REV7 is required for processing AID initiated DNA lesions in activated B cells.
ABSTRACT: Activation-induced cytidine deaminase (AID) initiates both antibody class switch recombination (CSR) and somatic hypermutation (SHM) in antibody diversification. DNA double-strand break response (DSBR) factors promote rearrangement in CSR, while translesion synthesis (TLS) polymerases generate mutations in SHM. REV7, a component of TLS polymerase zeta, is also a downstream effector of 53BP1-RIF1 DSBR pathway. Here, we study the multi-functions of REV7 and find that REV7 is required for the B cell survival upon AID-deamination, which is independent of its roles in DSBR, G2/M transition or REV1-mediated TLS. The cell death in REV7-deficient activated B cells can be fully rescued by AID-deficiency in vivo. We further identify that REV7-depedent TLS across UNG-processed apurinic/apyrimidinic sites is required for cell survival upon AID/APOBEC deamination. This study dissects the multiple roles of Rev7 in antibody diversification, and discovers that TLS is not only required for sequence diversification but also B cell survival upon AID-initiated lesions.
Project description:Activation-induced cytidine deaminase (AID) is essential for the DNA cleavage that initiates both somatic hypermutation (SHM) and class switch recombination (CSR) of the Ig gene. Two alternative mechanisms of DNA cleavage by AID have been proposed: RNA editing and DNA deamination. In support of the latter, AID has DNA deamination activity in cell-free systems that is assumed to represent its physiological function. To test this hypothesis, we generated various mouse AID mutants and compared their DNA deamination, CSR, and SHM activities. Here, we compared DNA deamination, CSR, and SHM activities of various AID mutants and found that most of their CSR or SHM activities were disproportionate with their DNA deamination activities. Specifically, we identified a cluster of mutants (H48A, L49A, R50A, and N51A) with low DNA deamination activity but relatively intact CSR activity. Of note is an AID mutant (N51A) that retained CSR function but lost DNA deamination activity. In addition, an APOBEC1 mutation at N57, homologous to N51 of AID, also abolished DNA deamination activity but retained RNA editing activity. These results indicate that DNA deamination activity does not represent the physiological function of AID.
Project description:Activation-induced cytidine deaminase (AID) initiates both class switch recombination (CSR) and somatic hypermutation (SHM) in antibody diversification. Mechanisms of AID targeting and catalysis remain elusive despite its critical immunological roles and off-target effects in tumorigenesis. Here, we produced active human AID and revealed its preferred recognition and deamination of structured substrates. G-quadruplex (G4)-containing substrates mimicking the mammalian immunoglobulin switch regions are particularly good AID substrates in vitro. By solving crystal structures of maltose binding protein (MBP)-fused AID alone and in complex with deoxycytidine monophosphate, we surprisingly identify a bifurcated substrate-binding surface that explains structured substrate recognition by capturing two adjacent single-stranded overhangs simultaneously. Moreover, G4 substrates induce cooperative AID oligomerization. Structure-based mutations that disrupt bifurcated substrate recognition or oligomerization both compromise CSR in splenic B cells. Collectively, our data implicate intrinsic preference of AID for structured substrates and uncover the importance of G4 recognition and oligomerization of AID in CSR.
Project description:Activation-induced cytidine deminase (AID) is crucial for controlling the immunoglobulin (Ig) diversification processes of somatic hypermutation (SHM) and class switch recombination (CSR). AID initiates these processes by deamination of cytosine, ultimately resulting in mutations or double strand DNA breaks needed for SHM and CSR. Levels of AID control mutation rates, and off-target non-Ig gene mutations can contribute to lymphomagenesis. Therefore, factors that control AID levels in the nucleus can regulate SHM and CSR, and may contribute to disease. We previously showed that transcription factor YY1 can regulate the level of AID in the nucleus and Ig CSR. Therefore, we hypothesized that conditional knock-out of YY1 would lead to reduction in AID localization at the Ig locus, and reduced AID-mediated mutations. Using mice that overexpress AID (Ig?AID yy1f/f ) or that express normal AID levels (yy1f/f ), we found that conditional knock-out of YY1 results in reduced AID nuclear levels, reduced localization of AID to the S? switch region, and reduced AID-mediated mutations. We find that the mechanism of YY1 control of AID nuclear accumulation is likely due to YY1-AID physical interaction which blocks AID ubiquitination.
Project description: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.
Project description:In activated B lymphocytes, AID initiates antibody variable (V) exon somatic hypermutation (SHM) for affinity maturation in germinal centers (GCs) and IgH switch (S) region DNA breaks (DSBs) for class-switch recombination (CSR). To resolve long-standing questions, we have developed an in vivo assay to study AID targeting of passenger sequences replacing a V exon. First, we find AID targets SHM hotspots within V exon and S region passengers at similar frequencies and that the normal SHM process frequently generates deletions, indicating that SHM and CSR employ the same mechanism. Second, AID mutates targets in diverse non-Ig passengers in GC B cells at levels similar to those of V exons, definitively establishing the V exon location as "privileged" for SHM. Finally, Peyer's patch GC B cells generate a reservoir of V exons that are highly mutated before selection for affinity maturation. We discuss the implications of these findings for harnessing antibody diversification mechanisms.
