FAM72A antagonizes UNG2 to promote mutagenic repair during antibody maturation.

Activation-induced cytidine deaminase (AID) catalyses the deamination of deoxycytidines to deoxyuracils within immunoglobulin genes to induce somatic hypermutation and class-switch recombination1,2. AID-generated deoxyuracils are recognized and processed by subverted base-excision and mismatch repair pathways that ensure a mutagenic outcome in B cells3-6. However, why these DNA repair pathways do not accurately repair AID-induced lesions remains unknown. Here, using a genome-wide CRISPR screen, we show that FAM72A is a major determinant for the error-prone processing of deoxyuracils. Fam72a-deficient CH12F3-2 B cells and primary B cells from Fam72a-/- mice exhibit reduced class-switch recombination and somatic hypermutation frequencies at immunoglobulin and Bcl6 genes, and reduced genome-wide deoxyuracils. The somatic hypermutation spectrum in B cells from Fam72a-/- mice is opposite to that observed in mice deficient in uracil DNA glycosylase 2 (UNG2)7, which suggests that UNG2 is hyperactive in FAM72A-deficient cells. Indeed, FAM72A binds to UNG2, resulting in reduced levels of UNG2 protein in the G1 phase of the cell cycle, coinciding with peak AID activity. FAM72A therefore causes U·G mispairs to persist into S phase, leading to error-prone processing by mismatch repair. By disabling the DNA repair pathways that normally efficiently remove deoxyuracils from DNA, FAM72A enables AID to exert its full effects on antibody maturation. This work has implications in cancer, as the overexpression of FAM72A that is observed in many cancers8 could promote mutagenesis.

APOBEC2 safeguards skeletal muscle cell fate through binding chromatin and regulating transcription of non-muscle genes during myoblast differentiation.

The apolipoprotein B messenger RNA editing enzyme, catalytic polypeptide (APOBEC) family is composed of nucleic acid editors with roles ranging from antibody diversification to RNA editing. APOBEC2, a member of this family with an evolutionarily conserved nucleic acid-binding cytidine deaminase domain, has neither an established substrate nor function. Using a cellular model of muscle differentiation where APOBEC2 is inducibly expressed, we confirmed that APOBEC2 does not have the attributed molecular functions of the APOBEC family, such as RNA editing, DNA demethylation, and DNA mutation. Instead, we found that during muscle differentiation APOBEC2 occupied a specific motif within promoter regions; its removal from those regions resulted in transcriptional changes. Mechanistically, these changes reflect the direct interaction of APOBEC2 with histone deacetylase (HDAC) transcriptional corepressor complexes. We also found that APOBEC2 could bind DNA directly, in a sequence-specific fashion, suggesting that it functions as a recruiter of HDAC to specific genes whose promoters it occupies. These genes are normally suppressed during muscle cell differentiation, and their suppression may contribute to the safeguarding of muscle cell fate. Altogether, our results reveal a unique role for APOBEC2 within the APOBEC family.

Conserved role of hnRNPL in alternative splicing of epigenetic modifiers enables B cell activation.

The multifunctional RNA-binding protein hnRNPL is implicated in antibody class switching but its broader function in B cells is unknown. Here, we show that hnRNPL is essential for B cell activation, germinal center formation, and antibody responses. Upon activation, hnRNPL-deficient B cells show proliferation defects and increased apoptosis. Comparative analysis of RNA-seq data from activated B cells and another eight hnRNPL-depleted cell types reveals common effects on MYC and E2F transcriptional programs required for proliferation. Notably, while individual gene expression changes are cell type specific, several alternative splicing events affecting histone modifiers like KDM6A and SIRT1, are conserved across cell types. Moreover, hnRNPL-deficient B cells show global changes in H3K27me3 and H3K9ac. Epigenetic dysregulation after hnRNPL loss could underlie differential gene expression and upregulation of lncRNAs, and explain common and cell type-specific phenotypes, such as dysfunctional mitochondria and ROS overproduction in mouse B cells. Thus, hnRNPL is essential for the resting-to-activated B cell transition by regulating transcriptional programs and metabolism, at least in part through the alternative splicing of several histone modifiers.

