RESUMO
Efficient humoral responses rely on DNA damage, mutagenesis and error-prone DNA repair. Diversification of B cell receptors through somatic hypermutation and class-switch recombination are initiated by cytidine deamination in DNA mediated by activation-induced cytidine deaminase (AID)1 and by the subsequent excision of the resulting uracils by uracil DNA glycosylase (UNG) and by mismatch repair proteins1-3. Although uracils arising in DNA are accurately repaired1-4, how these pathways are co-opted to generate mutations and double-strand DNA breaks in the context of somatic hypermutation and class-switch recombination is unknown1-3. Here we performed a genome-wide CRISPR-Cas9 knockout screen for genes involved in class-switch recombination and identified FAM72A, a protein that interacts with the nuclear isoform of UNG (UNG2)5 and is overexpressed in several cancers5. We show that the FAM72A-UNG2 interaction controls the levels of UNG2 and that class-switch recombination is defective in Fam72a-/- B cells due to the upregulation of UNG2. Moreover, we show that somatic hypermutation is reduced in Fam72a-/- B cells and that its pattern is skewed upon upregulation of UNG2. Our results are consistent with a model in which FAM72A interacts with UNG2 to control its physiological level by triggering its degradation, regulating the level of uracil excision and thus the balance between error-prone and error-free DNA repair. Our findings have potential implications for tumorigenesis, as reduced levels of UNG2 mediated by overexpression of Fam72a would shift the balance towards mutagenic DNA repair, rendering cells more prone to acquire mutations.
Assuntos
Linfócitos B , Reparo de Erro de Pareamento de DNA , Switching de Imunoglobulina , Região de Troca de Imunoglobulinas , Mutação , Hipermutação Somática de Imunoglobulina , Animais , Feminino , Masculino , Camundongos , Linfócitos B/metabolismo , Sistemas CRISPR-Cas/genética , Genoma/genética , Switching de Imunoglobulina/genética , Região de Troca de Imunoglobulinas/genética , Hipermutação Somática de Imunoglobulina/genética , Regulação para Cima , Uracila/metabolismoRESUMO
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.
Assuntos
Linfócitos B , DNA Glicosilases , Reparo de Erro de Pareamento de DNA , Switching de Imunoglobulina , Proteínas de Membrana , Mutação , Proteínas de Neoplasias , Hipermutação Somática de Imunoglobulina , Animais , Feminino , Humanos , Camundongos , Linfócitos B/metabolismo , Sistemas CRISPR-Cas , DNA Glicosilases/antagonistas & inibidores , DNA Glicosilases/metabolismo , Epistasia Genética , Células HEK293 , Switching de Imunoglobulina/genética , Região de Troca de Imunoglobulinas/genética , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos Endogâmicos C57BL , Proteína 2 Homóloga a MutS/genética , Proteína 2 Homóloga a MutS/metabolismo , Proteínas de Neoplasias/deficiência , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Hipermutação Somática de Imunoglobulina/genéticaRESUMO
Class switch recombination (CSR) in B cells involves deletion-recombination at switch (S) region DNA and is important for the diversification of antibody isotypes during an immune response. Here, we identify two NME [NM23/NDPK (nucleoside diphosphate kinase)] isoforms, NME1 and NME2, as novel players in this process. Knockdown of NME2 leads to decreased CSR, while knockdown of the highly homologous NME1 results in increased CSR. Interestingly, these NME proteins also display differential occupancy at S regions during CSR despite their homology; NME1 binds to S regions prior to stimulation, while NME2 binds to S regions only after stimulation. To the best of our knowledge, this represents the first report of a role for these proteins in the regulation of CSR.
