Proteasomal degradation restricts the nuclear lifespan of AID.
ABSTRACT: Activation-induced cytidine deaminase (AID) initiates all postrearrangement processes that diversify the immunoglobulin repertoire by specific deamination of cytidines at the immunoglobulin (Ig) locus. As uncontrolled expression of AID is potentially mutagenic, different types of regulation, particularly nucleocytoplasmic shuttling, restrict the likelihood of AID-deoxyribonucleic acid encounters. We studied additional mechanisms of regulation affecting the stability of the AID protein. No modulation of protein accumulation according to the cell cycle was observed in a Burkitt's lymphoma cell line. In contrast, the half-life of AID was markedly reduced in the nucleus, and this destabilization was accompanied by a polyubiquitination that was revealed in the presence of proteasome inhibitors. The same compartment-specific degradation was observed in activated mouse B cells, and also in a non-B cell line. No specific lysine residues could be linked to this degradation, so it remains unclear whether polyubiquitination proceeds through several alternatives sites or through the protein N terminus. The nuclear-restricted form of AID displayed enhanced mutagenicity at both Ig and non-Ig loci, most notably at TP53, suggesting that modulation of nuclear AID content through proteasomal degradation may represent another level of control of AID activity.
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:Immunoglobulin (Ig) isotype switching is a recombination event that changes the constant domain of antibody genes and is catalyzed by activation-induced cytidine deaminase (AID). Upon recruitment to Ig genes, AID deaminates cytidines at switch (S) recombination sites, leading to the formation of DNA breaks. In addition to their role in isotype switching, AID-induced lesions promote Igh-cMyc chromosomal translocations and tumor development. However, cMyc translocations are also present in lymphocytes from healthy humans and mice, and thus, it remains unclear whether AID directly contributes to the dynamics of B cell transformation. Using a plasmacytoma mouse model, we show that AID(+/-) mice have reduced AID expression levels and display haploinsufficiency both in the context of isotype switching and plasmacytomagenesis. At the Ig loci, AID(+/-) lymphocytes show impaired intra- and inter-switch recombination, and a substantial decrease in the frequency of S mutations and chromosomal breaks. In AID(+/-) mice, these defects correlate with a marked decrease in the accumulation of B cell clones carrying Igh-cMyc translocations during tumor latency. These results thus provide a causality link between the extent of AID enzymatic activity, the number of emerging Igh-cMyc-translocated cells, and the incidence of B cell transformation.
Project description: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.
Project description:Upon activation, B cells divide, form a germinal center, and express the activation induced deaminase (AID), an enzyme that triggers somatic hypermutation of the variable regions of immunoglobulin (Ig) loci. Recent evidence indicates that at least 25% of expressed genes in germinal center B cells are mutated or deaminated by AID. One of the most deaminated genes, c-Myc, frequently appears as a translocation partner with the Ig heavy chain gene (Igh) in mouse plasmacytomas and human Burkitt's lymphomas. This indicates that the two genes or their double-strand break ends come into close proximity at a biologically relevant frequency. However, the proximity of c-Myc and Igh has never been measured in germinal center B cells, where many such translocations are thought to occur. We hypothesized that in germinal center B cells, not only is c-Myc near Igh, but other mutating non-Ig genes are deaminated by AID because they are near Ig genes, the primary targets of AID. We tested this "collateral damage" model using 3D-fluorescence in situ hybridization (3D-FISH) to measure the distance from non-Ig genes to Ig genes in germinal center B cells. We also made mice transgenic for human MYC and measured expression and mutation of the transgenes. We found that there is no correlation between proximity to Ig genes and levels of AID targeting or gene mutation, and that c-Myc was not closer to Igh than were other non-Ig genes. In addition, the human MYC transgenes did not accumulate mutations and were not deaminated by AID. We conclude that proximity to Ig loci is unlikely to be a major determinant of AID targeting or mutation of non-Ig genes, and that the MYC transgenes are either missing important regulatory elements that allow mutation or are unable to mutate because their new nuclear position is not conducive to AID deamination.
