHV-mutated CLL (M-CLL) correlate strongly with better clinical outcomes, while, IGHV -unmutated (U-CLL) cases are associated with worse patient outcome. Paradoxically, U-CLL cells express significantly higher levels of AID than M- CLL cells.Eμ-T-cell leukemia-1 (TCL1) transgenic (Tg) mice are a valuable model of CLL. However, because SHM and CSR occur rarely in the leukemic clones of these animals, TCL1 Tg mice mimic only U-CLL and hence may not provide an understanding of the roles of SHM and CSR in disease evolution. To address this issue, we developed a TCL1 strain by interbreeding mice over-expressing AID in B lymphocytes (Eμ-TCL1xVκ-AID).Methods. B-cell clonal expansions were identified in spleen cells from 14 TCL1 and 23 TCL1xAID (DT) mice by amplifying cDNAs by PCR using consensus IGHV FR and isotype-specific primers for IgH chains and Vκ FR and Cκ for IgL chain genes. Ig sequences were compared to murine germline IGHV s and IGKV s, and IGHV-D-JH and IGKV-JK rearrangements by IMGT V-Quest. Six DT and 2 TCL1 tumors were transferred to SCID mice (5 mice/donor)Results.Clonal expansions: Monoclonal/oligoclonal expansions were detected in all TCL1 mice; these used only µ H and κ L chains. Similar expansions were detected in 21 of 23 DT mice; each animal bore an IgMκ+ clone and 5 also exhibited a concomitant IgGκ+ clone. Approximately 50% used VH1-55, VH11-2, or VH12-3.SHM: The IgM+ TCL1 clones had a mutation frequency of 0.05% for IGHV and 0.09% for IGKV . In contrast, the IGHV and IGKV mutation frequencies were considerably higher in DT mice: IGHV 0.47% in IgM+ and 3.0% in IgG+ clones, and IGKV 0.9% for IgM+ and IgG+ combined. These mutations localized more frequently in AID hotspots than coldspots at a ratio of ≥7:1.However, SHM did not affect all clones equally. Although the mutation frequency in VH12-3 and VH11-2 clones was higher (0.38%) than the TCL1 level (0.05%), it was considerably less than that found in the DT clones using other IGHV s (0.80%). In addition, no mutations were detected in VH1-55 clones from DT mice.IGHV gene use and SHM in clones that underwent CSR: Notably, in 2 of 5 instances the same IGHV-D-J rearrangement was found in IgMκ+ and IgGκ+ clones from the same mice. For the remaining 3, only the IgG+ version was detected. Also, within the IgG-only group, IGHV1-47 was used by 2 different clones and these were highly mutated (8.9%).Stereotyped IGHV-D-J and IGkV-Jk rearrangements: Among 32 IgM clones from DT mice, 4 expressed VH11-2 and Vκ14-126 and 8 expressed VH12-3 and Vκ4-91; these rearrangements were very similar to canonical anti-phosphatidylcholine-producing clones.DT mice develop more aggressive disease: we observed shorter median survival in DT mice comparing to TCL1 mice (279 days vs 356 days, P =0.0034)Transfer tumor development: Spleen cells from 6 CLL-bearing mice were transferred into SCID recipients, leading to a selected outgrowth of VH12-3/Vκ4-91 and VH11-2/Vκ14-126 clones. Survival in these mice inversely correlated with IGHV mutations (Figure).Summary and conclusions.Over-expression of AID in TCL1 mice leads to markedly increased SHM and CSR. However, SHM is not equivalent for all IGHV genes: certain IGHV s and IGK Vs are less susceptible to having major increases in the extent of SHM and the occurrence of CSR. This property resembles some human CLL IGHV s that rarely develop SHMs or undergo CSR despite the B-cell's ability to synthesize AID (e.g., many IGHV1-69+ clones in U-CLL patients). AID overexpression also led to IgG+ clones for which an IgM precursor was not found. This resembles those human stereotyped CLL clones that are only found as IgGs (e.g., stereotyped subsets 4 and 8). Finally, transfer of DT clones to SCID mice led to more IGHV and IGKV mutations and shorter survival opposite to that in M-CLL patients. Possibly this adverse effect might be a consequence of AID's aberrant action outside the IG loci which is facilitated in DT mice. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Activation-Induced Cytidine Deaminase Promotes Proliferation and Enhances Chemoresistance and Migration in B-cell Lymphoma.