Assuntos
Linfócitos B/metabolismo , Cadeias Pesadas de Imunoglobulinas/química , Nucleosídeo NM23 Difosfato Quinases/metabolismo , Animais , Proliferação de Células , Células Cultivadas , Técnicas de Silenciamento de Genes , Switching de Imunoglobulina , Cadeias Pesadas de Imunoglobulinas/metabolismo , Região de Troca de Imunoglobulinas , Camundongos , Nucleosídeo NM23 Difosfato Quinases/genética , Ligação ProteicaRESUMO
Chimeric antigen receptor (CAR) T cells with a long-lived memory phenotype are correlated with durable, complete remissions in patients with leukemia. However, not all CAR T cell products form robust memory populations, and those that do can induce chronic B cell aplasia in patients. To address these challenges, we previously developed a switchable CAR (sCAR) T cell system that allows fully tunable, on/off control over engineered cellular activity. To further evaluate the platform, we generated and assessed different murine sCAR constructs to determine the factors that afford efficacy, persistence, and expansion of sCAR T cells in a competent immune system. We find that sCAR T cells undergo significant in vivo expansion, which is correlated with potent antitumor efficacy. Most importantly, we show that the switch dosing regimen not only allows control over B cell populations through iterative depletion and repopulation, but that the "rest" period between dosing cycles is the key for induction of memory and expansion of sCAR T cells. These findings introduce rest as a paradigm in enhancing memory and improving the efficacy and persistence of engineered T cell products.
Assuntos
Bioengenharia/métodos , Imunoterapia Adotiva/métodos , Animais , Antígenos CD19/imunologia , Linfócitos B/imunologia , Citocinas/metabolismo , Citotoxicidade Imunológica/imunologia , Feminino , Região de Troca de Imunoglobulinas/genética , Região de Troca de Imunoglobulinas/imunologia , Ativação Linfocitária/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Modelos Animais , Modelos Biológicos , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/imunologiaRESUMO
Class switch recombination (CSR) at the immunoglobulin heavy-chain (IgH) locus is associated with the formation of R-loop structures over switch (S) regions. While these often occur co-transcriptionally between nascent RNA and template DNA, we now show that they also form as part of a post-transcriptional mechanism targeting AID to IgH S-regions. This depends on the RNA helicase DDX1 that is also required for CSR in vivo. DDX1 binds to G-quadruplex (G4) structures present in intronic switch transcripts and converts them into S-region R-loops. This in turn targets the cytidine deaminase enzyme AID to S-regions so promoting CSR. Notably R-loop levels over S-regions are diminished by chemical stabilization of G4 RNA or by the expression of a DDX1 ATPase-deficient mutant that acts as a dominant-negative protein to reduce CSR efficiency. In effect, we provide evidence for how S-region transcripts interconvert between G4 and R-loop structures to promote CSR in the IgH locus.
Assuntos
Adenosina Trifosfatases/metabolismo , RNA Helicases DEAD-box/fisiologia , Quadruplex G , Cadeias Pesadas de Imunoglobulinas/genética , Região de Troca de Imunoglobulinas/genética , RNA/química , Adenosina Trifosfatases/genética , Animais , Linfócitos B/citologia , Linfócitos B/metabolismo , Citidina Desaminase/genética , Citidina Desaminase/metabolismo , Replicação do DNA , Switching de Imunoglobulina , Cadeias Pesadas de Imunoglobulinas/química , Cadeias Pesadas de Imunoglobulinas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , RNA/genética , Recombinação GenéticaRESUMO
In B cells, Ig class switch recombination (CSR) is initiated by activation-induced cytidine deaminase (AID), the activity of which leads to DNA double-strand breaks (DSBs) within IgH switch (S) regions. Preferential targeting of AID-mediated DSBs to S sequences is critical for allowing diversification of antibody functions, while minimizing potential off-target oncogenic events. Here, we used gene targeted inactivation of histone methyltransferase (HMT) multiple myeloma SET domain (MMSET) in mouse B cells and the CH12F3 cell line to explore its role in CSR. We find that deletion of MMSET-II, the isoform containing the catalytic SET domain, inhibits CSR without affecting either IgH germline transcription or joining of DSBs within S regions by classical nonhomologous end joining (C-NHEJ). Instead, we find that MMSET-II inactivation leads to decreased AID recruitment and DSBs at the upstream donor Sµ region. Our findings suggest a role for the HMT MMSET in promoting AID-mediated DNA breaks during CSR.