RPA guides UNG to uracil in ssDNA to facilitate antibody class switching and repair of mutagenic uracil at the replication fork.

Activation-induced cytidine deaminase (AID) interacts with replication protein A (RPA), the major ssDNA-binding protein, to promote deamination of cytosine to uracil in transcribed immunoglobulin (Ig) genes. Uracil-DNA glycosylase (UNG) acts in concert with AID during Ig diversification. In addition, UNG preserves genome integrity by base-excision repair (BER) in the overall genome. How UNG is regulated to support both mutagenic processing and error-free repair remains unknown. UNG is expressed as two isoforms, UNG1 and UNG2, which both contain an RPA-binding helix that facilitates uracil excision from RPA-coated ssDNA. However, the impact of this interaction in antibody diversification and genome maintenance has not been investigated. Here, we generated B-cell clones with targeted mutations in the UNG RPA-binding motif, and analysed class switch recombination (CSR), mutation frequency (5' Ig Sµ), and genomic uracil in clones representing seven Ung genotypes. We show that the UNG:RPA interaction plays a crucial role in both CSR and repair of AID-induced uracil at the Ig loci. By contrast, the interaction had no significant impact on total genomic uracil levels. Thus, RPA coordinates UNG during CSR and pre-replicative repair of mutagenic uracil in ssDNA but is not essential in post-replicative and canonical BER of uracil in dsDNA.

AID overexpression leads to aggressive murine CLL and nonimmunoglobulin mutations that mirror human neoplasms.

Most cancers become more dangerous by the outgrowth of malignant subclones with additional DNA mutations that favor proliferation or survival. Using chronic lymphocytic leukemia (CLL), a disease that exemplifies this process and is a model for neoplasms in general, we created transgenic mice overexpressing the enzyme activation-induced deaminase (AID), which has a normal function of inducing DNA mutations in B lymphocytes. AID not only allows normal B lymphocytes to develop more effective immunoglobulin-mediated immunity, but is also able to mutate nonimmunoglobulin genes, predisposing to cancer. In CLL, AID expression correlates with poor prognosis, suggesting a role for this enzyme in disease progression. Nevertheless, direct experimental evidence identifying the specific genes that are mutated by AID and indicating that those genes are associated with disease progression is not available. To address this point, we overexpressed Aicda in a murine model of CLL (Eµ-TCL1). Analyses of TCL1/AID mice demonstrate a role for AID in disease kinetics, CLL cell proliferation, and the development of cancer-related target mutations with canonical AID signatures in nonimmunoglobulin genes. Notably, our mouse models can accumulate mutations in the same genes that are mutated in human cancers. Moreover, some of these mutations occur at homologous positions, leading to identical or chemically similar amino acid substitutions as in human CLL and lymphoma. Together, these findings support a direct link between aberrant AID activity and CLL driver mutations that are then selected for their oncogenic effects, whereby AID promotes aggressiveness in CLL and other B-cell neoplasms.

AID in Antibody Diversification: There and Back Again.

Activation-Induced cytidine Deaminase (AID) initiates affinity maturation and isotype switching by deaminating deoxycytidines within immunoglobulin genes, leading to somatic hypermutation (SHM) and class switch recombination (CSR). AID thus potentiates the humoral response to clear pathogens. Marking the 20th anniversary of the discovery of AID, we review the current understanding of AID function. We discuss AID biochemistry and how error-free forms of DNA repair are co-opted to prioritize mutagenesis over accuracy during antibody diversification. We discuss the regulation of DNA double-strand break (DSB) repair pathways during CSR. We describe genomic targeting of AID as a multilayered process involving chromatin architecture, cis- and trans-acting factors, and determining mutagenesis - distinct from AID occupancy at loci that are spared from mutation.

