Activation-induced Deaminase Activity Research Articles

Modular cytosine base editing promotes epigenomic and genomic modifications.

Prokaryotic and eukaryotic adaptive immunity differ considerably. Yet, their fundamental mechanisms of gene editing via Cas9 and activation-induced deaminase (AID), respectively, can be conveniently complimentary. Cas9 is an RNA targeted dual nuclease expressed in several bacterial species. AID is a cytosine deaminase expressed in germinal centre B cells to mediate genomic antibody diversification. AID can also mediate epigenomic reprogramming via active DNA demethylation. It is known that sequence motifs, nucleic acid structures, and associated co-factors affect AID activity. But despite repeated attempts, deciphering AID's intrinsic catalytic activities and harnessing its targeted recruitment to DNA is still intractable. Even recent cytosine base editors are unable to fully recapitulate AID's genomic and epigenomic editing properties. Here, we describe the first instance of a modular AID-based editor that recapitulates the full spectrum of genomic and epigenomic editing activity. Our 'Swiss army knife' toolbox will help better understand AID biology per se as well as improve targeted genomic and epigenomic editing.

Deep learning model of somatic hypermutation reveals importance of sequence context beyond hotspot targeting

SummaryB cells undergo somatic hypermutation (SHM) of the Immunoglobulin (Ig) variable region to generate high-affinity antibodies. SHM relies on the activity of activation-induced deaminase (AID), which mutates C>U preferentially targeting WRC (W=A/T, R=A/G) hotspots. Downstream mutations at WA Polymerase η hotspots contribute further mutations. Computational models of SHM can describe the probability of mutations essential for vaccine responses. Previous studies using short subsequences (k-mers) failed to explain divergent mutability for the same k-mer. We developed the DeepSHM (Deep learning on SHM) model using k-mers of size 5–21, improving accuracy over previous models. Interpretation of DeepSHM identified an extended WWRCT motif with particularly high mutability. Increased mutability was further associated with lower surrounding G content. Our model also discovered a conserved AGYCTGGGGG (Y=C/T) motif within FW1 of IGHV3 family genes with unusually high T>G substitution rates. Thus, a wider sequence context increases predictive power and identifies features that drive mutational targeting.

Small Molecule Inhibitors of Activation-Induced Deaminase Decrease Class Switch Recombination in B Cells.

Activation-induced deaminase (AID) not only mutates DNA within the immunoglobulin loci to generate antibody diversity, but it also promotes development of B cell lymphomas. To tame this mutagen, we performed a quantitative high-throughput screen of over 90 000 compounds to see if AID activity could be mitigated. The enzymatic activity was assessed in biochemical assays to detect cytosine deamination and in cellular assays to measure class switch recombination. Three compounds showed promise via inhibition of switching in a transformed B cell line and in murine splenic B cells. These compounds have similar chemical structures, which suggests a shared mechanism of action. Importantly, the inhibitors blocked AID, but not a related cytosine DNA deaminase, APOBEC3B. We further determined that AID was continually expressed for several days after B cell activation to induce switching. This first report of small molecules that inhibit AID can be used to gain regulatory control over base editors.

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.

Interplay between UNG and AID governs intratumoral heterogeneity in mature B cell lymphoma.

Most B cell lymphomas originate from B cells that have germinal center (GC) experience and bear chromosome translocations and numerous point mutations. GC B cells remodel their immunoglobulin (Ig) genes by somatic hypermutation (SHM) and class switch recombination (CSR) in their Ig genes. Activation Induced Deaminase (AID) initiates CSR and SHM by generating U:G mismatches on Ig DNA that can then be processed by Uracyl-N-glycosylase (UNG). AID promotes collateral damage in the form of chromosome translocations and off-target SHM, however, the exact contribution of AID activity to lymphoma generation and progression is not completely understood. Here we show using a conditional knock-in strategy that AID supra-activity alone is not sufficient to generate B cell transformation. In contrast, in the absence of UNG, AID supra-expression increases SHM and promotes lymphoma. Whole exome sequencing revealed that AID heavily contributes to lymphoma SHM, promoting subclonal variability and a wider range of oncogenic variants. Thus, our data provide direct evidence that UNG is a brake to AID-induced intratumoral heterogeneity and evolution of B cell lymphoma.

