Independent roles of switching and hypermutation in the development and persistence of B lymphocyte memory [Nurr77]
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE76865 | GEO | 2016/10/19
SECONDARY ACCESSION(S): PRJNA308821
REPOSITORIES: GEO
Similar Datasets
Project description:Somatic hypermutation (SHM) and class switch recombination (CSR) increase the affinity and diversify the effector functions of antibodies during immune responses. Although SHM and CSR are fundamentally different, their independent roles in regulating B cell fate have been difficult to uncouple because a single enzyme, activation-induced cytidine deaminase (encoded by Aicda), initiates both reactions. Here, we used a combination of Aicda and antibody mutant alleles that separate the effects of CSR and SHM on polyclonal immune responses. We found that class-switching to IgG1 biased the fate choice made by B cells, favoring the plasma cell over memory cell fate without significantly affecting clonal expansion in the germinal center (GC). In contrast, SHM reduced the longevity of memory B cells by creating polyreactive specificities that were selected against over time. Our data define the independent contributions of SHM and CSR to the generation and persistence of memory in the antibody system.
2016-10-19 | GSE76864 | GEO
Project description:Secondary diversification of antibodies through somatic hypermutation (SHM) and class switch recombination (CSR) is a critical component of the immune response. Activation-induced deaminase (AID) initiates both processes by deaminating cytosine residues in immunoglobulin genes. The resulting U:G mismatch can be processed by alternative pathways to give rise to a mutation (SHM) or a DNA double-strand break (CSR). Central to this processing is the activity of uracil-N-glycosylase (UNG), an enzyme normally involved in error-free base excision repair. We used next generation sequencing to analyze the contribution of UNG to the resolution of AID-induced lesions. Loss- and gain-of-function experiments showed that UNG activity can promote both error-prone and high fidelity repair of U:G lesions. Unexpectedly, the balance between these alternative outcomes was influenced by the sequence context of the deaminated cytosine, with individual hotspots exhibiting higher susceptibility to UNG-triggered error-free or error-prone resolution. These results reveal UNG as a new molecular layer that shapes the specificity of AID-induced mutations and may provide new insights into the role of AID in cancer development. Next Generation Sequencing analysis of mutations introduced by AID in activated B lymphocytes from WT and UNG-/- mice (n=4). Activated B cells from AID-/- mice (n=2) were used as negative controls.
2012-07-04 | E-GEOD-39114 | biostudies-arrayexpress
Project description:The activation induced cytosine deaminase (AID) mediates diversification of B cell immunoglobulin genes by the three distinct yet related processes of somatic hypermutation (SHM), class switch recombination (CSR), and gene conversion (GCV). SHM occurs in germinal center B cells, and the transcription factor Bcl6 is a key regulator of the germinal center B cell gene expression program, including expression of AID. To test the hypothesis that Bcl6 function is important for the process of SHM, we compared WT chicken DT40 B cells, which constitutively perform SHM/GCV, to their Bcl6-deficient counterparts. We found that Bcl6-deficient DT40 cells were unable to perform SHM and GCV despite enforced high level expression of AID and substantial levels of AID in the nucleus of the cells. To gain mechanistic insight into the GCV/SHM dependency on Bcl6, transcriptional features of a highly expressed SHM target gene were analyzed in Bcl6-sufficient and -deficient DT40 cells. No defect was observed in the accumulation of single stranded DNA in the target gene as a result of Bcl6 deficiency. In contrast, association of Spt5, an RNA polymerase II (Pol II) and AID binding factor, was strongly reduced at the target gene body relative to the transcription start site in Bcl6-deficient cells as compared to WT cells. However, partial reconstitution of Bcl6 function substantially reconstituted Spt5 association with the target gene body but did not restore detectable SHM. Our observations suggest that in the absence of Bcl6, Spt5 fails to associate efficiently with Pol II at SHM targets, perhaps precluding robust AID action on the SHM target DNA. Our data also suggest, however, that Spt5 binding is not sufficient for SHM of a target gene even in DT40 cells with strong expression of AID. Sequencing of the IgL V region and mutationally active GFP transgene in WT, Bcl6-/- Pax5R, and Bcl6-/- Pax5R Bcl6R chicken DT40 cells for evidence of AID dependent mutations. ChIP-seq proiles of RNA Pol II, Spt5, and pSer5 Pol II at a GFP transgene in WT, Bcl6-/- Pax5R, and Bcl6-/- Pax5R Bcl6R chicken DT40 cells.