Characterization of ADAT2/3 molecules in Trypanosoma cruzi and regulation of mucin gene expression by tRNA editing.

Adenosine-to-inosine conversion at position 34 (A34-to-I) of certain tRNAs is essential for expanding their decoding capacity. This reaction is catalyzed by the adenosine deaminase acting on tRNA (ADAT) complex, which in Eukarya is formed by two subunits: ADAT2 and ADAT3. We herein identified and thoroughly characterized the ADAT molecules from the protozoan pathogen Trypanosoma cruzi, the causative agent of Chagas Disease. TcADAT2 and TcADAT3 spontaneously form a catalytically active complex, as shown by expression in engineered bacteria and/or by the increased ex vivo tRNA A-to-I deamination activity of T. cruzi epimastigotes overexpressing TcADAT subunits. Importantly, enhanced TcADAT2/3 activity in transgenic parasites caused a shift in their in vivo tRNAThrAGU signature, which correlated with significant changes in the expression of the Thr-rich TcSMUG proteins. To our knowledge, this is the first evidence indicating that T. cruzi tRNA editing can be modulated in vivo, in turn post-transcriptionally changing the expression of specific genes. Our findings suggest tRNA editing/availability as a forcible step in controlling gene expression and driving codon adaptation in T. cruzi. Moreover, we unveil certain differences between parasite and mammalian host tRNA editing and processing, such as cytosine-to-uridine conversion at position 32 of tRNAThrAGU in T. cruzi, that may be exploited for the identification of novel druggable targets of intervention.

Ibrutinib therapy downregulates AID enzyme and proliferative fractions in chronic lymphocytic leukemia.

Activation-induced cytidine deaminase (AID) initiates somatic hypermutation and class switch recombination of the immunoglobulin genes. As a trade-off for its physiological function, AID also contributes to tumor development through its mutagenic activity. In chronic lymphocytic leukemia (CLL), AID is overexpressed in the proliferative fractions (PFs) of the malignant B lymphocytes, and its anomalous expression has been associated with a clinical poor outcome. Recent preclinical data suggested that ibrutinib and idelalisib, 2 clinically approved kinase inhibitors, increase AID expression and genomic instability in normal and neoplastic B cells. These results raise concerns about a potential mutagenic risk in patients receiving long-term therapy. To corroborate these findings in the clinical setting, we analyzed AID expression and PFs in a CLL cohort before and during ibrutinib treatment. We found that ibrutinib decreases the CLL PFs and, interestingly, also reduces AID expression, which correlates with dampened AKT and Janus Kinase 1 signaling. Moreover, although ibrutinib increases AID expression in a CLL cell line, it is unable to do so in primary CLL samples. Our results uncover a differential response to ibrutinib between cell lines and the CLL clone and imply that ibrutinib could differ from idelalisib in their potential to induce AID in treated patients. Possible reasons for the discrepancy between preclinical and clinical findings, and their effect on treatment safety, are discussed.

Roles for APRIN (PDS5B) in homologous recombination and in ovarian cancer prediction.

APRIN (PDS5 cohesin associated factor B) interacts with both the cohesin complex and the BRCA2 tumor suppressor. How APRIN influences cohesion and DNA repair processes is not well understood. Here, we show that APRIN is recruited to DNA damage sites. We find that APRIN interacts directly with RAD51, PALB2 and BRCA2. APRIN stimulates RAD51-mediated DNA strand invasion. APRIN also binds DNA with an affinity for D-loop structures and single-strand (ss) DNA. APRIN is a new homologous recombination (HR) mediator as it counteracts the RPA inhibitory effect on RAD51 loading to ssDNA. We show that APRIN strongly improves the annealing of complementary-strand DNA and that it can stimulate this process in synergy with BRCA2. Unlike cohesin constituents, its depletion has no impact on class switch recombination, supporting a specific role for this protein in HR. Furthermore, we show that low APRIN expression levels correlate with a better survival in ovarian cancer patients and that APRIN depletion sensitizes cells to the PARP inhibitor Olaparib in xenografted zebrafish. Our findings establish APRIN as an important and specific actor of HR, with cohesin-independent functions.