Project description:Activation-induced cytidine deaminase (AID) mediates the somatic hypermutation (SHM) of immunoglobulin (Ig) variable (V) regions that is required for the generation of antibody diversity and for the affinity maturation of the antibody response against infectious agents and toxic substances. AID preferentially targets WRC (W = A/T, R = A/G) hot spot motifs, particularly WGCW motifs that create overlapping hot spots on both strands. In order to gain a better understanding of the generation of antibody diversity and to create a platform for the in vitro generation of affinity-matured antibodies, we have established a system involving recombinase-mediated cassette exchange (RMCE) to replace the V region and its flanking sequences. This makes it possible to easily manipulate the sequence of the Ig gene within the endogenous heavy chain of the Ramos human Burkitt's lymphoma cell line. Here we show that the newly integrated wild-type (WT) VH regions introduced by RMCE undergo SHM similarly to non-RMCE-modified Ramos cells. Most importantly, we have shown that introducing a cluster of WGCW motifs into the complementary determining region 2 (CDR2) of the human heavy chain V region significantly raised the mutation frequency and number of mutations per sequence compared to WT controls. Thus, we have demonstrated a novel platform in Ramos cells whereby we can easily and quickly manipulate the endogenous human VH region to further explore the regulation and targeting of SHM. This platform will be useful for generating human antibodies with changes in affinity and specificity in vitro.An effective immune response requires a highly diverse repertoire of affinity-matured antibodies. Activation-induced cytidine deaminase (AID) is required for somatic hypermutation (SHM) of immunoglobulin (Ig) genes. Although a great deal has been learned about the regulation of AID, it remains unclear how it is preferentially targeted to particular motifs, to certain locations within the Ig gene and not to other highly expressed genes in the germinal center B cell. This is an important question because AID is highly mutagenic and is sometimes mistargeted to other highly expressed genes, including proto-oncogenes, leading to B cell lymphomas. Here we describe how we utilize recombinase-mediated cassette exchange (RMCE) to modify the sequence of the endogenous heavy chain locus in the Ramos Burkitt's lymphoma cell line. This platform can be used to explore the regulation and targeting of SHM and to generate human antibodies with changes in affinity and specificity in vitro.
Project description:Somatic hypermutation introduces base substitutions into the rearranged and expressed immunoglobulin (Ig) variable regions to promote immunity. This pathway requires and is initiated by the Activation Induced Deaminase (AID) protein, which deaminates cytidine to produce uracils and UG mismatches at the Ig genes. Subsequent processing of uracil by mismatch repair and base excision repair factors contributes to mutagenesis. While selective for certain genomic targets, the chromatin modifications which distinguish hypermutating from non-hypermutating loci are not defined.Here, we show that AID-targeted loci in mammalian B cells contain ubiquitinated chromatin. Chromatin immunoprecipitation (ChIP) analysis of a constitutively hypermutating Burkitt's B cell line, Ramos, revealed the presence of monoubiquitinated forms of both histone H2A and H2B at two AID-associated loci, but not at control loci which are expressed but not hypermutated. Similar analysis using LPS activated primary murine splenocytes showed enrichment of the expressed V(H) and Sgamma3 switch regions upon ChIP with antibody specific to AID and to monoubiquitinated H2A and H2B. In the mechanism of mammalian hypermutation, AID may interact with ubiquitinated chromatin because confocal immunofluorescence microscopy visualized AID colocalized with monoubiquitinated H2B within discrete nuclear foci.Our results indicate that monoubiquitinated histones accompany active somatic hypermutation, revealing part of the histone code marking AID-targeted loci. This expands the current view of the chromatin state during hypermutation by identifying a specific nucleosome architecture associated with somatic hypermutation.
Project description:Activation-induced cytidine deaminase (AID) is a single-stranded (ss) DNA-specific cytidine deaminase that initiates Ig heavy chain (IgH) class switch recombination (CSR) and Ig somatic hypermutation (SHM) by deaminating cytidines within, respectively, IgH switch (S) regions and Ig variable region (V) exons. AID that is phosphorylated on serine residue 38 interacts with replication protein A (RPA), a ssDNA binding protein, to promote deamination of transcribed double-stranded DNA in vitro, which, along with other evidence, suggests that AID may similarly gain access to transcribed S regions and V exons in vivo. However, the physiological role of AID phosphorylation at serine residue 38 (S38), and even the requirement for the S38 residue, with respect to CSR or SHM has been debated. To address this issue, we used gene targeting to generate an endogenous mouse AID locus that produces AID in which S38 is substituted with alanine (AID(S38A)), a mutant form of AID that retains similar catalytic activity on ssDNA as WT AID (AID(WT)). B cells homozygous for the AID(S38A) mutation show substantially impaired CSR and SHM, correlating with inability of AID(S38A) to interact with endogenous RPA. Moreover, mice haploinsufficient for AID(S38A) have even more severely impaired CSR when compared with mice haploinsufficient for AID(WT), with CSR levels reduced to nearly background levels. These results unequivocally demonstrate that integrity of the AID S38 phosphorylation site is required for normal CSR and SHM in mice and strongly support a role for AID phosphorylation at S38 and RPA interaction in regulating CSR and SHM.