Assuntos
Citidina Desaminase/genética , DNA/genética , Histona-Lisina N-Metiltransferase/genética , Switching de Imunoglobulina , Região de Troca de Imunoglobulinas , Imunoglobulinas/genética , Animais , Linfócitos B/citologia , Linfócitos B/imunologia , Domínio Catalítico , Citidina Desaminase/imunologia , DNA/imunologia , Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades , Regulação da Expressão Gênica , Inativação Gênica , Histona-Lisina N-Metiltransferase/antagonistas & inibidores , Histona-Lisina N-Metiltransferase/imunologia , Imunoglobulinas/metabolismo , Isoenzimas/antagonistas & inibidores , Isoenzimas/genética , Isoenzimas/imunologia , Camundongos , Camundongos Knockout , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Recombinação Genética , Transdução de SinaisRESUMO
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.
Assuntos
Citidina Desaminase/metabolismo , DNA/metabolismo , Switching de Imunoglobulina , Região de Troca de Imunoglobulinas , Recombinação Genética , Desaminases APOBEC/genética , Desaminases APOBEC/metabolismo , Animais , Diversidade de Anticorpos , Linfócitos B/enzimologia , Linfócitos B/imunologia , Citidina Desaminase/química , Citidina Desaminase/genética , DNA/química , DNA/genética , Humanos , Camundongos , Modelos Moleculares , Mutação , Conformação de Ácido Nucleico , Ligação Proteica , Conformação Proteica , Baço/enzimologia , Baço/imunologia , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
IgH class switch recombination (CSR) occurs through the deliberate introduction of activation-induced cytidine deaminase (AID)-instigated DNA double-strand breaks into the IgH loci. Because double-strand breaks are generally highly toxic, mechanisms that regulate AID expression are of much relevance to CSR and genomic integrity; however, effectors of such regulatory processes are still poorly understood. In this article, we show that the transcription factor sex determining region Y-box 2 (Sox2) is expressed in activated B cells, but almost exclusively in those that have undergone CSR. We demonstrate that enforced expression of Sox2 in splenic B cells severely inhibits AID expression and CSR, whereas deletion of Sox2 increases the frequency of IgH:c-Myc translocations. These results suggest that Sox2 may regulate AID expression in class-switched B cells to suppress genomic instability associated with CSR.
Assuntos
Linfócitos B/imunologia , Citidina Desaminase/metabolismo , Genes myc/genética , Fatores de Transcrição SOXB1/metabolismo , Baço/imunologia , Animais , Diferenciação Celular , Células Cultivadas , Citidina Desaminase/genética , Quebras de DNA de Cadeia Dupla , Instabilidade Genômica , Região de Troca de Imunoglobulinas , Ativação Linfocitária/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fatores de Transcrição SOXB1/genética , Translocação GenéticaRESUMO
Females and males differ in antibody isotype expression patterns and in immune responses to foreign- and self-antigens. For example, systemic lupus erythematosus is a condition that associates with the production of isotype-skewed anti-self antibodies, and exhibits a 9:1 female:male disease ratio. To explain differences between B cell responses in males and females, we sought to identify direct interactions of the estrogen receptor (ER) with the immunoglobulin heavy chain locus. This effort was encouraged by our previous identification of estrogen response elements (ERE) in heavy chain switch (S) regions. We conducted a full-genome chromatin immunoprecipitation analysis (ChIP-seq) using DNA from LPS-activated B cells and an ERα-specific antibody. Results revealed ER binding to a wide region of DNA, spanning sequences from the JH cluster to Cδ, with peaks in Eµ and Sµ sites. Additional peaks of ERα binding were coincident with hs1,2 and hs4 sites in the 3' regulatory region (3'RR) of the heavy chain locus. This first demonstration of direct binding of ER to key regulatory elements in the immunoglobulin locus supports our hypothesis that estrogen and other nuclear hormone receptors and ligands may directly influence antibody expression and class switch recombination (CSR). Our hypothesis encourages the conduct of new experiments to evaluate the consequences of ER binding. A better understanding of ER:DNA interactions in the immunoglobulin heavy chain locus, and respective mechanisms, may ultimately translate to better control of antibody expression, better protection against pathogens, and prevention of pathologies caused by auto-immune disease.