Project description:Activation-induced deoxycytidine deaminase (AID) and Apobec 3G (Apo3G) cause mutational diversity by initiating mutations on regions of single-stranded (ss) DNA. Expressed in B cells, AID deaminates C ? U in actively transcribed immunoglobulin (Ig) variable and switch regions to initiate the somatic hypermutation (SHM) and class switch recombination (CSR) that are essential for antibody diversity. Apo3G expressed in T cells catalyzes C deaminations on reverse transcribed cDNA causing HIV-1 retroviral inactivation. When operating properly, AID- and Apo3G-initiated mutations boost human fitness. Yet, both enzymes are potentially powerful somatic cell "mutators". Loss of regulated expression and proper genome targeting can cause human cancer. Here, we review well-established biological roles of AID and Apo3G. We provide a synopsis of AID partnering proteins during SHM and CSR, and describe how an Apo2 crystal structure provides "surrogate" insight for AID and Apo3G biochemical behavior. However, large gaps remain in our understanding of how dC deaminases search ssDNA to identify trinucleotide motifs to deaminate. We discuss two recent methods to analyze ssDNA scanning and deamination. Apo3G scanning and deamination is visualized in real-time using single-molecule FRET, and AID deamination efficiencies are determined with a random walk analysis. AID and Apo3G encounter many candidate deamination sites while scanning ssDNA. Generating mutational diversity is a principal aim of AID and an important ancillary property of Apo3G. Success seems likely to involve hit and miss deamination motif targeting, biased strongly toward miss.
Project description:Activation-induced cytidine deaminase (AID) initiates immunoglobulin (Ig) heavy-chain (IgH) class switch recombination (CSR) and Ig variable region somatic hypermutation (SHM) in B lymphocytes by deaminating cytidines on template and nontemplate strands of transcribed DNA substrates. However, the mechanism of AID access to the template DNA strand, particularly when hybridized to a nascent RNA transcript, has been an enigma. We now implicate the RNA exosome, a cellular RNA-processing/degradation complex, in targeting AID to both DNA strands. In B lineage cells activated for CSR, the RNA exosome associates with AID, accumulates on IgH switch regions in an AID-dependent fashion, and is required for optimal CSR. Moreover, both the cellular RNA exosome complex and a recombinant RNA exosome core complex impart robust AID- and transcription-dependent DNA deamination of both strands of transcribed SHM substrates in vitro. Our findings reveal a role for noncoding RNA surveillance machinery in generating antibody diversity.
Project description:Activation-induced cytidine deaminase (AID) initiates both somatic hypermutation (SHM) for antibody affinity maturation and DNA breakage for antibody class switch recombination (CSR) via transcription-dependent cytidine deamination of single-stranded DNA targets. Though largely specific for immunoglobulin genes, AID also acts on a limited set of off-targets, generating oncogenic translocations and mutations that contribute to B cell lymphoma. How AID is recruited to off-targets has been a long-standing mystery. Based on deep GRO-seq studies of mouse and human B lineage cells activated for CSR or SHM, we report that most robust AID off-target translocations occur within highly focal regions of target genes in which sense and antisense transcription converge. Moreover, we found that such AID-targeting "convergent" transcription arises from antisense transcription that emanates from super-enhancers within sense transcribed gene bodies. Our findings provide an explanation for AID off-targeting to a small subset of mostly lineage-specific genes in activated B cells.
Project description:Activation-induced cytidine deaminase (AID) is essential for antibody diversification, namely somatic hypermutation (SHM) and class switch recombination (CSR). The deficiency of apurinic/apyrimidinic endonuclease 1 (Ape1) in CH12F3-2A B cells reduces CSR to ?20% of wild-type cells, whereas the effect of APE1 loss on SHM has not been examined. Here we show that, although APE1's endonuclease activity is important for CSR, it is dispensable for SHM as well as IgH/c-myc translocation. Importantly, APE1 deficiency did not show any defect in AID-induced S-region break formation, but blocked both the recruitment of repair protein Ku80 to the S region and the synapse formation between S? and S?. Knockdown of end-processing factors such as meiotic recombination 11 homolog (MRE11) and carboxy-terminal binding protein (CtBP)-interacting protein (CtIP) further reduced the remaining CSR in Ape1-null CH12F3-2A cells. Together, our results show that APE1 is dispensable for SHM and AID-induced DNA breaks and may function as a DNA end-processing enzyme to facilitate the joining of broken ends during CSR.
Project description:Activation-induced deaminase (AID) mutates the immunoglobulin (Ig) genes to initiate somatic hypermutation (SHM) and class switch recombination (CSR) in B cells, thus underpinning antibody responses. AID mutates a few hundred other loci, but most AID-occupied genes are spared. The mechanisms underlying productive deamination versus non-productive AID targeting are unclear. Here we show that three clustered arginine residues define a functional AID domain required for SHM, CSR, and off-target activity in B cells without affecting AID deaminase activity or Escherichia coli mutagenesis. Both wt AID and mutants with single amino acid replacements in this domain broadly associate with Spt5 and chromatin and occupy the promoter of AID target genes. However, mutant AID fails to occupy the corresponding gene bodies and loses association with transcription elongation factors. Thus AID mutagenic activity is determined not by locus occupancy but by a licensing mechanism, which couples AID to transcription elongation.