Project description:Herpes simplex virus (HSV) immediate-early protein ICP0 is a transcriptional activator with E3 ubiquitin ligase activity that induces the degradation of ND10 proteins, including the promyelocytic leukemia protein (PML) and Sp100. Moreover, ICP0 has a role in the derepression of viral genomes and in the modulation of the host interferon response to virus infection. Here, we report that ICP0 interacts with SIAH-1, a cellular E3 ubiquitin ligase that is involved in multiple cellular pathways and is itself capable of mediating PML degradation. This novel virus-host interaction profoundly stabilized SIAH-1 and recruited this cellular E3 ligase into ICP0-containing nuclear bodies. Moreover, SIAH-1 mediated the polyubiquitination of HSV ICP0 in vitro and in vivo. After infection of SIAH-1 knockdown cells with HSV, higher levels of ICP0 were produced, ICP0 was less ubiquitinated, and the half-life of this multifunctional viral regulatory protein was increased. These results indicate an inhibitory role of SIAH-1 during lytic infection by targeting ICP0 for proteasomal degradation.
Project description:BACKGROUND: Activation induced deaminase (AID) mediates class switch recombination and somatic hypermutation of immunoglobulin (Ig) genes in germinal centre B cells. In order to regulate its specific activity and as a means to keep off-target mutations low, several mechanisms have evolved, including binding to specific cofactors, phosphorylation and destabilization of nuclear AID protein. Although ubiquitination at lysine residues of AID is recognized as an essential step in initiating degradation of nuclear AID, any functional relevance of lysine modifications has remained elusive. METHODOLOGY/PRINCIPAL FINDINGS: Here, we report functional implications of lysine modifications of the human AID protein by generating a panel of lysine to arginine mutants of AID and assessment of their catalytic class switch activity. We found that only mutation of Lys22 to Arg resulted in a significant reduction of class switching to IgG1 in transfected primary mouse B cells. This decrease in activity was neither reflected in reduced hypermutation of Ig genes in AID-mutant transfected DT40 B cell lines nor recapitulated in bacterial deamination assays, pointing to involvement of post-translational modification of Lys22 for AID activity in B cells. CONCLUSIONS/SIGNIFICANCE: Our results imply that lysine modification may represent a novel level of AID regulation and that Lys22 is important for effective AID activity.
Project description:Chromosome translocations between Ig (Ig) and non-Ig genes are frequently associated with B-cell lymphomas in humans and mice. The best characterized of these is c-myc/IgH translocation, which is associated with Burkitt's lymphoma. These translocations are caused by activation-induced cytidine deaminase (AID), which produces double-strand DNA breaks in both genes. c-myc/IgH translocations are rare events, in part because ATM, p53, and p19 actively suppress them. To further define the mechanism of protection against the accumulation of cells that bear c-myc/IgH translocation, we assayed B cells from mice that carry mutations in cell-cycle and apoptosis regulator proteins that act downstream of p53. We find that PUMA, Bim, and PKCdelta are required for protection against c-myc/IgH translocation, whereas Bcl-XL and BAFF enhance c-myc/IgH translocation. Whether these effects are general or specific to c-myc/IgH translocation and whether AID produces dsDNA breaks in genes other than c-myc and Ig is not known. To examine these questions, we developed an assay for translocation between IgH and Igbeta, both of which are somatically mutated by AID. Igbeta/IgH, like c-myc/IgH translocations, are AID-dependent, and AID is responsible for lesions on IgH and the non-IgH translocation partners. However, ATM, p53, and p19 do not protect against Igbeta/IgH translocations. Instead, B cells are protected against Igbeta/IgH translocations by a BAFF- and PKCdelta-dependent pathway. We conclude that AID-induced double-strand breaks in non-Ig genes other than c-myc lead to their translocation, and that at least two nonoverlapping pathways protect against translocations in primary B cells.