Assuntos
Linfócitos B/imunologia , Regulação da Expressão Gênica/imunologia , Cadeias Pesadas de Imunoglobulinas/imunologia , Região de Troca de Imunoglobulinas/imunologia , Receptores de Estrogênio/imunologia , Elementos de Resposta/imunologia , Animais , Formação de Anticorpos/genética , Formação de Anticorpos/imunologia , Imunoprecipitação da Cromatina , Feminino , Humanos , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Reação em Cadeia da Polimerase , Receptores de Estrogênio/metabolismoRESUMO
Activation-induced cytidine deaminase (AID) is essential for class switch recombination (CSR) and somatic hypermutation (SHM) of Ig genes. The C terminus of AID is required for CSR but not for SHM, but the reason for this is not entirely clear. By retroviral transduction of mutant AID proteins into aid-/- mouse splenic B cells, we show that 4 amino acids within the C terminus of mouse AID, when individually mutated to specific amino acids (R190K, A192K, L196S, F198S), reduce CSR about as much or more than deletion of the entire C terminal 10 amino acids. Similar to ΔAID, the substitutions reduce binding of UNG to Ig Sµ regions and some reduce binding of Msh2, both of which are important for introducing S region DNA breaks. Junctions between the IgH donor switch (S)µ and acceptor Sα regions from cells expressing ΔAID or the L196S mutant show increased microhomology compared to junctions in cells expressing wild-type AID, consistent with problems during CSR and the use of alternative end-joining, rather than non-homologous end-joining (NHEJ). Unlike deletion of the AID C terminus, 3 of the substitution mutants reduce DNA double-strand breaks (DSBs) detected within the Sµ region in splenic B cells undergoing CSR. Cells expressing these 3 substitution mutants also have greatly reduced mutations within unrearranged Sµ regions, and they decrease with time after activation. These results might be explained by increased error-free repair, but as the C terminus has been shown to be important for recruitment of NHEJ proteins, this appears unlikely. We hypothesize that Sµ DNA breaks in cells expressing these C terminus substitution mutants are poorly repaired, resulting in destruction of Sµ segments that are deaminated by these mutants. This could explain why these mutants cannot undergo CSR.
Assuntos
Citidina Desaminase/genética , Switching de Imunoglobulina/genética , Região de Troca de Imunoglobulinas/genética , Imunoglobulinas/genética , Recombinação Genética , Substituição de Aminoácidos/genética , Animais , Linfócitos B/imunologia , Linfócitos B/metabolismo , Citidina Desaminase/imunologia , DNA/genética , Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades/genética , Humanos , Camundongos , Camundongos Knockout , Mutação de Sentido IncorretoRESUMO
Immunoglobulin (Ig) heavy chains undergo class switch recombination (CSR) to change the heavy chain isotype from IgM to IgG, A or E. The switch regions are several kilobases long, repetitive, and G-rich on the nontemplate strand. They are also relatively depleted of CpG (also called CG) sites for unknown reasons. Here we use synthetic switch regions at the IgH switch alpha (Sα) locus to test the effect of CpG sites and to try to understand why the IgH switch sequences evolved to be relatively depleted of CpG. We find that even just two CpG sites within an 80 bp synthetic switch repeat iterated 15 times (total switch region length of 1200 bp containing 30 CpG sites) are sufficient to dramatically reduce both Ig CSR and transcription through the switch region from the upstream Iα sterile transcript promoter, which is the promoter that directs transcripts through the Sα region. De novo DNA methylation occurs at the four CpG sites in and around the Iα promoter when each 80 bp Iα switch repeat contains the two CpG sites. Thus, a relatively low density of CpG sites within the switch repeats can induce upstream CpG methylation at the IgH alpha locus, and cause a substantial decrease in transcription from the sterile transcript promoter. This effect is likely the reason that switch regions evolved to contain very few CpG sites. We discuss these findings as they relate to DNA methylation and to Ig CSR.
