Similar Datasets
Project description:Antibody class switch recombination (CSR) requires the ligation of DNA breaks in transcribed and juxtaposed donor and acceptor switch (S) sequences (S-S synapsis) spaced 50-200 kb apart within the immunoglobulin heavy chain locus (Igh). CSR is believed to occur via the association of active S regions with the 3’ regulatory region (3’RR) super-enhancer thereby coupling transcription with S-S synapsis. Here, we map the multi-way interactome underlying CSR using Tri-C. Surprisingly, S-S synapsis is strongly associated with the 3` CTCF binding element (3`CBE), but not the 3’RR, a finding also confirmed by Micro-C measurements. Instead, the 3’RR forms a self-interacting domain independently engaging with Igh genes. Additionally, transcription at acceptor S regions creates a topological barrier that insulates S-S synapsis from loop extrusion complexes. Thus, Igh gene activation and S-S synapsis occur via distinct topological states created by the interaction between transcription and the loop extrusion machinery.
2023-01-20 | GSE198176 | GEO
Project description:Antibody class switch recombination (CSR) requires the ligation of DNA breaks in transcribed and juxtaposed donor and acceptor switch (S) sequences (S-S synapsis) spaced 50-200 kb apart within the immunoglobulin heavy chain locus (Igh). CSR is believed to occur via the association of active S regions with the 3’ regulatory region (3’RR) super-enhancer thereby coupling transcription with S-S synapsis. Here, we map the multi-way interactome underlying CSR using Tri-C. Surprisingly, S-S synapsis is strongly associated with the 3` CTCF binding element (3`CBE), but not the 3’RR, a finding also confirmed by Micro-C measurements. Instead, the 3’RR forms a self-interacting domain independently engaging with Igh genes. Additionally, transcription at acceptor S regions creates a topological barrier that insulates S-S synapsis from loop extrusion complexes. Thus, Igh gene activation and S-S synapsis occur via distinct topological states created by the interaction between transcription and the loop extrusion machinery.
2023-01-20 | GSE198181 | GEO
Project description:Antibody class switch recombination (CSR) requires the ligation of DNA breaks in transcribed and juxtaposed donor and acceptor switch (S) sequences (S-S synapsis) spaced 50-200 kb apart within the immunoglobulin heavy chain locus (Igh). CSR is believed to occur via the association of active S regions with the 3’ regulatory region (3’RR) super-enhancer thereby coupling transcription with S-S synapsis. Here, we map the multi-way interactome underlying CSR using Tri-C. Surprisingly, S-S synapsis is strongly associated with the 3` CTCF binding element (3`CBE), but not the 3’RR, a finding also confirmed by Micro-C measurements. Instead, the 3’RR forms a self-interacting domain independently engaging with Igh genes. Additionally, transcription at acceptor S regions creates a topological barrier that insulates S-S synapsis from loop extrusion complexes. Thus, Igh gene activation and S-S synapsis occur via distinct topological states created by the interaction between transcription and the loop extrusion machinery.
2023-01-20 | GSE198180 | GEO
Project description:Antibody class switch recombination (CSR) requires the ligation of DNA breaks in transcribed and juxtaposed donor and acceptor switch (S) sequences (S-S synapsis) spaced 50-200 kb apart within the immunoglobulin heavy chain locus (Igh). CSR is believed to occur via the association of active S regions with the 3’ regulatory region (3’RR) super-enhancer thereby coupling transcription with S-S synapsis. Here, we map the multi-way interactome underlying CSR using Tri-C. Surprisingly, S-S synapsis is strongly associated with the 3` CTCF binding element (3`CBE), but not the 3’RR, a finding also confirmed by Micro-C measurements. Instead, the 3’RR forms a self-interacting domain independently engaging with Igh genes. Additionally, transcription at acceptor S regions creates a topological barrier that insulates S-S synapsis from loop extrusion complexes. Thus, Igh gene activation and S-S synapsis occur via distinct topological states created by the interaction between transcription and the loop extrusion machinery.
2023-01-20 | GSE198179 | GEO
Project description:Activation-induced cytidine deaminase (AID) is required for class-switch recombination (CSR), somatic hypermutation, and gene conversion of Ig genes. Although AID has sequence similarity to an RNA-editing enzyme Apobec-1, how AID functions in CSR and somatic hypermutation is unknown. Because involvement of RNA-editing but not DNA-editing in CSR requires de novo protein synthesis after AID expression, we examined whether protein synthesis inhibitors could block CSR in the presence of the AID activity. For this purpose we constructed AID fused with the hormone-binding domain of the estrogen receptor (AID-ER), which was introduced into AID-deficient spleen B cells. When such transfectants were treated with an estrogen analogue, 4-hydroxytamoxifen (OHT), CSR was induced within 1 h. Cycloheximide or puromycin drastically suppressed OHT-induced CSR in AID-ER expressing AID-/- B cells when added 1 h before OHT but not after OHT, suggesting that de novo protein synthesis is required for an event downstream to AID expression in CSR. The results lend the weight to RNA-editing hypothesis for the function of AID.