Activation-induced cytidine deaminase (AID) is a DNA modifying enzyme which has an essential function in promoting antibody diversification. Its overexpression is strongly associated with B-cell derived malignancies including Burkitt lymphoma, where AID is required for the characteristic c-MYC/IGH translocation. This study aimed at defining AID's oncopathogenic role which is still poorly understood. We created over-expressing and knock-down cell culture models of AID, and used cellular assays to provide insight into its contribution to lymphomagenesis. We showed that AID expression is highly specific to, and abundantly expressed in B-cell-derived cancers and that ectopic overexpression of AID leads to rapid cell death. Using a knock-down model, we revealed that AID expression significantly impacts genomic stability, proliferation, migration and drug resistance. AID is an important driver of lymphoma, impacting multiple cellular events, and is potentially a strong candidate for targeted therapy in lymphoma.

Activation-induced cytidine deaminase overexpression in double-hit lymphoma: potential target for novel anticancer therapy

Activation-induced cytidine deaminase (AID) is one kind of the mutant enzymes, which target regulating the immunoglobulin (Ig) gene in Burkitt’s lymphoma to initiate class switch recombination (CSR), resulting in c-Myc chromosomal translocation. However, it is not clear that whether AID induces c-Myc/IgH translocation in double-hit lymphoma (DHL) with c-Myc gene translocation. In this study, the AID in DHL tissues and classical diffuse large b-cell lymphoma (DLBCL) tissues were compared. The results suggested that AID is of important value in predicting DHL, stronger CSR of AID was observed in DHL patients, which exhibited AID overexpression and c-Myc gene translocation of DHL after CSR induction. It is concluded that AID directly induces CSR in DHL and may result in c-Myc gene translocation. Targeting AID may be a good treatment regimen for DHL.

Abstract A49: Induction of the lymphomagenic enzyme activation-induced cytidine deaminase (AID) upon in vitro exposure to phenylurea herbicides

Abstract Many studies have drawn associations between pesticide exposures and lymphomagenesis, both in occupational settings and in the general population. The specific compounds responsible for an increased risk of non-Hodgkin lymphoma in pesticide-exposed individuals have not been entirely established. Furthermore, the biologic mechanisms by which pesticides act as lymphomagens are unknown. One mechanism of B-cell genomic instability that plays a role in lymphomas derived from germinal center (GC)-type B cells, including follicular lymphoma, diffuse large B cell lymphoma, and Burkitt lymphoma, is the off-target/aberrant activity of activation-induced cytidine deaminase (AID). AID damages DNA through deamination of cytidine. This occurs on a physiologic basis within GC B cells and is responsible for both class switch recombination (CSR) and somatic hypermutation (SHM). AID can also target nonimmunoglobulin loci, including several proto-oncogenes, resulting in both mutations (aberrant somatic hypermutation; aSHM) and chromosomal translocations. Overexpression of AID has previously been found to increase B-cell genomic instability. In this study, GC-type B cell lines (Ramos, Raji, and CL-01) were treated with pesticides, including herbicides and insecticides from various classes, to determine the effect on AID expression. Real-time RT-PCR was used to determine the degree of AID mRNA expression after chemical treatment. Two representative phenylurea herbicides, linuron and diuron, were found to induce AID expression, in a time- and dose-dependent manner, at concentrations ranging from 50-200 micromolar, with maximal induction of 2- to 6-fold depending on the compound and time point. Additional pesticides that were tested but not found to induce AID expression, at concentrations that were minimally toxic in vitro, included representative organochlorine, organophosphate, phenoxy, and phosphanoglycine pesticides. The findings raise the possibility of increased AID-mediated genomic instability in GC type B cells exposed to phenylurea herbicides. Future studies will investigate the possible link between the observed phenylurea herbicide-related induction of AID, B-cell genomic instability, and lymphomagenesis. Citation Format: Rebecca J. Leeman-Neill, Uttiya Basu. Induction of the lymphomagenic enzyme activation-induced cytidine deaminase (AID) upon in vitro exposure to phenylurea herbicides [abstract]. In: Proceedings of the AACR Special Conference on Environmental Carcinogenesis: Potential Pathway to Cancer Prevention; 2019 Jun 22-24; Charlotte, NC. Philadelphia (PA): AACR; Can Prev Res 2020;13(7 Suppl): Abstract nr A49.