Assuntos
Linfócitos B/imunologia , Ilhas de CpG , Switching de Imunoglobulina/genética , Região de Troca de Imunoglobulinas , Recombinação Genética/imunologia , Transcrição Gênica/imunologia , Animais , Linfócitos B/citologia , Sequência de Bases , Linhagem Celular Tumoral , Metilação de DNA , Regulação da Expressão Gênica , Camundongos , Dados de Sequência Molecular , Regiões Promotoras GenéticasRESUMO
The activation-induced cytidine deaminase (AID) mediates somatic hypermutation and class switch recombination of the Ig genes by directly deaminating cytosines to uracils. As AID causes a substantial amount of off-target mutations, its activity has been associated with lymphomagenesis and clonal evolution of B-cell malignancies. Although it has been shown that AID is expressed in B-cell chronic lymphocytic leukemia (CLL), a clear analysis of in vivo AID activity in this B-cell malignancy remained elusive. In this study performed on primary human CLL samples, we report that, despite the presence of a dominant VDJ heavy chain region, a substantial intraclonal diversity was observed at VDJ as well as at IgM switch regions (Sµ), showing ongoing AID activity in vivo during disease progression. This AID-mediated heterogeneity was higher in CLL subclones expressing CD86, which we identified as the proliferative CLL fraction. Finally, CD86 expression correlated with shortened time to first treatment and increased γ-H2AX focus formation. Our data demonstrate that AID is active in CLL in vivo and thus, AID likely contributes to clonal evolution of CLL.
Assuntos
Antígeno B7-2/imunologia , Proliferação de Células , Citidina Desaminase/imunologia , Dano ao DNA/imunologia , Leucemia Linfocítica Crônica de Células B/imunologia , Proteínas de Neoplasias/imunologia , Feminino , Regulação Leucêmica da Expressão Gênica/imunologia , Genes de Cadeia Pesada de Imunoglobulina/imunologia , Histonas/imunologia , Humanos , Região de Troca de Imunoglobulinas/imunologia , Cadeias mu de Imunoglobulina/imunologia , Leucemia Linfocítica Crônica de Células B/patologia , MasculinoRESUMO
R loops exist at the murine IgH switch regions and possibly other locations, but their functional importance is unclear. In biochemical systems, R loop initiation requires DNA sequence regions containing clusters of G nucleotides, but cellular studies have not been done. Here, we vary the G-clustering, total switch region length, and the number of target sites (WGCW sites for the activation-induced deaminase) at synthetic switch regions in a murine B cell line to determine the effect on class switch recombination (CSR). G-clusters increase CSR regardless of their immediate proximity to the WGCW sites. This increase is accompanied by an increase in R loop formation. CSR efficiency correlates better with the absolute number of WGCW sites in the switch region rather than the total switch region length or density of WGCW sites. Thus, the overall strength of the switch region depends on G-clusters, which initiate R loop formation, and on the number of WGCW sites.
Assuntos
Switching de Imunoglobulina , Região de Troca de Imunoglobulinas , Animais , Linfócitos B/metabolismo , Linhagem Celular Tumoral , Sequência Rica em GC , Camundongos , Conformação de Ácido NucleicoRESUMO
Fanconi anemia is a rare genetic disorder that can lead to bone marrow failure, congenital abnormalities, and increased risk for leukemia and cancer. Cells with loss-of-function mutations in the FANC pathway are characterized by chromosome fragility, altered mutability, and abnormal regulation of the nonhomologous end-joining (NHEJ) pathway. Somatic hypermutation (SHM) and immunoglobulin (Ig) class switch recombination (CSR) enable B cells to produce high-affinity antibodies of various isotypes. Both processes are initiated after the generation of dG:dU mismatches by activation-induced cytidine deaminase. Whereas SHM involves an error-prone repair process that introduces novel point mutations into the Ig gene, the mismatches generated during CSR are processed to create double-stranded breaks (DSBs) in DNA, which are then repaired by the NHEJ pathway. As several lines of evidence suggest a possible role for the FANC pathway in SHM and CSR, we analyzed both processes in B cells derived from Fanca(-/-) mice. Here we show that Fanca is required for the induction of transition mutations at A/T residues during SHM and that despite globally normal CSR function in splenic B cells, Fanca is required during CSR to stabilize duplexes between pairs of short microhomology regions, thereby impeding short-range recombination downstream of DSB formation.