2016-02-21 | E-GEOD-77141 | biostudies-arrayexpress
Project description:Activation-induced cytidine deaminase (AID) plays a vital role in adaptive immunity by enabling class switch recombination (CSR) and somatic hypermutation (SHM). While many molecular factors affect AID targeting and function, mounting evidence implicates G-quadruplex (G4) nucleic acid structures as critical effectors of AID biology. To examine the function of AID-G4 binding in vivo, we generated a knock-in mouse with a mutation in Aicda (AIDG133V) that disrupts AID-G4 binding without altering DNA deaminase activity. AIDG133V mice completely lack CSR and SHM, modeling the pathology of hyper-IgM syndrome patients with an orthologous mutation. ChIP-sequencing experiments reveal a broad defect in AIDG133V chromatin localization, implicating AID-G4 binding in genome-wide AID targeting. We find that major histocompatibility complex (MHC) class II genes are AID targets, and that increased AID expression in diffuse large B-cell lymphomas (DLBCLs) correlates with decreased MHC class II expression. Given that loss of MHC class II expression in DLBCLs is highly correlated with adverse outcomes, AID-dependent gene regulation may provide a novel therapeutic target.
2020-10-14 | GSE136959 | GEO
Project description:The transcription factor Bach2 is required for germinal center formation and somatic hypermutation (SHM) of immunoglobulins, both central to an efficient antibody-mediated immune response. Activation-induced cytidine deaminase (AID) initiates SHM and CSR in germinal centers and has potential to induce human B cell lymphoma. To understand the role of Bach2 in AID-mediated immunoglobulin gene diversification processes, we established a Bach2-deficient DT40 B cell line. We show that in addition to allowing SHM, Bach2 drives immunoglobulin gene conversion (GCV), an important AID-dependent antibody gene diversification process. We demonstrate that Bach2 promotes GCV by increasing the expression of AID. Importantly, we found that the regulation of AID is independent of Blimp-1 and that Bach2-deficient cells have altered expression of several genes regulating AID expression, stability and function. These results demonstrate that Bach2 has a previously unappreciated role in the production of high-affinity antibodies.
2017-03-27 | GSE92430 | GEO
Project description:During germinal center (GC) reactions, activated B cells undergo clonal expansion and functional maturation to produce high-affinity antibodies, and differentiate into plasma and memory cells, accompanied with class-switching recombination (CSR) and somatic hypermutation (SHM). Activation-induced deaminase (AID) is responsible for both CSR and SHM in GC B cells. Transcriptional mechanisms underlying AID regulation and GC B cell reactions are still not well understood. Here, we show that expression of Ascl2 transcription factor is upregulated in GC B cells. Ectopic expression of Ascl2 promotes GC B cell development and enhances antibody production as well as affinity maturation. Conversely, deletion of Ascl2 in B cells impairs the germinal center response. Genome-wide analysis reveals that Ascl2 directly regulates GC B cell-related genes, including AID; ectopic expression of AID in Ascl2-deficient B cells rescues their antibody defects. Thus, Ascl2 regulates AID transcription and promotes germinal center B cell responses.
2021-03-30 | GSE169416 | GEO
Project description:Class switch recombination (CSR) and somatic hypermutation (SHM) are antibody gene diversification reactions occurring in mature B cells that are essential for protective humoral immunity. CSR and SHM are initiated following activation by the antigen, and are dependent on efficient induction of activation-induced cytidine deaminase (AID). Therefore, mature B cell viability and fitness are crucial to mount effective immune responses. Here, we identified PDGFA-associated protein 1 (Pdap1) as an essential regulator of cellular homeostasis in mature B cells. Pdap1 deficiency leads to sustained expression of the integrated stress response (ISR) effector activating transcription factor 4 (Atf4) and induction of the ISR transcriptional program, increased cell death, and defective AID expression. As a consequence, loss of Pdap1 reduces germinal center B cell formation and impairs CSR and SHM. Thus, Pdap1 protects mature B cells against chronic ISR activation, and ensures efficient antibody diversification by promoting their survival and optimal function.