AICDA drives epigenetic heterogeneity and accelerates germinal center-derived lymphomagenesis

Epigenetic heterogeneity is emerging as a feature of tumors. In diffuse large B-cell lymphoma (DLBCL), increased cytosine methylation heterogeneity is associated with poor clinical outcome, yet the underlying mechanisms remain unclear. Activation-induced cytidine deaminase (AICDA), an enzyme that mediates affinity maturation and facilitates DNA demethylation in germinal center (GC) B cells, is required for DLBCL pathogenesis and linked to inferior outcome. Here we show that AICDA overexpression causes more aggressive disease in BCL2-driven murine lymphomas. This phenotype is associated with increased cytosine methylation heterogeneity, but not with increased AICDA-mediated somatic mutation burden. Reciprocally, the cytosine methylation heterogeneity characteristic of normal GC B cells is lost upon AICDA depletion. These observations are relevant to human patients, since DLBCLs with high AICDA expression manifest increased methylation heterogeneity vs. AICDA-low DLBCLs. Our results identify AICDA as a driver of epigenetic heterogeneity in B-cell lymphomas with potential significance for other tumors with aberrant expression of cytidine deaminases.

Investigation of AID, Dicer, and Drosha Expressions in Patients with Chronic Lymphocytic Leukemia

ABSTRACTChronic lymphocytic leukemia (CLL) is the most common leukemia in Western countries. Cytogenetic lesions such as del13q14, del11q22.3, and del17p13 are identified in 50–60% of patients. Activation-induced cytidine deaminase (AID) plays a central role in somatic hyper mutation (SHM) and class switch recombination (CSR) and functions on Ig genes, but also target non-Ig genes, and over-expression of AID can lead to point mutations or translocations in non-Ig genes such as IgH/Myc translocation. Dicer and Drosha, which have a role in activation process of miRNA, also act in a double-strand DNA break (DSB) repair mechanism. In this study, whether the changes of AID, Dicer and Drosha expressions may be associated with both deletions and clinical outcomes in patients with CLL were investigated. AID expressions were increased in patients with CLL. However, cell lysate AID protein levels were only increased in patients with del17p or del11q who have poor prognosis. Decreased Dicer expressions were found in patients with deletion, whereas increased Drosha expressions were found in patients without deletion and with del13q. According to Rai and Binet staging systems, advanced-stage patients showed increased AID protein levels, decreased Dicer and Drosha expressions. Our findings may suggest that high AID expression and lower Dicer expression were observed in patients with CLL especially del17p and del11q and might associated with deletions such as del17p and del11q. AID, Dicer, and Drosha expressions might be used as an indicator of prognosis for CLL.

Activation-induced cytidine deaminase selectively catalyzed active DNA demethylation in pluripotency gene and improved cell reprogramming in bovine SCNT embryo.