Timing the initiation of multiple myeloma

The evolution and progression of multiple myeloma and its precursors over time is poorly understood. Here, we investigate the landscape and timing of mutational processes shaping multiple myeloma evolution in a large cohort of 89 whole genomes and 973 exomes. We identify eight processes, including a mutational signature caused by exposure to melphalan. Reconstructing the chronological activity of each mutational signature, we estimate that the initial transformation of a germinal center B-cell usually occurred during the first 2nd-3rd decades of life. We define four main patterns of activation-induced deaminase (AID) and apolipoprotein B mRNA editing catalytic polypeptide-like (APOBEC) mutagenesis over time, including a subset of patients with evidence of prolonged AID activity during the pre-malignant phase, indicating antigen-responsiveness and germinal center reentry. Our findings provide a framework to study the etiology of multiple myeloma and explore strategies for prevention and early detection.

Restriction of AID activity and somatic hypermutation by PARP-1.

Affinity maturation of the humoral immune response depends on somatic hypermutation (SHM) of immunoglobulin (Ig) genes, which is initiated by targeted lesion introduction by activation-induced deaminase (AID), followed by error-prone DNA repair. Stringent regulation of this process is essential to prevent genetic instability, but no negative feedback control has been identified to date. Here we show that poly(ADP-ribose) polymerase-1 (PARP-1) is a key factor restricting AID activity during somatic hypermutation. Poly(ADP-ribose) (PAR) chains formed at DNA breaks trigger AID-PAR association, thus preventing excessive DNA damage induction at sites of AID action. Accordingly, AID activity and somatic hypermutation at the Ig variable region is decreased by PARP-1 activity. In addition, PARP-1 regulates DNA lesion processing by affecting strand biased A:T mutagenesis. Our study establishes a novel function of the ancestral genome maintenance factor PARP-1 as a critical local feedback regulator of both AID activity and DNA repair during Ig gene diversification.

A broad atlas of somatic hypermutation allows prediction of activation-induced deaminase targets.

Activation-induced deaminase (AID) initiates antibody diversification in germinal center (GC) B cells through the deamination of cytosines on immunoglobulin genes. AID can also target other regions in the genome, triggering mutations or chromosome translocations, with major implications for oncogenic transformation. However, understanding the specificity of AID has proved extremely challenging. We have sequenced at very high depth >1,500 genomic regions from GC B cells and identified 275 genes targeted by AID, including 30 of the previously known 35 AID targets. We have also identified the most highly mutated hotspot for AID activity described to date. Furthermore, integrative analysis of the molecular features of mutated genes coupled to machine learning has produced a powerful predictive tool for AID targets. We also have found that base excision repair and mismatch repair back up each other to faithfully repair AID-induced lesions. Finally, our data establish a novel link between AID mutagenic activity and lymphomagenesis.

R Loops in the Regulation of Antibody Gene Diversification.

For nearly three decades, R loops have been closely linked with class switch recombination (CSR), the process that generates antibody isotypes and that occurs via a complex cascade initiated by transcription-coupled mutagenesis in switch recombination sequences. R loops form during transcription of switch recombination sequences in vitro and in vivo, and there is solid evidence that R loops are required for efficient class switching. The classical model of R loops posits that they boost mutation rates by generating stable and long tracts of single-stranded DNA that serve as the substrate for activation induced deaminase (AID), the enzyme that initiates the CSR reaction cascade by co-transcriptionally mutating ssDNA in switch recombination sequences. Though logical and compelling, this model has not been supported by in vivo evidence. Indeed, several reports suggest that R loops may not be involved in recruiting AID activity to switch regions, meaning that R loops probably serve other unanticipated roles in CSR. Here, I review the key findings in this field to date and propose hypotheses that could help towards elucidating the precise function of R loops in CSR.

Molecular Mechanisms of Somatic Hypermutation and Class Switch Recombination.

In order to promote an efficient humoral immune response, germinal center B cells modify both the antigen recognition and effector domains by programmed genetic alterations of their antibody genes. To do so, B cells use the enzyme activation-induced deaminase (AID), which transforms deoxycytidine into deoxyuridine at the immunoglobulin genes, triggering mutagenic DNA repair. Data accumulated during the past decade have significantly advanced our understanding of how AID activity is regulated and preferentially targeted to the immunoglobulin genes. There is also a better understanding of the ways by which AID-catalyzed uracil is recognized and the ensuing downstream processing underpinning the mechanisms of somatic hypermutation and class switch recombination. Here, we critically review these advances in the context of their relevance for the humoral immune response. A detailed understanding of these molecular mechanisms is paramount to uncover the basis of B cell intrinsic immunodeficiency, as well as to suggest tools and strategies that might allow boosting antibody gene diversification in the context of immunizations or infections that require the elicitation of rare or highly mutated antibody variants.