| S-EPMC151392 | biostudies-literature
Project description:Activation-induced cytidine deaminase is required for the DNA cleavage step of Ig class switch recombination (CSR). However, its molecular mechanism is controversial. RNA-editing hypothesis postulates that activation-induced cytidine deaminase deaminates cytosine in an unknown mRNA to generate a new mRNA encoding an endonuclease for CSR and thus predicts that DNA cleavage depends on de novo protein synthesis. On the other hand, DNA deamination hypothesis proposes that DNA cleavage is initiated by cytosine deamination in DNA, followed by uracil removal by uracil DNA glycosylase. By using the chromatin immunoprecipitation assay to detect gamma-H2AX focus formation as a marker for DNA cleavage, we found that cycloheximide inhibited DNA cleavage in the Ig heavy-chain locus during CSR. Requirement of protein synthesis in the DNA cleavage step of CSR strengthens the RNA-editing hypothesis.
| S-EPMC516508 | biostudies-other
Project description:Antibody class switching is mediated by somatic recombination between switch regions of the immunoglobulin heavy chain gene locus. Targeting of recombination to particular switch regions is strictly regulated by cytokines through the induction of switch transcripts starting 5' of the repetitive switch regions. However, switch transcription as such is not sufficient to target switch recombination. This has been shown in mutant mice, in which the I-exon and its promoter upstream of the switch region were replaced with heterologous promoters. Here we show that, in the murine germline targeted replacement of the endogenous gamma1 promoter, I-exon, and I-exon splice donor site by heterologous promoter and splice donor sites directs switch recombination in activated B lymphocytes constitutively to the gamma1 switch region. In contrast, switch recombination to IgG1 is inhibited in mutant mice, in which the replacement does not include the heterologous splice donor site. Our data unequivocally demonstrate that targeting of switch recombination to IgG1 in vivo requires processing of the Igamma1 switch transcripts. Either the processing machinery or the processed transcripts are involved in class switch recombination.
| S-EPMC2212419 | biostudies-literature
Project description:R-loops form at Sgamma3 and Sgamma2b Ig class switch regions in the chromosomes of stimulated murine primary B cells and are suspected to be a general feature of mammalian class switch regions. The in vivo upstream boundary of the R-loops is known to begin within the switch repeats. To determine how precisely the R-loop structure conforms to the repetitive zone of the murine Sgamma3 and Sgamma2b switch regions, a chemical probing method was used to obtain structural information on the downstream boundary. We find that only 61-67% of the R-loops terminate within the Sgamma3 and the Sgamma2b repetitive zones, and the remainder terminate downstream, usually within the first 600 bp immediately downstream of the core switch repeats. Interestingly, the nontemplate strand G density falls to the random level gradually through this same region. Hence, the R-loops terminate as the G-richness of the nascent RNA strand falls. This finding is consistent with thermodynamic predictions for RNA:DNA duplex strength relative to that of DNA:DNA duplexes. This result contrasts with the location of known recombination breakpoints, which correlate not with G-richness and R-loop location but rather with AGCT density. The implications of these findings are discussed in the context of models for the targeting of class switch recombination.
| S-EPMC1458789 | biostudies-literature
Project description:Mismatch repair proteins participate in antibody class switch recombination, although their roles are unknown. Previous nucleotide sequence analyses of switch recombination junctions indicated that the roles of Msh2 and the MutL homologues, Mlh1 and Pms2, differ. We now asked if Msh2 and Mlh1 function in the same pathway during switch recombination. Splenic B cells from mice deficient in both these proteins were induced to undergo switching in culture. The frequency of switching is reduced, similarly to that of B cells singly deficient in Msh2 or Mlh1. However, the nucleotide sequences of the Smu-Sgamma3 junctions resemble junctions from Mlh1- but not from Msh2-deficient cells, suggesting Mlh1 functions either independently of or before Msh2. The substitution mutations within S regions that are known to accompany switch recombination are increased in Msh2- and Mlh1 single-deficient cells and further increased in the double-deficient cells, again suggesting these proteins function independently in class switch recombination. The finding that MMR functions to reduce mutations in switch regions is unexpected since MMR proteins have been shown to contribute to somatic hypermutation of antibody variable region genes.
| S-EPMC2193780 | biostudies-literature
Project description:Class switch DNA recombinations change the constant (C) region of the antibody heavy (H) chain expressed by a B cell and thereby change the antibody effector function. Unusual tandemly repeated sequence elements located upstream of H chain gene exons have long been thought to be important in the targeting and/or mechanism of the switch recombination process. We have deleted the entire switch tandem repeat element (S(mu)) from the murine (mu) H chain gene. We find that the S(mu) tandem repeats are not required for class switching in the mouse immunoglobulin H-chain locus, although the efficiency of switching is clearly reduced. Our data demonstrate that sequences outside of the S(mu) tandem repeats must be capable of directing the class switch mechanism. The maintenance of the highly repeated S(mu) element during evolution appears to reflect selection for a highly efficient switching process rather than selection for a required sequence element.
| S-EPMC2193334 | biostudies-literature