2020-06-19 | GSE141876 | GEO
Project description:Secondary diversification of antibodies through somatic hypermutation (SHM) and class switch recombination (CSR) is a critical component of the immune response. Activation-induced deaminase (AID) initiates both processes by deaminating cytosine residues in immunoglobulin genes. The resulting U:G mismatch can be processed by alternative pathways to give rise to a mutation (SHM) or a DNA double-strand break (CSR). Central to this processing is the activity of uracil-N-glycosylase (UNG), an enzyme normally involved in error-free base excision repair. We used next generation sequencing to analyze the contribution of UNG to the resolution of AID-induced lesions. Loss- and gain-of-function experiments showed that UNG activity can promote both error-prone and high fidelity repair of U:G lesions. Unexpectedly, the balance between these alternative outcomes was influenced by the sequence context of the deaminated cytosine, with individual hotspots exhibiting higher susceptibility to UNG-triggered error-free or error-prone resolution. These results reveal UNG as a new molecular layer that shapes the specificity of AID-induced mutations and may provide new insights into the role of AID in cancer development. Next Generation Sequencing analysis of mutations introduced by AID in non B cells. NIH-3T3 cells were co-transduced with mOrangeSTOP and AID-ERM-bM-^@M-^Sexpressing vectors, together with Ugi (UNG inhibitor), UNG, or empty vector as control (n=3). Transduced cells were cultured in the presence of OHT during 11 d. AID-E58Q-ER vector (catalytically inactive form of AID) was used as a negative control in combination with the previously described constructions (n=3).
2012-07-04 | E-GEOD-39108 | biostudies-arrayexpress
Project description:The activation induced cytosine deaminase (AID) mediates diversification of B cell immunoglobulin genes by the three distinct yet related processes of somatic hypermutation (SHM), class switch recombination (CSR), and gene conversion (GCV). SHM occurs in germinal center B cells, and the transcription factor Bcl6 is a key regulator of the germinal center B cell gene expression program, including expression of AID. To test the hypothesis that Bcl6 function is important for the process of SHM, we compared WT chicken DT40 B cells, which constitutively perform SHM/GCV, to their Bcl6-deficient counterparts. We found that Bcl6-deficient DT40 cells were unable to perform SHM and GCV despite enforced high level expression of AID and substantial levels of AID in the nucleus of the cells. To gain mechanistic insight into the GCV/SHM dependency on Bcl6, transcriptional features of a highly expressed SHM target gene were analyzed in Bcl6-sufficient and -deficient DT40 cells. No defect was observed in the accumulation of single stranded DNA in the target gene as a result of Bcl6 deficiency. In contrast, association of Spt5, an RNA polymerase II (Pol II) and AID binding factor, was strongly reduced at the target gene body relative to the transcription start site in Bcl6-deficient cells as compared to WT cells. However, partial reconstitution of Bcl6 function substantially reconstituted Spt5 association with the target gene body but did not restore detectable SHM. Our observations suggest that in the absence of Bcl6, Spt5 fails to associate efficiently with Pol II at SHM targets, perhaps precluding robust AID action on the SHM target DNA. Our data also suggest, however, that Spt5 binding is not sufficient for SHM of a target gene even in DT40 cells with strong expression of AID.
2016-02-21 | GSE77141 | GEO
Project description:Somatic hypermutation (SHM) and class switch recombination (CSR) of the immunoglobulin (Ig) genes allow B cells to make antibodies that protect us against a wide variety of pathogens. SHM is mediated by activation induced deaminase (AID), occurs at a million times higher frequency than other mutations in the mammalian genome and is largely restricted to the variable (V) and switch (S) regions of Ig genes. Using the Ramos human Burkitt’s lymphoma cell line, we find that H3K79me2/3 and its methyltransferase Dot1L are more abundant on the V region than the constant (C) region which does not undergo mutation. In primary naïve mouse B cells examined ex vivo, the H3K79me2/3 modification appears constitutively in the donor Sμ and is inducible in the recipient Sγ1 upon CSR stimulation. Knockout (KO) and inhibition of Dot1L in Ramos cells significantly reduces V region mutation and the abundance of H3K79me2/3 on the V region and is associated with a decrease of Pol II and its S2 phosphorylated form at the IgH locus. KO of Dot1L also decreases the abundance of BRD4 and CDK9 (a subunit of P-TEFb complex) on the V region and this is accompanied by decreased nascent transcripts throughout the IgH gene. Treatment with JQ1 (inhibitor of BRD4) or DRB (inhibitor of CDK9) decreased SHM and the abundance of Pol II S2P at the IgH locus. With all these factors playing a role in transcription elongation, our studies extend our insights into the chromatin context and dynamics of transcription required for SHM.
2021-02-28 | GSE167873 | GEO