DNA methylation in mammals is an epigenetic marker and necessary for normal embryogenesis. The global genomic demethylation of 5-methylcytosine occurs during the first cell cycle following fertilization. Activation-induced cytidine deaminase (AID), which is well-known for the function in antibody diversification, has been implicated to play a role in active demethylation, but its role in cell reprogramming and its crosstalk with other DNA demethylation mechanism need to be clarified. In this study, the dynamic epigenetic regulation of cell pluripotency and embryo development by AID in bovine preimplantation embryos was investigated. The analysis of an AID overexpressing transgenic cell line showed that AID overexpression did not change the global genomic methylation but did change the methylation status of the promoters of the OCT4, NANOG and SOX2 genes, thereby causing changes in their expression. The siRNA-mediated AID knockdown in early embryonic development indicated that AID interference did not affect oocyte maturation or the following embryo development after in vitro fertilization but influenced the DNA methylation status of OCT4 and NANOG. To clarify the role of AID in preimplantation embryos, SCNT embryos were obtained using AID-overexpressing cells as nuclear donors. Compared to the control group, the cleavage and blastocyst rates were both significantly improved in the AID-overexpression group. The expression of OCT4 and NANOG was increased in the SCNT embryos, whereas the methylation levels of their promoters were reduced. In conclusion, this study demonstrated that AID selectively catalyzes DNA demethylation of pluripotency genes to play a role in regulation their expression, improves bovine SCNT embryo development by increased expression levels.

Overexpression of Activation-Induced Deaminase in TCL1 Mice Leads to the Development of IGHV -Mutated and -Unmutated CLL Clones That Resemble Unique Subsets of Human CLL

Introduction. Somatic hypermutation (SHM) and class-switch recombination (CSR) are critical physiologic events in an effective normal B-cell immune response. Notably, both SHM and CSR are initiated by activation-induced cytidine deaminase (AID). In CLL, IGHV clonal mutations correlate strongly with better clinical outcomes. Eμ-T-cell leukemia-1 (TCL1) transgenic (Tg) mice are a valuable model of CLL. However because SHM and CSR occur rarely in the leukemic clones from these animals, TCL1 Tg mice mimic only IGHV -unmutated CLL and hence may not provide an understanding of the roles of SHM and CSR in disease evolution. To address these issues, we developed two new TCL1 strains by interbreeding mice over-expressing AID in all cells (Em-TCL1xActin-AID) or only in B lymphocytes (Em-TCL1xVκ-AID).Methods. B-cell clonal expansions were identified in spleen cells from 22 TCL1 and 33 TCL1xAID Tg (10 Em-TCL1xActin-AID plus 23 Em-TCL1xVκ-AID) mice at 10-20 months of age. This was done by amplifying cDNAs by PCR using consensus IGHV FR and IgM-, IgG-, and IgA-specific primers for IgH chains and Vκ FR and Cκ for IgL chain genes. DNA sequences of these amplicons were compared to murine germline IGHV s and IGKV s, and IGHV-D-JH and IGKV-JK rearrangements by IMGT V-Quest. Because there were no major differences in the parameters listed below for the two TCL1xAID Tg mouse strains, data were combined.Results.Clonal expansions: Monoclonal/oligoclonal expansions were detected in all TCL1 mice; these used only µ H and κ L chains. Similar expansions were detected in 31 of 33 TCL1xAID mice; each animal bore an IgMκ+ clone and 7 also exhibited an IgGκ+ clone.IGHV gene use in IgMκ+ clones: IGHV use did not differ significantly between IgMκ+ TCL1 and IgMκ+ TCL1xAID clones. Approximately 50% used VH1-55, VH11-2, or VH12-3, some of which encoded stereotyped anti-phosphatidylcholine antibodies.SHM: Among the IgM+ TCL1 clones, there was a mutation frequency of 0.05% for IGHV and 0.09% IGKV. In contrast, the IGHV and IGKV mutation frequencies were considerably higher in TCL1xAID mice. Specifically, for IGHV, the TCL1xAID animals displayed a frequency of 0.47% in IgM+ and 3.0% in IgG+ clones; for IGKV, the frequency was 0.9% for IgM+ and IgG+ combined. Consistent with these mutations being mediated by AID, mutations localized more frequently in AID hotspots than coldspots at a ratio of ≥7:1.However, SHM did not affect all clones equally. Although the mutation frequency in VH12-3 and VH11-2 clones was higher (0.38%, range: 0-1.9%) than the TCL1 level (0.05%), it was considerably less than that found in the TCL1xAID clones using other IGHV s (0.80%, range: 0-3.8%). In addition, no mutations were detected in VH1-55 clones. Consistent with a reduced AID mutational activity in clones expressing these specific genes, none of the genes were found in the isotype-switched, IgG-expressing clones.IGHV gene use and SHM in clones that underwent CSR: Notably, in only 2 of 9 instances was the same IGHV-D-J rearrangement found in IgMκ+ and IgGκ+ clones from the same mice; these IgM+ and IgG+ shared clones used VH5 genes. For the remaining 7, only the IgG+ version was detected; all but one of these used a VH1 gene. Also, within the IgG-only group, IGHV1-47 was used by 2 different clones and these were highly mutated (8.9%).Stereotyped IGHV-D-J and IGκ V-Jκ rearrangements: Among 43 IgM clones from TCL1xAID mice, we found 8 clones expressing VH11-2 and Vκ14-126 and 10 clones expressing VH12-3 and Vκ4-91; these IGHV-D-J and IGκV-Jκ rearrangements were very similar to canonical anti-phosphatidylcholine-producing clones.Summary and conclusions.Over-expression of AID in TCL1 mice leads to markedly increased SHM and CSR. However, SHM is not equivalent for all IGHV genes since despite AID over-expression certain IGHVs and IGKVs appeared less sensitive to major increases in the extent of SHM and the occurrence of CSR. This property resembles some human CLL IGHV s that rarely develop SHMs or undergo CSR despite the B-cell's ability to synthesize AID (e.g., many IGHV1-69+ clones). AID overexpression also led to IgG+ clones for which an IgM precursor was not found. This resembles those human stereotyped CLL clones that are only found as IgGs (e.g., stereotyped subsets 4 and 8). Finally, the two new TCL1xAID mouse strains described provide new models to study IGHV -mutated and IGHV -unmutated CLL and represent novel tools to evaluate the role of AID in leukemic progression. DisclosuresNo relevant conflicts of interest to declare.