UNG protects B cells from AID-induced telomere loss.

Activation-induced deaminase (AID) initiates antibody gene diversification by creating G:U mismatches in the immunoglobulin loci. However, AID also deaminates nonimmunoglobulin genes, and failure to faithfully repair these off-target lesions can cause B cell lymphoma. In this study, we identify a mechanism by which processing of G:U produced by AID at the telomeres can eliminate B cells at risk of genomic instability. We show that telomeres are off-target substrates of AID and that B cell proliferation depends on protective repair by uracil-DNA glycosylase (UNG). In contrast, in the absence of UNG activity, deleterious processing by mismatch repair leads to telomere loss and defective cell proliferation. Indeed, we show that UNG deficiency reduces B cell clonal expansion in the germinal center in mice and blocks the proliferation of tumor B cells expressing AID. We propose that AID-induced damage at telomeres acts as a fail-safe mechanism to limit the tumor promoting activity of AID when it overwhelms uracil excision repair.

Regulation of Activation Induced Deaminase (AID) by Estrogen.

Regulation of Activation Induced Deaminase (AID) by the hormone estrogen has important implications for understanding adaptive immune responses as well as the involvement of AID in autoimmune diseases and tumorigenesis. This chapter describes the general laboratory techniques for analyzing AID expression and activity induced by estrogen, focusing on the isolation and preparation of cells for hormone treatment and the subsequent analysis of AID responsiveness to estrogen at the RNA level and for determining the regulation of AID activity via estrogen by analyzing Ig switch circle transcripts and mutations in switch region loci.

AID-expressing epithelium is protected from oncogenic transformation by an NKG2D surveillance pathway.

Activation-induced deaminase (AID) initiates secondary antibody diversification in germinal center B cells, giving rise to higher affinity antibodies through somatic hypermutation (SHM) or to isotype-switched antibodies through class switch recombination (CSR). SHM and CSR are triggered by AID-mediated deamination of cytosines in immunoglobulin genes. Importantly, AID activity in B cells is not restricted to Ig loci and can promote mutations and pro-lymphomagenic translocations, establishing a direct oncogenic mechanism for germinal center-derived neoplasias. AID is also expressed in response to inflammatory cues in epithelial cells, raising the possibility that AID mutagenic activity might drive carcinoma development. We directly tested this hypothesis by generating conditional knock-in mouse models for AID overexpression in colon and pancreas epithelium. AID overexpression alone was not sufficient to promote epithelial cell neoplasia in these tissues, in spite of displaying mutagenic and genotoxic activity. Instead, we found that heterologous AID expression in pancreas promotes the expression of NKG2D ligands, the recruitment of CD8+ T cells, and the induction of epithelial cell death. Our results indicate that AID oncogenic potential in epithelial cells can be neutralized by immunosurveillance protective mechanisms.

AIDing cancer treatment: Reducing AID activity via HSP90 inhibition.

The activation induced deaminase (AID) catalyses the two key events underlying humoral adaptive immunity: class switch recombination and somatic hypermutation of antibody genes in B lymphocytes. AID accomplishes this task by directly deaminating cytosines within the genomic immunoglobulin locus, thereby triggering a complex mutagenic process eventually leading to improved effector function of antibodies. However, it has long been noticed that AID can be aberrantly expressed in cancer and that its activity is not absolutely restricted to antibody genes, as substantial genome‐wide off‐target mutations have been observed, which contribute to tumorigenesis and clonal evolution of AID‐expressing malignancies. In this issue of the European Journal of Immunology, Montamat‐Sicotte et al. [Eur. J. Immunol. 2015. 45: 2365–2376] investigate the feasibility and efficacy of in vivo inhibition of AID with HSP90 inhibitors in a mouse model of B‐cell leukemia and in vitro with a human breast cancer cell line, thereby demonstrating that cancer patients may benefit from preventing noncanonical AID functions.

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.

Splice variants of activation induced deaminase (AID) do not affect the efficiency of class switch recombination in murine CH12F3 cells.

Activation Induced Deaminase (AID) triggers the antigen-driven antibody diversification processes through its ability to edit DNA. AID dependent DNA damage is also the cause of genetic alterations often found in mature B cell tumors. A number of splice variants of AID have been identified, for which a role in the modulation of its activity has been hypothesized. We have thus tested two of these splice variants, which we find catalytically inactive, for their ability to modulate the activity of endogenous AID in CH12F3 cells, a murine lymphoma cell line in which Class Switch Recombination (CSR) can be induced. In contrast to full-length AID, neither these splice variants or a catalytically impaired AID mutant affect the efficiency of Class Switch Recombination. Thus, while a role for these splice variants at the RNA level remains possible, it is unlikely that they exert any regulatory effect on the function of AID.