Assuntos
Linfócitos B/metabolismo , Proteína do Grupo de Complementação A da Anemia de Fanconi/metabolismo , Switching de Imunoglobulina/genética , Mutação Puntual , Hipermutação Somática de Imunoglobulina/genética , Animais , Linfócitos B/imunologia , Sequência de Bases , Western Blotting , Linhagem Celular , Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades , Proteína do Grupo de Complementação A da Anemia de Fanconi/deficiência , Proteína do Grupo de Complementação A da Anemia de Fanconi/genética , Proteína do Grupo de Complementação G da Anemia de Fanconi/deficiência , Proteína do Grupo de Complementação G da Anemia de Fanconi/genética , Proteína do Grupo de Complementação G da Anemia de Fanconi/metabolismo , Humanos , Região de Troca de Imunoglobulinas/genética , Camundongos da Linhagem 129 , Camundongos Knockout , Dados de Sequência Molecular , Proteína 2 Homóloga a MutS/metabolismo , Reação em Cadeia da Polimerase , Recombinação GenéticaRESUMO
Class switch DNA recombination (CSR) of the immunoglobulin heavy chain (IgH) locus crucially diversifies antibody biological effector functions. CSR involves the induction of activation-induced cytidine deaminase (AID) expression and AID targeting to switch (S) regions by 14-3-3 adaptors. 14-3-3 adaptors specifically bind to 5'-AGCT-3' repeats, which make up for the core of all IgH locus S regions. They selectively target the upstream and downstream S regions that are set to undergo S-S DNA recombination. We hypothesized that 14-3-3 adaptors function as scaffolds to stabilize CSR enzymatic elements on S regions. Here we demonstrate that all seven 14-3-3ß, 14-3-3ε, 14-3-3γ, 14-3-3η, 14-3-3σ, 14-3-3τ and 14-3-3ζ adaptors directly interacted with AID, PKA-Cα (catalytic subunit) and PKA-RIα (regulatory inhibitory subunit) and uracil DNA glycosylase (Ung). 14-3-3 adaptors, however, did not interact with AID C-terminal truncation mutant AIDΔ(180-198) or AIDF193A and AIDL196A point-mutants (which have been shown not to bind to S region DNA and fail to mediate CSR). 14-3-3 adaptors colocalized with AID and replication protein A (RPA) in B cells undergoing CSR. 14-3-3 and AID binding to S region DNA was disrupted by viral protein R (Vpr), an accessory protein of human immunodeficiency virus type-1 (HIV-1), which inhibited CSR without altering AID expression or germline IH-CH transcription. Accordingly, we demonstrated that 14-3-3 directly interact with Vpr, which in turn, also interact with AID, PKA-Cα and Ung. Altogether, our findings suggest that 14-3-3 adaptors play important scaffold functions and nucleate the assembly of multiple CSR factors on S regions. They also show that such assembly can be disrupted by a viral protein, thereby allowing us to hypothesize that small molecule compounds that specifically block 14-3-3 interactions with AID, PKA and/or Ung can be used to inhibit unwanted CSR.
Assuntos
Proteínas 14-3-3/fisiologia , Linfócitos B/imunologia , Switching de Imunoglobulina/genética , Recombinação Genética , Proteínas 14-3-3/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Citidina Desaminase/metabolismo , Produtos do Gene vpr/metabolismo , Humanos , Região de Troca de Imunoglobulinas , Modelos Genéticos , Modelos Moleculares , Imagem Óptica , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/fisiologia , Uracila-DNA Glicosidase/metabolismoRESUMO
Programmed mutagenesis of the immunoglobulin locus of B lymphocytes during class switch recombination (CSR) and somatic hypermutation requires RNA polymerase II (polII) transcription complex-dependent targeting of the DNA mutator activation-induced cytidine deaminase (AID). AID deaminates cytidine residues on substrate sequences in the immunoglobulin (Ig) locus via a transcription-dependent mechanism, and this activity is stimulated by the RNA polII stalling cofactor Spt5 and the 11-subunit cellular noncoding RNA 3'-5' exonucleolytic processing complex RNA exosome. The mechanism by which the RNA exosome recognizes immunoglobulin locus RNA substrates to stimulate AID DNA deamination activity on its in vivo substrate sequences is an important question. Here we report that E3-ubiquitin ligase Nedd4 destabilizes AID-associated RNA polII by a ubiquitination event, leading to generation of 3' end free RNA exosome RNA substrates at the Ig locus and other AID target sequences genome-wide. We found that lack of Nedd4 activity in B cells leads to accumulation of RNA exosome substrates at AID target genes and defective CSR. Taken together, our study links noncoding RNA processing following RNA polII pausing with regulation of the mutator AID protein. Our study also identifies Nedd4 as a regulator of noncoding RNAs that are generated by stalled RNA polII genome-wide.