Increased frequencies of memory and activated B cells and follicular helper T cells are positively associated with high levels of activation‑induced cytidine deaminase in patients with immunoglobulin A nephropathy.

The aim of the present study was to examine the frequencies of different subsets of B and follicular helper T (Tfh) lymphocytes in patients with immunoglobulin A nephropathy (IgAN), and investigate the potential underlying mechanism. A total of 27 patients with IgAN and 10 healthy controls (HC) were recruited for analysis of the frequencies of different subsets of B and Tfh cells. ELISA was used to analyze the concentration of serum interleukin (IL)‑21. The transcriptional levels of activation‑induced cytidine deaminase (AID) in the B cells were determined using reverse transcription‑quantitative polymerase chain reaction, while the translational levels of AID were analyzed using western blotting. The frequencies of circulating memory and activated B cells and Tfh cells were found to be significantly increased in the IgAN groups, compared with those of the HC group, although the number of plasma cells were not significantly different between the two IgAN groups. In addition, the serum levels of IL‑21 were found to be higher in the patients with IgAN, and correlated with 24‑h proteinuria. IL‑21 also enhanced the expression levels of AID in the B cells. The data of the present study revealed that the high levels of memory and activated B cells and Tfh cells were positively associated with the progression of IgAN, and that this may be mediated by the overexpression of AID, which is potentially regulated by IL‑21.

AID expression increased by TNF-α is associated with class switch recombination of Igα gene in cancers.

Recently, immunoglobulins (Igs) were unexpectedly found to be expressed in epithelial cancers. Immunoglobulin class switching or class switch recombination (CSR) is a natural biological process that alters a B cell's production of antibodies (immunoglobulins) from one class to another. However, the mechanism of CSR of Ig genes in cancer is still unknown. Here, we confirmed by detecting the hallmark of CSR that the Igα gene in cancer underwent CSR. Then we focused on activation-induced cytidine deaminase (AID), a crucial factor for initiating CSR. Further studies using tumor necrosis factor (TNF)-α stimulation and specific inhibitor of NF-κB revealed that TNF-α could increase AID expression through NF-κB signaling. Finally, we demonstrated that AID could co-localize with protein kinase A and bind to the switching (Sα) region of the Igα gene. Overexpression of AID obviously enhanced Igα heavy chain expression and its binding ability to the Sα region. These findings indicated that TNF-α-induced AID expression is involved with CSR in cancer.