Activation induced deaminase C-terminal domain links DNA breaks to end protection and repair during class switch recombination.

Activation-induced deaminase (AID) triggers antibody class switch recombination (CSR) in B cells by initiating DNA double strand breaks that are repaired by nonhomologous end-joining pathways. A role for AID at the repair step is unclear. We show that specific inactivation of the C-terminal AID domain encoded by exon 5 (E5) allows very efficient deamination of the AID target regions but greatly impacts the efficiency and quality of subsequent DNA repair. Specifically eliminating E5 not only precludes CSR but also, causes an atypical, enzymatic activity-dependent dominant-negative effect on CSR. Moreover, the E5 domain is required for the formation of AID-dependent Igh-cMyc chromosomal translocations. DNA breaks at the Igh switch regions induced by AID lacking E5 display defective end joining, failing to recruit DNA damage response factors and undergoing extensive end resection. These defects lead to nonproductive resolutions, such as rearrangements and homologous recombination that can antagonize CSR. Our results can explain the autosomal dominant inheritance of AID variants with truncated E5 in patients with hyper-IgM syndrome 2 and establish that AID, through the E5 domain, provides a link between DNA damage and repair during CSR.

Tumor suppressor and deubiquitinase BAP1 promotes DNA double-strand break repair.

The cellular response to highly genotoxic DNA double-strand breaks (DSBs) involves the exquisite coordination of multiple signaling and repair factors. Here, we conducted a functional RNAi screen and identified BAP1 as a deubiquitinase required for efficient assembly of the homologous recombination (HR) factors BRCA1 and RAD51 at ionizing radiation (IR) -induced foci. BAP1 is a chromatin-associated protein frequently inactivated in cancers of various tissues. To further investigate the role of BAP1 in DSB repair, we used a gene targeting approach to knockout (KO) this deubiquitinase in chicken DT40 cells. We show that BAP1-deficient cells are (i) sensitive to IR and other agents that induce DSBs, (ii) defective in HR-mediated immunoglobulin gene conversion, and (iii) exhibit an increased frequency of chromosomal breaks after IR treatment. We also show that BAP1 is recruited to chromatin in the proximity of a single site-specific I-SceI-induced DSB. Finally, we identified six IR-induced phosphorylation sites in BAP1 and showed that mutation of these residues inhibits BAP1 recruitment to DSB sites. We also found that both BAP1 catalytic activity and its phosphorylation are critical for promoting DNA repair and cellular recovery from DNA damage. Our data reveal an important role for BAP1 in DSB repair by HR, thereby providing a possible molecular basis for its tumor suppressor function.

Optimal functional levels of activation-induced deaminase specifically require the Hsp40 DnaJa1.

The enzyme activation-induced deaminase (AID) deaminates deoxycytidine at the immunoglobulin genes, thereby initiating antibody affinity maturation and isotype class switching during immune responses. In contrast, off-target DNA damage caused by AID is oncogenic. Central to balancing immunity and cancer is AID regulation, including the mechanisms determining AID protein levels. We describe a specific functional interaction between AID and the Hsp40 DnaJa1, which provides insight into the function of both proteins. Although both major cytoplasmic type I Hsp40s, DnaJa1 and DnaJa2, are induced upon B-cell activation and interact with AID in vitro, only DnaJa1 overexpression increases AID levels and biological activity in cell lines. Conversely, DnaJa1, but not DnaJa2, depletion reduces AID levels, stability and isotype switching. In vivo, DnaJa1-deficient mice display compromised response to immunization, AID protein and isotype switching levels being reduced by half. Moreover, DnaJa1 farnesylation is required to maintain, and farnesyltransferase inhibition reduces, AID protein levels in B cells. Thus, DnaJa1 is a limiting factor that plays a non-redundant role in the functional stabilization of AID.