Assuntos
Linfócitos B/enzimologia , Citidina Desaminase/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , RNA Polimerase II/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Linhagem Celular Tumoral , Células Cultivadas , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Complexo Multienzimático de Ribonucleases do Exossomo/metabolismo , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Região de Troca de Imunoglobulinas/genética , Camundongos , Ubiquitina-Proteína Ligases Nedd4 , Proteínas Nucleares/metabolismo , Ligação Proteica , Fatores de Elongação da Transcrição/metabolismo , Ubiquitina-Proteína Ligases/genética , UbiquitinaçãoRESUMO
To further unravel the molecular pathogenesis of diffuse large B-cell lymphoma (DLBCL), we performed high-resolution comparative genomic hybridization on lymph node biopsies from 70 patients. With this strategy, we identified microdeletions of genes involved in the mutation mismatch repair (MMR) pathway in two samples. The first patient presented with a homozygous deletion of MSH2-MSH6 due to duplication of an unbalanced pericentric inversion of chromosome 2. The other case showed a PMS2 heterozygous deletion. PMS2 and MSH2-MSH6 abnormalities, respectively, resulted in a decrease and complete loss of gene expression. However, unlike tumors associated with the hereditary non-polyposis colorectal cancer syndrome or immunodeficiency-related lymphomas, no microsatellite instability was detected. Mutational profiles revealed especially in one patient an aberrant hypermutation without a clear activation-induced cytidine deaminase signature, indicating a breakdown of the high-fidelity repair in favor of the error-prone repair pathway. Our findings suggest that in a rare subset of patients, inactivation of the genes of the MMR pathway is likely an important step in the molecular pathogenesis of DLBCL and does not involve the same molecular mechanisms as other common neoplasms with MMR deficiency.
Assuntos
Adenosina Trifosfatases/genética , Reparo de Erro de Pareamento de DNA/genética , Enzimas Reparadoras do DNA/genética , Proteínas de Ligação a DNA/genética , Deleção de Genes , Linfoma Difuso de Grandes Células B/genética , Mutação , Biópsia , Cromossomos Humanos Par 2 , Hibridização Genômica Comparativa , Expressão Gênica , Loci Gênicos , Instabilidade Genômica , Humanos , Cadeias Pesadas de Imunoglobulinas/genética , Região de Troca de Imunoglobulinas/genética , Linfoma Difuso de Grandes Células B/patologia , Endonuclease PMS2 de Reparo de Erro de Pareamento , Fator de Transcrição PAX5/genética , Proteínas Proto-Oncogênicas c-bcl-6 , Estudos RetrospectivosRESUMO
By diversifying the biological effector functions of antibodies, class switch DNA recombination (CSR) plays a critical role in the maturation of the immune response. It is initiated by activation-induced cytidine deaminase (AID)-mediated deoxycytosine deamination, yielding deoxyuridine (dU), and dU glycosylation by uracil DNA glycosylase (Ung) in antibody switch (S) region DNA. Here we showed that the translesion DNA synthesis polymerase Rev1 directly interacted with Ung and targeted in an AID-dependent and Ung-independent fashion the S regions undergoing CSR. Rev1(-/-)Ung(+/+) B cells reduced Ung recruitment to S regions, DNA-dU glycosylation, and CSR. Together with an S region spectrum of mutations similar to that of Rev1(+/+)Ung(-/-) B cells, this suggests that Rev1 operates in the same pathway as Ung, as emphasized by further decreased CSR in Rev1(-/-)Msh2(-/-) B cells. Rescue of CSR in Rev1(-/-) B cells by a catalytically inactive Rev1 mutant shows that the important role of Rev1 in CSR is mediated by Rev1's scaffolding function, not its enzymatic function.