Overexpression of Activation-Induced Cytidine Deaminase in MTX- and Age-Related Epstein-Barr Virus-Associated B-Cell Lymphoproliferative Disorders of the Head and Neck.

Recent research has shown that activation-induced cytidine deaminase (AID) triggers somatic hypermutation and recombination, in turn contributing to lymphomagenesis. Such aberrant AID expression is seen in B-cell leukemia/lymphomas, including Burkitt lymphoma which is associated with c-myc translocation. Moreover, Epstein-Barr virus (EBV) latent membrane protein-1 (LMP-1) increases genomic instability through early growth transcription response-1 (Egr-1) mediated upregulation of AID in B-cell lymphoma. However, few clinicopathological studies have focused on AID expression in lymphoproliferative disorders (LPDs). Therefore, we conducted an immunohistochemical study to investigate the relationship between AID and LMP-1 expression in LPDs (MTX-/Age-related EBV-associated), including diffuse large B-cell lymphomas (DLBCLs). More intense AID expression was detected in LPDs (89.5%) than in DLBCLs (20.0%), and the expression of LMP-1 and EBER was more intense in LPDs (68.4% and 94.7%) than in DLBCLs (10.0% and 20.0%). Furthermore, stronger Egr-1 expression was found in MTX/Age-EBV-LPDs (83.3%) than in DLBCLs (30.0%). AID expression was significantly constitutively overexpressed in LPDs as compared with DLBCLs. These results suggest that increased AID expression in LPDs may be one of the processes involved in lymphomagenesis, thereby further increasing the survival of genetically destabilized B-cells. AID expression may be a useful indicator for differentiation between LPDs and DLBCLs.

GANP regulates the choice of DNA repair pathway by DNA-PKcs interaction in AID-dependent IgV region diversification.

RNA export factor germinal center-associated nuclear protein (GANP) interacts with activation-induced cytidine deaminase (AID) and shepherds it from the cytoplasm to the nucleus and toward the IgV region loci in B cells. In this study, we demonstrate a role for GANP in the repair of AID-initiated DNA damage in chicken DT40 B cells to generate IgV region diversity by gene conversion and somatic hypermutation. GANP plays a positive role in IgV region diversification of DT40 B cells in a nonhomologous end joining-proficient state. DNA-PKcs physically interacts with GANP, and this interaction is dissociated by dsDNA breaks induced by a topoisomerase II inhibitor, etoposide, or AID overexpression. GANP affects the choice of DNA repair mechanism in B cells toward homologous recombination rather than nonhomologous end joining repair. Thus, GANP presumably plays a critical role in protection of the rearranged IgV loci by favoring homologous recombination of the DNA breaks under accelerated AID recruitment.

Identification of chromosomal translocation hotspots via scan statistics.

The detection of genomic regions unusually rich in a given pattern is an important undertaking in the analysis of next-generation sequencing data. Recent studies of chromosomal translocations in activated B lymphocytes have identified regions that are frequently translocated to c-myc oncogene. A quantitative method for the identification of translocation hotspots was crucial to this study. Here we improve this analysis by using a simple probabilistic model and the framework provided by scan statistics to define the number and location of translocation breakpoint hotspots. A key feature of our method is that it provides a global chromosome-wide nominal control level to clustering, as opposed to previous methods based on local criteria. While being motivated by a specific application, the detection of unusual clusters is a widespread problem in bioinformatics. We expect our method to be useful in the analysis of data from other experimental approaches such as of ChIP-seq and 4C-seq. The analysis of translocations from B lymphocytes with the method described here reveals the presence of longer hotspots when compared with those defined previously. Further, we show that the hotspot size changes substantially in the absence of DNA repair protein 53BP1. When 53BP1 deficiency is combined with overexpression of activation-induced cytidine deaminase, the hotspot length increases even further. These changes are not detected by previous methods that use local significance criteria for clustering. Our method is also able to identify several exclusive translocation hotspots located in genes of known tumor supressors. The detection of translocation hotspots is done with hot_scan, a program implemented in R and Perl. Source code and documentation are freely available for download at https://github.com/itojal/hot_scan.