HSP90 inhibitors decrease AID levels and activity in mice and in human cells.

Activation induced deaminase (AID) initiates somatic hypermutation and class switch recombination of the Ig genes in antigen-activated B cells, underpinning antibody affinity maturation and isotype switching. AID can also be pathogenic by contributing to autoimmune diseases and oncogenic mutations. Moreover, AID can exert noncanonical functions when aberrantly expressed in epithelial cells. The lack of specific inhibitors prevents therapeutic applications to modulate AID functions. Here, we have exploited our previous finding that the HSP90 molecular chaperoning pathway stabilizes AID in B cells, to test whether HSP90 inhibitors could target AID in vivo. We demonstrate that chronic administration of HSP90 inhibitors decreases AID protein levels and isotype switching in immunized mice. HSP90 inhibitors also reduce disease severity in a mouse model of acute B-cell lymphoblastic leukemia in which AID accelerates disease progression. We further show that human AID protein levels are sensitive to HSP90 inhibition in normal and leukemic B cells, and that HSP90 inhibition prevents AID-dependent epithelial to mesenchymal transition in a human breast cancer cell line in vitro. Thus, we provide proof-of-concept that HSP90 inhibitors indirectly target AID in vivo and that endogenous human AID is widely sensitive to them, which could have therapeutic applications.

Activation induced deaminase: how much and where?

Activation induced deaminase (AID) plays a central role in adaptive immunity by initiating the processes of somatic hypermutation (SHM) and class switch recombination (CSR). On the other hand, AID also predisposes to lymphoma and plays a role in some autoimmune diseases, for which reasons AID expression and activity are regulated at various levels. Post-translational mechanisms regulating the amount and subcellular localization of AID are prominent in balancing AID physiological and pathological functions in B cells. Mechanisms regulating AID protein levels include stabilizing chaperones in the cytoplasm and proteins efficiently targeting AID to the proteasome within the nucleus. Nuclear export and cytoplasmic retention contribute to limit the amount of AID accessing the genome. Additionally, a number of factors have been implicated in AID active nuclear import. We review these intertwined mechanisms proposing two scenarios in which they could interact as a network or as a cycle for defining the optimal amount of AID protein. We also comparatively review the expression levels of AID necessary for its function during the immune response, present in different cancers as well as in those tissues in which AID has been implicated in epigenetic remodeling of the genome by demethylating DNA.

Activation-induced cytidine deaminase (AID) is necessary for the epithelial-mesenchymal transition in mammary epithelial cells.

Activation-induced cytidine deaminase (AID), which functions in antibody diversification, is also expressed in a variety of germ and somatic cells. Evidence that AID promotes DNA demethylation in epigenetic reprogramming phenomena, and that it is induced by inflammatory signals, led us to investigate its role in the epithelial-mesenchymal transition (EMT), a critical process in normal morphogenesis and tumor metastasis. We find that expression of AID is induced by inflammatory signals that induce the EMT in nontransformed mammary epithelial cells and in ZR75.1 breast cancer cells. shRNA-mediated knockdown of AID blocks induction of the EMT and prevents cells from acquiring invasive properties. Knockdown of AID suppresses expression of several key EMT transcriptional regulators and is associated with increased methylation of CpG islands proximal to the promoters of these genes; furthermore, the DNA demethylating agent 5 aza-2'deoxycytidine (5-Aza-dC) antagonizes the effects of AID knockdown on the expression of EMT factors. We conclude that AID is necessary for the EMT in this breast cancer cell model and in nontransformed mammary epithelial cells. Our results suggest that AID may act near the apex of a hierarchy of regulatory steps that drive the EMT, and are consistent with this effect being mediated by cytosine demethylation. This evidence links our findings to other reports of a role for AID in epigenetic reprogramming and control of gene expression.