Assuntos
DNA/metabolismo , Nucleotidiltransferases/metabolismo , Uracila-DNA Glicosidase/metabolismo , Animais , Linfócitos B/imunologia , Linfócitos B/metabolismo , Citidina Desaminase/genética , Citidina Desaminase/metabolismo , DNA/genética , DNA Polimerase Dirigida por DNA , Desoxiuridina/metabolismo , Glicosilação , Humanos , Switching de Imunoglobulina , Região de Troca de Imunoglobulinas , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutação , Nucleotidiltransferases/deficiência , Nucleotidiltransferases/genética , Ligação Proteica , Recombinação Genética , Uracila-DNA Glicosidase/genéticaRESUMO
Activation-induced deaminase (AID) catalyses class switch recombination (CSR) and somatic hypermutation (SHM) in B lymphocytes to enhance Ab diversity. CSR involves breaking and rejoining highly repetitive switch (S) regions in the IgH (Igh) locus. S regions appear to be preferential targets of AID. To determine whether S region sequence per se, independent of Igh cis regulatory elements, can influence AID targeting efficiency and mutation frequency, we established a knock-in mouse model by inserting a core Sγ1 region into the first intron of proto-oncogene Bcl6, which is a non-Ig target of SHM. We found that the mutation frequency of the inserted Sγ1 region was dramatically higher than that of the adjacent Bcl6 endogenous sequence. Mechanistically, S region-enhanced SHM was associated with increased recruitment of AID and RNA polymerase II, together with Spt5, albeit to a lesser extent. Our studies demonstrate that target DNA sequences influence mutation frequency via regulating AID recruitment. We propose that the nucleotide sequence preference may serve as an additional layer of AID regulation by restricting its mutagenic activity to specific sequences despite the observation that AID has the potential to access the genome widely.
Assuntos
Citidina Desaminase/genética , Técnicas de Introdução de Genes , Hipermutação Somática de Imunoglobulina/genética , Animais , Citidina Desaminase/metabolismo , Análise Mutacional de DNA , Proteínas de Ligação a DNA/genética , Técnicas de Introdução de Genes/métodos , Humanos , Região de Troca de Imunoglobulinas/genética , Camundongos , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas c-bcl-6 , RNA Polimerase II/genética , Recombinação Genética/imunologiaRESUMO
The entirety of all protein coding sequences is reported to represent a small fraction (~2%) of the mouse and human genomes; the vast majority of the rest of the genome is presumed to be repetitive elements (REs). In this study, the C57BL/6J mouse reference genome was subjected to an unbiased RE mining to establish a whole-genome profile of RE occurrence and arrangement. The C57BL/6J mouse genome was fragmented into an initial set of 5,321 units of 0.5 Mb, and surveyed for REs using unbiased self-alignment and dot-matrix protocols. The survey revealed that individual chromosomes had unique profiles of RE arrangement structures, named RE arrays. The RE populations in certain genomic regions were arranged into various forms of complexly organized structures using combinations of direct and/or inverse repeats. Some of these RE arrays spanned stretches of over 2 Mb, which may contribute to the structural configuration of the respective genomic regions. There were substantial differences in RE density among the 21 chromosomes, with chromosome Y being the most densely populated. In addition, the RE array population in the mouse chromosomes X and Y was substantially different from those of the reference human chromosomes. Conversion of the dot-matrix data pertaining to a tandem 13-repeat structure within the Ch7.032 genome unit into a line map of known REs revealed a repeat unit of ~11.3 Kb as a mosaic of six different RE types. The data obtained from this study allowed for a comprehensive RE profiling, including the establishment of a library of RE arrays, of the reference mouse genome. Some of these RE arrays may participate in a spectrum of normal and disease biology that are specific for mice.