BCR-ABL1–positive microvesicles transform normal hematopoietic transplants through genomic instability: implications for donor cell leukemia

Malignant transformation of normal hematopoietic transplants induced by residual leukemia cells is considered as a pivotal mechanism of donor cell leukemia (DCL). The effects of leukemia cell-derived microvesicles (MVs) in this transformation were examined. We found that MVs derived from K562 leukemia cells contained the breakpoint cluster region-Abelson leukemia gene human homolog 1 (BCR-ABL1) mRNA. Following incubation with BCR-ABL1-positive MVs, mononuclear cells derived from normal transplants exhibited a leukemia-like malignant phenotype both in vitro and in vivo. Horizontal transfer of BCR-ABL1 mRNA from MVs into the recipient cells was critical to the transformation. Relative genomic instability was observed and considered the main mechanism in the recipient cells. MVs contributed to genomic instability by two distinct pathways: via consequent overexpression of activation-induced cytidine deaminase and reactive oxygen species, which mediated DNA breakage and recombination; and via upregulation of methyltransferases and global DNA hypermethylation. We demonstrated that BCR-ABL1-positive MVs could initiate malignant transformation of normal hematopoietic transplants through genomic instability, which might serve as a convenient and operable model for investigating leukemogenesis, especially for DCL. Furthermore, MVs themselves could act as an early warning indicator and a novel tool to detect and prevent the occurrence of DCL.

Interleukin-1 and Tumor Necrosis Factor-α Trigger Restriction of Hepatitis B Virus Infection via a Cytidine Deaminase Activation-induced Cytidine Deaminase (AID)

Virus infection is restricted by intracellular immune responses in host cells, and this is typically modulated by stimulation of cytokines. The cytokines and host factors that determine the host cell restriction against hepatitis B virus (HBV) infection are not well understood. We screened 36 cytokines and chemokines to determine which were able to reduce the susceptibility of HepaRG cells to HBV infection. Here, we found that pretreatment with IL-1β and TNFα remarkably reduced the host cell susceptibility to HBV infection. This effect was mediated by activation of the NF-κB signaling pathway. A cytidine deaminase, activation-induced cytidine deaminase (AID), was up-regulated by both IL-1β and TNFα in a variety of hepatocyte cell lines and primary human hepatocytes. Another deaminase APOBEC3G was not induced by these proinflammatory cytokines. Knockdown of AID expression impaired the anti-HBV effect of IL-1β, and overexpression of AID antagonized HBV infection, suggesting that AID was one of the responsible factors for the anti-HBV activity of IL-1/TNFα. Although AID induced hypermutation of HBV DNA, this activity was dispensable for the anti-HBV activity. The antiviral effect of IL-1/TNFα was also observed on different HBV genotypes but not on hepatitis C virus. These results demonstrate that proinflammatory cytokines IL-1/TNFα trigger a novel antiviral mechanism involving AID to regulate host cell permissiveness to HBV infection.

Are 8-oxoguanine (8-oxoGua) and 5-hydroxymethyluracil (5-hmUra) oxidatively damaged DNA bases or transcription (epigenetic) marks?