Somatic hypermutation at A.T pairs: polymerase error versus dUTP incorporation.

Somatic hypermutation of immunoglobulin genes occurs at both C.G pairs and A.T pairs. Mutations at C.G pairs are created by activation-induced deaminase (AID)-catalysed deamination of C residues to U residues. Mutations at A.T pairs are probably produced during patch repair of the AID-generated U.G lesion, but they occur through an unknown mechanism. Here, we compare the popular suggestion of nucleotide mispairing through polymerase error with an alternative possibility, mutation through incorporation of dUTP (or another non-canonical nucleotide).

Autoimmunity and antibody affinity maturation are modulated by genetic variants on mouse chromosome 12.

Autoimmune diseases result from a break in immune tolerance leading to an attack on self-antigens. Autoantibody levels serve as a predictive tool for the early diagnosis of many autoimmune diseases, including type 1 diabetes. We find that a genetic locus on mouse chromosome 12 influences the affinity maturation of antibodies as well as autoantibody production. Thus, we generated a NOD.H2(k) congenic strain bearing B10 alleles at the locus comprised within the D12Mit184 and D12Mit12 markers, which we named NOD.H2(k)-Chr12. We determined the biological relevance of the Chr12 locus on the autoimmune process using an antigen-specific TCR transgenic autoimmune mouse model. Specifically, the 3A9 TCR transgene, which recognizes a peptide from hen egg lysozyme (HEL) in the context of I-A(k), and the HEL transgene, which is expressed under the rat-insulin promoter (iHEL), were bred into the NOD.H2(k)-Chr12 congenic strain. In the resulting 3A9 TCR:iHEL NOD.H2(k)-Chr12 mice, we observed a significant decrease in diabetes incidence as well as a decrease in both the quantity and affinity of HEL-specific IgG autoantibodies relative to 3A9 TCR:iHEL NOD.H2(k) mice. Notably, the decrease in autoantibodies due to the Chr12 locus was not restricted to the TCR transgenic model, as it was also observed in the non-transgenic NOD.H2(k) setting. Of importance, antibody affinity maturation upon immunization and re-challenge was also impeded in NOD.H2(k)-Chr12 congenic mice relative to NOD.H2(k) mice. Together, these results demonstrate that a genetic variant(s) present within the Chr12 locus plays a global role in modulating antibody affinity maturation.

Alternative end-joining and classical nonhomologous end-joining pathways repair different types of double-strand breaks during class-switch recombination.

Classical nonhomologous end-joining (C-NHEJ) and alternative end-joining (A-EJ) are the main DNA double-strand break (DSB) repair pathways when a sister chromatid is not available. However, it is not clear how one pathway is chosen over the other to process a given DSB. To address this question, we studied in mouse splenic B cells and CH12F3 cells how C-NHEJ and A-EJ repair DSBs initiated by the activation-induced deaminase during IgH (Igh) class-switch recombination (CSR). We show in this study that lowering the deamination density at the Igh locus increases DSB resolution by microhomology-mediated repair while decreasing C-NHEJ activity. This process occurs without affecting 53BP1 and γH2AX levels during CSR. Mechanistically, lowering deamination density increases exonuclease I recruitment and single-stranded DNA at the Igh locus and promotes C-terminal binding protein interacting protein and MSH2-dependent DSB repair during CSR. Indeed, reducing activation-induced deaminase levels increases CSR efficiency in C-NHEJ-defective cells, suggesting enhanced use of an A-EJ pathway. Our results establish a mechanism by which C-NHEJ and this C-terminal binding protein interacting protein/MSH2-dependent pathway that relies on microhomology can act concurrently but independently to repair different types of DSBs and reveal that the density of DNA lesions influences the choice of DSB repair pathway during CSR.