Intraclonal diversity in follicular lymphoma analyzed by quantitative ultradeep sequencing of noncoding regions.

Cancers are characterized by genomic instability, and the resulting intraclonal diversity is a prerequisite for tumor evolution. Therefore, metrics of tumor heterogeneity may prove to be clinically meaningful. Intraclonal heterogeneity in follicular lymphoma (FL) is apparent from studies of somatic hypermutation (SHM) caused by activation-induced deaminase (AID) in IGH. Aberrant SHM (aSHM), defined as AID activity outside of the IG loci, predominantly targets noncoding regions causing numerous "passenger" mutations, but it has the potential to generate rare significant "driver" mutations. The quantitative relationship between SHM and aSHM has not been defined. To measure SHM and aSHM, ultradeep sequencing (>20,000-fold coverage) was performed on IGH (~1650 nt) and nine other noncoding regions potentially targeted by AID (combined 9411 nt), including the 5' untranslated region of BCL2. Single-nucleotide variants (SNVs) were found in 12/12 FL specimens (median 136 SHMs and 53 aSHMs). The aSHM SNVs were associated with AID motifs (p < 0.0001). The number of SNVs at BCL2 varied widely among specimens and correlated with the number of SNVs at eight other potential aSHM sites. In contrast, SHM at IGH was not predictive of aSHM. Tumor heterogeneity is apparent from SNVs at low variant allele frequencies; the relative number of SNVs with variable allele frequency < 5% varied with clinical grade, indicating that tumor heterogeneity based on aSHM reflects a clinically meaningful parameter. These data suggest that genome-wide aSHM may be estimated from aSHM of BCL2 but not SHM of IGH. The results demonstrate a practical approach to the quantification of intratumoral genetic heterogeneity for clinical specimens.

AID-induced remodeling of immunoglobulin genes and B cell fate.

Survival and phenotype of normal and malignant B lymphocytes are critically dependent on constitutive signals by the B cell receptor (BCR) for antigen. In addition, either antigen ligation of the BCR or various mitogenic stimuli result in B cell activation and induction of activation-induced deaminase (AID). AID activity can in turn mediate somatic hypermutation (SHM) of immunoglobulin (Ig) V regions and also deeply remodel the Ig heavy chain locus through class switch recombination (CSR) or locus suicide recombination (LSR). In addition to changes linked to affinity for antigen, modifying the class/isotype (i.e. the structure and function) of the BCR or suddenly deleting BCR expression also modulates the fate of antigen-experienced B cells.

Regulation of AID, the B-cell genome mutator.

The mechanisms by which B cells somatically engineer their genomes to generate the vast diversity of antibodies required to challenge the nearly infinite number of antigens that immune systems encounter are of tremendous clinical and academic interest. The DNA cytidine deaminase activation-induced deaminase (AID) catalyzes two of these mechanisms: class switch recombination (CSR) and somatic hypermutation (SHM). Recent discoveries indicate a significant promiscuous targeting of this B-cell mutator enzyme genome-wide. Here we discuss the various regulatory elements that control AID activity and prevent AID from inducing genomic instability and thereby initiating oncogenesis.

A critical role for AID in the initiation of reprogramming to induced pluripotent stem cells

Mechanistic insights into the reprogramming of fibroblasts to induced pluripotent stem cells (iPSCs) are limited, particularly for early acting molecular regulators. Here we use an acute loss of function approach to demonstrate that activation-induced deaminase (AID) activity is necessary for the initiation of reprogramming to iPSCs. While AID is well known for antibody diversification, it has also recently been shown to have a role in active DNA demethylation in reprogramming toward pluripotency and development. These findings suggested a potential role for AID in iPSC generation, yet, iPSC yield from AID-knockout mouse fibroblasts was similar to that of wild-type (WT) fibroblasts. We reasoned that an acute loss of AID function might reveal effects masked by compensatory mechanisms during development, as reported for other proteins. Accordingly, we induced an acute reduction (>50%) in AID levels using 4 different shRNAs and determined that reprogramming to iPSCs was significantly impaired by 79 ± 7%. The deaminase activity of AID was critical, as coexpression of WT but not a catalytic mutant AID rescued reprogramming. Notably, AID was required only during a 72-h time window at the onset of iPSC reprogramming. Our findings show a critical role for AID activity in the initiation of reprogramming to iPSCs.