The oxidatively modified DNA base 8-oxo-7,8-dihydroguanine (8-oxoGua) is nontoxic and weakly mutagenic. Here we report on new data suggesting a potential for 8-oxoGua to affect the expression of several genes via epigenetic changes resulting in chromatin relaxation. Using pig thymus extract, we analyzed the distribution of 8-oxoGua among different nuclei fractions representative of transcriptionally active and silenced regions. The levels of 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) found in transcriptionally active euchromatin (4.37/106 nucleotides) and in the matrix fraction (4.16/106 nucleotides) were about 5 times higher than in transcriptionally silenced heterochromatin (0.91/106 nucleotides). Other experimental data are presented which suggest that 8-oxoGua present in specific DNA sequences may be widely used for transcription regulation.Like 8-oxoGua, 5-hydroxymethyluracil (5-hmUra) is another oxidatively modified DNA base (the derivative is formed by thymine oxidation). Recent experimental evidence supports the notion that 5-hmUra plays an important role in active DNA demethylation. This involves overexpression of activation-induced cytidine deaminase (AID) and ten-eleven translocation 1 (TET1) protein (the key proteins involved in active demethylation), which leads to global accumulation of 5-hmUra. Our preliminary data demonstrate a significant increase of the 5-hmUra levels in pig brain extract when compared with liver extract. The lack of 5-hmUra in Escherichia coli DNA also speaks for a role of this modification in the active demethylation process. It is concluded that 8-oxodG and 5-hmUra in DNA may be considered as epigenetic marks.

Concerted action of activation-induced cytidine deaminase and uracil-DNA glycosylase reduces covalently closed circular DNA of duck hepatitis B virus

Covalently closed circular DNA (cccDNA) forms a template for the replication of hepatitis B virus (HBV) and duck HBV (DHBV). Recent studies suggest that activation-induced cytidine deaminase (AID) functions in innate immunity, although its molecular mechanism of action remains unclear, particularly regarding HBV restriction. Here we demonstrated that overexpression of chicken AID caused hypermutation and reduction of DHBV cccDNA levels. Inhibition of uracil-DNA glycosylase (UNG) by UNG inhibitor protein (UGI) abolished AID-induced cccDNA reduction, suggesting that the AID/UNG pathway triggers the degradation of cccDNA via cytosine deamination and uracil excision.

Zebrafish AID is capable of deaminating methylated deoxycytidines

Activation-induced cytidine deaminase (AID) deaminates deoxycytidine (dC) to deoxyuracil (dU) at immunoglobulin loci in B lymphocytes to mediate secondary antibody diversification. Recently, AID has been proposed to also mediate epigenetic reprogramming by demethylating methylated cytidines (mC) possibly through deamination. AID overexpression in zebrafish embryos was shown to promote genome demethylation through G:T lesions, implicating a deamination-dependent mechanism. We and others have previously shown that mC is a poor substrate for human AID. Here, we examined the ability of bony fish AID to deaminate mC. We report that zebrafish AID was unique among all orthologs in that it efficiently deaminates mC. Analysis of domain-swapped and mutant AID revealed that mC specificity is independent of the overall high-catalytic efficiency of zebrafish AID. Structural modeling with or without bound DNA suggests that efficient deamination of mC by zebrafish AID is likely not due to a larger catalytic pocket allowing for better fit of mC, but rather because of subtle differences in the flexibility of its structure.

53BP1 Alters the Landscape of DNA Rearrangements and Suppresses AID-Induced B Cell Lymphoma

Deficiencies in factors that regulate the DNA damage response enhance the incidence of malignancy by destabilizing the genome. However, the precise influence of the DNA damage response on regulation of cancer-associated rearrangements is not well defined. Here we examine the genome-wide impact of tumor protein P53-binding protein 1 (53BP1) deficiency in lymphoma and translocation. While both activation-induced cytidine deaminase (AID) and 53BP1 have been associated with cancer in humans, neither AID overexpression nor loss of 53BP1 is sufficient to produce malignancy. However, the combination of 53BP1 deficiency and AID deregulation results in B cell lymphoma. Deep sequencing of the genome of 53BP1(-/-) cancer cells and translocation capture sequencing (TC-Seq) of primary 53BP1(-/-) B cells revealed that their chromosomal rearrangements differ from those found in wild-type cells in that they show increased DNA end resection. Moreover, loss of 53BP1 alters the translocatome by increasing rearrangements to intergenic regions.